Adipose-derived mesenchymal stem cells (RNL Bio)

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.