FeederFreeCultureofhESCs

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