Isolation of Umbilical Cord Blood Stem Cells

Umbilical cord blood samples were lysed in BD PharmLyse Lysing Solution (BD Biosciences, San Jose, CA, USA) for 15 min at room temperature in the dark and washed twice in phosphate–buffered saline (PBS). The obtained suspension of UCB nucleated cells (NCs) was subjected to immunomagnetic separation procedures. Each cell population, lineage-negative SPCs, CD34⁺ or CD133⁺ cells enriched in SPCs were isolated from non-separated NCs using immunomagnetic isolation and a Lineage Cell Depletion Kit, CD34 MicroBead Kit or CD133 MicroBead Kit (Miltenyi Biotec, Auburn, CA, USA). Isolation procedures were performed according to the manufacturer's instructions. Briefly, lineage-negative cells were isolated through negative selection using a MidiMACS separator (Miltenyi Biotec, Auburn, CA, USA). To isolate a lineage-negative cell population, a Lineage Cell Depletion Kit (Miltenyi Biotec, Auburn, CA, USA) was used. One hundred microliters of biotin-antibody cocktail recognizing the lineage-specific cell antigens were added per 10⁸ cells according to the manufacturer's recommendations. After washing in PBS, 100 µL of anti-biotin MicroBeads for magnetic cell labeling was added. Labeled cell suspension was loaded onto a MACS LS column (Miltenyi Biotec), and unlabeled cells passing through the column were collected (lineage-negative population).
CD34⁺ and CD133⁺ cells were isolated through positive selection using a MidiMACS separator (Miltenyi Biotec). To label CD34⁺ cells, a CD34 MicroBead Kit was used, and CD133⁺ cells were labeled using the CD133 MicroBead Kit (Miltenyi Biotec). One hundred microliters of FcR blocking reagent, which inhibits nonspecific or Fc-receptor-mediated binding, and 100 µL of CD34/CD133 microbeads for magnetic cell labeling were added per 10⁸ cells according to the manufacturer's recommendations. Labeled cell suspensions were subjected to immunomagnetic separation, in which magnetically labeled cells were retained in the LS MACS affinity column (Miltenyi Biotec) and unlabeled cells passed through the column. After several washes, the column was removed from the magnet, and the retained CD34⁺ or CD133⁺ cells were eluted with 1-5 mL of PBS supplemented with 0.5% bovine serum albumin and 2 mM EDTA.
From UCB units with relatively high volumes (at least 450×10⁹ NCs), we simultaneously isolated three SPC populations immunomagnetically, but from units with a lower UCB volume, we only isolated lineage-negative SPCs for a more detailed analysis. The isolation procedures were performed immediately after obtaining NCs from the UCB units. From the 56 UCB units processed, lineage-negative cells were isolated 56 times, and CD34⁺ and CD133⁺ cells were isolated 25 times. Total numbers of isolated cells were determined using a TC Automated Cell Counter (Bio-Rad Inc., Philadelphia, PA, USA).