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Adipose-derived mesenchymal stem cells (adipose-MSCs) are spindle-shaped cells with a fibroblast-like morphology, which attach to tissue culture plates. These characteristics are well preserved during repeated subculture. Cultured adipose-MSCs show the typical immunophenotype and differentiation capacities of MSCs. The cells express MSC markers (CD90, CD105, CD44 and CD29), but do not express hematopoietic or endothelial markers (CD31, CD34 and CD45) and differentiate to adipogenic, osteogenic, neurogenic, myogenic and chondrogenic lineages in vitro. Culture-expanded adipose-MSCs are genetically stable for at least 12 passages, as determined by karyotype and single nucleotide polymorphism (SNP) assays.
Human adipose tissues were obtained by liposuction from the abdominal subcutaneous fat, following informed consent. Subcutaneous adipose tissues were digested with collagenase I (1 mg/mL) under gentle agitation, for 60 min at 370C. The digested tissues were filtered through a 100-um nylon sieve, to remove cellular debris, and were centrifuged at 470 g for 5 min. The pellet was resuspended in DMEM containing ascorbic acid (0.2 mM) and fetal bovine serum (FBS, 10%) obtained from bovine spongiform encephalopathy-free herds. The cell suspension was recentrifuged at 470 g for 5 min. The supernatant was discarded and the cell pellet was collected. The cell fraction was cultured overnight (370C/5% CO2) in DMEM supplemented with ascorbic acid (0.2 mM) and FBS (10%). After 24h, cell adhesion was assessed under an inverted microscope, and nonadherent cells were removed by washing with PBS. The cell medium was changed to Keratinocyte-SFM-based medium containing ascorbic acid (0.2 mM), calcium (0.09 mM), rEGF (5 ng/mL), and FBS (5%). The cells were maintained for 4-5 days until confluent (passage 0). When the cells reached 90% confluency, they were subculture-expanded in Keratinocyte-SFM-based medium containing ascorbic acid (0.02 mM), calcium (0.09 mM), rEGF (5 ng/mL), and FBS (5%), until passage 3. FBS from cultured MSCs were completely removed by several washing with PBS and was verified by testing for albumin levels below the measurement limit, using a bovine albumin ELISA quantitiation kit.