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Maldonado VV, Jensen H, Barnes CL, Samsonraj RM. Phenotypic changes associated with continuous long term in vitro expansion of bone marrow-derived mesenchymal stem cells. Biochimie 2025; 234:62-75. [PMID: 40209891 DOI: 10.1016/j.biochi.2025.04.002] [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: 01/28/2025] [Revised: 03/26/2025] [Accepted: 04/07/2025] [Indexed: 04/12/2025]
Abstract
In vitro expansion of mesenchymal stem cells is necessary to obtain a higher cell number for clinical applications. However, long-term expansion can produce significant phenotypic changes on these cells, decreasing their therapeutic utility. Therefore, understanding the phenotypic changes that long-term expansion triggers in mesenchymal stem cells will allow for better and more consistent cell therapy results. Here, we evaluate the phenotypic changes caused by continuous passaging through colony forming unit-fibroblast assay, senescence beta-galactosidase staining, morphology examination, secretome analysis, surface marker expression, protein quantification, osteogenic and adipogenic differentiation, and CD4+ T lymphocyte immunosuppressive potential. Long-term in vitro culture decreases mesenchymal stem cell osteogenic potential and self-renewal, increases cell size, and senescence, but does not consistently affect adipogenic differentiation. Surface marker expression remains similar for positive and negative markers, while secretory phenotype shifts with decreased p14ARF, MMP-3, p21 Waf1/Cip1,ENA-78, GCP-2, GROα, IL-3, IL-7, IL-8, RANTES, TNFβ, and VEGF-A expression, and increased p53, p16 INK4a, MCP-1, and SDF-1 expression. Immunomodulatory potential remains unchanged. These findings can help better understand the phenotypic changes that mesenchymal stem cells undergo while expanded in vitro.
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Affiliation(s)
- Vitali V Maldonado
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, 72701, USA
| | - Hanna Jensen
- Department of Surgery, University of Arkansas for Medical Sciences, Northwest Regional Campus, Fayetteville, AR, 72701, USA
| | - C Lowry Barnes
- Department of Orthopedic Surgery, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA
| | - Rebekah M Samsonraj
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, AR, 72701, USA; Department of Orthopedic Surgery, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA.
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2
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Riazuelo L, Planat-Bénard V, Vinel A, Laurencin S, Casteilla L, Kémoun P, Marty M, Monsarrat P. Acceptability of Allogeneic Mesenchymal Stromal Cell-Based Tissue Engineering for the Treatment of Periodontitis: A Qualitative Study in France. Int Dent J 2025; 75:840-848. [PMID: 39245621 PMCID: PMC11976543 DOI: 10.1016/j.identj.2024.07.1208] [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: 05/27/2024] [Revised: 07/15/2024] [Accepted: 07/20/2024] [Indexed: 09/10/2024] Open
Abstract
INTRODUCTION AND AIMS Periodontitis, the main cause of tooth loss in adults, is a public health concern; its incidence increases with age, and its prevalence increases with increasing life expectancy of the population. Innovative therapies such as cell therapy represent promising future solutions for guided tissue regeneration. However, these therapies may be associated with fears and mistrust from the general public. The aim of this study was to estimate the acceptability of an advanced therapy medicinal product combining allogeneic mesenchymal stromal cells from adipose tissue with a natural fibrin hydrogel in the treatment of periodontitis. METHODS The methodology was based on a qualitative study conducted through semi-structured interviews with patients followed for periodontitis in the Oral Medicine Department of the Toulouse University Hospital, Toulouse, France. Qualitative studies are essential methodologies to understand the patterns of health behaviours, describe illness experiences, and design health interventions in a humanistic and person-centred way of discovering. RESULTS Eleven interviews (with 4 men and 7 women) were required to reach thematic saturation. Analysis allowed 4 main themes to emerge: (1) perception of new treatments, science, and caregivers; (2) conditions that the treatment must meet; (3) patient perception of the disease; and (4) factors related to the content of the treatment. CONCLUSIONS Patients find cell therapy for periodontitis to be acceptable. If they express a need to be informed about the benefit/risk ratio, they are not particularly worried about side effects of the treatment, for either allogeneic or blood-derived products. Periodontitis is a prototypical model of chronic inflammatory pathology and is multitissular, with hard- and soft-tissue lesions. In a patient-centred approach, the success of cell therapy will require a bilateral, informed decision, taking into account potential therapeutic effectiveness and patient expectations for regeneration.
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Affiliation(s)
- Lucas Riazuelo
- Oral Medicine Department and CHU de Toulouse, Toulouse Institute of Oral Medicine and Science, Toulouse, France
| | - Valérie Planat-Bénard
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Université P. Sabatier, Toulouse, France
| | - Alexia Vinel
- Oral Medicine Department and CHU de Toulouse, Toulouse Institute of Oral Medicine and Science, Toulouse, France; I2MC, INSERM UMR 1297, University of Toulouse III, Toulouse, France
| | - Sara Laurencin
- Oral Medicine Department and CHU de Toulouse, Toulouse Institute of Oral Medicine and Science, Toulouse, France; Center for Epidemiology and Research in POPulation Health (CERPOP), UMR 1295, Paul Sabatier University, Toulouse, France
| | - Louis Casteilla
- RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Université P. Sabatier, Toulouse, France
| | - Philippe Kémoun
- Oral Medicine Department and CHU de Toulouse, Toulouse Institute of Oral Medicine and Science, Toulouse, France; RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Université P. Sabatier, Toulouse, France
| | - Mathieu Marty
- Oral Medicine Department and CHU de Toulouse, Toulouse Institute of Oral Medicine and Science, Toulouse, France; LIRDEF, Faculty of Educational Sciences, Paul Valery University, Montpellier, France
| | - Paul Monsarrat
- Oral Medicine Department and CHU de Toulouse, Toulouse Institute of Oral Medicine and Science, Toulouse, France; RESTORE Research Center, Université de Toulouse, INSERM, CNRS, EFS, ENVT, Université P. Sabatier, Toulouse, France; Artificial and Natural Intelligence Toulouse Institute ANITI, Toulouse, France.
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3
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Mahmod AI, Govindaraju K, Lokanathan Y, Said NABM, Ibrahim B. Exploring the Potential of Stem Cells in Modulating Gut Microbiota and Managing Hypertension. Stem Cells Dev 2025; 34:99-116. [PMID: 39836384 DOI: 10.1089/scd.2024.0195] [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: 01/22/2025] Open
Abstract
Hypertension, commonly known as high blood pressure, is a significant health issue that increases the risk of cardiovascular diseases, stroke, and renal failure. This condition broadly encompasses both primary and secondary forms. Despite extensive research, the underlying mechanisms of systemic arterial hypertension-particularly primary hypertension, which has no identifiable cause and is affected by genetic and lifestyle agents-remain complex and not fully understood. Recent studies indicate that an imbalance in gut microbiota, referred to as dysbiosis, may promote hypertension, affecting blood pressure regulation through metabolites such as short-chain fatty acids and trimethylamine N-oxide. Current antihypertensive medications face limitations, including resistance and adherence issues, highlighting the need for novel therapeutic approaches. Stem cell therapy, an emerging field in regenerative medicine, shows promise in addressing these challenges. Stem cells, with mesenchymal stem cells being a prime example, have regenerative, anti-inflammatory, and immunomodulatory properties. Emerging research indicates that stem cells can modulate gut microbiota, reduce inflammation, and improve vascular health, potentially aiding in blood pressure management. Research has shown the positive impact of stem cells on gut microbiota in various disorders, suggesting their potential therapeutic role in treating hypertension. This review synthesizes the recent studies on the complex interactions between gut microbiota, stem cells, and systemic arterial hypertension. By offering a thorough analysis of the current literature, it highlights key insights, uncovers critical gaps, and identifies emerging trends that will inform and guide future investigations in this rapidly advancing field.
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Affiliation(s)
- Asma Ismail Mahmod
- Department of Pharmaceutical Life Sciences, Faculty of Pharmacy, University of Malaya, Kuala Lumpur, Malaysia
| | - Kayatri Govindaraju
- Department of Pharmaceutical Life Sciences, Faculty of Pharmacy, University of Malaya, Kuala Lumpur, Malaysia
| | - Yogeswaran Lokanathan
- Department of Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
- Advance Bioactive Materials-Cells UKM Research Group, Universiti Kebangsaan Malaysia, Bangi, Malaysia
| | - Nur Akmarina B M Said
- Department of Pharmaceutical Life Sciences, Faculty of Pharmacy, University of Malaya, Kuala Lumpur, Malaysia
| | - Baharudin Ibrahim
- Department of Clinical Pharmacy and Pharmacy Practices, Faculty of Pharmacy, University Malaya, Kuala Lumpur, Malaysia
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Nunes OBDS, Buranello TW, Farias FDA, Rosero J, Recchia K, Bressan FF. Can cell-cultured meat from stem cells pave the way for sustainable alternative protein? Curr Res Food Sci 2025; 10:100979. [PMID: 40040753 PMCID: PMC11878651 DOI: 10.1016/j.crfs.2025.100979] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Revised: 01/09/2025] [Accepted: 01/18/2025] [Indexed: 03/06/2025] Open
Abstract
As the global population grows, the demand for food and animal-derived products rises significantly, posing a notable challenge to the progress of society in general. Alternative protein production may adequately address such a challenge, and cell-based meat production emerges as a promising solution. This review investigates methodologies for in vitro myogenesis and adipogenesis from stem cells (adult, embryonic, or induced pluripotent stem cells - iPSCs) across different animal species, as well as the remaining challenges for scalability, the possibility of genetic modification, along with safety concerns regarding the commercialization of cell-cultured meat. Regarding such complexities, interdisciplinary approaches will be vital for assessing the potential of cell-cultured meat as a sustainable protein source, mimicking the sensory and nutritional attributes of conventional livestock meat whilst meeting the demands of a growing global population while mitigating environmental impacts.
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Affiliation(s)
- Octavio Bignardi da Silva Nunes
- Department of Food Engineering, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
| | - Tiago Willian Buranello
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
| | - Fabiana de Andrade Farias
- Department of Food Engineering, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
| | - Jenyffer Rosero
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
| | - Kaiana Recchia
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
| | - Fabiana Fernandes Bressan
- Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engineering, University of São Paulo 13635-000, Pirassununga, SP, Brazil
- Postgraduate Program in Anatomy of Domestic and Wils Species, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo 01001-010, SP, Brazil
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5
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De Miguel MP, Cadenas-Martin M, Stokking M, Martin-Gonzalez AI. Biomedical Application of MSCs in Corneal Regeneration and Repair. Int J Mol Sci 2025; 26:695. [PMID: 39859409 PMCID: PMC11766311 DOI: 10.3390/ijms26020695] [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: 11/29/2024] [Revised: 01/10/2025] [Accepted: 01/11/2025] [Indexed: 01/27/2025] Open
Abstract
The World Health Organization estimates that approximately 285 million people suffer from visual impairments, around 5% of which are caused by corneal pathologies. Currently, the most common clinical treatment consists of a corneal transplant (keratoplasty) from a human donor. However, worldwide demand for donor corneas amply exceeds the available supply. Lamellar keratoplasty (transplantation replacement of only one of the three layers of the cornea) is partially solving the problem of cornea undersupply. Obviously, cell therapy applied to every one of these layers will expand current therapeutic options, reducing the cost of ophthalmological interventions and increasing the effectiveness of surgery. Mesenchymal stem cells (MSCs) are adult stem cells with the capacity for self-renewal and differentiation into different cell lineages. They can be obtained from many human tissues, such as bone marrow, umbilical cord, adipose tissue, dental pulp, skin, and cornea. Their ease of collection and advantages over embryonic stem cells or induced pluripotent stem cells make them a very practical source for experimental and potential clinical applications. In this review, we focus on recent advances using MSCs from different sources to replace the damaged cells of the three corneal layers, at both the preclinical and clinical levels for specific corneal diseases.
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Affiliation(s)
- Maria P. De Miguel
- Cell Engineering Laboratory, La Paz University Hospital Health Research Institute, IdiPAZ, 28046 Madrid, Spain; (M.C.-M.); (M.S.); (A.I.M.-G.)
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Gurel M, Rathod N, Cabrera LY, Voyton S, Yeo M, Ozogul F, Ozbolat IT. A narrative review: 3D bioprinting of cultured muscle meat and seafood products and its potential for the food industry. Trends Food Sci Technol 2024; 152:104670. [PMID: 39309029 PMCID: PMC11412102 DOI: 10.1016/j.tifs.2024.104670] [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] [Indexed: 09/25/2024]
Abstract
The demand for meat and seafood products has been globally increasing for decades. To address the environmental, social, and economic impacts of this trend, there has been a surge in the development of three-dimensional (3D) food bioprinting technologies for lab-grown muscle food products and their analogues. This innovative approach is a sustainable solution to mitigate the environmental risks associated with climate change caused by the negative impacts of indiscriminative livestock production and industrial aquaculture. This review article explores the adoption of 3D bioprinting modalities to manufacture lab-grown muscle food products and their associated technologies, cells, and bioink formulations. Additionally, various processing techniques, governing the characteristics of bioprinted food products, nutritional compositions, and safety aspects as well as its relevant ethical and social considerations, were discussed. Although promising, further research and development is needed to meet standards and translate into several industrial areas, such as the food and renewable energy industries. In specific, optimization of animal cell culture conditions, development of serum-free media, and bioreactor design are essential to eliminate the risk factors but achieve the unique nutritional requirements and consumer acceptance. In short, the advancement of 3D bioprinting technologies holds great potential for transforming the food industry, but achieving widespread adoption will require continued innovation, rigorous research, and adherence to ethical standards to ensure safety, nutritional quality, and consumer acceptance.
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Affiliation(s)
- Mediha Gurel
- Biotechnology Research and Application Center, Cukurova University, 01330, Adana, Turkey
- Electronic and Automation Department, Bitlis Eren University, Bitlis, 13000, Turkey
| | - Nikheel Rathod
- Department of Post Harvest Management of Meat, Poultry and Fish, Post-graduate Institute of Post-harvest Management (Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth), Raigad, 402116, India
| | - Laura Y. Cabrera
- Rock Ethics Institute, Penn State University, University Park, 16802, USA
- Engineering Science and Mechanics Department, Penn State University, University Park, PA, 16802, USA
| | - Stephen Voyton
- Engineering Science and Mechanics Department, Penn State University, University Park, PA, 16802, USA
| | - Miji Yeo
- Engineering Science and Mechanics Department, Penn State University, University Park, PA, 16802, USA
- The Huck Institutes of the Life Sciences, Penn State University; University Park, PA, 16802, USA
| | - Fatih Ozogul
- Biotechnology Research and Application Center, Cukurova University, 01330, Adana, Turkey
| | - Ibrahim T. Ozbolat
- Engineering Science and Mechanics Department, Penn State University, University Park, PA, 16802, USA
- The Huck Institutes of the Life Sciences, Penn State University; University Park, PA, 16802, USA
- Department of Biomedical Engineering, Penn State University, University Park, PA 16802, USA
- Materials Research Institute, Penn State University, University Park, PA, 16802, USA
- Department of Neurosurgery, Pennsylvania State College of Medicine, Hershey, PA, 17033, USA
- Penn State Cancer Institute, Penn State University, Hershey, PA, 17033, USA
- Department of Medical Oncology, Cukurova University, Adana, 01130, Turkey
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7
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Wang J, Zhang M, Wang H. Emerging Landscape of Mesenchymal Stem Cell Senescence Mechanisms and Implications on Therapeutic Strategies. ACS Pharmacol Transl Sci 2024; 7:2306-2325. [PMID: 39144566 PMCID: PMC11320744 DOI: 10.1021/acsptsci.4c00284] [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: 05/15/2024] [Revised: 07/05/2024] [Accepted: 07/09/2024] [Indexed: 08/16/2024]
Abstract
Mesenchymal stem cells (MSCs) hold significant promise for regenerative medicine and tissue engineering due to their unique multipotent differentiation ability and immunomodulatory properties. MSC therapy is widely discussed and utilized in clinical treatment. However, during both in vitro expansion and in vivo transplantation, MSCs are prone to senescence, an irreversible growth arrest characterized by morphological, gene expression, and functional changes in genomic regulation. The microenvironment surrounding MSCs plays a crucial role in modulating their senescence phenotype, influenced by factors such as hypoxia, inflammation, and aging status. Numerous strategies targeting MSC senescence have been developed, including senolytics and senomorphic agents, antioxidant and exosome therapies, mitochondrial transfer, and niche modulation. Novel approaches addressing replicative senescence have also emerged. This paper comprehensively reviews the current molecular manifestations of MSC senescence, addresses the environmental impact on senescence, and highlights potential therapeutic strategies to mitigate senescence in MSC-based therapies. These insights aim to enhance the efficacy and understanding of MSC therapies.
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Affiliation(s)
- Jing Wang
- Department
of Cellular and Molecular Medicine, University
of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, United States
| | - Muqing Zhang
- Institute
of Cell Engineering, School of Medicine, Johns Hopkins University, Baltimore, Maryland, 21215, United States
| | - Hu Wang
- Institute
of Cell Engineering, School of Medicine, Johns Hopkins University, Baltimore, Maryland, 21215, United States
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8
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Tsujimoto M, Moon S, Ito Y. Effect of conditioned media on the angiogenic activity of mesenchymal stem cells. J Biosci Bioeng 2024; 138:163-170. [PMID: 38821758 DOI: 10.1016/j.jbiosc.2024.04.004] [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: 12/26/2023] [Revised: 04/16/2024] [Accepted: 04/17/2024] [Indexed: 06/02/2024]
Abstract
Mesenchymal stem cells (MSCs) are promising candidates for use in novel cell therapies, although such live cell products are highly complex compared with traditional drugs. For example, difficulties such as the control of manufacturing conditions hinder the manufacture of stable cell populations that maintain their therapeutic potency. Here, assuming that medium selection significantly affects cell potency, we focused on the culture media as a critical manufacturing factor influencing the therapeutic efficacy of MSCs. We therefore performed a tube formation assay to quantify the angiogenic activities of conditioned media used to culture human umbilical vein endothelial cells compared with unconditioned media. Comprehensive molecular genetic analysis using microarrays was applied to determine the effects of these media on signal transduction pathways. We found that activation of the vascular endothelial growth factor (VEGF) signaling pathway differed, and that VEGF concentration was dependent on the composition of the conditioned media. These results indicate that the activation level of cell signaling pathways which contribute to therapeutic efficacy may vary depending on the media components affecting MSCs during their cultivation. Moreover, they indicate that therapeutic efficacy will likely depend on how cells are handled during manufacture. These findings will enhance our understanding of the quality control measures required to ensure the efficacy and safety of cell therapy products.
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Affiliation(s)
- Mami Tsujimoto
- Faculty of Life and Environmental Sciences (Bioindustrial Sciences), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8972, Japan
| | - SongHo Moon
- Faculty of Life and Environmental Sciences (Bioindustrial Sciences), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8972, Japan
| | - Yuzuru Ito
- Faculty of Life and Environmental Sciences (Bioindustrial Sciences), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8972, Japan; Life Science Development Department, Frontier Business Division, Chiyoda Corporation, 13 Moriya-cho 3-chome, Kanagawa-ku, Yokohama 221-0022, Japan.
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9
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Kılıç P, Özdemir C, Coşar B, Savran BN, Sarıkaya A, Sargon B, Toprakkale A, Songür İ, Kandemir Seçgin Ö, Akpınar Oktar P, Çetindağ EN, Yurtsever Sarıca D, Taşdelen S, Ezer Ü, Kürekçi AE, Gürman G. Upstream Process Protocol for MSCs Isolated from Different Human-Based Tissue Origins. Methods Mol Biol 2024. [PMID: 38967911 DOI: 10.1007/7651_2024_553] [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: 07/06/2024]
Abstract
This chapter introduces the increasing significance of mesenchymal stromal/stem cell (MSC) production in regenerative medicine and cellular therapeutics, outlines the growing interest in MSCs for various medical applications, and highlights their potential in advanced therapy medicinal products (ATMPs) and the advancements in cell culture technologies that have facilitated large-scale MSC production under Good Manufacturing Practices (GMP), ensuring safety and efficacy. This chapter describes an optimized upstream protocol for laboratory-scale MSC production from different tissue sources. This protocol, conducted in flasks, controls critical parameters and lays the foundation for downstream processing to generate ATMPs. This comprehensive approach underscores the potential of MSCs in clinical applications and the importance of tailored production processes.
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Affiliation(s)
- Pelin Kılıç
- Department of Stem Cells and Regenerative Medicine, Stem Cell Institute, Ankara University, Ankara, Türkiye.
- HücreCELL® Biotechnology Development and Commerce, Inc., Ankara, Türkiye.
| | - Cansu Özdemir
- Center for Stem Cell Research and Development (PEDI-STEM), Hacettepe University, Ankara, Türkiye
- Department of Stem Cell Sciences, Hacettepe University Graduate School of Health Sciences, Ankara, Türkiye
| | - Begüm Coşar
- HücreCELL® Biotechnology Development and Commerce, Inc., Ankara, Türkiye
- Department of Molecular Biology and Genetics, Institute of Science, Başkent University, Ankara, Türkiye
| | - Büşra Nigar Savran
- HücreCELL® Biotechnology Development and Commerce, Inc., Ankara, Türkiye
| | | | | | | | | | | | | | - Elif NazIı Çetindağ
- LÖSEV LÖSANTE Hospital, Ankara, Türkiye
- Ankara University, Graduate School of Health Sciences, Urogynecology Doctorate Program, Ankara, Türkiye
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10
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Lee NK, Chang JW. Manufacturing Cell and Gene Therapies: Challenges in Clinical Translation. Ann Lab Med 2024; 44:314-323. [PMID: 38361427 PMCID: PMC10961620 DOI: 10.3343/alm.2023.0382] [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: 09/26/2023] [Revised: 11/24/2023] [Accepted: 01/29/2024] [Indexed: 02/17/2024] Open
Abstract
The safety and efficacy of both cell and gene therapies have been demonstrated in numerous preclinical and clinical trials. Chimeric antigen receptor T (CAR-T) cell therapy, which leverages the technologies of both cell and gene therapies, has also shown great promise for treating various cancers. Advancements in pertinent fields have also highlighted challenges faced while manufacturing cell and gene therapy products. Potential problems and obstacles must be addressed to ease the clinical translation of individual therapies. Literature reviews of representative cell-based, gene-based, and cell-based gene therapies with regard to their general manufacturing processes, the challenges faced during manufacturing, and QC specifications are limited. We review the general manufacturing processes of cell and gene therapies, including those involving mesenchymal stem cells, viral vectors, and CAR-T cells. The complexities associated with the manufacturing processes and subsequent QC/validation processes may present challenges that could impede the clinical progression of the products. This article addresses these potential challenges. Further, we discuss the use of the manufacturing model and its impact on cell and gene therapy.
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Affiliation(s)
- Na Kyung Lee
- Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, Seoul, Korea
- Cell and Gene Therapy Institute (CGTI), Research Institute for Future Medicine, Samsung Medical Center, Seoul, Korea
| | - Jong Wook Chang
- Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology (SAIHST), Sungkyunkwan University, Seoul, Korea
- Cell and Gene Therapy Institute (CGTI), Research Institute for Future Medicine, Samsung Medical Center, Seoul, Korea
- Cell and Gene Therapy Institute, ENCell Co. Ltd., Seoul, Korea
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11
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Winston T, Song Y, Shi H, Yang J, Alsudais M, Kontaridis MI, Wu Y, Gaborski TR, Meng Q, Cooney RN, Ma Z. Lineage-Specific Mesenchymal Stromal Cells Derived from Human iPSCs Showed Distinct Patterns in Transcriptomic Profile and Extracellular Vesicle Production. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2308975. [PMID: 38757640 PMCID: PMC11267277 DOI: 10.1002/advs.202308975] [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: 11/21/2023] [Revised: 04/16/2024] [Indexed: 05/18/2024]
Abstract
Over the past decades, mesenchymal stromal cells (MSCs) have been extensively investigated as a potential therapeutic cell source for the treatment of various disorders. Differentiation of MSCs from human induced pluripotent stem cells (iMSCs) has provided a scalable approach for the biomanufacturing of MSCs and related biological products. Although iMSCs shared typical MSC markers and functions as primary MSCs (pMSCs), there is a lack of lineage specificity in many iMSC differentiation protocols. Here, a stepwise hiPSC-to-iMSC differentiation method is employed via intermediate cell stages of neural crest and cytotrophoblast to generate lineage-specific MSCs with varying differentiation efficiencies and gene expression. Through a comprehensive comparison between early developmental cell types (hiPSCs, neural crest, and cytotrophoblast), two lineage-specific iMSCs, and six source-specific pMSCs, are able to not only distinguish the transcriptomic differences between MSCs and early developmental cells, but also determine the transcriptomic similarities of iMSC subtypes to postnatal or perinatal pMSCs. Additionally, it is demonstrated that different iMSC subtypes and priming conditions affected EV production, exosomal protein expression, and cytokine cargo.
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Affiliation(s)
- Tackla Winston
- Department of Biomedical & Chemical EngineeringSyracuse University329 Link HallSyracuseNY13244USA
- BioInspired Institute for Materials and Living SystemsSyracuse University318 Bowne HallSyracuseNY13244USA
| | - Yuanhui Song
- Department of Biomedical & Chemical EngineeringSyracuse University329 Link HallSyracuseNY13244USA
- BioInspired Institute for Materials and Living SystemsSyracuse University318 Bowne HallSyracuseNY13244USA
| | - Huaiyu Shi
- Department of Biomedical & Chemical EngineeringSyracuse University329 Link HallSyracuseNY13244USA
- BioInspired Institute for Materials and Living SystemsSyracuse University318 Bowne HallSyracuseNY13244USA
| | - Junhui Yang
- Department of Biomedical & Chemical EngineeringSyracuse University329 Link HallSyracuseNY13244USA
- BioInspired Institute for Materials and Living SystemsSyracuse University318 Bowne HallSyracuseNY13244USA
| | - Munther Alsudais
- Departments of Biomedical and Chemical EngineeringRochester Institute of TechnologyOne Lomb Memorial DriveRochesterNY14623USA
| | - Maria I. Kontaridis
- Department of Biomedical Research and Translational MedicineMasonic Medical Research Institute2150 Bleecker StreetUticaNY13501USA
- Department of Medicine, Division of Cardiology, Beth Israel Deaconess Medical CenterHarvard Medical School330 Brookline AveBostonMA02215USA
- Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBuilding C, 240 Longwood AveBostonMA02115USA
| | - Yaoying Wu
- Department of Biomedical & Chemical EngineeringSyracuse University329 Link HallSyracuseNY13244USA
- BioInspired Institute for Materials and Living SystemsSyracuse University318 Bowne HallSyracuseNY13244USA
- Department of Microbiology & ImmunologySUNY Upstate Medical University766 Irving AvenueSyracuseNY13210USA
| | - Thomas R. Gaborski
- Departments of Biomedical and Chemical EngineeringRochester Institute of TechnologyOne Lomb Memorial DriveRochesterNY14623USA
| | - Qinghe Meng
- Department of SurgeryState University of New York Upstate Medical University750 East Adams StreetSyracuseNY13210USA
- Sepsis Interdisciplinary Research CenterState University of New York Upstate Medical University766 Irving AvenueSyracuseNY13210USA
| | - Robert N. Cooney
- Department of SurgeryState University of New York Upstate Medical University750 East Adams StreetSyracuseNY13210USA
- Sepsis Interdisciplinary Research CenterState University of New York Upstate Medical University766 Irving AvenueSyracuseNY13210USA
| | - Zhen Ma
- Department of Biomedical & Chemical EngineeringSyracuse University329 Link HallSyracuseNY13244USA
- BioInspired Institute for Materials and Living SystemsSyracuse University318 Bowne HallSyracuseNY13244USA
- Department of BiologySyracuse University107 College PlSyracuseNY13210USA
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12
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Li J, Liu Y, Zhang R, Yang Q, Xiong W, He Y, Ye Q. Insights into the role of mesenchymal stem cells in cutaneous medical aesthetics: from basics to clinics. Stem Cell Res Ther 2024; 15:169. [PMID: 38886773 PMCID: PMC11184751 DOI: 10.1186/s13287-024-03774-5] [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: 02/08/2024] [Accepted: 05/27/2024] [Indexed: 06/20/2024] Open
Abstract
With the development of the economy and the increasing prevalence of skin problems, cutaneous medical aesthetics are gaining more and more attention. Skin disorders like poor wound healing, aging, and pigmentation have an impact not only on appearance but also on patients with physical and psychological issues, and even impose a significant financial burden on families and society. However, due to the complexities of its occurrence, present treatment options cannot produce optimal outcomes, indicating a dire need for new and effective treatments. Mesenchymal stem cells (MSCs) and their secretomics treatment is a new regenerative medicine therapy that promotes and regulates endogenous stem cell populations and/or replenishes cell pools to achieve tissue homeostasis and regeneration. It has demonstrated remarkable advantages in several skin-related in vivo and in vitro investigations, aiding in the improvement of skin conditions and the promotion of skin aesthetics. As a result, this review gives a complete description of recent scientific breakthroughs in MSCs for skin aesthetics and the limitations of their clinical applications, aiming to provide new ideas for future research and clinical transformation.
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Affiliation(s)
- Junyi Li
- Center of Regenerative Medicine, Renmin Hospital of Wuhan University, Wuhan, 430060, China
| | - Ye Liu
- Center of Regenerative Medicine, Renmin Hospital of Wuhan University, Wuhan, 430060, China
| | - Rui Zhang
- Center of Regenerative Medicine, Renmin Hospital of Wuhan University, Wuhan, 430060, China
| | - Qianyu Yang
- Center of Regenerative Medicine, Renmin Hospital of Wuhan University, Wuhan, 430060, China
| | - Wei Xiong
- Center of Regenerative Medicine, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
| | - Yan He
- Institute of Regenerative and Translational Medicine, Tianyou Hospital, Wuhan University of Science and Technology, Wuhan, 430030, China.
| | - Qingsong Ye
- Center of Regenerative Medicine, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
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13
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Marquez-Curtis LA, Elliott JAW. Mesenchymal stromal cells derived from various tissues: Biological, clinical and cryopreservation aspects: Update from 2015 review. Cryobiology 2024; 115:104856. [PMID: 38340887 DOI: 10.1016/j.cryobiol.2024.104856] [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: 11/28/2023] [Revised: 01/26/2024] [Accepted: 01/30/2024] [Indexed: 02/12/2024]
Abstract
Mesenchymal stromal cells (MSCs) have become one of the most investigated and applied cells for cellular therapy and regenerative medicine. In this update of our review published in 2015, we show that studies continue to abound regarding the characterization of MSCs to distinguish them from other similar cell types, the discovery of new tissue sources of MSCs, and the confirmation of their properties and functions that render them suitable as a therapeutic. Because cryopreservation is widely recognized as the only technology that would enable the on-demand availability of MSCs, here we show that although the traditional method of cryopreserving cells by slow cooling in the presence of 10% dimethyl sulfoxide (Me2SO) continues to be used by many, several novel MSC cryopreservation approaches have emerged. As in our previous review, we conclude from these recent reports that viable and functional MSCs from diverse tissues can be recovered after cryopreservation using a variety of cryoprotectants, freezing protocols, storage temperatures, and periods of storage. We also show that for logistical reasons there are now more studies devoted to the cryopreservation of tissues from which MSCs are derived. A new topic included in this review covers the application in COVID-19 of MSCs arising from their immunomodulatory and antiviral properties. Due to the inherent heterogeneity in MSC populations from different sources there is still no standardized procedure for their isolation, identification, functional characterization, cryopreservation, and route of administration, and not likely to be a "one-size-fits-all" approach in their applications in cell-based therapy and regenerative medicine.
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Affiliation(s)
- Leah A Marquez-Curtis
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada, T6G 1H9; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada, T6G 1C9
| | - Janet A W Elliott
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada, T6G 1H9; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada, T6G 1C9.
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14
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Pignatti E, Maccaferri M, Pisciotta A, Carnevale G, Salvarani C. A comprehensive review on the role of mesenchymal stromal/stem cells in the management of rheumatoid arthritis. Expert Rev Clin Immunol 2024; 20:463-484. [PMID: 38163928 DOI: 10.1080/1744666x.2023.2299729] [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: 09/18/2023] [Accepted: 12/21/2023] [Indexed: 01/03/2024]
Abstract
INTRODUCTION Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease with systemic manifestations. Although the success of immune modulatory drug therapy is considerable, about 40% of patients do not respond to treatment. Mesenchymal stromal/stem cells (MSCs) have been demonstrated to have therapeutic potential for inflammatory diseases. AREAS COVERED This review provides an update on RA disease and on pre-clinical and clinical studies using MSCs from bone marrow, umbilical cord, adipose tissue, and dental pulp, to regulate the immune response. Moreover, the clinical use, safety, limitations, and future perspective of MSCs in RA are discussed. Using the PubMed database and ClincalTrials.gov, peer-reviewed full-text papers, abstracts and clinical trials were identified from 1985 through to April 2023. EXPERT OPINION MSCs demonstrated a satisfactory safety profile and potential for clinical efficacy. However, it is mandatory to deepen the investigations on how MSCs affect the proinflammatory deregulated RA patients' cells. MSCs are potentially good candidates for severe RA patients not responding to conventional therapies but a long-term follow-up after stem cells treatment and standardized protocols are needed. Future research should focus on well-designed multicenter randomized clinical trials with adequate sample sizes and properly selected patients satisfying RA criteria for a valid efficacy evaluation.
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Affiliation(s)
- Elisa Pignatti
- Department of Surgery, Medicine Dentistry and Morphological Sciences with Interest in Transplant, Oncological and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy
| | - Monia Maccaferri
- Department of Surgery, Medicine Dentistry and Morphological Sciences with Interest in Transplant, Oncological and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy
| | - Alessandra Pisciotta
- Department of Surgery, Medicine Dentistry and Morphological Sciences with Interest in Transplant, Oncological and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy
| | - Gianluca Carnevale
- Department of Surgery, Medicine Dentistry and Morphological Sciences with Interest in Transplant, Oncological and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy
| | - Carlo Salvarani
- Department of Surgery, Medicine Dentistry and Morphological Sciences with Interest in Transplant, Oncological and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy
- Rheumatology Unit, Azienda USL-IRCCS di Reggio Emilia, Reggio Emilia, Italy
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15
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López-Fernández A, Codinach M, Coca MI, Prat-Vidal C, Castaño J, Torrents S, Aran G, Rodríguez L, Querol S, Vives J. Comparability exercise of critical quality attributes of clinical-grade human mesenchymal stromal cells from the Wharton's jelly: single-use stirred tank bioreactors versus planar culture systems. Cytotherapy 2024; 26:418-426. [PMID: 37715777 DOI: 10.1016/j.jcyt.2023.08.008] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2023] [Revised: 08/14/2023] [Accepted: 08/21/2023] [Indexed: 09/18/2023]
Abstract
BACKGROUND AIMS The increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice-compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. METHODS MSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. RESULTS After 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 × 108 ± 1.07 × 107 cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 × 108 ± 2.36 × 108 cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. CONCLUSIONS In summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.
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Affiliation(s)
- Alba López-Fernández
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain; Musculoskeletal Tissue Engineering Group, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain.
| | - Margarita Codinach
- Laboratori Cel·lular, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Maria Isabel Coca
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Cristina Prat-Vidal
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Julio Castaño
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Sílvia Torrents
- Laboratori Cel·lular, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Gemma Aran
- Laboratori Cel·lular, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Luciano Rodríguez
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Sergi Querol
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Joaquim Vives
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits (BST), Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain; Musculoskeletal Tissue Engineering Group, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain; Departament de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain.
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16
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Puistola P, Kethiri A, Nurminen A, Turkki J, Hopia K, Miettinen S, Mörö A, Skottman H. Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering. ACS APPLIED MATERIALS & INTERFACES 2024; 16:15761-15772. [PMID: 38513048 PMCID: PMC10995904 DOI: 10.1021/acsami.3c17803] [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: 11/28/2023] [Revised: 01/23/2024] [Accepted: 01/23/2024] [Indexed: 03/23/2024]
Abstract
Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human- or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC-CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC-CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.
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Affiliation(s)
- Paula Puistola
- Eye
Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
| | - Abhinav Kethiri
- Eye
Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
| | - Antti Nurminen
- Eye
Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
| | - Johannes Turkki
- Eye
Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
| | - Karoliina Hopia
- Eye
Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
| | - Susanna Miettinen
- Adult
Stem Cell Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
- Tays
Research Services, Wellbeing Services County of Pirkanmaa, Tampere University Hospital, 33520 Tampere, Finland
| | - Anni Mörö
- Eye
Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
| | - Heli Skottman
- Eye
Regeneration Group, Faculty of Medicine and Health Technology, Tampere University, Tampere 33520, Finland
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17
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Lee J, Kim H, Lim HR, Kim YS, Hoang TTT, Choi J, Jeong GJ, Kim H, Herbert R, Soltis I, Kim KR, Lee SH, Kwon Y, Lee Y, Jang YC, Yeo WH. Large-scale smart bioreactor with fully integrated wireless multivariate sensors and electronics for long-term in situ monitoring of stem cell culture. SCIENCE ADVANCES 2024; 10:eadk6714. [PMID: 38354246 PMCID: PMC10866562 DOI: 10.1126/sciadv.adk6714] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Accepted: 01/17/2024] [Indexed: 02/16/2024]
Abstract
Achieving large-scale, cost-effective, and reproducible manufacturing of stem cells with the existing devices is challenging. Traditional single-use cell-bag bioreactors, limited by their rigid and single-point sensors, struggle with accuracy and scalability for high-quality cell manufacturing. Here, we introduce a smart bioreactor system that enables multi-spatial sensing for real-time, wireless culture monitoring. This scalable system includes a low-profile, label-free thin-film sensor array and electronics integrated with a flexible cell bag, allowing for simultaneous assessment of culture properties such as pH, dissolved oxygen, glucose, and temperature, to receive real-time feedback for up to 30 days. The experimental results show the accurate monitoring of time-dynamic and spatial variations of stem cells and myoblast cells with adjustable carriers from a plastic dish to a 2-liter cell bag. These advances open up the broad applicability of the smart sensing system for large-scale, lower-cost, reproducible, and high-quality engineered cell manufacturing for broad clinical use.
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Affiliation(s)
- Jimin Lee
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Hojoong Kim
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Hyo-Ryoung Lim
- Major of Human Biocovergence, Division of Smart Healthcare, College of Information Technology and Convergence, Pukyong National University, Busan 48513, Republic of Korea
| | - Yun Soung Kim
- Biomedical Engineering and Imaging Institute, Department of Radiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Thi Thai Thanh Hoang
- Department of Orthopaedics, Musculoskeletal Institute, Emory University, Atlanta, GA 30329, USA
- Atlanta VA Medical Center, Decatur, GA 30033, USA
| | - Jeongmoon Choi
- Department of Orthopaedics, Musculoskeletal Institute, Emory University, Atlanta, GA 30329, USA
- Altos Labs-San Diego Institute of Science, San Diego, CA 92121, USA
| | - Gun-Jae Jeong
- Department of Orthopaedics, Musculoskeletal Institute, Emory University, Atlanta, GA 30329, USA
- Institute of Cell and Tissue Engineering, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea
| | - Hodam Kim
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Robert Herbert
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
- Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213 USA
| | - Ira Soltis
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Ka Ram Kim
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Sung Hoon Lee
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
- School of Electrical and Computer Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Youngjin Kwon
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Yunki Lee
- Department of Orthopaedics, Musculoskeletal Institute, Emory University, Atlanta, GA 30329, USA
- Atlanta VA Medical Center, Decatur, GA 30033, USA
| | - Young Charles Jang
- Department of Orthopaedics, Musculoskeletal Institute, Emory University, Atlanta, GA 30329, USA
- Atlanta VA Medical Center, Decatur, GA 30033, USA
| | - Woon-Hong Yeo
- George W. Woodruff School of Mechanical Engineering, College of Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA
- IEN Center for Wearable Intelligent Systems and Healthcare at the Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA
- Parker H. Petit Institute for Bioengineering and Biosciences, Institute for Materials, Institute for Robotics and Intelligent Machines, Georgia Institute of Technology, Atlanta, GA 30332, USA
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18
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Ramachandran B, Sabbatier G, Bowden OM, Campbell K, Fekete N, Girard-Lauriault PL, Hoesli CA. Human mesenchymal stromal cell adhesion and expansion on fluoropolymer surfaces modified with oxygen and nitrogen-rich plasma polymers. Colloids Surf B Biointerfaces 2024; 234:113740. [PMID: 38199188 DOI: 10.1016/j.colsurfb.2023.113740] [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: 09/25/2023] [Revised: 12/12/2023] [Accepted: 12/28/2023] [Indexed: 01/12/2024]
Abstract
Fluorinated ethylene propylene (FEP) vessels are of significant interest for therapeutic cell biomanufacturing applications due to their chemical inertness, hydrophobic surface, and high oxygen permeability. However, these properties also limit the adhesion and survival of anchorage-dependent cells. Here, we develop novel plasma polymer coatings to modify FEP surfaces, enhancing the adhesion and expansion of human mesenchymal stromal cells (hMSCs). Similar to commercially available tissue culture polystyrene vessels, oxygen-rich or nitrogen-rich surface chemistries can be achieved using this approach. While steam sterilization increased the roughness of the coatings and altered the surface chemistry, the overall wettability and oxygen or nitrogen-rich nature of the coatings were maintained. In the absence of proteins during initial cell attachment, cells adhered to surfaces even in the presence of chelators, whereas adhesion was abrogated with chelator in a protein-containing medium, suggesting that integrin-mediated adhesion predominates over physicochemical tethering in normal protein-containing cell seeding conditions. Albumin adsorption was more elevated on nitrogen-rich coatings compared to the oxygen-rich coatings, which was correlated with a higher extent of hMSC expansion after 3 days. Both the oxygen and nitrogen-rich coatings significantly improved hMSC adhesion and expansion compared to untreated FEP. FEP surfaces with nitrogen-rich coatings were practically equivalent to commercially available standard tissue culture-treated polystyrene surfaces in terms of hMSC yields. Plasma polymer coatings show significant promise in expanding the potential usage of FEP-based culture vessels for cell therapy applications.
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Affiliation(s)
| | - Gad Sabbatier
- Department of Chemical Engineering, McGill University, Montréal, Canada
| | - Olivia M Bowden
- Department of Chemical Engineering, McGill University, Montréal, Canada
| | - Katie Campbell
- Saint-Gobain Ceramics & Plastics, Inc., Northboro R&D Center, Northborough, MA, USA
| | - Natalie Fekete
- Saint-Gobain Ceramics & Plastics, Inc., Northboro R&D Center, Northborough, MA, USA
| | | | - Corinne A Hoesli
- Department of Chemical Engineering, McGill University, Montréal, Canada.
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19
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Hade MD, Suire CN, Suo Z. Significant Enhancement of Fibroblast Migration, Invasion, and Proliferation by Exosomes Loaded with Human Fibroblast Growth Factor 1. ACS APPLIED MATERIALS & INTERFACES 2024; 16:1969-1984. [PMID: 38181175 DOI: 10.1021/acsami.3c10350] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2024]
Abstract
Exosomes possess several inherent properties that make them ideal for biomedical applications, including robust stability, biocompatibility, minimal immunogenicity, and the ability to cross biological barriers. These natural nanoparticles have recently been developed as drug delivery vesicles. To do so, therapeutic molecules must be efficiently loaded into exosomes first. Very recently, we developed a cell-penetrating peptide (CPP)-based platform for loading of nucleic acids and small molecules into exosomes by taking advantage of the membrane-penetration power of CPPs. Here, we extended this simple but effective platform by loading a protein cargo into exosomes isolated from either mesenchymal stem cells from three different sources or two different cancer cell lines. The protein cargo is a fusion protein YARA-FGF1-GFP through the covalent conjugation of a model CPP called YARA to human fibroblast growth factor 1 (FGF1) and green fluorescence protein (GFP). Loading of YARA-FGF1-GFP into exosomes was time-dependent and reached a maximum of about 1600 YARA-FGF1-GFP molecules in each exosome after 16 h. The ladened exosomes were effectively internalized by mammalian cells, and subsequently, the loaded protein cargo YARA-FGF1-GFP was delivered intracellularly. In comparison to YARA, YARA-FGF1-GFP, the unloaded exosomes, and the exosomes loaded with YARA, the exosomes loaded with YARA-FGF1-GFP substantially promoted the migration, proliferation, and invasion capabilities of mouse and human fibroblasts, which are important factors for wound repair. The work extended our CPP-based exosomal cargo loading platform and established a foundation for developing novel wound-healing therapies using exosomes loaded with FGF1 and other growth factors.
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Affiliation(s)
- Mangesh D Hade
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida 32306, United States
| | - Caitlin N Suire
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida 32306, United States
| | - Zucai Suo
- Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, Florida 32306, United States
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20
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León-Moreno LC, Reza-Zaldívar EE, Hernández-Sapiéns MA, Villafaña-Estarrón E, García-Martin M, Ojeda-Hernández DD, Matias-Guiu JA, Gomez-Pinedo U, Matias-Guiu J, Canales-Aguirre AA. Mesenchymal Stem Cell-Based Therapies in the Post-Acute Neurological COVID Syndrome: Current Landscape and Opportunities. Biomolecules 2023; 14:8. [PMID: 38275749 PMCID: PMC10813738 DOI: 10.3390/biom14010008] [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: 11/15/2023] [Revised: 12/15/2023] [Accepted: 12/19/2023] [Indexed: 01/27/2024] Open
Abstract
One of the main concerns related to SARS-CoV-2 infection is the symptoms that could be developed by survivors, known as long COVID, a syndrome characterized by persistent symptoms beyond the acute phase of the infection. This syndrome has emerged as a complex and debilitating condition with a diverse range of manifestations affecting multiple organ systems. It is increasingly recognized for affecting the Central Nervous System, in which one of the most prevalent manifestations is cognitive impairment. The search for effective therapeutic interventions has led to growing interest in Mesenchymal Stem Cell (MSC)-based therapies due to their immunomodulatory, anti-inflammatory, and tissue regenerative properties. This review provides a comprehensive analysis of the current understanding and potential applications of MSC-based interventions in the context of post-acute neurological COVID-19 syndrome, exploring the underlying mechanisms by which MSCs exert their effects on neuroinflammation, neuroprotection, and neural tissue repair. Moreover, we discuss the challenges and considerations specific to employing MSC-based therapies, including optimal delivery methods, and functional treatment enhancements.
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Affiliation(s)
- Lilia Carolina León-Moreno
- Unidad de Evaluación Preclínica, Biotecnología Médica Farmacéutica, CONACYT Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Guadalajara 44270, Mexico; (L.C.L.-M.); (M.A.H.-S.); (E.V.-E.)
| | | | - Mercedes Azucena Hernández-Sapiéns
- Unidad de Evaluación Preclínica, Biotecnología Médica Farmacéutica, CONACYT Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Guadalajara 44270, Mexico; (L.C.L.-M.); (M.A.H.-S.); (E.V.-E.)
| | - Erika Villafaña-Estarrón
- Unidad de Evaluación Preclínica, Biotecnología Médica Farmacéutica, CONACYT Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Guadalajara 44270, Mexico; (L.C.L.-M.); (M.A.H.-S.); (E.V.-E.)
| | - Marina García-Martin
- Laboratorio de Neurobiología, Instituto de Investigación Sanitaria, Hospital Clínico San Carlos, IdISSC, Universidad Complutense de Madrid, 28040 Madrid, Spain; (M.G.-M.); (D.D.O.-H.); (J.A.M.-G.); (U.G.-P.)
| | - Doddy Denise Ojeda-Hernández
- Laboratorio de Neurobiología, Instituto de Investigación Sanitaria, Hospital Clínico San Carlos, IdISSC, Universidad Complutense de Madrid, 28040 Madrid, Spain; (M.G.-M.); (D.D.O.-H.); (J.A.M.-G.); (U.G.-P.)
| | - Jordi A. Matias-Guiu
- Laboratorio de Neurobiología, Instituto de Investigación Sanitaria, Hospital Clínico San Carlos, IdISSC, Universidad Complutense de Madrid, 28040 Madrid, Spain; (M.G.-M.); (D.D.O.-H.); (J.A.M.-G.); (U.G.-P.)
| | - Ulises Gomez-Pinedo
- Laboratorio de Neurobiología, Instituto de Investigación Sanitaria, Hospital Clínico San Carlos, IdISSC, Universidad Complutense de Madrid, 28040 Madrid, Spain; (M.G.-M.); (D.D.O.-H.); (J.A.M.-G.); (U.G.-P.)
| | - Jorge Matias-Guiu
- Departamento de Neurología, Instituto de Investigación Sanitaria, Hospital Clínico San Carlos, IdISSC, Universidad Complutense de Madrid, 28040 Madrid, Spain
| | - Alejandro Arturo Canales-Aguirre
- Unidad de Evaluación Preclínica, Biotecnología Médica Farmacéutica, CONACYT Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Guadalajara 44270, Mexico; (L.C.L.-M.); (M.A.H.-S.); (E.V.-E.)
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21
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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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22
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Wang Z, Zhang X, Xue L, Wang G, Li X, Chen J, Xu R, Xu T. A controllable gelatin-based microcarriers fabrication system for the whole procedures of MSCs amplification and tissue engineering. Regen Biomater 2023; 10:rbad068. [PMID: 37638061 PMCID: PMC10458456 DOI: 10.1093/rb/rbad068] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Revised: 07/06/2023] [Accepted: 07/30/2023] [Indexed: 08/29/2023] Open
Abstract
Biopolymer microbeads present substantial benefits for cell expansion, tissue engineering, and drug release applications. However, a fabrication system capable of producing homogeneous microspheres with high precision and controllability for cell proliferation, passaging, harvesting and downstream application is limited. Therefore, we developed a co-flow microfluidics-based system for the generation of uniform and size-controllable gelatin-based microcarriers (GMs) for mesenchymal stromal cells (MSCs) expansion and tissue engineering. Our evaluation of GMs revealed superior homogeneity and efficiency of cellular attachment, expansion and harvest, and MSCs expanded on GMs exhibited high viability while retaining differentiation multipotency. Optimization of passaging and harvesting protocols was achieved through the addition of blank GMs and treatment with collagenase, respectively. Furthermore, we demonstrated that MSC-loaded GMs were printable and could serve as building blocks for tissue regeneration scaffolds. These results suggested that our platform held promise for the fabrication of uniform GMs with downstream application of MSC culture, expansion and tissue engineering.
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Affiliation(s)
- Zixian Wang
- Precision Medicine and Healthcare Research Center, Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, Shenzhen 518055, People’s Republic of China
| | - Xiuxiu Zhang
- Precision Medicine and Healthcare Research Center, Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, Shenzhen 518055, People’s Republic of China
| | - Limin Xue
- Department of Research and Development, Huaqing Zhimei (Shenzhen) Biotechnology Co., Ltd., Shenzhen 518107, People’s Republic of China
| | - Gangwei Wang
- Department of Emergency, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, People’s Republic of China
| | - Xinda Li
- Department of Neurosurgery, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu 610072, People’s Republic of China
| | - Jianwei Chen
- Bio-intelligent Manufacturing and Living Matter Bioprinting Center, Research Institute of Tsinghua University in Shenzhen, Tsinghua University, Shenzhen 518057, People’s Republic of China
| | - Ruxiang Xu
- Department of Neurosurgery, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu 610072, People’s Republic of China
| | - Tao Xu
- Precision Medicine and Healthcare Research Center, Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, Shenzhen 518055, People’s Republic of China
- Department of Neurosurgery, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu 610072, People’s Republic of China
- Bio-intelligent Manufacturing and Living Matter Bioprinting Center, Research Institute of Tsinghua University in Shenzhen, Tsinghua University, Shenzhen 518057, People’s Republic of China
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23
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Chiou SH, Ong HKA, Chou SJ, Aldoghachi AF, Loh JK, Verusingam ND, Yang YP, Chien Y. Current trends and promising clinical utility of IPSC-derived MSC (iMSC). PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 199:131-154. [PMID: 37678969 DOI: 10.1016/bs.pmbts.2023.04.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/09/2023]
Abstract
Mesenchymal stem cells (MSCs) differentiated from human induced pluripotent stem cells (iPSC) or induced MSC (iMSCs) are expected to address issues of scalability and safety as well as the difficulty in producing homogenous clinical grade MSCs as demonstrated by the promising outcomes from preclinical and clinical trials, currently ongoing. The assessment of iMSCs based in vitro and in vivo studies have thus far showed more superior performance as compared to that of the primary or native human MSCs, in terms of cell proliferation, expansion capacity, immunomodulation properties as well as the influence of paracrine signaling and exosomal influence in cell-cell interaction. In this chapter, an overview of current well-established methods in generating a sustainable source of iMSCs involving well defined culture media is discussed followed by the properties of iMSC as compared to that of MSC and its promising prospects for continuous development into potential clinical grade applications.
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Affiliation(s)
- Shih-Hwa Chiou
- Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan; Department of Medical Research, Taipei Veteran General Hospital, Taipei, Taiwan
| | - Han Kiat Alan Ong
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
| | - Shih-Jie Chou
- Institute of Pharmacology, College of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan; Department of Medical Research, Taipei Veteran General Hospital, Taipei, Taiwan
| | - A F Aldoghachi
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
| | - Jit Kai Loh
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
| | - Nalini Devi Verusingam
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Cheras, Malaysia
| | - Yi-Ping Yang
- Department of Medical Research, Taipei Veteran General Hospital, Taipei, Taiwan.
| | - Yueh Chien
- Department of Medical Research, Taipei Veteran General Hospital, Taipei, Taiwan
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24
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Pincela Lins PM, Pirlet E, Szymonik M, Bronckaers A, Nelissen I. Manufacture of extracellular vesicles derived from mesenchymal stromal cells. Trends Biotechnol 2023; 41:965-981. [PMID: 36750391 DOI: 10.1016/j.tibtech.2023.01.003] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2022] [Revised: 12/09/2022] [Accepted: 01/05/2023] [Indexed: 02/08/2023]
Abstract
Mesenchymal stromal cells (MSCs) are a promising therapy for various diseases ranging from ischemic stroke to wound healing and cancer. Their therapeutic effects are mainly mediated by secretome-derived paracrine factors, with extracellular vesicles (EVs) proven to play a key role. This has led to promising research on the potential of MSC-EVs as regenerative, off-the-shelf therapeutic agents. However, the translation of MSC-EVs into the clinic is hampered by the poor scalability of their production. Recently, new advanced methods have been developed to upscale MSC cultivation and EV production yields, ranging from new cell culture devices to priming procedures. This review gives an overview of these innovative strategies for manufacturing MSC-EVs.
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Affiliation(s)
- Paula M Pincela Lins
- Hasselt University, Faculty of Medicine and Life Sciences, Biomedical Research Institute (BIOMED), Agoralaan, 3590 Diepenbeek, Belgium; Flemish Institute for Technological Research (VITO), Health Department, Boeretang, 2400 Mol, Belgium
| | - Elke Pirlet
- Hasselt University, Faculty of Medicine and Life Sciences, Biomedical Research Institute (BIOMED), Agoralaan, 3590 Diepenbeek, Belgium
| | - Michal Szymonik
- Flemish Institute for Technological Research (VITO), Health Department, Boeretang, 2400 Mol, Belgium
| | - Annelies Bronckaers
- Hasselt University, Faculty of Medicine and Life Sciences, Biomedical Research Institute (BIOMED), Agoralaan, 3590 Diepenbeek, Belgium.
| | - Inge Nelissen
- Flemish Institute for Technological Research (VITO), Health Department, Boeretang, 2400 Mol, Belgium.
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25
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Gao T, Zhao X, Hao J, Tian Y, Ma H, Liu W, An B, Sun F, Liu S, Guo B, Niu S, Li Z, Wang C, Wang Y, Feng G, Wang L, Li W, Wu J, Guo M, Zhou Q, Gu Q. A scalable culture system incorporating microcarrier for specialised mesenchymal stem cells from human embryonic stem cells. Mater Today Bio 2023; 20:100662. [PMID: 37214547 PMCID: PMC10196860 DOI: 10.1016/j.mtbio.2023.100662] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2023] [Revised: 04/20/2023] [Accepted: 05/05/2023] [Indexed: 05/24/2023] Open
Abstract
Mesenchymal stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are a desirable cell source for cell therapy owing to their capacity to be produced stably and homogeneously in large quantities. However, a scalable culture system for hPSC-derived MSCs is urgently needed to meet the cell quantity and quality requirements of practical clinical applications. In this study, we developed a new microcarrier with hyaluronic acid (HA) as the core material, which allowed scalable serum-free suspension culture of hESC-derived MSCs (IMRCs). We used optimal microcarriers with a coating collagen concentration of 100 μg/mL or concave-structured surface (cHAMCs) for IMRC amplification in a stirred bioreactor, expanding IMRCs within six days with the highest yield of over one million cells per milliliter. In addition, the harvested cells exhibited high viability, immunomodulatory and regenerative therapeutic promise comparable to monolayer cultured MSCs while showing more increased secretion of extracellular matrix (ECM), particularly collagen-related proteins. In summary, we have established a scalable culture system for hESC-MSCs, providing novel approaches for future cell therapies.
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Affiliation(s)
- Tingting Gao
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiyuan Zhao
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jie Hao
- National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yao Tian
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Huike Ma
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
| | - Wenjing Liu
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Bin An
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Faguo Sun
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shasha Liu
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Baojie Guo
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shuaishuai Niu
- National Stem Cell Resource Center, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Zhongwen Li
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Chenxin Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Yukai Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Guihai Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Liu Wang
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jun Wu
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
| | - Meijin Guo
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
| | - Qi Zhou
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Qi Gu
- State Key Laboratory of Stem Cell and Reproductive Biology, State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
- Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
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26
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Shou Y, Liu L, Liu Q, Le Z, Lee KL, Li H, Li X, Koh DZ, Wang Y, Liu TM, Yang Z, Lim CT, Cheung C, Tay A. Mechano-responsive hydrogel for direct stem cell manufacturing to therapy. Bioact Mater 2023; 24:387-400. [PMID: 36632503 PMCID: PMC9817177 DOI: 10.1016/j.bioactmat.2022.12.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2022] [Revised: 12/05/2022] [Accepted: 12/20/2022] [Indexed: 01/04/2023] Open
Abstract
Bone marrow-derived mesenchymal stem cell (MSC) is one of the most actively studied cell types due to its regenerative potential and immunomodulatory properties. Conventional cell expansion methods using 2D tissue culture plates and 2.5D microcarriers in bioreactors can generate large cell numbers, but they compromise stem cell potency and lack mechanical preconditioning to prepare MSC for physiological loading expected in vivo. To overcome these challenges, in this work, we describe a 3D dynamic hydrogel using magneto-stimulation for direct MSC manufacturing to therapy. With our technology, we found that dynamic mechanical stimulation (DMS) enhanced matrix-integrin β1 interactions which induced MSCs spreading and proliferation. In addition, DMS could modulate MSC biofunctions including directing MSC differentiation into specific lineages and boosting paracrine activities (e.g., growth factor secretion) through YAP nuclear localization and FAK-ERK pathway. With our magnetic hydrogel, complex procedures from MSC manufacturing to final clinical use, can be integrated into one single platform, and we believe this 'all-in-one' technology could offer a paradigm shift to existing standards in MSC therapy.
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Affiliation(s)
- Yufeng Shou
- Department of Biomedical Engineering, National University of Singapore, 117583, Singapore
- Institute for Health Innovation & Technology, National University of Singapore, 117599, Singapore
| | - Ling Liu
- Institute for Health Innovation & Technology, National University of Singapore, 117599, Singapore
- NUS Tissue Engineering Program, National University of Singapore, 117510, Singapore
| | - Qimin Liu
- School of Civil Engineering and Architecture, Wuhan University of Technology, 430070, Wuhan, China
| | - Zhicheng Le
- Department of Biomedical Engineering, National University of Singapore, 117583, Singapore
- Institute for Health Innovation & Technology, National University of Singapore, 117599, Singapore
| | - Khang Leng Lee
- Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore
| | - Hua Li
- School of Mechanical and Aerospace Engineering, Nanyang Technological University, 639798, Singapore
| | - Xianlei Li
- Department of Biomedical Engineering, National University of Singapore, 117583, Singapore
- Institute for Health Innovation & Technology, National University of Singapore, 117599, Singapore
| | - Dion Zhanyun Koh
- Department of Biomedical Engineering, National University of Singapore, 117583, Singapore
| | - Yuwen Wang
- Department of Biomedical Engineering, National University of Singapore, 117583, Singapore
| | - Tong Ming Liu
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 138648, Singapore
| | - Zheng Yang
- NUS Tissue Engineering Program, National University of Singapore, 117510, Singapore
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 119288, Singapore
| | - Chwee Teck Lim
- Department of Biomedical Engineering, National University of Singapore, 117583, Singapore
- Institute for Health Innovation & Technology, National University of Singapore, 117599, Singapore
- Mechanobiology Institute, National University of Singapore, 117411, Singapore
| | - Christine Cheung
- Lee Kong Chian School of Medicine, Nanyang Technological University, 636921, Singapore
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 138648, Singapore
| | - Andy Tay
- Department of Biomedical Engineering, National University of Singapore, 117583, Singapore
- Institute for Health Innovation & Technology, National University of Singapore, 117599, Singapore
- NUS Tissue Engineering Program, National University of Singapore, 117510, Singapore
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27
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Teale MA, Schneider S, Eibl D, van den Bos C, Neubauer P, Eibl R. Mesenchymal and induced pluripotent stem cell-based therapeutics: a comparison. Appl Microbiol Biotechnol 2023:10.1007/s00253-023-12583-4. [PMID: 37246986 DOI: 10.1007/s00253-023-12583-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2023] [Revised: 05/07/2023] [Accepted: 05/08/2023] [Indexed: 05/30/2023]
Abstract
Stem cell-based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed. KEY POINTS: • Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation • Single-use stirred bioreactor systems are preferred for the cultivation of both cell types • Future research should adapt and modify downstream processes to available single-use devices.
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Affiliation(s)
- Misha A Teale
- Centre for Biochemical Engineering and Cell Cultivation Techniques, Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Grüentalstrasse 14, 8820, Wädenswil, Switzerland.
| | - Samuel Schneider
- Centre for Biochemical Engineering and Cell Cultivation Techniques, Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Grüentalstrasse 14, 8820, Wädenswil, Switzerland
| | - Dieter Eibl
- Centre for Biochemical Engineering and Cell Cultivation Techniques, Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Grüentalstrasse 14, 8820, Wädenswil, Switzerland
| | | | - Peter Neubauer
- Institute of Biotechnology, Chair of Bioprocess Engineering, Technical University of Berlin, ACK24, Ackerstraße 76, 13355, Berlin, Germany
| | - Regine Eibl
- Centre for Biochemical Engineering and Cell Cultivation Techniques, Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Grüentalstrasse 14, 8820, Wädenswil, Switzerland
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Simão VA, Brand H, da Silveira-Antunes RN, Fukasawa JT, Leme J, Tonso A, Ribeiro-Paes JT. Adipose-derived stem cells (ASCs) culture in spinner flask: improving the parameters of culture in a microcarrier-based system. Biotechnol Lett 2023:10.1007/s10529-023-03367-x. [PMID: 37171697 DOI: 10.1007/s10529-023-03367-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Revised: 03/17/2023] [Accepted: 03/24/2023] [Indexed: 05/13/2023]
Abstract
Prior to clinical use, extensive in vitro proliferation of human adipose-derived stem cells (ASCs) is required. Among the current options, spinner-type stirred flasks, which use microcarriers to increase the yield of adherent cells, are recommended. Here, we propose a methodology for ASCs proliferation through cell suspension culture using Cultispher-S® microcarriers (MC) under agitation in a spinner flask, with the aim of establishing a system that reconciles the efficiency of cell yield with high viability of the culture during two distinct phases: seeding and proliferation. The results showed that cell adhesion was potentiated under intermittent stirring at 70 rpm in the presence of 10% FBS for an initial cell concentration of 2.4 × 104 cells/mL in the initial 24 h of cultivation. In the proliferation phase, kinetic analysis showed that cell growth was higher under continuous agitation at 50 rpm with a culture medium renewal regime of 50% every 72 h, which was sufficient to maintain the culture at optimal levels of nutrients and metabolites for up to nine days of cultivation, representing an 11.1-fold increase and a maximum cell productivity of 422 cells/mL/h (1.0 × 105 viable cells/mL). ASCs maintained the immunophenotypic characteristics and mesodermal differentiation potential of both cell lines from different donors. The established protocol represents a more efficient and cost-effective method to obtain a high proliferation rate of ASCs in a microcarrier-based system, which is necessary for large-scale use in cell therapy, highlighting that the manipulation of critical parameters optimizes the ASCs production process.
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Affiliation(s)
- Vinícius Augusto Simão
- Department of Genetics, School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.
| | - Heloisa Brand
- Department of Biotechnology, School of Sciences and Letters, São Paulo State University (UNESP), Assis, São Paulo, Brazil
| | | | | | - Jaci Leme
- Center for Development and Innovation, Laboratory of Viral Biotechnology, Butantan Institute, São Paulo, São Paulo, Brazil
| | - Aldo Tonso
- Department of Chemical Engineering, Polytechnic School, University of São Paulo, São Paulo, São Paulo, Brazil
| | - João Tadeu Ribeiro-Paes
- Department of Biotechnology, School of Sciences and Letters, São Paulo State University (UNESP), Assis, São Paulo, Brazil
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Hoang DM, Nguyen QT, Phan TT, Ngo AT, Pham PT, Bach TQ, Le PT, Bui HT, Thanh LN. Advanced cell-based products generated via automated and manual manufacturing platforms under the quality by design principle: Are they equivalent or different? Heliyon 2023; 9:e15946. [PMID: 37229156 PMCID: PMC10205494 DOI: 10.1016/j.heliyon.2023.e15946] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2022] [Revised: 04/25/2023] [Accepted: 04/27/2023] [Indexed: 05/27/2023] Open
Abstract
Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells that can be isolated from bone marrow, adipose tissue, the umbilical cord, dental pulp, etc. These cells have unique properties that give them excellent therapeutic potential, including immunoregulation, immunomodulation, and tissue regeneration functions. MSC-based products are considered advanced therapy medicinal products (ATMPs) under European regulations (1394/2007); thus, they must be manufactured under good manufacturing practices and via effective manufacturing methods. The former can be achieved via a proper laboratory design and compliance with manufacturing protocols, whereas the latter requires an approach that ensures that the quality of the products is consistent regardless of the manufacturing procedure. To meet these daunting requirements, this study proposes an exchangeable approach that combines optimized and equivalent manufacturing processes under the Quality by Design (QbD) principle, allowing investigators to convert from small laboratory-scale to large-scale manufacturing of MSC-based products for clinical applications without altering the quality and quantity of the cell-based products.
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Affiliation(s)
- Duc M. Hoang
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Quyen T. Nguyen
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Trang T.K. Phan
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Anh T.L. Ngo
- Vinmec High Tech Center, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Phuong T. Pham
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Trung Q. Bach
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Phuong T.T. Le
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Hoa T.P. Bui
- Vinmec High Tech Center, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
| | - Liem Nguyen Thanh
- Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
- College of Health Science, Vin University, Vinhomes Ocean Park, Gia Lam District, Hanoi 12400, Viet Nam
- Vinmec International Hospital – Times City, Vinmec Healthcare System, 458 Minh Khai, Hanoi 11622, Viet Nam
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Mori T, Igarashi M, Onodera Y, Takehara T, Itokazu M, Teramura T. Fibrinogen supports self-renewal of mesenchymal stem cells under serum-reduced condition through autophagy activation. Biochem Biophys Res Commun 2023; 651:70-78. [PMID: 36796212 DOI: 10.1016/j.bbrc.2023.02.009] [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: 12/26/2022] [Revised: 01/05/2023] [Accepted: 02/02/2023] [Indexed: 02/12/2023]
Abstract
Mesenchymal stem cells (MSCs) are somatic stem cells used in cell transplantation therapy for tissue injuries and inflammatory diseases because of their ability to support tissue regeneration and to suppress inflammation. While their applications are expanding, needs for automation of culture procedures with reduction of animal-derived materials to meet stable quality and suppliability are also increasing. On the other hand, the development of molecules that safely support cell adherence and expansion on a variety of interfaces under the serum-reduced culture condition remains a challenge. We report here that fibrinogen enables MSC culture on various materials with low cell adhesion property even under serum-reduced culture conditions. Fibrinogen promoted MSC adhesion and proliferation by stabilizing basic fibroblast growth factor (bFGF), which was secreted in the culture medium by autocrine, and also activated autophagy to suppress cellar senescence. Fibrinogen coating allowed MSCs expansion even on the polyether sulfone membrane that represents very low cell adhesion, and the MSCs showed therapeutic effects in a pulmonary fibrosis model. This study demonstrates that fibrinogen is currently the safest and most widely available extracellular matrix and can be used as a versatile scaffold for cell culture in regenerative medicine.
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Affiliation(s)
| | | | - Yuta Onodera
- Institute of Advanced Clinical Medicine, Kindai University Hospital, Japan
| | - Toshiyuki Takehara
- Institute of Advanced Clinical Medicine, Kindai University Hospital, Japan
| | - Maki Itokazu
- Department of Rehabilitation Medicine, Kindai University Faculty of Medicine, Japan
| | - Takeshi Teramura
- Institute of Advanced Clinical Medicine, Kindai University Hospital, Japan.
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31
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Jankovic MG, Stojkovic M, Bojic S, Jovicic N, Kovacevic MM, Ivosevic Z, Juskovic A, Kovacevic V, Ljujic B. Scaling up human mesenchymal stem cell manufacturing using bioreactors for clinical uses. Curr Res Transl Med 2023; 71:103393. [PMID: 37163885 DOI: 10.1016/j.retram.2023.103393] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 03/13/2023] [Accepted: 04/26/2023] [Indexed: 05/12/2023]
Abstract
Human mesenchymal stem cells (hMSCs) are multipotent cells and an attractive therapeutic agent in regenerative medicine and intensive clinical research. Despite the great potential, the limitation that needs to be overcome is the necessity of ex vivo expansion because of insufficient number of hMSCs presented within adult organs and the high doses required for a transplantation. As a result, numerous research studies aim to provide novel expansion methods in order to achieve appropriate numbers of cells with preserved therapeutic quality. Bioreactor-based cell expansion provide high-level production of hMSCs in accordance with good manufacturing practice (GMP) and quality standards. This review summarizes current knowledge about the hMSCs manufacturing platforms with a main focus to the application of bioreactors for large-scale production of GMP-grade hMSCs.
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Affiliation(s)
- Marina Gazdic Jankovic
- University of Kragujevac, Serbia, Faculty of Medical Sciences, Department of Genetics, Serbia.
| | | | - Sanja Bojic
- Newcastle University, School of Computing, Newcastle upon Tyne, UK
| | - Nemanja Jovicic
- University of Kragujevac, Serbia, Faculty of Medical Sciences, Department of Histology and Embryology, Serbia
| | - Marina Miletic Kovacevic
- University of Kragujevac, Serbia, Faculty of Medical Sciences, Department of Histology and Embryology, Serbia
| | - Zeljko Ivosevic
- University of Kragujevac, Serbia, Faculty of Medical Sciences, Department of Genetics, Serbia
| | - Aleksandar Juskovic
- Department of Orthopaedic Surgery, Clinical Centre of Montenegro, 81110 Podgorica, Montenegro
| | - Vojin Kovacevic
- University of Kragujevac, Serbia, Faculty of Medical Sciences, Department of Surgery, Serbia
| | - Biljana Ljujic
- University of Kragujevac, Serbia, Faculty of Medical Sciences, Department of Genetics, Serbia
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A Multi-Stage Bioprocess for the Expansion of Rodent Skin-Derived Schwann Cells in Computer-Controlled Bioreactors. Int J Mol Sci 2023; 24:ijms24065152. [PMID: 36982227 PMCID: PMC10049355 DOI: 10.3390/ijms24065152] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Revised: 02/03/2023] [Accepted: 02/14/2023] [Indexed: 03/11/2023] Open
Abstract
Regenerative therapies for the treatment of peripheral nerve and spinal cord injuries can require hundreds of millions of autologous cells. Current treatments involve the harvest of Schwann cells (SCs) from nerves; however, this is an invasive procedure. Therefore, a promising alternative is using skin-derived Schwann cells (Sk-SCs), in which between 3–5 million cells can be harvested from a standard skin biopsy. However, traditional static planar culture is still inefficient at expanding cells to clinically relevant numbers. As a result, bioreactors can be used to develop reproducible bioprocesses for the large-scale expansion of therapeutic cells. Here, we present a proof-of-concept SC manufacturing bioprocess using rat Sk-SCs. With this integrated process, we were able to simulate a feasible bioprocess, taking into consideration the harvest and shipment of cells to a production facility, the generation of the final cell product, and the cryopreservation and shipment of cells back to the clinic and patient. This process started with 3 million cells and inoculated and expanded them to over 200 million cells in 6 days. Following the harvest and post-harvest cryopreservation and thaw, we were able to maintain 150 million viable cells that exhibited a characteristic Schwann cell phenotype throughout each step of the process. This process led to a 50-fold expansion, producing a clinically relevant number of cells in a 500 mL bioreactor in just 1 week, which is a dramatic improvement over current methods of expansion.
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Utilizing Additive Manufacturing to Produce Organ Mimics and Imaging Phantoms. SURGERIES 2023. [DOI: 10.3390/surgeries4010008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The complex geometries and material properties necessary for generating accurate organ mimics require new procedures and methods to fully utilize current technologies. The increased accessibility of 3D printers, along with more specialized bioprinters, allow the creation of highly tunable models of various body parts. Three-dimensional printing can reduce lead-time on custom parts, produce structures based on imaging data in patients, and generate a test bench for novel surgical methods. This technical note will cover three unique case studes and offer insights for how 3D printing can be used for lab research. Each case follows a unique design process in comparison to traditional manufacturing workflows as they required significantly more iterative design. The strengths of different printing technologies, design choices, and structural/chemical requirements all influence the design process. Utilization of in-house manufacturing allows for greater flexibility and lower lead-times for novel research applications. Detailed discussions of these design processes will help reduce some of the major barriers to entry for these technologies and provide options for researchers working in the field.
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34
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Mogha P, Iyer S, Majumder A. Extracellular matrix protein gelatin provides higher expansion, reduces size heterogeneity, and maintains cell stiffness in a long-term culture of mesenchymal stem cells. Tissue Cell 2023; 80:101969. [PMID: 36403499 DOI: 10.1016/j.tice.2022.101969] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 10/17/2022] [Accepted: 10/29/2022] [Indexed: 11/08/2022]
Abstract
Extracellular matrices (ECM) present in our tissues play a significant role in maintaining tissue homeostasis through various physical and chemical cues such as topology, stiffness, and secretion of biochemicals. They are known to influence the behavior of resident stem cells. It is also known that ECM type and coating density on cell culture plates strongly influence in vitro cellular behavior. However, the influence of ECM protein coating on long-term mesenchymal stem cell expansion has not been studied yet. To address this gap, we cultured bone-marrow derived hMSCs for multiple passages on the tissue culture plastic plates coated with 25 μg/ml of various ECM proteins. We found that cells on plates coated with ECM proteins had much higher proliferation compared to the regular tissue culture plates. Further, gelatin-coated plates helped the cells to grow faster compared to collagen, fibronectin, and laminin coated plates. Additionally, the use of gelatin showed less size heterogeneity among the cells when expanded from passages 3 to 9 (P3 to P9). Gelatin also helped in maintaining cellular stiffness which was not observed across other ECM proteins. In summary, in this research, we have shown that gelatin which is the least expensive compared to other ECM proteins, provides a better platform for mesenchymal stem cell expansion.
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Affiliation(s)
- Pankaj Mogha
- Chemical Engineering Department, IIT Bombay, Mumbai 400076 India.
| | - Shruti Iyer
- Chemical Engineering Department, IIT Bombay, Mumbai 400076 India
| | - Abhijit Majumder
- Chemical Engineering Department, IIT Bombay, Mumbai 400076 India.
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35
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Yang P, Zhang S, Yan T, Li F, Zhang S. The Therapeutic Application of Stem Cells and Their Derived Exosomes in the Treatment of Radiation-Induced Skin Injury. Radiat Res 2023; 199:182-201. [PMID: 36630584 DOI: 10.1667/rade-22-00023.1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2022] [Accepted: 12/05/2022] [Indexed: 01/13/2023]
Abstract
Radiation-induced skin injury (RISI) is a serious concern for nuclear accidents and cancer radiotherapy, which seriously affects the quality of life of patients. This injury differs from traditional wounds due to impaired healing and the propensity to recurrence and is divided into acute and chronic phases on the basis of the injury time. Unfortunately, there are few effective therapies for preventing or mitigating this injury. Over the last few decades, various studies have focused on the effects of stem cell-based therapies to address the tissue repair and regeneration of irradiated skin. These stem cells modulate inflammation and instigate tissue repair by differentiating into specific kinds of cells or releasing paracrine factors. Stem cell-based therapies, including bone marrow-derived stem cells (BMSCs), adipose-derived stem cells (ADSCs) and stromal vascular fraction (SVF), have been reported to facilitate wound healing after radiation exposure. Moreover, stem cell-derived exosomes have recently been suggested as an effective and cell-free approach to support skin regeneration, circumventing the concerns respecting direct application of stem cells. Based on the literature on stem cell-based therapies for radiation-induced skin injury, we summarize the characteristics of different stem cells and describe their latest animal and clinical applications, as well as potential mechanisms. The promise of stem-cell based therapies against radiation-induced skin injury contribute to our response to nuclear events and smooth progress of cancer radiotherapy.
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Affiliation(s)
- Ping Yang
- Laboratory of Radiation Medicine, West China Second University Hospital, Sichuan University, Chengdu 610041, China.,Laboratory of Radiation Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China
| | - Shuaijun Zhang
- Laboratory of Radiation Medicine, West China Second University Hospital, Sichuan University, Chengdu 610041, China
| | - Tao Yan
- Laboratory of Radiation Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China.,Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital, Chengdu 610051, China
| | - Fengsheng Li
- PLA Rocket Rorce Characteristic Medical Center, Beijing 100088, China
| | - Shuyu Zhang
- Laboratory of Radiation Medicine, West China Second University Hospital, Sichuan University, Chengdu 610041, China.,Laboratory of Radiation Medicine, West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu 610041, China.,Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital, Chengdu 610051, China.,NHC Key Laboratory of Nuclear Technology Medical Transformation, Mianyang Central Hospital, Mianyang 621099, China
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36
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Katahira Y, Murakami F, Inoue S, Miyakawa S, Sakamoto E, Furusaka Y, Watanabe A, Sekine A, Kuroda M, Hasegawa H, Mizoguchi I, Yoshimoto T. Protective effects of conditioned media of immortalized stem cells from human exfoliated deciduous teeth on pressure ulcer formation. Front Immunol 2023; 13:1010700. [PMID: 36713359 PMCID: PMC9881429 DOI: 10.3389/fimmu.2022.1010700] [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: 08/03/2022] [Accepted: 12/23/2022] [Indexed: 01/14/2023] Open
Abstract
Pressure ulcers (PUs) are increasing with aging worldwide, but there is no effective causal therapy. Although mesenchymal stem cells (MSCs) promote cutaneous wound healing, the effects of the conditioned medium (CM) of MSCs on cutaneous PU formation induced by ischemia-reperfusion injury have been poorly investigated. To address this issue, herein, we first established an immortalized stem cell line from human exfoliated deciduous teeth (SHED). This cell line was revealed to have superior characteristics in that it grows infinitely and vigorously, and stably and consistently secretes a variety of cytokines. Using the CM obtained from the immortalized SHED cell line, we investigated the therapeutic potential on a cutaneous ischemia-reperfusion mouse model for PU formation using two magnetic plates. This is the first study to show that CM from immortalized SHEDs exerts therapeutic effects on PU formation by promoting angiogenesis and oxidative stress resistance through vascular endothelial growth factor and hepatocyte growth factor. Thus, the CM of MSCs has potent therapeutic effects, whereas these therapies have not been implemented in human medicine. To try to meet the regulatory requirements for manufacturing and quality control as much as possible, it is necessary to produce CM that is consistently safe and effective. The immortalization of stem cells could be one of the breakthroughs to meet the regulatory requirements and consequently open up a novel avenue to create a novel type of cell-free regenerative medicine, although further investigation into the quality control is warranted.
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Affiliation(s)
- Yasuhiro Katahira
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Fumihiro Murakami
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Shinya Inoue
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Satomi Miyakawa
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Eri Sakamoto
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Yuma Furusaka
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Aruma Watanabe
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Ami Sekine
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Masahiko Kuroda
- Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan
| | - Hideaki Hasegawa
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Izuru Mizoguchi
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
| | - Takayuki Yoshimoto
- Department of Immunoregulation, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan,*Correspondence: Takayuki Yoshimoto,
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37
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The Role of Process Systems Engineering in Applying Quality by Design (QbD) in Mesenchymal Stem Cell Production. Comput Chem Eng 2023. [DOI: 10.1016/j.compchemeng.2023.108144] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
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38
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Scholz BX, Hayashi Y, Udugama IA, Kino-oka M, Sugiyama H. A CFD model-based design of seeding processes for two-dimensional mesenchymal stem cell cultivation. Comput Chem Eng 2023. [DOI: 10.1016/j.compchemeng.2023.108157] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
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39
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Tuomisto HL, Allan SJ, Ellis MJ. Prospective life cycle assessment of a bioprocess design for cultured meat production in hollow fiber bioreactors. THE SCIENCE OF THE TOTAL ENVIRONMENT 2022; 851:158051. [PMID: 35985596 DOI: 10.1016/j.scitotenv.2022.158051] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Revised: 07/18/2022] [Accepted: 08/11/2022] [Indexed: 06/15/2023]
Abstract
The aim of cellular agriculture is to use cell-culturing technologies to produce alternatives to agricultural products. Cultured meat is an example of a cellular agriculture product, made by using tissue engineering methods. This study aims to improve the understanding of the potential environmental impacts of cultured meat production by comparing between different bioprocess design scenarios. This was done by carrying out a life cycle assessment (LCA) for a bioprocess system using hollow fiber bioreactors, and utilizing bench-scale experimental data for C2C12 cell proliferation, differentiation and media metabolism. Scenario and sensitivity analyses were used to test the impact of changes in the system design, data sources, and LCA methods on the results to support process design decision making. We compared alternative scenarios to a baseline of C2C12 cells cultured in hollow fiber bioreactors using media consisting of DMEM with serum, for a 16-day proliferation stage and 7-day differentiation stage. The baseline LCA used the average UK electricity mix as the energy source, and heat treatment for wastewater sterilization. The greatest reduction in environmental impacts were achieved with the scenarios using CHO cell metabolism instead of C2C12 cell metabolisim (64-67 % reduction); achieving 128 % cell biomass increase during differentiation instead of no increase (42-56 % reduction); using wind electricity instead of average UK electricity (6-39 % reduction); and adjusting the amino acid use based on experimental data (16-27 % reduction). The use of chemical wastewater treatment instead of heat treatment increased all environmental impacts, except energy demand, by 1-16 %. This study provides valuable insights for the cultured meat field to understand the effects of different process design scenarios on environmental impacts, and therefore provides a framework for deciding where to focus development efforts for improving the environmental performance of the production system.
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Affiliation(s)
- Hanna L Tuomisto
- Future Sustainable Food Systems Research Group, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, P.O. Box 27, University of Helsinki, 00014 Helsinki, Finland; Helsinki Institute of Sustainability Science (HELSUS), P.O. Box 4, University of Helsinki, 00014 Helsinki, Finland; Natural Resources Institute Finland (Luke), Latokartanonkaari 9, 00790 Helsinki, Finland.
| | - Scott J Allan
- EPSRC Centre for Doctoral Training, Centre for Sustainable Chemical Technologies, University of Bath, Claverton Down, BA2 7AY Bath, UK; Department of Chemical Engineering, University of Bath, Claverton Down, BA2 7AY Bath, UK
| | - Marianne J Ellis
- Department of Chemical Engineering, University of Bath, Claverton Down, BA2 7AY Bath, UK
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40
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Stem cell sheet fabrication from human umbilical cord mesenchymal stem cell and Col-T scaffold. Stem Cell Res 2022; 65:102960. [PMID: 36399925 DOI: 10.1016/j.scr.2022.102960] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/13/2022] [Revised: 11/02/2022] [Accepted: 11/04/2022] [Indexed: 11/09/2022] Open
Abstract
Today, stem cell therapy has been shown to be a remarkable progress and an important application in the regeneration of defective tissues and organs. To deliver stem cells to the injured area, several methods have been proposed such as an intravenous infusion, direct damaged tissue injection, or stem cell sheet transplantation. In this study, we aimed to fabricate a stem cell sheet by culturing human umbilical cord mesenchymal stem cells (hUC-MSCs) on a Col-T scaffold to recover the structure and function of damaged tissues. The results showed that cells reach confluent on the scaffold surface 18 h after seeding. These stem cells were able to survive and proliferate on Col-T scaffold. The average tensile strength of the stem cell sheet was 2.65 MPa. The sheet reached the sterile standards when tested for total bacteria, Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus according to Circular number 06/2011/TT-BYT of Vietnam Ministry of Health. In addition, the stem cell sheet was non-toxic when evaluated for exposure toxicity and fluid toxicity according to iSO-10993. Importantly, 5 days after culturing on the Col-T scaffold, the seeded hUC-MSCs were still possessed all properties of MSC such as spindle-shaped, adhesive, could differentiate into mesoderm-derived cells, showed to be CD90, CD105, CD73 positive and CD45, CD34, CD11b, CD19, HLA-DR negative. In summary, our study was successful in creating a stem cell sheet from hUC-MSCs and Col-T scaffold for subsequent in vivo transplantation in the future.
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41
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Katiyar S, Singh D, Kumari S, Srivastava P, Mishra A. Novel strategies for designing regenerative skin products for accelerated wound healing. 3 Biotech 2022; 12:316. [PMID: 36276437 PMCID: PMC9547767 DOI: 10.1007/s13205-022-03331-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2022] [Accepted: 08/23/2022] [Indexed: 11/01/2022] Open
Abstract
Healthy skin protects from pathogens, water loss, ultraviolet rays, and also maintains homeostasis conditions along with sensory perceptions in normal circumstances. Skin wound healing mechanism is a multi-phased biodynamic process that ultimately triggers intercellular and intracellular mechanisms. Failure to implement the normal and effective healing process may result in chronic injuries and aberrant scarring. Chronic wounds lead to substantial rising healthcare expenditure, and innovative methods to diagnose and control severe consequences are urgently needed. Skin tissue engineering (STE) has achieved several therapeutic accomplishments during the last few decades, demonstrating tremendous development. The engineered skin substitutes provide instant coverage for extensive wounds and facilitate the prevention of microbial infections and fluid loss; furthermore, they help in fighting inflammation and allow rapid neo-tissue formation. The current review primarily focused on the wound recovery and restoration process and the current conditions of STE with various advancements and complexities associated with different strategies such as cell sources, biopolymers, innovative fabrication techniques, and growth factors delivery systems.
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Affiliation(s)
- Soumya Katiyar
- School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005 India
| | - Divakar Singh
- School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005 India
| | - Shikha Kumari
- School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005 India
| | - Pradeep Srivastava
- School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005 India
| | - Abha Mishra
- School of Biochemical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi, 221005 India
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Exploring the Concept of In Vivo Guided Tissue Engineering by a Single-Stage Surgical Procedure in a Rodent Model. Int J Mol Sci 2022; 23:ijms232012703. [PMID: 36293558 PMCID: PMC9604108 DOI: 10.3390/ijms232012703] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2022] [Revised: 10/09/2022] [Accepted: 10/15/2022] [Indexed: 11/17/2022] Open
Abstract
In severe malformations with a lack of native tissues, treatment options are limited. We aimed at expanding tissue in vivo using the body as a bioreactor and developing a sustainable single-staged procedure for autologous tissue reconstruction in malformation surgery. Autologous micro-epithelium from skin was integrated with plastically compressed collagen and a degradable knitted fabric mesh. Sixty-three scaffolds were implanted in nine rats for histological and mechanical analyses, up to 4 weeks after transplantation. Tissue integration, cell expansion, proliferation, inflammation, strength, and elasticity were evaluated over time in vivo and validated in vitro in a bladder wound healing model. After 5 days in vivo, we observed keratinocyte proliferation on top of the transplant, remodeling of the collagen, and neovascularization within the transplant. At 4 weeks, all transplants were fully integrated with the surrounding tissue. Tensile strength and elasticity were retained during the whole study period. In the in vitro models, a multilayered epithelium covered the defect after 4 weeks. Autologous micro-epithelial transplants allowed for cell expansion and reorganization in vivo without conventional pre-operative in vitro cell propagation. The method was easy to perform and did not require handling outside the operating theater.
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Suyama T, Takemoto Y, Miyauchi H, Kato Y, Matsuzaki Y, Kato R. Morphology-based noninvasive early prediction of serial-passage potency enhances the selection of clone-derived high-potency cell bank from mesenchymal stem cells. Inflamm Regen 2022; 42:30. [PMID: 36182958 PMCID: PMC9526913 DOI: 10.1186/s41232-022-00214-w] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Accepted: 05/29/2022] [Indexed: 11/12/2022] Open
Abstract
Background Rapidly expanding clones (RECs) are one of the single-cell-derived mesenchymal stem cell clones sorted from human bone marrow mononuclear cells (BMMCs), which possess advantageous features. The RECs exhibit long-lasting proliferation potency that allows more than 10 repeated serial passages in vitro, considerably benefiting the manufacturing process of allogenic MSC-based therapeutic products. Although RECs aid the preparation of large-variation clone libraries for a greedy selection of better-quality clones, such a selection is only possible by establishing multiple-candidate cell banks for quality comparisons. Thus, there is a high demand for a novel method that can predict “low-risk and high-potency clones” early and in a feasible manner given the excessive cost and effort required to maintain such an establishment. Methods LNGFR and Thy-1 co-positive cells from BMMCs were single-cell-sorted into 96-well plates, and only fast-growing clones that reached confluency in 2 weeks were picked up and passaged as RECs. Fifteen RECs were prepared as passage 3 (P3) cryostock as the primary cell bank. From this cryostock, RECs were passaged until their proliferation limitation; their serial-passage limitation numbers were labeled as serial-passage potencies. At the P1 stage, phase-contrast microscopic images were obtained over 6–90 h to identify time-course changes of 24 morphological descriptors describing cell population information. Machine learning models were constructed using the morphological descriptors for predicting serial-passage potencies. The time window and field-of-view-number effects were evaluated to identify the most efficient image data usage condition for realizing high-performance serial-passage potency models. Results Serial-passage test results indicated variations of 7–13-repeated serial-passage potencies within RECs. Such potency values were predicted quantitatively with high performance (RMSE < 1.0) from P1 morphological profiles using a LASSO model. The earliest and minimum effort predictions require 6–30 h with 40 FOVs and 6–90 h with 15 FOVs, respectively. Conclusion We successfully developed a noninvasive morphology-based machine learning model to enhance the efficiency of establishing cell banks with single-cell-derived RECs for quantitatively predicting the future serial-passage potencies of clones. Conventional methods that can make noninvasive and quantitative predictions without wasting precious cells in the early stage are lacking; the proposed method will provide a more efficient and robust cell bank establishment process for allogenic therapeutic product manufacturing. Supplementary Information The online version contains supplementary material available at 10.1186/s41232-022-00214-w.
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Affiliation(s)
- Takashi Suyama
- Department of Life Science, Faculty of Medicine, Shimane University, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan.,PuREC Co. Ltd, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan
| | - Yuto Takemoto
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Tokai National Higher Education and Research System, Furocho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan
| | - Hiromi Miyauchi
- PuREC Co. Ltd, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan
| | - Yuko Kato
- Department of Life Science, Faculty of Medicine, Shimane University, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan.,PuREC Co. Ltd, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan
| | - Yumi Matsuzaki
- Department of Life Science, Faculty of Medicine, Shimane University, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan. .,PuREC Co. Ltd, 89-1 Enya-cho, Izumo, Shimane, 693-8501, Japan.
| | - Ryuji Kato
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Tokai National Higher Education and Research System, Furocho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan. .,Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Tokai National Higher Education and Research System, Furocho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan.
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Mawji I, Roberts EL, Dang T, Abraham B, Kallos MS. Challenges and Opportunities in Downstream Separation Processes for Mesenchymal Stromal Cells Cultured in Microcarrier-based Stirred Suspension Bioreactors. Biotechnol Bioeng 2022; 119:3062-3078. [PMID: 35962467 DOI: 10.1002/bit.28210] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Revised: 07/27/2022] [Accepted: 08/11/2022] [Indexed: 11/08/2022]
Abstract
Mesenchymal stromal cells (MSC) are a promising platform for regenerative medicine applications because of their multi-lineage differentiation abilities and ease of collection, isolation, and growth ex-vivo. To meet the demand for clinical applications, large scale manufacturing will be required using three-dimension culture platforms in vessels such as stirred suspension bioreactors. As MSCs are an adherent cell type, microcarriers are added to the culture to increase the available surface area for attachment and growth. Although extensive research has been performed on efficiently culturing MSCs using microcarriers, challenges persist in downstream processing including harvesting, filtration, and volume reduction which all play a critical role for the translation of cell therapies to the clinic. The objective of this review is to assess the current state of downstream technologies available for microcarrier-based MSC cultures. This includes a review of current research within the three stages: harvesting, filtration, and volume reduction. Using this information, a downstream process for MSCs is proposed which can be applied for a wide range of applications. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Inaara Mawji
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.,Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada
| | - Erin L Roberts
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.,Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada
| | - Tiffany Dang
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.,Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada
| | - Brett Abraham
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.,Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada
| | - Michael S Kallos
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada.,Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada
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Wiese DM, Wood CA, Ford BN, Braid LR. Cytokine Activation Reveals Tissue-Imprinted Gene Profiles of Mesenchymal Stromal Cells. Front Immunol 2022; 13:917790. [PMID: 35924240 PMCID: PMC9341285 DOI: 10.3389/fimmu.2022.917790] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2022] [Accepted: 06/10/2022] [Indexed: 11/30/2022] Open
Abstract
Development of standardized metrics to support manufacturing and regulatory approval of mesenchymal stromal cell (MSC) products is confounded by heterogeneity of MSC populations. Many reports describe fundamental differences between MSCs from various tissues and compare unstimulated and activated counterparts. However, molecular information comparing biological profiles of activated MSCs across different origins and donors is limited. To better understand common and source-specific mechanisms of action, we compared the responses of 3 donor populations each of human umbilical cord (UC) and bone marrow (BM) MSCs to TNF-α, IL-1β or IFN-γ. Transcriptome profiles were analysed by microarray and select secretome profiles were assessed by multiplex immunoassay. Unstimulated (resting) UC and BM-MSCs differentially expressed (DE) 174 genes. Signatures of TNF-α-stimulated BM and UC-MSCs included 45 and 14 new DE genes, respectively, while all but 7 of the initial 174 DE genes were expressed at comparable levels after licensing. After IL-1β activation, only 5 of the 174 DE genes remained significantly different, while 6 new DE genes were identified. IFN-γ elicited a robust transcriptome response from both cell types, yet nearly all differences (171/174) between resting populations were attenuated. Nine DE genes predominantly corresponding to immunogenic cell surface proteins emerged as a BM-MSC signature of IFN-γ activation. Changes in protein synthesis of select analytes correlated modestly with transcript levels. The dynamic responses of licensed MSCs documented herein, which attenuated heterogeneity between unstimulated populations, provide new insight into common and source-imprinted responses to cytokine activation and can inform strategic development of meaningful, standardized assays.
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Affiliation(s)
| | | | - Barry N. Ford
- Defence Research and Development Canada Suffield Research Centre, Casualty Management Section, Medicine Hat, AB, Canada
| | - Lorena R. Braid
- Aurora BioSolutions Inc., Medicine Hat, AB, Canada
- Simon Fraser University, Department of Molecular Biology and Biochemistry, Burnaby, BC, Canada
- *Correspondence: Lorena R. Braid, ;
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46
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Skibber MA, Olson SD, Prabhakara KS, Gill BS, Cox CS. Enhancing Mesenchymal Stromal Cell Potency: Inflammatory Licensing via Mechanotransduction. Front Immunol 2022; 13:874698. [PMID: 35874742 PMCID: PMC9297916 DOI: 10.3389/fimmu.2022.874698] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2022] [Accepted: 06/02/2022] [Indexed: 11/22/2022] Open
Abstract
Mesenchymal stromal cells (MSC) undergo functional maturation upon their migration from bone marrow and introduction to a site of injury. This inflammatory licensing leads to heightened immune regulation via cell-to-cell interaction and the secretion of immunomodulatory molecules, such as anti-inflammatory mediators and antioxidants. Pro-inflammatory cytokines are a recognized catalyst of inflammatory licensing; however, biomechanical forces, such as fluid shear stress, are a second, distinct class of stimuli that incite functional maturation. Here we show mechanotransduction, achieved by exposing MSC to various grades of wall shear stress (WSS) within a scalable conditioning platform, enhances the immunomodulatory potential of MSC independent of classical pro-inflammatory cytokines. A dose-dependent effect of WSS on potency is evidenced by production of prostaglandin E2 (PGE2) and indoleamine 2,3 dioxygenase 1 (IDO1), as well as suppression of tumor necrosis factor-α (TNF- α) and interferon-γ (IFN-γ) production by activated immune cells. Consistent, reproducible licensing is demonstrated in adipose tissue and bone marrow human derived MSC without significant impact on cell viability, cellular yield, or identity. Transcriptome analysis of WSS-conditioned BM-MSC elucidates the broader phenotypic implications on the differential expression of immunomodulatory factors. These results suggest mechanotransduction as a viable, scalable pre-conditioning alternative to pro-inflammatory cytokines. Enhancing the immunomodulatory capacity of MSC via biomechanical conditioning represents a novel cell therapy manufacturing approach.
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Affiliation(s)
- Max A. Skibber
- Department of Pediatric Surgery, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States
| | - Scott D. Olson
- Department of Pediatric Surgery, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States
- *Correspondence: Scott D. Olson, ; Brijesh S. Gill, ; Charles S. Cox Jr,
| | - Karthik S. Prabhakara
- Department of Pediatric Surgery, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States
| | - Brijesh S. Gill
- Department of Surgery, McGovern Medical School, University of Texas Health Science Center At Houston, Houston, TX, United States
- *Correspondence: Scott D. Olson, ; Brijesh S. Gill, ; Charles S. Cox Jr,
| | - Charles S. Cox
- Department of Pediatric Surgery, McGovern Medical School, University of Texas Health Science Center at Houston, Houston, TX, United States
- *Correspondence: Scott D. Olson, ; Brijesh S. Gill, ; Charles S. Cox Jr,
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47
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GMP Compliant Production of a Cryopreserved Adipose-Derived Stromal Cell Product for Feasible and Allogeneic Clinical Use. Stem Cells Int 2022; 2022:4664917. [PMID: 35769340 PMCID: PMC9236818 DOI: 10.1155/2022/4664917] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 05/20/2022] [Accepted: 05/31/2022] [Indexed: 12/13/2022] Open
Abstract
The emerging field of advanced therapy medicinal products (ATMP) holds promise of treating a variety of diseases. Adipose-derived stromal cells (ASCs) are currently being marketed or tested as cell-based therapies in numerous clinical trials. To ensure safety and efficacy of treatments, high-quality products must be manufactured. A good manufacturing practice (GMP) compliant and consistent manufacturing process including validated quality control methods is critical. Product design and formulation are equally important to ensure clinical feasibility. Here, we present a GMP-compliant, xeno-free, and semiautomated manufacturing process and quality controls, used for large-scale production of a cryopreserved off-the-shelf ASC product and tested in several phase I and II allogeneic clinical applications.
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48
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Ivanovska A, Wang M, Arshaghi TE, Shaw G, Alves J, Byrne A, Butterworth S, Chandler R, Cuddy L, Dunne J, Guerin S, Harry R, McAlindan A, Mullins RA, Barry F. Manufacturing Mesenchymal Stromal Cells for the Treatment of Osteoarthritis in Canine Patients: Challenges and Recommendations. Front Vet Sci 2022; 9:897150. [PMID: 35754551 PMCID: PMC9230578 DOI: 10.3389/fvets.2022.897150] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Accepted: 04/14/2022] [Indexed: 12/28/2022] Open
Abstract
The recent interest in advanced biologic therapies in veterinary medicine has opened up opportunities for new treatment modalities with considerable clinical potential. Studies with mesenchymal stromal cells (MSCs) from animal species have focused on in vitro characterization (mostly following protocols developed for human application), experimental testing in controlled studies and clinical use in veterinary patients. The ability of MSCs to interact with the inflammatory environment through immunomodulatory and paracrine mechanisms makes them a good candidate for treatment of inflammatory musculoskeletal conditions in canine species. Analysis of existing data shows promising results in the treatment of canine hip dysplasia, osteoarthritis and rupture of the cranial cruciate ligament in both sport and companion animals. Despite the absence of clear regulatory frameworks for veterinary advanced therapy medicinal products, there has been an increase in the number of commercial cell-based products that are available for clinical applications, and currently the commercial use of veterinary MSC products has outpaced basic research on characterization of the cell product. In the absence of quality standards for MSCs for use in canine patients, their safety, clinical efficacy and production standards are uncertain, leading to a risk of poor product consistency. To deliver high-quality MSC products for veterinary use in the future, there are critical issues that need to be addressed. By translating standards and strategies applied in human MSC manufacturing to products for veterinary use, in a collaborative effort between stem cell scientists and veterinary researchers and surgeons, we hope to facilitate the development of quality standards. We point out critical issues that need to be addressed, including a much higher level of attention to cell characterization, manufacturing standards and release criteria. We provide a set of recommendations that will contribute to the standardization of cell manufacturing methods and better quality assurance.
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Affiliation(s)
- Ana Ivanovska
- Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, Galway, Ireland
| | - Mengyu Wang
- Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, Galway, Ireland
| | - Tarlan Eslami Arshaghi
- Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, Galway, Ireland
| | - Georgina Shaw
- Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, Galway, Ireland
| | | | | | | | - Russell Chandler
- Orthopaedic Referral Service, Alphavet Veterinary Centre, Newport, United Kingdom
| | - Laura Cuddy
- Small Animal Surgery, Canine Sports Medicine and Rehabilitation, Veterinary Specialists Ireland, Summerhill, Ireland
| | - James Dunne
- Knocknacarra Veterinary Clinic, Ark Vets Galway, Galway, Ireland
| | - Shane Guerin
- Small Animal Surgery, Gilabbey Veterinary Hospital, Cork, Ireland
| | | | - Aidan McAlindan
- Northern Ireland Veterinary Specialists, Hillsborough, United Kingdom
| | - Ronan A Mullins
- Department of Small Animal Surgery, School of Veterinary Medicine, University College Dublin, Dublin, Ireland
| | - Frank Barry
- Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, Galway, Ireland
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Clinical Application of Induced Hepatocyte-like Cells Produced from Mesenchymal Stromal Cells: A Literature Review. Cells 2022; 11:cells11131998. [PMID: 35805080 PMCID: PMC9265349 DOI: 10.3390/cells11131998] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 06/15/2022] [Accepted: 06/16/2022] [Indexed: 11/17/2022] Open
Abstract
Liver disease is a leading cause of mortality worldwide, resulting in 1.3 million deaths annually. The vast majority of liver disease is caused by metabolic disease (i.e., NASH) and alcohol-induced hepatitis, and to a lesser extent by acute and chronic viral infection. Furthermore, multiple insults to the liver is becoming common due to the prevalence of metabolic and alcohol-related liver diseases. Despite this rising prevalence of liver disease, there are few treatment options: there are treatments for viral hepatitis C and there is vaccination for hepatitis B. Aside from the management of metabolic syndrome, no direct liver therapy has shown clinical efficacy for metabolic liver disease, there is very little for acute alcohol-induced liver disease, and liver transplantation remains the only effective treatment for late-stage liver disease. Traditional pharmacologic interventions have failed to appreciably impact the pathophysiology of alcohol-related liver disease or end-stage liver disease. The difficulties associated with developing liver-specific therapies result from three factors that are common to late-stage liver disease arising from any cause: hepatocyte injury, inflammation, and aberrant tissue healing. Hepatocyte injury results in tissue damage with inflammation, which sensitizes the liver to additional hepatocyte injury and stimulates hepatic stellate cells and aberrant tissue healing responses. In the setting of chronic liver insults, there is progressive scarring, the loss of hepatocyte function, and hemodynamic dysregulation. Regenerative strategies using hepatocyte-like cells that are manufactured from mesenchymal stromal cells may be able to correct this pathophysiology through multiple mechanisms of action. Preclinical studies support their effectiveness and recent clinical studies suggest that cell replacement therapy can be safe and effective in patients with liver disease for whom there is no other option.
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50
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Zheng D, Bhuvan T, Payne NL, Heng TSP. Secondary Lymphoid Organs in Mesenchymal Stromal Cell Therapy: More Than Just a Filter. Front Immunol 2022; 13:892443. [PMID: 35784291 PMCID: PMC9243307 DOI: 10.3389/fimmu.2022.892443] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Accepted: 05/19/2022] [Indexed: 11/13/2022] Open
Abstract
Mesenchymal stromal cells (MSCs) have demonstrated therapeutic potential in inflammatory models of human disease. However, clinical translation has fallen short of expectations, with many trials failing to meet primary endpoints. Failure to fully understand their mechanisms of action is a key factor contributing to the lack of successful commercialisation. Indeed, it remains unclear how the long-ranging immunomodulatory effects of MSCs can be attributed to their secretome, when MSCs undergo apoptosis in the lung shortly after intravenous infusion. Their apoptotic fate suggests that efficacy is not based solely on their viable properties, but also on the immune response to dying MSCs. The secondary lymphoid organs (SLOs) orchestrate immune responses and play a key role in immune regulation. In this review, we will discuss how apoptotic cells can modify immune responses and highlight the importance of MSC-immune cell interactions in SLOs for therapeutic outcomes.
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Affiliation(s)
- Di Zheng
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
| | - Tejasvini Bhuvan
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
| | - Natalie L. Payne
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
- Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, Australia
| | - Tracy S. P. Heng
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
- ARC Training Centre for Cell and Tissue Engineering Technologies, Monash University, Clayton, VIC, Australia
- *Correspondence: Tracy S. P. Heng,
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