Effects of long-term cryopreservation on hematopoietic progenitor cells in umbilical cord blood

Abstract

Summary:There is considerable interest in developing banks of frozen umbilical cord blood cells for transplants but it is uncertain how long frozen cells survive. Our objective was to determine the recovery of frozen umbilical cord blood cells. We quantitated recovery of hematopoietic progenitor cells (CFU-GM, BFU-E, and CFU-GEMM) from frozen umbilical cord blood cells stored for up to 12 years. Decay rates of CFU-GM, BFU-E and CFU-GEMM Umbilical cord blood cells are increasingly used for transplant, especially when no HLA-identical sibling donor is available. Consequently, there is growing interest in developing frozen HLA-typed cord blood banks.Umbilical cord blood cells in these banks may be stored for several years before use. However, there are few data on long-term survival of frozen cord blood cells; most reports focus on relatively short storage intervals. 1 We studied viability of hematopoietic progenitor cells in 12 umbilical cord blood samples frozen for up to 12 years. Materials and methodsBetween July 1985 and September 1985, cord blood samples were obtained from the placenta of 12 healthy volunteer mothers at delivery (38 to 41 weeks of gestation). Informed consent was obtained from all. Immediately after delivery of the baby, while the placenta was still in utero, the umbilical cord was double-clamped 6 to 8 cm from the baby and cord blood collected. Before collection, the venepuncture site was cleaned with alcohol and betadine. An 18 gauge needle was inserted into the umbilical vein and cord blood was aspirated into a 50 ml syringe containing 5 ml of ACD. The volume of cord blood collected ranged from 28 ml to 45 ml. The umbilical cord blood was processed using standard techniques. 2 Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation was used to isolate the mononuclear cell fraction, which was resuspended at concentration of 2 ϫ 10 7 /ml. Mean (s.d.) mononuclear cell recovery was 48 Ϯ 11%. Umbilical cord blood mononuclear cells were cryopreserved using a programmable freezer (model 801 CryoMed, Mt Clemens, Ml, USA), using standard cryopreservation programs, in RPMI-1640 media, 10% DMSO and 20% fetal calf serum 3 and stored in the liquid phase of liquid nitrogen.Cells were thawed after 1 and 6 months and 1, 2 and 12 years and a small aliquot removed to assess viable cell recovery (trypan-blue exclusion test) and to assay hematopoietic progenitor cells (CFU-GM, BFU-E and CFU-GEMM) by standard techniques. 3 Recovery was calculated by comparing numbers of cells to 1 month values. StatisticsA linear model was applied to the logarithm of cell viability results. Logarithmic transformation was used to stabilize variance in observed recoveries. For a given progenitor cell assay, the model is:Log (viability) ϭ a ϩ b interval cryopreserved when the estimate of slope (b) ϭ 0, there is no trend for cell loss over time. Results were expressed as a decay factor (d) with 95% confidence intervals.Decay over time (k) was calculated as d k .

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.…”

Polychromatic flow cytometry analysis of CD34+ hematopoietic stem cells in cryopreserved early preterm human cord blood samples

“…We decided to exploit this technology to propose a polychromatic FCM characterization of HSCs from long-term cryopreserved hEPCB samples and to compare their immunophenotypic pattern with counterparts from cryopreserved full-term CB. Regarding to long-term cryopreservation, current practice indicates that standard cryopreservation protocols of CB freezing in 10% DMSO in controlled-rate freezers and storage below 21358C give an average of 80% recovery of multipotent hematopoietic progenitor cell subpopulation, as demonstrated by functional studies in terms of colony forming unit assay (21,22).…”

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

“…The 15-yr study (40) reported on recovery of relatively mature subsets of CFU-GM (as detected by colony formation after stimulation with 5637 conditioned medium), but, because no prefreeze data were available for these same samples, the efficiency of recovery of the CFU-GM could not be calculated. The 12-yr study (41) reported recovery of CFU-GM, BFU-E, and CFU-GEMM based on comparing numbers of cells to 1-mo defrost values from the same samples. These authors noted Ͼ90% recovery of HPC after 10 yr. Because the recovery calculations were not based on prefreeze control numbers, the actual efficiency of recovery could not be absolutely calculated.…”