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Original Articles: Experimental Transplantation

Prevention of Apoptosis as a Possible Mechanism behind Improved Cryoprotection of Hematopoietic Cells by Catalase and Trehalose

Sasnoor, Lalita M.; Kale, Vaijayanti P.; Limaye, Lalita S.

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doi: 10.1097/01.tp.0000169028.01327.90
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Abstract

Biological metabolism in living cells dramatically diminishes at low temperatures, a fact that permits the long-term preservation of living cells and tissues for either scientific research, medical, or industrial applications (e.g., blood transfusion, bone marrow transplantation, artificial insemination, in vitro fertilization, food storage) (1).The emergence of engineered tissues utilized in clinical applications, basic research and product safety testing has increased the demand for cryopreservation techniques that support the improved preservation of both cells and engineered tissues (2). Cryopreservation of hematopoietic cells is considered as an integral part of their use in stem cell banks, clinical transplantations, and biological research (3, 4).

We have earlier shown that antioxidants like catalase, alpha tocopheryl acetate, and ascorbic acid—when used singly as additives in conventional freezing medium—help in better protection of mouse bone marrow cells and adult human bone marrow (5). We have further shown that a combination of trehalose and catalase in conventional freezing medium helps in preserving human hematopoietic cells isolated from cord blood and fetal liver. This was studied mainly by in vitro colony forming unit assay as read out system. The results obtained with the combination were more striking than those obtained when the two additives were used singly (6). Therefore, all our further studies were done using a combination rather than single additives. The combination was found to be effective in preserving LTC forming ability, surface molecule expression (7), and in vitro adhesion and chemotaxis (8) of frozen human hematopoietic cells. Many other investigators have also used trehalose for freezing of hematopoietic cells either alone or in combination with dimethyl sulfoxide (DMSO) (9–11). Similarly, there are reports on use of antioxidants for freezing of various cells and tissues (12, 13).

In the present investigation, we tested the efficacy of this combination on engraftment potential of frozen marrow using a murine model. It was observed that when mouse bone marrow was frozen with catalase and trehalose as additives to the conventional freezing medium, there was better protection of the cells as compared to those frozen with 10% DMSO alone. A battery of in vitro and in vivo assays was employed to detect the beneficial effect. We studied viability of the revived cells by two different methods via Trypan blue dye exclusion and PI staining. In vitro colony forming unit (CFU) assays were done to assess the content of committed and pluripotent cells in the frozen bone marrow. The efficacy of additives was also tested by in vivo assays like CFU-spleen (CFU-S), pre-CFU-S, short-term and long-term engraftment in irradiated mice. Our results indicate that there is improvement in protection of cells in the test set as compared to control set with respect to all parameters tested.

Reactive oxygen species (ROS) generation (12, 13) and apoptosis (14–20) have been implicated as two of the multiple causes of damage to cells during freezing. To get an insight into the possible mechanism of protection afforded by the two additives, we detected the level of apoptosis in both the sets. Three commonly used methods were employed (i.e., TUNEL, DNA ladder, and Annexin V staining). It was found that there was reduction in level of apoptosis when the cells were frozen with additives. Detection of ROS levels in both sets pointed to more efficient scavenging of ROS by additives. Our studies indicate that probably the two additives exert their protective effect by scavenging the free radicals and stabilizing the membranes leading to reduced apoptotic cell death during freezing and thawing. Thus we propose that reduction in apoptosis is perhaps the mechanism behind the enhanced engraftment potential of cells cryopreserved with catalase and trehalose as additives.

MATERIALS AND METHODS

Materials

Dimethyl sulphoxide (DMSO), deoxyribonuclease 1 (DNase1), trehalose, catalase (all cell culture tested), methylcellulose of viscosity 4000 cps, trypan blue, crystal violet, Wright’s and Giemsa stains, and camptothecin, were purchased from Sigma (St. Louis, MO). Recombinant murine growth factors, namely stem cell factor (SCF), interleukin (IL)-3, granulocyte monocyte colony stimulating factor (GM-CSF), erythropoietin (EPO), and fetal calf serum for colony forming unit assay were obtained from Stem Cell Technologies (Vancouver, Canada). All flow cytometry qualified antibodies, Annexin V, PI, were purchased from Pharmingen (Becton Dickinson; San Jose, California). Media like Iscove’s modified Dulbecco’s medium (IMDM) and fetal calf serum (FCS) were from GIBCO (Grandisland). TUNEL kit was from Roch Applied Science, Mannheim, Germany. DCFHDA was from Molecular Probes (Eugene, OR). Plates for CFU assays were from Stem Cell Technologies and other tissue culture grade plasticware was from Nunc and Falcon.

Animals

Protocols for all animal experiments were approved by the Institutional Animal Ethics Committee. Swiss albino mice 8-10 weeks old of either sex were used for CFU-S, pre-CFU-S and short-term engraftment experiments. C57BL/6 (Ly5.2) (donor) and C57Bl/6 Ly5.1:Pep3b (Ly 5.1) (recipient) mice obtained from Jackson Laboratories (Bar Harbor, ME) were bred in our animal facility and used for chimera experiments at age 10–12 weeks.

BM and MNC Preparation

BM cells were flushed from femurs with Iscove’s modified Dulbecco’s medium (IMDM) and pooled for further use. Low-density mononuclear cells were obtained by centrifugation at 1000 rpm for 15 min on Ficoll/Hypaque (1.077 gm/ml) density gradient. The cells were harvested from interface, washed, and resuspended in complete medium containing 20% FCS (5).

Freezing of Mouse Bone Marrow

Isolated MNCs were frozen at a cell density of 107 cells/ml/vial in Nunc cryovials in a portable programmable freezer (Freeze Control, Australia) as described earlier (8). Control cells were frozen in a medium containing IMDM + 20% FCS + 10% DMSO. Test cells were frozen in the same medium with additives (trehalose at a final concentration of 25 μg/ml and catalase at a final concentration of 100 μg/ml).

Irradiation of Mice

For CFU-S, pre-CFU-S, engraftment, and chimera experiments, the recipient mice were subjected to total body irradiation (TBI). The dose of irradiation was 900 cGy as a split dose of 450–450 cGy 4 hours apart. The source of irradiation was Co60 gamma chamber 5000 from BRIT, Mumbai, India.

Cell Recovery and Viability

Nucleated cell recovery and viability by trypan blue dye exclusion of revived cells was estimated as described earlier (5). Viability was also quantitated on FACS by staining the cells with PI and analyzing the fluorescence on FACS.

CFU Assay

This was done as described earlier (6). Briefly, 2×105 mouse bone marrow MNCs were plated in methyl cellulose along with growth factors: mouse interleukin (mIL)-3 (10 ng), mouse stem cell factor (mSCF) (10 ng), mGM CSF (5 ng), EPO (2U). The colonies were counted as BFU E, GM, GEMM on the 10th day postincubation.

CFU-S Assay

This assay was performed as described (21). Briefly, 2×105 frozen mouse bone marrow derived MNCs suspended in 200 μl PBS were infused through tail vein in syngeneic lethally irradiated Swiss albino recipients. Twelve days later, mice were killed and spleens were excised and fixed in Tellyesniczky’s solution (64% ethanol, 5% acetic acid, and 2% formaldehyde in water). Colonies on spleen were then counted under dissection microscope. Freshly isolated bone marrow was infused in one group of mice as positive control and only PBS was infused in another set of mice which served as radiation control.

Pre-CFU-S Assay

Fresh/frozen mouse bone marrow (BM) MNCs were injected intravenously (IV) into TBI-treated mice. After 12 days, BM MNCs were isolated from femurs and 2×105 MNCs were injected into secondary TBI-treated recipients. After 12 days, recipient mice were sacrificed, spleens were fixed in Teleyesnizky’s solution, and pre-CFU-S was counted (21).

Short-term Engraftment

Briefly, 2×105 fresh or frozen mouse bone marrow cells were infused into TBI mice as described above. The number of mice surviving in each group at different time intervals posttransplantation was recorded.

Blood Cell Analysis of Recipient

Transplanted mice were bled at intervals by retro-orbital plexus and 20 μl of the free flowing blood was collected in heparin (40 IU) containing medium. This was used for MNC count and platelet count. Smears were prepared for neutrophil count. The smears were stained with Wright’s Giemsa method. Neutrophil count was performed by counting at least 100 leukocytes per peripheral blood smear under oil immersion as described by Ulich et al. (22). Platelets were counted under inverted microscope after dilution in platelet dilution fluid.

CFU Assay

Mice from each group were sacrificed at defined intervals. Femurs of individual mouse were flushed and bone marrow collected in plain medium. MNCs were isolated by F/H density gradient centrifugation as described above. MNCs were plated for CFU assays in methyl cellulose as described above. Colony number was normalized to total MNCs recovered per two femurs/mouse.

Detection of Long-term Donor Cell Engraftment by Chimera Model

Frozen ly5.2 cells were revived from test and control sets. They were mixed with recipient bone marrow cells (8 × 105 donor cells + 2×105 recipient cells) and infused through tail vein in Ly 5.1 irradiated recipient mice. Two groups of animals received PBS only or fresh bone marrow cells to serve as negative and positive controls. The mice were given sterile acidified water supplemented with 2 mg/ml neomycin sulfate. Over the course of the study, the mice were followed for up to 1 year. Percent donor engraftment in the survivors was compared. The stability of donor engraftment was used to reflect long term marrow repopulating ability (21).

Percent donor engraftment was determined by two-color fluorescence-activated cell sorting (FACS) analysis with antibodies against the leukocyte markers CD45.1 and CD45.2. Whole blood (10–100 μl) was collected from orbital plexus in heparin containing medium. Cells were stained using fluorescein isothicyanate (FITC)-conjugated mouse antimouse CD45.2 and phycoerythrin (PE)-conjugated CD45.1 monoclonal Ab (Pharmingen).Control studies were performed with FITC or PE-conjugated mouse IgG2a isotype controls (Pharmingen) (21).

Measurement of ROS

Peroxide sensitive fluorescent probe DCFH-DA (23) was used to assess levels of net intracellular generation of ROS. Fresh or frozen thawed cells were washed once and incubated with 5 μM DCFH-DA for 30 min. After incubation cells were washed with phosphate buffered saline, and suspended in ice cold phosphate buffered saline. Levels of intracellular ROS were measured with a FACS Vantage (Becton Dickinson, Mountain View, CA). In each analysis, 10,000 events were recorded.

ROS were measured using a previously described method with some modifications (24). Fresh or revived cells were suspended at a density of 106/ml in 2.5 μM DCFH-DA in PBS. Samples were incubated at 37°C for 30 min in dark, and the 2′,7′-dichlorofluorescein (DCF) fluorescence intensity was measured with a fluorescence plate reader (Fluoroskan) (excitation wavelength, 485 nm; emission wavelength, 530 nm).

Apoptosis Detection

Apoptosis was detected by annexin V/PI staining, TUNEL, and DNA laddering.

Annexin V is a Ca+ dependent phospholipid binding protein (30–36 KDa) that has a high affinity for phoshatidylserine (Kd, 5×10−2) and binds to cells with exposed phoshatidylserine. Fluorescencin-bound annexin V (green dye) serves as a fluorescence probe for apoptotic cells. The staining was performed on freshly isolated mouse bone marrow MNCs as well as on frozen test and control revived cells and Annexin V positive cells were quantitated on FACS.

For TUNEL assay, 2×106 fresh or revived cells were washed twice with PBS then fixed with 1 ml of 4% paraformaldehyde for 20 min at RT. The cells were spun down and permeabilized with 1 ml of 70% ethanol for 30 min in ice. The fixed cells were stained by TUNEL assay kit as per manufacturer’s instructions and were analyzed on FACS.

DNA laddering for detection of apoptosis was carried out as described by Zhu Ning et al. (25) Briefly, 106 cells of different sets were washed once with ice-cold PBS and resupended in 30 μl of lysis buffer (10 mM Tris, pH 7.4; 100 mM Nacl; 25 mM EDTA; 1% SDS) by gentle vortexing, and 4 μl of proteinase K (10 μg/μl) was added. The cell lysates were incubated at 45°C for 1-2 hr. Two μl of RNase (10 μg/μl) was added to the supernatant and incubated for 1 hr at RT. Samples were spun briefly to pellet any further cell debris and the supernatant was collected. Four μl of 6× DNA sample dye was mixed in the lysate (the final volume of each sample was about 40 μl). The genomic DNA was eletrophoresed on a 2% agarose gel containing 0.2 μg/ml ethidium bromide at 7 V/cm and then visualized under UV light. DNA marker used was the 1-kb DNA ladder from GIBCO.

Calculations and Statistical Analysis

The values obtained for cells frozen with additives (test) were compared with those obtained with cells frozen without additives (control). One-way repeated-measure analysis of variance (ANOVA) test was used for statistical analysis. SIGMASTAT (Jandel Scientific Corporation; San Rafael, CA) software program was used for statistical analysis. A P value ≤0.05 was considered to be statistically significant. The data obtained with fresh cells served as a positive reference point and were not used for statistical analysis.

RESULTS

Cell Recovery and Viability

Frozen mouse bone marrow cells were revived and the nucleated cells recovered from test and control sets were counted (n=10). Data is shown in Fig. 1A. There is a statistically significant difference in the recovery of cells from test set as compared to control set. Viability as assessed by trypan blue dye exclusion and PI positive cells quantitated by FACS also showed significant differences between test and control cells. Values obtained with fresh mouse bone marrow cells are also shown in Fig. 1A.

F1-17
FIGURE 1.:
Cell recovery, viability and CFU assay of revived mouse bone marrow MNCs frozen with or without catalase and trehalose were revived and tested for various parameters. (A) Nucleated cell recovery, viability by trypan blue dye exclusion and PI staining. Test cells showed better cell recovery and viability. (B) Progenitor content of fresh cells as detected by methyl cellulose cultures (values normalized for 107 MNCs; i.e. the number frozen/vial). (C) Progenitor content of revived cells of both test and control sets (values normalized for number of MNCs recovered). **P≤0.01; ***P≤0.001.

CFU Assay

To test the effect of the combination of additives on progenitor content of frozen marrow, we carried out CFU assay as described in methods. Results of CFU assay showed higher recoveries of BFU (E), GM, GEMM and total colonies from test cells as compared to control cells. The difference between the two sets was statistically significant for all types of colonies. Thus the results showed that the combination of the two additives in the conventional freezing medium helps to preserve the progenitor compartment in a better fashion and there is no bias towards protection of any specific colony type (Fig 1C). Figure 1B shows progenitor content of fresh cells normalized to initial number of cells frozen per vial.

CFU-S Assay

Spleen colony formation (i.e., CFU-S in irradiated mice) is used as a short-term in vivo assay for pluripotent hematopoietic stem cells. We set up in vivo CFU-S assay using fresh or frozen mouse bone marrow. The mouse bone marrow cells were infused in irradiated syngenic recipients via tail vein and the colonies formed on spleens on 12th day were scored as described in methods. Figure 2A represents a mean ± SD of colonies formed on spleen of 10 animals in each group (n=3). Results showed that indeed the test cells formed significantly higher number of CFU-S than control cells. Fresh mouse bone marrow cells were infused in one group of animals as positive control and PBS in another group as vehicle control. The former showed a mean of 17.12±4.32 colonies and the latter group showed no endogenous colonies.

F2-17
FIGURE 2.:
CFU-S and PreCFU-S formed after infusion of test and control frozen mouse bone marrow MNCs. (A) Primary repopulating ability of frozen cells was assessed by infusing them in TBI recipients and the colonies formed on spleen were scored on 12th day posttransplantation as CFU-S. (B) Secondary repopulating ability of frozen mouse bone marrow MNCs was quantitated by pre-CFU-S assay as described in Methods. In both the assays, cells that were frozen with additives formed significantly higher number of colonies on spleen than those that were frozen without additives. ***P≤0.001. CFU-S and pre-CFU-S data obtained with fresh mouse bone marrow cells are also shown in (A) and (B) respectively.

Pre-CFU-S

The efficacy of catalase and trehalose in better protection of freezing of mouse bone marrow cells was further evaluated by pre-CFU-S assay which detects a more primitive population than CFU-S (21). The assay was performed as described in Methods. There were 10 animals in each group (n=2). Results of one representative experiment are shown in Fig. 2B. The results indicate that there is a statistically significant difference in number of pre-CFU-S colonies formed by cells frozen with additives than those frozen without additives. Thus the additives protect the primary as well as secondary repopulating ability of frozen marrow cells. Figure 2B also shows data obtained when fresh cells were infused.

Short-term Engraftment

Engraftment of fresh or frozen marrow in irradiated recipient was assessed at intervals for a short period of 2 months by monitoring percent survival. Initially, survival was studied using different cell doses. It was found that we got more than 50% survival on the 60th day posttransplantation in all three groups (range 60–90%) at higher cell doses than at lower doses. However, for these studies we selected the cell dose of 2×105 for further experiments because at this limiting cell dose the difference between the control and test cells became more striking particularly at 60th day posttransplantation.

The parameters chosen for detection of donor cell engraftment was survival of mice in control and test groups, as well as recovery of MNC, platelet, and neutrophils in peripheral blood of mice of both groups. The colonies formed from bone marrow in methyl cellulose cultures were also scored. There were 15 animals in each group at the start of the experiment (n=2). Results of one representative experiment are shown in Fig. 3.

F3-17
FIGURE 3.:
Short-term engraftment in irradiated recipients. Fresh or frozen marrow cells were infused in TBI syngeneic mice and recovery assessed at different time points until two months as described in Methods. (A) Percent survival of mice was higher in the group that received test frozen cells at each time point as compared to control group. (B) Hematology of PBL of recipient mice showed significantly higher recovery of MNCs, platelets and neutrophils in the test group. This was also true for progenitor content of femur bone marrow. *P ≤ 0.05, **P ≤ 0.01. Data obtained when fresh mouse bone marrow cells were used for infusion are also depicted in (A) and (B).

It is clear from Fig. 3B that there is a comparatively speedy recovery of MNC, platelets and neutrophils in the mice that received infusion from test cells as compared to control cells. Even the number of CFC per two femurs was statistically higher in test group than control at all three intervals (Fig. 3B).

Donor Cell Engraftment

Mice from each group were bled at various intervals posttransplantation as shown in Figure 4. Peripheral blood samples were stained with Ly5.1 and 5.2 MAbs to detect donor cell engraftment as described in methods. Initially, after 15 days posttransplantation, there was no donor cell engraftment in any group and the percent of recipient cells was also very low (data not shown). Later on from month 3 to 1 year posttransplantation, donor cell engraftment was observed in all groups as detected by Ly5.2 FITC positive cells (Fig. 4). In the group receiving test cells the percentage of donor cells was consistently higher than the group receiving control cells at all time points tested. Dot plots from one representative experiment are shown in Fig. 4. Percent donor cell engraftment in mice that received fresh marrow is also depicted in Fig. 4. Mice from vehicle control group died between 15th to 30th day posttransplantation.

F4-17
FIGURE 4.:
Long term engraftment of frozen mouse bone marrow by competitive repopulation assay in chimera model. Ly 5.2(FITC-FL1) frozen test and control cells were revived mixed with recipient marrow in the proportion of 4:1 and infused in TBI recipient Ly5.1 mice (PE-FL2). PBL samples of recipient were analyzed for donor cell engraftment at 3, 6, 9 and 12 months posttransplantation as described in methods. The donor cell engraftment is represented by the population in lower right quadrant (green). Percent engraftment data of fresh mouse bone marrow is also shown.

Estimation of Free Radical Level

The levels of free radicals generated in fresh or frozen cells were estimated immediately after revival of frozen cells by two methods, fluorimetry and FACS, using the free radical detection probe DCFHDA. It was found that in test group the levels of free radicals were lower than those in control group. This was true of both methods employed for detection of free radicals. Figure 5A and B (mean ± SD of five samples). One representative histogram plot of the FACS data is depicted in Fig. 5C. The figures show negligible level of ROS generation in fresh marrow cells.

F5-17
FIGURE 5.:
Estimation of ROS in mouse bone marrow MNCs. ROS generated in fresh mouse bone marrow MNCs and during freezing and thawing were estimated by fluorescent probe DCFHDA in test and control cells by fluorimetry (A) and FACS (B) as described in Methods. By both methods, it was seen that there was a significant reduction in level of ROS in test cells.*P≤0.05, ***P≤0.001. (C) Histogram plot of one representative sample after FACS analysis of DCF+ cells.

Detection of Apoptosis

The level of apoptosis in control and test group was quantitated on flow cytometer by annexin V staining and TUNEL as described in Methods. The data obtained by both methods indicated that there was reduction in level of apoptosis in the test group as compared to control group as shown in Fig. 6A and B (n = 5). Figure 6C shows histogram plots of one representative experiment of each method. DNA ladder method also showed less DNA fragmentation in test group as compared to control group (Fig. 6D). Camptothecin-treated cells served as positive control in this experiment. Thus the results suggest that the additives are exerting their protective effect probably by reducing the apoptotic cell death that occurs during freezing thawing process. It is clear from Figs. 6A–D that there is insignificant level of apoptosis in fresh marrow cells.

F6-17
FIGURE 6.:
Apoptosis level in fresh cells and cells frozen with or without additives. Apoptosis was detected by Annexin V staining, TUNEL, and DNA ladder as described in Methods. (A) Annexin+ cells (n=5). (B) TUNEL-positive cells (n=5). (C) Histogram plot of one representative sample showing Annexin and TUNEL. (D) DNA gel electrophoresis. Lane 1: 1 Kb marker; lane 2: fresh mouse bone marrow; lane 3: mouse bone marrow frozen in conventional medium containing 10% DMSO alone; lane 4: mouse bone marrow frozen with 10% D+C+T; lane 5: camptothecin treated cells.

DISCUSSION

Previously we have reported the benefits of using catalase and trehalose as additives to conventional freezing medium containing 10% DMSO for freezing of human hematopoietic cells (6–8). One of our goals in the present study was to see whether the protection evident in in vitro assays could be supported by in vivo assays. Therefore, we used mouse bone marrow system for the purpose. We herein report better cryoprotection in test frozen cells as compared to control frozen cells as assessed by standard in vitro assays like cell recovery, viability, and CFU assays and also by in vivo assays like CFU-S, pre-CFU-S, short- and long-term engraftment in irradiated host.

The colony forming spleen assays devised by Till and McCulloch (21) have been considered a means of measuring HSC contents. Because the colonies derive from single progenitor and because the stromal cells in the spleen are found to maintain long-term hematopoiesis (21), spleen colony formation seems to be a useful tool for analyzing the interaction between hematopoietic progenitors and stromal microenvironments in vivo. Using frozen mouse bone marrow, we have shown that indeed the two additives protect the CFU-S content of the frozen marrow. However, day 12 CFU-S is derived from primitive multipotent progenitor and therefore in vivo assays to detect precursors of CFU-S must incorporate delayed end points (21). One approach to extend the time of hematopoietic reconstitution assays has been to use a double transplant procedure to measure the number of CFU-S in the marrow of mice transplanted two weeks previously with the test cells under consideration. This assay termed as pre-CFU-S assay, quantitates secondary repopulating ability of primitive hematopoietic stem cell population (21). We observed a higher number of both CFU-S and pre-CFU-S with test cells thereby suggesting more efficient homing of test frozen hematopoietic precursors to spleen and bone marrow respectively.

Another approach to detect cells more primitive to CFU-S is by quantitation of survival (21), hematopoietic reconstitution as well as femoral progenitors of recipient mice (26) at different time points posttransplantation. In our studies, femoral progenitor cells of recipient mice showed consistently higher numbers in the group that was infused with test frozen cells as compared to mice that were transplanted with control cells. Hematological examinations of recipient mice confirmed this observation. Survival was found to be dose-dependent and there was more than 50% survival in all groups, at 60 days posttransplantation at high cell dose (data not shown). However the difference in percent survival between the test and control groups was more striking when a limiting cell dose (2×105) was infused.

In mice, in vivo competitive repopulation assays using Ly 5.1-5.2 chimera models is considered as a powerful tool for measuring the functional potential of stem cell population (21). In our studies, we could get better donor cell engraftment in mice that received test cells during transplantation. Thus by carrying out a comprehensive study using in vitro and in vivo functional assays each of which probably detects overlapping subsets of pluripotent cells from the hierarchial pattern of hematopoietic cells, we conclude that catalase and trehalose are useful additives to conventional freezing medium. Though we (5) and others (27, 28) have shown that a good correlation exists in freezing protocols of mouse and human hematopoietic cells, it still remains to be seen whether human cells frozen with additives will behave in similar fashion when infused in NOD /SCID mice. If so, our observations may have significant clinical implications in transplantation settings. We propose to carry out these experiments in near future.

To dissect out the possible mechanism of protection afforded by additives we carried out apoptosis and ROS assays and found that there is reduction in the levels of both parameters in the test frozen cells. Many cell types are known to require modification of freezing solutions or culture systems for optimal growth or survival. Addition of apoptosis inhibitors in the cryopreservation solution or cold storage solution has been shown to enhance the survival of MDCK cell line and skin tissue (12, 14). In human bone marrow cells, cryopreservation can activate intracellular proteases and the activated proteases in turn cause cleavage of apoptosis related proteins (29). Apoptosis has also been reported in frozen thawed CD34+ cells of PBL stem cell transplants (16–18) resulting in loss of viable cells leading to decrease in quality of graft. Therefore control for viability of CD34+ cells in terms of early apoptosis, after freeze thawing, immediately before reinfusion seems to be an advisable step to maintain the graft quality.

In addition to activation of intracellular protease, the physical stress to cell membrane as a consequence of shrinkage and swelling of a cell during the freeze thaw process may also be responsible for apoptotic cell death. This structural stress in combination with profound hyperosmolality may be activating death receptors (FAS, TNF, and JNK) (30) on a cells surface resulting in the onset of apoptosis. The reduction in apoptosis in our system may be attributed to trehalose which is a known membrane stabilizer and natural cryoprotectant (31). Yet another possible mechanism behind induction of apoptosis might be accumulation of free radicals in the cytosol of frozen cells causing the activation of mitochondrial permeability transition pore (MPTp) (30). Improvement of cryopreservation outcome with the inclusion of catalase in the freezing medium and reduced level of ROS seen in the test sample indicates that accumulation of free radicals indeed leads to apoptotic death in cryopreserved hematopoietic cells. Similar strategy of free radical scavenging by α-tocopherol has been used with success by Baust et al. (12) for freezing MDCK cells.

Taken together our results indicate that effective scavenging of ROS and stabilization of membrane leading to reduction in apoptosis during freezing thawing underlies the better engraftment of mouse bone marrow cells that were frozen with additives. We propose to focus our subsequent studies on identification of specific causes leading to apoptotic induction pathways.

Our present studies on murine system clearly show that an improved engraftment can be achieved if the cells are frozen in a medium supplemented with a combination of catalase and trehalose than those frozen in conventional medium. It appears therefore that use of the two additives to conventional freezing medium may result in improved quality of frozen graft in clinical transplantation settings.

ACKNOWLEDGMENTS

We thank Nikhat Firdaus A. Siddiqui for technical assistance, DRDO (LSRB) for financial support and Dr. G.C. Mishra for support.

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Keywords:

Cryopreservation; Mouse bone marrow; Engraftment; Apoptosis; Reactive oxygen species

© 2005 Lippincott Williams & Wilkins, Inc.