Medium: 90% of MEM (with Earle's salts) with 10% FCS + 2 mM L-glutamine + non-essential amino acids. Can also use RPMI-1640 and 5-10% FBS.
Subculture: split confluent culture 1:4 or 1:6 every 3-5 days depending on confluency using trypsin/EDTA. Rate of doubling time is 24 to 48 hours. Seed at 1-2 x 106 cells/80 cm2. Incubate at 37 °C with 5% CO2
Storage: frozen with 70% medium, 20% FCS with 10% DMSO at about 1 x 106cells/ampoule