This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.
Abstract
This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.
Protocol
hESC culture daily maintenance hESC culture media must be changed every 24 hrs. From the ES media stock bottle stored at 4°C, take out the amount of solution needed (~20ml per 6-well plate), place in a 50ml falcon tube, and warm to 37°C in a water bath. Once the media is warmed, add bFGF stored at 4°C to a final concentration of 10ng/ml. Put the bFGF stock back at 4°C immediately after use! Remove from the 6-well plates all but ~500µl of media. Make sure to swirl the culture dis…
Discussion
This video demonstrates how to passage hESCs from MEF plates to feeder cell-free Matrigel plates. Note that the colony density on Matrigel is higher than the density of colonies by usual splitting on MEFs. Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct-4 and SSEA-4, are needed to confirm maintenance of hESCs in an undifferentiated state in feeder-free culture conditions.
Acknowledgements
Human embryonic stem cell studies in the Teitell lab are supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank members of the Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, especially Dr. Amander Clark, Dr. Jerome Zack, and members of the UCLA Broad Institute Stem Cell Core Facility for their support of our studies.
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