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Optimized mouse ES cell culture system by suspension growth in a fully defined medium

Nature Protocols volume 3, pages 10131017 (2008) | Download Citation



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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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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  1. Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm 171 77, Sweden.

    • Michael Andäng
    •  & Patrik Ernfors
  2. Department of Neuroscience, Karolinska Institutet, Stockholm 171 77, Sweden.

    • Annalena Moliner
    •  & Carlos F Ibañez
  3. Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, USA.

    • Claudia A Doege


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Correspondence to Michael Andäng or Patrik Ernfors.

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