Feeder-Free Culture of hESCs
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MEF Conditioned medium
- Plate MEFs (p4 or p5) onto gelatin-coated plates at a density of 1x106 cells/10cm culture dish.
- The next day, wash cells with PBS and add 10ml normal hESC medium containing 4ng/ml bFGF. This is collected the next day and each subsequent day for 7 days.
- CM may be stored frozen for several months.
- Before use, add 4ng/ml bFGF to the medium and filter.
Culture of hESCs
- Coat 6-well tissue culture plates (Falcon) with 5% Matrigel in DMEM/F12 overnight at 4°C using 1.5ml Matrigel suspension per well.
- Allow the Matrigel plate to warm to room temperature in the hood for approx 30min prior to use. DO NOT WARM IN INCUBATOR.
- Remove hESC cells from MEFs using collagenase treatment and sediment as usual. Aspirate the Matrigel and plate triturated colonies in conditioned medium supplemented with an additional 4ng/ml bFGF or in normal hESC medium containing 100ng/ml bFGF.
- Feed cells every day up to 7 days. Note: Colonies can grow bigger and more densely on Matrigel without losing morphology than on MEFs.
Passaging
- Wash cells once with PBS and incubate at 37°C with 1ml/well of 2mg/ml dispase in DMEM/F12.
- Colonies should detach intact within 10�15 minutes upon tapping smartly on the side of the plate. DO NOT SCRAPE.
- Remove the colonies in dispase to a 15ml tube and rinse wells with an additional 1ml/well growth medium.
- Sediment and wash twice more as usual.
- Triturate VERY gently as colonies grow flat and dissociate readily in dispase. Note: Small colonies and single cells do not survive well on Matrigel especially in 100ng/ml bFGF only.
- Plate as usual on fresh Matrigel
