Abstract
Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time efficiency and typically requires only 20 min of effective hands-on time per week. This protocol maintains a very high degree of pluripotent cells partly by mechanical separation of spontaneously differentiating cells. mES cells can be cultured for extended periods (>6 months) without the loss of pluripotency markers. High passage (>20) adherent mES cultures containing contaminating differentiated cells can be rescued and enriched in undifferentiated ES cells.
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Acknowledgements
This work was supported by the Swedish Research Council, the Swedish foundation for strategic research, CEDB and DBRM grants (to P.E., C.F.I.), Marcus and Marianne Wallenberg Foundation (to C.F.I.), the Swedish Cancer Foundation, the Swedish Brain Foundation and the Bertil Hållsten Research Foundation (to P.E.). M.A. was supported by grants from the Karolinska Institutet and the Swedish Brain Foundation.
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Andäng, M., Moliner, A., Doege, C. et al. Optimized mouse ES cell culture system by suspension growth in a fully defined medium. Nat Protoc 3, 1013–1017 (2008). https://doi.org/10.1038/nprot.2008.65
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DOI: https://doi.org/10.1038/nprot.2008.65
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