Abstract
Background. Cryopreserved cord blood may be stored for decades before being used for allogeneic stem cell transplantation. Little is known about the effect of long-term cryopreservation in liquid nitrogen on the viability and function of cord blood cells. We examined the recovery, viability, clonogenic capacity, and T-cell reactivity to HLA alloantigens of cord blood samples cryopreserved up to 15 years.
Methods. Progenitor cell recoveries were studied by (colony-forming unit-granulocyte-macrophage) clonogenic assays from 18 cord blood samples short-term frozen for 2-8 weeks and from 8 samples cryopreserved for 15 years. Proliferative and cytotoxic responses against HLA antigens of thawed cord blood mononuclear cells after short-term or long-term cryopreservation were tested in standard mixed lymphocyte cultures and cell-mediated lympholysis assays.
Results. After thawing, the mononuclear cell recovery from long-term frozen cord blood low-density fractions averaged 80% (range, 64% to 92%). The presented data show that long-term frozen cord blood cells keep their clonogenic potential. No damaging effect was seen on the proliferative and cytotoxic capacities of long-term frozen cord blood T cells.
Conclusions. The results support the possibility of long-term storage of progenitor cells from umbilical cord blood for future bone marrow reconstitution.
In recent years, several centers have started to establish cord blood banks for the application of cord blood as a source of hematopoietic stem cells for allogenic bone marrow (BM*) restoration, mostly for children with genetic diseases and hematological malignancies (1, 2). The cord blood banks are organizing a worldwide register of stored HLA-typed cord blood cells, immediately available for allogeneic stem cell transplantation (3).
According to the complexity of the HLA system, it must be anticipated that some cryopreserved cord blood units will be stored for several years or decades until needed for an HLA-matched patient. Thus, the question arises whether long-term cryopreservation in liquid nitrogen in any way impairs the viability and function of cord blood cells.
In Amsterdam, we started in 1980 to examine the reactivity of cord blood T lymphocytes against HLA alloantigens. This gave us the opportunity to examine the effect of long-term (15 years) storage on viability, progenitor cell recovery, and T lymphocyte function of frozen cord blood. Cord blood mononuclear cells (CBMC) were isolated from heparinized cord blood samples by Ficoll-Hypaque gradient centrifugation. CBMC were resuspended(8-16×106/ml) in ice-cold freezing medium. Just before controlled rate freezing, an equal volume of ice-cold freezing medium containing 20% dimethylsulfoxide (DMSO) was added dropwise to the cells, while shaking. CBMC were stored in 2-ml sterile vials in liquid nitrogen. Cryopreservation was completed within 24 hr of cord blood collection. To investigate the immunological properties of cord blood T lymphocytes, CBMC were thawed quickly in a 37 °C water bath and then washed twice before counting and evaluating the viability by trypan blue exclusion.
Recently, 18 units of cord blood were collected in Prague. Aliquots of unseparated cord blood were cryopreserved using a programmed nitrogen freezer within 6 hr after collection. DMSO was used as cryoprotectant at a final concentration of 10% (vol/vol). Before examining the effect of short-term freezing on viability and clonogenic activity of progenitor cells, the cryopreserved cord blood samples were stored in liquid nitrogen for 2-8 weeks.
For testing the clonogenic capacities of progenitors, frozen unseparated cord blood cells were thawed quickly in a 37 °C water bath. Two hundred microliters of the cell suspensions (containing DMSO) were diluted in 5 ml of minimal essential medium. After centrifugation, the supernatants were discarded and the cells were resuspended in Iscove's modified Dulbecco's medium.
The numbers of nucleated cells (NC) and hematopoietic progenitors given per milliliter of cord blood of fresh unseparated cord blood samples (n=18) before freezing and after short-term (2-8 weeks) cryopreservation are shown in Table 1. After thawing, the mean NC recovery of short-term cryopreserved cord blood was 90% (range, 78%-97%). The viability in all samples of fresh and frozen cord blood cells was >90% (data not shown). The numbers of NC in the different cord blood samples are clearly variable, ranging from 4.5 to 15.9 (mean 9.5) ×106 NC per milliliter.
Table 1. Numbers of NC and myeloid hematopoietic progenitors in unseparated cord blood before and after short-term freezing
Table 2 shows the numbers of mononuclear cells (MC) per milliliter of cord blood in the low-density fractions of eight fresh cord blood samples. Also shown are the MC contents per milliliter of cell suspension in the ampoules before freezing. The mean CBMC recovery from the different ampoules that had been stored for 15 years in liquid nitrogen was 80% (range, 64%-92%). The viability of the thawed cells was >95% (data not shown). Thus, the majority of CBMC survived long-term storage in liquid nitrogen.
Table 2. Numbers of MC in fresh low-density cord blood fractions, the MC recoveries after long-term freezing, and CFU-GM values
To test the clonogenic capacities of hematopoietic progenitor cells, colony-forming units for granulocytes-macrophages (CFU-GM) were assayed in triplicate in 1-ml cultures containing 2×105 NC of unseparated cord blood samples in Iscove's modified Dulbecco's medium supplemented with 20% fetal bovine serum, 5% of the human bladder carcinoma cell line 5637-conditioned medium as a source of hematopoietic growth factors (4), and 0.3% Bacto-Agar (Difco, Detroit, MI). After 7 days of incubation at 37 °C in a humidified atmosphere consisting of 5% CO2 in air, the number of colonies (consisting of 40 and more cells) and clusters (2-39 cells) were counted, according to the method of Eaves et al. (5). Clearly different numbers of hematopoietic progenitor cells were found in the samples of fresh unseparated cord blood, varying from 19 to 432 (mean 176) ×103 CFU-GM colonies per milliliter of cord blood (Table 1). After short-term cryopreservation, the clonogenic activity declined to 10-52% of the values found before freezing, corresponding to 3-178 (mean 52.5) ×103 CFU-GM colonies per milliliter of cord blood, as recovery was directly related to the number of NC per milliliter in fresh samples.
The clonogenic capacity of myeloid progenitors of the long-term frozen CBMC samples were tested only after cryopreservation, by seeding 100 µl of thawed CBMC suspension directly into CFU-GM assay medium. Viable progenitor cells were found in all cultures, although with much variation per sample (Table 2). The results show that in two of eight samples, no CFU-GM colonies were counted, however, in these samples numerous clusters were clearly present. Most important, the data show that the potential of CFU-GM formation remained preserved after 15 years of cryopreservation in liquid nitrogen.
We also examined the effects of long-term storage in liquid nitrogen on the ability of CBMC to respond to HLA alloantigens. The proliferative and cytotoxic responses of HLA-typed short- and long-term cryopreserved CBMC were tested against HLA-mismatched adult peripheral blood mononuclear cells (PBMC) in mixed lymphocyte cultures. For proliferation assays, 50,000 CBMC were stimulated with 50,000 irradiated adult PBMC in microculture plates.[3H]thymidine incorporation was measured at day 6. Cord blood T lymphocytes proliferated strongly to HLA alloantigens. As shown in Figure 1, similar proliferative alloresponses were obtained from short-term (n=4) and long-term (n=7) cryopreserved CBMC. For cytotoxicity assays, CBMC were stimulated with irradiated adult PBMC in a ratio of 1:1 for 6 days in tissue culture flasks. Cytotoxicity was tested in an 8-hr 51Cr-release assay as described previously (6). Different concentrations of effector cells were mixed with 5000 51Cr-labeled stimulator target cells, cultured for 6 days without any mitogen. In contrast to the strong proliferative alloresponses of cord T lymphocytes, we obtained regularly weak cytotoxic responses against HLA antigens (Fig. 2). In 30-40% of the combinations with cord blood lymphocytes as responder and adult PBMC as stimulator, the lysis of stimulator target cells did not exceed 10%, indicating the low cytotoxic potential of cord blood T lymphocytes to respond to HLA alloantigens. Essentially similar alloreactive cytotoxic T lymphocyte responses were found with thawed short-term (n=7) and long-term (n=10) cryopreserved CBMC. Taken together, the presented data demonstrate that prolonged frozen storage had no damaging effects on the alloreactive capacities of cord blood T lymphocytes. Proliferative and cytotoxic responses against HLA antigens of T lymphocytes from cord blood in comparison to those of adult blood will be evaluated separately (B.S. Breur-Vriesendorp et al., manuscript in preparation).
Figure 1. Proliferative responses against HLA antigens, expressed in stimulation indices. Mean values are indicated by the line. HLA-typed CBMC were stimulated with four to five different HLA-mismatched adult irradiated PBMC in primary mixed lymphocyte cultures. The reactivities of seven short-term and four long-term cryopreserved CBMC samples are compared.
Figure 2. Cytotoxic responses against HLA antigens expressed as percentage of 51Cr release of stimulator target cells (effector to target ratio is 10:1). Mean values are indicated by the line. HLA-typed CBMC from 6 short-term and 10 long-term cryopreserved samples were stimulated with 1-3 different one HLA-A and/or HLA-B antigen-mismatched irradiated PBMC. The mixed lymphocyte reaction was positive in all combinations.
Similar results with long-term storage were reported by others, although the “longest” storage of cord blood cells in liquid nitrogen recorded with minimal effects on cell viability, cytotoxicity, and clonogenic capacity was 10 years (7). Most groups that examined the effects of processing and cryopreservation on CBMC detected some loss of progenitor cells (8-10) with differential effects on burst-forming uniterythroid, CFU-GM, and CFU-granulocyte, erythroid, monocyte, megakaryocyte, respectively. In this study of 15-year-old cryopreserved cord blood, only CFU-GM were assayed because they are widely used as a criterion for BM repopulation capacity of the graft.
In conclusion, the presented data support the possibility of long-term storage in liquid nitrogen of hematopoietic progenitor cells from umbilical cord blood for future BM reconstitution.
Acknowledgments. The authors acknowledge the skillful technical assistance of José Vingerhoed and Pammy Treep-van Leeuwen, and thank Dr. C.M. Slaper-Cortenbach for critical discussions.
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Accession Number: 00007890-199805150-00024