Hair follicle stem cells (HFSC) were isolated from human scalp skin. HFSCs rapidly adhere to collagen IV-treated plates, are stationary and can be propagated for 6 passages.
Bulb hair fragments dissected from human scalp skin were digested for 10 minutes in trypsin (0.05%) containing ethylene diamine tetraacetic acid (EDTA). Versene (1.33:1) was added and cells were incubated for an additional 10 minutes. Cells were plated on a feeder layer of mitomycin C (15 mg/mL), treated J2-3T3 fibroblasts and grown in keratinocyte medium (KCM). KCM is comprised of Dulbecco's modified Eagles medium/Nutrient Mixture F-12 (DMEM/F12, 4:1), fetal bovine serum (FBS, 10%), cholera toxin (0.1 nM), penicillin (100 U/ml), streptomycin (100 mg/ml), hydrocortisone (0.4 mg/ml), transferrin (5 µg/ml), triiodo-l-thyronine (T3, 2 nM), insulin (5 mg/ml), epidermal growth factor (EGF, 10 ng/ml), pH 7.2.