4.2. Cell Culture and Optimization of Extracellular Factors

The H9 hESCs were cultured on mitotically arrested CF1 MEF feeder layers using 10 µg/mL of mitomycin C in knockout DMEM/F12 supplemented with 20% knockout serum replacement, 2 mM L-glutamine, 1% penicillin–streptomycin, 0.1 mM non-essential amino acid, 0.1 mM 2-mercaptoethanol, and 4 ng/mL of hFGF-2. For feeder-free culture, H9 hESCs were cultured on hESC-qualified matrigel in commercial E8 medium [5]. Once H9 hESCs attained 70–80% confluence, colonies were manually split or enzymatically split using 1 mg/mL of collagenase IV. The cells were subcultured further in E8 medium containing 10 µg/mL Y-27632. Subsequently, H9 hESCs were maintained in homemade E8 media consisting of DMEM/F12 supplemented with 64 µg/mL L-ascorbic acid-2-phosphate, 14 ng/mL sodium selenite, 10 µg/mL holo-transferrin, 20 µg/mL insulin, 1 mg/mL NaCl, 1% penicillin–streptomycin, 2 ng/mL TGF-β, and 100 ng/mL of either hFGF-2 or hDJ-1. The media was changed every day except for the next day after the split. All experiments using hESCs were conducted under the approval of the Institutional Ethics Committee, University of Ulsan College of Medicine, Seoul, Korea.