SERUM-FREE CULTURE OF RAT KERATINOCYTES

Procedures for the serum-free culture of rat keratinocytes have been e stablished. Basal cells prepared from epidermis of newborn rat were st ored in liquid nitrogen and used for primary culture. Among the availa ble media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. T o grow rat keratinocytes, bovine serum albumin was arbitrarily substit uted for the macromolecule supplements needed for HK culture, i.e. fet al bovine serum protein or bovine pituitary extract. Qualitative and q uantitative adjustment of supplements was thereafter made to support r apid cell growth. Satisfactory cell growth was achieved in the optimiz ed medium of MCDB 153 supplemented with growth factors and amino acids : insulin (10 mu g/ml), hydrocortisone (0.1 mu g/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), is oleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyros ine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimiz ed culture system was superior to the original HK culture condition fo r rapid growth of rat keratinocytes. Under our condition, cells grew a s a monolayer, becoming confluent, but without stratification, and wer e passaged 2 to 3 times without any changes in morphology. The serum-f ree formulation allows us to control more accurately the concentration s of biomolecules in the medium including lipids and hormones, and the refore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.