Perfect count microbeads

UCB samples were obtained from the Blood and Tissue Research Biobank (Barcelona, Spain). All samples were obtained between 20 and 72 h after delivery. To isolate CD34⁺ cells, UCB were incubated with RosetteSep (STEMCELL Technologies, Vancouver, Canada) for 20 min prior to density gradient purification using Ficoll-Paque (GE Healthcare, Uppsala, Sweden) and centrifugation at 700g for 30 min. CD34⁺ cells were selected from the mononuclear cells fraction using the EasySep human cord blood CD34⁺ selection kit II (STEMCELL Technologies, Vancouver, Canada). Briefly, mononuclear cells were incubated with anti-human CD34 antibodies and FcR-blocking antibodies for 10 min. Then, dextran microbeads were added to the cell suspension and incubated for 1 min at room temperature. The immunomagnetic selection was performed using EasySep Magnets (STEMCELL Technologies) according to the manufacturer’s instructions. Purity, viability, and cell number were then assessed by flow cytometry using anti-human CD34-PE (BD Biosciences, San Jose, CA, USA), anti-human CD45-FITC (BD Biosciences), 7-amino-actinomycin-D (7-AAD, BD Biosciences) and Perfect Count Microbeads (Cytognos, Salamanca, Spain). Only samples with cell purity higher than 75% and viability higher than 90% were used.

CD34⁺ stem cells were cultured at a density of 1 × 10⁵ CD34⁺/mL with RPMI-1640 (Biowest, Nuaille, France) complete medium (CM) containing 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA), penicillin (100 U/ml, Normon SA, Madrid, Spain), streptomycin (100 ug/mL, Reig Jofre, Sant Joan Despí, Spain), glutamine (2 mmol/mL, Sigma-Aldrich, St. Louis, MO, USA), sodium pyruvate (1 mmol/L, Gibco) and beta-mercaptoethanol (1 mmol/L, Sigma-Aldrich). Moreover, CM was supplemented with the following human growth factors: Stem cell factor (100 ng/mL), FMS-related tyrosine kinase 3 ligand (100 ng/mL), IL-6 (50 ng/mL) and thrombopoietin (40 ng/mL; Preprotech, London UK). Depending on the group condition, cells were cultured with betamethasone (100 nM, Sigma-Aldrich), Fluticasone propionate (100 nM, Fluticasone, Selleckchem, Houston, TX, USA), or phosphate-buffered saline (PBS). Glucocorticoid concentration was chosen based on our previous results (Perna-Barrull et al. 2019 (link)). Cells were incubated for 20 h at 37 ºC in a humidified atmosphere containing 5% CO₂. Before transplantation, viability and CD34⁺ purity were assessed by flow cytometry using anti-human CD34-PE (BD Biosciences), anti-human CD45-FITC (BD Biosciences), and 7-AAD (BD Biosciences). In addition, cell number was determined using Perfect Count Microbeads (Cytognos, Salamanca, Spain).