Umbilical Cord Blood-Derived Mesenchymal Stem Cell Protocol

As previously described [15 (link)], umbilical cord blood (UCB) was collected from umbilical veins after neonatal delivery with maternal informed consent. UCB harvests were processed within 24 h of collection. UCB was separated by isolating mononuclear cells with Ficoll-Hypaque solution (density, 1.077g/cm3; Sigma, St. Louis, MO, USA). The separated mononuclear cells were washed, suspended in Minimum Essential Medium a modification (MEM a; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), and seeded at a concentration of 5 x 10⁵ cells/cm². Cultures were maintained at 37°C in a humidified atmosphere (21% O₂) containing 5% CO₂ with a change of culture medium twice a week. One batch of cells was used to inject all pups. As described previously [16 (link)], human UCB derived MSCs from a single donor at passage 6 manufactured and also used for various preclinical studies of neonatal intractable disorders including BPD [17 (link)], IVH [18 (link)] and HIE [19 (link)] were obtained from Medipost Co., Ltd. (Seoul, Korea).