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.
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.
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
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