Keratinocytes should be cultured in a 50%-50% mixture of DMEM and Ham’s F12. Use a 1:1 ratio of the two medias. The mixture should be modified by addition of 10% FBS. Make sure that F12 contains 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone. Incubate cells at 37.0°C. Cell cultures will double approximately every 30 hours. This will vary depending on the keratinocyte cells source and culturing conditions.
Split the cells 1:4 to 1:8 once just confluent, and renew the medium ever 2 to 3 days.
- Remove medium
- Rinse cells with solution containing 0.05% Trypsin and 0.03% EDTA
- Add an additional 1-2 mL of Trypsin-EDTA and allow cells to disperse
- If cells are not dispersing, place cells in incubator for 3-5 minutes to encourage rounding up
- Centrifuge cells at 125 x g for 5-10 minutes
- Remove supernatant (Trypsin-EDTA)
- Add fresh culture medium
- Split and dispense cells into new flasks
Freeze cells in conditioned growth medium supplemented with 5-10% (v/v) DMSO by freezing at 1 degree (C) per minute, and store in the liquid nitrogen vapor phase or at -135 °C.