Feeder-free hiPSC Expansion and Banking

The WTSli020 hiPSC line from fibroblasts of dermis of a healthy female that was provided by EBiSC (European Bank for induced pluripotent Stem Cells) was cultured in feeder-free conditions using Vitronectin-coated culture vessels (VTN-N; Thermo Fisher Scientific, Waltham, MA, USA) and Essential 8 Flex medium (Gibco, Grand-Island, NY, USA) supplemented with Penicillin/Streptomycin (1:1000 PenStrep; Gibco). Another hiPSC line derived from a healthy female was also used as previously described [38 (link)]. Briefly, cells were thawed and manually expanded over five supplementary passages. For manual passaging, StemPro EZPassage tool (Thermo Fisher Scientific) was used. The automated cell culture system CompacT SelecT (Sartorius, Gottingen, Germany) was then used to generate a working cell bank using 0.25 mM EDTA (Thermo Fisher Scientific) in Phosphate-Buffered Saline (PBS; Gibco) without calcium or magnesium for cell passaging. Finally, cells were dispensed into cryovials using the automated system Fill-It (Sartorius) and frozen using CryoMed Controlled-Rate Freezer (Thermo Fisher Scientific). Quality controls (mycoplasma detection, pluripotency marker expression, genomic integrity) were performed before and after amplification.