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Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation |
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| Authors: | Breanna S Borys Tania So Erin L Roberts Leah Ferrie Leila Larijani Brett Abraham Roman Krawetz Derrick E Rancourt Michael S Kallos |
| Institution: | 1. Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada
Biomedical Engineering Graduate Program, University of Calgary, Calgary, Alberta, Canada;2. Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada;3. Biomedical Engineering Graduate Program, University of Calgary, Calgary, Alberta, Canada;4. Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada;5. Department of Chemical and Petroleum Engineering, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada;6. Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada;7. Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, Alberta, Canada |
| Abstract: | 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. |
| Keywords: | bioprocess cryopreservation embryonic stem cells scale-up stirred suspension bioreactors |