Biolaminin 521 ctg

The derived hESCs were cultured under hypoxic culture conditions (5% O₂, 5% CO₂, 37 °C) in a NutriStem^® hPSC XF Medium (Biological Industries) with a daily medium change. The cells were passaged mechanically by an insulin syringe (B.Braun) and cultured on 16.6 µg/mL (1.9 µg/cm²) Biolaminin 521 CTG (BioLamina) with 1.7 µg/mL (0.27 µg/cm²) E-cadherin for the first three passages when colonies reached sufficient size. Then, the non-enzymatic passaging by 0.5mM EDTA was performed when the cells reached 70% confluence. The cells were exposed to 10 µM ROCK inhibitor (Y27632, GMP, Bio-techne) 1 h before and 24 h after passage. After an one-hour ROCK inhibitor treatment, the cells were rinsed with phosphate-buffered saline (PBS, Gibco), dissociated by 0.5mM EDTA (Invitrogen) in clumps, and placed on 10.0 µg/mL (1.1 µg/cm²) Biolaminin 521 CTG (BioLamina). The media change and cell culture evaluation were conducted by a trained staff member on a daily basis.

Differentiation to the three germ layers was supported by the medium: DMEM/F12 (Gibco), 15% knockout-serum replacement (Gibco), 1% non-essential amino acids (Sigma), 1% Glutamax (Gibco), 1% ZellShield (Minerva Biolabs), and 0.2% 2-Mercaptoethanol (Gibco). The cells were passaged in a low attachment 96-well dish (20 × 10³ cells/well) and cultured for 1 week until embryoid bodies were formed. The embryoid bodies were then transferred to 4-well dishes coated with 10.0 µg/mL Biolaminin 521 CTG (BioLamina), where they were allowed to attach and culture for another 14 days.