Neonatal Umbilical Cord Blood Mesenchymal Stem Cell Spheroid

Neonatal UCB samples were collected from the umbilical vein after obtaining informed consent from the 5 different donors. This protocol was approved by the Institutional Review Board of MEDIPOST Co. Ltd. (MP-2014-04). The UCB samples were isolated by separating mononuclear cells (MNCs) using a Ficoll-Hypaque solution (Sigma, St. Louis, MO, USA). Separated mononuclear cells were suspended in a minimum essential medium (a-MEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 50 μg/mL gentamicin (Gibco, Carlsbad, CA, USA), respectively. The cells were cultured and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. The culture medium was replaced with fresh medium twice a week, as previously described [34 (link)]. Neutralizing antibody treatment was performed to block E-cadherin expression. After trypsinization of the monolayered UCB-MSCs, as described above, they were then cultured in hanging drops that contained 20 μL of spheroid medium containing 100,000 cells/drop with E-cadherin neutralizing antibody (Sigma, St. Louis, MO, USA) or immunoglobulin G (R&D Systems, Minneapolis, MN, USA). The spheroid was incubated at 37 °C for 72 h. UCB-MSCs used in all experiments were at passage 6.