Large‐scale expansion of feeder‐free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation. Issue 5 (11th February 2020)

• Record Type: Journal Article
• Authors: Borys, Breanna S.; So, Tania; Roberts, Erin L.; Ferrie, Leah; Larijani, Leila; Abraham, Brett; Krawetz, Roman; Rancourt, Derrick E.; Kallos, Michael S.
• Is Part Of: Biotechnology and bioengineering. Volume 117:Issue 5(2020)
• Page Start: 1316
• Page End: 1328
• Publication Date: 2020-02-11
• Subjects: bioprocess -- cryopreservation -- embryonic stem cells -- scale‐up -- stirred suspension bioreactors; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27279
• Languages: English
• ISSNs: 0006-3592

Abstract

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 (10 9 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. Abstract : We have demonstrated that mouse ESCs cryopreserved in the absence of feeder cells can 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.