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Francis N, Aho J, Ben-Nun IF, Bharti K, Dianat N, Makovoz B, Nouri P, Rothberg J, Song H, Zamilpa R, Lakshmipathy U, Allickson J. Scaling up pluripotent stem cell-based therapies - considerations, current challenges and emerging technologies: perspectives from the ISCT Emerging Regenerative Medicine Working Group. Cytotherapy 2025:S1465-3249(25)00678-4. [PMID: 40353785 DOI: 10.1016/j.jcyt.2025.04.058] [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: 02/11/2025] [Revised: 03/21/2025] [Accepted: 04/03/2025] [Indexed: 05/14/2025]
Abstract
Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after more than 20 years of research and development. However, manufacturing these therapies at the scale required for patient treatment, as well as for key clinical trial enabling activities such as preclinical and stability studies, remains a challenge. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSC-derived technologies are expected to play a key role. In this article, the Working Group provides an overview of the considerations and challenges facing stem cell therapy developers, including development-stage specific manufacturing processes, the decision on when to implement automation, the choice of technology and different requirements of expansion and differentiation aspects of the process, and the integration of automation for both manufacturing and analytics in an end-to-end manufacturing process. The role of scalable manufacturing technologies in the application of quality-by-design approaches to product development, and the use of design-of-experiment approaches for increased product characterization, is discussed. Finally, we provide an in-depth review of the different technologies that have been used for expansion and differentiation of iPSC-derived therapies to date, including compatibility with good manufacturing practice requirements and process analytical technologies. We hope that this overview will summarize the existing knowledge in the field and reduce the challenges that therapy developers face in translating their research into clinical and commercial scale manufacturing.
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Affiliation(s)
- Natalie Francis
- Centre for Gene Therapy and Regenerative Medicine, King's College London, London, UK.
| | - Joy Aho
- Product Management and Strategy, NMDP BioTherapies, Minneapolis, USA
| | | | - Kapil Bharti
- National Eye Institute, National Institute of Health, Bethesda, Maryland, USA
| | - Noushin Dianat
- R&D Cell Therapy, Sartorius Stedim Biotech, Göttingen, Germany
| | - Bar Makovoz
- Product Management, Cellino, Cambridge, Massachusetts, USA
| | - Parivash Nouri
- Pluripotent Stem Cells Product Management, Miltenyi Biotec, Bergisch, Gladbach, Germany
| | - Janet Rothberg
- Process & Analytical Development, CCRM, MaRS Centre, West Tower, Toronto, Ontario, Canada
| | - Hannah Song
- Center for Cellular Engineering, National Institute of Health, Rockville, Maryland, USA
| | | | - Uma Lakshmipathy
- Patheon Cell & Gene Translation Services, Thermo Fisher Scientific, San Diego, California, USA
| | - Julie Allickson
- Clinic's Center for Regenerative Biotherapeutics, Mayo Clinic, Rochester, Minnesota, USA
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Miller RC, Temenoff JS. Biomaterials for Cell Manufacturing. ACS Macro Lett 2024; 13:1521-1530. [PMID: 39466845 PMCID: PMC11580378 DOI: 10.1021/acsmacrolett.4c00634] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2024] [Revised: 10/18/2024] [Accepted: 10/22/2024] [Indexed: 10/30/2024]
Abstract
Cell therapies, potent populations of cells used to treat disease and injury, can be strategically manufactured with biomaterial intervention to improve clinical translation. In this viewpoint, we discuss biomaterial design and integration into cell manufacturing steps to achieve three main goals: scale-up, phenotype control, and selection of potent cells. Material properties can be engineered to influence the cell-biomaterial interface and, therefore, impart desirable cell behavior such as growth, secretory activity, and differentiation. Future directions for the field should capitalize on the combinatorial design of biomaterial properties to yield highly specific and potent cell populations. Furthermore, future biomaterials could contribute to novel high-throughput cell separation technologies that can individually select the most therapeutically relevant cells within a produced batch.
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Affiliation(s)
- Ryan C. Miller
- Wallace
H. Coulter Department of Biomedical Engineering, Georgia Tech/Emory University, Atlanta, Georgia 30332, United States
| | - Johnna S. Temenoff
- Wallace
H. Coulter Department of Biomedical Engineering, Georgia Tech/Emory University, Atlanta, Georgia 30332, United States
- Parker
H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
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Padmanaban AM, Ganesan K, Ramkumar KM. A Co-Culture System for Studying Cellular Interactions in Vascular Disease. Bioengineering (Basel) 2024; 11:1090. [PMID: 39593750 PMCID: PMC11591305 DOI: 10.3390/bioengineering11111090] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2024] [Revised: 10/17/2024] [Accepted: 10/21/2024] [Indexed: 11/28/2024] Open
Abstract
Cardiovascular diseases (CVDs) are leading causes of morbidity and mortality globally, characterized by complications such as heart failure, atherosclerosis, and coronary artery disease. The vascular endothelium, forming the inner lining of blood vessels, plays a pivotal role in maintaining vascular homeostasis. The dysfunction of endothelial cells contributes significantly to the progression of CVDs, particularly through impaired cellular communication and paracrine signaling with other cell types, such as smooth muscle cells and macrophages. In recent years, co-culture systems have emerged as advanced in vitro models for investigating these interactions and mimicking the pathological environment of CVDs. This review provides an in-depth analysis of co-culture models that explore endothelial cell dysfunction and the role of cellular interactions in the development of vascular diseases. It summarizes recent advancements in multicellular co-culture models, their physiological and therapeutic relevance, and the insights they provide into the molecular mechanisms underlying CVDs. Additionally, we evaluate the advantages and limitations of these models, offering perspectives on how they can be utilized for the development of novel therapeutic strategies and drug testing in cardiovascular research.
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Affiliation(s)
- Abirami M. Padmanaban
- Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur 603 203, Tamil Nadu, India;
| | - Kumar Ganesan
- School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong 999077, China;
| | - Kunka Mohanram Ramkumar
- Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur 603 203, Tamil Nadu, India;
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Zheng F, Tian R, Lu H, Liang X, Shafiq M, Uchida S, Chen H, Ma M. Droplet Microfluidics Powered Hydrogel Microparticles for Stem Cell-Mediated Biomedical Applications. SMALL (WEINHEIM AN DER BERGSTRASSE, GERMANY) 2024; 20:e2401400. [PMID: 38881184 DOI: 10.1002/smll.202401400] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/21/2024] [Revised: 05/21/2024] [Indexed: 06/18/2024]
Abstract
Stem cell-related therapeutic technologies have garnered significant attention of the research community for their multi-faceted applications. To promote the therapeutic effects of stem cells, the strategies for cell microencapsulation in hydrogel microparticles have been widely explored, as the hydrogel microparticles have the potential to facilitate oxygen diffusion and nutrient transport alongside their ability to promote crucial cell-cell and cell-matrix interactions. Despite their significant promise, there is an acute shortage of automated, standardized, and reproducible platforms to further stem cell-related research. Microfluidics offers an intriguing platform to produce stem cell-laden hydrogel microparticles (SCHMs) owing to its ability to manipulate the fluids at the micrometer scale as well as precisely control the structure and composition of microparticles. In this review, the typical biomaterials and crosslinking methods for microfluidic encapsulation of stem cells as well as the progress in droplet-based microfluidics for the fabrication of SCHMs are outlined. Moreover, the important biomedical applications of SCHMs are highlighted, including regenerative medicine, tissue engineering, scale-up production of stem cells, and microenvironmental simulation for fundamental cell studies. Overall, microfluidics holds tremendous potential for enabling the production of diverse hydrogel microparticles and is worthy for various stem cell-related biomedical applications.
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Affiliation(s)
- Fangqiao Zheng
- School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, P. R. China
| | - Ruizhi Tian
- Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China
- Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
| | - Hongxu Lu
- Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China
- Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
| | - Xiao Liang
- School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, P. R. China
| | - Muhammad Shafiq
- Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, Kawasaki-ku, Kawasaki, Kanagawa, 210-0821, Japan
| | - Satoshi Uchida
- Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, Kawasaki-ku, Kawasaki, Kanagawa, 210-0821, Japan
- Department of Advanced Nanomedical Engineering, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan
| | - Hangrong Chen
- School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, P. R. China
- Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China
- Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
| | - Ming Ma
- School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, 310024, P. R. China
- Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China
- Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing, 100049, P. R. China
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Bandarra-Tavares H, Franchi-Mendes T, Ulpiano C, Morini S, Kaur N, Harris-Becker A, Vemuri MC, Cabral JMS, Fernandes-Platzgummer A, da Silva CL. Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system. Cytotherapy 2024; 26:749-756. [PMID: 38506771 DOI: 10.1016/j.jcyt.2024.03.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2023] [Revised: 01/29/2024] [Accepted: 03/02/2024] [Indexed: 03/21/2024]
Abstract
BACKGROUND & AIMS Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. METHODS MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. RESULTS Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). CONCLUSIONS Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.
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Affiliation(s)
- Hélder Bandarra-Tavares
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Teresa Franchi-Mendes
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cristiana Ulpiano
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Sara Morini
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Navjot Kaur
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Abigail Harris-Becker
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Mohan C Vemuri
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
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Major GS, Doan VK, Longoni A, Bilek MMM, Wise SG, Rnjak-Kovacina J, Yeo GC, Lim KS. Mapping the microcarrier design pathway to modernise clinical mesenchymal stromal cell expansion. Trends Biotechnol 2024; 42:859-876. [PMID: 38320911 DOI: 10.1016/j.tibtech.2024.01.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2023] [Revised: 01/08/2024] [Accepted: 01/08/2024] [Indexed: 02/08/2024]
Abstract
Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs.
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Affiliation(s)
- Gretel S Major
- School of Medical Sciences, University of Sydney, Sydney, Australia
| | - Vinh K Doan
- School of Medical Sciences, University of Sydney, Sydney, Australia
| | - Alessia Longoni
- Department of Orthopedics, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Marcela M M Bilek
- School of Biomedical Engineering, University of Sydney, Sydney, Australia; School of Physics, University of Sydney, Sydney, Australia; Charles Perkins Centre, University of Sydney, Sydney, Australia; Sydney Nano Institute, University of Sydney, Sydney, Australia
| | - Steven G Wise
- School of Medical Sciences, University of Sydney, Sydney, Australia; Charles Perkins Centre, University of Sydney, Sydney, Australia
| | - Jelena Rnjak-Kovacina
- Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Australia; Tyree Institute of Health Engineering, University of New South Wales, Sydney, Australia
| | - Giselle C Yeo
- Charles Perkins Centre, University of Sydney, Sydney, Australia; School of Life and Environmental Sciences, University of Sydney, Sydney, Australia.
| | - Khoon S Lim
- School of Medical Sciences, University of Sydney, Sydney, Australia; Charles Perkins Centre, University of Sydney, Sydney, Australia; Sydney Nano Institute, University of Sydney, Sydney, Australia.
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Ali EAM, Smaida R, Meyer M, Ou W, Li Z, Han Z, Benkirane-Jessel N, Gottenberg JE, Hua G. iPSCs chondrogenic differentiation for personalized regenerative medicine: a literature review. Stem Cell Res Ther 2024; 15:185. [PMID: 38926793 PMCID: PMC11210138 DOI: 10.1186/s13287-024-03794-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2024] [Accepted: 06/08/2024] [Indexed: 06/28/2024] Open
Abstract
Cartilage, an important connective tissue, provides structural support to other body tissues, and serves as a cushion against impacts throughout the body. Found at the end of the bones, cartilage decreases friction and averts bone-on-bone contact during joint movement. Therefore, defects of cartilage can result from natural wear and tear, or from traumatic events, such as injuries or sudden changes in direction during sports activities. Overtime, these cartilage defects which do not always produce immediate symptoms, could lead to severe clinical pathologies. The emergence of induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine, providing a promising platform for generating various cell types for therapeutic applications. Thus, chondrocytes differentiated from iPSCs become a promising avenue for non-invasive clinical interventions for cartilage injuries and diseases. In this review, we aim to highlight the current strategies used for in vitro chondrogenic differentiation of iPSCs and to explore their multifaceted applications in disease modeling, drug screening, and personalized regenerative medicine. Achieving abundant functional iPSC-derived chondrocytes requires optimization of culture conditions, incorporating specific growth factors, and precise temporal control. Continual improvements in differentiation methods and integration of emerging genome editing, organoids, and 3D bioprinting technologies will enhance the translational applications of iPSC-derived chondrocytes. Finally, to unlock the benefits for patients suffering from cartilage diseases through iPSCs-derived technologies in chondrogenesis, automatic cell therapy manufacturing systems will not only reduce human intervention and ensure sterile processes within isolator-like platforms to minimize contamination risks, but also provide customized production processes with enhanced scalability and efficiency.
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Affiliation(s)
- Eltahir Abdelrazig Mohamed Ali
- Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 1260, Regenerative NanoMedicine (RNM), 1 Rue Eugène Boeckel, 67000, Strasbourg, France
- Université de Strasbourg, 67000, Strasbourg, France
| | - Rana Smaida
- Lamina Therapeutics, 1 Rue Eugène Boeckel, 67000, Strasbourg, France
| | - Morgane Meyer
- Université de Strasbourg, 67000, Strasbourg, France
- Lamina Therapeutics, 1 Rue Eugène Boeckel, 67000, Strasbourg, France
| | - Wenxin Ou
- Université de Strasbourg, 67000, Strasbourg, France
- Centre National de Référence des Maladies Auto-Immunes et Systémiques Rares, Est/Sud-Ouest (RESO), Service de Rhumatologie, Centre Hospitalier Universitaire de Strasbourg, 67000, Strasbourg, France
- Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing, 400016, China
| | - Zongjin Li
- Nankai University School of Medicine, Tianjin, 300071, China
| | - Zhongchao Han
- Beijing Engineering Laboratory of Perinatal Stem Cells, Beijing Institute of Health and Stem Cells, Health & Biotech Co, Beijing, 100176, China
| | - Nadia Benkirane-Jessel
- Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 1260, Regenerative NanoMedicine (RNM), 1 Rue Eugène Boeckel, 67000, Strasbourg, France.
- Université de Strasbourg, 67000, Strasbourg, France.
- Lamina Therapeutics, 1 Rue Eugène Boeckel, 67000, Strasbourg, France.
| | - Jacques Eric Gottenberg
- Université de Strasbourg, 67000, Strasbourg, France.
- Centre National de Référence des Maladies Auto-Immunes et Systémiques Rares, Est/Sud-Ouest (RESO), Service de Rhumatologie, Centre Hospitalier Universitaire de Strasbourg, 67000, Strasbourg, France.
| | - Guoqiang Hua
- Institut National de la Santé et de la Recherche Médicale (INSERM), UMR 1260, Regenerative NanoMedicine (RNM), 1 Rue Eugène Boeckel, 67000, Strasbourg, France.
- Université de Strasbourg, 67000, Strasbourg, France.
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Benavides OR, White BP, Gibbs HC, Kaunas R, Gregory CA, Maitland KC, Walsh AJ. Comparison of polystyrene and hydrogel microcarriers for optical imaging of adherent cells. JOURNAL OF BIOMEDICAL OPTICS 2024; 29:S22708. [PMID: 38872791 PMCID: PMC11175462 DOI: 10.1117/1.jbo.29.s2.s22708] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Revised: 05/10/2024] [Accepted: 05/28/2024] [Indexed: 06/15/2024]
Abstract
Significance The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition. Aim Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells. Approach Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto, three-dimensional, high-resolution visualization of cells cultured on microcarriers. Results The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography. Conclusions The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes.
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Affiliation(s)
- Oscar R. Benavides
- Texas A&M University, Department of Biomedical Engineering, College Station, Texas, United States
| | - Berkley P. White
- Texas A&M University, Department of Biomedical Engineering, College Station, Texas, United States
| | - Holly C. Gibbs
- Texas A&M University, Department of Biomedical Engineering, College Station, Texas, United States
- Texas A&M University, Microscopy and Imaging Center, College Station, Texas, United States
| | - Roland Kaunas
- Texas A&M University, Department of Biomedical Engineering, College Station, Texas, United States
| | - Carl A. Gregory
- Texas A&M Health Science Center, School of Medicine, Bryan, Texas, United States
| | - Kristen C. Maitland
- Texas A&M University, Department of Biomedical Engineering, College Station, Texas, United States
- Texas A&M University, Microscopy and Imaging Center, College Station, Texas, United States
| | - Alex J. Walsh
- Texas A&M University, Department of Biomedical Engineering, College Station, Texas, United States
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9
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Haskell A, White BP, Rogers RE, Goebel E, Lopez MG, Syvyk AE, de Oliveira DA, Barreda HA, Benton J, Benavides OR, Dalal S, Bae E, Zhang Y, Maitland K, Nikolov Z, Liu F, Lee RH, Kaunas R, Gregory CA. Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost. Cytotherapy 2024; 26:372-382. [PMID: 38363250 PMCID: PMC11057043 DOI: 10.1016/j.jcyt.2024.01.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Revised: 01/23/2024] [Accepted: 01/27/2024] [Indexed: 02/17/2024]
Abstract
BACKGROUND AIMS Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.
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Affiliation(s)
- Andrew Haskell
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Berkley P White
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA
| | - Robert E Rogers
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Erin Goebel
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA; Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA
| | - Megan G Lopez
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Andrew E Syvyk
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA
| | - Daniela A de Oliveira
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA; Biological and Agricultural Engineering, Texas A&M University, College Station, Texas, USA
| | - Heather A Barreda
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Joshua Benton
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Oscar R Benavides
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA
| | - Sujata Dalal
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - EunHye Bae
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Yu Zhang
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Kristen Maitland
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA; Imaging Program, Chan Zuckerberg Initiative, Redwood City, California, USA
| | - Zivko Nikolov
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA; Biological and Agricultural Engineering, Texas A&M University, College Station, Texas, USA
| | - Fei Liu
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Ryang Hwa Lee
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Roland Kaunas
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA.
| | - Carl A Gregory
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA.
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10
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Ding L, Oh S, Shrestha J, Lam A, Wang Y, Radfar P, Warkiani ME. Scaling up stem cell production: harnessing the potential of microfluidic devices. Biotechnol Adv 2023; 69:108271. [PMID: 37844769 DOI: 10.1016/j.biotechadv.2023.108271] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2023] [Revised: 10/08/2023] [Accepted: 10/13/2023] [Indexed: 10/18/2023]
Abstract
Stem cells are specialised cells characterised by their unique ability to both self-renew and transform into a wide array of specialised cell types. The widespread interest in stem cells for regenerative medicine and cultivated meat has led to a significant demand for these cells in both research and practical applications. Despite the growing need for stem cell manufacturing, the industry faces significant obstacles, including high costs for equipment and maintenance, complicated operation, and low product quality and yield. Microfluidic technology presents a promising solution to the abovementioned challenges. As an innovative approach for manipulating liquids and cells within microchannels, microfluidics offers a plethora of advantages at an industrial scale. These benefits encompass low setup costs, ease of operation and multiplexing, minimal energy consumption, and the added advantage of being labour-free. This review presents a thorough examination of the prominent microfluidic technologies employed in stem cell research and explores their promising applications in the burgeoning stem cell industry. It thoroughly examines how microfluidics can enhance cell harvesting from tissue samples, facilitate mixing and cryopreservation, streamline microcarrier production, and efficiently conduct cell separation, purification, washing, and final cell formulation post-culture.
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Affiliation(s)
- Lin Ding
- Smart MCs Pty Ltd, Ultimo, Sydney, 2007, Australia.
| | - Steve Oh
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, 138668, Singapore
| | - Jesus Shrestha
- School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW 2007, Australia
| | - Alan Lam
- Stem Cell Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, 138668, Singapore
| | - Yaqing Wang
- School of Biomedical Engineering, University of Science and Technology of China, Hefei 230026, China; Suzhou Institute for Advanced Research, University of Science and Technology of China, Suzhou 215123, China
| | - Payar Radfar
- Smart MCs Pty Ltd, Ultimo, Sydney, 2007, Australia
| | - Majid Ebrahimi Warkiani
- School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW 2007, Australia..
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11
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Lee RH, Boregowda SV, Shigemoto-Kuroda T, Bae E, Haga CL, Abbery CA, Bayless KJ, Haskell A, Gregory CA, Ortiz LA, Phinney DG. TWIST1 and TSG6 are coordinately regulated and function as potency biomarkers in human MSCs. SCIENCE ADVANCES 2023; 9:eadi2387. [PMID: 37948519 PMCID: PMC10637745 DOI: 10.1126/sciadv.adi2387] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Accepted: 10/11/2023] [Indexed: 11/12/2023]
Abstract
Mesenchymal stem/stromal cells (MSCs) have been evaluated in >1500 clinical trials, but outcomes remain suboptimal because of knowledge gaps in quality attributes that confer potency. We show that TWIST1 directly represses TSG6 expression that TWIST1 and TSG6 are inversely correlated across bone marrow-derived MSC (BM-MSC) donor cohorts and predict interdonor differences in their proangiogenic, anti-inflammatory, and immune suppressive activity in vitro and in sterile inflammation and autoimmune type 1 diabetes preclinical models. Transcript profiling of TWIST1HiTSG6Low versus TWISTLowTSG6Hi BM-MSCs revealed previously unidentified roles for TWIST1/TSG6 in regulating cellular oxidative stress and TGF-β2 in modulating TSG6 expression and anti-inflammatory activity. TWIST1 and TSG6 levels also correlate to donor stature and predict differences in iPSC-derived MSC quality attributes. These results validate TWIST1 and TSG6 as biomarkers that predict interdonor differences in potency across laboratories and assay platforms, thereby providing a means to manufacture MSC products tailored to specific diseases.
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Affiliation(s)
- Ryang Hwa Lee
- Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, 77845, USA
| | - Siddaraju V. Boregowda
- Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, 33458, USA
| | - Taeko Shigemoto-Kuroda
- Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, 77845, USA
| | - EunHye Bae
- Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, 77845, USA
| | - Christopher L. Haga
- Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, 33458, USA
| | - Colette A. Abbery
- Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, 77845, USA
| | - Kayla J. Bayless
- Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, 77845, USA
| | - Andrew Haskell
- Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, 77845, USA
| | - Carl A. Gregory
- Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, 77845, USA
| | - Luis A. Ortiz
- Department of Environmental Health, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Donald G. Phinney
- Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, 33458, USA
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12
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Ando Y, Chang FC, James M, Zhou Y, Zhang M. Chitosan Scaffolds as Microcarriers for Dynamic Culture of Human Neural Stem Cells. Pharmaceutics 2023; 15:1957. [PMID: 37514142 PMCID: PMC10384976 DOI: 10.3390/pharmaceutics15071957] [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: 06/12/2023] [Revised: 07/03/2023] [Accepted: 07/11/2023] [Indexed: 07/30/2023] Open
Abstract
Human neural stem cells (hNSCs) possess remarkable potential for regenerative medicine in the treatment of presently incurable diseases. However, a key challenge lies in producing sufficient quantities of hNSCs, which is necessary for effective treatment. Dynamic culture systems are recognized as a powerful approach to producing large quantities of hNSCs required, where microcarriers play a critical role in supporting cell expansion. Nevertheless, the currently available microcarriers have limitations, including a lack of appropriate surface chemistry to promote cell adhesion, inadequate mechanical properties to protect cells from dynamic forces, and poor suitability for mass production. Here, we present the development of three-dimensional (3D) chitosan scaffolds as microcarriers for hNSC expansion under defined conditions in bioreactors. We demonstrate that chitosan scaffolds with a concentration of 4 wt% (4CS scaffolds) exhibit desirable microstructural characteristics and mechanical properties suited for hNSC expansion. Furthermore, they could also withstand degradation in dynamic conditions. The 4CS scaffold condition yields optimal metabolic activity, cell adhesion, and protein expression, enabling sustained hNSC expansion for up to three weeks in a dynamic culture. Our study introduces an effective microcarrier approach for prolonged expansion of hNSCs, which has the potential for mass production in a three-dimensional setting.
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Affiliation(s)
- Yoshiki Ando
- Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA
- Materials Department, Medical R&D Center, Corporate R&D Group, KYOCERA Corporation, Yasu 520-2362, Shiga, Japan
| | - Fei-Chien Chang
- Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA
| | - Matthew James
- Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA
| | - Yang Zhou
- Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA
| | - Miqin Zhang
- Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA
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13
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Licata JP, Schwab KH, Har-El YE, Gerstenhaber JA, Lelkes PI. Bioreactor Technologies for Enhanced Organoid Culture. Int J Mol Sci 2023; 24:11427. [PMID: 37511186 PMCID: PMC10380004 DOI: 10.3390/ijms241411427] [Citation(s) in RCA: 28] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2023] [Revised: 07/07/2023] [Accepted: 07/10/2023] [Indexed: 07/30/2023] Open
Abstract
An organoid is a 3D organization of cells that can recapitulate some of the structure and function of native tissue. Recent work has seen organoids gain prominence as a valuable model for studying tissue development, drug discovery, and potential clinical applications. The requirements for the successful culture of organoids in vitro differ significantly from those of traditional monolayer cell cultures. The generation and maturation of high-fidelity organoids entails developing and optimizing environmental conditions to provide the optimal cues for growth and 3D maturation, such as oxygenation, mechanical and fluidic activation, nutrition gradients, etc. To this end, we discuss the four main categories of bioreactors used for organoid culture: stirred bioreactors (SBR), microfluidic bioreactors (MFB), rotating wall vessels (RWV), and electrically stimulating (ES) bioreactors. We aim to lay out the state-of-the-art of both commercial and in-house developed bioreactor systems, their benefits to the culture of organoids derived from various cells and tissues, and the limitations of bioreactor technology, including sterilization, accessibility, and suitability and ease of use for long-term culture. Finally, we discuss future directions for improvements to existing bioreactor technology and how they may be used to enhance organoid culture for specific applications.
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Affiliation(s)
- Joseph P Licata
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA 19122, USA
| | - Kyle H Schwab
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA 19122, USA
- Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Yah-El Har-El
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA 19122, USA
| | - Jonathan A Gerstenhaber
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA 19122, USA
| | - Peter I Lelkes
- Department of Bioengineering, College of Engineering, Temple University, Philadelphia, PA 19122, USA
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14
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Park S, Gwon Y, Khan SA, Jang KJ, Kim J. Engineering considerations of iPSC-based personalized medicine. Biomater Res 2023; 27:67. [PMID: 37420273 DOI: 10.1186/s40824-023-00382-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Accepted: 04/19/2023] [Indexed: 07/09/2023] Open
Abstract
Personalized medicine aims to provide tailored medical treatment that considers the clinical, genetic, and environmental characteristics of patients. iPSCs have attracted considerable attention in the field of personalized medicine; however, the inherent limitations of iPSCs prevent their widespread use in clinical applications. That is, it would be important to develop notable engineering strategies to overcome the current limitations of iPSCs. Such engineering approaches could lead to significant advances in iPSC-based personalized therapy by offering innovative solutions to existing challenges, from iPSC preparation to clinical applications. In this review, we summarize how engineering strategies have been used to advance iPSC-based personalized medicine by categorizing the development process into three distinctive steps: 1) the production of therapeutic iPSCs; 2) engineering of therapeutic iPSCs; and 3) clinical applications of engineered iPSCs. Specifically, we focus on engineering strategies and their implications for each step in the development of iPSC-based personalized medicine.
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Affiliation(s)
- Sangbae Park
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea
- Institute of Nano-Stem Cells Therapeutics, NANOBIOSYSTEM Co, Ltd, Gwangju, 61011, Republic of Korea
| | - Yonghyun Gwon
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea
| | - Shahidul Ahmed Khan
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea
| | - Kyoung-Je Jang
- Department of Bio-Systems Engineering, Institute of Smart Farm, Gyeongsang National University, Jinju, 52828, Republic of Korea.
- Institute of Agriculture & Life Science, Gyeongsang National University, Jinju, 52828, Republic of Korea.
| | - Jangho Kim
- Department of Convergence Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea.
- Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju, 61186, Republic of Korea.
- Interdisciplinary Program in IT-Bio Convergence System, Chonnam National University, Gwangju, 61186, Republic of Korea.
- Institute of Nano-Stem Cells Therapeutics, NANOBIOSYSTEM Co, Ltd, Gwangju, 61011, Republic of Korea.
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15
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Benavides OR, Gibbs HC, White BP, Kaunas R, Gregory CA, Walsh AJ, Maitland KC. Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth. PLoS One 2023; 18:e0282298. [PMID: 36976801 PMCID: PMC10047548 DOI: 10.1371/journal.pone.0282298] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2022] [Accepted: 02/11/2023] [Indexed: 03/29/2023] Open
Abstract
The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures.
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Affiliation(s)
- Oscar R. Benavides
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America
- * E-mail:
| | - Holly C. Gibbs
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America
- Microscopy and Imaging Center, Texas A&M University, College Station, Texas, United States of America
| | - Berkley P. White
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America
| | - Roland Kaunas
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America
| | - Carl A. Gregory
- School of Medicine, Texas A&M Health Science Center, Bryan, Texas, United States of America
| | - Alex J. Walsh
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America
| | - Kristen C. Maitland
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, United States of America
- Microscopy and Imaging Center, Texas A&M University, College Station, Texas, United States of America
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16
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Peng L, Nadal C, Gautrot JE. Growth of mesenchymal stem cells at the surface of silicone, mineral and plant-based oils. Biomed Mater 2023; 18. [PMID: 36808917 DOI: 10.1088/1748-605x/acbdda] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2022] [Accepted: 02/21/2023] [Indexed: 02/23/2023]
Abstract
Bioemulsions are attractive platforms for the expansion of adherent cells in bioreactors. Their design relies on the self-assembly of protein nanosheets at liquid-liquid interfaces, displaying strong interfacial mechanical properties and promoting integrin-mediated cell adhesion. However, most systems developed to date have focused on fluorinated oils, which are unlikely to be accepted for direct implantation of resulting cell products for regenerative medicine, and protein nanosheets self-assembly at other interfaces has not been investigated. In this report, the composition of aliphatic pro-surfactants palmitoyl chloride and sebacoyl chloride, on the assembly kinetics of poly(L-lysine) at silicone oil interfaces and characterisation of ultimate interfacial shear mechanics and viscoelasticity is presented. The impact of the resulting nanosheets on the adhesion of mesenchymal stem cells (MSCs) is investigated via immunostaining and fluorescence microscopy, demonstrating the engagement of the classic focal adhesion-actin cytoskeleton machinery. The ability of MSCs to proliferate at the corresponding interfaces is quantified. In addition, expansion of MSCs at other non-fluorinated oil interfaces, based on mineral and plant-based oils is investigated. Finally, the proof-of-concept of such non-fluorinated oil systems for the formulation of bioemulsions supporting stem cell adhesion and expansion is demonstrated.
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Affiliation(s)
- Lihui Peng
- Institute of Bioengineering, University of London, Mile End Road, London E1 4NS, United Kingdom
- School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom
| | - Clémence Nadal
- Institute of Bioengineering, University of London, Mile End Road, London E1 4NS, United Kingdom
- School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom
| | - Julien E Gautrot
- Institute of Bioengineering, University of London, Mile End Road, London E1 4NS, United Kingdom
- School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, United Kingdom
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17
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Dai J, Huang C, Zhang H, Samuel R, Li Y, Jayaraman A, de Figueiredo P, Han A. Microfluidic Dielectrophoretic Method Enables On-Demand Spatial Arrangement of Bacteria-Encapsulated Agarose Gel Microparticles. Anal Chem 2022; 94:13197-13204. [DOI: 10.1021/acs.analchem.2c02724] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Jing Dai
- Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas 77843, United States
| | - Can Huang
- Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas 77843, United States
| | - Han Zhang
- Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas 77843, United States
| | - Ryan Samuel
- Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas 77843, United States
| | - Yuwen Li
- Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas 77843, United States
| | - Arul Jayaraman
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas 77843, United States
- Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843, United States
| | - Paul de Figueiredo
- Department of Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, Bryan, Texas 77807, United States
- Department of Veterinary Pathobiology, Texas A&M University, College Station, Texas 77843, United States
| | - Arum Han
- Department of Electrical and Computer Engineering, Texas A&M University, College Station, Texas 77843, United States
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas 77843, United States
- Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843, United States
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18
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Hu H, Zhai X, Li W, Ji S, Dong W, Chen W, Wei W, Lu Z. A photo-triggering double cross-linked adhesive, antibacterial, and biocompatible hydrogel for wound healing. iScience 2022; 25:104619. [PMID: 35789848 PMCID: PMC9250026 DOI: 10.1016/j.isci.2022.104619] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Revised: 06/01/2022] [Accepted: 06/09/2022] [Indexed: 11/27/2022] Open
Abstract
Full-thickness wounds, lacking the epidermis and entire dermis and extending into subcutaneous fat, represent a common treatment challenge. Due to the loss of adnexal structures as a source of keratinocytes, full-thickness wounds healing can only be achieved by re-epithelialization from the wound edge and contraction. Here, we developed a hydrogel composed of chitosan methacrylate (CSMA) and o-nitrosobenzaldehyde-modified gelatin (GelNB) for promoting full-thickness wound healing. The CSMA/GelNB (CM/GN) hydrogels exhibited superior mechanical and adhesive properties than that of pure CSMA hydrogel. In vivo experiments confirmed that CM/GN could promote wound healing by generating more hair follicles and mutual blood vessels, high fibroblasts density, and thicker granulation tissue thickness. In addition, reduced secretions of tumor necrosis factor-α (TNF-α) and enhanced secretions of vascular endothelial growth factor (VEGF) could be observed in regenerated tissues after CM/GN treatment. These results suggested that CM/GN hydrogels could be promising candidates to promote wound healing.
The CM/GN hydrogels exhibited tissue adhesive properties CM/GN hydrogel facilitated the proliferation of bone marrow stem cells CM/GN hydrogel efficiently promote full-thickness wound healing More hair follicles and mutual blood vessels were generated during wound healing
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19
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Croughan MS, Giroux D, Agbojo OM, McCain E, Starkweather N, Guerra S, Hashimura Y, Lee B, Jung S. Initial Power Measurements for a Family of Novel
Vertical‐Wheel
Bioreactors. CAN J CHEM ENG 2022. [DOI: 10.1002/cjce.24434] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Affiliation(s)
| | | | | | | | | | | | | | - Brian Lee
- PBS Biotech Inc., Camarillo California USA
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20
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Choudhery MS, Mahmood R. Insight into generation of induced mesenchymal stem cells from induced pluripotent cells. World J Stem Cells 2022; 14:142-145. [PMID: 35126833 PMCID: PMC8788181 DOI: 10.4252/wjsc.v14.i1.142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/09/2021] [Revised: 10/25/2021] [Accepted: 12/23/2021] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem cells (MSCs) have the potential for use in cell-based regenerative therapies. Currently, hundreds of clinical trials are using MSCs for the treatment of various diseases. However, MSCs are low in number in adult tissues; they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures. These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications. As a result, novel methods to generate induced MSCs (iMSCs) from induced pluripotent stem cells have been explored. The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro. In addition, it is important to compare iMSCs with primary MSCs (isolated from adult tissues) in terms of their safety and efficacy. Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.
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Affiliation(s)
- Mahmood S Choudhery
- Department of Biomedical Sciences, King Edward Medical University, Lahore 54000, Punjab, Pakistan
- Department of Genetics and Molecular Biology, University of Health Sciences, Lahore 54600, Punjab, Pakistan.
| | - Ruhma Mahmood
- Stem Cells Laboratory, Allama Iqbal Medical College, Lahore 54000, Punjab, Pakistan
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21
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Rogers RE, Haskell A, White BP, Dalal S, Lopez M, Tahan D, Pan S, Kaur G, Kim H, Barreda H, Woodard SL, Benavides OR, Dai J, Zhao Q, Maitland KC, Han A, Nikolov ZL, Liu F, Lee RH, Gregory CA, Kaunas R. A scalable system for generation of mesenchymal stem cells derived from induced pluripotent cells employing bioreactors and degradable microcarriers. Stem Cells Transl Med 2021; 10:1650-1665. [PMID: 34505405 PMCID: PMC8641084 DOI: 10.1002/sctm.21-0151] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2021] [Revised: 07/21/2021] [Accepted: 08/11/2021] [Indexed: 02/06/2023] Open
Abstract
Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3‐fold expansion of ihMSCs was achieved over 8 days. Complete recovery by 5‐minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost‐effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential.
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Affiliation(s)
- Robert E Rogers
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Andrew Haskell
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Berkley P White
- Department of Biomedical Engineering, Texas A&M University, Emerging Technologies Building, College Station, Texas, USA
| | - Sujata Dalal
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Megan Lopez
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Daniel Tahan
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Simin Pan
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Gagandeep Kaur
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Hyemee Kim
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Heather Barreda
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Susan L Woodard
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA
| | - Oscar R Benavides
- Department of Biomedical Engineering, Texas A&M University, Emerging Technologies Building, College Station, Texas, USA
| | - Jing Dai
- Department of Electrical and Computer Engineering, Texas A&M University, Wisenbaker Engineering Building, College Station, Texas, USA
| | - Qingguo Zhao
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Kristen C Maitland
- Department of Biomedical Engineering, Texas A&M University, Emerging Technologies Building, College Station, Texas, USA
| | - Arum Han
- Department of Biomedical Engineering, Texas A&M University, Emerging Technologies Building, College Station, Texas, USA.,Department of Electrical and Computer Engineering, Texas A&M University, Wisenbaker Engineering Building, College Station, Texas, USA
| | - Zivko L Nikolov
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA.,Biological and Agricultural Engineering, Texas A&M University, Scoates Hall, College Station, Texas, USA
| | - Fei Liu
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Ryang Hwa Lee
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Carl A Gregory
- Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College of Medicine, Bryan, Texas, USA
| | - Roland Kaunas
- Department of Biomedical Engineering, Texas A&M University, Emerging Technologies Building, College Station, Texas, USA
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