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Ulpiano C, Salvador W, Franchi-Mendes T, Huang MC, Lin YH, Lin HT, Rodrigues CAV, Fernandes-Platzgummer A, Cabral JMS, Monteiro GA, da Silva CL. Continuous collection of human mesenchymal-stromal-cell-derived extracellular vesicles from a stirred tank reactor operated under xenogeneic-free conditions for therapeutic applications. Stem Cell Res Ther 2025; 16:210. [PMID: 40275409 PMCID: PMC12023423 DOI: 10.1186/s13287-025-04341-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2024] [Accepted: 04/11/2025] [Indexed: 04/26/2025] Open
Abstract
BACKGROUND Mesenchymal-stromal-cell-derived extracellular vesicles (MSC-EVs) play a key role in the paracrine effects of MSC and have demonstrated therapeutic potential in various preclinical models. However, clinical translation is hindered by manufacturing practices relying on planar culture systems, fetal bovine serum (FBS)-supplemented media, and non-scalable, low-purity EV isolation methods that fail to meet dose and safety requirements, underscoring the need for innovative approaches. In this study, we developed a scalable platform to manufacture human MSC-EVs at clinically relevant numbers, integrating continuous collection of EV-enriched conditioned media (CM) using a stirred-tank reactor (STR) under xenogeneic-free conditions and a scalable downstream process. METHODS Wharton's jelly-derived MSC (MSC(WJ)) were expanded using microcarriers in a controlled STR using human platelet lysate (hPL)-supplemented medium. Then, a 3-day EV production stage, featuring continuous harvesting of the CM, was established using a novel serum-/xeno(geneic)-free exosome depleted-hPL supplement. For the isolation of MSC-EVs, a scalable process was implemented by pairing tangential flow filtration and anion exchange chromatography. Isolated MSC-EVs were characterised using nanoparticle tracking analysis, protein and zeta potential quantification, western blot analysis of EV protein markers, transmission electron microscopy and uptake studies of fluorescently labelled-EVs. RESULTS The system sustained the efficient expansion of MSC(WJ), reaching a total of (6.03 ± 0.181) x 107 cells after 7 days, which corresponds to a 30.1 ± 0.740-fold expansion. Upon a 3-day continuous CM harvesting, a total of (2.13 ± 0.301) x 1012 EVs were isolated corresponding to a particle yield factor of (1.26 ± 0.186) x 104 EVs/cell/day. MSC-EVs presented high purity levels ((5.53 ± 1.55) x 109 particles/µg), a homogeneous small size distribution (mean diameter of 115 ± 4.88 nm), a surface charge of -23.4 ± 6.23 mV, positive detection of tetraspanins CD9 and CD63 and syntenin-1 and displayed a typical cup-shaped morphology. MSC-EVs were readily incorporated by endothelial cells and two human breast cancer cell lines. CONCLUSIONS Overall, the scalable and Good Manufacturing Practices (GMP)-compliant platform established herein enabled the reproducible manufacturing of MSC-EVs with high purity and generally accepted characteristics concerning size, protein markers, surface charge, morphology, and cellular internalization, validating its potential for future clinical applications.
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Affiliation(s)
- Cristiana Ulpiano
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - William Salvador
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Teresa Franchi-Mendes
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | | | | | | | - Carlos A V Rodrigues
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Gabriel A Monteiro
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
- Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
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Hassan MNF, Yazid MD, Yunus MHBM, Lokanathan Y, Ng MH, Idrus RBH, Tang YL, Ng SN, Law JX. Comparing the growth kinetics and characteristics of Wharton's jelly derived mesenchymal stem cells expanded using different culture mediums. Cytotechnology 2025; 77:24. [PMID: 39711971 PMCID: PMC11659549 DOI: 10.1007/s10616-024-00682-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Accepted: 12/07/2024] [Indexed: 12/24/2024] Open
Abstract
Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) can be isolated from umbilical cords which is abundant and easy to obtain. Due to their potent immunosuppressive properties, multilineage differentiation potential, and lack of ethical issues, WJ-MSCs are considered a promising candidate for therapeutic applications. However, large-scale in vitro expansion is necessary to obtain enough cells for therapeutic purposes. Therefore, this study aimed to optimize cell culture conditions and determine the characteristics of expanded WJ-MSCs. WJ-MSCs were seeded in 6-well plates at a density of 5000 cells/cm2 and cultured with different mediums, including DMEM-LG+10% FBS, DMEM-LG+10% HPL, serum-free commercial medium 1, serum-free GMP grade commercial medium 2, and HPL supplemented commercial medium 3. The cell morphology and growth kinetics were compared, and the three most suitable mediums were selected for further experiments. WJ-MSCs were then cultured in the selected mediums at seeding densities ranging from 1000 to 5000 cells/cm2, and cell growth kinetics were analysed. WJ-MSCs cultured in the selected mediums were characterized by their immunophenotype, tri-lineage differentiation potential and immunosuppression property. WJ-MSCs cultured with DMEM-LG+10% HPL, commercial medium 1 and commercial medium 2 showed smaller size, significantly higher cell yield, and shorter population doubling time than those cultured in other mediums. Hence, these three mediums were selected for further experiments. Only DMEM-LG + 10% HPL medium maintained high cell yields (1.48 ± 0.14 × 106 with bFGF and 1.56 ± 0.17 × 106 without bFGF) at the lowest seeding density tested. However, WJ-MSCs cultured in all three mediums expressed the MSC surface markers, were able to suppress PBMC proliferation, and could differentiate into adipogenic, chondrogenic and osteogenic lineages. In summary, DMEM-LG+10% HPL is the best medium for WJ-MSC expansion, as it provides the highest cell yield without compromising cell characteristics and functionality. The potential of this medium for large-scale expansion using a bioreactor or multilayered flask should be investigated in the future.
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Affiliation(s)
- Muhammad Najib Fathi Hassan
- Department of Tissue Engineering and Regenerative Medicine (DTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Muhammad Dain Yazid
- Department of Tissue Engineering and Regenerative Medicine (DTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | | | - Yogeswaran Lokanathan
- Department of Tissue Engineering and Regenerative Medicine (DTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Min Hwei Ng
- Department of Tissue Engineering and Regenerative Medicine (DTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Ruszymah Bt Hj Idrus
- Department of Tissue Engineering and Regenerative Medicine (DTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Yee Loong Tang
- Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - See Nguan Ng
- Ming Medical Sdn Bhd, D3-3 (2Nd Floor), Block D3 Dana 1 Commercial Centre, Jalan PJU 1a/46, 47301 Petaling Jaya, Selangor Malaysia
| | - Jia Xian Law
- Department of Tissue Engineering and Regenerative Medicine (DTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
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Strecanska M, Sekelova T, Smolinska V, Kuniakova M, Nicodemou A. Automated Manufacturing Processes and Platforms for Large-scale Production of Clinical-grade Mesenchymal Stem/ Stromal Cells. Stem Cell Rev Rep 2025; 21:372-389. [PMID: 39546186 PMCID: PMC11872983 DOI: 10.1007/s12015-024-10812-5] [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] [Accepted: 10/24/2024] [Indexed: 11/17/2024]
Abstract
Mesenchymal stem/stromal cells (MSCs) hold immense potential for regenerative medicine due to their remarkable regenerative and immunomodulatory properties. However, their therapeutic application requires large-scale production under stringent regulatory standards and Good Manufacturing Practice (GMP) guidelines, presenting significant challenges. This review comprehensively evaluates automated manufacturing processes and platforms for the scalable production of clinical-grade MSCs. Various large-scale culture vessels, including multilayer flasks and bioreactors, are analyzed for their efficacy in MSCs expansion. Furthermore, automated MSCs production platforms, such as Quantum® Cell Expansion System, CliniMACS Prodigy®, NANT001/ XL, CellQualia™, Cocoon® Platform, and Xuri™ Cell Expansion System W25 are reviewed and compared as well. We also underscore the importance of optimizing culture media specifically emphasizing the shift from fetal bovine serum to humanized or serum-free alternatives to meet GMP standards. Moreover, advances in alternative cryopreservation methods and controlled-rate freezing systems, that offer promising improvements in MSCs preservation, are discussed as well. In conclusion, advancing automated manufacturing processes and platforms is essential for realizing the full potential of MSCs-based regenerative medicine and accomplishing the increasing demand for cell-based therapies. Collaborative initiatives involving industry, academia, and regulatory bodies are emphasized to accelerate the translation of MSCs-based therapies into clinical practice.
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Affiliation(s)
- Magdalena Strecanska
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, Bratislava, Bratislava, 811 08, Slovakia
- National Institute of Rheumatic Diseases, Nabrezie I. Krasku 4, Piestany, 921 12, Slovakia
| | - Tatiana Sekelova
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, Bratislava, Bratislava, 811 08, Slovakia
- National Institute of Rheumatic Diseases, Nabrezie I. Krasku 4, Piestany, 921 12, Slovakia
| | - Veronika Smolinska
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, Bratislava, Bratislava, 811 08, Slovakia
- National Institute of Rheumatic Diseases, Nabrezie I. Krasku 4, Piestany, 921 12, Slovakia
| | - Marcela Kuniakova
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, Bratislava, Bratislava, 811 08, Slovakia
| | - Andreas Nicodemou
- Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, Bratislava, Bratislava, 811 08, Slovakia.
- National Institute of Rheumatic Diseases, Nabrezie I. Krasku 4, Piestany, 921 12, Slovakia.
- GAMMA-ZA, Kollarova 8, Trencin, 911 01, Slovakia.
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Da Silva K, Kumar P, Choonara YE. The paradigm of stem cell secretome in tissue repair and regeneration: Present and future perspectives. Wound Repair Regen 2025; 33:e13251. [PMID: 39780313 PMCID: PMC11711308 DOI: 10.1111/wrr.13251] [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: 08/01/2024] [Revised: 12/04/2024] [Accepted: 12/13/2024] [Indexed: 01/11/2025]
Abstract
As the number of patients requiring organ transplants continues to rise exponentially, there is a dire need for therapeutics, with repair and regenerative properties, to assist in alleviating this medical crisis. Over the past decade, there has been a shift from conventional stem cell treatments towards the use of the secretome, the protein and factor secretions from cells. These components may possess novel druggable targets and hold the key to profoundly altering the field of regenerative medicine. Despite the progress in this field, clinical translation of secretome-containing products is limited by several challenges including but not limited to ensuring batch-to-batch consistency, the prevention of further heterogeneity, production of sufficient secretome quantities, product registration, good manufacturing practice protocols and the pharmacokinetic/pharmacodynamic profiles of all the components. Despite this, the secretome may hold the key to unlocking the regenerative blockage scientists have encountered for years. This review critically analyses the secretome derived from different cell sources and used in several tissues for tissue regeneration. Furthermore, it provides an overview of the current delivery strategies and the future perspectives for the secretome as a potential therapeutic. The success and possible shortcomings of the secretome are evaluated.
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Affiliation(s)
- Kate Da Silva
- Wits Advanced Drug Delivery Platform (WADDP) Research Unit, Department of Pharmacy and Pharmacology, School of Therapeutic Sciences, Faculty of Health SciencesUniversity of the WitwatersrandJohannesburgSouth Africa
| | - Pradeep Kumar
- Wits Advanced Drug Delivery Platform (WADDP) Research Unit, Department of Pharmacy and Pharmacology, School of Therapeutic Sciences, Faculty of Health SciencesUniversity of the WitwatersrandJohannesburgSouth Africa
| | - Yahya E. Choonara
- Wits Advanced Drug Delivery Platform (WADDP) Research Unit, Department of Pharmacy and Pharmacology, School of Therapeutic Sciences, Faculty of Health SciencesUniversity of the WitwatersrandJohannesburgSouth Africa
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Soder RP, Dudley NR, Dawn B. Microcarrier-based clinical-grade manufacturing of therapeutic Wharton's jelly mesenchymal stromal cells. Cytotherapy 2024; 26:1556-1565. [PMID: 39093227 DOI: 10.1016/j.jcyt.2024.07.003] [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: 05/22/2024] [Revised: 07/01/2024] [Accepted: 07/02/2024] [Indexed: 08/04/2024]
Abstract
Due to their immunomodulatory and anti-inflammatory properties, tissue repair capabilities and regenerative potential, Wharton's jelly mesenchymal stem/stromal cells (WJMSCs) have been widely investigated as potential treatment for diverse clinical indications. WJMSCs have been found to be well-tolerated and safe, positioning them as a promising candidate for cellular therapy. To address the commercial need for manufacturing WJMSCs for clinical applications, the production scale should be capable of generating large quantities of cells that retain their expected identity, purity and potency. This study aimed to establish a current Good Manufacturing Practice (cGMP) compliant robust and scalable expansion process representing a critical step towards a cGMP-compliant large-scale production platform for WJMSC-based clinical applications. Using our in-house cGMP-manufactured WJMSCs, which are currently being tested in a Phase Ib clinical trial (NCT03158896) using two-dimensional (2D) planar systems, we optimized various culture parameters including type of microcarrier, seeding density, agitation and culture feed regime in a 3D microcarrier-based culture system in spinner flasks. The results showed that cell adhesion was potentiated under intermittent stirring (3 min of agitation at 25 rpm followed by a period of non-agitation for 30 min), with reduced supplementation (0.05%) during the initial 8 h of cultivation with an initial cell concentration of 0.45 × 105 cells/mL. Microcarrier-based WJMSC expansion in spinner flasks achieved greater cell densities of 1.67 × 106 cells/mL with a maximum of 37-fold expansion, yielding ∼84 × 106 cells after 6 days of culture with a 95% harvest efficiency. Additionally, post 3D expansion, WJMSCs maintained their phenotypic characteristics, differentiation potential, normal karyotype, functional properties and sterility in the culture systems evaluated. This cGMP-compliant expansion process described herein demonstrates a successful transition of an established 2D planar culture process of clinical grade WJMSCs to 3D microcarrier-based suspension process generating higher cell yields, is cost-effective and represents an important step toward fulfilling the commercial demand of clinical grade mesenchymal stromal cells.
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Affiliation(s)
- Rupal P Soder
- Midwest Stem Cell Therapy Center, University of Kansas Medical Center, Kansas City, Kansas, USA.
| | - Nathaniel R Dudley
- Midwest Stem Cell Therapy Center, University of Kansas Medical Center, Kansas City, Kansas, USA
| | - Buddhadeb Dawn
- Department of Internal Medicine, Kirk Kerkorian School of Medicine at the University of Nevada, Las Vegas, Nevada, USA
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Bandarra-Tavares H, Franchi-Mendes T, Ulpiano C, Morini S, Kaur N, Harris-Becker A, Vemuri MC, Cabral JMS, Fernandes-Platzgummer A, da Silva CL. Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system. Cytotherapy 2024; 26:749-756. [PMID: 38506771 DOI: 10.1016/j.jcyt.2024.03.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2023] [Revised: 01/29/2024] [Accepted: 03/02/2024] [Indexed: 03/21/2024]
Abstract
BACKGROUND & AIMS Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium. METHODS MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 106 cells per spinner flask, i.e., 2.8 × 103 cells per cm2 of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h. RESULTS Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1). CONCLUSIONS Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.
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Affiliation(s)
- Hélder Bandarra-Tavares
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Teresa Franchi-Mendes
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cristiana Ulpiano
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Sara Morini
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Navjot Kaur
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Abigail Harris-Becker
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Mohan C Vemuri
- Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Ana Fernandes-Platzgummer
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
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Dos Santos NCD, Bruzadelle-Vieira P, de Cássia Noronha N, Mizukami-Martins A, Orellana MD, Bentley MVLB, Covas DT, Swiech K, Malmegrim KCR. Transitioning from static to suspension culture system for large-scale production of xeno-free extracellular vesicles derived from mesenchymal stromal cells. Biotechnol Prog 2024; 40:e3419. [PMID: 38247123 DOI: 10.1002/btpr.3419] [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: 06/03/2023] [Revised: 11/10/2023] [Accepted: 11/27/2023] [Indexed: 01/23/2024]
Abstract
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC-EV in suspension cultures using spinner flasks and human collagen-coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T-flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold-increase was 5.7-fold for the 3D culture system and 4.6-fold for the 2D culture system, signifying a fold-change of 1.2 (calculated as the ratio of fold-increase 3D to fold-increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T-flask cultures. The total cell production in the spinner flask cultures was 5.2-fold higher than that in T-flask cultures. While the EV specific production (particles/cell) in T-flask cultures (4.40 ± 1.21 × 108 particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 108 particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC-EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC-EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno-free conditions.
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Affiliation(s)
| | - Paula Bruzadelle-Vieira
- Department of Pharmaceutical Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Nádia de Cássia Noronha
- School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
- Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Amanda Mizukami-Martins
- Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Maristela Delgado Orellana
- Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Maria Vitória L B Bentley
- Department of Pharmaceutical Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Dimas Tadeu Covas
- Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Kamilla Swiech
- Department of Pharmaceutical Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
- Center for Cell-Based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
| | - Kelen Cristina Ribeiro Malmegrim
- Department of Pharmaceutical Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
- Department of Clinical Analysis, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil
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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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9
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Rajput SN, Naeem BK, Ali A, Salim A, Khan I. Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine. World J Stem Cells 2024; 16:410-433. [PMID: 38690517 PMCID: PMC11056638 DOI: 10.4252/wjsc.v16.i4.410] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Revised: 02/01/2024] [Accepted: 03/18/2024] [Indexed: 04/25/2024] Open
Abstract
BACKGROUND Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application. AIM To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols. METHODS Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated β-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection. RESULTS Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and β-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination. CONCLUSION This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.
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Affiliation(s)
- Shafiqa Naeem Rajput
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
| | - Bushra Kiran Naeem
- Surgical Unit 4, Dr. Ruth KM Pfau Civil Hospital, Karachi 74400, Pakistan
| | - Anwar Ali
- Department of Physiology, University of Karachi, Karachi 75270, Pakistan
| | - Asmat Salim
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
| | - Irfan Khan
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Sindh, Pakistan
- Center for Regenerative Medicine and Stem Cells Research, and Department of Ophthalmology and Visual Sciences, The Aga Khan University, Karachi 74800, Sindh, Pakistan.
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10
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López-Fernández A, Garcia-Gragera V, Lecina M, Vives J. Identification of critical process parameters for expansion of clinical grade human Wharton's jelly-derived mesenchymal stromal cells in stirred-tank bioreactors. Biotechnol J 2024; 19:e2300381. [PMID: 38403461 DOI: 10.1002/biot.202300381] [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: 08/01/2023] [Revised: 12/11/2023] [Accepted: 01/02/2024] [Indexed: 02/27/2024]
Abstract
Cell therapies based on multipotent mesenchymal stromal cells (MSCs) are traditionally produced using 2D culture systems and platelet lysate- or serum-containing media (SCM). Although cost-effective for single-dose autologous treatments, this approach is not suitable for larger scale manufacturing (e.g., multiple-dose autologous or allogeneic therapies with banked MSCs); automated, scalable and Good Manufacturing Practices (GMP)-compliant platforms are urgently needed. The feasibility of transitioning was evaluated from an established Wharton's jelly MSCs (WJ-MSCs) 2D production strategy to a new one with stirred-tank bioreactors (STRs). Experimental conditions included four GMP-compliant xeno- and serum-free media (XSFM) screened in 2D conditions and two GMP-grade microcarriers assessed in 0.25 L-STRs using SCM. From the screening, a XSFM was selected and compared against SCM using the best-performing microcarrier. It was observed that SCM outperformed the 2D-selected medium in STRs, reinforcing the importance of 2D-to-3D transition studies before translation into clinical production settings. It was also found that attachment efficiency and microcarrier colonization were essential to attain higher fold expansions, and were therefore defined as critical process parameters. Nevertheless, WJ-MSCs were readily expanded in STRs with both media, preserving critical quality attributes in terms of identity, viability and differentiation potency, and yielding up to 1.47 × 109 cells in a real-scale 2.4-L batch.
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Affiliation(s)
- Alba López-Fernández
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
| | - Víctor Garcia-Gragera
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Barcelona, Spain
- Engineering Materials Group (GEMAT), Bioprocessing Lab, IQS School of Engineering, Universitat Ramón Llull, Barcelona, Spain
| | - Martí Lecina
- Engineering Materials Group (GEMAT), Bioprocessing Lab, IQS School of Engineering, Universitat Ramón Llull, Barcelona, Spain
| | - Joaquim Vives
- Servei de Teràpia Cel·lular i Avançada, Banc de Sang i Teixits, 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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11
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Cifuentes SJ, Domenech M. Heparin-collagen I bilayers stimulate FAK/ERK½ signaling via α2β1 integrin to support the growth and anti-inflammatory potency of mesenchymal stromal cells. J Biomed Mater Res A 2024; 112:65-81. [PMID: 37723658 DOI: 10.1002/jbm.a.37614] [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: 05/15/2023] [Revised: 08/25/2023] [Accepted: 09/01/2023] [Indexed: 09/20/2023]
Abstract
Understanding mesenchymal stromal cells (MSCs) growth mechanisms in response to surface chemistries is essential to optimize culture methods for high-quality and robust cell yields in cell manufacturing applications. Heparin (HEP) and collagen 1 (COL) substrates have been reported to enhance cell adhesion, growth, viability, and secretory potential in MSCs. However, the biomolecular mechanisms underlying the benefits of combined HEP/COL substrates are unknown. This work used HEP/COL bilayered surfaces to investigate the role of integrin-HEP interactions in the advantages of MSC culture. The layer-by-layer approach (LbL) was used to create HEP/COL bilayers, which were made up of stacks of 8 and 9 layers that combined HEP and COL in an alternate arrangement. Surface spectroscopic investigations and laser scanning microscopy evaluations verified the biochemical fingerprint of each component and a total stacked bilayer thickness of roughly 150 nm. Cell growth and apoptosis in response to IC50 and IC75 levels of BTT-3033 and Cilengitide, α2β1 and αvβ3 integrin inhibitors respectively, were evaluated on HEP/COL coated surfaces using two bone marrow-derived MSC donors. While integrin activity did not affect cell growth rates, it significantly affected cell adhesion and apoptosis on HEP/COL surfaces. HEP-ending HEP/COL surfaces significantly increased FAK-ERK½ phosphorylation and endogenous cell COL deposition compared to COL, COL-ending HEP/COL and uncoated surfaces. BTT-3033 but not Cilengitide treatment markedly affected FAK-ERK½ activity levels on HEP-ending HEP/COL surfaces supporting a major role for α2β1 activity. BTT-3033 treatment on HEP-ending bilayers reduced MSC-mediated macrophage inhibitory activity and altered the cytokine profile of co-cultures. Overall, this study supports a novel role for HEP in regulating the survival and potency of MSCs via enhancing the α2β1-FAK-ERK½ signaling mechanism.
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Affiliation(s)
- Said J Cifuentes
- Bioengineering Graduate Program, University of Puerto Rico Mayaguez, Mayaguez, Puerto Rico, USA
| | - Maribella Domenech
- Bioengineering Graduate Program, University of Puerto Rico Mayaguez, Mayaguez, Puerto Rico, USA
- Department of Chemical Engineering, University of Puerto Rico Mayaguez, Mayaguez, Puerto Rico, USA
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12
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Ebrahimi F, Pirouzmand F, Cosme Pecho RD, Alwan M, Yassen Mohamed M, Ali MS, Hormozi A, Hasanzadeh S, Daei N, Hajimortezayi Z, Zamani M. Application of mesenchymal stem cells in regenerative medicine: A new approach in modern medical science. Biotechnol Prog 2023; 39:e3374. [PMID: 37454344 DOI: 10.1002/btpr.3374] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Revised: 06/14/2023] [Accepted: 06/21/2023] [Indexed: 07/18/2023]
Abstract
Mesenchymal Stem Cells (MSCs) are non-hematopoietic and multipotent stem cells, which have been considered in regenerative medicine. These cells are easily separated from different sources, such as bone marrow (BM), umbilical cord (UC), adipose tissue (AT), and etc. MSCs have the differentiation capability into chondrocytes, osteocytes, and adipocytes; This differentiation potential along with the paracrine properties have made them a key choice for tissue repair. MSCs also have various advantages over other stem cells, which is why they have been extensively studied in recent years. The effectiveness of MSCs-based therapies depend on several factors, including differentiation status at the time of use, concentration per injection, delivery method, the used vehicle, and the nature and extent of the damage. Although, MSCs have emerged promising sources for regenerative medicine, there are potential risks regarding their safety in their clinical use, including tumorigenesis, lack of availability, aging, and sensitivity to toxic environments. In this study, we aimed to discuss how MSCs may be useful in treating defects and diseases. To this aim, we will review recent advances of MSCs action mechanisms in regenerative medicine, as well as the most recent clinical trials. We will also have a brief overview of MSCs resources, differences between their sources, culture conditions, extraction methods, and clinical application of MSCs in various fields of regenerative medicine.
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Affiliation(s)
- Faezeh Ebrahimi
- Medical Laboratory, Student Research Committee, Gonabad University of Medical Sciences, Gonabad, Iran
| | - Farzaneh Pirouzmand
- Infectious Diseases Research Center, Gonabad University of Medical Sciences, Gonabad, Iran
| | | | - Mariam Alwan
- Medical Technical College, Al-Farahidi University, Baghdad, Iraq
| | | | | | - Arezoo Hormozi
- Medical Laboratory, Student Research Committee, Gonabad University of Medical Sciences, Gonabad, Iran
| | - Sajedeh Hasanzadeh
- Medical Laboratory, Student Research Committee, Gonabad University of Medical Sciences, Gonabad, Iran
| | - Narges Daei
- Medical Laboratory, Student Research Committee, Gonabad University of Medical Sciences, Gonabad, Iran
| | - Zahra Hajimortezayi
- Medical Laboratory, Student Research Committee, Gonabad University of Medical Sciences, Gonabad, Iran
| | - Majid Zamani
- Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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13
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Roberts EL, Abraham BD, Dang T, Gysel E, Mehrpouyan S, Alizadeh AH, Koch TG, Kallos MS. Computer controlled expansion of equine cord blood mesenchymal stromal cells on microcarriers in 3 L vertical-wheel ® bioreactors. Front Bioeng Biotechnol 2023; 11:1250077. [PMID: 37929186 PMCID: PMC10622666 DOI: 10.3389/fbioe.2023.1250077] [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: 06/29/2023] [Accepted: 09/11/2023] [Indexed: 11/07/2023] Open
Abstract
Mesenchymal stromal cells (MSCs) are an ideal cell source for allogenic cell therapy due to their immunomodulatory and differentiation properties. Equine MSCs (eMSCs) have been found to be a promising treatment for equine joint injuries including meniscal injuries, cartilage degradation, and osteoarthritis. Although the use of eMSCs has shown efficacy in preliminary studies, challenges associated with biomanufacturing remain. To achieve the required cell numbers for clinical application, bioreactor-based processes are required. Initial studies have shown that eMSCs can be cultivated in microcarrier-based, stirred suspension bioreactor culture at the laboratory 0.1 L scale using a Vertical-Wheel® (VW) bioreactor. However, investigations regarding scale up of these processes to the required biomanufacturing scales are required. This study investigated the scale-up of a equine cord blood MSC (eCB-MSC) bioprocess in VW bioreactors at three scales. This included scale-up from the 0.1-0.5 L bioreactor, scale-up from static culture to the 3 L computer-controlled bioreactor, and scale-up into the 3 L computer-controlled bioreactor using a mock clinical trial process. Results from the various scale-up experiments demonstrated similar cell expansion at the various tested scales. The 3 L computer-controlled system resulted in a final cell densities of 1.5 × 105 cells/cm2 on average, achieving 1.5 × 109 harvested cells. Biological testing of the cells showed that cell phenotype and functionality were maintained after scale-up. These findings demonstrate the scalability of an eCB-MSC bioprocess using microcarriers in VW bioreactors to achieve clinically relevant cell numbers, a critical step to translate MSC treatments from research to clinical applications. This study also represents the first known published study expanding any cell type in the 3 L VW bioreactor.
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Affiliation(s)
- E. L. Roberts
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
- Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
| | - B. D. Abraham
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
- Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
| | - T. Dang
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
- Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
| | - E. Gysel
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
- Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
| | - S. Mehrpouyan
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
| | - A. H. Alizadeh
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
| | - T. G. Koch
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
- eQcell Inc, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
| | - M. S. Kallos
- Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
- Department of Biomedical Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada
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14
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Wang X, Ouyang L, Chen W, Cao Y, Zhang L. Efficient expansion and delayed senescence of hUC-MSCs by microcarrier-bioreactor system. Stem Cell Res Ther 2023; 14:284. [PMID: 37794520 PMCID: PMC10552362 DOI: 10.1186/s13287-023-03514-1] [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: 06/06/2023] [Accepted: 09/25/2023] [Indexed: 10/06/2023] Open
Abstract
BACKGROUND Human umbilical cord mesenchymal stem cells (hUC-MSCs) are widely used in cell therapy due to their robust immunomodulatory and tissue regenerative capabilities. Currently, the predominant method for obtaining hUC-MSCs for clinical use is through planar culture expansion, which presents several limitations. Specifically, continuous cell passaging can lead to cellular aging, susceptibility to contamination, and an absence of process monitoring and control, among other limitations. To overcome these challenges, the technology of microcarrier-bioreactor culture was developed with the aim of ensuring the therapeutic efficacy of cells while enabling large-scale expansion to meet clinical requirements. However, there is still a knowledge gap regarding the comparison of biological differences in cells obtained through different culture methods. METHODS We developed a culture process for hUC-MSCs using self-made microcarrier and stirred bioreactor. This study systematically compares the biological properties of hUC-MSCs amplified through planar culture and microcarrier-bioreactor systems. Additionally, RNA-seq was employed to compare the differences in gene expression profiles between the two cultures, facilitating the identification of pathways and genes associated with cell aging. RESULTS The findings revealed that hUC-MSCs expanded on microcarriers exhibited a lower degree of cellular aging compared to those expanded through planar culture. Additionally, these microcarrier-expanded hUC-MSCs showed an enhanced proliferation capacity and a reduced number of cells in the cell cycle retardation period. Moreover, bioreactor-cultured cells differ significantly from planar cultures in the expression of genes associated with the cytoskeleton and extracellular matrix. CONCLUSIONS The results of this study demonstrate that our microcarrier-bioreactor culture method enhances the proliferation efficiency of hUC-MSCs. Moreover, this culture method exhibits the potential to delay the process of cell aging while preserving the essential stem cell properties of hUC-MSCs.
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Affiliation(s)
- Xia Wang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China
| | - Liming Ouyang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
| | - Wenxia Chen
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China
| | - Yulin Cao
- Beijing Tang Yi Hui Kang Biomedical Technology Co., LTD, Beijing, 100032, People's Republic of China
| | - Lixin Zhang
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, People's Republic of China
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15
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Couto PS, Stibbs DJ, Rotondi MC, Takeuchi Y, Rafiq QA. Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks. Appl Microbiol Biotechnol 2023; 107:5669-5685. [PMID: 37470820 PMCID: PMC10439856 DOI: 10.1007/s00253-023-12634-w] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Revised: 06/06/2023] [Accepted: 06/11/2023] [Indexed: 07/21/2023]
Abstract
Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 105 cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck. KEY POINTS: • hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF • Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days • The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics.
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Affiliation(s)
- Pedro Silva Couto
- Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT UK
| | - Dale J. Stibbs
- Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT UK
| | - Marco C. Rotondi
- Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT UK
| | - Yasuhiro Takeuchi
- Division of Infection and Immunity, University College London, Gower Street, London, WC1E 6BT UK
- Biotherapeutics and Advanced Therapies, Scientific Research and Innovation, Medicines, and Healthcare Products Regulatory Agency, South Mimms, EN6 3QG UK
| | - Qasim A. Rafiq
- Department of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT UK
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16
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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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17
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Li J, Wu Y, Yao X, Tian Y, Sun X, Liu Z, Ye X, Wu C. Preclinical Research of Stem Cells: Challenges and Progress. Stem Cell Rev Rep 2023:10.1007/s12015-023-10528-y. [PMID: 37097496 DOI: 10.1007/s12015-023-10528-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/06/2023] [Indexed: 04/26/2023]
Abstract
In recent years, great breakthroughs have been made in basic research and clinical applications of stem cells in regenerative medicine and other fields, which continue to inspire people to explore the field of stem cells. With nearly unlimited self-renewal ability, stem cells can generate at least one type of highly differentiated daughter cell, which provides broad development prospects for the treatment of human organ damage and other diseases. In the field of stem cell research, related technologies for inducing or isolating stem cells are relatively mature, and a variety of stable stem cell lines have been successfully constructed. To realize the full clinical application of stem cells as soon as possible, it is more and more important to further optimize each stage of stem cell research while conforming to Current Good Manufacture Practices (cGMP) standards. Here, we synthesized recent developments in stem cell research and focus on the introduction of xenogenicity in the preclinical research process and the remaining problems of various cell bioreactors. Our goal is to promote the development of technologies for xeno-free culture and clinical expansion of stem cells through in-depth discussion of current research. This review will provide new insight into stem cell research protocols and will contribute to the creation of efficient and stable stem cell expansion systems.
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Affiliation(s)
- Jinhu Li
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yurou Wu
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xiang Yao
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yao Tian
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xue Sun
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Zibo Liu
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Xun Ye
- School of Pharmacy, School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Chunjie Wu
- Innovative Institute of Chinese Medicine and Pharmacy/Academy for Interdiscipline, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
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18
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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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19
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Jeske R, Chen X, Ma S, Zeng EZ, Driscoll T, Li Y. Bioreactor Expansion Reconfigures Metabolism and Extracellular Vesicle Biogenesis of Human Adipose-derived Stem Cells In Vitro. Biochem Eng J 2022; 188:108711. [PMID: 36540623 PMCID: PMC9762695 DOI: 10.1016/j.bej.2022.108711] [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: 11/05/2022]
Abstract
Human mesenchymal stem cells (hMSCs), including human adipose tissue-derived stem cells (hASCs), as well as the secreted extracellular vesicles (EVs), are promising therapeutics in treating inflammatory and neural degenerative diseases. However, prolonged expansion can lead to cellular senescence characterized by a gradual loss of self-renewal ability while altering secretome composition and EV generation. Additionally, hMSCs are highly sensitive to biophysical microenvironment in bioreactor systems utilized in scaling production. In this study, hASCs grown on Plastic Plus or Synthemax II microcarriers in a spinner flask bioreactor (SFB) system were compared to traditional 2D culture. The SFB microenvironment was found to increase the expression of genes associated with hASC stemness, nicotinamide adenine dinucleotide (NAD+) metabolism, glycolysis, and the pentose phosphate pathway as well as alter cytokine secretion (e.g., PGE2 and CXCL10). Elevated reactive oxidative species levels in hASCs of SFB culture were observed without increasing rates of cellular senescence. Expression levels of Sirtuins responsible for preventing cellular senescence through anti-oxidant and DNA repair mechanisms were also elevated in SFB cultures. In particular, the EV biogenesis genes were significantly upregulated (3-10 fold) and the EV production increased 40% per cell in SFB cultures of hASCs. This study provides advanced understanding of hASC sensitivity to the bioreactor microenvironment for EV production and bio-manufacturing towards the applications in treating inflammatory and neural degenerative diseases.
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Affiliation(s)
- Richard Jeske
- Department of Chemical and Biomedical Engineering, FAMU-FSU college of engineering, Florida state university, USA
| | - Xingchi Chen
- Department of Chemical and Biomedical Engineering, FAMU-FSU college of engineering, Florida state university, USA
| | - Shaoyang Ma
- Department of Chemical and Biomedical Engineering, FAMU-FSU college of engineering, Florida state university, USA
| | - Eric Z Zeng
- Department of Chemical and Biomedical Engineering, FAMU-FSU college of engineering, Florida state university, USA
| | - Tristan Driscoll
- Department of Chemical and Biomedical Engineering, FAMU-FSU college of engineering, Florida state university, USA
| | - Yan Li
- Department of Chemical and Biomedical Engineering, FAMU-FSU college of engineering, Florida state university, USA
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20
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Padhiar C, Aruni AW, Abhaya M, Muthuchamy M, Dhanraj AK, Ganesan V, Bovas FB, Rajakani SN. GMP compliant clinical grade and xenofree manufacturing of human Wharton’s jelly derived mesenchymal stem cell from pooled donors. Biochem Eng J 2022. [DOI: 10.1016/j.bej.2022.108470] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
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21
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Srinivasan A, Sathiyanathan P, Yin L, Liu TM, Lam A, Ravikumar M, Smith RAA, Loh HP, Zhang Y, Ling L, Ng SK, Yang YS, Lezhava A, Hui J, Oh S, Cool SM. Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion. Cytotherapy 2022; 24:456-472. [PMID: 35227601 DOI: 10.1016/j.jcyt.2021.11.009] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Revised: 10/24/2021] [Accepted: 11/08/2021] [Indexed: 02/06/2023]
Abstract
Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.
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Affiliation(s)
- Akshaya Srinivasan
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | | | - Lu Yin
- Bioprocessing Technology Institute, A*STAR, Singapore
| | - Tong Ming Liu
- Institute of Molecular and Cell Biology, A*STAR, Singapore
| | - Alan Lam
- Bioprocessing Technology Institute, A*STAR, Singapore
| | - Maanasa Ravikumar
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Institute of Molecular and Cell Biology, A*STAR, Singapore
| | | | - Han Ping Loh
- Bioprocessing Technology Institute, A*STAR, Singapore
| | - Ying Zhang
- Institute of Molecular and Cell Biology, A*STAR, Singapore
| | - Ling Ling
- Institute of Molecular and Cell Biology, A*STAR, Singapore
| | - Say Kong Ng
- Bioprocessing Technology Institute, A*STAR, Singapore
| | | | - Alexander Lezhava
- Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore
| | - James Hui
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
| | - Steve Oh
- Bioprocessing Technology Institute, A*STAR, Singapore.
| | - Simon M Cool
- Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Institute of Molecular and Cell Biology, A*STAR, Singapore.
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22
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Dai Y, Cui X, Zhang G, Mohsin A, Xu H, Zhuang Y, Guo M. Development of a novel feeding regime for large scale production of human umbilical cord mesenchymal stem/stromal cells. Cytotechnology 2022; 74:351-369. [DOI: 10.1007/s10616-022-00523-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Accepted: 01/23/2022] [Indexed: 12/21/2022] Open
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23
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Shaw TD, Krasnodembskaya AD, Schroeder GN, Zumla A, Maeurer M, O’Kane CM. Mesenchymal Stromal Cells: an Antimicrobial and Host-Directed Therapy for Complex Infectious Diseases. Clin Microbiol Rev 2021; 34:e0006421. [PMID: 34612662 PMCID: PMC8510528 DOI: 10.1128/cmr.00064-21] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
There is an urgent need for new antimicrobial strategies for treating complex infections and emerging pathogens. Human mesenchymal stromal cells (MSCs) are adult multipotent cells with antimicrobial properties, mediated through direct bactericidal activity and modulation of host innate and adaptive immune cells. More than 30 in vivo studies have reported on the use of human MSCs for the treatment of infectious diseases, with many more studies of animal MSCs in same-species models of infection. MSCs demonstrate potent antimicrobial effects against the major classes of human pathogens (bacteria, viruses, fungi, and parasites) across a wide range of infection models. Mechanistic studies have yielded important insight into their immunomodulatory and bactericidal activity, which can be enhanced through various forms of preconditioning. MSCs are being investigated in over 80 clinical trials for difficult-to-treat infectious diseases, including sepsis and pulmonary, intra-abdominal, cutaneous, and viral infections. Completed trials consistently report MSCs to be safe and well tolerated, with signals of efficacy against some infectious diseases. Although significant obstacles must be overcome to produce a standardized, affordable, clinical-grade cell therapy, these studies suggest that MSCs may have particular potential as an adjunct therapy in complex or resistant infections.
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Affiliation(s)
- Timothy D. Shaw
- Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University, Belfast, United Kingdom
| | - Anna D. Krasnodembskaya
- Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University, Belfast, United Kingdom
| | - Gunnar N. Schroeder
- Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University, Belfast, United Kingdom
| | - Alimuddin Zumla
- Center for Clinical Microbiology, Division of Infection and Immunity, University College London, NIHR Biomedical Research Centre, UCL Hospitals NHS Foundation Trust, London, United Kingdom
| | - Markus Maeurer
- Immunosurgery Unit, Champalimaud Centre for the Unknown, Lisbon, Portugal
- Department of Oncology and Haematology, Krankenhaus Nordwest, Frankfurt, Germany
| | - Cecilia M. O’Kane
- Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University, Belfast, United Kingdom
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24
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Le Clainche T, Moisan A, Coll JL, Martel-Frachet V. The disc-shaped microcarriers: A new tool for increasing harvesting of adipose-derived mesenchymal stromal cells. Biochem Eng J 2021. [DOI: 10.1016/j.bej.2021.108082] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
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25
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Merimi M, El-Majzoub R, Lagneaux L, Moussa Agha D, Bouhtit F, Meuleman N, Fahmi H, Lewalle P, Fayyad-Kazan M, Najar M. The Therapeutic Potential of Mesenchymal Stromal Cells for Regenerative Medicine: Current Knowledge and Future Understandings. Front Cell Dev Biol 2021; 9:661532. [PMID: 34490235 PMCID: PMC8416483 DOI: 10.3389/fcell.2021.661532] [Citation(s) in RCA: 97] [Impact Index Per Article: 24.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Accepted: 05/28/2021] [Indexed: 12/11/2022] Open
Abstract
In recent decades, research on the therapeutic potential of progenitor cells has advanced considerably. Among progenitor cells, mesenchymal stromal cells (MSCs) have attracted significant interest and have proven to be a promising tool for regenerative medicine. MSCs are isolated from various anatomical sites, including bone marrow, adipose tissue, and umbilical cord. Advances in separation, culture, and expansion techniques for MSCs have enabled their large-scale therapeutic application. This progress accompanied by the rapid improvement of transplantation practices has enhanced the utilization of MSCs in regenerative medicine. During tissue healing, MSCs may exhibit several therapeutic functions to support the repair and regeneration of injured tissue. The process underlying these effects likely involves the migration and homing of MSCs, as well as their immunotropic functions. The direct differentiation of MSCs as a cell replacement therapeutic mechanism is discussed. The fate and behavior of MSCs are further regulated by their microenvironment, which may consequently influence their repair potential. A paracrine pathway based on the release of different messengers, including regulatory factors, chemokines, cytokines, growth factors, and nucleic acids that can be secreted or packaged into extracellular vesicles, is also implicated in the therapeutic properties of MSCs. In this review, we will discuss relevant outcomes regarding the properties and roles of MSCs during tissue repair and regeneration. We will critically examine the influence of the local microenvironment, especially immunological and inflammatory signals, as well as the mechanisms underlying these therapeutic effects. Importantly, we will describe the interactions of local progenitor and immune cells with MSCs and their modulation during tissue injury. We will also highlight the crucial role of paracrine pathways, including the role of extracellular vesicles, in this healing process. Moreover, we will discuss the therapeutic potential of MSCs and MSC-derived extracellular vesicles in the treatment of COVID-19 (coronavirus disease 2019) patients. Overall, this review will provide a better understanding of MSC-based therapies as a novel immunoregenerative strategy.
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Affiliation(s)
- Makram Merimi
- Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Bruxelles, Belgium
- LBBES Laboratory, Genetics and Immune-Cell Therapy Unit, Faculty of Sciences, University Mohammed Premier, Oujda, Morocco
| | - Rania El-Majzoub
- Department of Biomedical Sciences, School of Pharmacy, Lebanese International University, Beirut, Lebanon
- Laboratory of Cancer Biology and Molecular Immunology, Faculty of Sciences-I, Lebanese University, Beirut, Lebanon
| | - Laurence Lagneaux
- Laboratory of Clinical Cell Therapy, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Douâa Moussa Agha
- Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Bruxelles, Belgium
| | - Fatima Bouhtit
- Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Bruxelles, Belgium
- LBBES Laboratory, Genetics and Immune-Cell Therapy Unit, Faculty of Sciences, University Mohammed Premier, Oujda, Morocco
| | - Nathalie Meuleman
- Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Bruxelles, Belgium
| | - Hassan Fahmi
- Osteoarthritis Research Unit, University of Montreal Hospital Research Center (CRCHUM), Montreal, QC, Canada
| | - Philippe Lewalle
- Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Bruxelles, Belgium
| | - Mohammad Fayyad-Kazan
- Laboratory of Cancer Biology and Molecular Immunology, Faculty of Sciences-I, Lebanese University, Beirut, Lebanon
- Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, Beirut, Lebanon
| | - Mehdi Najar
- Laboratory of Clinical Cell Therapy, Institut Jules Bordet, Université Libre de Bruxelles (ULB), Brussels, Belgium
- Osteoarthritis Research Unit, University of Montreal Hospital Research Center (CRCHUM), Montreal, QC, Canada
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26
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Sion C, Ghannoum D, Ebel B, Gallo F, de Isla N, Guedon E, Chevalot I, Olmos E. A new perfusion mode of culture for WJ-MSCs expansion in a stirred and online monitored bioreactor. Biotechnol Bioeng 2021; 118:4453-4464. [PMID: 34387862 DOI: 10.1002/bit.27914] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 08/04/2021] [Accepted: 08/04/2021] [Indexed: 01/22/2023]
Abstract
As a clinical dose requires a minimum of 106 cells per kilogram of patients, it is, therefore, crucial to develop a scalable method of production of Wharton Jelly mesenchymal stem cells (WJ-MSCs) with maintained inner characteristics. Scalable expansion of WJ-MSCs on microcarriers usually found in cell culture, involves specific cell detachment using trypsin and could have harmful effects on cells. In this study, the performance of batch, fed-batch, and perfused-continuous mode of culture were compared. The batch and fed-batch modes resulted in expansion factors of 5 and 43, respectively. The perfused-continuous mode strategy consisted of the implementation of a settling tube inside the bioreactor. The diameter of the tube was calculated to maintain microcarriers colonized by cells in the bioreactor whereas empty microcarriers (responsible for potentially damaging collisions) were removed, using a continuous flow rate based on MSCs physiological requirements. Thanks to this strategy, a maximal number of 800 million cells was obtained in a 1.5 L bioreactor in 10 days. Lastly, online dielectric spectroscopy was implemented in the bioreactor and indicated that cell growth could be monitored during the culture.
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Affiliation(s)
- Caroline Sion
- Laboratoire Réactions et Génie des Procédés, Université de Lorraine, CNRS UMR 7274, Vandoeuvre les Nancy, France
| | - Dima Ghannoum
- Ingénierie Moléculaire et Physiopathologie Articulaire, Université de Lorraine, CNRS UMR 7365, Vandœuvre-lès-Nancy, France
| | - Bruno Ebel
- Laboratoire Réactions et Génie des Procédés, Université de Lorraine, CNRS UMR 7274, Vandoeuvre les Nancy, France
| | - Fanny Gallo
- Laboratoire Réactions et Génie des Procédés, Université de Lorraine, CNRS UMR 7274, Vandoeuvre les Nancy, France
| | - Natalia de Isla
- Ingénierie Moléculaire et Physiopathologie Articulaire, Université de Lorraine, CNRS UMR 7365, Vandœuvre-lès-Nancy, France
| | - Emmanuel Guedon
- Laboratoire Réactions et Génie des Procédés, Université de Lorraine, CNRS UMR 7274, Vandoeuvre les Nancy, France
| | - Isabelle Chevalot
- Laboratoire Réactions et Génie des Procédés, Université de Lorraine, CNRS UMR 7274, Vandoeuvre les Nancy, France
| | - Eric Olmos
- Laboratoire Réactions et Génie des Procédés, Université de Lorraine, CNRS UMR 7274, Vandoeuvre les Nancy, France
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27
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Petry F, Salzig D. Impact of Bioreactor Geometry on Mesenchymal Stem Cell Production in Stirred‐Tank Bioreactors. CHEM-ING-TECH 2021. [DOI: 10.1002/cite.202100041] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Affiliation(s)
- Florian Petry
- University of Applied Sciences Mittelhessen Institute of Bioprocess Engineering and Pharmaceutical Technology Wiesenstraße 14 35390 Giessen Germany
| | - Denise Salzig
- University of Applied Sciences Mittelhessen Institute of Bioprocess Engineering and Pharmaceutical Technology Wiesenstraße 14 35390 Giessen Germany
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28
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Tsai AC, Pacak CA. Bioprocessing of Human Mesenchymal Stem Cells: From Planar Culture to Microcarrier-Based Bioreactors. Bioengineering (Basel) 2021; 8:bioengineering8070096. [PMID: 34356203 PMCID: PMC8301102 DOI: 10.3390/bioengineering8070096] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2021] [Revised: 07/02/2021] [Accepted: 07/02/2021] [Indexed: 01/14/2023] Open
Abstract
Human mesenchymal stem cells (hMSCs) have demonstrated great potential to be used as therapies for many types of diseases. Due to their immunoprivileged status, allogeneic hMSCs therapies are particularly attractive options and methodologies to improve their scaling and manufacturing are needed. Microcarrier-based bioreactor systems provide higher volumetric hMSC production in automated closed systems than conventional planar cultures. However, more sophisticated bioprocesses are necessary to successfully convert from planar culture to microcarriers. This article summarizes key steps involved in the planar culture to microcarrier hMSC manufacturing scheme, from seed train, inoculation, expansion and harvest. Important bioreactor parameters, such as temperature, pH, dissolved oxygen (DO), mixing, feeding strategies and cell counting techniques, are also discussed.
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Affiliation(s)
- Ang-Chen Tsai
- Department of Pediatrics, University of Florida, Gainesville, FL 32603, USA
- Correspondence: (A.-C.T.); (C.A.P.)
| | - Christina A. Pacak
- Department of Neurology, University of Minnesota, Minneapolis, MN 55455, USA
- Correspondence: (A.-C.T.); (C.A.P.)
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29
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Kurogi H, Takahashi A, Isogai M, Sakumoto M, Takijiri T, Hori A, Furuno T, Koike T, Yamada T, Nagamura-Inoue T, Sakaki-Yumoto M. Umbilical cord derived mesenchymal stromal cells in microcarrier based industrial scale culture sustain the immune regulatory functions. Biotechnol J 2021; 16:e2000558. [PMID: 33545746 DOI: 10.1002/biot.202000558] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Revised: 02/03/2021] [Accepted: 02/04/2021] [Indexed: 12/22/2022]
Abstract
Mesenchymal stromal cells (MSCs) have been isolated from numerous sources and are potentially therapeutic against various diseases. Umbilical cord-derived MSCs (UC-MSCs) are considered superior to other tissue-derived MSCs since they have a higher proliferation rate and can be procured using less invasive surgical procedures. However, it has been recently reported that 2D culture systems, using conventional cell culture flasks, limit the mass production of MSCs for cell therapy. Therefore, the development of alternative technologies, including microcarrier-based cell culture in bioreactors, is required for the large-scale production and industrialization of MSC therapy. In this study, we aimed to optimize the culture conditions for UC-MSCs by using a good manufacturing practice (GMP)-compatible serum-free medium, developed in-house, and a small-scale (30 mL) bioreactor, which was later scaled up to 500 mL. UC-MSCs cultured in microcarrier-based bioreactors (MC-UC-MSCs) showed characteristics equivalent to those cultured statically in conventional cell culture flasks (ST-UC-MSCs), fulfilling the minimum International Society for Cellular Therapy criteria for MSCs. Additionally, we report, for the first time, the equivalent therapeutic effect of MC-UC-MSCs and ST-UC-MSCs in immunodeficient mice (graft-versus-host disease model). Lastly, we developed a semi-automated cell dispensing system, without bag-to-bag variation in the filled volume or cell concentration. In summary, our results show that the combination of our GMP-compatible serum-free and microcarrier-based culture systems is suitable for the mass production of MSCs at an industrial scale. Further improvements in this microcarrier-based cell culture system can contribute to lowering the cost of therapy and satisfying several unmet medical needs.
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Affiliation(s)
- Hikari Kurogi
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
| | - Atsuko Takahashi
- Department of Cell Processing and Transfusion, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Maya Isogai
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
| | - Marimu Sakumoto
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
| | - Takashi Takijiri
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
| | - Akiko Hori
- Department of Cell Processing and Transfusion, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Tetsuo Furuno
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
| | - Tetsuo Koike
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
| | - Tetsumasa Yamada
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
| | - Tokiko Nagamura-Inoue
- Department of Cell Processing and Transfusion, Research Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
| | - Masayo Sakaki-Yumoto
- Regenerative Medicine Research & Planning Division, Rohto Pharmaceutical Co., Ltd., Osaka, Japan
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30
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Silva Couto P, Rotondi M, Bersenev A, Hewitt C, Nienow A, Verter F, Rafiq Q. Expansion of human mesenchymal stem/stromal cells (hMSCs) in bioreactors using microcarriers: lessons learnt and what the future holds. Biotechnol Adv 2020; 45:107636. [DOI: 10.1016/j.biotechadv.2020.107636] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2020] [Revised: 08/01/2020] [Accepted: 09/22/2020] [Indexed: 02/06/2023]
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31
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Gonzalez Gil LV, Singh H, da Silva JDS, dos Santos DP, Covas DT, Swiech K, Torres Suazo CA. Feasibility of the taylor vortex flow bioreactor for mesenchymal stromal cell expansion on microcarriers. Biochem Eng J 2020. [DOI: 10.1016/j.bej.2020.107710] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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32
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Umbilical Cord-Derived Mesenchymal Stem Cells Are Able to Use bFGF Treatment and Represent a Superb Tool for Immunosuppressive Clinical Applications. Int J Mol Sci 2020; 21:ijms21155366. [PMID: 32731615 PMCID: PMC7432622 DOI: 10.3390/ijms21155366] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 07/15/2020] [Accepted: 07/22/2020] [Indexed: 12/17/2022] Open
Abstract
Mesenchymal stem cells (MSCs) have become a promising tool in cellular therapy for restoring immune system haemostasis; however, the success of clinical trials has been impaired by the lack of standardized manufacturing processes. This study aims to determine the suitability of source tissues and culture media for the production of MSC-based advanced therapy medicinal products (ATMPs) and to define parameters to extend the set of release criteria. MSCs were isolated from umbilical cord (UC), bone marrow and lipoaspirate and expanded in three different culture media. MSC phenotype, proliferation capacity and immunosuppressive parameters were evaluated in normal MSCs compared to primed MSCs treated with cytokines mimicking an inflammatory environment. Compared to bone marrow and lipoaspirate, UC-derived MSCs (UC-MSCs) showed the highest proliferative capacity, which was further enhanced by media supplemented with bFGF, while the cells maintained their immunosuppressive characteristics. Moreover, UC-MSCs expanded in the bFGF-enriched medium were the least sensitive to undesirable priming-induced changes in the MSC phenotype. Surface markers and secreted factors were identified to reflect the cell response to inflammatory priming and to be variable among MSCs from different source tissues. This study demonstrates that UC is a favorable cell source for manufacturing MSC-based ATMPs for immunosuppressive applications. UC-MSCs are able to use the bFGF-enriched medium for higher cell yields without the impairment of immunosuppressive parameters and undesirable phenotype changes after inflammatory preconditioning of MSCs before transplantation. Additionally, immunosuppressive parameters were identified to help finding predictors of clinically efficient MSCs in the following clinical trials.
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Large-Scale Expansion of Human Mesenchymal Stem Cells. Stem Cells Int 2020; 2020:9529465. [PMID: 32733574 PMCID: PMC7378617 DOI: 10.1155/2020/9529465] [Citation(s) in RCA: 57] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2020] [Revised: 06/07/2020] [Accepted: 07/01/2020] [Indexed: 12/15/2022] Open
Abstract
Mesenchymal stem cells (MSCs) are multipotent stem cells with strong immunosuppressive property that renders them an attractive source of cells for cell therapy. MSCs have been studied in multiple clinical trials to treat liver diseases, peripheral nerve damage, graft-versus-host disease, autoimmune diseases, diabetes mellitus, and cardiovascular damage. Millions to hundred millions of MSCs are required per patient depending on the disease, route of administration, frequency of administration, and patient body weight. Multiple large-scale cell expansion strategies have been described in the literature to fetch the cell quantity required for the therapy. In this review, bioprocessing strategies for large-scale expansion of MSCs were systematically reviewed and discussed. The literature search in Medline and Scopus databases identified 26 articles that met the inclusion criteria and were included in this review. These articles described the large-scale expansion of 7 different sources of MSCs using 4 different bioprocessing strategies, i.e., bioreactor, spinner flask, roller bottle, and multilayered flask. The bioreactor, spinner flask, and multilayered flask were more commonly used to upscale the MSCs compared to the roller bottle. Generally, a higher expansion ratio was achieved with the bioreactor and multilayered flask. Importantly, regardless of the bioprocessing strategies, the expanded MSCs were able to maintain its phenotype and potency. In summary, the bioreactor, spinner flask, roller bottle, and multilayered flask can be used for large-scale expansion of MSCs without compromising the cell quality.
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Wyrobnik TA, Ducci A, Micheletti M. Advances in human mesenchymal stromal cell-based therapies - Towards an integrated biological and engineering approach. Stem Cell Res 2020; 47:101888. [PMID: 32688331 DOI: 10.1016/j.scr.2020.101888] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Revised: 06/17/2020] [Accepted: 06/19/2020] [Indexed: 12/14/2022] Open
Abstract
Recent advances of stem cell-based therapies in clinical trials have raised the need for large-scale manufacturing platforms that can supply clinically relevant doses to meet an increasing demand. Promising results have been reported using stirred-tank bioreactors, where human Mesenchymal Stromal Cells (hMSCs) were cultured in suspension on microcarriers (MCs), although the formation of microcarrier-cell-aggregates might still limit mass transfer and determine a heterogeneous distribution of hMSCs. A variety of MCs, bioreactor-impeller configurations, and agitation conditions have been established in an attempt to overcome the trade-off of ensuring good suspension while keeping the stresses to a minimum. While understanding and controlling the fluid flow environment of bioreactors has been initially under-appreciated, it has recently gained in popularity in the mission of providing ideal culture environments across different scales. This review article aims to provide a comprehensive overview of how rigorous engineering characterisation studies improved the outcome of biological process development and scale-up efforts. Reconciling these two disciplines is crucial to propose tailored bioprocessing solutions that can provide improved growth environments across a range of scales for the allogeneic cell therapies of the future.
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Affiliation(s)
- Tom A Wyrobnik
- Department of Biochemical Engineering, UCL, Gower Street, London WC1E 6BT, UK
| | - Andrea Ducci
- Department of Mechanical Engineering, UCL, Torrington Place, London WC1E 7JE, UK
| | - Martina Micheletti
- Department of Biochemical Engineering, UCL, Gower Street, London WC1E 6BT, UK.
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Cherian DS, Bhuvan T, Meagher L, Heng TSP. Biological Considerations in Scaling Up Therapeutic Cell Manufacturing. Front Pharmacol 2020; 11:654. [PMID: 32528277 PMCID: PMC7247829 DOI: 10.3389/fphar.2020.00654] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Accepted: 04/22/2020] [Indexed: 12/12/2022] Open
Abstract
Cell therapeutics - using cells as living drugs - have made advances in many areas of medicine. One of the most clinically studied cell-based therapy products is mesenchymal stromal cells (MSCs), which have shown promising results in promoting tissue regeneration and modulating inflammation. However, MSC therapy requires large numbers of cells, the generation of which is not feasible via conventional planar tissue culture methods. Scale-up manufacturing methods (e.g., propagation on microcarriers in stirred-tank bioreactors), however, are not specifically tailored for MSC expansion. These processes may, in principle, alter the cell secretome, a vital component underlying the immunosuppressive properties and clinical effectiveness of MSCs. This review outlines our current understanding of MSC properties and immunomodulatory function, expansion in commercial manufacturing systems, and gaps in our knowledge that need to be addressed for effective up-scaling commercialization of MSC therapy.
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Affiliation(s)
- Darshana S Cherian
- 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
| | - Laurence Meagher
- Department of Materials Science and Engineering, Monash University, Clayton, VIC, Australia
| | - Tracy S P Heng
- Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
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Moreira F, Mizukami A, de Souza LEB, Cabral JMS, da Silva CL, Covas DT, Swiech K. Successful Use of Human AB Serum to Support the Expansion of Adipose Tissue-Derived Mesenchymal Stem/Stromal Cell in a Microcarrier-Based Platform. Front Bioeng Biotechnol 2020; 8:307. [PMID: 32373600 PMCID: PMC7184110 DOI: 10.3389/fbioe.2020.00307] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2019] [Accepted: 03/20/2020] [Indexed: 12/15/2022] Open
Abstract
Mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and for the promotion of tissue repair, hence the increase of clinical trials in a worldwide scale. In particular, adipose tissue-derived stem/stromal cells (AT MSC) present easy accessibility and a rather straightforward process of isolation, providing a clear advantage over other sources. The high demand of cell doses (millions of cells/kg), needed for infusion in clinical settings, requires a scalable and efficient manufacturing of AT MSC under xenogeneic(xeno)-free culture conditions. Here we describe the successful use of human AB serum [10%(v/v)] as a culture supplement, as well as coating substrate for the expansion of these cells in microcarriers using (i) a spinner flask and (ii) a 500-mL mini-bioreactor (ApplikonTM Biotechnology). Cells were characterized by immunophenotype and multilineage differentiation potential. Upon an initial cell adhesion in the spinner flask of 35 ± 2.5%, culture reached a maximal cell density of 2.6 ± 0.1 × 105 at day 7, obtaining a 15 ± 1-fold increase. The implementation of the culture in the 500-mL mini-bioreactor presented an initial cell adhesion of 22 ± 5%, but it reached maximal cell density of 2.7 ± 0.4 × 105 at day 7, obtaining a 27 ± 8-fold increase. Importantly, in both stirred systems, cells retained their immunophenotype and multilineage differentiation potential (osteo-, chondro- and adipogenic lineages). Overall, the scalability of this microcarrier-based system presented herein is of major importance for the purpose of achieving clinically meaningful cell numbers.
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Affiliation(s)
- Francisco Moreira
- Department of Bioengineering, Instituto Superior Técnico, iBB-Institute for Bioengineering and Biosciences, Universidade de Lisboa, Lisbon, Portugal
| | - Amanda Mizukami
- Center for Cell-based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, São Paulo, Brazil
| | | | - Joaquim M S Cabral
- Department of Bioengineering, Instituto Superior Técnico, iBB-Institute for Bioengineering and Biosciences, Universidade de Lisboa, Lisbon, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering, Instituto Superior Técnico, iBB-Institute for Bioengineering and Biosciences, Universidade de Lisboa, Lisbon, Portugal
| | - Dimas T Covas
- Center for Cell-based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, São Paulo, Brazil
| | - Kamilla Swiech
- Center for Cell-based Therapy, Regional Blood Center of Ribeirão Preto, University of São Paulo, São Paulo, Brazil.,Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, São Paulo, Brazil
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Fuoco NL, de Oliveira RG, Marcelino MY, Stessuk T, Sakalem ME, Medina DAL, Modotti WP, Forte A, Ribeiro-Paes JT. Efficient isolation and proliferation of human adipose-derived mesenchymal stromal cells in xeno-free conditions. Mol Biol Rep 2020; 47:2475-2486. [PMID: 32124173 DOI: 10.1007/s11033-020-05322-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2019] [Accepted: 02/13/2020] [Indexed: 12/15/2022]
Abstract
Classical methods used for culture of adipose-derived mesenchymal stromal cells (ADSCs) use xenobiotic components, which may present a potential risk for biological contamination and/or elicit immunological reactions. Therefore, the aim of this study was to establish a xeno-free methodology for the isolation and proliferation of human ADSCs (hADSCs). hADSCs were isolated by enzymatic digestion or mechanical dissociation and cultured in the presence of fetal bovine serum or human platelet lysate. Proliferation curves were performed as a function of time from the cell culture and used to calculate the population doubling time. Immunophenotyping and differentiation tests were used to identify and characterize the hADSCs. Human ADSCs isolated and cultured in conventional or xenobiotic-free conditions peaked at different days but achieved similar maximum proliferation. The hADSCs differentiation ability was similar in all groups. The characterization of hADSCs by flow cytometry showed low contamination of the cultures by other cell types. The xenobiotic-free methodology described in this study is a feasible and reproducible alternative for isolation and proliferation of hADSCs. This methodology is in accordance with the recommendations of the National Health Surveillance Agency, which proposes avoidance of xenobiotic products.
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Affiliation(s)
- Natalia Langenfeld Fuoco
- Biotechnology Interunits Post-Graduation Program, Biomedical Science Institute, University of São Paulo (USP), São Paulo, SP, Brazil
| | - Rafael Guilen de Oliveira
- Biotechnology Interunits Post-Graduation Program, Biomedical Science Institute, University of São Paulo (USP), São Paulo, SP, Brazil
| | - Monica Yonashiro Marcelino
- Biotechnology Interunits Post-Graduation Program, Biomedical Science Institute, University of São Paulo (USP), São Paulo, SP, Brazil
| | - Talita Stessuk
- Biotechnology Interunits Post-Graduation Program, Biomedical Science Institute, University of São Paulo (USP), São Paulo, SP, Brazil
| | - Marna Eliana Sakalem
- Genetics and Cell Therapy Laboratory (GenTe Cel), São Paulo State University (Unesp), São Paulo, SP, Brazil
| | | | | | - Andresa Forte
- São Lucas - Cell Therapy Group, São Paulo, SP, Brazil
| | - João Tadeu Ribeiro-Paes
- Genetics and Cell Therapy Laboratory (GenTe Cel), São Paulo State University (Unesp), São Paulo, SP, Brazil. .,Laboratório de Genética e Terapia Celular - GenTe Cel, Departamento de Biotecnologia - Unesp, Av. Dom Antonio, 2100, Assis, SP, CEP 19806-330, Brasil.
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Bodiou V, Moutsatsou P, Post MJ. Microcarriers for Upscaling Cultured Meat Production. Front Nutr 2020; 7:10. [PMID: 32154261 PMCID: PMC7045063 DOI: 10.3389/fnut.2020.00010] [Citation(s) in RCA: 121] [Impact Index Per Article: 24.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2019] [Accepted: 01/28/2020] [Indexed: 12/19/2022] Open
Abstract
Due to the considerable environmental impact and the controversial animal welfare associated with industrial meat production, combined with the ever-increasing global population and demand for meat products, sustainable production alternatives are indispensable. In 2013, the world's first laboratory grown hamburger made from cultured muscle cells was developed. However, coming at a price of $300.000, and being produced manually, substantial effort is still required to reach sustainable large-scale production. One of the main challenges is scalability. Microcarriers (MCs), offering a large surface/volume ratio, are the most promising candidates for upscaling muscle cell culture. However, although many MCs have been developed for cell lines and stem cells typically used in the medical field, none have been specifically developed for muscle stem cells and meat production. This paper aims to discuss the MCs' design criteria for skeletal muscle cell proliferation and subsequently for meat production based on three scenarios: (1) MCs are serving only as a temporary substrate for cell attachment and proliferation and therefore they need to be separated from the cells at some stage of the bioprocess, (2) MCs serve as a temporary substrate for cell proliferation but are degraded or dissolved during the bioprocess, and (3) MCs are embedded in the final product and therefore need to be edible. The particularities of each of these three bioprocesses will be discussed from the perspective of MCs as well as the feasibility of a one-step bioprocess. Each scenario presents advantages and drawbacks, which are discussed in detail, nevertheless the third scenario appears to be the most promising one for a production process. Indeed, using an edible material can limit or completely eliminate dissociation/degradation/separation steps and even promote organoleptic qualities when embedded in the final product. Edible microcarriers could also be used as a temporary substrate similarly to scenarios 1 and 2, which would limit the risk of non-edible residues.
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Affiliation(s)
- Vincent Bodiou
- Department of Physiology, Faculty of Health, Medicine and Life Sciences, School for Cardiovascular Diseases, Maastricht University, Maastricht, Netherlands
- Mosa Meat BV, Maastricht, Netherlands
- CARIM, Faculty of Health, Medicine and Life Sciences, School for Cardiovascular Diseases, Maastricht University, Maastricht, Netherlands
| | - Panagiota Moutsatsou
- Department of Physiology, Faculty of Health, Medicine and Life Sciences, School for Cardiovascular Diseases, Maastricht University, Maastricht, Netherlands
- Mosa Meat BV, Maastricht, Netherlands
| | - Mark J. Post
- Department of Physiology, Faculty of Health, Medicine and Life Sciences, School for Cardiovascular Diseases, Maastricht University, Maastricht, Netherlands
- Mosa Meat BV, Maastricht, Netherlands
- CARIM, Faculty of Health, Medicine and Life Sciences, School for Cardiovascular Diseases, Maastricht University, Maastricht, Netherlands
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Large-Scale Automated Hollow-Fiber Bioreactor Expansion of Umbilical Cord-Derived Human Mesenchymal Stromal Cells for Neurological Disorders. Neurochem Res 2019; 45:204-214. [PMID: 31828497 DOI: 10.1007/s11064-019-02925-y] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 11/07/2019] [Accepted: 11/27/2019] [Indexed: 12/11/2022]
Abstract
Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.
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Derakhti S, Safiabadi-Tali SH, Amoabediny G, Sheikhpour M. Attachment and detachment strategies in microcarrier-based cell culture technology: A comprehensive review. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2019; 103:109782. [DOI: 10.1016/j.msec.2019.109782] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Revised: 04/11/2019] [Accepted: 05/20/2019] [Indexed: 12/27/2022]
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The mesenchymal stem cell secretome: A new paradigm towards cell-free therapeutic mode in regenerative medicine. Cytokine Growth Factor Rev 2019; 46:1-9. [PMID: 30954374 DOI: 10.1016/j.cytogfr.2019.04.002] [Citation(s) in RCA: 310] [Impact Index Per Article: 51.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Revised: 04/01/2019] [Accepted: 04/01/2019] [Indexed: 12/15/2022]
Abstract
Mesenchymal Stem Cells (MSCs) have been shown to be a promising candidate for cell-based therapy. The therapeutic potential of MSCs, towards tissue repair and wound healing is essentially based on their paracrine effects. Numerous pre-clinical and clinical studies of MSCs have yielded encouraging results. Further, these cells have been shown to be relatively safe for clinical applications. MSCs harvested from numerous anatomical locations including the bone marrow, adipose tissue, Wharton's jelly of the umbilical cord etc., display similar immunophenotypic profiles. However, there is a large body of evidence showing that MSCs secrete a variety of biologically active molecules such as growth factors, chemokines, and cytokines. Despite the similarity in their immunophenotype, the secretome of MSCs appears to vary significantly, depending on the age of the host and niches where the cells reside. Thus, by implication, proteomics-based profiling suggests that the therapeutic potential of the different MSC populations must also be different. Analysis of the secretome points to its influence on varied biological processes such as angiogenesis, neurogenesis, tissue repair, immunomodulation, wound healing, anti-fibrotic and anti-tumour for tissue maintenance and regeneration. Though MSC based therapy has been shown to be relatively safe, from a clinical standpoint, the use of cell-free infusions can altogether circumvent the administration of viable cells for therapy. Understanding the secretome of in vitro cultured MSC populations, by the analysis of the corresponding conditioned medium, will enable us to evaluate its utility as a new therapeutic option. This review will focus on the accumulating evidence that points to the therapeutic potential of the conditioned medium, both from pre-clinical and clinical studies. Finally, this review will emphasize the importance of profiling the conditioned medium for assessing its potential for cell-free therapy therapy.
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Tirughana R, Metz MZ, Li Z, Hall C, Hsu D, Beltzer J, Annala AJ, Oganesyan D, Gutova M, Aboody KS. GMP Production and Scale-Up of Adherent Neural Stem Cells with a Quantum Cell Expansion System. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2018; 10:48-56. [PMID: 29992178 PMCID: PMC6037686 DOI: 10.1016/j.omtm.2018.05.006] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/07/2017] [Accepted: 05/31/2018] [Indexed: 12/15/2022]
Abstract
Cell-based therapies hold great promise for a myriad of clinical applications. However, as these therapies move from phase I to phase II and III trials, there is a need to improve scale-up of adherent cells for the production of larger good manufacturing practice (GMP) cell banks. As we advanced our neural stem cell (NSC)-mediated gene therapy trials for glioma to include dose escalation and multiple treatment cycles, GMP production using cell factories (CellStacks) generated insufficient neural stem cell (NSC) yields. To increase yield, we developed an expansion method using the hollow fiber quantum cell expansion (QCE) system. Seeding of 5.2 × 107 NSCs in a single unit yielded up to 3 × 109 cells within 10 days. These QCE NSCs showed genetic and functional stability equivalent to those expanded by conventional flask-based methods. We then expanded the NSCs in 7 units simultaneously to generate a pooled GMP-grade NSC clinical lot of more than 1.5 × 1010 cells in only 9 days versus 8 × 109 over 6 weeks in CellStacks. We also adenovirally transduced our NSCs within the QCE. We found the QCE system enabled rapid cell expansion and increased yield while maintaining cell properties and reducing process time, labor, and costs with improved efficiency and reproducibility.
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Affiliation(s)
- Revathiswari Tirughana
- Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
| | - Marianne Z Metz
- Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
| | - Zhongqi Li
- Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
| | - Christine Hall
- Center for Biomedicine and Genetics, City of Hope, Duarte, CA, USA
| | - David Hsu
- Center for Biomedicine and Genetics, City of Hope, Duarte, CA, USA
| | | | - Alexander J Annala
- Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
| | - Diana Oganesyan
- Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
| | - Margarita Gutova
- Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
| | - Karen S Aboody
- Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA
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Inwood S, Xu H, Black MA, Betenbaugh MJ, Feldman S, Shiloach J. Continuous production process of retroviral vector for adoptive T- cell therapy. Biochem Eng J 2018; 132:145-151. [PMID: 29977134 DOI: 10.1016/j.bej.2018.01.010] [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: 10/18/2022]
Abstract
Adoptive T-Cell therapy is being considered as a promising method for cancer treatment. In this approach, patient's T cells are isolated, modified, expanded, and administered back to the patient. Modifications may include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) to the isolated cells by using retroviral vectors. PG13 cells, derivatives of NIH3T3 mouse fibroblasts, are being used to stably produce retroviral vectors that transduce the T cells. PG13 cells are anchorage-dependent cells that grow in roller bottles or cell factories and lately also in fixed bed bioreactors to produce the needed viral vector. To scale up viral vector production, PG13 cells were propagated on microcarriers in a stirred tank bioreactor utilizing an alternating tangential flow perfusion system. Microcarriers are 10 µm - 0.5 mm beads that support the attachment of cells and are suspended in the bioreactor that provides controlled growth conditions. As a result, growth parameters, such as dissolved oxygen concentration, pH, and nutrients are monitored and continuously controlled. There were no detrimental effects on the specific viral vector titer or on the efficacy of the vector in transducing the T cells of several patients. Viral vector titer increased throughout the 11 days perfusion period, a total of 4.8 × 1011 transducing units (TU) were obtained with an average titer of 4.4 × 107 TU/mL and average specific productivity of 10.3 (TU) per cell, suggesting that this method can be an efficient way to produce large quantities of active vector suitable for clinical use.
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Affiliation(s)
- Sarah Inwood
- Biotechnology Core Laboratory NIDDK, NIH Bethesda Maryland 20892, USA.,Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore Maryland 21218, USA
| | - Hui Xu
- Surgery Branch Vector Production Facility, Center For Cancer Research, NCI, NIH Bethesda Maryland 20892, USA
| | - Mary A Black
- Surgery Branch Vector Production Facility, Center For Cancer Research, NCI, NIH Bethesda Maryland 20892, USA
| | - Michael J Betenbaugh
- Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore Maryland 21218, USA
| | - Steven Feldman
- Surgery Branch Vector Production Facility, Center For Cancer Research, NCI, NIH Bethesda Maryland 20892, USA
| | - Joseph Shiloach
- Biotechnology Core Laboratory NIDDK, NIH Bethesda Maryland 20892, USA
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Mesenchymal Stromal Cells: From Discovery to Manufacturing and Commercialization. Stem Cells Int 2018; 2018:4083921. [PMID: 30057622 PMCID: PMC6051015 DOI: 10.1155/2018/4083921] [Citation(s) in RCA: 89] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2017] [Revised: 03/01/2018] [Accepted: 03/11/2018] [Indexed: 02/07/2023] Open
Abstract
Over the last decades, mesenchymal stromal cells (MSC) have been the focus of intense research by academia and industry due to their unique features. MSC can be easily isolated and expanded through in vitro culture by taking full advantage of their self-renewing capacity. In addition, MSC exert immunomodulatory effects and can be differentiated into various lineages, which makes them highly attractive for clinical applications in cell-based therapies. In this review, we attempt to provide a brief historical overview of MSC discovery, characterization, and the first clinical studies conducted. The current MSC manufacturing platforms are reviewed with special attention regarding the use of bioreactors for the production of GMP-compliant clinically relevant cell numbers. The first commercial MSC-based products are also addressed, as well as the remaining challenges to the widespread use of MSC-derived products.
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Balikov DA, Crowder SW, Lee JB, Lee Y, Ko UH, Kang ML, Kim WS, Shin JH, Sung HJ. Aging Donor-Derived Human Mesenchymal Stem Cells Exhibit Reduced Reactive Oxygen Species Loads and Increased Differentiation Potential Following Serial Expansion on a PEG-PCL Copolymer Substrate. Int J Mol Sci 2018; 19:ijms19020359. [PMID: 29370101 PMCID: PMC5855581 DOI: 10.3390/ijms19020359] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Revised: 01/22/2018] [Accepted: 01/23/2018] [Indexed: 12/13/2022] Open
Abstract
Human mesenchymal stem cells (hMSCs) have been widely studied for therapeutic development in tissue engineering and regenerative medicine. They can be harvested from human donors via tissue biopsies, such as bone marrow aspiration, and cultured to reach clinically relevant cell numbers. However, an unmet issue lies in the fact that the hMSC donors for regenerative therapies are more likely to be of advanced age. Their stem cells are not as potent compared to those of young donors, and continue to lose healthy, stemness-related activities when the hMSCs are serially passaged in tissue culture plates. Here, we have developed a cheap, scalable, and effective copolymer film to culture hMSCs obtained from aged human donors over several passages without loss of reactive oxygen species (ROS) handling or differentiation capacity. Assays of cell morphology, reactive oxygen species load, and differentiation potential demonstrate the effectiveness of copolymer culture on reduction in senescence-related activities of aging donor-derived hMSCs that could hinder the therapeutic potential of autologous stem cell therapies.
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Affiliation(s)
- Daniel A Balikov
- Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USA.
| | - Spencer W Crowder
- Department of Materials and Department of Bioengineering, Imperial College London, London SW7 2AZ, UK.
| | - Jung Bok Lee
- Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USA.
- Severance Biomedical Science Institute, College of Medicine, Yonsei University, Seoul 03722, Korea.
| | - Yunki Lee
- Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USA.
- Department of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
| | - Ung Hyun Ko
- Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea.
| | - Mi-Lan Kang
- Severance Biomedical Science Institute, College of Medicine, Yonsei University, Seoul 03722, Korea.
| | - Won Shik Kim
- Department of Otorhinolaryngology, College of Medicine, Yonsei University, Seoul 03722, Korea.
| | - Jennifer H Shin
- Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea.
| | - Hak-Joon Sung
- Severance Biomedical Science Institute, College of Medicine, Yonsei University, Seoul 03722, Korea.
- Department of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
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Wang X, Schröder HC, Müller WEG. Amorphous polyphosphate, a smart bioinspired nano-/bio-material for bone and cartilage regeneration: towards a new paradigm in tissue engineering. J Mater Chem B 2018; 6:2385-2412. [DOI: 10.1039/c8tb00241j] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Physiological amorphous polyphosphate nano/micro-particles, injectable and implantable, attract and stimulate MSCs into implants for tissue regeneration.
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Affiliation(s)
- Xiaohong Wang
- ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry
- University Medical Center of the Johannes Gutenberg University
- 55128 Mainz
- Germany
| | - Heinz C. Schröder
- ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry
- University Medical Center of the Johannes Gutenberg University
- 55128 Mainz
- Germany
| | - Werner E. G. Müller
- ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry
- University Medical Center of the Johannes Gutenberg University
- 55128 Mainz
- Germany
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