Large‐scale expansion of feeder‐free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation
| Autor: | Breanna S. Borys, Derrick E. Rancourt, Michael S. Kallos, Leah Ferrie, Erin L. Roberts, Tania So, Brett Abraham, Leila Larijani, Roman Krawetz |
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| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
0301 basic medicine Homeobox protein NANOG Cell Culture Techniques Cell Count Bioengineering Mice SCID 01 natural sciences Applied Microbiology and Biotechnology Cryopreservation Flow cytometry Mice 03 medical and health sciences Laboratory flask Bioreactors Feeder Layer 010608 biotechnology Bioreactor medicine Animals Bioprocess Cells Cultured Embryonic Stem Cells medicine.diagnostic_test Chemistry equipment and supplies Embryonic stem cell Cell biology 030104 developmental biology embryonic structures Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 117:1316-1328 |
| ISSN: | 1097-0290 0006-3592 |
| Popis: | Embryonic stem cells (ESCs) have almost unlimited proliferation capacity in vitro and can retain the ability to contribute to all cell lineages, making them an ideal platform material for cell-based therapies. ESCs are traditionally cultured in static flasks on a feeder layer of murine embryonic fibroblast cells. Although sufficient to generate cells for research purposes, this approach is impractical to achieve large quantities for clinical applications. In this study, we have developed protocols that address a variety of challenges that currently bottleneck clinical translation of ESCs expanded in stirred suspension bioreactors. We demonstrated that mouse ESCs (mESCs) cryopreserved in the absence of feeder cells could be thawed directly into stirred suspension bioreactors at extremely low inoculation densities (100 cells/ml). These cells sustained proliferative capacity through multiple passages and various reactor sizes and geometries, producing clinically relevant numbers (109 cells) and maintaining pluripotency phenotypic and functional properties. Passages were completed in stirred suspension bioreactors of increasing scale, under defined batch conditions which greatly improved resource efficiency. Output mESCs were analyzed for pluripotency marker expression (SSEA-1, SOX-2, and Nanog) through flow cytometry, and spontaneous differentiation and teratoma analysis was used to demonstrate functional maintenance of pluripotency. |
| Databáze: | OpenAIRE |
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