5.1. Cell Culture and Monitor of Cell Proliferation

hESC line H9 and human iPSC line IMR90 acquired from the WiCell Research Institute (Madison, WI, USA) were routinely maintained in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) on growth factor reduced Matrigel-coated tissue culture dishes at 37 °C and 5% CO₂ as described previously [4,5,39]. Cells were passaged by 1 mg/mL of dispase treatment and scraping every 3–4 days, with a split ratio of 1:3–1:6. The culture medium was exchanged daily. The morphology of cell colonies was examined daily, and spontaneously differentiated colonies were removed to ensure the maintenance of undifferentiated state of hPSCs. To characterize hESC and iPSC behavior under various conditions, ~5 × 10⁴ cells/cm² were seeded to the PET porous membrane surface with 2 × 10⁶ pores/cm² (EMD Millipore, Billerica, MA, USA) after Matrigel coating (Corning Inc., New York, NY, USA). Cells seeded onto Matrigel coated tissue culture polystyrene dishes, referred to as TCP, served as a control. Cell doubling time (t_d) was calculated as follows:

where x is the cell concentration and µ is the specific growth rate. hPSC numbers were counted at 24-h intervals during the culture using a hemocytometer after trypan blue staining.