Isolation and Culture of Mesenchymal Stromal Cells

Mesenchymal stromal cells were derived from lipo-aspirates obtained from consenting healthy donors with approval from the Mayo Clinic Institutional Review Board (IRB) as previously described [Crespo-Diaz et al., 2011 (link); Mader et al., 2013 (link)]. Samples were obtained from three consenting normal patients (respectively, male/41 yr, female/32 yr, male/54 yr) that underwent elective removal of subcutaneous adipose tissue. Fat tissue was enzymatically digested using collagenase (Type I at 0.075%; Worthington Biochemicals) for 1.5 h at 37°C. Adipocytes were separated from the stromal vascular fraction by low speed centrifugation (400 g for 5 min). After the adipose supernatant was removed, the cell pellet was rinsed with PBS and passed through cell strainers (70µm followed by 40µm) (BD Biosciences). The resulting cell fraction was incubated in T-175 cm² flasks at 37°C in 5% CO² at a cell density of 1.0–2.5 ×10³ cells/cm² in standard culture medium (Advanced MEM) with 5% PLTMax (a clinical grade commercial platelet lysate product [MillCreekLifeSciences]), 2 U/ml heparin (hospital pharmacy), 2 mM L-glutamine (Invitrogen) and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin). Cells were harvested while still actively proliferating or when they reached confluence (typically four days after plating).