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Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks
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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10439856/ https://www.ncbi.nlm.nih.gov/pubmed/37470820 http://dx.doi.org/10.1007/s00253-023-12634-w |
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author | Couto, Pedro Silva Stibbs, Dale J. Rotondi, Marco C. Takeuchi, Yasuhiro Rafiq, Qasim A. |
author_facet | Couto, Pedro Silva Stibbs, Dale J. Rotondi, Marco C. Takeuchi, Yasuhiro Rafiq, Qasim A. |
author_sort | Couto, Pedro Silva |
collection | PubMed |
description | 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 × 10(5) 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 SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-023-12634-w. |
format | Online Article Text |
id | pubmed-10439856 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-104398562023-08-21 Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks Couto, Pedro Silva Stibbs, Dale J. Rotondi, Marco C. Takeuchi, Yasuhiro Rafiq, Qasim A. Appl Microbiol Biotechnol Biotechnological Products and Process Engineering 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 × 10(5) 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 SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00253-023-12634-w. Springer Berlin Heidelberg 2023-07-20 2023 /pmc/articles/PMC10439856/ /pubmed/37470820 http://dx.doi.org/10.1007/s00253-023-12634-w Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Biotechnological Products and Process Engineering Couto, Pedro Silva Stibbs, Dale J. Rotondi, Marco C. Takeuchi, Yasuhiro Rafiq, Qasim A. Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks |
title | Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks |
title_full | Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks |
title_fullStr | Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks |
title_full_unstemmed | Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks |
title_short | Scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks |
title_sort | scalable manufacturing of gene-modified human mesenchymal stromal cells with microcarriers in spinner flasks |
topic | Biotechnological Products and Process Engineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10439856/ https://www.ncbi.nlm.nih.gov/pubmed/37470820 http://dx.doi.org/10.1007/s00253-023-12634-w |
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