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Nat Protoc. 2008;3(9):1435-43. doi: 10.1038/nprot.2008.140.

Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.

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Hubrecht Institute, Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.


Realizing the potential of human embryonic stem cells (hESCs) in research and commercial applications requires generic protocols for culture, expansion and genetic modification that function between multiple lines. Here we describe a feeder-free hESC culture protocol that was tested in 13 independent hESC lines derived in five different laboratories. The procedure is based on Matrigel adaptation in mouse embryonic fibroblast conditioned medium (CM) followed by monolayer culture of hESC. When combined, these techniques provide a robust hESC culture platform, suitable for high-efficiency genetic modification via plasmid transfection (using lipofection or electroporation), siRNA knockdown and viral transduction. In contrast to other available protocols, it does not require optimization for individual lines. hESC transiently expressing ectopic genes are obtained within 9 d and stable transgenic lines within 3 weeks.

[Indexed for MEDLINE]

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