2.2. Stem Cell Isolation, Culture, and Identification

rASCs were isolated from the rats weighing 60 g~80 g. The inguinal fat tissues were mechanically dissected into pieces, washed three times in phosphate buffered-saline (PBS, Gibco), and then digested by collagenase I (Sigma) and trypsin (Hyclone) in 37°C for 40 min. After filtration through a 100 μm cell strainer and resuspended in DMEM (Hyclone) containing 10% fetal bovine serum (FBS) and 1% antibiotics (Hyclone), the cell cultures were incubated in 37°C. The culture media were changed every 3 days, and rASCs applied for all experimental use were passage 3 or 4.

The multidifferentiation abilities were verified by inducing differentiation assay (Cyagen). rASCs were seeded at 2 × 10⁴/cm² in 6-well cell culture plates in the presence of adipogenic, chondrogenic, or osteogenic differentiation mediums according to the manufacturer's instructions. The induction medium was changed every 3 d for 3 w. After removing the induction medium and fixing by 4% formaldehyde solution, the cells were stained by freshly prepared Oil red O, Alcian blue, and Alizarin red working solutions for specially stained adipocytes, chondrocytes, and osteocytes, respectively. All the results were observed on an inverted-phase contrast microscope (Olympus).

The characteristic surface markers of rASCs were analyzed on the FACScan flow cytometry (BD). The third passage of rASCs was trypsinized, washed once, resuspended in PBS, and incubated with 5 μL monoclonal antibodies at 4°C avoiding light: fluorescein isothiocyanate- (FITC-) conjugated CD45, phycoerythrin- (PE-) conjugated CD31, FITC-conjugated CD34, FITC-conjugated CD44, PerCP-conjugated CD90.1, AF647-conjugated CD29, and Ig isotype antibodies were applied to set the fluorescence background. All the antibodies used for surface marker analysis were purchased from BD.