Skin-derived fibroblasts (Fibrocell Technologies, Inc.)

Subjects undergo three 3-mm punch biopsies in one retroauricular area to harvest  fibroblasts. After an antibiotic wash containing gentamicin and amphotericin B, biopsy tissue is subjected to enzymatic dissociation in a collagenase enzyme cocktail at 37°C. After the dissociation is complete, cells are seeded into a T-175 culture flask within Iscove's modified Dulbecco's medium with phenol red (IMDM) supplemented with antibiotics and fetal bovine serum (FBS) and stored in a humidified incubator at 37°C with 5% carbon dioxide. Routine feeding of the fibroblasts is performed using IMDM with FBS, by removing half of the volume of spent medium and adding back the volume with fresh medium. Once the culture reaches confluence, cells are rinsed with PBS and exposed to trypsin–ethylenediaminetetraacetic acid to remove cells from the surface of the flask. Cells are cryopreserved in IMDM and freezing medium supplemented with dimethyl sulfoxide. Cells are stored in the vapor phase of liquid nitrogen. No serum is present in the cryopreservative formulation.