Although non-frozen storage of peripheral blood stem cells (PBSC) has been extensively studied and utilized clinically, the optimal storage conditions have not been determined. In order to improve the maintenance of clonogenic capacity during storage, we evaluated the feasibility of subzero non-freezing preservation of PBSC and attempted to determine the optimal conditions. Human PBSC were stored in different non-cryopreserved conditions. University of Wisconsin (UW) solution was used as the storage medium for PBSC. The stem cell integrity was optimally maintained when PBSC were preserved in a supercooled state at −2°C in UW solution without any cryoprotectants, and the highest values for nucleated cell survival (91.6%), CFU-GM survival (67.3%) and trypan blue viability (92%) were achieved at 72 h. CFU-GM survival in our storage conditions was significantly better than the survival achieved with hypothermic preservation in autologous serum and ACD-A solution at 4°C (67.3 ± 9.2% vs 42.9 ± 15.3%; P < 0.01) or cryopreservation at −80°C (67.3 ± 9.2% vs 52.7 ± 10.7%; P < 0.01). Thus, the combination of supercooling and UW solution was the optimal non-freezing method of preserving transplantable PBSC tested here. This method is of clinical utility in peripheral blood stem cell transplantation (PBSCT) for its simplicity and storage efficiency, and has value as a short-term storage method for PBSC to support dose-intensive multicyclic chemotherapy.
Autologous peripheral blood stem cell transplantation (PBSCT) has been widely performed to support high-dose chemotherapy not only for hematological malignancies but also for some chemosensitive solid tumors, including breast cancer1 and small cell lung cancer.2,3 Moreover, we and others have demonstrated that sequential reinfusion of peripheral blood stem cells (PBSC) can increase the dose intensity of the chemotherapy.3,4
For successful transplantation, PBSC must be stored in a way that preserves their viability. Despite the enormous progress that has been made, there are still major problems with regard to this cryopreservation. Even for simplified cryopreservation procedures, however, a −80°C deep freezer system is necessary, and besides, multiple freeze–thaw procedures are needed for tandem transplantation. These problems are major factors impeding the spread of PBSCT to general hospitals.
Recently, hypothermic non-freezing preservation has been used to store viable stem cells in whole blood and successfully applied after high-dose chemotherapy for multiple myeloma,5 non-Hodgkin's lymphoma6 and lung cancer.4,7 A temperature of around 4°C has been recommended for hypothermic liquid storage to avoid intracellular ice crystal formation caused by freezing. However, there is no evidence indicating that this temperature is optimal. All aqueous solutions and organic matter have specific freezing points, and above those temperatures they can be chilled without freezing. For example, the freezing point of human plasma is −0.8°C.8
Subzero non-freezing temperatures are defined as temperatures ranging from 0°C to the freezing point of a given solution. Biological materials preserved at subzero non-freezing temperatures can be kept unfrozen without damage due to ice crystal formation. This storage method, in theory, drastically reduces cellular metabolism, and is thus expected to prolong cell survival compared to that at higher temperatures.
Furthermore, there are supercooling temperatures that stabilize materials unfrozen at subzero temperatures below the freezing point. When certain solutions or organic materials are cooled slowly in a freezing compartment, their temperatures follow a cooling curve (time–temperature relationship, Figure 1). With cooling, the materials can be chilled to a certain temperature below the freezing point, and then the temperature rapidly rises exothermically to the specific freezing point. Ice crystals will begin to form at this point. The lowest temperature which the material finally reaches on the cooling curve without freezing is called the supercooling threshold temperature. The range from the freezing point to the supercooling threshold temperature is below the freezing point but not yet frozen, and is known as the ‘supercooled state’. For example, the specific freezing point of distilled water is 0°C, but distilled water does not freeze spontaneously at 0°C and remains in a liquid condition unless cooled to about −3.0°C. In that condition, the water is supercooled without freezing, and the supercooling threshold temperature of distilled water is about −3.0°C.
In the supercooled state, the material is at a risk of freezing as the temperature continues to drop. Although it is a metastable condition, the supercooled state can be maintained stably due to the remarkable progress that has been made in hypothermic preservation technology. The preservation method using temperatures below the subzero non-freezing temperature is called ‘supercooling storage’, when marked reduction of cellular metabolism is expected, and biological materials can presumably be preserved with little deterioration until the time that they are used.
Thus far, extensive attempts have been made to apply this special preservative technology to organ transplantation. It has been demonstrated that rat hearts9 and murine limbs10 can be stored with minimum damage to the tissue using this technology, and that subzero non-freezing storage is significantly superior to 4°C storage in preserving the organ functions in these materials.
However, the application of subzero non-freezing preservation in the medical field is still limited, and to our knowledge, no previous reports have been made about the effects of such preservation on the viability of hematopoietic progenitors. Use of subzero non-freezing preservation of PBSC in place of conventional cryopreservation in PBSCT has the potential advantage of greater simplicity. We designed the present study to evaluate the feasibility of subzero and supercooled storage as non-freezing methods for preservation of PBSC, and attempted to determine the optimal storage conditions.
Materials and methods
Study samples and chemotherapy regimen
Human PBSC were obtained from patients with small cell lung cancer who were entered in a study of multicyclic, dose-intensive chemotherapy with peripheral blood stem cell support.3 The treatment regimen is shown in Table 1. Briefly, patients were treated with multiple cycles of ICE (ifosfamide 7 g/m2 i.v. on day 1, carboplatin 400 mg/m2 i.v. on day 1, etoposide 100 mg/m2 i.v. on days 1 to 3) and granulocyte-colony stimulating factor (lenograstim; Chugai, Tokyo, Japan) 2 μg/kg subcutaneously injected daily from day 4 until the day of leukapheresis. Patients received ICE chemotherapy with a median interval of 17 days. Leukapheresis was performed when the peak of progenitor release was predicted (peripheral-blood WBC count reached at least 5000/μl). After stem cell collection, the leukapheresis product was cryopreserved until reinfusion by supplementation with 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) cryoprotectant mixture (CP-1; Kyokuto, Tokyo, Japan) and simple immersion at −80°C in a mechanical freezer. Human serum albumin (Bayer, Osaka, Japan) was added at a final concentration of 4%. The next ICE cycle was given 2 days after the last leukapheresis. On day 4 (the first day after chemotherapy), the frozen PBSC were thawed rapidly at 37°C and reinfused into the patient. Study samples were obtained from each leukapheresis cell suspension prior to cryopreservation. In order to exclude differences due to inter-patient variation, each leukapheresis cell suspension was divided into 10 samples. These cell suspensions were centrifuged at 1500 r.p.m. for 15 min and suspended in the various storage media described below. One milliliter aliquots of cell suspension at 2 × 107 cells/ml were transferred into sterile plastic tubes (1ml/tube, Sumitomo, Tokyo, Japan) for the storage. Informed consent was obtained according to the Declaration of Helsinki.
The storage media used for the experiments were autologous serum (AS)-ACD group: individual's own plasma anticoagulated with 7% ACD-A solution and preservative-free heparin (25 U/ml); UW group: University of Wisconsin (UW) solution (Belzer UW cold storage solution; Viaspan; Dupont Pharmaceuticals, Wilmington, DE, USA), containing preservative-free heparin (25 U/ml). The composition of UW solution was as follows: potassium lactobionate 100 mM, KH2PO4 25 mM, MgSO4 5 mM, raffinose 30 mM, adenosin 5 mM, glutathione 3 mM, allopurinol 1 mM, hydroxyethyl starch 50 g/l, and the pH was adjusted by the addition of NaOH to 7.4. AS-ACD-albumin or UW-albumin (4% or 8%) groups: human serum albumin was added to the above storage medium at the concentrations of albumin 4 g/100 ml or 8 g/100 ml.
Determination of storage temperatures
For subzero or supercooled storage, a refrigerator that precisely maintained accurate low temperatures was used (NH-60 refrigerator; Ninomiya, Chiba, Japan) throughout the study. This refrigerator was specially developed for studies at subzero non-freezing temperatures, and maintains subzero temperatures extremely precisely down to −5°C with fluctuations of less than ±0.5°C relative to the preset temperature.
Each stem cell sample was placed in the refrigerator previously set at the required temperature, and preserved at the same temperature for 24 to 72 h, or more. Each sample was capped tightly at the end of storage to prevent bacterial contamination or hemoconcentration due to evaporation. Quadruplicate storage tubes containing 1 ml of cell suspension were set up for every sample and subjected to analysis at the time points of 0, 24, 48, and 72 h.
Samples refrigerated at 4°C and samples cryopreserved by non rate-controlled freezing (REVCO, ULT 1386DOC) served as control samples.
Nucleated cell counts and cell viability
At the end of the preservation, nucleated cell counts of each sample were manually estimated using a hemocytometer. Percent survival of nucleated cells was calculated as follows:
Trypan blue dye exclusion was used to estimate the percent viability of nucleated cells.
Colony assays for CFU-GM
CFU-GM (colony-forming unit granulocyte–macrophage) assays were performed daily to assess the efficacy of the storage methods. Prior to stem cell assays, liquid storage samples were allowed to settle for 30–60 min at room temperature, while frozen PBSC were prepared for culture after being thawed by slightly shaking the storage ampule in a 37°C water bath.
CFU-GM were evaluated using a commercially available assay kit (Methocult GFH4434V, Stem Cell Technologies, Vancouver, BC, Canada), which utilizes methylcellulose-based semisolid medium (0.9% methylcellulose in Iscove's modified Dulbecco's medium) supplemented with recombinant human (rh) interleukin 3, rh G-CSF, rh GM-CSF (granulocyte–macrophage colony-stimulating factor), rh erythropoietin, and rh stem cell factor as colony-stimulating factors.
For the colony assays, a final concentration of 1 × 105 nucleated cells/dish were plated in standard 35 mm plastic tissue culture dishes (Falcon, Becton Dickinson Labware, NJ, USA). The dishes were then incubated at 37°C for 14 days in a humidified atmosphere containing 5% CO2 in air (ESPEC BNA-121D). After 14 days of culture, all colonies (CFU-GM) containing more than 40 cells were counted using an inverted microscope. Triplicate assays were performed on each specimen, and the average CFU-GM counts of three dishes were recorded. For each specimen, the number of CFU-GM after preservation was compared to the number of CFU-GM before preservation. Percent survival of CFU-GM was calculated as follows:
The results were expressed as the mean ± standard deviation (s.d.). Statistical analyses among groups were carried out using Student's t-test, with P values of 0.05 or less taken to indicate significance.
Determination of freezing point
Initially, the freezing point of each sample was measured. The study samples were poured into plastic tubes and a thermometer was inserted. Then each tube was placed into the chamber of the refrigerator set at −14°C and monitored until the temperature of the sample reached that of the chamber.
The specific freezing point of AS-ACD samples was −0.7°C (average of three measurements), so the optimal preset temperature was taken as 0°C for subzero storage. AS-ACD samples did not freeze when supercooled to and kept at −5°C, and, therefore, −1, −2, −3, −4 and −5°C were evaluated as supercooled temperatures for the ACD-AS group. Human plasma can generally be supercooled to −16°C if chilled in a controlled manner.11 In contrast, the freezing point of UW solution was −0.7°C (average of three measurements), so that 0°C was taken as the optimal subzero storage temperature. UW solution could be supercooled if it had not been chilled to about −4°C (the supercooling threshold temperature); thus, to avoid freezing, the suitable supercooled temperatures for the UW group were taken as −1 and −2°C. Ten samples were preserved at each storage temperature.
Effects of simple subzero non-freezing storage (AS-ACD group)
We investigated the effects of simple subzero non-freezing storage in the AS-ACD group. In this group, leukapheresis samples were stored without further manipulation, ie PBSC were preserved with ACD-A solution and heparin in autologous serum. Figure 2 shows the preservation characteristics of the AS-ACD group. At 0 h (baseline values) the nucleated cell count was (3.86 ± 1.73) × 107/ml, and the CFU-GM colony count was (3.1 ± 2.7) × 104/ml. After 72 h the percent survival of nucleated cells was 93.4 ± 10.7% at 4°C, 53.2 ± 30.6% at 0°C, 70.4 ± 28.5% at −1°C, and 65.2 ± 19.3% at −2°C. Cell viability as assessed by trypan blue exclusion after 72 h was 93 ± 5% at 4°C, 80 ± 6% at 0°C, 76 ± 6% at −1°C, and 78 ± 8% at −2°C. The percent survival of CFU-GM after 72 h was 42.9 ± 15.3% at 4°C, 16.2 ± 13.0% at 0°C, 13.9 ± 9.5% at −1°C, and 15.8 ± 8.0% at −2°C. CFU-GM survival in the subzero groups was significantly lower than in the 4°C group (P < 0.01). Further cooling studies at temperatures below −2°C were not carried out because of these unpromising data. In short, 4°C was a better storage temperature than subzero temperatures in these preservative conditions. At 4°C, the percent survival of CFU-GM was 66.8% at 24 h, 54.2% at 48 h, and 42.9% at 72 h.
Effects of UW solution (UW group)
At 72 h, the percent survival of nucleated cells was over 90%, with cell viability of approximately 90% at every storage temperature. Although we found no significant difference between the survival of nucleated cells stored in UW solution at various storage temperatures, CFU-GM survival at supercooled temperatures was significantly superior to that at 4°C, ie after 72 h of preservation, the percent survival of CFU-GM at 4°C was 46.2 ± 18.7%, while in the subzero groups, CFU-GM survival was 51.2 ± 11.2% at 0°C, 60.7 ± 9.2% at −1°C, and 67.3 ± 9.2% at −2°C (Figure 3), with a significant difference between the supercooled UW group (−1 or −2°C) and the 4°C and 0°C UW groups (P < 0.01∼0.025). The two supercooled UW groups (−1 and −2°C) had similar values for percent survival of CFU-GM after 72 h. In addition, supercooled storage in UW solution at −1°C or −2°C was superior to hypothermic preservation in autologous serum and ACD-A solution at 4°C (60.7 ± 9.2%, 67.3 ± 9.2% vs 42.9 ± 15.3%; P < 0.01).
Effects of albumin addition
Figure 4 shows the percent survival of nucleated cells, CFU-GM, and cell viability in the presence of 4% or 8% human albumin. In contrast to previously published observations,12 addition of albumin did not improve the survival of the cells in either the As-ACD or UW groups stored at 4°C or −2°C.
Comparison of supercooled storage and cryopreservation
The percent survival of nucleated cells, CFU-GM, and trypan blue exclusion of PBSC cryopreserved at −80°C were compared with those of PBSC stored at supercooled temperatures. Before cryopreservation, the leukapheresis product contained (9.24 ± 3.35) × 107 nucleated cells/ml, of which 1.9 ± 1.4% were CD34 positive. The mean nucleated cell numbers did not decrease significantly after cryopreservation (P = 0.43). The survival of CFU-GM was 52.7 ± 10.7% (100% = (8.1 ± 4.8) × 104/ml), and the mean viability, assessed by trypan blue exclusion, was 80 ± 8% after thawing. As shown in Figure 5, trypan blue viability was significantly better with supercooled storage (P < 0.01). Furthermore, with respect to CFU-GM survival after 72 h of storage, the survival with −2°C supercooled storage in UW solution was significantly better compared to that with cryopreservation at −80°C (67.3 ± 9.2% vs 52.7 ± 10.7%; P < 0.01).
Prolonged supercooled storage in UW solution
Because supercooled storage at −2°C in UW solution resulted in the best preservative effects, we further examined the possibility of extending the preservation time of PBSC in this storage condition compared with the results with those of AS-ACD at 4°C (Figure 6). We found that no CFU-GM were detectable after 240 h at 4°C in AS-ACD, compared with 14 ± 10% survival at −2°C in UW solution. After 168 h, the survival of CFU-GM at −2°C in UW solution was 38% which was significantly better than that of AS-ACD at 4°C. Therefore, the maximum tolerated time of prolonged storage at −2°C is approximately 168 h.
The increased use of stem cell transplantation in response to clinical demand has made it necessary to investigate new methods of stem cell storage.
The simplified method of cryopreservation at −80°C using the cryoprotectant combination of DMSO, an intracellular cryoprotectant, and HES, an extracellular cryoprotectant, has been used successfully until now to freeze PBSC for transplantation. It has been reported that the mean CFU-GM recovery rate after 18 months of simplified cryopreservation was 73.8 ± 4.1%.11 Deep-freezing may be the only method for long-term (from months to years) preservation of PBSC. In the clinical setting, though, short-term (approximately 72 h) preservative methods for stem cells may be sufficient because high-dose chemotherapy can be adjusted to be completed within 72 h. Besides, repeated stem cell collection is necessary in tandem transplantation; therefore, the development of simple methods and less expensive equipment for storage of PBSC have long been awaited.
Some trials of non-freezing preservation of hematopoietic stem cells have been reported by several investigators. Pettengell et al4,7 examined non-freezing preservation of hematopoietic stem cells from bone marrow, leukapheresis products and whole blood. They found the survival of CFU-GM at 48 h to be 68% and at 72 h to be 47% in autologous serum and citrate phosphate dextrose at 4°C. Our results of storage at 4°C are in agreement with the results of Pettengell.
Since the rate of enzymatic activity is related to temperature (according to van't Hoff's rule), the effectiveness of hypothermic preservation is related to how it retards metabolic deterioration and reduces cellular energy consumption of stored cells. However, in hypothermic preservation, the activity of the Na+-K+ ATPase which participates in active Na+-K+ membrane transport is suppressed, and Na+ in the extracellular solution enters the cells dependent on the concentration gradient. As a result, the cells swell because Na+ accumulates water (hypothermic-induced cell swelling). To minimize cell swelling, storage media should have a composition close to that of the intracellular electrolyte composition, and have a component to which the cell membrane is impermeable, in order to counteract the colloid oncotic pressure derived from the intracellular proteins or impermeable anions. Such substances in the cold storage solution are called impermeants. Next, to prevent intracellular acidosis induced by anaerobic glycolysis, storage media should not contain glucose. And the third important consideration regards energy metabolism. Intracellular adenosine triphosphate(ATP) is rapidly degraded during hypothermic storage. However, when the cold storage ends, rapid recovery of Na+-K+ pump activity, which requires ATP, is necessary, and therefore it is important to provide ATP precursors for ATP synthesis. On the basis of the above considerations, we used UW solution as a storage medium for subzero non-freezing preservation to avoid various disadvantages of hypothermia.
UW solution (developed by Belzer et al) is a cold storage solution used to preserve many transplantable organs such as liver, pancreas and kidney, and its successful preservative capacity has now been confirmed in clinical organ transplantation.13 UW solution is brought to pH 7.4 at room temperature by the addition of NaOH, and the osmolality is 320 ± 10 mOsm/l. The solution contains a number of components. Potassium-lactobionic acid is a key agent which acts as an impermeant to suppress hypothermic-induced cell swelling. Raffinose is added for additional osmotic support. Other substances in the UW solution are adenosine, which is an effective substrate for the resynthesis of ATP, and glutathione as a scavenger of oxygen-free radicals.14 The electrolyte concentrations are adjusted to resemble the intracellular electrolyte composition (25 mM sodium and 120 mM potassium) to reduce ion exchange across the cell membrane during the hypothermic phase. The only point that requires attention is the high concentration of potassium, which should be removed by washing it out before reinfusion to patients.
The concept and advantages of using UW solution for subzero non-freezing storage have been reported by several investigators in the field of transplant surgery. The effects of subzero non-freezing storage in UW solution have been examined for the preservation of whole hearts15 and liver.16 The results indicated that storage at subzero non-freezing temperatures is better than storage at 4°C. In the field of supercooled storage, Matsuda et al17 evaluated the superiority of supercooled storage using isolated rat hepatocytes suspended in UW solution at −4°C. After 48 h of storage, the supercooled group showed significantly better results in the trypan blue exclusion test, ATP content, mitochondrial activity and morphological examination compared with the 4°C group.
We have now shown that the stem cell integrity was optimally maintained when PBSC were preserved in a supercooled state at −2°C in UW solution without any cryoprotectants and the highest values for nucleated cell survival (91.6%), CFU-GM survival (67.3%) and trypan blue exclusion (92%) were achieved at 72 h. For up to 72 h, PBSC can be preserved at −2°C with only about one-third loss of CFU-GM, which was a lower loss of hematopoietic potential than that of cryopreserved PBSC in this study.
Further attempts to improve this preservative method will, if possible, involve storing cells at much lower temperatures where biochemical deterioration can be minimized. For this purpose, the freezing point of the storage media should be low enough to avoid freezing. When freezing occurs in the extracellular solution, it is lethal for stored PBSC; as ice forms in the solution, the remaining extracellular liquid becomes highly concentrated, and the increased osmolality draws intracellular water out of the cell. Consequently, stored cells collapse and their structures break down.
The freezing point of a medium depends on its osmolality. The predicted freezing point is calculated as follows:18
The storage media contain some osmotic substances such as sucrose, and therefore it is expected that these media will have increased osmolality and depressed freezing point, thus improving storage efficacy. To lower the freezing point of the storage media, however, we would need to adjust the solution such that it become extremely hyperosmotic. Actually, 538 mOsm escalation is needed for every 1°C freezing point depression. Such a tremendous osmotic stress would be likely to harm cells and, moreover, it is assumed that osmotic substances increasing the osmolality of the solution penetrate slowly into the cells during storage, and lead to cell swelling caused by high osmotic pressure of the intracellular space.19 From these points of view, hyperosmolar solutions are thought to be unsuitable for subzero non-freezing storage of PBSC.
On the other hand, the usefulness of serum as a component of storage media was emphasized by Grilli et al20 in the cryopreservation of bone marrow cells. They confirmed that homologous serum is required for optimal stem cell cryopreservation, because macromolecules such as serum proteins can protect stem cells against handling injury during the actual freezing–thawing process. In the liquid storage of stem cells, the usefulness of serum was also documented by Kohsaki et al,12 who observed a higher survival of colony forming cells when large amounts of serum were added to the storage media. In their study, bone marrow cells were stored in alpha-medium with FCS, HEPES and CPD at 4°C. The preservative effects were significantly improved when FCS was increased from 20% to 40%. Although the mechanism by which serum exerts its preservative effects is still unknown, one of the possible effects of serum is that, serum albumin elevates the colloid oncotic pressure of storage media. Increased colloid oncotic pressure of the solution leads to cellular dehydration, which reduces cell swelling during storage.21 Based on this consideration, we tried to improve the preservation results by increasing the albumin concentration of the media. However, we did not find significant increased preservative capacity upon the addition of human albumin. Further increase of the albumin concentration may be impractical.
Regarding the cooling rate in supercooled storage, there are some problems that should be discussed. It has not been proven whether there are any differences in preservative efficacy between simple immersion at supercooling temperatures and cooling down in a rate-controlled manner. Rate-controlled supercooling may be useful for stabilizing the supercooled state by preventing ice crystal formation in the samples.
We concluded from the present experiments that the combination of supercooling and UW solution provides the optimal non-freezing preservation method for transplantable PBSC. This method should be of clinical utility in PBSCT for its simplicity and storage efficiency, and has value as a short-term storage method for PBSC to support dose-intensive multicyclic chemotherapy. Hitherto, a preservation period of more than 168 h could not be attained by the use of this method. The reasons for the deterioration of stored PBSC after prolonged preservation are not clear. It will be necessary to elucidate the mechanism by which PBSC are damaged during hypothermic storage, and this information should lead to the further improvement of preservation of PBSC.
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We thank Hyo-On Laboratories for help with this study.
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Cite this article
Matsumoto, N., Yoshizawa, H., Kagamu, H. et al. Successful liquid storage of peripheral blood stem cells at subzero non-freezing temperature. Bone Marrow Transplant 30, 777–784 (2002) doi:10.1038/sj.bmt.1703692
- peripheral blood stem cell
- dose-intensive chemotherapy
- hematopoietic stem cell transplantation
- subzero non-freezing preservation
- supercooled storage
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