Adipose-derived spheroid cells (Kyung Hee University)

Rat subcutaneous tissue was carefully dissected from beneath the dermis, mechanically minced, and treated with 0.15% (w/v) collagenase type I. Collagenase was inactivated by addition of the same volume of DMEM/F12 containing  FBS (10% ). After passage through a filter of 40 -μm pore size, cells were resuspended in spheroid induction medium consisting of DMEM/F12 (3:1 ratio, v/v) supplemented with  N2 (X1),  EGF (20 ng/ml),  bFGF (20 ng/ml), and heparin (2 μg/ml ), and plated in 75 cm² flasks.  Medium was replaced every 3 days. After 7–10 days, spheroids were harvested by centrifugation, dissociated into single cells, and resuspended in DMEM/F12 (3:1 ratio, v/v) with  FBS (10%) for monolayer culturing. When reaching high confluence, cells were harvested and replated at a ratio of 1:3 and can be cultured up to Passage 5. In order to reform spheroids, cells were incubated with spheroid induction medium for 3 days and next in neurobasal medium with G5 supplements, laminin (2 μg/ml), and  heparin (2 μg/ml), for 3–4 days.