Culture-expanded adipose-derived stromal cells (ADSCs) are stem cells isolated from adipose tissue. ADSCs are similar to bone marrow-derived mesenchymal stem cells in their extensive proliferative and multilineage potential. In order to obtain a sufficient number of ADSCs for clinical use, it is necessary to culture and expand the cells for several weeks. Several studies have demonstrated the ability of ADSCs to undergo differentiation, when cultivated under lineage-specific conditions, along classical mesenchymal lineages, such as adipocytes, chondrocytes and osteocytes. Moreover, there is evidence that ADSCs can differentiate into other cell lines, such as neurons, hepatocytes, smooth muscle cells, endothelial cells and cardiomyocytes.
Lipoaspirates (100–150 ml) obtained from patients are washed 2-3 times with phosphate-buffered saline (PBS) to remove residual blood and free lipids. Then digestion by collagenase NB 6 GMP grade (0.3 U/ml), dissolved in Hanks’ balanced salt solution (+ CaCl2 + MgCl2 + 2 mM calcium) at 37 ± 1°C for 45 min, under gentle rotation. The collagenase is neutralized with an equal amount of complete medium consisting of DMEM, low glucose (1 g/l) supplemented with hydroxyethyl piperazineethanesulfonic acid (25 mM) and L-glutamine, fetal bovine serum pharma grade (10%) and penicillin/streptomycin (1%). The digested tissue is filtered through a 100 µm mesh to remove tissue remnants and is centrifugated at 1200 g. The supernatant containing adipocytes is discarded and the cell pellet is resuspended in an appropriate volume of complete medium. ASCs are plated at a density of 6 × 104 cells/cm2 in T-75 culture flasks containing 15 ml complete medium, in humid air with 5% CO2 at 37°C. Medium is changed every 3–4 days. The cells are passed once (P1) when they reach 80–90% confluence.