Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation
Abstract Background Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) h...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2024-03-01
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| Series: | Stem Cell Research & Therapy |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13287-024-03699-z |
| _version_ | 1797233753488097280 |
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| author | Kevin Ullmann Felix Manstein Wiebke Triebert Nils Kriedemann Annika Franke Jana Teske Mira Mertens Victoria Lupanow Gudrun Göhring Alexandra Haase Ulrich Martin Robert Zweigerdt |
| author_facet | Kevin Ullmann Felix Manstein Wiebke Triebert Nils Kriedemann Annika Franke Jana Teske Mira Mertens Victoria Lupanow Gudrun Göhring Alexandra Haase Ulrich Martin Robert Zweigerdt |
| author_sort | Kevin Ullmann |
| collection | DOAJ |
| description | Abstract Background Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. Methods and results STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs’ pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. Conclusions Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale. |
| first_indexed | 2024-04-24T16:21:11Z |
| format | Article |
| id | doaj.art-68aa172d3c3c41398fe5f1d1b2655b03 |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-04-24T16:21:11Z |
| publishDate | 2024-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-68aa172d3c3c41398fe5f1d1b2655b032024-03-31T11:12:04ZengBMCStem Cell Research & Therapy1757-65122024-03-0115112010.1186/s13287-024-03699-zMatrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservationKevin Ullmann0Felix Manstein1Wiebke Triebert2Nils Kriedemann3Annika Franke4Jana Teske5Mira Mertens6Victoria Lupanow7Gudrun Göhring8Alexandra Haase9Ulrich Martin10Robert Zweigerdt11Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Department of Human Genetics, Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic Transplantation and Vascular Surgery (HTTG), Hannover Medical School (MHH)Abstract Background Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. Methods and results STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs’ pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. Conclusions Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.https://doi.org/10.1186/s13287-024-03699-zhPSCSTBRSuspension cultureIntermediate cryopreservationSeed trainAggregate dissociation |
| spellingShingle | Kevin Ullmann Felix Manstein Wiebke Triebert Nils Kriedemann Annika Franke Jana Teske Mira Mertens Victoria Lupanow Gudrun Göhring Alexandra Haase Ulrich Martin Robert Zweigerdt Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation Stem Cell Research & Therapy hPSC STBR Suspension culture Intermediate cryopreservation Seed train Aggregate dissociation |
| title | Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation |
| title_full | Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation |
| title_fullStr | Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation |
| title_full_unstemmed | Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation |
| title_short | Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation |
| title_sort | matrix free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation |
| topic | hPSC STBR Suspension culture Intermediate cryopreservation Seed train Aggregate dissociation |
| url | https://doi.org/10.1186/s13287-024-03699-z |
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