Isolation and Culture of Adipose-Derived Cells

Adipose tissue was transferred to the lab in medium 199 at room temperature. After mincing into approximately 5 to 10 mg fragments, tissue was digested with collagenase (type 1, 1 mg/ml in HBSS) for 2 hours at 37ºC with shaking (100 rpm) (9 (link)). The mixture was passed through a 250 micron mesh, centrifuged at 500g for 10 minutes and floating adipocytes were discarded. Cell pellets were treated with erythrocyte lysis buffer (0.154mM NH₄Cl, 10 mM K₂HPO₄ and 0.1 mM EDTA, pH 7.3) for 10 min at room temperature. After discarding the supernatant, cells were repelleted with centrifugation, resuspended in growth media (α-MEM with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin) and then plated. On the following day, cells were washed with PBS and replenished with growth media. At 70~80% confluence, cells were either subcultured or frozen (10% DMSO supplemented growth media) for cryopreservation.