Growth and characterization of normal human keratinocytes in F12 serum-free medium

In a F12 medium supplemented with epidermal growth factor (20 ng/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (1 microM), cholera toxin (40 ng/ml), endothelial cell growth supplement (15 ng/ml) and retinoic acid (1 x 10(-7) M) on Vitrogen coated culture dishes, normal adult and newborn human foreskin keratinocytes were cultured for 4- and 2-time with population doublings (PD) accumulated as 8 and 12, respectively. The cells grown in this medium possessed a basaloid, undifferentiated and hyperproliferating nature, with a population doubling time of about 24 hours at early passage. Between the 1st and 2nd subcultures, cell proliferation was the most active. Delaying the time of first subculture lowered the rate of cell proliferation. The keratin of the cultured cells was studied by immunoblotting and revealed the presence of permanent keratin markers of the human skin (AE1 50 kDa and AE3 58kDa) as well as a relatively high intensity of a proliferating marker (AE1 48 kDa) and a relatively low intensity of a differentiation marker (AE3 67 kDa).