Complete Growth Medium
Dulbecco's modified Eagle's medium with 4.5 g/L glucose, 50%;
Ham's F12 medium, 50%,
Supplements: 0.01 mg/ml bovine insulin, 0.025 mg/ml human transferrin, 0.02 mg/ml human high density lipoprotein (HDL), 100 ng/ml mouse epidermal growth factor (EGF), 10 nM sodium selenite, 1 unit/ml human platelet derived growth factor (PDGF),
and 15 mM HEPES.
NOTE: HDL is available from Sigma Chemical Co. (catalog number L2014).
Subcultivation Ratio: A subcultivation ratio of 1:10 to 1:100 is recommended
Medium Renewal: Twice per week
Remove medium, and collect suspended clusters by centrifugation.
To disperse clusters or to remove adherent cells, add fresh 0.25% Trypsin, 0.02% EDTA at room temperature until the cells disperse (2 to 3 minutes).
Add an equal volume of fresh serum free medium plus 0.1% soybean trypsin inhibitor and centrifuge at 1000 X g.
Resuspend the cell pellet in growth medium, aspirate and dispense into new (fibronectin coated, if adherence is desired) flasks.
Floating clusters may also be remove using a pipette and transferred to a flask containing fresh growth medium.
culture medium, 80%; fetal bovine serum, 10%; DMSO, 10%