Insulin transferrin selenium media supplement

CD34 negative UCBSC were isolated and grown as previous described^{1 (link)}. Briefly, umbilical cord blood was collected after delivery and, within 4 h, mononuclear cells were separated by centrifugation at 500 × g for 30 min in a Ficoll-hypaque density gradient (1.077 g/cm³) (Sigma, St. Louis, MO, USA). The cells were then grown in Dulbecco’s modified Eagle medium DMEM (Invitrogen, Waltham, MA, USA) / MCDB-201(Sigma) mix with 10% fetal bovine serum (Invitrogen), 10^–4 M of l-ascorbic-acid-2-PO₄ (Sigma), 10^–9 M of dexamethasone (Sigma), insulin-transferrin-selenium media supplement (Sigma), 1 mg/mL of linoleic acid/bovine serum albumin (Sigma) and 10 ng/ml of epidermal growth factor (R&D Systems, Minneapolis, MN, USA) and recombinant human basic fibroblast growth factor (R&D Systems).

CD34-negative human nh-UCBSCs were isolated and grown as in our previous publications^{23 –26 (no link found, link, no link found)}. Briefly, UCB was collected after delivery, and within 4 h, MNCs were separated by centrifugation at 500 × g for 30 min in a Ficoll-hypaque density gradient (1.077 g/cm³) (Sigma, St. Louis, MO, USA). The cells were then grown in Dulbecco’s modified Eagle medium DMEM (Invitrogen, Waltham, MA, USA)/MCDB-201(Sigma) mix with 10% fetal bovine serum (Invitrogen), 10⁻⁴ M of l-ascorbic-acid-2-PO₄ (Sigma), 10⁻⁹ M of dexamethasone (Sigma), insulin-transferrin-selenium media supplement (Sigma), 1 mg/ml of linoleic acid/bovine serum albumin (Sigma) and 10 ng/ml of epidermal growth factor (R&D Systems, Minneapolis, MN, USA), and recombinant human basic fibroblast growth factor (R&D Systems). The cells were expanded to 20 passages as in our previous studies^{23 ,24} . The final cell suspension contained 5 × 10⁶ cells/ml.