Collagenase type 2 clostridium histolyticum

The skin biopsy samples were put in sterile culture DMEM medium (Lonza, catalog BE12-614F), 20% FBS (Biowest, catalog S181B-500), 50 IU/ml penicillin, and 50 μg/ml streptomycin (Gibco). The skin biopsies were cut into small fragments in PBS on a Petri dish and transferred to Falcon tubes containing 3 ml PBS (without Mg and Ca) and 1000 U/ml Collagenase Type II: Clostridium histolyticum (Gibco). The tubes were incubated at +37°C for 2 hours. Collagenase inactivation was performed by addition of 3 ml ice-cold medium. The tubes were mixed by vortexing, and tissue lysates were centrifuged for 10 minutes, 150 g at +4 °C. The supernatants were removed, the cell pellets were resuspended in 900 μl DMEM and transferred to T-25 flasks (Corning Life Sciences). 4 ml of DMEM was added to each flask, and they were incubated in a 5% CO₂ humidified incubator at 37°C for 1–2 weeks.