Collagen-producing cells derived from skin fibroblasts (RepliCel Life Sciences Inc.)

A 4-mm skin sample was obtained from the lateral side of the hip using a 4-mm punch biopsy needle. The skin sample was placed in cell transport medium (DMEM/F12 with gentamicin) and transported to a GMP facility. Under GMP conditions, the skin sample was washed in PBS, disintegrated, and digested by adding HEPES-containing medium with collagenase type 1 and FCS. PBS was added during the next day and the connective tissue was pelleted by centrifugation. The pellet was resuspended by adding a fibroblast medium to each 50 ml tube and  transferred to tissue culture flasks, which were incubated in a 37° C, 5% CO₂ incubator for 48-72 hours. FCS served as a medium additive for the propagation of connective tissue cells. Medium was changed every 2-4 days.