Immortalized hair follicle stem cells (University of Massachusetts Medical School)

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 20 minutes. Cells were plated on mitomycin C (15 mg/mL), treated J2-3T3 fibroblasts and grown in keratinocyte medium (KCM). Cells were fed every 2 days. Primary cells were immortalized by transduction with a retroviral vector (LXSN- 16E6E7). Briefly, primary cells were co-cultured with mitomycin C-treated virus-producing fibroblasts (PA317/L(E6E7)) in KCM for 6 days. Virus-producing cells were then removed and tranduced cells were plated on a feeder layer of mitomycin-treated 3T3-J2 NHP cells and selected by treatment with G418 (0.2 mg/ml) for 6 days.  The resulting Tel-E6E7 cells were maintained on a feeder layer in 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.