Cargando…

Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors

The conventional planar culture of adherent cells is inefficient for large‐scale manufacturing of cell and gene therapy products. We developed a facile and efficient bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells (hMSCs) with microca...

Descripción completa

Detalles Bibliográficos
Autores principales: Chen, Shangwu, Sato, Yushi, Tada, Yasuhiko, Suzuki, Yuma, Takahashi, Ryosuke, Okanojo, Masahiro, Nakashima, Katsuhiko
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley & Sons, Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380445/
https://www.ncbi.nlm.nih.gov/pubmed/34008349
http://dx.doi.org/10.1002/sctm.20-0501
_version_ 1783741199333457920
author Chen, Shangwu
Sato, Yushi
Tada, Yasuhiko
Suzuki, Yuma
Takahashi, Ryosuke
Okanojo, Masahiro
Nakashima, Katsuhiko
author_facet Chen, Shangwu
Sato, Yushi
Tada, Yasuhiko
Suzuki, Yuma
Takahashi, Ryosuke
Okanojo, Masahiro
Nakashima, Katsuhiko
author_sort Chen, Shangwu
collection PubMed
description The conventional planar culture of adherent cells is inefficient for large‐scale manufacturing of cell and gene therapy products. We developed a facile and efficient bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells (hMSCs) with microcarriers in bioreactors. We first compared culture medium with and without nucleosides and found the former maintained the expression of surface markers of hMSCs during their prolonged culture and enabled faster cell proliferation. Subsequently, we developed our bead‐to‐bead cell transfer method to subculture hMSCs and found that intermittent agitation after adding fresh microcarriers to cell‐populated microcarriers could promote spontaneous cell migration to fresh microcarriers, reduce microcarrier aggregation, and improve cell yield. This method enabled serial subculture of hMSCs in spinner flasks from passage 4 to passage 9 without using proteolytic enzymes, which showed faster cell proliferation than the serial planar cultures undergoing multiple enzyme treatment. Finally, we used the medium containing nucleosides and our bead‐to‐bead cell transfer method for cell culture scale‐up from 4‐ to 50‐L cultures in single‐use bioreactors. We achieved a 242‐fold increase in the number of cells to 1.45 × 10(10) after 27‐day culture and found that the cells harvested from the bioreactors maintained proliferation ability, expression of their surface markers, tri‐lineage differentiation potential and immunomodulatory property. This study shows the promotive effect of nucleosides on hMSC expansion and the potential of using our bead‐to‐bead transfer method for larger‐scale manufacturing of hMSCs for cell therapy.
format Online
Article
Text
id pubmed-8380445
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher John Wiley & Sons, Inc.
record_format MEDLINE/PubMed
spelling pubmed-83804452021-08-27 Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors Chen, Shangwu Sato, Yushi Tada, Yasuhiko Suzuki, Yuma Takahashi, Ryosuke Okanojo, Masahiro Nakashima, Katsuhiko Stem Cells Transl Med Manufacturing for Regenerative Medicine The conventional planar culture of adherent cells is inefficient for large‐scale manufacturing of cell and gene therapy products. We developed a facile and efficient bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells (hMSCs) with microcarriers in bioreactors. We first compared culture medium with and without nucleosides and found the former maintained the expression of surface markers of hMSCs during their prolonged culture and enabled faster cell proliferation. Subsequently, we developed our bead‐to‐bead cell transfer method to subculture hMSCs and found that intermittent agitation after adding fresh microcarriers to cell‐populated microcarriers could promote spontaneous cell migration to fresh microcarriers, reduce microcarrier aggregation, and improve cell yield. This method enabled serial subculture of hMSCs in spinner flasks from passage 4 to passage 9 without using proteolytic enzymes, which showed faster cell proliferation than the serial planar cultures undergoing multiple enzyme treatment. Finally, we used the medium containing nucleosides and our bead‐to‐bead cell transfer method for cell culture scale‐up from 4‐ to 50‐L cultures in single‐use bioreactors. We achieved a 242‐fold increase in the number of cells to 1.45 × 10(10) after 27‐day culture and found that the cells harvested from the bioreactors maintained proliferation ability, expression of their surface markers, tri‐lineage differentiation potential and immunomodulatory property. This study shows the promotive effect of nucleosides on hMSC expansion and the potential of using our bead‐to‐bead transfer method for larger‐scale manufacturing of hMSCs for cell therapy. John Wiley & Sons, Inc. 2021-05-18 /pmc/articles/PMC8380445/ /pubmed/34008349 http://dx.doi.org/10.1002/sctm.20-0501 Text en © 2021 Showa Denko Materials Co., Ltd. stem cells translational medicine published by Wiley Periodicals LLC on behalf of AlphaMed Press. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Manufacturing for Regenerative Medicine
Chen, Shangwu
Sato, Yushi
Tada, Yasuhiko
Suzuki, Yuma
Takahashi, Ryosuke
Okanojo, Masahiro
Nakashima, Katsuhiko
Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors
title Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors
title_full Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors
title_fullStr Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors
title_full_unstemmed Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors
title_short Facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors
title_sort facile bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells in bioreactors
topic Manufacturing for Regenerative Medicine
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380445/
https://www.ncbi.nlm.nih.gov/pubmed/34008349
http://dx.doi.org/10.1002/sctm.20-0501
work_keys_str_mv AT chenshangwu facilebeadtobeadcelltransfermethodforserialsubcultureandlargescaleexpansionofhumanmesenchymalstemcellsinbioreactors
AT satoyushi facilebeadtobeadcelltransfermethodforserialsubcultureandlargescaleexpansionofhumanmesenchymalstemcellsinbioreactors
AT tadayasuhiko facilebeadtobeadcelltransfermethodforserialsubcultureandlargescaleexpansionofhumanmesenchymalstemcellsinbioreactors
AT suzukiyuma facilebeadtobeadcelltransfermethodforserialsubcultureandlargescaleexpansionofhumanmesenchymalstemcellsinbioreactors
AT takahashiryosuke facilebeadtobeadcelltransfermethodforserialsubcultureandlargescaleexpansionofhumanmesenchymalstemcellsinbioreactors
AT okanojomasahiro facilebeadtobeadcelltransfermethodforserialsubcultureandlargescaleexpansionofhumanmesenchymalstemcellsinbioreactors
AT nakashimakatsuhiko facilebeadtobeadcelltransfermethodforserialsubcultureandlargescaleexpansionofhumanmesenchymalstemcellsinbioreactors