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Stem Cells, Vol. 15, No. 5, 353-358, September 1997
© 1997 AlphaMed Press

Bone Marrow Cryopreservation: Improved Recovery Due to Bioantioxidant Additives in the Freezing Solution

Lalita S. Limaye

National Centre for Cell Science, Pune, India

Key Words. Mouse bone marrow • Human bone marrow • DMSO • Antioxidants • GM • GEMM

Dr. Lalita S. Limaye, National Centre for Cell Science, Ganeshkhind, Pune 411007, India.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Summary
 References
 
One hypothesis of cryoinjury is the damaging effect of oxygen-free radicals formed during freezing and thawing. Addition of physiologically acceptable antioxidants into the preservation solution improved the cryoprotection of bone marrow cells. Bone marrow nucleated cells were frozen using rate-controlled freezing devices. Antioxidants used in combination with 10% dimethylsulfoxide were {alpha} tocopheryl acetate, catalase, ascorbic acid, superoxide dismutase and reduced glutathione. The parameters used to assess the efficacy of cryopreservation were viability, nucleated cell recovery, and colony-forming unit assays: granulocyte-macrophage and granulocyte-erythroid-macrophage-megakaryocyte. Results obtained indicate that the first three antioxidants increase the post-thaw recovery of cells, particularly in terms of early and late progenitors. Superoxide dismutase and reduced glutathione, however, have no beneficial effect on the preservation. The response of cryopreserved cells to suboptimal concentrations of colony-stimulating factors in in vitro assays was also restored to some extent when the cells were frozen with antioxidants.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Summary
 References
 
Ultra-low-temperature storage of cells, embryos, and some human tissues for banking purposes is now of common practice. Freezing is an insult to cells where physical disruption leading to chemical changes takes place. The rate at which cells are cooled and the concentration of cryoprotectant are two of the main factors governing the survival of frozen cells [1]. The use of various freezing devices allows a better control of the cooling rate and protects the cells at critical freezing stages [2]. The commonly used cryoprotectants glycerol and dimethylsulfoxide (DMSO) protect on a molar basis and act by reducing electrolyte concentration in the residual unfrozen solution in and around a cell at any given temperature [3, 4].

One proposed hypothesis of injury during hypothermia and freezing is the formation of oxygen-free radicals [5-8]. Whitley et al. [9] have shown a rise in MDA levels of rat liver homogenates stored at high subzero temperatures. Killian et al. [10] have used a synthetic antioxidant butylated hydroxytoluene (BHT) as a cryoprotectant to whole or skim milk diluent for preservation of bull semen. They report higher sperm motility upon thawing in samples frozen with BHT than in samples without it. Thus, free radical damage has been implicated as one of the causes of loss of viability of cells during or just after freezing. [11]

Methods of cryopreservation of bone marrow (BM) have been well established [12-17]. Though DMSO is a known hydroxyl-free radical scavenger [18], it has been reported to be ineffective in protecting V-79 Chinese hamster cells exposed to hypothermia [19]. DMSO is also known for its pharmacological toxicity; therefore, in the present investigation we have used certain physiologically acceptable bioantioxidants in the freezing mixture in combination with 10% DMSO to see whether they give enhanced protection in terms of viability, cell recovery, and proliferative potential. It was also observed that the poor growth factor responsiveness of cryopreserved bone marrow cells was considerably restored when they were frozen in the presence of antioxidants.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Summary
 References
 
Chemicals and Cell Lines
Collagenase Type IV, DMSO (cell culture tested), ascorbic acid, catalase, reduced glutathione, {alpha} tocopheryl acetate, superoxide dismutase, bovine pancreas DNase I, methylcellulose viscosity 4,000 cps and recombinant erythropoietin (EPO) were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal calf serum (FCS) and Iscove's modified Dulbecco's medium (IMDM) were from GIBCO (Grand Island, NY). Recombinant mouse interleukin 3 (IL-3) and recombinant mouse GM-CSF were obtained from Boehringer Mannheim (Indianapolis, IN). Giant cell tumor (GCT) and 5637 bladder carcinoma cell lines were originally from ATCC, now belonging to the National Centre for Cell Science repository. Conditioned medium collected from these cell lines was concentrated and used for the assay.

Isolation of Mononuclear Cells (MNCs)

Mouse BM   Swiss albino mice, six to eight weeks old, of either sex, were sacrificed. Their femurs were dissected, cleaned, and the BM flushed out. MNCs were isolated by loading on Ficoll/ Hypaque (1.077 gm/ml) density gradient.

Human BM   Resected ribs were obtained from the Surgery Department of Sassoon Hospital, Pune, India. BM cells were isolated by collagenase (0.1%) digestion at 37°C for 20 min. In our laboratory, we found that this treatment helps in loosening the firmly bound hematopoietic cells from the bone tissue and thus facilitates the recovery. Protocols of all human and animal studies were approved by the Institutional Review Board.

Freezing   The washed MNCs, either from mouse or human, were frozen at a density of 107 cells/ml/vial. The final volume including medium, serum, DMSO, antioxidants, and cells was 1 ml. Nunc (Naperville, IL) cryo vials were used for freezing. The freezing was carried out in a computer-controlled programmable freezer (Cryomed; New Baltimore, MI), using a cooling rate of 1°C/min down to –40°C followed by 10°C/min down to –90°C. Finally, the vials were stored in the liquid phase of liquid N2 (–196°C). The freezing medium contained IMDM 70%, FCS 20%, and DMSO 10% with or without antioxidant additives. (FCS, though reported to cause allergic reactions after i.v. infusion, was used here by convention). Incidentally, there was no change in pH after addition of antioxidants, and the pH ranged between 6.8 and 7.2. All antioxidants were water-soluble except {alpha} tocopheryl acetate, the stock of which was prepared as follows: 10 mg were dissolved in 100 µl of absolute alcohol; to this, 4 ml of FCS were added and stirred for 48 h at 4° C; and then 16 ml of IMDM were added. The requisite aliquot was then taken from the stock. Appropriate alcohol control was kept in the experiment.

Thawing   After 15 days of storage, the vials from liquid N2 were removed and thawed quickly in a water bath at 37°C. The cells were diluted with thawing medium (1:5; IMDM + 10% FCS + 20 U/ml DNaseI). After centrifugation at 4°C, the pellet was resuspended in complete medium (IMDM + 20% FCS).

Studies Performed on Fresh and Frozen Cells

Viability   Erythrocin B dye exclusion test was used. Those cells which took up the red stain were considered nonviable.

Nucleated Cell Recovery   This count was obtained by using Turk's solution (0.01% Crystal violet/3% Acetic acid/water) to lyse the red blood cells [20]. The cell count was determined by employing a hemocytometer and an inverted microscope.

Colony-Forming Unit (CFU) Assays   Committed BM progenitor cells were assayed using a modification of the method described by Fausner and Messner [21]. A combination of agar and methylcellulose cultures was used. The number of cells plated before and after freezing was kept constant (2 x 105 MNCs/plate) irrespective of volume. Growth factors used were recombinant (r)GM-CSF (mouse GM), rIL-3 + rEPO (mouse granulocyte-erythroid-macrophage-megakaryocyte [GEMM]) GCT-conditioned medium (CM; human GM), 5637 CM + rEPO (human GEMM). Plates were incubated at 37°C in 5% CO2 and colonies were scored after 14 days of incubation. Clones >40 cells were considered as colonies. The CFU plates were prepared in triplicates for all experiments. Some colonies were picked up after fixation with paraformaldehyde and identification of cell type was confirmed using Leishman-Giemsa staining of cytospin preparation. For suboptimal assays, the concentrations of colony stimulating factors (CSFs) were reduced as follows:

Optimal concentrations of CSFs used were as follows:

EPO = 2 U/ml, IL-3 = 20 U/ml, GM-CSF = 20 U/ml 5637 CM = 15% and GCT CM = 15%

(EPO is a hormone which is responsible for erythroid differentiation of progenitor cells. Various CSFs used in the CFU assays direct the progenitor cells to the respective lineage differentiation.)

Calculations and Statistical Evaluation of Data   Precryopreservation values for all parameters were taken as 100% and results were expressed as a percentage of these values. Number of CFUs has been calculated according to Rowley and Anderson [22]. Briefly, colony numbers in the total sample were calculated according to the following formula:


(1)

One-way Repeated Measure Analysis of Variance test was used for statistical analysis of CFU numbers. This was done using a computerized statistical analysis software program, Sigma Stat (Jandel Scientific Corp; San Rafael, CA). p values less than 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Summary
 References
 
Freezing of Mouse BM with 10% DMSO and Antioxidant Additives
Mouse BM was cryopreserved using 10% DMSO and different concentrations of each of the five antioxidants. From these studies, the optimum concentrations selected were ascorbic acid (80 µg/ml), catalase (100 µg/ml), and {alpha} tocopheryl acetate (40 µg/ml). Reduced glutathione and superoxide dismutase had no beneficial effect on the post-thaw recoveries (data not shown). Results of a representative experiment using optimum concentrations from the earlier experiments are shown in Figure 1. Samples cryopreserved in the presence of antioxidants show a statistically significant increase in both late (CFU-GM) and early (CFU-GEMM) progenitors as compared to those cryopreserved in the absence of antioxidants. (p < 0.01, except for ascorbic acid, CFU-GM p < 0.05). It is unlikely that trace amounts of antioxidants that may remain after washing give this enhanced recovery. We had treated the fresh cells with 10% DMSO alone and in combination with the antioxidants for 30 min, then washed the cells and performed the GM and GEMM assays. There was no significant difference in the colony numbers between treated and untreated cells (data not shown). Leishman-Giemsa stained smears of representative colonies showed presence of different cell types, indicating, thereby, that there was no bias toward differentiation of a particular cell lineage with antioxidants in the freezing mixture (data not shown).



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  		Figure 1. Cryopreservation of mouse BM using 10% DMSO (10% D) with or without the three antioxidants in the freezing solution. Concentration of additives: ascorbic acid (80 µg/ml), catalase (100 µg/ml); and {alpha} tocopheryl acetate (40 µg/ml). Values are expressed as a percentage of precryopreservation values. {diamondsuit}p < 0.05 and {diamondsuit}{diamondsuit}p < 0.01 for 10% D alone versus 10% D + additive.

 
Freezing of Human BM with 10% DMSO and Antioxidant Additives
Using the optimum concentrations of additives that have worked on preservation of mouse BM, experiments (n = 5) were repeated on human BM samples. Figure 2 shows data from one sample where the cell yield was sufficient to test all three antioxidant additives. The viability of samples ranged from 86% to 89% and nucleated cell recovery ranged from 40% to 47%. Both the early and late progenitor numbers increased for cells frozen in the presence of catalase and {alpha} tocopheryl acetate. With the latter, the CFU-GM (p < 0.05) and CFU-GEMM (p < 0.01) rise was statistically significant. The absolute colony numbers/2 x 105 cells plated were as follows: 50 ± 2.44 (GM) and 163.66 ± 7.40 (GEMM) for cells frozen without {alpha} tocopheryl acetate and 72.66 ± 6.12 (GM) and 281.33 ± 7.58 (GEMM) for cells frozen with {alpha} tocopheryl acetate (values expressed as mean ± SD). In the presence of ascorbic acid, there was an increase in recovery of the committed progenitors but a slight decrease in recovery of immature progenitors.



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  		Figure 2. Cryopreservation of human BM using 10% DMSO (10% D) with or without optimum concentrations of the three antioxidants in the freezing solution. Concentration of the additives are the same as described in the legend of Figure 1. All values are expressed as a percentage of precryopreservation values. {diamondsuit}p < 0.05 and {diamondsuit}{diamondsuit}p < 0.01 for 10% D alone versus 10% D + additive.

 
Cryopreservation of Mouse BM with 10% DMSO and Antioxidants and Response of the Revived Cells to Suboptimal Concentrations of CSFs
It has been our frequent observation that the response of frozen BM cells to suboptimal concentrations of CSFs is very poor. It was therefore thought worthwhile to see whether this response is restored by freezing the cells in the presence of antioxidants. Figure 3 shows that the cells frozen in the presence of antioxidants responded better than cells preserved in their absence (except ascorbic acid); catalase was particularly effective in this respect. Our preliminary data using mouse stem cell factor also show similar findings (data not shown), suggesting a similar poor response of frozen cells to suboptimal concentrations of the early-acting cytokine.



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  		Figure 3. Response of cryopreserved mouse BM with or without antioxidants in the freezing solution to suboptimal 1 assays. Values of GM and GEMM are expressed as a percentage of precryopreservation values. {diamondsuit}p < 0.05 for 10% DMSO alone versus 10% DMSO + catalase for suboptimal 1 GM.

 
Cryopreservation of Mouse BM with 10% DMSO with or without Catalase and Response of Revived Cells to Various Concentrations of CSFs
The experiments with catalase as an additive in the freezing mixture were further carried out in detail. The revived cells were titrated by using various concentrations of CSFs in the in vitro assays (optimum, suboptimum 1, suboptimum 2 and suboptimum 3). Figure 4 clearly shows that when catalase was present in the freezing mixture, the yield of both late (CFU-GM) and early (CFU-GEMM) progenitors at each suboptimal concentration was more than that of cells frozen in its absence. This difference was statistically significant (p < 0.05 except for suboptimal 3 GM p < 0.01) except for suboptimal 2 GM (not significant). The cells stored with 10% DMSO alone did not form any GEMM colonies at suboptimal 3 concentration of CSFs.



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  		Figure 4. Response of cryopreserved mouse BM with or without catalase in the freezing solution to suboptimal CFU assays. Values of GM and GEMM are expressed as a percentage of precryopreservation values. The value for 10% DMSO alone in suboptimal 3 GEMM is zero. {diamondsuit}p < 0.05 and {diamondsuit}{diamondsuit}p < 0.01 for 10% DMSO alone versus 10% DMSO + catalase.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Summary
 References
 
The stability of free radicals which is low in systems of high water content is considerably increased under low moisture and subfreezing conditions [23]. If not properly scavenged, it results in oxidative damage such as lipid peroxidation, protein oxidation, and DNA damage. A cell's major defense against free-radical-mediated damage includes antioxidants such as ascorbic acid, {alpha} Tocopheryl acetate (vitamin E), and reduced glutathione and enzymes such as superoxide dismutase, catalase, and peroxidases [24]. Matthes et al. [25, 26] have reported increased CFU-GM through use of antioxidant drugs such as methionine, selenomethionine, {alpha} tocopheryl acetate, and penicillin Fe2+ in the freezing solution. CFU-GM evaluation is a quantitative, simple, reliable, and reproducible index of the capacity of cryopreserved marrow to restore normal hemopoiesis, and this kind of study may be useful in predicting delayed kinetics or absence of engraftment following autologous BM transplantation [27]. CFU-GEMM multipotent progenitor cells give rise to erythroid, granulocyte-macrophage, and megakaryocytic cells. Roodman et al. [28] have found that the CFU-GEMM assay may be useful for determining the repopulating ability of cryopreserved BM. In the present study, it was found that {alpha} tocopheryl acetate, catalase, and ascorbic acid improved significantly the recoveries of mouse BM cells in terms of multipotential (GEMM) and committed (GM) stem cells. A similar trend was also shown by a few human BM samples tested. {alpha} tocopheryl acetate was found to be particularly useful for cryopreservation of human BM cells. The reasons for superoxide dismutase and reduced glutathione failing to protect the BM cells during freezing require further investigation. One possible explanation for this failure may be the generation of radicals other than superoxide during the freezing of marrow. Since there were no pH changes after addition of antioxidants, the results cannot be attributed to pH changes. One wonders, in regard to getting more than 100% CFU recovery in some cases, if it may be due to preferential loss of matured cells resulting in the appearance of more colonies after freezing. Here our results agree with those of Zaheer et al. [29]. We report only a marginal rise in the viability of cells frozen in the presence of antioxidants. The recovery of MNCs post-thaw varied considerably. Although a wide variety of techniques have been used for freezing of marrows with varying degrees of success, recoveries ranging from 9% to 118% have been reported [30]. Loss of MNCs post-thaw has also been reported by Zaheer et al. [29]. Similarly, the overall low recoveries of CFU-GM and CFU-GEMM in these experiments compared with other studies may be because we are calculating the CFU numbers in relation to MNC recovery. However, since it is the washed and thawed sample that is used for transfusion under clinical situations, it is important to know the CFU content in this sample. Further, in traditional clonogenic assays such as CFU-GM, interlaboratory comparisons are difficult owing to variation in methodology [31]. Though not expected, it was observed that recoveries with human BM are somewhat higher than with mouse BM. At the moment, we do not have any concrete explanation; however, it may be owing to cell loss taking place during processing due to the sticky nature of mouse BM cells. We have experienced this innumerable times, and therefore it may be viewed as an experimental fact.

The mechanism of action of the three antioxidants in scavenging free radicals is different [32-34]. Still, it was observed that the magnitude of protection afforded by all three antioxidant agents was almost similar. In order to delineate the mechanism of action of various agents in affording protection, we carried out experiments to see the growth factor responsiveness of cryopreserved cells. We changed the culture conditions by using suboptimal concentrations of CSFs in the in vitro CFU assays. Here, only catalase was found to be very effective and resulted in better protection of GM and GEMM at each suboptimal concentration used. The results suggest that probably the peroxide radicals scavenged by catalase may be causing damage at the CSF receptor level, and perhaps the presence of exogenous catalase protects the receptors.

The use of these antioxidants under clinical situations can be recommended only after toxicity testing of i.v. infusion of the antioxidants. The protective effect of the three antioxidants needs to be further confirmed by in vitro assays such as long-term culture initiating cells, high proliferative potential colony-forming cells, and in vivo animal experiments to study engraftment kinetics, and also by extending the storage period.


   Summary
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Summary
References
 
Our results show that the three antioxidants, ascorbic acid, catalase, and {alpha} tocopheryl acetate, helped to preserve the functionality of cells as seen by enhanced in vitro CFU formation of cryopreserved mouse BM cells. Our data further indicates that catalase probably exerts its cryoprotective effect by preventing the damage at the CSF receptor level. Thus, these three antioxidants show potential as valuable additives for storage of BM in BM transplantation studies.


   Acknowledgments
 
I thank my colleague Dr. Vaijayanti Kale for her fruitful suggestions in experimental planning and Mr. Sarang Satoor, Technician B, for his help in computer work. I am grateful to Dr. A.V. Jamkar, Surgery Department, Sassoon Hospital, Pune, India, for providing human rib samples. I acknowledge Dr. R.L. Marathe, hematologist, Jehangir Hospital, Pune, India, for his kind help in morphological identification of cell types. I am grateful to Ms. Shashi Verma of National Institute of Virology for assistance in statistical analysis of my data. I am indebted to Dr. U.V. Wagh (former Director, National Centre for Cell Science) for his constant support and encouragement. I thank Dr. G.C. Mishra (Director, National Centre for Cell Science) for his valuable suggestions during preparation of the manuscript.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Summary
 References
 

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accepted for publication June 4, 1997.




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