High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years

Transplanted cord blood (CB) hematopoietic stem cells (HSC) and progenitor cells (HPC) can treat malignant and nonmalignant disorders. Because long-term cryopreservation is critical for CB banking and transplantation, we assessed the efficiency of recovery of viable HSC/HPC from individual CBs stored frozen for 15 yr. Average recoveries (± 1 SD) of defrosted nucleated cells, colony-forming unit-granulocyte, -macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte, -erythrocyte, -monocyte, and -megakaryocyte (CFU-GEMM) were, respectively, 83 ± 12, 95 ± 16, 84 ± 25, and 85 ± 25 using the same culture conditions as for prefreeze samples. Proliferative capacities of CFU-GM, BFU-E, and CFU-GEMM were intact as colonies generated respectively contained up to 22,500, 182,500, and 292,500 cells. Self-renewal of CFU-GEMM was also retained as replating efficiency of single CFU-GEMM colonies into 2° dishes was >96% and yielded 2° colonies of CFU-GM, BFU-E, and CFU-GEMM. Moreover, CD34(+)CD38(−) cells isolated by FACS after thawing yielded >250-fold ex vivo expansion of HPC. To assess HSC capability, defrosts from single collections were bead-separated into CD34(+) cells and infused into sublethally irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. CD45(+) human cell engraftment with multilineage phenotypes was detected in mice after 11–13 wk; engrafting levels were comparable to that reported with fresh CB. Thus, immature human CB cells with high proliferative, replating, ex vivo expansion and mouse NOD/SCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.