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Zhang G, Gu T, Wang Y. A Safe and Convenient Method to Isolate Bone Marrow Mononuclear Cells in Clinical Practice. Aesthetic Plast Surg 2025; 49:2085-2096. [PMID: 39638905 DOI: 10.1007/s00266-024-04581-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2024] [Accepted: 11/20/2024] [Indexed: 12/07/2024]
Abstract
BACKGROUND Bone marrow mononuclear cells (BMMNCs) are becoming a promising cell therapy in regeneration medicine. BMMNCs are now obtained by density gradient centrifugation (DGC) in clinical practice, which is complicated and greatly influenced by human manipulation. OBJECTIVE Our objective is to develop a simple and safe method to isolate BMMNCs. METHODS Bone marrow was aspirated from nine minipigs. The optimal hypotonic sodium chloride (NaCl) concentration was first investigated based on the BMMNCs viability and lysis efficiency tests. Afterward, three different methods (ammonium chloride (NH4Cl) lysis, hypotonic NaCl lysis, and DGC) were used for BMMNCs isolation. Nucleated cell yield, residual red blood cells (RBCs) level, BMMNCs viability, apoptotic cell percentage, and colony-forming ability were measured in three groups. Cell morphology, cell phenotype, proliferative capacity, and osteogenic, adipogenic, and chondrogenic lineage differentiation potential of the bone marrow mesenchymal stem cells (BMSCs) were compared in three groups. RESULTS 0.3% NaCl lysis group had optimal cell viability and lysis efficiency and the 0.3% NaCl lysis group had higher cell yield and lower RBCs remaining in BMMNCs compared to the DGC group. The BMSCs harvested from the NH4Cl lysis group had the worst proliferation ability. The NaCl lysis group was not inferior to the other two groups in terms of other biological characteristics of BMMNCs/BMSCs. CONCLUSIONS The optimal concentration for hypotonic NaCl lysis to obtain BMMNCs is 0.3%. Compared with NH4Cl lysis and DGC, the 0.3% NaCl lysis may be a safe, appropriate, and low-cost method for BMMNCs isolation. NO LEVEL ASSIGNED This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .
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Affiliation(s)
- Guang Zhang
- Cleft Lip and Palate Center, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 33 Ba-Da-Chu Road, Shi-Jing-Shan District, Beijing, 100144, China
| | - Tianyi Gu
- The Second Department of Craniomaxillofacial Surgery, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 33 Ba-Da-Chu Road, Shi-Jing-Shan District, Beijing, 100144, China.
| | - Yongqian Wang
- Cleft Lip and Palate Center, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 33 Ba-Da-Chu Road, Shi-Jing-Shan District, Beijing, 100144, China
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Pacini S. Mesangiogenic progenitor cells: a mesengenic and vasculogenic branch of hemopoiesis? A story of neglected plasticity. Front Cell Dev Biol 2025; 13:1513440. [PMID: 40196849 PMCID: PMC11973335 DOI: 10.3389/fcell.2025.1513440] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2024] [Accepted: 02/20/2025] [Indexed: 04/09/2025] Open
Abstract
Mesangiogenic progenitor cells (MPCs) are mesengenic and vasculogenic cells isolated from human bone marrow mononuclear cell cultures. Although MPCs were first described over two decades ago and have demonstrated promising differentiation capabilities, these cells did not attract sufficient attention from experts in the field of tissue regeneration. Several reports from the first decade of the 2000s showed MPC-like cells co-isolated in primary mesenchymal stromal cell (MSC) cultures, applying human serum. However, in most cases, these rounded and firmly attached cells were described as "contaminating" cells of hemopoietic origin. Indeed, MPC morphology, phenotype, and functional features evoke but do not completely overlap with those of cultured peripheral macrophages, and their hemopoietic origin should not be excluded. The plasticity of cells from the monocyte lineage is surprising but not completely unprecedented. Underestimated data demonstrated that circulating monocyte/macrophages could acquire broader plasticity under specific and different culture conditions, and this plasticity could be a consequence of in vitro de-differentiation. The evidence discussed here suggests that MPCs could represent the cell identity toward which the de-differentiation process reprograms the circulating mature phagocytic compartment.
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Affiliation(s)
- Simone Pacini
- Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
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Allouh MZ, Rizvi SFA, Alamri A, Jimoh Y, Aouda S, Ouda ZH, Hamad MIK, Perez-Cruet M, Chaudhry GR. Mesenchymal stromal/stem cells from perinatal sources: biological facts, molecular biomarkers, and therapeutic promises. Stem Cell Res Ther 2025; 16:127. [PMID: 40055783 PMCID: PMC11889844 DOI: 10.1186/s13287-025-04254-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 02/25/2025] [Indexed: 05/13/2025] Open
Abstract
The use of mesenchymal stem cells (MSCs) from perinatal tissue sources has gained attention due to their availability and lack of significant ethical or moral concerns. These cells have a higher proliferative capability than adult MSCs and less immunogenic or tumorigenesis risk than fetal and embryonic stem cells. Additionally, they do not require invasive isolation methods like fetal and adult MSCs. We reviewed the main biological and therapeutic aspects of perinatal MSCs in a three-part article. In the first part, we revised the main biological features and characteristics of MSCs and the advantages of perinatal MSCs over other types of SCs. In the second part, we provided a detailed molecular background for the main biomarkers that can be used to identify MSCs. In the final part, we appraised the therapeutic application of perinatal MSCs in four major degenerative disorders: degenerative disc disease, retinal degenerative diseases, ischemic heart disease, and neurodegenerative diseases. In conclusion, there is no single specific molecular marker to identify MSCs. We recommend using at least two positive markers of stemness (CD29, CD73, CD90, or CD105) and two negative markers (CD34, CD45, or CD14) to exclude the hematopoietic origin. Moreover, utilizing perinatal MSCs for managing degenerative diseases presents a promising therapeutic approach. This review emphasizes the significance of employing more specialized progenitor cells that originated from the perinatal MSCs. The review provides scientific evidence from the literature that applying these progenitor cells in therapeutic procedures provides a greater regenerative capacity than the original primitive MSCs. Finally, this review provides a valuable reference for researchers exploring perinatal MSCs and their therapeutic applications.
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Affiliation(s)
- Mohammed Z Allouh
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, P. O. Box: 15551, Al Ain, UAE.
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA.
| | - Syed Faizan Ali Rizvi
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - Ali Alamri
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - Yusuf Jimoh
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA
| | - Salma Aouda
- College of Medicine and Health Sciences, Khalifa University, Abu Dhabi, UAE
| | - Zakaria H Ouda
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, P. O. Box: 15551, Al Ain, UAE
| | - Mohammad I K Hamad
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, P. O. Box: 15551, Al Ain, UAE
| | - Mick Perez-Cruet
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA
- Department of Neurosurgery, Corewell Health, Royal Oak, MI, USA
| | - G Rasul Chaudhry
- OU-WB Institute for Stem Cell and Regenerative Medicine, Oakland University, Rochester, MI, 48309, USA.
- Department of Biological Sciences, Oakland University, Rochester, MI, 48309, USA.
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4
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Qiu D, Wang L, Wang L, Dong Y. Human platelet lysate: a potential therapeutic for intracerebral hemorrhage. Front Neurosci 2025; 18:1517601. [PMID: 39881806 PMCID: PMC11774881 DOI: 10.3389/fnins.2024.1517601] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2024] [Accepted: 12/30/2024] [Indexed: 01/31/2025] Open
Abstract
Intracerebral hemorrhage (ICH) is a major public health challenge worldwide, and is associated with elevated rates of mortality, disability, and morbidity, especially in low- and middle-income nations. However, our knowledge of the detailed molecular processes involved in ICH remains insufficient, particularly those involved in the secondary injury stage, resulting in a lack of effective treatments for ICH. Human platelet lysates (HPL) are abundant in bioactive factors, and numerous studies have demonstrated their beneficial effects on neurological diseases, including their anti-neuroinflammatory ability, anti-oxidant effects, maintenance of blood-brain barrier integrity, and promotion of neurogenesis. In this review, we thoroughly explore the potential of HPL for treating ICH from three critical perspectives: the rationale for selecting HPL as a treatment for ICH, the mechanisms through which HPL contributes to ICH management, and the additional measures necessary for HPL as a treatment for ICH. We elucidate the role of platelets in ICH pathophysiology and highlight the limitations of the current treatment options and advancements in preclinical research on the application of HPL in neurological disorders. Furthermore, historical developments and preparation methods of HPL in the field of biomedicine are discussed. Additionally, we summarize the bioactive molecules present in HPL and their potential therapeutic effects in ICH. Finally, we outline the issues that must be addressed regarding utilizing HPL as a treatment modality for ICH.
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Affiliation(s)
- Dachang Qiu
- Wuxi School of Medicine, Jiangnan University, Wuxi, China
| | - Lin Wang
- Wuxi School of Medicine, Jiangnan University, Wuxi, China
| | - Lanlan Wang
- Department of Geriatrics, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China
| | - Yongfei Dong
- Department of Neurosurgery, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China
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Oeller M, Schally T, Zimmermann G, Lauth W, Schallmoser K, Rohde E, Laner-Plamberger S. Heparin Differentially Regulates the Expression of Specific miRNAs in Mesenchymal Stromal Cells. Int J Mol Sci 2024; 25:12589. [PMID: 39684301 DOI: 10.3390/ijms252312589] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2024] [Revised: 11/13/2024] [Accepted: 11/21/2024] [Indexed: 12/18/2024] Open
Abstract
In regenerative medicine, stromal cells are supposed to play an important role by modulating immune responses and differentiating into various tissue types. The aim of this study was to investigate the influence of heparin, frequently used as an anticoagulant in human platelet lysate (HPL)-supplemented cell cultures, on the expression of non-coding RNA species, particularly microRNAs (miRNA), which are pivotal regulators of gene expression. Through genomic analysis and quantitative RT-PCR, we assessed the differential impact of heparin on miRNA expression in various stromal cell types, derived from human bone marrow, umbilical cord and white adipose tissue. Our results demonstrate that heparin significantly alters miRNA expression, with distinct up- and downregulation patterns depending on the original tissue source of human stromal cells. Furthermore, our analyses indicate that these heparin-induced alterations in miRNA expression profiles influence critical cellular processes, including proliferation, apoptosis and differentiation. In conclusion, our study highlights that heparin not only fulfills its primary role as an efficient anticoagulant but can also modulate important regulatory pathways in stromal cells by influencing miRNA expression. This may alter cellular properties and thus influence stromal cell-based therapeutic applications in regenerative medicine.
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Affiliation(s)
- Michaela Oeller
- Department for Transfusion Medicine, University Hospital of Salzburg (SALK), Paracelsus Medical University (PMU) Salzburg, Muellner Hauptstraße 48, 5020 Salzburg, Austria
| | - Tanja Schally
- GMP Laboratory, PMU Salzburg, Strubergasse 21, 5020 Salzburg, Austria
| | - Georg Zimmermann
- Team Biostatistics and Big Medical Data, IDA Lab Salzburg, PMU Salzburg, Strubergasse 16, 5020 Salzburg, Austria
- Research Program Biomedical Data Science, PMU Salzburg, Strubergasse 16, 5020 Salzburg, Austria
- Department of Artificial Intelligence and Human Interfaces, Faculty of Digital and Analytical Sciences, Paris Lodron University Salzburg, Jakob Haringer Straße 2, 5020 Salzburg, Austria
| | - Wanda Lauth
- Team Biostatistics and Big Medical Data, IDA Lab Salzburg, PMU Salzburg, Strubergasse 16, 5020 Salzburg, Austria
- Research Program Biomedical Data Science, PMU Salzburg, Strubergasse 16, 5020 Salzburg, Austria
| | - Katharina Schallmoser
- Department for Transfusion Medicine, University Hospital of Salzburg (SALK), Paracelsus Medical University (PMU) Salzburg, Muellner Hauptstraße 48, 5020 Salzburg, Austria
| | - Eva Rohde
- Department for Transfusion Medicine, University Hospital of Salzburg (SALK), Paracelsus Medical University (PMU) Salzburg, Muellner Hauptstraße 48, 5020 Salzburg, Austria
- GMP Laboratory, PMU Salzburg, Strubergasse 21, 5020 Salzburg, Austria
| | - Sandra Laner-Plamberger
- Department for Transfusion Medicine, University Hospital of Salzburg (SALK), Paracelsus Medical University (PMU) Salzburg, Muellner Hauptstraße 48, 5020 Salzburg, Austria
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Mouloud Y, Staubach S, Stambouli O, Mokhtari S, Kutzner TJ, Zwanziger D, Hemeda H, Giebel B. Calcium chloride declotted human platelet lysate promotes the expansion of mesenchymal stromal cells and allows manufacturing of immunomodulatory active extracellular vesicle products. Cytotherapy 2024; 26:988-998. [PMID: 38819364 DOI: 10.1016/j.jcyt.2024.04.069] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2024] [Revised: 04/18/2024] [Accepted: 04/23/2024] [Indexed: 06/01/2024]
Abstract
BACKGROUND Mesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media. METHODS To circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl2-mediated declotting method as the most effective option. To determine whether CaCl2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay. RESULTS AND CONCLUSIONS Our findings conclusively show that CaCl2-declotted hPLs support the production of immunomodulatory-active MSC-EV products.
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Affiliation(s)
- Yanis Mouloud
- Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Simon Staubach
- Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; Sartorius Stedim Biotech GmbH, Göttingen, Germany
| | - Oumaima Stambouli
- Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Shakiba Mokhtari
- Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Tanja J Kutzner
- Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Denise Zwanziger
- Department of Endocrinology, Diabetes and Metabolism and Clinical Chemistry - Division of Laboratory Research, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Hatim Hemeda
- PL BioScience GmbH, Technology Centre Aachen, Aachen, Germany
| | - Bernd Giebel
- Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
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Larsen K, Petrovski G, Boix-Lemonche G. Alternative cryoprotective agent for corneal stroma-derived mesenchymal stromal cells for clinical applications. Sci Rep 2024; 14:15788. [PMID: 38982099 PMCID: PMC11233711 DOI: 10.1038/s41598-024-65469-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Accepted: 06/20/2024] [Indexed: 07/11/2024] Open
Abstract
Cryopreservation of human corneal stroma-derived mesenchymal stromal cells (hCS-MSCs) with dimethylsulfoxide (DMSO) as a cryoprotective agent (CPA) has not been previously compared to that with glycerol under standard conditions. The hCS-MSCs were hereby cryopreserved with both compounds using a freezing rate of 1 °C/minute. The CPAs were tested by different concentrations in complete Minimum Essential Medium (MEM) approved for good manufacturing practice, and a medium frequently used in cell laboratory culturing-Dulbecco's modified eagle serum. The hCS-MSCs were isolated from cadaveric human corneas obtained from the Norwegian Eye Bank, and immunophenotypically characterized by flow cytometry before and after cryopreservation. The survival rate, the cellular adhesion, proliferation and cell surface coverage after cryopreservation of hCS-MSCs has been studied. The hCS-MSCs were immunofluorescent stained and examined for their morphology microscopically. The results showed that cryopreservation of hCS-MSCs in MEM with 10% glycerol gives a higher proliferation rate compared to other cryopreserving media tested. Based on the results, hCS-MSCs can safely be cryopreserved using glycerol instead of the traditional use of DMSO.
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Affiliation(s)
- Kristoffer Larsen
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Goran Petrovski
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway
- Department of Ophthalmology, Oslo University Hospital, Oslo, Norway
- School of Medicine, University of Split, 21000, Split, Croatia
- UKLONetwork, University St. Kliment Ohridski -Bitola, 7000, Bitola, North Macedonia
| | - Gerard Boix-Lemonche
- Center for Eye Research and Innovative Diagnostics, Department of Ophthalmology, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
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Nguyen VVT, Welsh JA, Tertel T, Choo A, van de Wakker SI, Defourny KAY, Giebel B, Vader P, Padmanabhan J, Lim SK, Nolte‐'t Hoen ENM, Verhaar MC, Bostancioglu RB, Zickler AM, Hong JM, Jones JC, EL Andaloussi S, van Balkom BWM, Görgens A. Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures. J Extracell Vesicles 2024; 13:e12463. [PMID: 38868945 PMCID: PMC11170075 DOI: 10.1002/jev2.12463] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2023] [Revised: 04/15/2024] [Accepted: 05/21/2024] [Indexed: 06/14/2024] Open
Abstract
Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.
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Affiliation(s)
| | - Joshua A. Welsh
- Translational Nanobiology Section, Laboratory of Pathology, National Cancer InstituteNational Institutes of HealthBethesdaMarylandUSA
- The Measuring Stick, LtdPeterboroughUK
- Advanced Technology GroupBecton DickinsonSan JoseCaliforniaUSA
| | - Tobias Tertel
- Institute for Transfusion MedicineUniversity Hospital EssenUniversity of Duisburg‐EssenEssenGermany
| | - Andre Choo
- Bioprocessing Technology Institute (BTI)Agency for Science, Technology and Research (A*STAR)SingaporeSingapore
| | - Simonides I. van de Wakker
- Department of Cardiology, Experimental Cardiology LaboratoryUniversity Medical Center Utrecht, Utrecht UniversityUtrechtThe Netherlands
| | - Kyra A. Y. Defourny
- Division of Infectious Diseases & Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary MedicineUtrecht UniversityUtrechtThe Netherlands
| | - Bernd Giebel
- Institute for Transfusion MedicineUniversity Hospital EssenUniversity of Duisburg‐EssenEssenGermany
| | - Pieter Vader
- Department of Cardiology, Experimental Cardiology LaboratoryUniversity Medical Center Utrecht, Utrecht UniversityUtrechtThe Netherlands
- CDL ResearchUniversity Medical Center Utrecht, Utrecht UniversityUtrechtThe Netherlands
| | - Jayanthi Padmanabhan
- Bioprocessing Technology Institute (BTI)Agency for Science, Technology and Research (A*STAR)SingaporeSingapore
| | - Sai Kiang Lim
- Bioprocessing Technology Institute (BTI)Agency for Science, Technology and Research (A*STAR)SingaporeSingapore
| | - Esther N. M. Nolte‐'t Hoen
- Division of Infectious Diseases & Immunology, Department of Biomolecular Health Sciences, Faculty of Veterinary MedicineUtrecht UniversityUtrechtThe Netherlands
| | | | - R. Beklem Bostancioglu
- Division of Biomolecular and Cellular Medicine, Department of Laboratory MedicineKarolinska InstitutetStockholmSweden
- Department of Cellular Therapy and Allogeneic Stem Cell Transplantation (CAST)Karolinska University Hospital Huddinge and Karolinska Comprehensive Cancer CenterStockholmSweden
| | - Antje M. Zickler
- Division of Biomolecular and Cellular Medicine, Department of Laboratory MedicineKarolinska InstitutetStockholmSweden
- Department of Cellular Therapy and Allogeneic Stem Cell Transplantation (CAST)Karolinska University Hospital Huddinge and Karolinska Comprehensive Cancer CenterStockholmSweden
- Karolinska ATMP CenterANA FuturaHuddingeSweden
| | - Jia Mei Hong
- Bioprocessing Technology Institute (BTI)Agency for Science, Technology and Research (A*STAR)SingaporeSingapore
| | - Jennifer C. Jones
- Translational Nanobiology Section, Laboratory of Pathology, National Cancer InstituteNational Institutes of HealthBethesdaMarylandUSA
| | - Samir EL Andaloussi
- Division of Biomolecular and Cellular Medicine, Department of Laboratory MedicineKarolinska InstitutetStockholmSweden
- Department of Cellular Therapy and Allogeneic Stem Cell Transplantation (CAST)Karolinska University Hospital Huddinge and Karolinska Comprehensive Cancer CenterStockholmSweden
- Karolinska ATMP CenterANA FuturaHuddingeSweden
| | | | - André Görgens
- Institute for Transfusion MedicineUniversity Hospital EssenUniversity of Duisburg‐EssenEssenGermany
- Division of Biomolecular and Cellular Medicine, Department of Laboratory MedicineKarolinska InstitutetStockholmSweden
- Department of Cellular Therapy and Allogeneic Stem Cell Transplantation (CAST)Karolinska University Hospital Huddinge and Karolinska Comprehensive Cancer CenterStockholmSweden
- Karolinska ATMP CenterANA FuturaHuddingeSweden
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Tibourtine F, Canceill T, Marfoglia A, Lavalle P, Gibot L, Pilloux L, Aubry C, Medemblik C, Goudouneche D, Dupret-Bories A, Cazalbou S. Advanced Platelet Lysate Aerogels: Biomaterials for Regenerative Applications. J Funct Biomater 2024; 15:49. [PMID: 38391902 PMCID: PMC10890004 DOI: 10.3390/jfb15020049] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2023] [Revised: 02/01/2024] [Accepted: 02/15/2024] [Indexed: 02/24/2024] Open
Abstract
Human platelet lysate (HPL), rich in growth factors, is increasingly recognized for its potential in tissue engineering and regenerative medicine. However, its use in liquid or gel form is constrained by limited stability and handling difficulties. This study aimed to develop dry and porous aerogels from HPL hydrogel using an environmentally friendly supercritical CO2-based shaping process, specifically tailored for tissue engineering applications. The aerogels produced retained their three-dimensional structure and demonstrated significant mechanical robustness and enhanced manageability. Impressively, they exhibited high water absorption capacity, absorbing 87% of their weight in water within 120 min. Furthermore, the growth factors released by these aerogels showed a sustained and favourable biological response in vitro. They maintained the cellular metabolic activity of fibroblasts (BALB-3T3) at levels akin to conventional culture conditions, even after prolonged storage, and facilitated the migration of human umbilical vein endothelial cells (HUVECs). Additionally, the aerogels themselves supported the adhesion and proliferation of murine fibroblasts (BALB-3T3). Beyond serving as excellent matrices for cell culture, these aerogels function as efficient systems for the delivery of growth factors. Their multifunctional capabilities position them as promising candidates for various tissue regeneration strategies. Importantly, the developed aerogels can be stored conveniently and are considered ready to use, enhancing their practicality and applicability in regenerative medicine.
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Affiliation(s)
- Fahd Tibourtine
- CIRIMAT, Université Toulouse 3 Paul Sabatier, Toulouse INP, CNRS, Université de Toulouse, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France
| | - Thibault Canceill
- Département Odontologie, Faculté de Santé, Hôpitaux de Toulouse, Université Paul Sabatier, 3 Chemin des Maraichers, CEDEX 9, 31062 Toulouse, France
| | - Andrea Marfoglia
- CIRIMAT, Université Toulouse 3 Paul Sabatier, Toulouse INP, CNRS, Université de Toulouse, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France
- Laboratoire de Génie Chimique, Université Toulouse 3 Paul Sabatier, Toulouse INP, CNRS, Université de Toulouse, 31062 Toulouse, France
| | - Philippe Lavalle
- Institut National de la Santé et de la Recherche Médicale, Inserm UMR_S 1121 Biomaterials and Bioengineering, 67085 Strasbourg, France
| | - Laure Gibot
- Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III-Paul Sabatier, 31062 Toulouse, France
| | - Ludovic Pilloux
- Laboratoire de Génie Chimique, Université Toulouse 3 Paul Sabatier, Toulouse INP, CNRS, Université de Toulouse, 31062 Toulouse, France
| | - Clementine Aubry
- ARNA, Inserm U1212, CNRS 5320, University of Bordeaux, 146 Rue Léo Saignat, CEDEX, 33076 Bordeaux, France
| | - Claire Medemblik
- Institut National de la Santé et de la Recherche Médicale, Inserm UMR_S 1121 Biomaterials and Bioengineering, 67085 Strasbourg, France
| | - Dominique Goudouneche
- Centre de Microscopie Electronique Appliquée à la Biologie, Faculté de Médecine, 133 Route de Narbonne, 31062 Toulouse, France
| | - Agnès Dupret-Bories
- CIRIMAT, Université Toulouse 3 Paul Sabatier, Toulouse INP, CNRS, Université de Toulouse, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France
- Department of Surgery, University Cancer Institute of Toulouse-Oncopole, 1 Avenue Irène Joliot-Curie, 31100 Toulouse, France
- Department of Ear, Nose and Throat Surgery, Toulouse University Hospital-Larrey Hospital, 31400 Toulouse, France
| | - Sophie Cazalbou
- CIRIMAT, Université Toulouse 3 Paul Sabatier, Toulouse INP, CNRS, Université de Toulouse, 118 Route de Narbonne, CEDEX 9, 31062 Toulouse, France
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10
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De Korte D, Delabie W, Feys HB, Klei T, Larsen R, Sigurjónsson Ó, Sousa AP. Towards standardized human platelet lysate production in Europe: An initiative of the European Blood Alliance. Vox Sang 2024; 119:79-87. [PMID: 38049931 DOI: 10.1111/vox.13562] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2023] [Revised: 10/23/2023] [Accepted: 10/25/2023] [Indexed: 12/06/2023]
Abstract
Human platelet lysate (hPL) is a supplement for cell culture media that can be derived from platelet concentrates. As not-for-profit blood establishments, we endorse the evolution of maximally exploiting the potential of donated blood and its derived components, including platelets. The decision to use platelet concentrates to supply hPL as a cell culture supplement should align with the principles and values that blood establishments hold towards the use of donated blood components in transfusion. As a consequence, questions on ethics, practical standardization of hPL production and logistics as well as on assuring hPL quality and safety need careful consideration. We therefore propose an opinion on some of these matters based on available literature and on discussions within the proceedings of the Working Group on Innovation and New Products of the European Blood Alliance. In addition, we propose collaboration among European blood establishments to streamline efforts of hPL supply to maximize the potential of hPL and its application in the wider field of medicine.
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Affiliation(s)
- Dirk De Korte
- Department of Product and Process Development, Sanquin Blood Bank, Amsterdam, The Netherlands
| | - Willem Delabie
- Transfusion Research Center, Belgian Red Cross Flanders, Ghent, Belgium
| | - Hendrik B Feys
- Transfusion Research Center, Belgian Red Cross Flanders, Ghent, Belgium
- Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
| | - Thomas Klei
- Department of Product and Process Development, Sanquin Blood Bank, Amsterdam, The Netherlands
| | - Rune Larsen
- Department of Clinical Immunology, Zealand University Hospital, Køge, Denmark
| | - Ólafur Sigurjónsson
- The Blood Bank, Landspitali University Hospital, Reykjavik, Iceland
- School of Science and Engineering, Reykjavik University, Reykjavik, Iceland
| | - Ana Paula Sousa
- Blood and Transplantation Centre of Lisboa, Portuguese Institute for Blood and Transplantation (IPST), Lisbon, Portugal
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11
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Fitzgerald JC, Shaw G, Murphy JM, Barry F. Media matters: culture medium-dependent hypervariable phenotype of mesenchymal stromal cells. Stem Cell Res Ther 2023; 14:363. [PMID: 38087388 PMCID: PMC10717324 DOI: 10.1186/s13287-023-03589-w] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2023] [Accepted: 11/28/2023] [Indexed: 12/18/2023] Open
Abstract
BACKGROUND Despite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action. METHODS This work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers. RESULTS Significant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media. CONCLUSIONS These data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release.
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Affiliation(s)
- Joan C Fitzgerald
- Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland
| | - Georgina Shaw
- Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland
| | - J Mary Murphy
- Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland
| | - Frank Barry
- Regenerative Medicine Institute (REMEDI), University of Galway, Galway, Ireland.
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12
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Burnouf T, Chou ML, Lundy DJ, Chuang EY, Tseng CL, Goubran H. Expanding applications of allogeneic platelets, platelet lysates, and platelet extracellular vesicles in cell therapy, regenerative medicine, and targeted drug delivery. J Biomed Sci 2023; 30:79. [PMID: 37704991 PMCID: PMC10500824 DOI: 10.1186/s12929-023-00972-w] [Citation(s) in RCA: 50] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Accepted: 08/23/2023] [Indexed: 09/15/2023] Open
Abstract
Platelets are small anucleated blood cells primarily known for their vital hemostatic role. Allogeneic platelet concentrates (PCs) collected from healthy donors are an essential cellular product transfused by hospitals to control or prevent bleeding in patients affected by thrombocytopenia or platelet dysfunctions. Platelets fulfill additional essential functions in innate and adaptive immunity and inflammation, as well as in wound-healing and tissue-repair mechanisms. Platelets contain mitochondria, lysosomes, dense granules, and alpha-granules, which collectively are a remarkable reservoir of multiple trophic factors, enzymes, and signaling molecules. In addition, platelets are prone to release in the blood circulation a unique set of extracellular vesicles (p-EVs), which carry a rich biomolecular cargo influential in cell-cell communications. The exceptional functional roles played by platelets and p-EVs explain the recent interest in exploring the use of allogeneic PCs as source material to develop new biotherapies that could address needs in cell therapy, regenerative medicine, and targeted drug delivery. Pooled human platelet lysates (HPLs) can be produced from allogeneic PCs that have reached their expiration date and are no longer suitable for transfusion but remain valuable source materials for other applications. These HPLs can substitute for fetal bovine serum as a clinical grade xeno-free supplement of growth media used in the in vitro expansion of human cells for transplantation purposes. The use of expired allogeneic platelet concentrates has opened the way for small-pool or large-pool allogeneic HPLs and HPL-derived p-EVs as biotherapy for ocular surface disorders, wound care and, potentially, neurodegenerative diseases, osteoarthritis, and others. Additionally, allogeneic platelets are now seen as a readily available source of cells and EVs that can be exploited for targeted drug delivery vehicles. This article aims to offer an in-depth update on emerging translational applications of allogeneic platelet biotherapies while also highlighting their advantages and limitations as a clinical modality in regenerative medicine and cell therapies.
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Affiliation(s)
- Thierry Burnouf
- Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan.
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan.
- International Ph.D. Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
| | - Ming-Li Chou
- Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan
- Institute of Clinical Medicine, National Yang-Ming Chiao Tung University, Taipei, Taiwan
| | - David J Lundy
- Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan
| | - Er-Yuan Chuang
- Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan
| | - Ching-Li Tseng
- Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan
- International Ph.D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan
| | - Hadi Goubran
- Saskatoon Cancer Centre and College of Medicine, University of Saskatchewan, Saskatchewan, Canada
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13
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Jakl V, Popp T, Haupt J, Port M, Roesler R, Wiese S, Friemert B, Rojewski MT, Schrezenmeier H. Effect of Expansion Media on Functional Characteristics of Bone Marrow-Derived Mesenchymal Stromal Cells. Cells 2023; 12:2105. [PMID: 37626914 PMCID: PMC10453497 DOI: 10.3390/cells12162105] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Revised: 08/07/2023] [Accepted: 08/16/2023] [Indexed: 08/27/2023] Open
Abstract
The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFβ, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.
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Affiliation(s)
- Viktoria Jakl
- Institute for Transfusion Medicine, University Hospital Ulm, 89081 Ulm, Germany; (V.J.)
| | - Tanja Popp
- Bundeswehr Institute of Radiobiology, 80937 Munich, Germany (J.H.); (M.P.)
| | - Julian Haupt
- Bundeswehr Institute of Radiobiology, 80937 Munich, Germany (J.H.); (M.P.)
- Clinic for Trauma Surgery and Orthopedics, Army Hospital Ulm, 89081 Ulm, Germany
| | - Matthias Port
- Bundeswehr Institute of Radiobiology, 80937 Munich, Germany (J.H.); (M.P.)
| | - Reinhild Roesler
- Core Unit of Mass Spectrometry and Proteomics, Ulm University Medical Center, 89081 Ulm, Germany; (R.R.); (S.W.)
| | - Sebastian Wiese
- Core Unit of Mass Spectrometry and Proteomics, Ulm University Medical Center, 89081 Ulm, Germany; (R.R.); (S.W.)
| | - Benedikt Friemert
- Clinic for Trauma Surgery and Orthopedics, Army Hospital Ulm, 89081 Ulm, Germany
| | - Markus T. Rojewski
- Institute for Transfusion Medicine, University Hospital Ulm, 89081 Ulm, Germany; (V.J.)
- Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Donation Service Baden-Württemberg—Hessia and University Hospital Ulm, 89081 Ulm, Germany
| | - Hubert Schrezenmeier
- Institute for Transfusion Medicine, University Hospital Ulm, 89081 Ulm, Germany; (V.J.)
- Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Donation Service Baden-Württemberg—Hessia and University Hospital Ulm, 89081 Ulm, Germany
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14
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Yaja K, Aungsuchawan S, Narakornsak S, Pothacharoen P, Pantan R, Tancharoen W. Combination of human platelet lysate and 3D gelatin scaffolds to enhance osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells. Heliyon 2023; 9:e18599. [PMID: 37576189 PMCID: PMC10413082 DOI: 10.1016/j.heliyon.2023.e18599] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2023] [Revised: 07/14/2023] [Accepted: 07/21/2023] [Indexed: 08/15/2023] Open
Abstract
Bone disorders are major health issues requiring specialized care; however, the traditional bone grafting method had several limitations. Thus, bone tissue engineering has become a potential alternative. In therapeutic treatments, using fetal bovine serum (FBS) as a culture supplement may result in the risk of contamination and host immunological response; therefore, human platelet lysate (hPL) has been considered a viable alternative source. This study attempted to compare the effectiveness and safety of different culture supplements, either FBS or hPL, on the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid (hAF-MSCs) under a three-dimensional gelatin scaffold. The results indicate that hAF-MSCs have the potential to be used in clinical applications as they meet the criteria for mesenchymal stem cells based on their morphology, the expression of a particular surface antigen, their proliferation ability, and their capacity for multipotent differentiation. After evaluation by MTT and Alamar blue proliferation assay, 10% of hPL was selected. The osteogenic differentiation of hAF-MSCs under three-dimensional gelatin scaffold using osteogenic-induced media supplemented with hPL was achievable and markedly stimulated osteoblast differentiation. Moreover, the expressions of osteoblastogenic related genes, including OCN, ALP, and COL1A1, exhibited the highest degree of expression under hPL-supplemented circumstances when compared with the control and the FBS-supplemented group. The induced cells under hPL-supplemented conditions also presented the highest ALP activity level and the greatest degree of calcium accumulation. These outcomes would indicate that hPL is a suitable substitute for animal derived serum. Importantly, osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells using hPL-supplemented media and three-dimensional scaffolds may open the door to developing an alternative construct for repairing bone defects.
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Affiliation(s)
- Kantirat Yaja
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Sirinda Aungsuchawan
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Suteera Narakornsak
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Peraphan Pothacharoen
- Thailand Excellence Center for Tissue Engineering and Stem Cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Thailand
| | - Rungusa Pantan
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
| | - Waleephan Tancharoen
- Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
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15
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Secretome of Adipose-Derived Stem Cells Cultured in Platelet Lysate Improves Migration and Viability of Keratinocytes. Int J Mol Sci 2023; 24:ijms24043522. [PMID: 36834932 PMCID: PMC9962933 DOI: 10.3390/ijms24043522] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2022] [Revised: 02/05/2023] [Accepted: 02/07/2023] [Indexed: 02/12/2023] Open
Abstract
Chronic wounds depict a silent epidemic challenging medical professionals worldwide. Regenerative medicine uses adipose-derived stem cells (ADSC) in promising new therapies. In this study, platelet lysate (PL) as a xenogen-free substitute for foetal bovine serum (FBS) in ADSC culture was used to create an ADSC secretome containing cytokines for optimal wound healing conditions. The ADSC secretome was tested on keratinocytes for migrational behaviour and viability. Therefore, human ADSC were characterized under FBS (10%) and PL (5% and 10%) substitution, regarding morphology, differentiation, viability, gene and protein expression. ADSC were then cultured in 5% PL and their secretome was used for stimulation of keratinocyte migration and viability. To enhance the effect, ADSC were treated with Epithelial Growth Factor (EGF, 100 ng/mL) and hypoxia (1% O₂). In both PL and FBS groups, ADSC expressed typical stem cell markers. PL induced a significantly higher increase in cell viability compared to FBS substitution. ADSC secretome contained various beneficial proteins which enhance the wound healing capacity of keratinocytes. This could be optimized treating ADSC with hypoxia and EGF. In conclusion, the study shows that ADSC cultivated in 5% PL can effectively support wound healing conditions and can be considered as a promising new therapy for individual treatment of chronic wound disorders.
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16
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Jenner F, Wagner A, Gerner I, Ludewig E, Trujanovic R, Rohde E, von Rechenberg B, Gimona M, Traweger A. Evaluation of the Potential of Umbilical Cord Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles to Improve Rotator Cuff Healing: A Pilot Ovine Study. Am J Sports Med 2023; 51:331-342. [PMID: 36645050 DOI: 10.1177/03635465221145958] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
BACKGROUND Despite significant advancements in surgical techniques to repair rotator cuff (RC) injuries, failure rates remain high and novel approaches to adequately overcome the natural biological limits of tendon and enthesis regeneration of the RC are required. Small extracellular vesicles (sEVs) derived from the secretome of human multipotent mesenchymal stromal cells (MSCs) have been demonstrated to modulate inflammation and reduce fibrotic adhesions, and therefore their local application could improve outcomes after RC repair. PURPOSE In this pilot study, we evaluated the efficacy of clinical-grade human umbilical cord (hUC) MSC-derived sEVs (hUC-MSC-sEVs) loaded onto a type 1 collagen scaffold in an ovine model of acute infraspinatus tendon injury to improve RC healing. STUDY DESIGN Controlled laboratory study. METHODS sEVs were enriched from hUC-MSC culture media and were characterized by surface marker profiling. The immunomodulatory capacity was evaluated in vitro by T-cell proliferation assays, and particle count was determined by nanoparticle tracking analysis. Twelve skeletally mature sheep were subjected to partial infraspinatus tenotomy and enthesis debridement. The defects of 6 animals were treated with 2 × 1010 hUC-MSC-sEVs loaded onto a type 1 collagen sponge, whereas 6 animals received only a collagen sponge, serving as controls. Six weeks postoperatively, the healing of the infraspinatus tendon and the enthesis was evaluated by magnetic resonance imaging (MRI) and hard tissue histology. RESULTS CD3/CD28-stimulated T-cell proliferation was significantly inhibited by hUC-MSC-sEVs (P = .015) that displayed the typical surface marker profile, including the presence of the MSC marker proteins CD44 and melanoma-associated chondroitin sulfate proteoglycan. The local application of hUC-MSC-sEVs did not result in any marked systemic adverse events. Histologically, significantly improved Watkins scores (P = .031) indicated improved tendon and tendon-to-bone insertion repair after sEV treatment and lower postcontrast signal of the tendon and adjacent structures on MRI suggested less residual inflammation at the defect area. Furthermore, the formation of osteophytes at the injury site was significantly attenuated (P = .037). CONCLUSION A local, single-dose application of hUC-MSC-sEVs promoted tendon and enthesis healing in an ovine model of acute RC injury. CLINICAL RELEVANCE Surgical repair of RC tears generally results in a clinical benefit for the patient; however, considerable rerupture rates have been reported. sEVs have potential as a cell-free biotherapeutic to improve healing outcomes after RC injury.
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Affiliation(s)
- Florien Jenner
- VETERM, Equine Surgery Unit, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria.,Austrian Cluster for Tissue Regeneration, Vienna, Austria
| | - Andrea Wagner
- Institute of Tendon and Bone Regeneration, Spinal Cord Injury and Tissue Regeneration Centre Salzburg, Paracelsus Medical University, Salzburg, Austria.,Austrian Cluster for Tissue Regeneration, Vienna, Austria
| | - Iris Gerner
- VETERM, Equine Surgery Unit, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria.,Austrian Cluster for Tissue Regeneration, Vienna, Austria
| | - Eberhard Ludewig
- Diagnostic Imaging Unit, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Robert Trujanovic
- Clinical Unit of Anaesthesiology and Perioperative Intensive Care, Department for Companion Animals and Horses, University of Veterinary Medicine Vienna, Vienna, Austria
| | - Eva Rohde
- Department of Transfusion Medicine, Salzburger Landeskliniken GesmbH, Paracelsus Medical University, Salzburg, Austria.,GMP Unit, Spinal Cord Injury and Tissue Regeneration Centre Salzburg, Paracelsus Medical University, Salzburg, Austria
| | - Brigitte von Rechenberg
- Musculoskeletal Research Unit (MSRU), Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.,Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Zurich, Switzerland
| | - Mario Gimona
- GMP Unit, Spinal Cord Injury and Tissue Regeneration Centre Salzburg, Paracelsus Medical University, Salzburg, Austria.,Research Program "Nanovesicular Therapies," Paracelsus Medical University, Salzburg, Austria
| | - Andreas Traweger
- Institute of Tendon and Bone Regeneration, Spinal Cord Injury and Tissue Regeneration Centre Salzburg, Paracelsus Medical University, Salzburg, Austria.,Austrian Cluster for Tissue Regeneration, Vienna, Austria
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17
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Delabie W, De Bleser D, Vandewalle V, Vandekerckhove P, Compernolle V, Feys HB. Single step method for high yield human platelet lysate production. Transfusion 2023; 63:373-383. [PMID: 36426732 PMCID: PMC10099704 DOI: 10.1111/trf.17188] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2022] [Revised: 10/28/2022] [Accepted: 11/01/2022] [Indexed: 11/27/2022]
Abstract
BACKGROUND We aimed to develop a single step method for the production of human platelet lysate (hPL). The method must result in high hPL yields, be closed system and avoid heparin use. STUDY DESIGN AND METHODS The method aimed at using glass beads and calcium. An optimal concentration of calcium and glass beads was determined by serial dilution. This was translated to a novel method and compared to known methods: freeze-thawing and high calcium. Quality outcome measures were transmittance, fibrinogen and growth factor content, and cell doubling time. RESULTS An optimal concentration of 5 mM Ca2+ and 0.2 g/ml glass beads resulted in hPL with yields of 92% ± 1% (n = 50) independent of source material (apheresis or buffy coat-derived). The transmittance was highest (56% ± 9%) compared to known methods (<39%). The fibrinogen concentration (7.0 ± 1.1 μg/ml) was well below the threshold, avoiding the need for heparin. Growth factor content was similar across hPL production methods. The cell doubling time of adipose derived stem cells was 25 ± 1 h and not different across methods. Batch consistency was determined across six batches of hPL (each n = 25 constituting concentrates) and was <11% for all parameters including cell doubling time. Calcium precipitation formed after 4 days of culturing stem cells in media with hPL prepared by the high (15 mM) Ca2+ method, but not with hPL prepared by glass bead method. DISCUSSION The novel method transforms platelet concentrates to hPL with little hands-on time. The method results in high yield, is closed system, without heparin and non-inferior to published methods.
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Affiliation(s)
- Willem Delabie
- Transfusion Research Center, Belgian Red Cross-Flanders, Ghent, Belgium
| | - Dominique De Bleser
- Transfusion Innovation Center, Belgian Red Cross-Flanders, Ghent, Belgium.,Blood Services, Belgian Red Cross-Flanders, Mechelen, Belgium
| | - Vicky Vandewalle
- Transfusion Innovation Center, Belgian Red Cross-Flanders, Ghent, Belgium.,Blood Services, Belgian Red Cross-Flanders, Mechelen, Belgium
| | - Philippe Vandekerckhove
- Blood Services, Belgian Red Cross-Flanders, Mechelen, Belgium.,Department of Public Health and Primary Care, Faculty of Medicine, KU Leuven, Leuven, Belgium.,Department of Global Health, Stellenbosch University, Stellenbosch, South Africa
| | - Veerle Compernolle
- Transfusion Research Center, Belgian Red Cross-Flanders, Ghent, Belgium.,Transfusion Innovation Center, Belgian Red Cross-Flanders, Ghent, Belgium.,Blood Services, Belgian Red Cross-Flanders, Mechelen, Belgium.,Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
| | - Hendrik B Feys
- Transfusion Research Center, Belgian Red Cross-Flanders, Ghent, Belgium.,Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium
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18
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Canceill T, Jourdan G, Kémoun P, Guissard C, Monsef YA, Bourdens M, Chaput B, Cavalie S, Casteilla L, Planat-Bénard V, Monsarrat P, Raymond-Letron I. Characterization and Safety Profile of a New Combined Advanced Therapeutic Medical Product Platelet Lysate-Based Fibrin Hydrogel for Mesenchymal Stromal Cell Local Delivery in Regenerative Medicine. Int J Mol Sci 2023; 24:ijms24032206. [PMID: 36768532 PMCID: PMC9916739 DOI: 10.3390/ijms24032206] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2022] [Revised: 01/14/2023] [Accepted: 01/16/2023] [Indexed: 01/25/2023] Open
Abstract
Adipose-derived mesenchymal stromal cells (ASC) transplant to recover the optimal tissue structure/function relationship is a promising strategy to regenerate tissue lesions. Because filling local tissue defects by injection alone is often challenging, designing adequate cell carriers with suitable characteristics is critical for in situ ASC delivery. The aim of this study was to optimize the generation phase of a platelet-lysate-based fibrin hydrogel (PLFH) as a proper carrier for in situ ASC implantation and (1) to investigate in vitro PLFH biomechanical properties, cell viability, proliferation and migration sustainability, and (2) to comprehensively assess the local in vivo PLFH/ASC safety profile (local tolerance, ASC fate, biodistribution and toxicity). We first defined the experimental conditions to enhance physicochemical properties and microscopic features of PLFH as an adequate ASC vehicle. When ASC were mixed with PLFH, in vitro assays exhibited hydrogel supporting cell migration, viability and proliferation. In vivo local subcutaneous and subgingival PLFH/ASC administration in nude mice allowed us to generate biosafety data, including biodegradability, tolerance, ASC fate and engraftment, and the absence of biodistribution and toxicity to non-target tissues. Our data strongly suggest that this novel combined ATMP for in situ administration is safe with an efficient local ASC engraftment, supporting the further development for human clinical cell therapy.
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Affiliation(s)
- Thibault Canceill
- CIRIMAT, Université Toulouse III Paul Sabatier, CNRS UMR 5085, INPT, Faculté de Pharmacie, 35 Chemin des Maraichers, CEDEX 09, 31062 Toulouse, France
- Department of Oral Medicine and Toulouse University Hospital (CHU of Toulouse)—Toulouse Institute of Oral Medicine and Science, CEDEX 09, 31062 Toulouse, France
| | - Géraldine Jourdan
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
| | - Philippe Kémoun
- Department of Oral Medicine and Toulouse University Hospital (CHU of Toulouse)—Toulouse Institute of Oral Medicine and Science, CEDEX 09, 31062 Toulouse, France
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
| | - Christophe Guissard
- Department of Oral Medicine and Toulouse University Hospital (CHU of Toulouse)—Toulouse Institute of Oral Medicine and Science, CEDEX 09, 31062 Toulouse, France
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
| | - Yanad Abou Monsef
- LabHPEC, Histology and Pathology Department, Université de Toulouse, ENVT, CEDEX 03, 31076 Toulouse, France
| | - Marion Bourdens
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
| | - Benoit Chaput
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
- Service de Chirurgie Plastique, Reconstructrice et Esthétique, Centre Hospitalier Universitaire Rangueil, Avenue du Professeur Jean Poulhès, CEDEX 09, 31059 Toulouse, France
| | - Sandrine Cavalie
- CIRIMAT, Université Toulouse III Paul Sabatier, CNRS UMR 5085, INPT, Faculté de Pharmacie, 35 Chemin des Maraichers, CEDEX 09, 31062 Toulouse, France
| | - Louis Casteilla
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
| | - Valérie Planat-Bénard
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
| | - Paul Monsarrat
- Department of Oral Medicine and Toulouse University Hospital (CHU of Toulouse)—Toulouse Institute of Oral Medicine and Science, CEDEX 09, 31062 Toulouse, France
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
- Artificial and Natural Intelligence Toulouse Institute ANITI, 31400 Toulouse, France
- Correspondence:
| | - Isabelle Raymond-Letron
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Batiment INCERE, 4bis Avenue Hubert Curien, 31100 Toulouse, France
- LabHPEC, Histology and Pathology Department, Université de Toulouse, ENVT, CEDEX 03, 31076 Toulouse, France
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Transcription Factors STAT3 and MYC Are Key Players of Human Platelet Lysate-Induced Cell Proliferation. Int J Mol Sci 2022; 23:ijms232415782. [PMID: 36555426 PMCID: PMC9781157 DOI: 10.3390/ijms232415782] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 12/07/2022] [Accepted: 12/10/2022] [Indexed: 12/15/2022] Open
Abstract
Human platelet lysate (HPL) is an efficient alternative for animal serum supplements, significantly enhancing stromal cell proliferation. However, the molecular mechanism behind this growth-promoting effect remains elusive. The aim of this study was to investigate the effect of HPL on cell cycle gene expression in different human stromal cells and to identify the main key players that mediate HPL's growth-enhancing effect. RT-qPCR and an antibody array revealed significant upregulation of cell cycle genes in stromal cells cultured in HPL. As HPL is rich in growth factors that are ligands of tyrosine kinase receptor (TKR) pathways, we used TKR inhibitors and could significantly reduce cell proliferation. Genome profiling, RT-qPCR and Western blotting revealed an enhanced expression of the transcription factors signal transducer and activator of transcription 3 (STAT3) and MYC, both known TKR downstream effectors and stimulators of cell proliferation, in response to HPL. In addition, specifically blocking STAT3 resulted in reduced cell proliferation and expression of cell cycle genes. Our data indicate that HPL-enhanced cell proliferation can, at least in part, be explained by the TKR-enhanced expression of STAT3 and MYC, which in turn induce the expression of genes being involved in the promotion and control of the cell cycle.
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Almeria C, Kreß S, Weber V, Egger D, Kasper C. Heterogeneity of mesenchymal stem cell-derived extracellular vesicles is highly impacted by the tissue/cell source and culture conditions. Cell Biosci 2022; 12:51. [PMID: 35501833 PMCID: PMC9063275 DOI: 10.1186/s13578-022-00786-7] [Citation(s) in RCA: 43] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 04/10/2022] [Indexed: 12/19/2022] Open
Abstract
AbstractExtracellular vesicles (EVs) are cell-derived membrane structures exerting major effects in physiological as well as pathological processes by functioning as vehicles for the delivery of biomolecules to their target cells. An increasing number of effects previously attributed to cell-based therapies have been recognized to be actually mediated by EVs derived from the respective cells, suggesting the administration of purified EVs instead of living cells for cell-based therapies. In this review, we focus on the heterogeneity of EVs derived from mesenchymal stem/stromal cells (MSC) and summarize upstream process parameters that crucially affect the resulting therapeutic properties and biological functions. Hereby, we discuss the effects of the cell source, medium composition, 3D culture, bioreactor culture and hypoxia. Furthermore, aspects of the isolation and storage strategies influences EVs are described. Conclusively, optimization of upstream process parameters should focus on controlling MSC-derived EV heterogeneity for specific therapeutic applications.
Graphical Abstract
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Jaloux C, Bonnet M, Vogtensperger M, Witters M, Veran J, Giraudo L, Sabatier F, Michel J, Legré R, Guiraudie-Capraz G, Féron F. Human nasal olfactory stem cells, purified as advanced therapy medicinal products, improve neuronal differentiation. Front Neurosci 2022; 16:1042276. [PMID: 36466172 PMCID: PMC9713000 DOI: 10.3389/fnins.2022.1042276] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2022] [Accepted: 11/04/2022] [Indexed: 04/11/2024] Open
Abstract
BACKGROUND Olfactory ecto-mesenchymal stem cells (OE-MSC) are mesenchymal stem cells derived from the lamina propria of the nasal mucosa. They display neurogenic and immunomodulatory properties and were shown to induce recovery in animal models of spinal cord trauma, hearing loss, Parkinsons's disease, amnesia, and peripheral nerve injury. As a step toward clinical practice, we sought to (i) devise a culture protocol that meets the requirements set by human health agencies and (ii) assess the efficacy of stem cells on neuron differentiation. METHODS Nasal olfactory mucosa biopsies from three donors were used to design and validate the good manufacturing process for purifying stem cells. All processes and procedures were performed by expert staff from the cell therapy laboratory of the public hospital of Marseille (AP-HM), according to aseptic handling manipulations. Premises, materials and air were kept clean at all times to avoid cross-contamination, accidents, or even fatalities. Purified stem cells were cultivated for 24 or 48 h and conditioned media were collected before being added to the culture medium of the neuroblastoma cell line Neuro2a. RESULTS Compared to the explant culture-based protocol, enzymatic digestion provides higher cell numbers more rapidly and is less prone to contamination. The use of platelet lysate in place of fetal calf serum is effective in promoting higher cell proliferation (the percentage of CFU-F progenitors is 15.5%), with the optimal percentage of platelet lysate being 10%. Cultured OE-MSCs do not show chromosomal rearrangement and, as expected, express the usual phenotypic markers of mesenchymal stem cells. When incorporated in standard culture medium, the conditioned medium of purified OE-MSCs promotes cell differentiation of Neuro2a neuroblastoma cells. CONCLUSION We developed a safer and more efficient manufacturing process for clinical grade olfactory stem cells. With this protocol, human OE-MSCs will soon be used in a Phase I clinical based on their autologous transplantation in digital nerves with a neglected injury. However, further studies are required to unveil the underlying mechanisms of action.
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Affiliation(s)
- Charlotte Jaloux
- CNRS, INP, UMR 7051, Institut de Neuropathophysiologie, Equipe Nasal Olfactory Stemness and Epigenesis (NOSE), Aix Marseille University, Marseille, France
- Department of Hand Surgery and Reconstructive Surgery of the Limbs, La Timone University Hospital, Assistance Publique Hôpitaux de Marseille, Marseille, France
| | - Maxime Bonnet
- CNRS, INP, UMR 7051, Institut de Neuropathophysiologie, Equipe Nasal Olfactory Stemness and Epigenesis (NOSE), Aix Marseille University, Marseille, France
- Faculté des Sciences du Sport de Marseille, CNRS, ISM, UMR 7287, Institut des Sciences du Mouvement Etienne-Jules MAREY, Equipe Plasticité des Systèmes Nerveux et Musculaire (PSNM), Parc Scientifique et Technologique de Luminy, Aix Marseille University, Marseille, France
| | - Marie Vogtensperger
- Cell Therapy Department, Hôpital de la Conception, AP-HM, INSERM CIC BT 1409, Marseille, France
| | - Marie Witters
- CNRS, INP, UMR 7051, Institut de Neuropathophysiologie, Equipe Nasal Olfactory Stemness and Epigenesis (NOSE), Aix Marseille University, Marseille, France
- Department of Hand Surgery and Reconstructive Surgery of the Limbs, La Timone University Hospital, Assistance Publique Hôpitaux de Marseille, Marseille, France
| | - Julie Veran
- Cell Therapy Department, Hôpital de la Conception, AP-HM, INSERM CIC BT 1409, Marseille, France
| | - Laurent Giraudo
- Cell Therapy Department, Hôpital de la Conception, AP-HM, INSERM CIC BT 1409, Marseille, France
| | - Florence Sabatier
- Cell Therapy Department, Hôpital de la Conception, AP-HM, INSERM CIC BT 1409, Marseille, France
- Aix-Marseille Université, C2VN, UMR-1263, INSERM, INRA 1260, UFR de Pharmacie, Marseille, France
| | - Justin Michel
- Department of Otorhinolaryngology and Head and Neck Surgery, Assistance Publique des Hôpitaux de Marseille, Institut Universitaire des Systèmes Thermiques Industriels, La Conception University Hospital, Aix Marseille University, Marseille, France
| | - Regis Legré
- Department of Hand Surgery and Reconstructive Surgery of the Limbs, La Timone University Hospital, Assistance Publique Hôpitaux de Marseille, Marseille, France
| | - Gaëlle Guiraudie-Capraz
- CNRS, INP, UMR 7051, Institut de Neuropathophysiologie, Equipe Nasal Olfactory Stemness and Epigenesis (NOSE), Aix Marseille University, Marseille, France
| | - François Féron
- CNRS, INP, UMR 7051, Institut de Neuropathophysiologie, Equipe Nasal Olfactory Stemness and Epigenesis (NOSE), Aix Marseille University, Marseille, France
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22
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Qiu G, Huang M, Ke D, Liu J, Weir MD, Ma T, Wang P, Oates TW, Schneider A, Xia Y, Xu HHK, Zhao L. Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration. FRONTIERS IN MATERIALS 2022; 9. [DOI: 10.3389/fmats.2022.977853] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
Abstract
Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications.Methods: hPDLSCs were encapsulated in alginate-fibrin microbeads (DAMB + fibrin), alginate-hPL degradable microbeads (DAMB + hPL), or alginate-fibrin-hPL microbeads (DAMB + fibrin + hPL). The proliferation and osteogenic differentiation of hPDLSCs were investigated in culturing with the ICPC scaffold.Results: Flexural strength of ICPC was 8.4 ± 0.91 MPa, and elastic modulus was 1.56 ± 0.1 GPa, exceeding those of cancellous bone. hPDLSCs had higher viability in DAMB + fibrin + hPL group than in DAMB + fibrin. ALP was 69.97 ± 16.96 mU/mg for ICPC + DAMB + fibrin + hPL group, higher than 30.68 ± 2.86 mU/mg of ICPC + DAMB + fibrin (p < 0.05) and 4.12 ± 1.65 mU/mg of control (p < 0.01). At 7 days, osteogenic gene expressions (ALP, RUNX2, COL1, and OPN) in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + fibrin were 4–11 folds that of control. At 21 days, the hPDLSC-synthesized bone mineral amounts in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + fibrin were 13.2 folds and 11.1 folds that of control group, respectively.Conclusion: The novel injectable CPC scaffold encapsulating hPDLSCs and hPL is promising to protect and deliver hPDLSCs. The hPL-based medium significantly enhanced the osteogenic differentiation of hPDLSCs in ICPC + DAMB + fibrin + hPL construct, suggesting a promising xeno-free approach for bone tissue regeneration applications.
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Extra-hematopoietic immunomodulatory role of the guanine-exchange factor DOCK2. Commun Biol 2022; 5:1246. [DOI: 10.1038/s42003-022-04078-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Accepted: 10/06/2022] [Indexed: 11/16/2022] Open
Abstract
AbstractStromal cells interact with immune cells during initiation and resolution of immune responses, though the precise underlying mechanisms remain to be resolved. Lessons learned from stromal cell-based therapies indicate that environmental signals instruct their immunomodulatory action contributing to immune response control. Here, to the best of our knowledge, we show a novel function for the guanine-exchange factor DOCK2 in regulating immunosuppressive function in three human stromal cell models and by siRNA-mediated DOCK2 knockdown. To identify immune function-related stromal cell molecular signatures, we first reprogrammed mesenchymal stem/progenitor cells (MSPCs) into induced pluripotent stem cells (iPSCs) before differentiating these iPSCs in a back-loop into MSPCs. The iPSCs and immature iPS-MSPCs lacked immunosuppressive potential. Successive maturation facilitated immunomodulation, while maintaining clonogenicity, comparable to their parental MSPCs. Sequential transcriptomics and methylomics displayed time-dependent immune-related gene expression trajectories, including DOCK2, eventually resembling parental MSPCs. Severe combined immunodeficiency (SCID) patient-derived fibroblasts harboring bi-allelic DOCK2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. Conditional DOCK2 siRNA knockdown in iPS-MSPCs and fibroblasts also immediately reduced immunomodulatory capacity. Conclusively, CRISPR/Cas9-mediated DOCK2 knockout in iPS-MSPCs also resulted in significantly reduced immunomodulation, reduced CDC42 Rho family GTPase activation and blunted filopodia formation. These data identify G protein signaling as key element devising stromal cell immunomodulation.
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Ravanetti F, Saleri R, Martelli P, Andrani M, Ferrari L, Cavalli V, Conti V, Rossetti AP, De Angelis E, Borghetti P. Hypoxia and platelet lysate sustain differentiation of primary horse articular chondrocytes in xeno-free supplementation culture. Res Vet Sci 2022; 152:687-697. [DOI: 10.1016/j.rvsc.2022.09.031] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 09/16/2022] [Accepted: 09/27/2022] [Indexed: 11/29/2022]
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Qiu G, Huang M, Liu J, Ma T, Schneider A, Oates TW, Lynch CD, Weir MD, Zhang K, Zhao L, Xu HHK. Human periodontal ligament stem cell encapsulation in alginate-fibrin-platelet lysate microbeads for dental and craniofacial regeneration. J Dent 2022; 124:104219. [PMID: 35817226 DOI: 10.1016/j.jdent.2022.104219] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 05/20/2022] [Accepted: 05/25/2022] [Indexed: 11/24/2022] Open
Abstract
OBJECTIVE Tissue engineering is promising for dental and craniofacial regeneration. The objectives of this study were to develop a novel xeno-free alginate-fibrin-platelet lysate hydrogel with human periodontal ligament stem cells (hPDLSCs) for dental regeneration, and to investigate the proliferation and osteogenic differentiation of hPDLSCs using hPL as a cell culture nutrient supplement. METHODS hPDLSCs were cultured with Dulbecco's modified eagle medium (DMEM), DMEM + 10% fetal bovine serum (FBS), and DMEM + hPL (1%, 2.5%, and 5%). hPDLSCs were encapsulated in alginate-fibrin microbeads (Alg+Fib), alginate-hPL microbeads (Alg+hPL), or alginate-fibrin-hPL microbeads (Alg+Fib+hPL). hPDLSCs encapsulated in alginate microbeads were induced with an osteogenic medium containing hPL or FBS. Quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) activity, ALP staining, and alizarin red (ARS) staining was investigated. RESULTS hPDLSCs were released faster from Alg+Fib+hPL than from Alg+hPL. At 14 days, ALP activity was 44.1 ± 7.61 mU/mg for Alg+Fib+hPL group, higher than 28.07 ± 5.15 mU/mg of Alg+Fib (p<0.05) and 0.95 ± 0.2 mU/mg of control (p<0.01). At 7 days, osteogenic genes (ALP, RUNX2, COL1, and OPN) in Alg+Fib+hPL and Alg+Fib were 3-10 folds those of control. At 21 days, the hPDLSC-synthesized bone mineral amount in Alg+Fib+hPL and Alg+Fib was 7.5 folds and 4.3 folds that of control group, respectively. CONCLUSIONS The 2.5% hPL was determined to be optimal for hPDLSCs. Adding hPL into alginate hydrogel improved the viability of the hPDLSCs encapsulated in the microbeads. The hPL-based medium enhanced the osteogenic differentiation of hPDLSCs in Alg+Fib+hPL construct, showing a promising xeno-free approach for delivering hPDLSCs to enhance dental, craniofacial and orthopedic regenerations.
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Affiliation(s)
- Gengtao Qiu
- Department of Trauma and Joint Surgery, Shunde Hospital, Southern Medical University, Foshan, Guangdong, China; Department of Advanced Oral Sciences and Therapeutics, University of Maryland Dental School, Baltimore, MD 21201, United States of America; Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China
| | - Mingguang Huang
- Department of Trauma and Joint Surgery, Shunde Hospital, Southern Medical University, Foshan, Guangdong, China
| | - Jin Liu
- Department of Advanced Oral Sciences and Therapeutics, University of Maryland Dental School, Baltimore, MD 21201, United States of America; Key Laboratory of Shannxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, Shannxi, China
| | - Tao Ma
- Department of Oncology and Diagnostic Sciences, University of Maryland School of Dentistry, Baltimore, United States of America
| | - Abraham Schneider
- Department of Oncology and Diagnostic Sciences, University of Maryland School of Dentistry, Baltimore, United States of America; Member, Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America
| | - Thomas W Oates
- Department of Advanced Oral Sciences and Therapeutics, University of Maryland Dental School, Baltimore, MD 21201, United States of America
| | - Christopher D Lynch
- Restorative Dentistry, University Dental School and Hospital, University College Cork, Wilton, Cork, Ireland
| | - Michael D Weir
- Department of Advanced Oral Sciences and Therapeutics, University of Maryland Dental School, Baltimore, MD 21201, United States of America.
| | - Ke Zhang
- Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China.
| | - Liang Zhao
- Department of Trauma and Joint Surgery, Shunde Hospital, Southern Medical University, Foshan, Guangdong, China; Department of Orthopaedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.
| | - Hockin H K Xu
- Department of Trauma and Joint Surgery, Shunde Hospital, Southern Medical University, Foshan, Guangdong, China; Member, Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America; Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America
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Hagen A, Niebert S, Brandt VP, Holland H, Melzer M, Wehrend A, Burk J. Functional properties of equine adipose-derived mesenchymal stromal cells cultured with equine platelet lysate. Front Vet Sci 2022; 9:890302. [PMID: 36016806 PMCID: PMC9395693 DOI: 10.3389/fvets.2022.890302] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2022] [Accepted: 07/11/2022] [Indexed: 12/03/2022] Open
Abstract
Successful translation of multipotent mesenchymal stromal cell (MSC)-based therapies into clinical reality relies on adequate cell production procedures. These should be available not only for human MSC, but also for MSC from animal species relevant to preclinical research and veterinary medicine. The cell culture medium supplementation is one of the critical aspects in MSC production. Therefore, we previously established a scalable protocol for the production of buffy-coat based equine platelet lysate (ePL). This ePL proved to be a suitable alternative to fetal bovine serum (FBS) for equine adipose-derived (AD-) MSC culture so far, as it supported AD-MSC proliferation and basic characteristics. The aim of the current study was to further analyze the functional properties of equine AD-MSC cultured with the same ePL, focusing on cell fitness, genetic stability and pro-angiogenic potency. All experiments were performed with AD-MSC from n = 5 horses, which were cultured either in medium supplemented with 10% FBS, 10% ePL or 2.5% ePL. AD-MSC cultured with 2.5% ePL, which previously showed decreased proliferation potential, displayed higher apoptosis but lower senescence levels as compared to 10% ePL medium (p < 0.05). Non-clonal chromosomal aberrations occurred in 8% of equine AD-MSC cultivated with FBS and only in 4.8% of equine AD-MSC cultivated with 10% ePL. Clonal aberrations in the AD-MSC were neither observed in FBS nor in 10% ePL medium. Analysis of AD-MSC and endothelial cells in an indirect co-culture revealed that the ePL supported the pro-angiogenic effects of AD-MSC. In the 10% ePL group, more vascular endothelial growth factor (VEGF-A) was released and highest VEGF-A concentrations were reached in the presence of ePL and co-cultured cells (p < 0.05). Correspondingly, AD-MSC expressed the VEGF receptor-2 at higher levels in the presence of ePL (p < 0.05). Finally, AD-MSC and 10% ePL together promoted the growth of endothelial cells and induced the formation of vessel-like structures in two of the samples. These data further substantiate that buffy-coat-based ePL is a valuable supplement for equine AD-MSC culture media. The ePL does not only support stable equine AD-MSC characteristics as demonstrated before, but it also enhances their functional properties.
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Affiliation(s)
- Alina Hagen
- Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany
| | - Sabine Niebert
- Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany
| | - Vivian-Pascal Brandt
- Saxon Incubator for Clinical Translation (SIKT), University of Leipzig, Leipzig, Germany
| | - Heidrun Holland
- Saxon Incubator for Clinical Translation (SIKT), University of Leipzig, Leipzig, Germany
| | - Michaela Melzer
- Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany
| | - Axel Wehrend
- Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig-University Giessen, Giessen, Germany
| | - Janina Burk
- Equine Clinic (Surgery, Orthopedics), Justus-Liebig-University Giessen, Giessen, Germany
- *Correspondence: Janina Burk
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Effect of Human Platelet Lysate as Cultivation Nutrient Supplement on Human Natal Dental Pulp Stem Cell In Vitro Expansion. Biomolecules 2022; 12:biom12081091. [PMID: 36008985 PMCID: PMC9405745 DOI: 10.3390/biom12081091] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 08/04/2022] [Accepted: 08/06/2022] [Indexed: 11/29/2022] Open
Abstract
Despite several scientific or ethical issues, fetal bovine serum (FBS) remains the standard nutrient supplement in the mesenchymal stem cell cultivation medium. Cell amplification plays an important role in human stem cell therapies. Increasing interest in this field has supported attempts to find suitable human alternatives to FBS for in vitro cell propagation. Human platelet lysate (hPL) has recently been determined as one of them. Our study aimed to evaluate the influence of 2% hPL in the growth medium for in vitro expansion of human natal dental pulp stem cells (hNDP-SCs). The effect was determined on proliferation rate, viability, phenotype profile, expression of several markers, relative telomere length change, and differentiation potential of four lineages of hNDP-SCs. As a control, hNDP-SCs were simultaneously cultivated in 2% FBS. hNDP-SCs cultivated in hPL showed a statistically significantly higher proliferation rate in initial passages. We did not observe a statistically significant effect on mesenchymal stem cell marker (CD29, CD44, CD73, CD90) or stromal-associated marker (CD13, CD166) expression. The cell viability, relative telomere length, or multipotency remained unaffected in hNDP-SCs cultivated in hPL-medium. In conclusion, hPL produced under controlled and standardized conditions is an efficient serum supplement for in vitro expansion of hNDP-SCs.
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González MB, Cuerva RC, Muñoz BF, Rosell-Valle C, López MM, Arribas BA, Montiel MÁ, Sánchez GC, González MS. Optimization of human platelet lysate production and pathogen reduction in a public blood transfusion center. Transfusion 2022; 62:1839-1849. [PMID: 35924726 DOI: 10.1111/trf.17045] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2022] [Revised: 06/26/2022] [Accepted: 06/29/2022] [Indexed: 11/29/2022]
Abstract
BACKGROUND Human platelet lysate (HPL) has been proposed as a safe and efficient xeno-free alternative to fetal bovine serum (FBS) for large-scale culturing of cell-based medicinal products. However, the use of blood derivatives poses a potential risk of pathogen transmission. To mitigate this risk, different pathogen reduction treatment (PRT) practices can be applied on starting materials or on final products, but these methods might modify the final composition and the quality of the products. STUDY DESIGN AND METHODS We evaluated the impact of applying a PRT based on riboflavin and ultraviolet irradiation on the raw materials used to manufacture an improved Good Manufacturing Practices (GMP)-grade HPL product in a public blood center. Growth promotion and the levels of growth factors and proteins were compared between an inactivated product (HPL4-i) and a non-inactivated product (HPL4). Stability studies were performed at 4°C, -20°C, and -80°C. RESULTS The application of a PRT on the starting materials significantly altered the protein composition of HPL4-i as compared with HPL4. Despite this, the growth promoting rates were unaffected when compared with FBS used as a control. While all products were stable at -20°C and -80°C for 24 months, a significant decrease in the activity of HPL4-i was observed when stored at 4°C. CONCLUSION Our results show that the application of a PRT based on riboflavin and ultraviolet light on starting materials used in the manufacture of HPL modifies the final composition of the product, yet its cell growth promoting activity is maintained at levels similar to those of non-inactivated products.
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Affiliation(s)
- María Bermejo González
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain.,PhD Program in Biología Molecular, Biomedicina e Investigación Clínica, University of Seville, Seville, Spain
| | - Rafael Campos Cuerva
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain.,Centro de Transfusiones, Tejidos y Células de Sevilla (CTTS), Fundación Pública Andaluza para la Gestión de la Investigación en Salud en Sevilla (FISEVI), Seville, Spain
| | - Beatriz Fernández Muñoz
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain
| | - Cristina Rosell-Valle
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain
| | - María Martín López
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain
| | - Blanca Arribas Arribas
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain.,PhD Program in Pharmaceutical Technology and Medicine Sciences (Pharmacy), University of Seville, Seville, Spain
| | - Migue Ángel Montiel
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain.,PhD Program in Pharmaceutical Technology and Medicine Sciences (Pharmacy), University of Seville, Seville, Spain
| | - Gloria Carmona Sánchez
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain.,PhD Program in Biomedicine, University of Granada, Granada, Spain
| | - Mónica Santos González
- Unidad de Producción y Reprogramación Celular de Sevilla (UPRC) Red Andaluza de Diseño y, Traslación de Terapias Avanzadas (RADyTTA), Seville, Spain.,Centro de Transfusiones, Tejidos y Células de Sevilla (CTTS), Fundación Pública Andaluza para la Gestión de la Investigación en Salud en Sevilla (FISEVI), Seville, Spain
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Nebie O, Buée L, Blum D, Burnouf T. Can the administration of platelet lysates to the brain help treat neurological disorders? Cell Mol Life Sci 2022; 79:379. [PMID: 35750991 PMCID: PMC9243829 DOI: 10.1007/s00018-022-04397-w] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2022] [Revised: 05/09/2022] [Accepted: 05/23/2022] [Indexed: 11/03/2022]
Abstract
Neurodegenerative disorders of the central nervous system (CNS) and brain traumatic insults are characterized by complex overlapping pathophysiological alterations encompassing neuroinflammation, alterations of synaptic functions, oxidative stress, and progressive neurodegeneration that eventually lead to irreversible motor and cognitive dysfunctions. A single pharmacological approach is unlikely to provide a complementary set of molecular therapeutic actions suitable to resolve these complex pathologies. Recent preclinical data are providing evidence-based scientific rationales to support biotherapies based on administering neurotrophic factors and extracellular vesicles present in the lysates of human platelets collected from healthy donors to the brain. Here, we present the most recent findings on the composition of the platelet proteome that can activate complementary signaling pathways in vivo to trigger neuroprotection, synapse protection, anti-inflammation, antioxidation, and neurorestoration. We also report experimental data where the administration of human platelet lysates (HPL) was safe and resulted in beneficial neuroprotective effects in established rodent models of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, traumatic brain injury, and stroke. Platelet-based biotherapies, prepared from collected platelet concentrates (PC), are emerging as a novel pragmatic and accessible translational therapeutic strategy for treating neurological diseases. Based on this assumption, we further elaborated on various clinical, manufacturing, and regulatory issues that need to be addressed to ensure the ethical supply, quality, and safety of HPL preparations for treating neurodegenerative and traumatic pathologies of the CNS. HPL made from PC may become a unique approach for scientifically based treatments of neurological disorders readily accessible in low-, middle-, and high-income countries.
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Affiliation(s)
- Ouada Nebie
- College of Biomedical Engineering, Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan
- University of Lille, Inserm, CHU Lille, U1172 - LilNCog - Lille Neuroscience and Cognition, 59045, Lille, France
- Alzheimer and Tauopathies, LabEx DISTALZ, LiCEND, 59000, Lille, France
| | - Luc Buée
- University of Lille, Inserm, CHU Lille, U1172 - LilNCog - Lille Neuroscience and Cognition, 59045, Lille, France
- Alzheimer and Tauopathies, LabEx DISTALZ, LiCEND, 59000, Lille, France
- NeuroTMULille International Laboratory, Univ. Lille, Lille, France
| | - David Blum
- University of Lille, Inserm, CHU Lille, U1172 - LilNCog - Lille Neuroscience and Cognition, 59045, Lille, France.
- Alzheimer and Tauopathies, LabEx DISTALZ, LiCEND, 59000, Lille, France.
- NeuroTMULille International Laboratory, Univ. Lille, Lille, France.
- NeuroTMULille International Laboratory, Taipei Medical University, Taipei, 11031, Taiwan.
| | - Thierry Burnouf
- College of Biomedical Engineering, Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan.
- NeuroTMULille International Laboratory, Taipei Medical University, Taipei, 11031, Taiwan.
- International PhD Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, 11031, Taiwan.
- International PhD Program in Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.
- Brain and Consciousness Research Centre, Taipei Medical University Shuang-Ho Hospital, New Taipei City, 23561, Taiwan.
- Neuroscience Research Center, Taipei Medical University, Taipei, Taiwan.
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30
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Schepici G, Gugliandolo A, Mazzon E. Serum-Free Cultures: Could They Be a Future Direction to Improve Neuronal Differentiation of Mesenchymal Stromal Cells? Int J Mol Sci 2022; 23:ijms23126391. [PMID: 35742836 PMCID: PMC9223839 DOI: 10.3390/ijms23126391] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Revised: 06/02/2022] [Accepted: 06/06/2022] [Indexed: 02/04/2023] Open
Abstract
Mesenchymal stem/stromal cells (MSCs) are undifferentiated cells with multilinear potential, known for their immunomodulatory and regenerative properties. Although the scientific community is working to improve their application, concerns limit their use to repair tissues following neurological damage. One of these obstacles is represented by the use of culture media supplemented with fetal bovine serum (FBS), which, due to its xenogenic nature and the risk of contamination, has increased scientific, ethical and safety problems. Therefore, the use of serum-free media could improve MSC culture methods, avoiding infectious and immunogenic transmission problems as well as MSC bioprocesses, without the use of animal components. The purpose of our review is to provide an overview of experimental studies that demonstrate that serum-free cultures, along with the supplementation of growth factors or chemicals, can lead to a more defined and controlled environment, enhancing the proliferation and neuronal differentiation of MSCs.
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31
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Aussel C, Busson E, Vantomme H, Peltzer J, Martinaud C. Quality assessment of a serum and xenofree medium for the expansion of human GMP-grade mesenchymal stromal cells. PeerJ 2022; 10:e13391. [PMID: 35663525 PMCID: PMC9161815 DOI: 10.7717/peerj.13391] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Accepted: 04/15/2022] [Indexed: 01/14/2023] Open
Abstract
Background Cell-based therapies are emerging as a viable modality to treat challenging diseases, resulting in an increasing demand for their large-scale, high-quality production. Production facilities face the issue of batch-to-batch consistency while producing a safe and efficient cell-based product. Controlling culture conditions and particularly media composition is a key factor of success in this challenge. Serum and Xeno-Free Media (SXFM) represent an interesting option to achieve this goal. By reducing batch to batch variability, they increase Good Manufacturing Practices (GMP)-compliance and safety regarding xenogenic transmission, as compared to fetal bovine serum (FBS) supplemented-media or human platelet lysate supplemented medium. Methods In this study, the isolation, expansion and characteristics including the anti-inflammatory function of human mesenchymal stromal cells (MSC) are compared after culture in MEMα supplemented with human Concentrate Platelet Lysate (hCPL, reference medium) or in MSC-Brew GMP Medium. The latter is a GMP SXFM manufactured in bags under strictly controlled conditions in volumes suitable for expansion to a clinical scale and does not require neither pre-coating of the cell culture units nor the addition of blood derivatives at the isolation step. Results We showed that MSC derived from human bone-marrow and adipose tissue can be successfully isolated and expanded in this SXFM. Number and size of Colony-Forming Unit fibroblast (CFU-F) is increased compared to cells cultivated in hCPL medium. All cells retained a CD90+, CD73+, CD105+, HLADR-, CD34-, CD45- phenotype. Furthermore, the osteogenic and adipocyte potentials as well as the anti-inflammatory activity were comparable between culture conditions. All cells reached the release criteria established in our production facility to treat inflammatory pathologies. Conclusions The use of MSC-Brew GMP Medium can therefore be considered for clinical bioprocesses as a safe and efficient substitute for hCPL media.
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Affiliation(s)
- Clotilde Aussel
- Biomedical Research Institute of the Armed Forces, Clamart, France
| | - Elodie Busson
- Advanced Therapy Medicine Unit, French Military Blood Institute, Clamart, France
| | - Helene Vantomme
- Advanced Therapy Medicine Unit, French Military Blood Institute, Clamart, France
| | - Juliette Peltzer
- Biomedical Research Institute of the Armed Forces, Clamart, France
| | - Christophe Martinaud
- Advanced Therapy Medicine Unit, French Military Blood Institute, Clamart, France
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32
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Ladeira BMF, Gomes MC, Custódio CA, Mano JF. High-Throughput Production of Microsponges from Platelet Lysate for Tissue Engineering Applications. Tissue Eng Part C Methods 2022; 28:325-334. [PMID: 35343236 DOI: 10.1089/ten.tec.2022.0029] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Cell-based therapies require a large number of cells, as well as appropriate methods to deliver the cells to damaged tissue. Microcarriers provide an optimal platform for large-scale cell culture while also improving cell retention during cell delivery. However, this technology still presents significant challenges due to low-throughput fabrication methods and an inability of the microcarriers to recreate the properties of human tissue. This work proposes, for the first time, the use of methacryloyl platelet lysates (PLMA), a photocrosslinkable material derived from human platelet lysates, to produce porous microcarriers. Initially, high quantities of PLMA/alginate core-shell microcapsules are produced using coaxial electrospray. Subsequently, the microcapsules are collected, irradiated with ultraviolet light, washed, and freeze dried yielding PLMA microsponges. These microsponges are able to support the adhesion and proliferation of human adipose-derived stem cells, while also displaying potential in the assembly of autologous microtissues. Cell-laden microsponges were shown to self-organize into aggregates, suggesting possible applications in bottom-up tissue engineering applications. Impact Statement Microcarriers have increasingly been used as delivery platforms in cell therapy. Herein, the encapsulation of human-derived proteins in alginate microcapsules is proposed as a method to produce microcarriers from photopolymerizable materials. The capsules function as a template structure, which is then processed into spherical microparticles, which can be used in cell culture, cell delivery, and bottom-up assembly. As a proof of concept, this method was combined with lyophilization to process methacryloyl platelet lysates into injectable microsponges for cell delivery.
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Affiliation(s)
- Bruno M F Ladeira
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
| | - Maria C Gomes
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
| | - Catarina A Custódio
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
| | - João F Mano
- Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, Portugal
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33
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Palombella S, Perucca Orfei C, Castellini G, Gianola S, Lopa S, Mastrogiacomo M, Moretti M, de Girolamo L. Systematic review and meta-analysis on the use of human platelet lysate for mesenchymal stem cell cultures: comparison with fetal bovine serum and considerations on the production protocol. Stem Cell Res Ther 2022; 13:142. [PMID: 35379348 PMCID: PMC8981660 DOI: 10.1186/s13287-022-02815-1] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 03/10/2022] [Indexed: 11/24/2022] Open
Abstract
Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures.
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Affiliation(s)
- Silvia Palombella
- Cell and Tissue Engineering Laboratory, IRCCS Istituto Ortopedico Galeazzi, 20161, Milan, Italy
| | - Carlotta Perucca Orfei
- Laboratorio di Biotecnologie Applicate all'Ortopedia, IRCCS Istituto Ortopedico Galeazzi, 20161, Milan, Italy
| | - Greta Castellini
- Unit of Clinical Epidemiology, IRCCS Istituto Ortopedico Galeazzi, 20161, Milan, Italy
| | - Silvia Gianola
- Unit of Clinical Epidemiology, IRCCS Istituto Ortopedico Galeazzi, 20161, Milan, Italy
| | - Silvia Lopa
- Cell and Tissue Engineering Laboratory, IRCCS Istituto Ortopedico Galeazzi, 20161, Milan, Italy
| | | | - Matteo Moretti
- Cell and Tissue Engineering Laboratory, IRCCS Istituto Ortopedico Galeazzi, 20161, Milan, Italy.,Regenerative Medicine Technologies Laboratory, Ente Ospedaliero Cantonale, Laboratories for Translational Research (LRT), 6500, Bellinzona, Switzerland.,Department of Surgery, Ente Ospedaliero Cantonale, Service of Orthopaedics and Traumatology, 6962, Lugano, Switzerland.,Faculty of Biomedical Sciences, Euler Institute, USI, 6900, Lugano, Switzerland
| | - Laura de Girolamo
- Laboratorio di Biotecnologie Applicate all'Ortopedia, IRCCS Istituto Ortopedico Galeazzi, 20161, Milan, Italy.
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34
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Wolf M, Poupardin RW, Ebner‐Peking P, Andrade AC, Blöchl C, Obermayer A, Gomes FG, Vari B, Maeding N, Eminger E, Binder H, Raninger AM, Hochmann S, Brachtl G, Spittler A, Heuser T, Ofir R, Huber CG, Aberman Z, Schallmoser K, Volk H, Strunk D. A functional corona around extracellular vesicles enhances angiogenesis, skin regeneration and immunomodulation. J Extracell Vesicles 2022; 11:e12207. [PMID: 35398993 PMCID: PMC8994701 DOI: 10.1002/jev2.12207] [Citation(s) in RCA: 119] [Impact Index Per Article: 39.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Revised: 02/08/2022] [Accepted: 03/02/2022] [Indexed: 02/06/2023] Open
Abstract
Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell-derived extracellular vesicles (EVs). Here we demonstrate that nano-sized EVs from therapy-grade human placental-expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell-secreted soluble factors via tangential flow-filtration (TFF) and subtractive tandem mass-tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein-based flow cytometry, super-resolution and electron microscopy verified EV identity. PLX-EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose-dependently inhibited T cell proliferation in vitro. Corona removal by size-exclusion or ultracentrifugation abrogated angiogenesis. Re-establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super-resolution microscopy, electron microscopy and zeta-potential shift, and served as a proof-of-concept. Understanding EV corona formation will improve rational EV-inspired nano-therapy design.
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Affiliation(s)
- Martin Wolf
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Rodolphe W. Poupardin
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Patricia Ebner‐Peking
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - André Cronemberger Andrade
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Constantin Blöchl
- Department of BiosciencesParis Lodron University SalzburgSalzburgAustria
| | - Astrid Obermayer
- Department of BiosciencesParis Lodron University SalzburgSalzburgAustria
| | - Fausto Gueths Gomes
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
- Department of Transfusion Medicine and SCI‐TReCSPMUSalzburgAustria
| | - Balazs Vari
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Nicole Maeding
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Essi Eminger
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Heide‐Marie Binder
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Anna M. Raninger
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Sarah Hochmann
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Gabriele Brachtl
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
| | - Andreas Spittler
- Core Facility Flow Cytometry and Department of SurgeryResearch LaboratoriesMedical University of ViennaViennaAustria
| | | | | | - Christian G. Huber
- Department of BiosciencesParis Lodron University SalzburgSalzburgAustria
| | | | | | - Hans‐Dieter Volk
- Berlin Institute of Health at Charité – UniversitätsmedizinBIH Centre for Regenerative Therapies (BCRT)BerlinGermany
| | - Dirk Strunk
- Cell Therapy InstituteSpinal Cord Injury and Tissue Regeneration Centre Salzburg (SCI‐TReCS)Paracelsus Medical University (PMU)SalzburgAustria
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35
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Brachtl G, Poupardin R, Hochmann S, Raninger A, Jürchott K, Streitz M, Schlickeiser S, Oeller M, Wolf M, Schallmoser K, Volk HD, Geissler S, Strunk D. Batch Effects during Human Bone Marrow Stromal Cell Propagation Prevail Donor Variation and Culture Duration: Impact on Genotype, Phenotype and Function. Cells 2022; 11:946. [PMID: 35326396 PMCID: PMC8946746 DOI: 10.3390/cells11060946] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2022] [Revised: 03/04/2022] [Accepted: 03/08/2022] [Indexed: 02/01/2023] Open
Abstract
Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.
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Affiliation(s)
- Gabriele Brachtl
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (G.B.); (R.P.); (S.H.); (A.R.); (M.W.)
| | - Rodolphe Poupardin
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (G.B.); (R.P.); (S.H.); (A.R.); (M.W.)
| | - Sarah Hochmann
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (G.B.); (R.P.); (S.H.); (A.R.); (M.W.)
| | - Anna Raninger
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (G.B.); (R.P.); (S.H.); (A.R.); (M.W.)
| | - Karsten Jürchott
- Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.J.); (M.S.); (S.S.); (H.-D.V.); (S.G.)
| | - Mathias Streitz
- Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.J.); (M.S.); (S.S.); (H.-D.V.); (S.G.)
- Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Insel Riems, 17493 Greifswald, Germany
| | - Stephan Schlickeiser
- Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.J.); (M.S.); (S.S.); (H.-D.V.); (S.G.)
| | - Michaela Oeller
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (M.O.); (K.S.)
| | - Martin Wolf
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (G.B.); (R.P.); (S.H.); (A.R.); (M.W.)
| | - Katharina Schallmoser
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (M.O.); (K.S.)
| | - Hans-Dieter Volk
- Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.J.); (M.S.); (S.S.); (H.-D.V.); (S.G.)
- Berlin Center for Advanced Therapies (BeCAT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
- Institute of Medical Immunology, Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Sven Geissler
- Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.J.); (M.S.); (S.S.); (H.-D.V.); (S.G.)
- Berlin Center for Advanced Therapies (BeCAT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Dirk Strunk
- Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Cell Therapy Institute, Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (G.B.); (R.P.); (S.H.); (A.R.); (M.W.)
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Vicinanza C, Lombardi E, Da Ros F, Marangon M, Durante C, Mazzucato M, Agostini F. Modified mesenchymal stem cells in cancer therapy: A smart weapon requiring upgrades for wider clinical applications. World J Stem Cells 2022; 14:54-75. [PMID: 35126828 PMCID: PMC8788179 DOI: 10.4252/wjsc.v14.i1.54] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2021] [Revised: 08/06/2021] [Accepted: 12/23/2021] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem stromal cells (MSC) are characterized by the intriguing capacity to home toward cancer cells after systemic administration. Thus, MSC can be harnessed as targeted delivery vehicles of cytotoxic agents against tumors. In cancer patients, MSC based advanced cellular therapies were shown to be safe but their clinical efficacy was limited. Indeed, the amount of systemically infused MSC actually homing to human cancer masses is insufficient to reduce tumor growth. Moreover, induction of an unequivocal anticancer cytotoxic phenotype in expanded MSC is necessary to achieve significant therapeutic efficacy. Ex vivo cell modifications are, thus, required to improve anti-cancer properties of MSC. MSC based cellular therapy products must be handled in compliance with good manufacturing practice (GMP) guidelines. In the present review we include MSC-improving manipulation approaches that, even though actually tested at preclinical level, could be compatible with GMP guidelines. In particular, we describe possible approaches to improve MSC homing on cancer, including genetic engineering, membrane modification and cytokine priming. Similarly, we discuss appropriate modalities aimed at inducing a marked cytotoxic phenotype in expanded MSC by direct chemotherapeutic drug loading or by genetic methods. In conclusion, we suggest that, to configure MSC as a powerful weapon against cancer, combinations of clinical grade compatible modification protocols that are currently selected, should be introduced in the final product. Highly standardized cancer clinical trials are required to test the efficacy of ameliorated MSC based cell therapies.
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Affiliation(s)
- Carla Vicinanza
- Stem Cell Unit, Centro di Riferimento Oncologico di Aviano, IRCCS, Aviano 33081, Italy
| | - Elisabetta Lombardi
- Stem Cell Unit, Centro di Riferimento Oncologico di Aviano, IRCCS, Aviano 33081, Italy
| | - Francesco Da Ros
- Stem Cell Unit, Centro di Riferimento Oncologico di Aviano, IRCCS, Aviano 33081, Italy
| | - Miriam Marangon
- Stem Cell Unit, Centro di Riferimento Oncologico di Aviano, IRCCS, Aviano 33081, Italy
| | - Cristina Durante
- Stem Cell Unit, Centro di Riferimento Oncologico di Aviano, IRCCS, Aviano 33081, Italy
| | - Mario Mazzucato
- Stem Cell Unit, Centro di Riferimento Oncologico di Aviano, IRCCS, Aviano 33081, Italy
| | - Francesco Agostini
- Stem Cell Unit, Centro di Riferimento Oncologico di Aviano, IRCCS, Aviano 33081, Italy
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Gomes FG, Andrade AC, Wolf M, Hochmann S, Krisch L, Maeding N, Regl C, Poupardin R, Ebner-Peking P, Huber CG, Meisner-Kober N, Schallmoser K, Strunk D. Synergy of Human Platelet-Derived Extracellular Vesicles with Secretome Proteins Promotes Regenerative Functions. Biomedicines 2022; 10:238. [PMID: 35203448 PMCID: PMC8869293 DOI: 10.3390/biomedicines10020238] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 01/17/2022] [Accepted: 01/19/2022] [Indexed: 11/29/2022] Open
Abstract
Platelet-rich plasma is a promising regenerative therapeutic with controversial efficacy. We and others have previously demonstrated regenerative functions of human platelet lysate (HPL) as an alternative platelet-derived product. Here we separated extracellular vesicles (EVs) from soluble factors of HPL to understand the mode of action during skin-organoid formation and immune modulation as model systems for tissue regeneration. HPL-EVs were isolated by tangential-flow filtration (TFF) and further purified by size-exclusion chromatography (SEC) separating EVs from (lipo)protein-enriched soluble fractions. We characterized samples by tunable resistive pulse sensing, western blot, tandem mass-tag proteomics and super-resolution microscopy. We evaluated EV function during angiogenesis, wound healing, organoid formation and immune modulation. We characterized EV enrichment by TFF and SEC according to MISEV2018 guidelines. Proteomics showed three major clusters of protein composition separating TSEC-EVs from HPL clustering with TFF soluble fractions and TFF-EVs clustering with TSEC soluble fractions, respectively. HPL-derived TFF-EVs promoted skin-organoid formation and inhibited T-cell proliferation more efficiently than TSEC-EVs or TSEC-soluble fractions. Recombining TSEC-EVs with TSEC soluble fractions re-capitulated TFF-EV effects. Zeta potential and super-resolution imaging further evidenced protein corona formation on TFF-EVs. Corona depletion on SEC-EVs could be artificially reconstituted by TSEC late fraction add-back. In contrast to synthetic nanoparticles, which commonly experience reduced function after corona formation, the corona-bearing EVs displayed improved functionality. We conclude that permissive isolation technology, such as TFF, and better understanding of the mechanism of EV corona function are required to realize the complete potential of platelet-based regenerative therapies.
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Affiliation(s)
- Fausto Gueths Gomes
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - André Cronemberger Andrade
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Martin Wolf
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Sarah Hochmann
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Linda Krisch
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - Nicole Maeding
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Christof Regl
- Department for Biosciences and Medical Biology, Paris Lodron University, 5020 Salzburg, Austria; (C.R.); (C.G.H.); (N.M.-K.)
| | - Rodolphe Poupardin
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Patricia Ebner-Peking
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
| | - Christian G. Huber
- Department for Biosciences and Medical Biology, Paris Lodron University, 5020 Salzburg, Austria; (C.R.); (C.G.H.); (N.M.-K.)
| | - Nicole Meisner-Kober
- Department for Biosciences and Medical Biology, Paris Lodron University, 5020 Salzburg, Austria; (C.R.); (C.G.H.); (N.M.-K.)
| | - Katharina Schallmoser
- Department of Transfusion Medicine and SCI-TReCS, Paracelsus Medical University (PMU), 5020 Salzburg, Austria;
| | - Dirk Strunk
- Cell Therapy Institute, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), 5020 Salzburg, Austria; (F.G.G.); (A.C.A.); (M.W.); (S.H.); (L.K.); (N.M.); (R.P.); (P.E.-P.)
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38
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Effect of biomolecules derived from human platelet-rich plasma on the ex vivo expansion of human adipose-derived mesenchymal stem cells for clinical applications. Biologicals 2021; 75:37-48. [PMID: 34785135 DOI: 10.1016/j.biologicals.2021.11.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Revised: 11/02/2021] [Accepted: 11/06/2021] [Indexed: 11/20/2022] Open
Abstract
Mesenchymal stem cells are a tool in cell therapies but demand a large cell number per treatment, for that, suitable culture media is required which contains fetal bovine serum (FBS). However, for cell-based therapy applications, the use of FBS is problematic. Several alternatives to FBS have been explored, including human derivatives from platelet-rich plasma (hD-PRP). Although various studies have evaluated the impact of hD-PRP on MSC proliferation and differentiation, few of them have assessed their influence on processes, such as metabolism and gene expression. Here, we cultured human adipose-derived MSCs (hAD-MSCs) in media supplemented with either 10% hD-PRP (hD-PRP-SM) or 10% FBS (FBS-SM) in order to characterize them and evaluate the effect of hD-PRP on cell metabolism, gene expression of associated regenerative factors, as well as chromosome stability during cell expansion. We found that hAD-MSCs cultured in hD-PRP-SM have a greater cell elongation but express similar surface markers; in addition, hD-PRP-SM promoted a significant osteogenic differentiation in the absence of differentiation medium and increased the growth rate, maintaining chromosomal stability. In terms of cell metabolic profile, hAD-MSC behavior did not reveal any differences between both culture conditions. Conversely, significant differences in collagen I and angiopoietin 2 expression were observed between both conditions. The present results suggest that hD-PRP may influence hAD-MSC behavior.
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Laner-Plamberger S, Oeller M, Rohde E, Schallmoser K, Strunk D. Heparin and Derivatives for Advanced Cell Therapies. Int J Mol Sci 2021; 22:12041. [PMID: 34769471 PMCID: PMC8584295 DOI: 10.3390/ijms222112041] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 11/03/2021] [Accepted: 11/05/2021] [Indexed: 12/27/2022] Open
Abstract
Heparin and its derivatives are saving thousands of human lives annually, by successfully preventing and treating thromboembolic events. Although the mode of action during anticoagulation is well studied, their influence on cell behavior is not fully understood as is the risk of bleeding and other side effects. New applications in regenerative medicine have evolved supporting production of cell-based therapeutics or as a substrate for creating functionalized matrices in biotechnology. The currently resurgent interest in heparins is related to the expected combined anti-inflammatory, anti-thrombotic and anti-viral action against COVID-19. Based on a concise summary of key biochemical and clinical data, this review summarizes the impact for manufacturing and application of cell therapeutics and highlights the need for discriminating the different heparins.
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Affiliation(s)
- Sandra Laner-Plamberger
- Department of Transfusion Medicine, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria; (S.L.-P.); (M.O.); (E.R.)
- Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria;
| | - Michaela Oeller
- Department of Transfusion Medicine, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria; (S.L.-P.); (M.O.); (E.R.)
- Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria;
| | - Eva Rohde
- Department of Transfusion Medicine, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria; (S.L.-P.); (M.O.); (E.R.)
- Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria;
| | - Katharina Schallmoser
- Department of Transfusion Medicine, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria; (S.L.-P.); (M.O.); (E.R.)
- Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria;
| | - Dirk Strunk
- Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria;
- Cell Therapy Institute, Paracelsus Medical University of Salzburg, 5020 Salzburg, Austria
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40
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Schrom S, Hebesberger T, Wallner SA, Anders I, Richtig E, Brandl W, Hirschmugl B, Garofalo M, Bernecker C, Schlenke P, Kashofer K, Wadsack C, Aigelsreiter A, Heitzer E, Riedl S, Zweytick D, Kretschmer N, Richtig G, Rinner B. MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy. Int J Mol Sci 2021; 22:ijms222111318. [PMID: 34768746 PMCID: PMC8583216 DOI: 10.3390/ijms222111318] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2021] [Revised: 10/12/2021] [Accepted: 10/13/2021] [Indexed: 12/22/2022] Open
Abstract
Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options—especially during pregnancy.
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Affiliation(s)
- Silke Schrom
- Division of Biomedical Research, Medical University of Graz, 8036 Graz, Austria; (S.S.); (T.H.); (S.A.W.); (I.A.)
| | - Thomas Hebesberger
- Division of Biomedical Research, Medical University of Graz, 8036 Graz, Austria; (S.S.); (T.H.); (S.A.W.); (I.A.)
| | - Stefanie Angela Wallner
- Division of Biomedical Research, Medical University of Graz, 8036 Graz, Austria; (S.S.); (T.H.); (S.A.W.); (I.A.)
| | - Ines Anders
- Division of Biomedical Research, Medical University of Graz, 8036 Graz, Austria; (S.S.); (T.H.); (S.A.W.); (I.A.)
| | - Erika Richtig
- Department of Dermatology, Medical University of Graz, 8036 Graz, Austria;
| | - Waltraud Brandl
- Department of Obstetrics and Gynecology, Medical University of Graz, 8036 Graz, Austria; (W.B.); (B.H.); (C.W.)
| | - Birgit Hirschmugl
- Department of Obstetrics and Gynecology, Medical University of Graz, 8036 Graz, Austria; (W.B.); (B.H.); (C.W.)
- BioTechMed-Graz, 8010 Graz, Austria; (S.R.); (D.Z.)
| | - Mariangela Garofalo
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35122 Padova, Italy;
| | - Claudia Bernecker
- Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, 8036 Graz, Austria; (C.B.); (P.S.)
| | - Peter Schlenke
- Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz, 8036 Graz, Austria; (C.B.); (P.S.)
| | - Karl Kashofer
- Diagnostic and Research Institute of Pathology, Medical University of Graz, 8036 Graz, Austria; (K.K.); (A.A.)
| | - Christian Wadsack
- Department of Obstetrics and Gynecology, Medical University of Graz, 8036 Graz, Austria; (W.B.); (B.H.); (C.W.)
- BioTechMed-Graz, 8010 Graz, Austria; (S.R.); (D.Z.)
| | - Ariane Aigelsreiter
- Diagnostic and Research Institute of Pathology, Medical University of Graz, 8036 Graz, Austria; (K.K.); (A.A.)
| | - Ellen Heitzer
- Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, 8036 Graz, Austria;
| | - Sabrina Riedl
- BioTechMed-Graz, 8010 Graz, Austria; (S.R.); (D.Z.)
- Institute of Molecular Biosciences, Biophysics Division, University of Graz, 8010 Graz, Austria
- BioHealth, 8010 Graz, Austria
| | - Dagmar Zweytick
- BioTechMed-Graz, 8010 Graz, Austria; (S.R.); (D.Z.)
- Institute of Molecular Biosciences, Biophysics Division, University of Graz, 8010 Graz, Austria
- BioHealth, 8010 Graz, Austria
| | - Nadine Kretschmer
- Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, 8010 Graz, Austria;
| | - Georg Richtig
- Division of Oncology, Medical University of Graz, 8036 Graz, Austria;
| | - Beate Rinner
- Division of Biomedical Research, Medical University of Graz, 8036 Graz, Austria; (S.S.); (T.H.); (S.A.W.); (I.A.)
- BioTechMed-Graz, 8010 Graz, Austria; (S.R.); (D.Z.)
- Correspondence: ; Tel.: +43-316-3857-3524
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41
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Scaled preparation of extracellular vesicles from conditioned media. Adv Drug Deliv Rev 2021; 177:113940. [PMID: 34419502 DOI: 10.1016/j.addr.2021.113940] [Citation(s) in RCA: 76] [Impact Index Per Article: 19.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 07/13/2021] [Accepted: 08/16/2021] [Indexed: 12/15/2022]
Abstract
Extracellular vesicles (EVs) especially of mesenchymal stem/stomal cells (MSCs) are increasingly considered as biotherapeutic agents for a variety of different diseases. For translating them effectively into the clinics, scalable production processes fulfilling good manufacturing practice (GMP) are needed. Like for other biotherapeutic agents, the manufacturing of EV products can be subdivided in the upstream and downstream processing and the subsequent quality control, each of them containing several unit operations. During upstream processing (USP), cells are isolated, stored (cell banking) and expanded; furthermore, EV-containing conditioned media are produced. During downstream processing (DSP), conditioned media (CM) are processed to obtain concentrated and purified EV products. CM are either stored until DSP or are directly processed. As first unit operation in DSP, clarification removes remaining cells, debris and other larger impurities. The key operations of each EV DSP is volume-reduction combined with purification of the concentrated EVs. Most of the EV preparation methods used in conventional research labs including differential centrifugation procedures are limited in their scalability. Consequently, it is a major challenge in the therapeutic EV field to identify appropriate EV concentration and purification methods allowing scale up. As EVs share several features with enveloped viruses, that are used for more than two decades in the clinics now, several principles can be adopted to EV manufacturing. Here, we introduce and discuss volume reducing and purification methods frequently used for viruses and analyze their value for the manufacturing of EV-based therapeutics.
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42
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Seidelmann N, Duarte Campos DF, Rohde M, Johnen S, Salla S, Yam GHF, Mehta JS, Walter P, Fuest M. Human platelet lysate as a replacement for fetal bovine serum in human corneal stromal keratocyte and fibroblast culture. J Cell Mol Med 2021; 25:9647-9659. [PMID: 34486211 PMCID: PMC8505853 DOI: 10.1111/jcmm.16912] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2021] [Revised: 08/19/2021] [Accepted: 08/23/2021] [Indexed: 12/13/2022] Open
Abstract
The isolation and propagation of primary human corneal stromal keratocytes (CSK) are crucial for cellular research and corneal tissue engineering. However, this delicate cell type easily transforms into stromal fibroblasts (SF) and scar inducing myofibroblasts (Myo‐SF). Current protocols mainly rely on xenogeneic fetal bovine serum (FBS). Human platelet lysate (hPL) could be a viable, potentially autologous, alternative. We found high cell survival with both supplements in CSK and SF. Cell numbers and Ki67+ ratios increased with higher fractions of hPL and FBS in CSK and SF. We detected a loss in CSK marker expression (Col8A2, ALDH3A1 and LUM) with increasing fractions of FBS and hPL in CSK and SF. The expression of the Myo‐SF marker SMA increased with higher amounts of FBS but decreased with incremental hPL substitution in both cell types, implying an antifibrotic effect of hPL. Immunohistochemistry confirmed the RT‐PCR findings. bFGF and HGF were only found in hPL and could be responsible for suppressing the Myo‐SF conversion. Considering all findings, we propose 0.5% hPL as a suitable substitution in CSK culture, as this xeno‐free component efficiently preserved CSK characteristics, with non‐inferiority in terms of cell viability, cell number and proliferation in comparison to the established 0.5% FBS protocol.
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Affiliation(s)
- Nina Seidelmann
- Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
| | - Daniela F Duarte Campos
- Institute of Applied Medical Engineering, RWTH Aachen University Hospital, Aachen, Germany.,Center for Molecular Biology, Heidelberg University, Heidelberg, Germany
| | - Malena Rohde
- Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
| | - Sandra Johnen
- Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
| | - Sabine Salla
- Department of Ophthalmology, RWTH Aachen University, Aachen, Germany.,Cornea Bank Aachen, RWTH Aachen University, Aachen, Germany
| | - Gary Hin-Fai Yam
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.,Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore
| | - Jodhbir S Mehta
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore, Singapore.,Singapore National Eye Centre, Singapore, Singapore.,Eye-Academic Clinical Program, Duke-National University of Singapore (NUS) Graduate Medical School, Singapore, Singapore.,School of Material Science and Engineering, Nanyang Technological University, Singapore, Singapore
| | - Peter Walter
- Department of Ophthalmology, RWTH Aachen University, Aachen, Germany.,Cornea Bank Aachen, RWTH Aachen University, Aachen, Germany
| | - Matthias Fuest
- Department of Ophthalmology, RWTH Aachen University, Aachen, Germany
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Grangier A, Branchu J, Volatron J, Piffoux M, Gazeau F, Wilhelm C, Silva AKA. Technological advances towards extracellular vesicles mass production. Adv Drug Deliv Rev 2021; 176:113843. [PMID: 34147532 DOI: 10.1016/j.addr.2021.113843] [Citation(s) in RCA: 95] [Impact Index Per Article: 23.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 05/29/2021] [Accepted: 06/15/2021] [Indexed: 12/13/2022]
Abstract
Extracellular vesicles (EVs) are becoming essential actors in bio-therapeutics, as much for their regenerative or immunomodulatory properties as for their potential as cargo delivery vehicles. To enable the democratization of these EV-based therapies, many challenges remain such as large-scale production which is necessary to reduce costs of treatment. Herein, we review some advanced works on high-yield EV manufacturing. One approach consists in developing large-scale cell culture platforms, while others focus on cell stimulation to increase particle yield per cell. This can be done by moderate physico-chemical stresses or by disrupting cell membrane towards autoassembled vesicle-like particles. We critically compare these different techniques, keeping in mind that the field still lacks shared characterization standards, underline the importance of therapeutic potency assessment and discuss mass production strategies that have been identified in current clinical trials.
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Affiliation(s)
- Alice Grangier
- Laboratoire MSC Matière et Systèmes Complexes, CNRS UMR 7057, Université de Paris, 75013 and 75006 Paris, France
| | | | | | - Max Piffoux
- Laboratoire MSC Matière et Systèmes Complexes, CNRS UMR 7057, Université de Paris, 75013 and 75006 Paris, France; Everzom, 75006 Paris, France; Department of Medical Oncology, Centre Léon Bérard, Lyon, France
| | - Florence Gazeau
- Laboratoire MSC Matière et Systèmes Complexes, CNRS UMR 7057, Université de Paris, 75013 and 75006 Paris, France
| | - Claire Wilhelm
- Laboratoire MSC Matière et Systèmes Complexes, CNRS UMR 7057, Université de Paris, 75013 and 75006 Paris, France; Laboratoire PhysicoChimie Curie, Institut Curie, PSL Research University - Sorbonne Université - CNRS, 75005 Paris, France.
| | - Amanda K A Silva
- Laboratoire MSC Matière et Systèmes Complexes, CNRS UMR 7057, Université de Paris, 75013 and 75006 Paris, France.
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Mallis P, Sokolis DP, Katsimpoulas M, Kostakis A, Stavropoulos-Giokas C, Michalopoulos E. Improved Repopulation Efficacy of Decellularized Small Diameter Vascular Grafts Utilizing the Cord Blood Platelet Lysate. Bioengineering (Basel) 2021; 8:118. [PMID: 34562940 PMCID: PMC8467559 DOI: 10.3390/bioengineering8090118] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Revised: 08/18/2021] [Accepted: 08/24/2021] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND The development of functional bioengineered small-diameter vascular grafts (SDVGs), represents a major challenge of tissue engineering. This study aimed to evaluate the repopulation efficacy of biological vessels, utilizing the cord blood platelet lysate (CBPL). METHODS Human umbilical arteries (hUAs, n = 10) were submitted to decellularization. Then, an evaluation of decellularized hUAs, involving histological, biochemical and biomechanical analysis, was performed. Wharton's Jelly (WJ) Mesenchymal Stromal Cells (MSCs) were isolated and characterized for their properties. Then, WJ-MSCs (1.5 × 106 cells) were seeded on decellularized hUAs (n = 5) and cultivated with (Group A) or without the presence of the CBPL, (Group B) for 30 days. Histological analysis involving immunohistochemistry (against Ki67, for determination of cell proliferation) and indirect immunofluorescence (against activated MAP kinase, additional marker for cell growth and proliferation) was performed. RESULTS The decellularized hUAs retained their initial vessel's properties, in terms of key-specific proteins, the biochemical and biomechanical characteristics were preserved. The evaluation of the repopulation process indicated a more uniform distribution of WJ-MSCs in group A compared to group B. The repopulated vascular grafts of group B were characterized by greater Ki67 and MAP kinase expression compared to group A. CONCLUSION The results of this study indicated that the CBPL may improve the repopulation efficacy, thus bringing the biological SDVGs one step closer to clinical application.
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Affiliation(s)
- Panagiotis Mallis
- Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, 4 Soranou Ephessiou Street, 115 27 Athens, Greece; (C.S.-G.); (E.M.)
| | - Dimitrios P. Sokolis
- Laboratory of Biomechanics, Center for Experimental Surgery, Biomedical Research Foundation Academy of Athens, 4 Soranou Ephessiou Street, 115 27 Athens, Greece;
| | - Michalis Katsimpoulas
- Center of Experimental Surgery and Translational Research, Biomedical Research Foundation Academy of Athens, 4 Soranou Ephessiou Street, 115 27 Athens, Greece; (M.K.); (A.K.)
| | - Alkiviadis Kostakis
- Center of Experimental Surgery and Translational Research, Biomedical Research Foundation Academy of Athens, 4 Soranou Ephessiou Street, 115 27 Athens, Greece; (M.K.); (A.K.)
| | - Catherine Stavropoulos-Giokas
- Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, 4 Soranou Ephessiou Street, 115 27 Athens, Greece; (C.S.-G.); (E.M.)
| | - Efstathios Michalopoulos
- Hellenic Cord Blood Bank, Biomedical Research Foundation Academy of Athens, 4 Soranou Ephessiou Street, 115 27 Athens, Greece; (C.S.-G.); (E.M.)
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Dupuis V, Oltra E. Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells. World J Stem Cells 2021; 13:1094-1111. [PMID: 34567428 PMCID: PMC8422924 DOI: 10.4252/wjsc.v13.i8.1094] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Revised: 05/03/2021] [Accepted: 07/14/2021] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.
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Affiliation(s)
- Victoria Dupuis
- Faculté des Sciences et d’Ingénierie, Sorbonne Université, Paris 75252, France
| | - Elisa Oltra
- Department of Pathology, Universidad Católica de Valencia San Vicente Mártir, Valencia 46001, Spain
- Centro de Investigación Traslacional San Alberto Magno, Universidad Católica de Valencia San Vicente Mártir, Valencia 46001, Spain.
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Bone Marrow Mesenchymal Stem Cells in Acute-on-Chronic Liver Failure Grades 2 and 3: A Phase I-II Randomized Clinical Trial. Can J Gastroenterol Hepatol 2021; 2021:3662776. [PMID: 34395335 PMCID: PMC8357501 DOI: 10.1155/2021/3662776] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/08/2021] [Accepted: 07/24/2021] [Indexed: 12/16/2022] Open
Abstract
INTRODUCTION Acute-on-chronic liver failure (ACLF) is an acute liver decompensation in cirrhotic patients, which leads to organ failures and high short-term mortality. The treatment is based on the management of complications and, in severe cases, liver transplantation. Since specific treatment is unavailable, we aimed to evaluate the safety and initial efficacy of bone marrow mesenchymal stem cells (BM-MSC) in patients with ACLF Grades 2 and 3, a population excluded from previous clinical trials. METHODS This is a randomized placebo-controlled phase I-II single center study, which enrolled 9 cirrhotic patients from 2018 to 2020, regardless of the etiology. The control group (n = 5) was treated with standard medical therapy (SMT) and placebo infusion of saline. The intervention group (n = 4) received SMT plus 5 infusions of 1 × 106 cells/kg of BM-MSC for 3 weeks. Both groups were monitored for 90 days. A Chi-square test was used for qualitative variables, and the t-test and Mann-Whitney U test for quantitative variables. The Kaplan-Meier estimator was used to build survival curves. In this study, we followed the intention-to-treat analysis, with a significance of 5%. RESULTS Nine patients with a mean Child-Pugh (CP) of 12.3, MELD of 38.4, and CLIF-C score of 50.7 were recruited. Hepatitis C and alcohol were the main etiologies. The average infusion per patient was 2.9 and only 3 patients (2 in control and 1 in the BM-MSC group) received all the protocol infusions. There were no infusion-related side effects, although one patient in the intervention group presented hypernatremia and a gastric ulcer, after the third and fifth infusions, respectively. The survival rate after 90 days was 20% (1/5) for placebo versus 25% (1/4) for the BM-MSC. The patient who completed the entire MSC protocol showed a significant improvement in CP (C-14 to B-9), MELD (32 to 22), and ACLF (grade 3 to 0). CONCLUSION BM-MSC infusion is safe and feasible in patients with ACLF Grades 2 and 3.
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da Fonseca L, Santos GS, Huber SC, Setti TM, Setti T, Lana JF. Human platelet lysate - A potent (and overlooked) orthobiologic. J Clin Orthop Trauma 2021; 21:101534. [PMID: 34386346 PMCID: PMC8339333 DOI: 10.1016/j.jcot.2021.101534] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 04/25/2021] [Accepted: 07/25/2021] [Indexed: 01/03/2023] Open
Abstract
The knowledge of the essential role of platelets in tissue healing is gradually increasing and as regenerative medicine prompts new solutions, platelet-derived bioproducts have been proposed as a potential tool in this field. In orthopaedics and sports medicine, the use of PRP has been rapidly increasing in popularity as patients seek novel non-surgical approaches to acute and chronic musculoskeletal conditions. The concept of having platelets as a secretory organ other than a mere sponge-like coagulation component opens up new frontiers for the use of the platelet secretome. Platelet lysate is a solution saturated by growth factors, proteins, cytokines, and chemokines involved in crucial healing processes and is administered to treat different diseases such as alopecia, oral mucositis, radicular pain, osteoarthritis, and cartilage and tendon disorders. For this purpose, the abundant presence of growth factors and chemokines stored in platelet granules can be naturally released by different strategies, mostly through lyophilization, thrombin activation or ultrasound baths (ultrasonication). As a result, human platelet lysate can be produced and applied as a pure orthobiologic. This review outlines the current knowledge about human platelet lysate as a powerful adjuvant in the orthobiological use for the treatment of musculoskeletal injuries, without however failing to raise some of its most applicable basic science.
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Affiliation(s)
- Lucas da Fonseca
- Orthopaedic Department – UNIFESP/EPM, 715 Napoleão de Barros St – Vila Clementino, 04024-002, São Paulo, SP, Brazil
| | - Gabriel Silva Santos
- IOC – Instituto Do Osso e da Cartilagem/the Bone and Cartilage Institute, 1386 Presidente Kennedy Avenue – Cidade Nova I, 13334-170, Indaiatuba, SP, Brazil,Corresponding author. IOC – Instituto do Osso e da Cartilagem/The Bone and Cartilage Institute, 1386 Presidente Kennedy Avenue – 2nd floor, Room #29, Indaiatuba, São Paulo, 13334-170, Brazil. Tel.: +551930174366, +5519989283863.
| | - Stephany Cares Huber
- IOC – Instituto Do Osso e da Cartilagem/the Bone and Cartilage Institute, 1386 Presidente Kennedy Avenue – Cidade Nova I, 13334-170, Indaiatuba, SP, Brazil
| | - Taís Mazzini Setti
- Indolor - Centro Intervencionista de Controle da Dor, 583 Sul Brasil Avenue – Room #406 – Centro, 89814-210, Maravilha, SC, Brazil
| | - Thiago Setti
- Indolor - Centro Intervencionista de Controle da Dor, 583 Sul Brasil Avenue – Room #406 – Centro, 89814-210, Maravilha, SC, Brazil
| | - José Fábio Lana
- IOC – Instituto Do Osso e da Cartilagem/the Bone and Cartilage Institute, 1386 Presidente Kennedy Avenue – Cidade Nova I, 13334-170, Indaiatuba, SP, Brazil
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De Angelis E, Saleri R, Martelli P, Elviri L, Bianchera A, Bergonzi C, Pirola M, Romeo R, Andrani M, Cavalli V, Conti V, Bettini R, Passeri B, Ravanetti F, Borghetti P. Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate. Front Vet Sci 2021; 8:671776. [PMID: 34322533 PMCID: PMC8311290 DOI: 10.3389/fvets.2021.671776] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Accepted: 05/14/2021] [Indexed: 11/13/2022] Open
Abstract
Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.
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Affiliation(s)
- Elena De Angelis
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Roberta Saleri
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Paolo Martelli
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Lisa Elviri
- Food and Drug Department, University of Parma, Parma, Italy
| | | | - Carlo Bergonzi
- Food and Drug Department, University of Parma, Parma, Italy
| | - Marta Pirola
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Roberta Romeo
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Melania Andrani
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Valeria Cavalli
- Department of Veterinary Science, University of Parma, Parma, Italy
| | - Virna Conti
- Department of Veterinary Science, University of Parma, Parma, Italy
| | | | | | | | - Paolo Borghetti
- Department of Veterinary Science, University of Parma, Parma, Italy
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Developing a Glyoxal-Crosslinked Chitosan/Gelatin Hydrogel for Sustained Release of Human Platelet Lysate to Promote Tissue Regeneration. Int J Mol Sci 2021; 22:ijms22126451. [PMID: 34208633 PMCID: PMC8234746 DOI: 10.3390/ijms22126451] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2021] [Revised: 06/12/2021] [Accepted: 06/12/2021] [Indexed: 12/22/2022] Open
Abstract
The clinical application of human platelet lysate (HPL) holds promise for tissue regeneration, and the development of an efficient vehicle for its delivery is desired. Chitosan-based hydrogels are potential candidates, but they often exhibit weak mechanical properties. In this study, a chitosan/gelatin (CS-GE) hydrogel crosslinked by glyoxal was fabricated for sustained release of HPL. The influence of HPL on Hs68 fibroblast and human umbilical vein endothelial cell (HUVEC) culture was evaluated, and we found that supplementing 5% HPL in the medium could significantly improve cell proliferation relative to supplementing 10% fetal bovine serum (FBS). Moreover, HPL accelerated the in vitro wound closure of Hs68 cells and facilitated the tube formation of HUVECs. Subsequently, we fabricated CS-GE hydrogels crosslinked with different concentrations of glyoxal, and the release pattern of FITC-dextrans (4, 40 and 500 kDa) from the hydrogels was assessed. After an ideal glyoxal concentration was determined, we further characterized the crosslinked CS-GE hydrogels encapsulated with different amounts of HPL. The HPL-incorporated hydrogel was shown to significantly promote the proliferation of Hs68 cells and the migration of HUVECs. Moreover, the release pattern of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB) from hydrogel was examined in vitro, demonstrating a sustained release profile of the growth factors. Finally, the chick chorioallantoic membrane assay revealed that HPL encapsulation in the hydrogel significantly stimulated angiogenesis in ovo. These results demonstrate the great potential of the crosslinked CS-GE hydrogel to serve as an effective delivery system for HPL to promote tissue regeneration.
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Jeyaraman M, Muthu S, Khanna M, Jain R, Anudeep TC, Muthukanagaraj P, Siddesh SE, Gulati A, Satish AS, Jeyaraman N, Khanna V. Platelet lysate for COVID-19 pneumonia-a newer adjunctive therapeutic avenue. Stem Cell Investig 2021; 8:11. [PMID: 34268440 PMCID: PMC8256133 DOI: 10.21037/sci-2020-042] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Accepted: 04/27/2021] [Indexed: 02/05/2023]
Abstract
The linchpin for COVID-19 pathogenesis is the severe inflammatory process in the respiratory tract wherein the accumulation of excessive cytokines paves the way for a series of systemic hemodynamic alterations and mortality. The mortality rate is higher in individuals with co-morbidities and advancing age. The absence of a specific therapy is responsible for this uncontrolled spread and the significant mortality. This renders potential insight for considering biologics as a plausible option to repair and regenerate the affected lung tissue and pulverize the causative organism. The plausible role of megakaryocytes against invading microbes was not clearly understood. Platelet lysate is an acellular product consisting of regenerative molecules released from a cluster of platelets. It attenuates the changes caused by immune reactions in allogenic utility with the introduction of growth factors, cytokines, and proteins at supraphysiologic levels and thereby serves as a regenerative immunomodulatory agent to combat COVID-19. This platelet lysate can be used in nebulized form for such acute respiratory distress conditions in COVID-19 elderly patients. Platelet lysate may emerge as a pivotal player provided investigations pace up in this context. Here, we discuss how the platelet lysate can plausibly perquisite to relegate COVID-19. Undertaking prospective randomized controlled trials to prove its efficacy is the need of the hour in this pandemic scenario.
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Affiliation(s)
- Madhan Jeyaraman
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- Department of Orthopedics, School of Medical Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
- Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Sathish Muthu
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, Uttar Pradesh, India
- Department of Orthopedics, Government Medical College & Hospital, Dindigul, Tamil Nadu, India
| | - Manish Khanna
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- Department of Orthopedics, Prasad Institute of Medical Science and Hospital, Lucknow, Uttar Pradesh, India
| | - Rashmi Jain
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- School of Medical Sciences and Research, Sharda University, Greater Noida, Uttar Pradesh, India
| | - Talagavadi Channaiah Anudeep
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- Department of Plastic Surgery, Topiwala National Medical College and BYL Nair Ch. Hospital, Mumbai, Maharashtra, India
| | - Purushothaman Muthukanagaraj
- Department of Internal Medicine & Psychiatry, SUNY-Upstate Binghamton Clinical Campus, Binghamton, New York, USA
| | | | - Arun Gulati
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- Department of Orthopedics, Kalpana Chawla Government Medical College & Hospital, Karnal, Haryana, India
| | | | - Naveen Jeyaraman
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- Department of Orthopedics, Kasturba Medical College, MAHE Unievrsity, Manipal, Karnataka, India
| | - Venus Khanna
- Indian Stem Cell Study Group (ISCSG) Association, Lucknow, Uttar Pradesh, India
- Department of Pathology, Prasad Institute of Medical Science and Hospital, Lucknow, Uttar Pradesh, India
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