Large-scale expansion of feeder-free mouse embryonic stem cells serially passaged in stirred suspension bioreactors at low inoculation densities directly from cryopreservation.
Biotechnol Bioeng
; 117(5): 1316-1328, 2020 05.
Article
en En
| MEDLINE
| ID: mdl-31960947
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.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Criopreservación
/
Técnicas de Cultivo de Célula
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Reactores Biológicos
/
Células Madre Embrionarias
Tipo de estudio:
Guideline
Límite:
Animals
Idioma:
En
Revista:
Biotechnol Bioeng
Año:
2020
Tipo del documento:
Article
País de afiliación:
Canadá