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Aarattuthodi S, Kang D, Gupta SK, Chen P, Redel B, Matuha M, Mohammed H, Sinha AK. Cryopreservation of biological materials: applications and economic perspectives. In Vitro Cell Dev Biol Anim 2025:10.1007/s11626-025-01027-0. [PMID: 40266443 DOI: 10.1007/s11626-025-01027-0] [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: 10/23/2024] [Accepted: 02/09/2025] [Indexed: 04/24/2025]
Abstract
Cryopreservation is a transformative technology that allows for the long-term storage of biological materials by cooling them to extremely low temperatures at which metabolic and biochemical processes are effectively slowed or halted. Cryopreservation utilizes various techniques to minimize ice crystal formation and cellular damage during freezing and thawing processes. This technology has broad applications in the fields of medicine, agriculture, and conservation, spanning across stem cell research, reproductive and regenerative medicine, organ transplantation, and cell-based therapies, each with significant economic implications. While current techniques and their associated costs present certain challenges, ongoing research advancements related to cryoprotectants, cooling methods, and automation promise to enhance efficiency and accessibility, potentially broadening the technology's impact across various sectors. This review focuses on the applications of cryopreservation, research advancements, and economic implications, emphasizing the importance of continued research to overcome the current limitations.
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Affiliation(s)
- Suja Aarattuthodi
- Plant Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA.
| | - David Kang
- Biological Control of Insects Research Laboratory, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA
| | - Sanjay Kumar Gupta
- Indian Institute of Agricultural Biotechnology, Garhkhatanga, Ranchi, Jharkhand, 834003, India
| | - Paula Chen
- Plant Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA
| | - Bethany Redel
- Plant Genetics Research Unit, United States Department of Agriculture - Agricultural Research Service, Columbia, MO, 65211, USA
| | - Moureen Matuha
- Department of Agriculture and Environmental Sciences, Lincoln University of Missouri, Jefferson City, MO, 65101, USA
| | - Haitham Mohammed
- Department of Rangeland, Wildlife and Fisheries Management, Texas a&M University, College Station, TX, 77843, USA
| | - Amit Kumar Sinha
- Department of Aquaculture and Fisheries, University of Arkansas Pine Bluff, Pine Bluff, AR, 71601, USA
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Aabling RR, Rusan M, Møller AMJ, Munk-Pedersen N, Holm C, Elmengaard B, Pedersen M, Møller BK. A Narrative Review on Manufacturing Methods Employed in the Production of Mesenchymal Stromal Cells for Knee Osteoarthritis Therapy. Biomedicines 2025; 13:509. [PMID: 40002922 PMCID: PMC11853043 DOI: 10.3390/biomedicines13020509] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2024] [Revised: 02/06/2025] [Accepted: 02/10/2025] [Indexed: 02/27/2025] Open
Abstract
Knee osteoarthritis (OA) is a chronic, progressive, inflammatory, and degenerative whole-joint disease. Early-stage OA treatments typically include physiotherapy, weight-loss, pain relief medications, and intra-articular knee injections, such as corticosteroids, hyaluronic acid, or platelet-rich plasma. These treatments primarily provide symptomatic relief rather than reversing or halting disease progression. Recently, mesenchymal stromal cell (MSC) injections have garnered attention due to their immunomodulatory and regenerative capacities. MSCs, which can be derived from sources such as bone marrow, umbilical cord, or adipose tissue, and can be allogeneic or autologous, have demonstrated promising results in both animal models and several human studies. However, different protocols have been employed, presenting challenges for comparing outcomes. In this review, we address these variable settings, evaluate current practices, and identify key factors critical in optimizing MSC-based therapies by critically reviewing clinical trials of ex vivo expanded MSC therapies for OA undertaken between 2008 and 2023. Specific attention was given to two key aspects: (1) the cell culture process employed in manufacturing of autologous or allogeneic MSC products, and (2) the post-culture methods employed in storage, reconstitution and administration of the MSCs. Our findings suggest that standardizing MSC production for clinical applications remains a significant challenge, primarily due to variations in tissue sources, harvesting techniques, and manufacturing protocols, and due to broad discrepancies in reporting. Thus, we propose a set of minimal reporting criteria to guide future clinical trials. A common reporting guideline is a critical step towards a more standardized MSC production across different laboratories and clinical settings, thereby enhancing reproducibility and advancing the field of regenerative medicine for knee OA, as well as other disease settings.
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Affiliation(s)
- Rasmus Roost Aabling
- Comparative Medicine Lab, SDCA-Steno Diabetes Center Aarhus, Department of Clinical Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99 and 11, DK-8200 Aarhus, Denmark
| | - Maria Rusan
- Department of Molecular Medicine, Aarhus University Hospital, Brendstrupgårdsvej 21A, DK-8200 Aarhus, Denmark;
- Department of Clinical Pharmacology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark
- Department of Clinical Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark;
| | - Anaïs Marie Julie Møller
- Center for Gene and Cellular Therapy, Department of Clinical Immunology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark;
| | - Naija Munk-Pedersen
- Comparative Medicine Lab, Department of Clinical Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark; (N.M.-P.); (M.P.)
| | - Carsten Holm
- Department of Orthopedic Surgery, Elective Surgery Centre, Silkeborg Regional Hospital, Falkevej 1G, DK-8600 Silkeborg, Denmark; (C.H.); (B.E.)
| | - Brian Elmengaard
- Department of Orthopedic Surgery, Elective Surgery Centre, Silkeborg Regional Hospital, Falkevej 1G, DK-8600 Silkeborg, Denmark; (C.H.); (B.E.)
| | - Michael Pedersen
- Comparative Medicine Lab, Department of Clinical Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark; (N.M.-P.); (M.P.)
| | - Bjarne Kuno Møller
- Department of Clinical Medicine, Aarhus University, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark;
- Center for Gene and Cellular Therapy, Department of Clinical Immunology, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus, Denmark;
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Trufanova N, Hubenia O, Kot Y, Trufanov O, Kovalenko I, Kot K, Petrenko O. Metabolic Mode of Alginate-Encapsulated Human Mesenchymal Stromal Cells as a Background for Storage at Ambient Temperature. Biopreserv Biobank 2024. [PMID: 39723454 DOI: 10.1089/bio.2024.0103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2024] Open
Abstract
Introduction: Human mesenchymal stromal cells (MSCs) are attractive for both medical practice and biomedical research. Nonfreezing short-term storage may provide safe and simple transportation and promote the practical use of MSCs. Objectives: We aimed to determine the duration of efficient storage at ambient temperature (22°C) of human dermal MSCs in different three-dimensional organization and to investigate the role of cell metabolic mode in the resistance to the ambient storage damaging factors. Methods: MSCs in monolayer, suspension, and encapsulated in alginate microspheres (AMS) were stored in sealed containers at 22°С in culture medium. Viability (fluorescein diacetate /ethidium bromide) and metabolic activity (Alamar Blue assay) were assessed at 0, 3, 7, 10, and 14 days of the storage. Mitochondrial membrane potential (JC-1 test), cell cycle analysis, reactive oxygen species level, and resistance to hydrogen peroxide were analyzed under culture conditions. Results: Alginate encapsulation was shown to maintain viability (about 85%), metabolic activity, and adhesion ability during storage for 7 days. The storage of MSCs in both monolayer and suspension was less efficient. Culture of MSCs in AMS decreased basal metabolic activity, mitochondrial activity, and led to reversible cell cycle arrest compared to standard two-dimensional culture. MSCs in AMS have a lower basal level of reactive oxygen species and higher resistance to hydrogen peroxide compared with those in monolayer culture. Conclusion: Revealed shift into quiescent metabolic mode is essential for alginate-encapsulated MSCs resistance to storage at ambient temperature.
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Affiliation(s)
- Natalia Trufanova
- Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine
| | - Oleksandra Hubenia
- Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine
- Institute for Multiphase Processes, Leibniz University Hannover, Garbsen, Germany
| | - Yurii Kot
- V.N. Karazin Kharkiv National University, Kharkiv, Ukraine
| | - Oleh Trufanov
- Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine
| | - Ihor Kovalenko
- Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine
| | - Kateryna Kot
- V.N. Karazin Kharkiv National University, Kharkiv, Ukraine
| | - Oleksandr Petrenko
- Institute for Problems of Cryobiology and Cryomedicine of National Academy of Sciences of Ukraine, Kharkiv, Ukraine
- V.N. Karazin Kharkiv National University, Kharkiv, Ukraine
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Izaguirre-Pérez N, Ligero G, Aguilar-Solana PA, Carrillo-Ávila JA, Rodriguez-Reyes CR, Biunno I, Aguilar-Quesada R, Catalina P. Trehalose Cryopreservation of Human Mesenchymal Stem Cells from Cord Tissue. Biopreserv Biobank 2024. [PMID: 39723442 DOI: 10.1089/bio.2024.0025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2024] Open
Abstract
Adequate hypothermic storage of human mesenchymal stem cells (hMSCs) is of fundamental importance since they have been explored in several regenerative medicine initiatives. However, the actual clinical application of hMSCs necessitates hypothermic storage for long periods, a process that requires the use of non-toxic and efficient cryo-reagents capable of maintaining high viability and differentiating properties after thawing. Current cryopreservation methods are based on cryoprotectant agents (CPAs) containing dimethylsulphoxide (DMSO), which have been shown to be toxic for clinical applications. In this study, we describe a simple and effective trehalose (TRE)-based solution to cryo-store human umbilical cord-derived MSCs (UC-MSCs) in liquid nitrogen. Cells viability, identity, chromosomal stability, proliferative and migration capacity, and stress response were assessed after cryopreservation in TRE as CPA, testing different concentrations by itself or in combination with ethylene glycol (EG). Here we show that TRE-stored UC-MSCs provided lower cell recovery rates compared with DMSO-based solution, but maintained good functional properties, stability, and differentiating potential. The best cell recovery was obtained using 0.5 M TRE with 10% EG showing no differences in the osteogenic, adipogenic, and chondrogenic differentiation capacity. A second cycle of cryopreservation in this TRE-based solution had no additional impact on the viability and morphology, although slightly affected cell migration. Furthermore, the expression of the stress-related genes, HSPA1A, SOD2, TP53, BCL-2, and BAX, did not show a higher response in UC-MSCs cryopreserved in 0.5 M TRE + 10% EG compared with DMSO. Together these results, in addition to ascertained therapeutic properties of TRE, provide sufficient evidence to consider TRE-based medium as a low-cost and efficient solution for the storage of human UC-MSCs cells and potentially substitute DMSO-based cryo-reagents.
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Affiliation(s)
| | - Gertrudis Ligero
- Coordinating Node, Andalusian Public Health System Biobank, Granada, Spain
| | | | | | | | - Ida Biunno
- Integrated System Engineering, Bresso-Milano, Italy
- Department of Translational Medicine-Pavia, University of Pavia, Pavia, Italy
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Fang WH, Vangsness CT. Orthobiologic Products: Preservation Options for Orthopedic Research and Clinical Applications. J Clin Med 2024; 13:6577. [PMID: 39518716 PMCID: PMC11546119 DOI: 10.3390/jcm13216577] [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: 09/11/2024] [Revised: 10/14/2024] [Accepted: 10/28/2024] [Indexed: 11/16/2024] Open
Abstract
The biological products used in orthopedics include musculoskeletal allografts-such as bones, tendons, ligaments, and cartilage-as well as biological therapies. Musculoskeletal allografts support the body's healing process by utilizing preserved and sterilized donor tissue. These allografts are becoming increasingly common in surgical practice, allowing patients to avoid more invasive procedures and the risks associated with donor site morbidity. Bone grafting is one of the most frequently used procedures in orthopedics and traumatology. Biologic approaches aim to improve clinical outcomes by enhancing the body's natural healing capacity and reducing inflammation. They serve as an alternative to surgical interventions. While preliminary results from animal studies and small-scale clinical trials have been promising, the field of biologics still lacks robust clinical evidence supporting their efficacy. Biological therapies include PRP (platelet-rich plasma), mesenchymal stem cells (MSCs)/stromal cells/progenitor cells, bone marrow stem/stromal cells (BMSCs), adipose stem/stromal cells/progenitor cells (ASCs), cord blood (CB), and extracellular vesicles (EVs), including exosomes. The proper preservation and storage of these cellular therapies are essential for future use. Preservation techniques include cryopreservation, vitrification, lyophilization, and the use of cryoprotective agents (CPAs). The most commonly used CPA is DMSO (dimethyl sulfoxide). The highest success rates and post-thaw viability have been achieved by preserving PRP with a rate-controlled freezer using 6% DMSO and storing other cellular treatments using a rate-controlled freezer with 5% or 10% DMSO as the CPA. Extracellular vesicles (EVs) have shown the best results when lyophilized with 50 mM or 4% trehalose to prevent aggregation and stored at room temperature.
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Affiliation(s)
- William H. Fang
- Department of Orthopedic Surgery, Valley Health Systems, 620 Shadow Lane, Las Vegas, NV 89106, USA
| | - C. Thomas Vangsness
- Department of Orthopedic Surgery, Keck School of Medicine of USC, Los Angeles, CA 90033, USA
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Park JJ, Lee OH, Park JE, Cho J. Comparison of Cryopreservation Media for Mesenchymal Stem Cell Spheroids. Biopreserv Biobank 2024; 22:486-496. [PMID: 38011543 DOI: 10.1089/bio.2023.0057] [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] [Indexed: 11/29/2023] Open
Abstract
Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.
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Affiliation(s)
- Jin Ju Park
- Department of Dental Regenerative Biotechnology, School of Dentistry, Seoul National University, Seoul, Republic of Korea
- Dental Research Institute, Seoul National University, Seoul, Republic of Korea
| | - Ok-Hee Lee
- Department of Dental Regenerative Biotechnology, School of Dentistry, Seoul National University, Seoul, Republic of Korea
- Dental Research Institute, Seoul National University, Seoul, Republic of Korea
| | - Jie-Eun Park
- Department of Dental Regenerative Biotechnology, School of Dentistry, Seoul National University, Seoul, Republic of Korea
- Dental Research Institute, Seoul National University, Seoul, Republic of Korea
| | - Jaejin Cho
- Department of Dental Regenerative Biotechnology, School of Dentistry, Seoul National University, Seoul, Republic of Korea
- Dental Research Institute, Seoul National University, Seoul, Republic of Korea
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Gil-Chinchilla JI, Bueno C, Martínez CM, Ferrández-Múrtula A, García-Hernández AM, Blanquer M, Molina-Molina M, Zapata AG, Sackstein R, Moraleda JM, García-Bernal D. Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics. Front Immunol 2024; 15:1385691. [PMID: 38605955 PMCID: PMC11007032 DOI: 10.3389/fimmu.2024.1385691] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2024] [Accepted: 03/20/2024] [Indexed: 04/13/2024] Open
Abstract
Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.
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Affiliation(s)
- Jesús I. Gil-Chinchilla
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
| | - Carlos Bueno
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
| | - Carlos M. Martínez
- Experimental Pathology Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia, Murcia, Spain
| | - Ana Ferrández-Múrtula
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
| | - Ana M. García-Hernández
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
| | - Miguel Blanquer
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
- Department of Medicine, University of Murcia, Murcia, Spain
| | - Mar Molina-Molina
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
| | | | - Robert Sackstein
- Department of Translational Medicine, and the Translational Glycobiology Institute, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, United States
| | - Jose M. Moraleda
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
- Department of Medicine, University of Murcia, Murcia, Spain
| | - David García-Bernal
- Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria (IMIB) Pascual Parrilla, University of Murcia and Virgen de la Arrixaca University Hospital, Murcia, Spain
- Department of Biochemistry, Molecular Biology, and Immunology, University of Murcia, Murcia, Spain
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Guo Z, Zuchowicz N, Bouwmeester J, Joshi AS, Neisch AL, Smith K, Daly J, Etheridge ML, Finger EB, Kodandaramaiah SB, Hays TS, Hagedorn M, Bischof JC. Conduction-Dominated Cryomesh for Organism Vitrification. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2303317. [PMID: 38018294 PMCID: PMC10797434 DOI: 10.1002/advs.202303317] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Revised: 10/20/2023] [Indexed: 11/30/2023]
Abstract
Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction-dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105 °C min-1 ) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction-dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated.
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Affiliation(s)
- Zongqi Guo
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
| | - Nikolas Zuchowicz
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
| | - Jessica Bouwmeester
- Hawaii Institute of Marine BiologyUniversity of HawaiiKaneoheHI96744USA
- Smithsonian National Zoo and Conservation Biology InstituteFront RoyalVA22630USA
| | - Amey S. Joshi
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
| | - Amanda L. Neisch
- Department of GeneticsCell Biology and DevelopmentUniversity of MinnesotaMinneapolisMN55455USA
| | - Kieran Smith
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
| | - Jonathan Daly
- Taronga Conservation Society AustraliaMosmanNew South Wales2088Australia
- School of BiologicalEarth and Environmental SciencesUniversity of New South WalesKensingtonNew South Wales2033Australia
| | - Michael L. Etheridge
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
| | - Erik B. Finger
- Department of SurgeryUniversity of MinnesotaMinneapolisMN55455USA
| | - Suhasa B. Kodandaramaiah
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Department of Biomedical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Graduate Program in NeuroscienceUniversity of MinnesotaMinneapolisMN55455USA
| | - Thomas S. Hays
- Department of GeneticsCell Biology and DevelopmentUniversity of MinnesotaMinneapolisMN55455USA
| | - Mary Hagedorn
- Hawaii Institute of Marine BiologyUniversity of HawaiiKaneoheHI96744USA
- Smithsonian National Zoo and Conservation Biology InstituteFront RoyalVA22630USA
| | - John C. Bischof
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Department of Biomedical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Institute for Engineering in MedicineUniversity of MinnesotaMinneapolisMN55455USA
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9
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Azam M, Ghufran H, Tasneem S, Mehmood A, Malik K, Yousaf MA, Tarar MN, Akram SJ, Riazuddin S. Priming of adipose-derived stem cells with curcumin prior to cryopreservation preserves their functional potency: Towards an 'Off-the-shelf' therapy for burns. Cryobiology 2023; 110:69-78. [PMID: 36470459 DOI: 10.1016/j.cryobiol.2022.12.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2022] [Revised: 11/18/2022] [Accepted: 12/01/2022] [Indexed: 12/12/2022]
Abstract
Stem cells-based treatment for burn wounds require frozen cells as an off-the-shelf therapy; however, cryopreservation-induced oxidative stress resulted in post-thaw cell death or loss of cell functions, thus arrested their clinical practicality. Although antioxidant priming to stem cells increase their resistant to oxidative stress, but this strategy is still unexplored on cryopreserved cells. Herein, we investigated whether curcumin priming before cryopreservation could preserve the therapeutic potency of thawed stem cells. For this, unprimed and curcumin-primed adipose-derived stem cells (ASCs) were cryopreserved for one month. Post-thawing, cells were assessed for viability by trypan blue assay; metabolic activity by MTT assay; senescence by senescence-associated (SA)-β-galactosidase activity staining assay; migration by scratch healing assay and; mRNA expression by real-time PCR. Subsequently, the healing potential was examined by injecting cells around the wound periphery of acidic burn in rats. Post-healing, skin architecture was histologically examined. Results demonstrated that, curcumin-primed frozen cells (Cryo/Cur-ASCs) showed better post-thaw viability, metabolic activity, migration ability and lower percent of senescence comparative to unprimed frozen cells (Cryo/ASCs). Curcumin priming alleviated the oxidative damage by activating the ROS-reducing cellular antioxidant system as shown by the evident increase in GSH levels and upregulated mRNA expression of glutathione peroxidase (GPx), superoxide dismutases (SOD1, SOD2), and catalase (CAT). Further, invivo findings revealed that Cryo/Cur-ASCs-treated wounds exhibited earlier wound closure with an improved architecture comparative to Cryo/ASCs and depicted healing capacity almost similar to Fresh/ASCs. Our findings suggested that curcumin priming could be effective to alleviate the cryo-induced oxidative stress in post-thawed cells.
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Affiliation(s)
- Maryam Azam
- National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan
| | - Hafiz Ghufran
- National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan
| | - Saba Tasneem
- National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan
| | - Azra Mehmood
- National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan.
| | - Kausar Malik
- National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan
| | | | - Moazzam N Tarar
- Jinnah Burn and Reconstructive Surgery Centre, Lahore, Pakistan
| | | | - Sheikh Riazuddin
- National Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, University of the Punjab, Lahore, Pakistan; Jinnah Burn and Reconstructive Surgery Centre, Lahore, Pakistan
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10
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Rodrigues LLV, Moura YBF, Viana JVS, Praxedes ÉA, Oliveira LRM, Silva HVR, Pereira AF. Comparison between concentration and type of intracellular cryoprotectants and the presence of sucrose for cryobanks of somatic cells derived from captive Pumas. Zoo Biol 2022; 42:440-448. [PMID: 36515220 DOI: 10.1002/zoo.21748] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Revised: 10/12/2022] [Accepted: 12/06/2022] [Indexed: 12/15/2022]
Abstract
The loss of wild biodiversity has prompted the development of cryobanks, such as those of somatic cells. This is the reality of Pumas, wild felids of ecological importance that suffer from anthropogenic actions, population decline, and subsequent loss of genetic diversity. Somatic cell banks are a strategy for conserving population diversity. We compared different concentrations and types of intracellular cryoprotectants (dimethyl sulfoxide, DMSO; ethylene glycol, EG) associated with 0.2 M of sucrose (SUC) in the cryopreservation of the somatic cells of captive Pumas. The cells were cryopreserved by slow freezing with different solutions containing Dulbecco's modified Eagle's medium with 10% fetal bovine serum and varying concentrations of DMSO and EG in the absence or presence of SUC. The cells were analyzed for morphological characteristics, viability, proliferative activity, metabolic activity, and apoptosis levels. Cells maintained similar fusiform morphology before and after cryopreservation. There was no difference in viability, regardless of the reduction in the concentration and type of intracellular cryoprotectants and sucrose. Similarly, proliferative activity, metabolic activity, and apoptosis levels were not altered by the composition of the cryoprotectants. In summary, we demonstrate that reducing the concentration of DMSO or EG ensures adequate cryopreservation of Puma somatic cells, regardless of the presence of SUC.
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Affiliation(s)
- Luanna L V Rodrigues
- Laboratory of Animal Biotechnology, Department of Biosciences, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil
| | - Yasmin B F Moura
- Laboratory of Animal Biotechnology, Department of Biosciences, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil
| | - João V S Viana
- Laboratory of Animal Biotechnology, Department of Biosciences, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil
| | - Érika A Praxedes
- Laboratory of Animal Biotechnology, Department of Biosciences, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil
| | - Lhara R M Oliveira
- Laboratory of Animal Biotechnology, Department of Biosciences, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil
| | - Herlon V R Silva
- Laboratory of Reproduction of Carnivorous, Faculty of Veterinary, Ceara State University, Fortaleza, CE, Brazil
| | - Alexsandra F Pereira
- Laboratory of Animal Biotechnology, Department of Biosciences, Federal Rural University of Semi-Arid, Mossoro, RN, Brazil
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11
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Liu X, Gao S, Ren L, Yuan X. Achieving high intracellular trehalose in hRBCs by reversible membrane perturbation of maltopyranosides with synergistic membrane protection of macromolecular protectants. BIOMATERIALS ADVANCES 2022; 141:213114. [PMID: 36113360 DOI: 10.1016/j.bioadv.2022.213114] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Revised: 08/20/2022] [Accepted: 09/05/2022] [Indexed: 06/15/2023]
Abstract
Trehalose is considered as a biocompatible cryoprotectant for solvent-free cryopreservation of cells, but the difficulty of the current trehalose delivery platforms to human red blood cells (hRBCs) limits its wide applications. Due to cell injuries caused by incubation at 37 °C and low intracellular loading efficiency, development of novel methods to facilitate trehalose entry in hRBCs is essential. Herein, a reversible membrane perturbation and synergistic membrane stabilization system based on maltopyranosides and macromolecular protectants was constructed, demonstrating the ability of efficient trehalose loading in hRBCs at 4 °C. Results of confocal laser scanning microscopy exhibited that the intracellular loading with the assistance of maltopyranosides was a reversible process, while the membrane protective effect of macromolecular protectants on trehalose loading in hRBCs was necessary. It was suggested that introduction of 30 mM poly(vinyl pyrrolidone) 8000 combined with 1 mM dodecyl-β-D-maltopyranoside and 0.8 M trehalose could increase the intracellular trehalose to 84.0 ± 11.3 mM in hRBCs, whereas poly(ethylene glycol), dextran, human serum albumin or hydroxyethyl starch had a weak effect. All the macromolecular protectants could promote the cryosurvival of hRBCs, exhibiting membrane stabilization, and incubation and followed by cryopreservation did not change the basic functions and normal morphology of hRBCs substantially. This study provided an alternative strategy for glycerol-free cryopreservation of cells and the delivery of membrane-impermeable cargos.
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Affiliation(s)
- Xingwen Liu
- School of Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin University, Tianjin 300350, China
| | - Shuhui Gao
- School of Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin University, Tianjin 300350, China
| | - Lixia Ren
- School of Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin University, Tianjin 300350, China
| | - Xiaoyan Yuan
- School of Materials Science and Engineering, Tianjin Key Laboratory of Composite and Functional Materials, Tianjin University, Tianjin 300350, China.
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12
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Linkova DD, Rubtsova YP, Egorikhina MN. Cryostorage of Mesenchymal Stem Cells and Biomedical Cell-Based Products. Cells 2022; 11:cells11172691. [PMID: 36078098 PMCID: PMC9454587 DOI: 10.3390/cells11172691] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 08/15/2022] [Accepted: 08/24/2022] [Indexed: 11/16/2022] Open
Abstract
Mesenchymal stem cells (MSCs) manifest vast opportunities for clinical use due both to their ability for self-renewal and for effecting paracrine therapeutic benefits. At the same time, difficulties with non-recurrent generation of large numbers of cells due to the necessity for long-term MSC expansion ex vivo, or the requirement for repeated sampling of biological material from a patient significantly limits the current use of MSCs in clinical practice. One solution to these problems entails the creation of a biobank using cell cryopreservation technology. This review is aimed at analyzing and classifying literature data related to the development of protocols for the cryopreservation of various types of MSCs and tissue-engineered structures. The materials in the review show that the existing techniques and protocols for MSC cryopreservation are very diverse, which significantly complicates standardization of the entire process. Here, the selection of cryoprotectors and of cryoprotective media shows the greatest variability. Currently, it is the cryopreservation of cell suspensions that has been studied most extensively, whereas there are very few studies in the literature on the freezing of intact tissues or of tissue-engineered structures. However, even now it is possible to develop general recommendations to optimize the cryopreservation process, making it less traumatic for cells.
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13
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Dave C, Mei SHJ, McRae A, Hum C, Sullivan KJ, Champagne J, Ramsay T, McIntyre L. Comparison of freshly cultured versus cryopreserved mesenchymal stem cells in animal models of inflammation: A pre-clinical systematic review. eLife 2022; 11:75053. [PMID: 35838024 PMCID: PMC9286731 DOI: 10.7554/elife.75053] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Accepted: 06/05/2022] [Indexed: 12/09/2022] Open
Abstract
Background: Mesenchymal stem cells (MSCs) are multipotent cells that demonstrate therapeutic potential for the treatment of acute and chronic inflammatory-mediated conditions. Although controversial, some studies suggest that MSCs may lose their functionality with cryopreservation which could render them non-efficacious. Hence, we conducted a systematic review of comparative pre-clinical models of inflammation to determine if there are differences in in vivo measures of pre-clinical efficacy (primary outcomes) and in vitro potency (secondary outcomes) between freshly cultured and cryopreserved MSCs. Methods: A systematic search on OvidMEDLINE, EMBASE, BIOSIS, and Web of Science (until January 13, 2022) was conducted. The primary outcome included measures of in vivo pre-clinical efficacy; secondary outcomes included measures of in vitro MSC potency. Risk of bias was assessed by the SYRCLE ‘Risk of Bias’ assessment tool for pre-clinical studies. Results: Eighteen studies were included. A total of 257 in vivo pre-clinical efficacy experiments represented 101 distinct outcome measures. Of these outcomes, 2.3% (6/257) were significantly different at the 0.05 level or less; 2 favoured freshly cultured and 4 favoured cryopreserved MSCs. A total of 68 in vitro experiments represented 32 different potency measures; 13% (9/68) of the experiments were significantly different at the 0.05 level or less, with seven experiments favouring freshly cultured MSC and two favouring cryopreserved MSCs. Conclusions: The majority of preclinical primary in vivo efficacy and secondary in vitro potency outcomes were not significantly different (p<0.05) between freshly cultured and cryopreserved MSCs. Our systematic summary of the current evidence base may provide MSC basic and clinical research scientists additional rationale for considering a cryopreserved MSC product in their pre-clinical studies and clinical trials as well as help identify research gaps and guide future related research. Funding: Ontario Institute for Regenerative Medicine
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Affiliation(s)
- Chintan Dave
- Division of Critical Care Medicine, Department of Medicine, Western University, London, Canada
| | - Shirley H J Mei
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada
| | - Andrea McRae
- Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada
| | - Christine Hum
- Knowledge Synthesis Group, Ottawa Hospital Research Institute, Ottawa, Canada.,University of Ottawa, Ottawa, Canada
| | - Katrina J Sullivan
- Knowledge Synthesis Group, Ottawa Hospital Research Institute, Ottawa, Canada
| | - Josee Champagne
- Knowledge Synthesis Group, Ottawa Hospital Research Institute, Ottawa, Canada.,Clinical Epidemiology, Ottawa Hospital Research Institute, Ottawa, Canada
| | - Tim Ramsay
- Clinical Epidemiology, Ottawa Hospital Research Institute, Ottawa, Canada
| | - Lauralyn McIntyre
- Knowledge Synthesis Group, Ottawa Hospital Research Institute, Ottawa, Canada.,Division of Critical Care, Department of Medicine, University of Ottawa, Ottawa, Canada
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14
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Pakhomov O, Gurina T, Mazaeva V, Polyakova A, Deng B, Legach E, Bozhok G. Phase transitions and mechanisms of cryoprotection of serum-/xeno-free media based on dextran and dimethyl sulfoxide. Cryobiology 2022; 107:13-22. [PMID: 35753382 DOI: 10.1016/j.cryobiol.2022.06.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Revised: 06/15/2022] [Accepted: 06/21/2022] [Indexed: 11/19/2022]
Abstract
The development of serum-/xeno-free media may help avoid the drawbacks of using serum and its components, such as probable contamination, instability of composition, or difficulty in sterilization. The objectives of this research were to investigate the use of combinations of a permeating cryoprotective agent (Me2SO) and non-permeating (polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, hydroxyethyl starch, dextran) polymers for cryopreservation of interstitial cells (ICs) of rat testis, and to propose the mechanism of cryoprotection of such compositions. In the course of this study, the best combination was 100 mg/ml dextran (M.m. 40 kDa) (Dex40) with 0.7 M Me2SO in Ham's F12. The ICs were additionally cooled and warmed to different end temperatures (-30, -50, -50 and -196 °C) to determine which temperature intervals contributed most to the IC loss. Then, the cryoprotective action of this serum-/xeno-free medium was investigated in comparison with serum or albumin-containing media by differential scanning calorimetry (DSC) and thermomechanical analysis (TMA). The results showed that the medium based on Dex40 did not decrease the amount of ice formed. However, it could undergo other phase separation and phase transformation to form glassy states. Potential cell-damaging physical processes such as eutectic crystallization/melting, recrystallization of NaCl and/or Me2SO derivatives, found in serum-containing media and taking place in specific temperature intervals, were not observed in the Dex40 based media. This was in good correlation with indicators of cell survival. Additionally, the application of Dex40 allowed using Me2SO in lower concentrations (0.7 M) than required for serum-containing media (1.4 M), which may decrease the toxicity of serum-/xeno-free media.
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Affiliation(s)
- Oleksandr Pakhomov
- Department of Cryoendocrinology, Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, 23 Pereyaslavskaya St, Kharkiv, 61016, Ukraine.
| | - Tatyana Gurina
- Department of Cryoendocrinology, Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, 23 Pereyaslavskaya St, Kharkiv, 61016, Ukraine.
| | - Viktoria Mazaeva
- Department of Studies of Technology for Processing Oils and Fats, Ukrainian Research Institute of Oils and Fats, National Academy of Agrarian Sciences of Ukraine, 2a Dziuby Ave, Kharkiv, 61019, Ukraine.
| | - Anna Polyakova
- Department of Cryoendocrinology, Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, 23 Pereyaslavskaya St, Kharkiv, 61016, Ukraine.
| | - Bo Deng
- Department of Physiology, Henan University of Science and Technology, 263 Kaiyuan Ave, Henan, Luoyang, 471023, China.
| | - Evgeniy Legach
- Department of Cryoendocrinology, Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, 23 Pereyaslavskaya St, Kharkiv, 61016, Ukraine.
| | - Galyna Bozhok
- Department of Cryoendocrinology, Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, 23 Pereyaslavskaya St, Kharkiv, 61016, Ukraine.
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15
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Arutyunyan I, Elchaninov A, Sukhikh G, Fatkhudinov T. Cryopreservation of Tissue-Engineered Scaffold-Based Constructs: from Concept to Reality. Stem Cell Rev Rep 2022; 18:1234-1252. [PMID: 34761366 DOI: 10.1007/s12015-021-10299-4] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/01/2021] [Indexed: 02/07/2023]
Abstract
Creation of scaffold-based tissue-engineered constructs (SB TECs) is costly and requires coordinated qualified efforts. Cryopreservation enables longer shelf-life for SB TECs while enormously enhancing their availability as medical products. Regenerative treatment with cryopreserved SB TECs prepared in advance (possibly prêt-à-porter) can be started straight away on demand. Animal studies and clinical trials indicate similar levels of safety for cryopreserved and freshly prepared SB TECs. Although cryopreservation of such constructs is more difficult than that of cell suspensions or tissues, years of research have proved the principal possibility of using ready-to-transplant SB TECs after prolonged cryostorage. Cryopreservation efficiency depends not only on the sheer viability of adherent cells on scaffolds after thawing, but largely on the retention of proliferative and functional properties by the cells, as well as physical and mechanical properties by the scaffolds. Cryopreservation protocols require careful optimization, as their efficiency depends on multiple parameters including cryosensitivity of cells, chemistry and architecture of scaffolds, conditions of cell culture before freezing, cryoprotectant formulations, etc. In this review we discuss recent achievements in SB TEC cryopreservation as a major boost for the field of tissue engineering and biobanking.
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Affiliation(s)
- Irina Arutyunyan
- National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia
| | - Andrey Elchaninov
- National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia
- Research Institute of Human Morphology, Moscow, Russia
| | - Gennady Sukhikh
- National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov of Ministry of Healthcare of Russian Federation, Moscow, Russia
| | - Timur Fatkhudinov
- Research Institute of Human Morphology, Moscow, Russia.
- Department of Histology, Cytology and Embryology, Peoples' Friendship University of Russia (RUDN University, 6, Miklukho-Maklaya Street, 117198, Moscow, Russia.
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16
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Lira GPO, Borges AA, Nascimento MB, Aquino LVC, Moura LFMP, Silva HVR, Ribeiro LR, Silva AR, Pereira AF. Morphological, Ultrastructural, and Immunocytochemical Characterization and Assessment of Puma ( Puma concolor Linnaeus, 1771) Cell Lines After Extended Culture and Cryopreservation. Biopreserv Biobank 2022; 20:557-566. [PMID: 35049356 DOI: 10.1089/bio.2021.0117] [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: 11/02/2022] Open
Abstract
Cell lines are valuable tools to safeguard genetic material from species threatened with extinction that is mainly due to human action. In this scenario, the puma constitutes a species whose population is being rapidly reduced in the ecosystems it inhabits. For the first time, we characterized puma skin-derived cell lines and assessed these cells after extended culture (experiment 1) and cryopreservation (experiment 2). Initially, we identified and characterized four dermal fibroblast lines using morphology, ultrastructure, and immunofluorescence assays. Moreover, we evaluated the effects of culture time (1st, 3rd, and 10th passages) and cryopreservation on their morphology, ultrastructure, viability, metabolism, proliferative activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis. The cells showed a typical spindle-shaped morphology with centrally located oval nuclei. The cells were identified as fibroblasts by staining for vimentin. In vitro culture after the 1st, 3rd, and 10th passages did not alter most of the evaluated parameters. Cells in the 3rd and 10th passages showed a reduction in ROS levels (p < 0.05). The ultrastructure revealed morphological damage in the prolongments, and nuclei of cells derived from the 3rd and 10th passages. Moreover, cryopreservation resulted in a reduction in ΔΨm compared with that of noncryopreserved cells, suggesting that the optimization of cryopreservation methods for puma fibroblasts is essential. In conclusion, we found that viable fibroblasts could be obtained from puma skin, with slight changes after the 10th passage in in vitro culture and cryopreservation. This is the first report on the development of cell lines derived from pumas.
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Affiliation(s)
- Gabriela P O Lira
- Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoró, Brazil
| | - Alana A Borges
- Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoró, Brazil
| | - Matheus B Nascimento
- Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoró, Brazil
| | - Leonardo V C Aquino
- Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoró, Brazil
| | - Luiz F M P Moura
- Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoró, Brazil
| | - Herlon V R Silva
- Laboratory of Reproduction of Carnivores, Ceara State University (UECE), Fortaleza, Brazil
| | - Leandro R Ribeiro
- Laboratory of Reproduction of Carnivores, Ceara State University (UECE), Fortaleza, Brazil
| | - Alexandre R Silva
- Laboratory of Animal Germplasm Conservation, UFERSA, Mossoró, Brazil
| | - Alexsandra F Pereira
- Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoró, Brazil
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17
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Particles in Biopharmaceutical Formulations, Part 2: An Update on Analytical Techniques and Applications for Therapeutic Proteins, Viruses, Vaccines and Cells. J Pharm Sci 2021; 111:933-950. [PMID: 34919969 DOI: 10.1016/j.xphs.2021.12.011] [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: 12/07/2021] [Revised: 12/09/2021] [Accepted: 12/09/2021] [Indexed: 11/21/2022]
Abstract
Particles in biopharmaceutical formulations remain a hot topic in drug product development. With new product classes emerging it is crucial to discriminate particulate active pharmaceutical ingredients from particulate impurities. Technical improvements, new analytical developments and emerging tools (e.g., machine learning tools) increase the amount of information generated for particles. For a proper interpretation and judgment of the generated data a thorough understanding of the measurement principle, suitable application fields and potential limitations and pitfalls is required. Our review provides a comprehensive overview of novel particle analysis techniques emerging in the last decade for particulate impurities in therapeutic protein formulations (protein-related, excipient-related and primary packaging material-related), as well as particulate biopharmaceutical formulations (virus particles, virus-like particles, lipid nanoparticles and cell-based medicinal products). In addition, we review the literature on applications, describe specific analytical approaches and illustrate advantages and drawbacks of currently available techniques for particulate biopharmaceutical formulations.
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18
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Oyarzo R, Valderrama X, Valenzuela F, Bahamonde J. Bovine Fetal Mesenchymal Stem Cells Obtained From Omental Adipose Tissue and Placenta Are More Resistant to Cryoprotectant Exposure Than Those From Bone Marrow. Front Vet Sci 2021; 8:708972. [PMID: 34671660 PMCID: PMC8520992 DOI: 10.3389/fvets.2021.708972] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Accepted: 09/03/2021] [Indexed: 01/08/2023] Open
Abstract
Recent studies have shown promise for the development of cellular therapies with mesenchymal stem cells (MSCs) in livestock species, specifically bovines, and cryopreservation is highly relevant for the advancement of these applications. The use of permeable and/or non-permeable cryoprotectant solutions is necessary to reduce cell damage during freezing and thawing, but these same compounds can also cause negative effects on MSCs and their therapeutic properties. Another important factor to consider is the tissue source of MSCs, since it is now known that MSCs from different tissues of the same individual do not behave the same way, so optimizing the type and concentration of cryoprotectants for each cell type is essential to achieve a large and healthy population of MSCs after cryopreservation. Furthermore, sources of MSCs that could provide great quantities, non-invasively and without ethical concerns, such as placental tissue, have great potential for the development of regenerative medicine in livestock species, and have not been thoroughly evaluated. The objective of this study was to compare the viability of bovine fetal MSCs extracted from bone marrow (BM), adipose tissue (AT), and placenta (PT), following their exposure (15 and 30 min) to several solutions of permeable (dimethyl sulfoxide and ethylene glycol) and non-permeable (trehalose) cryoprotectants. Viability assays were performed with Trypan Blue to assess post-exposure plasma membrane integrity. The apoptotic potential was estimated analyzing the mRNA abundance of BAX and BCL-2 genes using quantitative rt-PCR. Based on the results of the study, BM-MSC exhibited significantly lower viability compared to AT-MSC and PT-MSC, at both 15 and 30 min of exposure to cryoprotectant solutions. Nevertheless, viability did not differ among treatments for any of the cell types or timepoints studied. BCL-2 expression was higher in BM-MSC compared to AT-MSC, however, BAX/BCL-2 ratio did not differ. In conclusion, AT-MSC and PT-MSC were more resistant that BM-MSC, which showed higher sensitivity to experimental conditions, regardless of the exposure times, and cryoprotectant solutions used in the study.
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Affiliation(s)
- Rudy Oyarzo
- Laboratory of Applied Morphology, Instituto de Farmacología y Morfofisiología, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile
| | - Ximena Valderrama
- Laboratory of Applied Morphology, Instituto de Farmacología y Morfofisiología, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile
- Instituto de Investigaciones Agropecuarias, INIA Remehue, Osorno, Chile
| | - Francisca Valenzuela
- Laboratory of Applied Morphology, Instituto de Farmacología y Morfofisiología, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile
| | - Javiera Bahamonde
- Laboratory of Applied Morphology, Instituto de Farmacología y Morfofisiología, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile
- Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, United States
- Escuela de Graduados, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile
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19
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Erol OD, Pervin B, Seker ME, Aerts-Kaya F. Effects of storage media, supplements and cryopreservation methods on quality of stem cells. World J Stem Cells 2021; 13:1197-1214. [PMID: 34630858 PMCID: PMC8474714 DOI: 10.4252/wjsc.v13.i9.1197] [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: 02/27/2021] [Revised: 04/21/2021] [Accepted: 08/24/2021] [Indexed: 02/06/2023] Open
Abstract
Despite a vast amount of different methods, protocols and cryoprotective agents (CPA), stem cells are often frozen using standard protocols that have been optimized for use with cell lines, rather than with stem cells. Relatively few comparative studies have been performed to assess the effects of cryopreservation methods on these stem cells. Dimethyl sulfoxide (DMSO) has been a key agent for the development of cryobiology and has been used universally for cryopreservation. However, the use of DMSO has been associated with in vitro and in vivo toxicity and has been shown to affect many cellular processes due to changes in DNA methylation and dysregulation of gene expression. Despite studies showing that DMSO may affect cell characteristics, DMSO remains the CPA of choice, both in a research setting and in the clinics. However, numerous alternatives to DMSO have been shown to hold promise for use as a CPA and include albumin, trehalose, sucrose, ethylene glycol, polyethylene glycol and many more. Here, we will discuss the use, advantages and disadvantages of these CPAs for cryopreservation of different types of stem cells, including hematopoietic stem cells, mesenchymal stromal/stem cells and induced pluripotent stem cells.
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Affiliation(s)
- Ozgur Dogus Erol
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Burcu Pervin
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Mehmet Emin Seker
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
| | - Fatima Aerts-Kaya
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara 06100, Turkey
- Center for Stem Cell Research and Development, Hacettepe University, Ankara 06100, Turkey
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20
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Zhan L, Guo S, Kangas J, Shao Q, Shiao M, Khosla K, Low WC, McAlpine MC, Bischof J. Conduction Cooling and Plasmonic Heating Dramatically Increase Droplet Vitrification Volumes for Cell Cryopreservation. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2021; 8:2004605. [PMID: 34141523 PMCID: PMC8188207 DOI: 10.1002/advs.202004605] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/28/2020] [Revised: 02/05/2021] [Indexed: 05/28/2023]
Abstract
Droplet vitrification has emerged as a promising ice-free cryopreservation approach to provide a supply chain for off-the-shelf cell products in cell therapy and regenerative medicine applications. Translation of this approach requires the use of low concentration (i.e., low toxicity) permeable cryoprotectant agents (CPA) and high post cryopreservation viability (>90%), thereby demanding fast cooling and warming rates. Unfortunately, with traditional approaches using convective heat transfer, the droplet volumes that can be successfully vitrified and rewarmed are impractically small (i.e., 180 picoliter) for <2.5 m permeable CPA. Herein, a novel approach to achieve 90-95% viability in micro-liter size droplets with 2 m permeable CPA, is presented. Droplets with plasmonic gold nanorods (GNRs) are printed onto a cryogenic copper substrate for improved cooling rates via conduction, while plasmonic laser heating yields >400-fold improvement in warming rates over traditional convective approach. High viability cryopreservation is then demonstrated in a model cell line (human dermal fibroblasts) and an important regenerative medicine cell line (human umbilical cord blood stem cells). This approach opens a new paradigm for cryopreservation and rewarming of dramatically larger volume droplets at lower CPA concentration for cell therapy and other regenerative medicine applications.
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Affiliation(s)
- Li Zhan
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Center for Advanced Technologies for the Preservation of Biological Systems (ATP‐Bio)University of MinnesotaMinneapolisMN55455USA
| | - Shuang‐Zhuang Guo
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- School of Materials Science and EngineeringSun Yat‐sen UniversityGuangzhou510275China
| | - Joseph Kangas
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Center for Advanced Technologies for the Preservation of Biological Systems (ATP‐Bio)University of MinnesotaMinneapolisMN55455USA
| | - Qi Shao
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
| | - Maple Shiao
- Department of NeurosurgeryUniversity of MinnesotaMinneapolisMN55455USA
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMN55455USA
| | - Kanav Khosla
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Center for Advanced Technologies for the Preservation of Biological Systems (ATP‐Bio)University of MinnesotaMinneapolisMN55455USA
| | - Walter C. Low
- Department of NeurosurgeryUniversity of MinnesotaMinneapolisMN55455USA
- Stem Cell InstituteUniversity of MinnesotaMinneapolisMN55455USA
| | - Michael C. McAlpine
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Center for Advanced Technologies for the Preservation of Biological Systems (ATP‐Bio)University of MinnesotaMinneapolisMN55455USA
| | - John Bischof
- Department of Mechanical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
- Center for Advanced Technologies for the Preservation of Biological Systems (ATP‐Bio)University of MinnesotaMinneapolisMN55455USA
- Department of Biomedical EngineeringUniversity of MinnesotaMinneapolisMN55455USA
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21
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García-Bernal D, García-Arranz M, Yáñez RM, Hervás-Salcedo R, Cortés A, Fernández-García M, Hernando-Rodríguez M, Quintana-Bustamante Ó, Bueren JA, García-Olmo D, Moraleda JM, Segovia JC, Zapata AG. The Current Status of Mesenchymal Stromal Cells: Controversies, Unresolved Issues and Some Promising Solutions to Improve Their Therapeutic Efficacy. Front Cell Dev Biol 2021; 9:650664. [PMID: 33796536 PMCID: PMC8007911 DOI: 10.3389/fcell.2021.650664] [Citation(s) in RCA: 78] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Accepted: 02/26/2021] [Indexed: 12/16/2022] Open
Abstract
Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.
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Affiliation(s)
- David García-Bernal
- Hematopoietic Transplant and Cellular Therapy Unit, Medicine Department, Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca, University of Murcia, Murcia, Spain.,Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain
| | - Mariano García-Arranz
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain
| | - Rosa M Yáñez
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain.,Centre for Cytometry and Fluorescence Microscopy, Complutense University, Madrid, Spain
| | - Rosario Hervás-Salcedo
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain.,Centre for Cytometry and Fluorescence Microscopy, Complutense University, Madrid, Spain
| | - Alfonso Cortés
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas and Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain
| | - María Fernández-García
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain.,Centre for Cytometry and Fluorescence Microscopy, Complutense University, Madrid, Spain
| | - Miriam Hernando-Rodríguez
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain.,Centre for Cytometry and Fluorescence Microscopy, Complutense University, Madrid, Spain
| | - Óscar Quintana-Bustamante
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain.,Centre for Cytometry and Fluorescence Microscopy, Complutense University, Madrid, Spain
| | - Juan A Bueren
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain.,Centre for Cytometry and Fluorescence Microscopy, Complutense University, Madrid, Spain
| | - Damián García-Olmo
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain
| | - Jose M Moraleda
- Hematopoietic Transplant and Cellular Therapy Unit, Medicine Department, Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca, University of Murcia, Murcia, Spain.,Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain
| | - José C Segovia
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, Autonomous University of Madrid (UAM)), Madrid, Spain.,Centre for Cytometry and Fluorescence Microscopy, Complutense University, Madrid, Spain
| | - Agustín G Zapata
- Spanish Network of Cell Therapy (TerCel), Instituto de Salud Carlos III, Madrid, Spain.,Department of Cell Biology, Complutense University, Madrid, Spain
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22
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Li QY, Zou T, Gong Y, Chen SY, Zeng YX, Gao LX, Weng CH, Xu HW, Yin ZQ. Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases. Exp Eye Res 2020; 202:108305. [PMID: 33080300 DOI: 10.1016/j.exer.2020.108305] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2020] [Revised: 09/29/2020] [Accepted: 10/13/2020] [Indexed: 01/04/2023]
Abstract
The biosafety and efficiency of transplanting retinal pigment epithelial (RPE) cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been evaluated in phase I and phase II clinical trials. For further large-scale application, cryopreserved RPE cells must be used; thus, it is highly important to investigate the influence of cryopreservation and thawing on the biological characteristics of hESC-RPE cells and their post-transplantation vision-restoring function. Here, via immunofluorescence, qPCR, transmission electron microscopy, transepithelial electrical resistance, and enzyme-linked immunosorbent assays (ELISAs), we showed that cryopreserved hESC-RPE cells retained the specific gene expression profile, morphology, ultrastructure, and maturity-related functions of induced RPE cells. Additionally, cryopreserved hESC-RPE cells exhibited a polarized monolayer, tight junction, and gap junction structure and an in vitro nanoparticle phagocytosis capability similar to those of induced hESC-RPE cells. However, the level of pigment epithelium-derived factor (PEDF) secretion was significantly decreased in cryopreserved hESC-RPE cells. Royal College of Surgeons rats with cryopreserved hESC-RPE cells engrafted into the subretinal space exhibited a significant decrease in the b-wave amplitude compared with rats engrafted with induced hESC-RPE cells at 4 weeks post transplantation. However, the difference disappeared at 8 weeks and 12 weeks post operation. No significant difference in the outer nuclear layer (ONL) thickness was observed between the two groups. Our data showed that even after cryopreservation and thawing, cryopreserved hESC-RPE cells are still qualified as a donor cell source for cell-based therapy of retinal degenerative diseases.
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Affiliation(s)
- Qi-You Li
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China
| | - Ting Zou
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China
| | - Yu Gong
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China
| | - Si-Yu Chen
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China
| | - Yu-Xiao Zeng
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China
| | - Li-Xiong Gao
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China; Department of Ophthalmology, The 6th Medical Center of PLA General Hospital, Beijing, China
| | - Chuan-Huang Weng
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China
| | - Hai-Wei Xu
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China.
| | - Zheng-Qin Yin
- Southwest Hospital/ Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, PR China; Key Lab of Visual Damage and Regeneration & Restoration of Chongqing, Chongqing, 400038, PR China.
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23
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Awan M, Buriak I, Fleck R, Fuller B, Goltsev A, Kerby J, Lowdell M, Mericka P, Petrenko A, Petrenko Y, Rogulska O, Stolzing A, Stacey GN. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen Med 2020; 15:1463-1491. [PMID: 32342730 DOI: 10.2217/rme-2019-0145] [Citation(s) in RCA: 116] [Impact Index Per Article: 23.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice for most animal cell systems since the early history of cryopreservation. It has been used for decades in many thousands of cell transplants. These treatments would not have taken place without suitable sources of DMSO that enabled stable and safe storage of bone marrow and blood cells until needed for transfusion. Nevertheless, its effects on cell biology and apparent toxicity in patients have been an ongoing topic of debate, driving the search for less cytotoxic cryoprotectants. This review seeks to place the toxicity of DMSO in context of its effectiveness. It will also consider means of reducing its toxic effects, the alternatives to its use and their readiness for active use in clinical settings.
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Affiliation(s)
- Maooz Awan
- Institute for Liver & Digestive Health, UCL Division of Medicine, Royal Free Hospital, UCL, London, NW3 2PF, UK
| | - Iryna Buriak
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Roland Fleck
- Centre for Ultrastructural Imaging, Kings College London, London, SE1 1UL, UK
| | - Barry Fuller
- Department of Surgical Biotechnology, UCL Division of Surgery, Royal Free Hospital, UCL, London, NW3 2QG, UK
| | - Anatoliy Goltsev
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Julie Kerby
- Cell & Gene Therapy Catapult, 12th Floor Tower Wing, Guy's Hospital, Great Maze Pond, London, SE1 9RT, UK
| | - Mark Lowdell
- Centre for Cell, Gene & Tissue Therapy, Royal Free London NHS FT & UCL, London, NW3 2PF, UK
| | - Pavel Mericka
- Tissue Bank, University Hospital Hradec Kralové, Czech Republic
| | - Alexander Petrenko
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Yuri Petrenko
- Department of Biomaterials & Biophysical Methods, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czech Republic
| | - Olena Rogulska
- Institute for Problems of Cryobiology & Cryomedicine, National Academy of Sciences of Ukraine, Pereyaslavska 23, 61016, Kharkiv
| | - Alexandra Stolzing
- University of Loughborough, Centre for Biological Engineering, Loughborough University, Holywell Park, Loughborough, UK
| | - Glyn N Stacey
- International Stem Cell Banking Initiative, 2 High Street, Barley, Hertfordshire, SG8 8HZ
- Beijing Stem Cell Bank, Institute of Zoology, Chinese Academy of Sciences, 25–2 Beishuan West, Haidan District, 100190 Beijing, China
- Institute of Stem Cells & Regeneration, Chinese Academy of Sciences, Beijing 100101, China
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24
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Vasudevan B, Chang Q, Wang B, Huang S, Sui Y, Zhu W, Fan Q, Song Y. Effect of intracellular uptake of nanoparticle-encapsulated trehalose on the hemocompatibility of allogeneic valves in the VS83 vitrification protocol. Nanobiomedicine (Rij) 2020; 7:1849543520983173. [PMID: 33447299 PMCID: PMC7780325 DOI: 10.1177/1849543520983173] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Accepted: 12/03/2020] [Indexed: 11/16/2022] Open
Abstract
Trehalose is a disaccharide molecule consisting of two molecules of glucose. Industrially, trehalose is derived from corn starch and utilized as a drug. This study aims to examine whether the integration of nanoparticle-encapsulated trehalose to the Ice-Free Cryopreservation (IFC) method for preserving heart valves has better cell viability, benefits to protect the extracellular matrix (ECM), and reduce immune response after storage. For the experiment to be carried out, we obtained materials, and the procedures were carried out in the following manner. The initial step was the preparation of hydroxyapatite nanoparticles, followed by precipitation to acquire Apatite colloidal suspensions. Animals were obtained, and their tissue isolation and grouping were done ethically. All samples were then divided into four groups, Control group, Conventional Frozen Cryopreservation (CFC) group, IFC group, and IFC + T (IFC with the addition of 0.2 M nanoparticle-encapsulated Trehalose) group. Histological analysis was carried out via H&E staining, ECM components were stained with Modified Weigert staining, and the Gomori Ammonia method was used to stain reticular fibers. Alamar Blue assay was utilized to assess cell viability. Hemocompatibility was evaluated, and samples were processed for immunohistochemistry (TNFα and IL-10). Hemocompatibility was quantified using Terminal Complement Complex (TCC) and Neutrophil elastase (NE) as an indicator. The results of the H&E staining revealed less formation of extracellular ice crystals and intracellular vacuoles in the IFC + T group compared with all other groups. The CFC group's cell viability showed better viability than the IFC group, but the highest viability was exhibited in the IFC + T group (70.96 ± 2.53, P < 0.0001, n = 6). In immunohistochemistry, TNFα levels were lowest in both IFC and IFC + T group, and IL-10 expression had significantly reduced in IFC and IFC + T group. The results suggested that the nanoparticle encapsulated trehalose did not show significant hemocompatibility issues on the cryopreserved heart valves.
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Affiliation(s)
| | - Qing Chang
- Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
| | - Bin Wang
- Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
| | - Siyang Huang
- Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
| | - Yulong Sui
- Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
| | - Wenjie Zhu
- Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
| | - Qing Fan
- Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
| | - Yisheng Song
- Affiliated Hospital of Medical College, Qingdao University, Qingdao, China
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25
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More than a Confinement: “Soft” and “Hard” Enzyme Entrapment Modulates Biological Catalyst Function. Catalysts 2019. [DOI: 10.3390/catal9121024] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Catalysis makes chemical and biochemical reactions kinetically accessible. From a technological point of view, organic, inorganic, and biochemical catalysis is relevant for several applications, from industrial synthesis to biomedical, material, and food sciences. A heterogeneous catalyst, i.e., a catalyst confined in a different phase with respect to the reagents’ phase, requires either its physical confinement in an immobilization matrix or its physical adsorption on a surface. In this review, we will focus on the immobilization of biological catalysts, i.e., enzymes, by comparing hard and soft immobilization matrices and their effect on the modulation of the catalysts’ function. Indeed, unlike smaller molecules, the catalytic activity of protein catalysts depends on their structure, conformation, local environment, and dynamics, properties that can be strongly affected by the immobilization matrices, which, therefore, not only provide physical confinement, but also modulate catalysis.
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26
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Bahsoun S, Coopman K, Akam EC. The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review. J Transl Med 2019; 17:397. [PMID: 31783866 PMCID: PMC6883667 DOI: 10.1186/s12967-019-02136-7] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2019] [Accepted: 11/12/2019] [Indexed: 12/12/2022] Open
Abstract
Mesenchymal stem cells (MSCs) represent an invaluable asset for the field of cell therapy. Human Bone marrow-derived MSCs (hBM-MSCs) are one of the most commonly used cell types in clinical trials. They are currently being studied and tested for the treatment of a wide range of diseases and conditions. The future availability of MSCs therapies to the public will require a robust and reliable delivery process. Cryopreservation represents the gold standard in cell storage and transportation, but its effect on BM-MSCs is still not well established. A systematic review was conducted to evaluate the impact of cryopreservation on BM-MSCs and to attempt to uncover the reasons behind some of the controversial results reported in the literature. Forty-one in vitro studies were analysed, and their results organised according to the cell attributes they assess. It was concluded that cryopreservation does not affect BM-MSCs morphology, surface marker expression, differentiation or proliferation potential. However, mixed results exist regarding the effect on colony forming ability and the effects on viability, attachment and migration, genomic stability and paracrine function are undefined mainly due to the huge variabilities governing the cryopreservation process as a whole and to the lack of standardised assays.
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Affiliation(s)
- Soukaina Bahsoun
- School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, Leicestershire, LE11 3TU, UK
| | - Karen Coopman
- Centre for Biological Engineering, Loughborough University, Loughborough, Leicestershire, LE11 3TU, UK
| | - Elizabeth C Akam
- School of Sport, Exercise and Health Sciences, Loughborough University, Loughborough, Leicestershire, LE11 3TU, UK.
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27
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Sun Y, Dos Santos A, Balayan A, Deng SX. Evaluation of Cryopreservation Media for the Preservation of Human Corneal Stromal Stem Cells. Tissue Eng Part C Methods 2019; 26:37-43. [PMID: 31686624 DOI: 10.1089/ten.tec.2019.0195] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Introduction: Human corneal stromal stem cells (CSSCs) have gained increasing attention in the treatment of corneal stromal scars. In view of this, the preparation and storage of CSSCs are critical to maintaining the regenerative potential of CSSCs. The goal of the study was to investigate the human serum (HS) concentration in the cryomedia that could best preserve CSSCs. Materials and Methods: Three different cryopreservation media, varying in HS concentration were evaluated in their ability to preserve the viability and phenotype of CSSCs: 2% HS (FS1), 4% HS (FS2), and 90% HS (FS3). After thawing, CSSCs morphology, recovery rate, cell proliferation, relative gene expression of CSSC markers (ABCG2, SOX2, NANOG, PAX6, and SIX3), and their anti-inflammatory response (level of TNFAIP6) were compared with those of unfrozen CSSCs (control). Results: Cryopreserved CSSCs had similar cell morphology as the control. Cell viability was significantly higher using FS2 (92.7 ± 1.3%) compared with FS1 (88 ± 0.8%, p = 0.018). Doubling times of CSSCs were maintained in all cryopreserved conditions, as in the control (p > 0.05), which were 0.9 ± 0.1 days and 1.8 ± 0.0 days at passages 3 and 4, then increased to 18.2 ± 1.9 days at passage 6 (p > 0.05). The expression level of stem cell/progenitor cell markers investigated was not affected by the cryopreservation with any of the three media. In addition, cryopreserved CSSCs have a similar expression level of TNFAIP6 after stimulation with proinflammatory cytokines as the control (p > 0.05). Conclusion: Our results indicated that all three cryopreservation media maintained CSSCs phenotype after undergoing one freezing/thawing cycle. Impact Statement Corneal stromal stem cells (CSSCs) offer an alternative for the treatment of corneal stromal scars. Cryopreservation of CSSCs is necessary as it enables feasibility of using CSSCs as a cell therapy candidate. The current study shows that media used to cryopreserve CSSCs could be optimized to maintain cell viability, phenotype, and potency of CSSCs after thawing.
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Affiliation(s)
- Yuzhao Sun
- Department of Ophthalmology, Stein Eye Institute, University of California, Los Angeles, Los Angeles, California.,Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning, P.R. China
| | - Aurelie Dos Santos
- Department of Ophthalmology, Stein Eye Institute, University of California, Los Angeles, Los Angeles, California
| | - Alis Balayan
- Department of Ophthalmology, Stein Eye Institute, University of California, Los Angeles, Los Angeles, California
| | - Sophie X Deng
- Department of Ophthalmology, Stein Eye Institute, University of California, Los Angeles, Los Angeles, California
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28
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Cryopreserved mesenchymal stem cells regain functional potency following a 24-h acclimation period. J Transl Med 2019; 17:297. [PMID: 31464641 PMCID: PMC6716839 DOI: 10.1186/s12967-019-2038-5] [Citation(s) in RCA: 46] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2019] [Accepted: 08/18/2019] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Mesenchymal stem cells (MSCs) are attractive cell-therapy candidates. Despite their popularity and promise, there is no uniform method of preparation of MSCs. Typically, cells are cryopreserved in liquid nitrogen, thawed, and subsequently administered to a patient with little to no information on their function post-thaw. We hypothesized that a short acclimation period post-thaw will facilitate the recovery of MSC's functional potency. METHODS Human bone-marrow-derived MSCs were divided into 3 groups: FC (fresh cells; from existing culture); TT (thawed + time; acclimated for 24 h post-thaw); and FT (freshly thawed; thawed and immediately used). The 3 groups were analyzed for their cellular and functional potency. RESULTS Phenotypic analysis demonstrated a decrease in CD44 and CD105 surface markers in FT MSCs, with no change in the other two groups. All MSCs were able to differentiate down the osteogenic and chondrogenic lineages. In FT cells, metabolic activity and apoptosis was significantly increased with concomitant decrease in cell proliferation; clonogenic capacity; and key regenerative genes. Following 24-h acclimation, apoptosis was significantly reduced in TT cells with a concomitant upregulation in angiogenic and anti-inflammatory genes. While all MSCs significantly arrested T-cell proliferation, the TT MSCs were significantly more potent. Similarly, although all MSCs maintained their anti-inflammatory properties, IFN-γ secretion was significantly diminished in FT cells. CONCLUSIONS These data demonstrate that FT MSCs maintain their multipotent differentiation capacity, immunomodulatory function, and anti-inflammatory properties; yet, various aspects of cell characteristics and function are deleteriously affected by cryopreservation. Importantly, a 24-h acclimation period 'reactivates' thawed cells to recover their diminished stem-cell function.
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29
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Duan W, Lopez MJ. Effects of enzyme and cryoprotectant concentrations on yield of equine adipose-derived multipotent stromal cells. Am J Vet Res 2019; 79:1100-1112. [PMID: 30256145 DOI: 10.2460/ajvr.79.10.1100] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44+, CD105+, and major histocompatability complex class II (MHCII; CD44+-CD105+-MHCII+ cells and CD44+-CD105+-MHCII- cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44+-CD105+-MHCII- CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.
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30
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Celikkan FT, Mungan C, Sucu M, Ulus AT, Cinar O, Ili EG, Can A. Optimizing the transport and storage conditions of current Good Manufacturing Practice -grade human umbilical cord mesenchymal stromal cells for transplantation (HUC-HEART Trial). Cytotherapy 2018; 21:64-75. [PMID: 30455106 DOI: 10.1016/j.jcyt.2018.10.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Revised: 10/01/2018] [Accepted: 10/13/2018] [Indexed: 01/12/2023]
Abstract
BACKGROUND The HUC-HEART Trial is a clinical study of intramyocardial delivery of current Good Manufacturing Practice (cGMP)-grade human umbilical cord multipotent stromal cells (HUC-MSCs) in ischemic cardiomyopathy where 2 × 107 cells are administered to peri-infarcted myocardium. Prior to the onset of the trial, we aimed to optimize the transport/storage conditions for obtaining the highest cell viability and proliferation rate of cells to be transplanted. METHODS Cells were tested after being transported in phosphate-buffered saline (PBS) or Ringer's lactate-based (RL) transport media supplemented with human serum albumin (HSA) and/or hydroxyethyl starch (HES) at two temperatures (2-10°C or 22-24°C). RESULTS The effects of transport conditions on cell viability following 6 h were found highest (93.4 ± 1.5) in RL-based media at 2-10°C. Karyotypes were found normal upon transportation in any of the formulations and temperatures. However, the highest proliferation rate was noted (3.1-fold increase) in RL (1% HSA) media at 2-10°C over 6 days in culture. From that point, RL (1% HSA) media at 2-10°C was used for further experiments. The maximum cell storage time was detected around 24 h at 2-10°C. Extended storage periods resulted in a decrease in cell viability but not in MSC marker expression. An increase in actin quantity was detected in hypoxia (5% O2) groups in early culture days; no difference was noted between hypoxic versus normoxic (21% O2) conditions in later days. DISCUSSION The overall results suggest that non-commercial, simple media formulations with extended storage intervals at 2-10°C temperatures are capable of retaining the characteristics of clinical-grade HUC-MSCs. The above findings led us to use RL (1% HSA) media at 2-10°C for transport and storage in the HUC-HEART Trial; 23 patients received HUC-MSCs by August 2018; no adverse effects were noted related to cell processing and transplantation.
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Affiliation(s)
- Ferda Topal Celikkan
- Department of Histology and Embryology, Faculty of Medicine, Ankara University, Ankara, Turkey
| | - Ceren Mungan
- Ankara University Biotechnology Institute, Ankara, Turkey
| | - Merve Sucu
- Ankara University Biotechnology Institute, Ankara, Turkey
| | - A Tulga Ulus
- Division of Cardiovascular Surgery, Acibadem Hospital, Ankara, Turkey
| | - Ozgur Cinar
- Department of Histology and Embryology, Faculty of Medicine, Ankara University, Ankara, Turkey
| | - Ezgi Gokpinar Ili
- Department of Medical Genetics, Faculty of Medicine, Ankara University, Ankara, Turkey
| | - Alp Can
- Department of Histology and Embryology, Faculty of Medicine, Ankara University, Ankara, Turkey.
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Park S, Lee DR, Nam JS, Ahn CW, Kim H. Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells. Cryobiology 2018; 81:65-73. [PMID: 29448017 DOI: 10.1016/j.cryobiol.2018.02.008] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2017] [Revised: 01/23/2018] [Accepted: 02/12/2018] [Indexed: 12/16/2022]
Abstract
The use of fetal bovine serum (FBS) as a cryopreservation supplement is not suitable for the banking of mesenchymal stem cells (MSCs) due to the risk of transmission of disease as well as xenogeneic immune reactions in the transplanted host. Here, we investigated if human serum albumin (HSA), human serum (HS), or knockout serum replacement (KSR) can replace FBS for the cryopreservation of MSCs. In addition, we examined the characteristics of MSCs after multiple rounds of cryopreservation. Human adipose-derived stem cells (ASCs) cryopreserved with three FBS replacements, 9% HSA, 90% HS, or 90% KSR, in combination with 10% dimethyl sulfoxide (Me2SO) maintained stem cell properties including growth, immunophenotypes, gene expression patterns, and the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages, similar to ASCs frozen with FBS. Moreover, the immunophenotype, gene expression, and differentiation capabilities of ASCs were not altered by up to four freeze-thaw cycles. However, the performance of three or four freeze-thaw cycles significantly reduced the proliferation ability of ASCs, as indicated by the longer population doubling time and reduced colony-forming unit-fibroblast frequency. Together, our results suggest that HSA, HS, or KSR can replace FBS for the cryopreservation of ASCs, without altering their stemness, and should be processed with no more than two freeze-thaw cycles for clinical approaches.
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Affiliation(s)
- Seah Park
- Department of Biotechnology, Seoul Women's University, 621 Hwarangro, Nowon-Gu, Seoul, Republic of Korea.
| | - Dong Ryul Lee
- Department of Biomedical Science, CHA University, 120 Haeryong-ro, Pocheon-shi, Gyeongghi-do, Republic of Korea.
| | - Ji Sun Nam
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea.
| | - Chul Woo Ahn
- Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea.
| | - Haekwon Kim
- Department of Biotechnology, Seoul Women's University, 621 Hwarangro, Nowon-Gu, Seoul, Republic of Korea.
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Gurgul A, Romanek J, Pawlina-Tyszko K, Szmatoła T, Opiela J. Evaluation of changes arising in the pig mesenchymal stromal cells transcriptome following cryopreservation and Trichostatin A treatment. PLoS One 2018; 13:e0192147. [PMID: 29390033 PMCID: PMC5794156 DOI: 10.1371/journal.pone.0192147] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Accepted: 01/17/2018] [Indexed: 12/27/2022] Open
Abstract
Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator—Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.
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Affiliation(s)
- Artur Gurgul
- National Research Institute of Animal Production, Department of Genomics and Molecular Biology, Balice, Poland
| | - Joanna Romanek
- National Research Institute of Animal Production, Department of Animal Reproduction Biotechnology, Balice, Poland
| | - Klaudia Pawlina-Tyszko
- National Research Institute of Animal Production, Department of Genomics and Molecular Biology, Balice, Poland
| | - Tomasz Szmatoła
- National Research Institute of Animal Production, Department of Genomics and Molecular Biology, Balice, Poland
| | - Jolanta Opiela
- National Research Institute of Animal Production, Department of Animal Reproduction Biotechnology, Balice, Poland
- * E-mail:
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Duan W, Lopez MJ, Hicok K. Adult multipotent stromal cell cryopreservation: Pluses and pitfalls. Vet Surg 2018; 47:19-29. [PMID: 29023790 PMCID: PMC5813167 DOI: 10.1111/vsu.12730] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2017] [Revised: 07/21/2017] [Accepted: 08/10/2017] [Indexed: 01/01/2023]
Abstract
Study and clinical testing of adult multipotent stromal cells (MSCs) are central to progressive improvements in veterinary regenerative medicine. Inherent limitations to long-term culture preclude use for storage. Until cell line creation from primary isolates becomes routine, MSC stasis at cryogenic temperatures is required for this purpose. Many protocols and reagents, including cryoprotectants, used for veterinary MSCs are derived from those for human and rodent cells. Dissimilarities in cryopreservation strategies play a role in variable MSC behaviors. Familiarity with contemporary cryopreservation reagents and processes is essential to an appreciation of their impact on MSC survival and post-cryopreservation behavior. In addition to these points, this review includes a brief history and description of current veterinary stem cell regulation.
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Affiliation(s)
- Wei Duan
- Department of Veterinary Clinical Sciences, School of Veterinary MedicineLouisiana State UniversityBaton RougeLouisiana
| | - Mandi J. Lopez
- Department of Veterinary Clinical Sciences, School of Veterinary MedicineLouisiana State UniversityBaton RougeLouisiana
| | - Kevin Hicok
- Research and Development, VetStem Biopharma IncPowayCalifornia
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Lopez MJ. Creative technology advances tissue preservation. ANNALS OF TRANSLATIONAL MEDICINE 2017; 5:463. [PMID: 29285496 DOI: 10.21037/atm.2017.09.08] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Mandi J Lopez
- Laboratory for Equine and Comparative Orthopedic Research, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA
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Haack-Sørensen M, Ekblond A, Kastrup J. Cryopreservation and Revival of Human Mesenchymal Stromal Cells. Methods Mol Biol 2017; 1416:357-74. [PMID: 27236683 DOI: 10.1007/978-1-4939-3584-0_21] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Cell-based therapy is a promising and innovative new treatment for different degenerative and autoimmune diseases, and mesenchymal stromal cells (MSCs) from the bone marrow have demonstrated great therapeutic potential due to their immunosuppressive and regenerative capacities.The establishment of methods for large-scale expansion of clinical-grade MSCs in vitro has paved the way for their therapeutic use in clinical trials. However, the clinical application of MSCs also requires cryopreservation and banking of the cell products. To preserve autologous or allogeneic MSCs for future clinical applications, a reliable and effective cryopreservation method is required.Developing a successful cryopreservation protocol for clinical stem cell products, cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate are all aspects which should be considered.A major challenge is the selection of a suitable cryoprotectant which is able to penetrate the cells and yet has low toxicity.This chapter focuses on recent technological developments relevant for the cryopreservation of MSCs using the most commonly used cryopreservation medium containing DMSO and animal serum or human-derived products for research use and the animal protein-free cryopreservation media CryoStor (BioLife Solutions) for clinical use.
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Affiliation(s)
- Mandana Haack-Sørensen
- Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Juliane Mariesvej 20, 9302, Copenhagen Ø, 2100, Denmark.
| | - Annette Ekblond
- Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Juliane Mariesvej 20, 9302, Copenhagen Ø, 2100, Denmark
| | - Jens Kastrup
- Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet Copenhagen University Hospital, Juliane Mariesvej 20, 9302, Copenhagen Ø, 2100, Denmark
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Yuan Z, Lourenco SDS, Sage EK, Kolluri KK, Lowdell MW, Janes SM. Cryopreservation of human mesenchymal stromal cells expressing TRAIL for human anti-cancer therapy. Cytotherapy 2017; 18:860-9. [PMID: 27260207 PMCID: PMC4906234 DOI: 10.1016/j.jcyt.2016.04.005] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2016] [Revised: 04/04/2016] [Accepted: 04/17/2016] [Indexed: 12/13/2022]
Abstract
BACKGROUND AIMS Mesenchymal stromal cells (MSCs) are being extensively researched for cell therapy and tissue engineering. We have engineered MSCs to express the pro-apoptotic protein tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and are currently preparing this genetically modified cell therapy for a phase 1/2a clinical trial in patients with metastatic lung cancer. To do this, we need to prepare a cryopreserved allogeneic MSCTRAIL cell bank for further expansion before patient delivery. The effects of cryopreservation on a genetically modified cell therapy product have not been clearly determined. METHODS We tested different concentrations of dimethyl sulfoxide (DMSO) added to the human serum albumin ZENALB 4.5 and measured post-thaw cell viability, proliferation ability and differentiation characteristics. In addition, we examined the homing ability, TRAIL expression and cancer cell-killing capacities of cryopreserved genetically modified MSCs compared with fresh, continually cultured cells. RESULTS We demonstrated that the post-thaw viability of MSCs in 5% DMSO (v/v) with 95% ZENALB 4.5 (v/v) is 85.7 ± 0.4%, which is comparable to that in conventional freezing media. We show that cryopreservation does not affect the long-term expression of TRAIL and that cryopreserved TRAIL-expressing MSCs exhibit similar levels of homing and, importantly, retain their potency in triggering cancer cell death. CONCLUSIONS This study shows that cryopreservation is unlikely to affect the therapeutic properties of MSCTRAIL and supports the generation of a cryopreserved master cell bank.
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Affiliation(s)
- Zhengqiang Yuan
- Lungs for Living Research Centre, UCL Respiratory, Division of Medicine, University College London, London, United Kingdom
| | - Sofia Da Silva Lourenco
- Lungs for Living Research Centre, UCL Respiratory, Division of Medicine, University College London, London, United Kingdom
| | - Elizabeth K Sage
- Lungs for Living Research Centre, UCL Respiratory, Division of Medicine, University College London, London, United Kingdom
| | - Krishna K Kolluri
- Lungs for Living Research Centre, UCL Respiratory, Division of Medicine, University College London, London, United Kingdom
| | - Mark W Lowdell
- Centre for Cell, Gene & Tissue Therapy, Royal Free London National Health Services Foundation Trust & University College London, London, United Kingdom
| | - Sam M Janes
- Lungs for Living Research Centre, UCL Respiratory, Division of Medicine, University College London, London, United Kingdom.
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Zhang L, Xue X, Yan J, Yan LY, Jin XH, Zhu XH, He ZZ, Liu J, Li R, Qiao J. Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation. Chin Med J (Engl) 2017; 129:1963-8. [PMID: 27503023 PMCID: PMC4989429 DOI: 10.4103/0366-6999.187846] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Background: L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods: The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results: L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions: It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.
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Affiliation(s)
- Lu Zhang
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing 100191; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China
| | - Xu Xue
- Beijing Key Laboratory of Cryo-Biomedical Engineering and Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China
| | - Jie Yan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing 100191; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China
| | - Li-Ying Yan
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing 100191; Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China
| | - Xiao-Hu Jin
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing 100191; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China
| | - Xiao-Hui Zhu
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing 100191; Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproduction, Beijing 100191, China
| | - Zhi-Zhu He
- Beijing Key Laboratory of Cryo-Biomedical Engineering and Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China
| | - Jing Liu
- Beijing Key Laboratory of Cryo-Biomedical Engineering and Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190; Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China
| | - Rong Li
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing 100191; Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China
| | - Jie Qiao
- Department of Obstetrics and Gynecology, Center for Reproductive Medicine, Peking University Third Hospital, Beijing 100191; Key Laboratory of Assisted Reproduction, Ministry of Education, Beijing 100191, China
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Abstract
Cryopreservation is the application of low temperatures to preserve the structural and functional integrity of cells and tissues. Conventional cooling protocols allow ice to form and solute concentrations to rise during the cryopreservation process. The damage caused by the rise in solute concentration can be mitigated by the use of compounds known as cryoprotectants. Such compounds protect cells from the consequences of slow cooling injury, allowing them to be cooled at cooling rates which avoid the lethal effects of intracellular ice. An alternative to conventional cooling is vitrification. Vitrification methods incorporate cryoprotectants at sufficiently high concentrations to prevent ice crystallization so that the system forms an amorphous glass thus avoiding the damaging effects caused by conventional slow cooling. However, vitrification too can impose damaging consequences on cells as the cryoprotectant concentrations required to vitrify cells at lower cooling rates are potentially, and often, harmful. While these concentrations can be lowered to nontoxic levels, if the cells are ultra-rapidly cooled, the resulting metastable system can lead to damage through devitrification and growth of ice during subsequent storage and rewarming if not appropriately handled.The commercial and clinical application of stem cells requires robust and reproducible cryopreservation protocols and appropriate long-term, low-temperature storage conditions to provide reliable master and working cell banks. Though current Good Manufacturing Practice (cGMP) compliant methods for the derivation and banking of clinical grade pluripotent stem cells exist and stem cell lines suitable for clinical applications are available, current cryopreservation protocols, whether for vitrification or conventional slow freezing, remain suboptimal. Apart from the resultant loss of valuable product that suboptimal cryopreservation engenders, there is a danger that such processes will impose a selective pressure on the cells selecting out a nonrepresentative, freeze-resistant subpopulation. Optimizing this process requires knowledge of the fundamental processes that occur during the freezing of cellular systems, the mechanisms of damage and methods for avoiding them. This chapter draws together the knowledge of cryopreservation gained in other systems with the current state-of-the-art for embryonic and induced pluripotent stem cell preservation in an attempt to provide the background for future attempts to optimize cryopreservation protocols.
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Affiliation(s)
- Charles J Hunt
- UK Stem Cell Bank, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK.
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Lauterboeck L, Saha D, Chatterjee A, Hofmann N, Glasmacher B. Xeno-Free Cryopreservation of Bone Marrow-Derived Multipotent Stromal Cells from Callithrix jacchus. Biopreserv Biobank 2016; 14:530-538. [DOI: 10.1089/bio.2016.0038] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Affiliation(s)
- Lothar Lauterboeck
- Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany
| | - Debapriya Saha
- Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany
| | - Anamika Chatterjee
- Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany
| | - Nicola Hofmann
- Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany
| | - Birgit Glasmacher
- Institute for Multiphase Processes, Leibniz Universität Hannover, Hannover, Germany
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Volbers JC, Lauterböck L, Hofmann N, Glasmacher B. Cryopreservation of cells using defined serum-free cryoprotective agents. CURRENT DIRECTIONS IN BIOMEDICAL ENGINEERING 2016. [DOI: 10.1515/cdbme-2016-0070] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Abstract
For regenerative purposes, there is a high demand for viable and active cells. A big issue is to have enough viable cells available at any given time. One solution is cryopreservation. In this context, DMSO is used as cryoprotective agent (CPA) along with fetal bovine serum for nutrient supply and stress shielding effects. To use these cells for human clinical studies, it is important to eliminate the serum to prevent foreign immune reactions and virus transmittance and DMSO for its toxic effect. In this study a serum free cryopreservation solution and protocol has been established. The combination of methylcellulose and poloxamer 188 provide the basis for the new CPA. Other additves are α-tocopherol, ectoine, prolin and ascorbic acid. The CPAs were examined with 3T3-cells and multipotent stromal cells from the common marmoset monkey (Callithrix jacchus). The cells were preserved with various CPA concentrations, incubation times and different cooling rates. To enable a higher throughput of encouraging conditions a fluorescence microscopy analysis was used. The use of methylcellulose, poloxamer 188 and α-tocopherol enables the reduction of DMSO [up to 2.5% (v/v)] and the elimination of serum without viability losses compared to control.
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Affiliation(s)
- Jan-Cedric Volbers
- Institute for Multiphase Processes (Leibniz Universität Hannover), Hannover, Germany
| | - Lothar Lauterböck
- Institute for Multiphase Processes (Leibniz Universität Hannover), Hannover, Germany
| | - Nicola Hofmann
- Institute for Multiphase Processes (Leibniz Universität Hannover), Hannover, Germany
| | - Birgit Glasmacher
- Institute for Multiphase Processes (Leibniz Universität Hannover), Hannover, Germany
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Kim SM, Yun CK, Park JH, Hwang JW, Kim ZH, Choi YS. Efficient cryopreservation of human mesenchymal stem cells using silkworm hemolymph-derived proteins. J Tissue Eng Regen Med 2016. [DOI: 10.1002/term.2116] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Sun-Mi Kim
- Department of Biotechnology; CHA University; Seongnam Republic of Korea
| | - Chang-Koo Yun
- Department of Biotechnology; CHA University; Seongnam Republic of Korea
| | - Jin-Ho Park
- Department of Biotechnology; CHA University; Seongnam Republic of Korea
| | - Jung Wook Hwang
- Department of Biotechnology; CHA University; Seongnam Republic of Korea
| | - Z-Hun Kim
- Department of Biotechnology; CHA University; Seongnam Republic of Korea
| | - Yong-Soo Choi
- Department of Biotechnology; CHA University; Seongnam Republic of Korea
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Short-term hypothermic preservation of human testicular tissue: the effect of storage medium and storage period. Fertil Steril 2016; 105:1162-1169.e5. [PMID: 26868991 DOI: 10.1016/j.fertnstert.2016.01.018] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2015] [Revised: 01/12/2016] [Accepted: 01/15/2016] [Indexed: 01/15/2023]
Abstract
OBJECTIVE To optimize the storage medium and period during short-term preservation of human testicular tissue. DESIGN First, human testicular tissue fragments from five patients were kept at 4°C for 3 days in different media (Dulbecco's modified Eagle's medium [DMEM]/F12, DMEM/F12 + 20% human serum albumin [HSA], DMEM/F12 + 50% HSA, and HSA). Secondly, fragments from four patients were kept in DMEM/F12 for 3, 5, or 8 days at 4°C. SETTING Laboratory research environment. PATIENT(S) Adult human testicular tissue. INTERVENTION(S) Biopsy and short-term storage of human testicular tissue at different conditions. MAIN OUTCOME MEASURE(S) Viability, general tissue morphology, Sertoli cell morphology, number of spermatogonia, and apoptosis. The experimental conditions were compared with fresh control samples. RESULT(S) Storing human testicular tissue in DMEM/F12 did not alter any of the investigated parameters. In most conditions containing HSA, tissue morphology was altered, and in all of them the Sertoli cell morphology was affected. The number of spermatogonia was only affected when tissue was stored in 100% HSA. In the second part of the study, tissue morphology deteriorated significantly as of 5 days of hypothermic storage, and Sertoli cell morphology after 8 days. CONCLUSION(S) Human testicular tissue can be preserved for 3 days at 4°C in DMEM/F12 without altering tissue morphology, Sertoli cell morphology, number of spermatogonia, or number of apoptotic cells.
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Shivakumar SB, Bharti D, Jang SJ, Hwang SC, Park JK, Shin JK, Byun JH, Park BW, Rho GJ. Cryopreservation of Human Wharton's Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study. Int J Stem Cells 2015; 8:155-69. [PMID: 26634064 PMCID: PMC4651280 DOI: 10.15283/ijsc.2015.8.2.155] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
OBJECTIVES To compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton's jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol. METHODS The mesenchymal stem cells isolated from human Wharton's jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation. RESULTS The cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2±0.58% whereas 10% PVP and cocktail solution have shown 62.87±0.35% and 72.2±0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic BAX and p53 genes were higher whilst p21 was lower in all the cryopreserved groups when compare to the control group of WJMSCs. CONCLUSION Although 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.
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Affiliation(s)
- Sharath Belame Shivakumar
- Department of Vet OBS/Theriogenology and Biotechnology, Gyeongsang National University, Jinju, Korea
| | - Dinesh Bharti
- Department of Vet OBS/Theriogenology and Biotechnology, Gyeongsang National University, Jinju, Korea
| | - Si-Jung Jang
- Department of Vet OBS/Theriogenology and Biotechnology, Gyeongsang National University, Jinju, Korea
| | - Sun-Chul Hwang
- Department of Orthopaedic Surgery, School of Medicine, Gyeongsang National University, Jinju, Korea
| | - Ji-Kwon Park
- Department of Obstetrics and Gynaecology, School of Medicine, Gyeongsang National University, Jinju, Korea
| | - Jeong-Kyu Shin
- Department of Obstetrics and Gynaecology, School of Medicine, Gyeongsang National University, Jinju, Korea
| | - June-Ho Byun
- Department of Oral and Maxillofacial Surgery, School of Medicine, Gyeongsang National University, Jinju, Korea
| | - Bong-Wook Park
- Department of Oral and Maxillofacial Surgery, School of Medicine, Gyeongsang National University, Jinju, Korea
| | - Gyu-Jin Rho
- Department of Vet OBS/Theriogenology and Biotechnology, Gyeongsang National University, Jinju, Korea ; Research Institute of Life Sciences, Gyeongsang National University, Jinju, Korea
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Hashemi M, Kalalinia F. Application of encapsulation technology in stem cell therapy. Life Sci 2015; 143:139-46. [DOI: 10.1016/j.lfs.2015.11.007] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2015] [Revised: 10/15/2015] [Accepted: 11/06/2015] [Indexed: 11/26/2022]
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Mitchell A, Rivas KA, Smith R, Watts AE. Cryopreservation of equine mesenchymal stem cells in 95% autologous serum and 5% DMSO does not alter post-thaw growth or morphology in vitro compared to fetal bovine serum or allogeneic serum at 20 or 95% and DMSO at 10 or 5. Stem Cell Res Ther 2015; 6:231. [PMID: 26611913 PMCID: PMC4661990 DOI: 10.1186/s13287-015-0230-y] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2015] [Revised: 09/18/2015] [Accepted: 11/09/2015] [Indexed: 12/17/2022] Open
Abstract
Introduction Equine superficial digital flexor tendon injury is a well-accepted model of human tendon injury and is routinely treated with local injections of autologous mesenchymal stem cells (MSCs). Identification of a clinically safe medium for short-term cryopreservation of MSCs prior to cell implantation would streamline laboratory and clinical procedures for autologous regenerative therapies. Veterinary experience with short-term (MSCs prepared after the injury has occurred) cryopreserved MSCs in naturally occurring injury in the horse will be of value to human practitioners. Methods Equine bone marrow derived MSCs were cryopreserved in 6 different solutions consisting of 20 % serum, 10 % DMSO and 70 % media or 95 % serum and 5 % DMSO. Serum was autologous serum, commercially available pooled equine serum or fetal bovine serum (FBS). Cell survival, morphology and growth kinetics were assessed by total cell number, measurement of growth kinetics, colony-forming-unit-assay and morphology of MSCs after monolayer culture post-thaw. Results There were no significant differences in post-thaw viability, total cell number, morphology scores or growth kinetics among the 6 solutions. Post thaw viabilities from each group ranged from 80-90 %. In all solutions, there were significantly fewer MSCs and the majority (99 %) of MSCs remained in the original generation 24 hours post-thaw. Seventy two hours post-thaw, the majority of MSCs (50 %) were proliferating in the fourth generation. Mean colony count in the CFU-F assay ranged from 72 to 115 colonies. Conclusions Each of the serum sources could be used for short-term cryopreservation of equine bone marrow derived MSCs. Prior to clinical use, clinicians may prefer autologous serum and a lower concentration of DMSO. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0230-y) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Alexis Mitchell
- Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, 77843, USA.
| | - Kristen A Rivas
- Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, 77843, USA.
| | - Roger Smith
- Department of Veterinary Pathobiology, Texas A&M University, College Station, TX, 77843, USA.
| | - Ashlee E Watts
- Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, 77843, USA.
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Miyagi-Shiohira C, Kurima K, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, Matsushita M, Noguchi H. Cryopreservation of Adipose-Derived Mesenchymal Stem Cells. CELL MEDICINE 2015; 8:3-7. [PMID: 26858903 DOI: 10.3727/215517915x689100] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Mesenchymal stem cells (MSCs) have the potential to differentiate into cells of mesodermal origin such as osteoblasts, adipocytes, myocytes, and chondrocytes. They possess an immunosuppressive effect, which makes them a viable cell population for the cell-based therapy of treatment-resistant immune diseases. Adipose-derived mesenchymal stem cells (ASCs) have been demonstrated to have the ability to acquire the properties of subcutaneous adipose tissue particularly easily, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. However, many studies have reported that cellular activity after freezing and thawing may be affected by the solutions used for cryopreservation. Dimethyl sulfoxide (DMSO) is commonly used as a cryopreservation medium as it diffuses into the cell through the plasma membrane and protects the cells from the damage caused by freezing. As substitutes for DMSO or animal-derived serum, cell banker series, polyvinylpyrrolidone (PVP), sericin and maltose, and methyl cellulose (MC) have been investigated for their clinical applications. It is critical to develop a reliable cell cryopreservation protocol for regenerative medicine using MSCs.
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Affiliation(s)
- Chika Miyagi-Shiohira
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus , Okinawa , Japan
| | - Kiyoto Kurima
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus , Okinawa , Japan
| | | | - Issei Saitoh
- ‡ Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University , Niigata , Japan
| | - Masami Watanabe
- § Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences , Okayama , Japan
| | - Yasufumi Noguchi
- ¶ Department of Socio-environmental Design, Hiroshima International University , Hiroshima , Japan
| | - Masayuki Matsushita
- # Department of Molecular and Cellular Physiology, Graduate School of Medicine, University of the Ryukyus , Okinawa , Japan
| | - Hirofumi Noguchi
- Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus , Okinawa , Japan
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Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects. Cryobiology 2015; 71:181-97. [PMID: 26186998 DOI: 10.1016/j.cryobiol.2015.07.003] [Citation(s) in RCA: 235] [Impact Index Per Article: 23.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2015] [Accepted: 07/13/2015] [Indexed: 12/11/2022]
Abstract
Originally isolated from bone marrow, mesenchymal stromal cells (MSCs) have since been obtained from various fetal and post-natal tissues and are the focus of an increasing number of clinical trials. Because of their tremendous potential for cellular therapy, regenerative medicine and tissue engineering, it is desirable to cryopreserve and bank MSCs to increase their access and availability. A remarkable amount of research and resources have been expended towards optimizing the protocols, freezing media composition, cooling devices and storage containers, as well as developing good manufacturing practices in order to ensure that MSCs retain their therapeutic characteristics following cryopreservation and that they are safe for clinical use. Here, we first present an overview of the identification of MSCs, their tissue sources and the properties that render them suitable as a cellular therapeutic. Next, we discuss the responses of cells during freezing and focus on the traditional and novel approaches used to cryopreserve MSCs. We conclude that viable MSCs from diverse tissues can be recovered after cryopreservation using a variety of freezing protocols, cryoprotectants, storage periods and temperatures. However, alterations in certain functions of MSCs following cryopreservation warrant future investigations on the recovery of cells post-thaw followed by expansion of functional cells in order to achieve their full therapeutic potential.
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Heathman TRJ, Glyn VAM, Picken A, Rafiq QA, Coopman K, Nienow AW, Kara B, Hewitt CJ. Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process. Biotechnol Bioeng 2015; 112:1696-707. [PMID: 25727395 PMCID: PMC5029583 DOI: 10.1002/bit.25582] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2014] [Revised: 01/02/2015] [Accepted: 02/18/2015] [Indexed: 02/06/2023]
Abstract
Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot‐sizes required for commercial production. The use of animal‐derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot‐to‐lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large‐scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum‐free hMSC manufacturing process. Human bone‐marrow derived hMSCs were expanded on fibronectin‐coated, non‐porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 105 cells.mL−1 in serum‐free medium. The hMSCs were successfully harvested by our recently‐developed technique using animal‐free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post‐harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony‐forming potential. The hMSCs were held in suspension post‐harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum‐free vehicle solution using a controlled‐rate freezing process. Post‐thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component‐free hMSC production process from expansion through to cryopreservation. Biotechnol. Bioeng. 2015;112: 1696–1707. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
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Affiliation(s)
- Thomas R J Heathman
- Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK
| | - Veronica A M Glyn
- Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK
| | - Andrew Picken
- Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK
| | - Qasim A Rafiq
- Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK.,Aston Medical Research Institute, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET
| | - Karen Coopman
- Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK.
| | - Alvin W Nienow
- Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK.,Centre for Bioprocess Engineering, University of Birmingham, B15 2TT, UK
| | - Bo Kara
- FUJIFILM Diosynth Biotechnologies, Billingham, TS23 1LH, UK
| | - Christopher J Hewitt
- Centre for Biological Engineering, Loughborough University, Leicestershire, LE11 3TU, UK.,Aston Medical Research Institute, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET
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Effects of trehalose supplementation on cell viability and oxidative stress variables in frozen-thawed bovine calf testicular tissue. Cryobiology 2015; 70:246-52. [PMID: 25818604 DOI: 10.1016/j.cryobiol.2015.03.004] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2015] [Revised: 03/10/2015] [Accepted: 03/11/2015] [Indexed: 11/23/2022]
Abstract
Trehalose is widely used for cryopreservation of various cells and tissues. Until now, the effect of trehalose supplementation on cell viability and antioxidant enzyme activity in frozen-thawed bovine calf testicular tissue remains unexplored. The objective of the present study was to compare the effect of varying doses of trehalose in cryomedia on cell viability and key antioxidant enzymes activities in frozen-thawed bovine calf testicular tissue. Bovine calf testicular tissue samples were collected and cryopreserved in the cryomedias containing varying doses (0, 5, 10, 15, 20 and 25%; v/v) of trehalose, respectively. Cell viability, total antioxidant capacity (T-AOC) activity, catalase (CAT) activity, superoxide dismutase (SOD) activity, glutathione (GSH) content and malondialdehyde (MDA) content were measured and analyzed. The results showed that cell viability, T-AOC activity, SOD activity, CAT activity and GSH content of frozen-thawed bovine calf testicular tissue was decreased compared with that of fresh group (P<0.05). MDA content in frozen-thawed bovine calf testicular tissue was significantly increased compared with that of fresh group (P<0.05). The cryomedia added 15% trehalose exhibited the greatest percentage of cell viability and antioxidant enzyme activity (SOD and CAT) among frozen-thawed groups (P<0.05). Meanwhile, GSH content was the lowest among frozen-thawed groups (P<0.05). However, there were no significance differences in MDA content among the groups added 10, 15 and 20% trehalose (P>0.05). In conclusion, the cryomedia added 15% trehalose reduced the oxidative stress and improved the cryoprotective effect of bovine calf testicular tissue. Further studies are required to obtain more concrete results on the determination of antioxidant capacity of trehalose in frozen-thawed bovine calf testicular tissue.
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Asghar W, El Assal R, Shafiee H, Anchan RM, Demirci U. Preserving human cells for regenerative, reproductive, and transfusion medicine. Biotechnol J 2015; 9:895-903. [PMID: 24995723 DOI: 10.1002/biot.201300074] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2013] [Revised: 05/21/2014] [Accepted: 05/30/2014] [Indexed: 12/15/2022]
Abstract
Cell cryopreservation maintains cellular life at sub-zero temperatures by slowing down biochemical processes. Various cell types are routinely cryopreserved in modern reproductive, regenerative, and transfusion medicine. Current cell cryopreservation methods involve freezing (slow/rapid) or vitrifying cells in the presence of a cryoprotective agent (CPA). Although these methods are clinically utilized, cryo-injury due to ice crystals, osmotic shock, and CPA toxicity cause loss of cell viability and function. Recent approaches using minimum volume vitrification provide alternatives to the conventional cryopreservation methods. Minimum volume vitrification provides ultra-high cooling and rewarming rates that enable preserving cells without ice crystal formation. Herein, we review recent advances in cell cryopreservation technology and provide examples of techniques that are utilized in oocyte, stem cell, and red blood cell cryopreservation.
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Affiliation(s)
- Waseem Asghar
- Bio-Acoustic-MEMS in Medicine (BAMM) Laboratories, Department of Radiology, Canary Center at Stanford for Cancer Early Detection, Stanford School of Medicine, Stanford University, Palo Alto, CA, USA
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