Cellbind cell culture dishes

RPChiPS771 human induced pluripotent stem cells (iPSCs; REPROCELL) were maintained in StemFit AK02N medium (Ajinomoto) on 100 mm CellBIND cell culture dishes (Corning, 3296) precoated with vitronectin (ThermoFisher Scientific, A31804). Cell passages were performed as follows. hiPSCs were washed, dissociated with TrypLE^TM Select (Gibco), and resuspended in StemFit AK02N medium supplemented with 10 µM ROCK inhibitor (Y-27632; Wako) and replated at 1-2×10⁶ cells per dish.

Umbilical cords were collected from healthy newborn children immediately after their birth with the fully informed consent from their legal guardians and approval from the Shandong Provincial Medical Ethics Council. After screening for human immunodeficiency virus (HIV), hepatitis C virus (HCV), cytomegalovirus (CMV), hepatitis B virus (HBV) and syphilis spirochete, the Wharton’s jelly was mechanically separated from the umbilical cords and dissected into small pieces, which were then resuspended in the MesenCult-SF culture medium (Stemcell Technologies, Vancouver, BC, Canada) supplemented with 2 mM Glutamax (Invitrogen, Carlsbad, CA, USA), plated onto CellBIND™ cell culture dishes (Corning, Corning, NY, USA) and cultured in a humidified 5% CO₂ atmosphere at 37 °C. The medium was replaced by half at a 3-day interval until the attached cells grew to 80% confluence. The cells were then detached with 0.05% trypsin and expanded for another passage before storage in liquid nitrogen. Prior to injection, the cells were resuscitated and screened for aerobes, mycoplasma, HIV, HBV, HCV, CMV, and the presence of endotoxins. The cells were also examined for growth capacity, differentiation capacity, and immune phenotype (CD34, CD44, CD45, CD90, CD105, and HLA-DR) (Additional file 1: Figure S3).