Optimizing Human Mesenchymal Stem Cell Culture

Human mesenchymal stem cells (hMSCs) were purchased from the Bioresource Collection and Research Center (BCRC, No. RM60596), Hsinchu, Taiwan. ATCC (PCS-500-010). Cells were grown in culture dishes containing 56% Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) and 37% MCDB-201 medium (Sigma Aldrich, St Louis, MO, USA), supplemented with 2% fetal bovine serum (Thermo, Logan, UT, USA), 1× Insulin-Transferrin-Selenium-A (Invitrogen, Carlsbad, CA, USA), 0.5 mg/mL of AlbuMax I (Invitrogen, Carlsbad, CA, USA), 10 ng/mL of epidermal growth factor (PeproTech, Rocky Hill, NJ, USA), 1 ng/mL of platelet-derived growth factor (PeproTech, Rocky Hill, NJ, USA), 10 nM dexamethasone (Sigma Aldrich, St Louis, MO, USA), and 50 μM L-ascorbic acid-2-phosphate (Sigma Aldrich, St Louis, MO, USA) [29 (link)]. The cells were maintained at 37 °C in a 5% CO₂ incubator. When cells reached 70–80% confluence, they were detached with HyQtase (Thermo-Fisher Scientific, Waltham, MA, USA), centrifuged, and resuspended for subculture or further experiments. Cells between Passages 6 and 10 were used in the experiments. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma Aldrich, St Louis, MO, USA) was dissolved in DMSO as a stock solution at 10 mM, further diluted in hMSC cultured medium, and then used at various concentrations of 0.1, 0.3, 1, 2, 3, 10, and 30 μM.