Cells are isolated from the stromal-vascular fraction of abdominal adipose tissues.
When cultured in a tissue culture plate, cells exhibit an elongated fibroblast-like morphology.
The subcutaneous adipose tissue is obtained by liposuction from the abdomens of patients. The tissue is then washed several times with Krebs Ringers buffer to remove any residual blood contamination. The tissue is then digested in the same buffer supplemented with bovine serum albumin (1%), and collagenase type I (0.025%). The floating adipocyte cells are separated by centrifugation, at 300g for 5 minutes. The isolated preadipocytes in the stromal-vascular fraction are cultured in DMEM– Ham’s F-12 medium supplemented with fetal bovine serum (FBS, 10%) and maintained for 1–3 days. After this period, the culture dishes are washed with phosphate-buffered saline to remove non-adherent cells and cultured in an expansion medium consisting of DMEM–Ham’s F-12 medium supplemented with FBS (10%), human transforming growth factor-b (0.25 ng/ml), human epidermal growth factor (5 ng/ml), and human bFGF (2.5 ng/ml).