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

Abstract

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 HSC͞HPC from individual CBs stored frozen for 15 yr. Average recoveries (؎ 1 SD) of defrosted nucleated cells, colony-forming unitgranulocyte, -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 NOD͞SCID engrafting ability can be stored frozen for >15 yr, can be efficiently retrieved, and most likely remain effective for clinical transplantation.C ord blood (CB) is a viable alternative to bone marrow for related and unrelated allogeneic hematopoietic stem cell (HSC)͞progenitor cell (HPC) transplantation (1-13). Since our initial preclinical (14-16) and clinical (1, 17-19) studies, there have been Ͼ2,000 CB transplants performed to treat a variety of malignant and nonmalignant disorders in children and adults (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(17)(18)(19). CB HSC͞HPC are frozen before use for transplantation (14,(20)(21)(22), but the longest that a CB collection has been stored frozen before use for clinical transplantation is in the 3-to 5-yr range. With Ͼ100,000 CBs stored frozen world-wide for prolonged periods in anticipation of their clinical use, information on longer-term storage of CB HSC and HPC is of critical importance.The capacity to freeze and retrieve CB HPC cells was first reported when we suggested that CB could serve as a source of transplantable and engrafting HSC and HPC (14). We subsequently evaluated effects of 5-yr (16) and 10-yr (23) storage on retrieval of HPC in which postfreeze HPC numbers were compared directly to prefreeze numbers from the exact same CB samples. Thus, a true recovery rate could be calculated. At those times, we assessed only numbers and proliferation of HPC in vitro. In the present report, we extended anal...

Recovery, viability and clinical toxicity of thawed and washed haematopoietic progenitor cells: analysis of 952 autologous peripheral blood stem cell transplantations

“…23 Moreover, the NC and CD34 þ cell recovery and the viability were not linked with the duration of freezing in this study, consistent with data showing that HSC retain engraftment potential after extended (5-14 years) cryostorage. 21,24,25 Interestingly, we found a significant decrease in NC recovery and viability in patients with myeloma. It could be related to the higher granulocyte count in the apheresis collection in myeloma than in other patients (48 versus 40%, P ¼ 0.0001).…”

Mesenchymal stem cells from cryopreserved human umbilical cord blood

“…UCB as the power of HSCs has been increasingly used for various clinical settings since 1988 [17]. Since then, hundreds of thousands of UCB collections have been frozen and stored throughout the world, in anticipation of their potential use to treat various disorders.…”

Relative recovery of haematopoietic stem cell products after cryogenic storage of up to 19 years

“…Although some data have been reported on the effects of long-term (up to 15 years) storage of stem cells obtained from cord blood, [5][6][7][8] there are few data to show how stem cells harvested from BM or PB fare with time. A study on BM stored for a median of 2.7 years and a separate study of both BM and PB stored for a median of 2.8 years demonstrated that the thawed products were capable of engraftment but did not examine how the markers of potential engraftment changed with time for each specimen.…”

“…Studies performed on cord blood stem cells have reported that storage for up to 15 years does not significantly decrease recovery. [5][6][7][8] However, studies on PB-or BM-derived stem cells have been limited and predominantly analysed the effects of relatively short (median p2 years) storage. [9][10][11][12] A single study has suggested that SCPs stored for longer periods (median 9 years) may be inadequate for transplant 13 but did not compare pre-and post-storage results for individual samples.…”