Cryopreservation of Hematopoietic Stem/Progenitor Cells for Therapeutic Use

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 HSCHPC 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 degrees dishes was >96% and yielded 2 degrees 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 NODSCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.

The ability of glycerol to protect cells from freezing injury was discovered accidentally. The subsequent development of cryopreservation techniques has had a huge impact in many fields, most notably in reproductive medicine. Freezing injury has been shown to have two components, direct damage from the ice crystals and secondary damage caused by the increase in concentration of solutes as progressively more ice is formed. Intracellular freezing is generally lethal but can be avoided by sufficiently slow cooling, and under usual conditions solute damage dominates. However, extracellular ice plays a major role in tissues. Cryoprotectants act primarily by reducing the amount of ice that is formed at any given subzero temperature. If sufficient cryoprotectant could be introduced, freezing would be avoided altogether and a glassy or vitreous state could be produced, but osmotic and toxic damage caused by the high concentrations of cryoprotectant that are required then become critical problems. The transport of cryoprotectants into and out of cells and tissues is sufficiently well understood to make optimization by calculation a practical possibility but direct experiment remains crucial to the development of other aspects of the cryopreservation process.