Isolation, culture and identification of human adipose-derived stem cells

The aim of the present study was to improve methods for the isolation and identification of adipose-derived stem cells (ASCs). Human subcutaneous adipose tissue was collected during liposuction surgery, without ultrasound-assisted liposuction and other assisted techniques, and digested with 0.075% collagenase I. First (P1) and second (P2) passage ASCs were applied to the subsequent experiments. ASCs were observed under a microscope, the growth curves of the cells were assessed using a cell counting kit-8 assay and the membrane expression of cell surface antigens, including cluster of differentiation (CD)44, CD105 and CD45, were detected by flow cytometry. In addition, ASCs were induced to differentiate into lipocytes and osteocytes. Oil red staining was applied to examine adipogenic induction, whereas alkaline phosphatase (ALP) staining was used to assess osteogenic induction. Primary ASCs adhered to the culture vessel wall after 72 h, were fusiform in appearance at 5 days and exhibited stable growth with active proliferation. In total, 1×10⁵ stem cells were gained per 50 ml of lipo-aspirate. ASCs were plated in a 25 cm² culture flask at a density of 5×10⁴/ml; the cells underwent the first logarithmic growth period after 72 h and grew to 90% confluence within 3 days. Flow cytometry demonstrated that the cells were highly positive for CD105 and CD44, and weakly positive for CD45; 18.6% of P1 cells and 90.7% of P2 cells were CD44⁺CD45^-CD105⁺. Oil red and ALP staining were positive. The results of the present study suggested that ASCs may be considered a promising cell type for tissue engineering. Furthermore, the present study established an effective method for the isolation and identification of ASCs, which reduced damage to the stem cells and simplified the identification procedure.