2.2. Cell Culture and Differentiation Model Construction

Primary sebocyte cultures were performed according to a previously described method [10, 11]. Briefly, the isolated sebaceous glands from the separated epidermis of normal scalp were transferred to a tissue culture dish. The isolated cells were cultivated in Sebomed basal medium (Biochrom GmbH, Berlin, Germany) containing 10% fetal bovine serum (FBS; Gibco BRL, Rockville, MD, USA) and 5 ng/ml recombinant human epidermal growth factor (Invitrogen) in a 5% CO₂ incubator at 37°C. The cells were harvested with 0.05% Trypsin-EDTA (Gibco BRL) and subcultured after they became subconfluent. The cells after the second passage were used in this study. Sebocytes grown to 50% confluence were serum starved for 24 h in Sebomed without FBS, and the cells at this time point were designated as day 0 (D0). To induce differentiation, the D0 sebocytes were switched into Sebomed supplemented with 2% FBS and cultured up to 7 days with medium change every day.