Isolation and Propagation of Human MSCs

Following informed consent and IRB approval at the authors’ institution, human MSCs were isolated from lipo-aspirates obtained from three representative donors (cell lines: 211, 258 and 283) using previously described methods [48 (link)–50 (link)]. The donor-derived MSCs selected in this study were propagated with platelet lysate containing zoonotic free media and have been previously shown to express standard MSC markers and to undergo multi-lineage differentiation [48 (link), 49 (link), 51 (link), 52 (link)]. In brief, lipo-aspirates were digested with Type I collagenase (Worthington Biochemicals, Lakewood, NJ) for 1.5 hours at 37°C, centrifuged at 400g for 5 min, rinsed with phosphate buffered saline (Life Technologies, Grand Island, NY), and strained using 70μm cell strainers (BD Biosciences, San Jose, CA). Following this, the aspirate was treated with 154 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA for erythrocyte lysis. The remaining MSCs were expanded in advanced minimum essential medium (Life Technologies, Grand Island, NY), supplemented with 5% (vol/vol) human platelet lysate (PLTMax; MillCreekLifeSciences, Rochester, MN), 2mM Glutamax (Life Technologies, Grand Island, NY), 2 U/mL heparin, and 1% Penn-Strep (100 U/mL penicillin, 100 μg/mL streptomycin; Cellgro, Corning, NY). All MSCs used in experimentation were of passage 7.