BACKGROUND: Umbilical cord blood (UCB) is a source of hematopoietic stem cells for transplantation, offering an alternative for patients unable to find a matched adult donor. UCB is also a versatile source of hematopoietic stem and progenitor cells (hCD34 + HSPCs) for research into hematologic diseases, in vitro expansion, ex vivo gene therapy, and adoptive immunotherapy. For these studies, there is a need to isolate hCD34 + HSPCs from cryopreserved units, and protocols developed for isolation from fresh cord blood are unsuitable. STUDY DESIGN:This study describes a modified method for isolating hCD34 + HSPCs from cryopreserved UCB. It uses the Plasmatherm system for thawing, followed by CD34 microbead magneticactivated cell sorting isolation with a cell separation kit (Whole Blood Columns, Miltenyi Biotec). hCD34 + HSPC phenotypes and functionality were assessed in vitro and hematologic reconstitution determined in vivo in immunodeficient mice. RESULTS:Total nucleated cell recovery after thawing and washing was 44.7 AE 11.7%. Recovery of hCD34 + HSPCs after application of thawed cells to Whole Blood Columns was 77.5 AE 22.6%. When assessed in two independent laboratories, the hCD34+ cell purities were 71.7 AE 10.7% and 87.8 AE 2.4%. Transplantation of the enriched hCD34 + HSPCs into NSG mice revealed the presence of repopulating hematopoietic stem cells (estimated frequency of 0.07%) and multilineage engraftment.CONCLUSION: This provides a simplified protocol for isolating high-purity human CD34 + HSPCs from banked UCB adaptable to current Good Manufacturing Practice. This protocol reduces the number of steps and associated risks and thus total production costs. Importantly, the isolated CD34 + HSPCs possess in vivo repopulating activity in immunodeficient mice, making them a suitable starting population for ex vivo culture and gene editing. ABBREVIATIONS: BMRs = batch manufacturing records; CFUs = colony-forming units; cGMP = current good manufacturing practice; HSC = hematopoietic stem cell; HSCT = hematopoietic stem cell transplantation; HSPCs = hematopoietic stem and progenitor cells; LMPP = lymphoid-primed multipotent; MPP = Multipotent progenitors; SOPs = standard operating procedures; SRCs = severe combined immunodeficiency repopulating cells; TNCs = total nucleated cells; UCB = umbilical cord blood.

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