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The role of caspases in cryoinjury: caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells¹

CHRISTOPHER STROH^*,, UWE CASSENS, AJOY K. SAMRAJ^*,, WALTER SIBROWSKI, KLAUS SCHULZE-OSTHOFF^*,,2 and MAREK LOS^*,23

^* Institute of Experimental Dermatology, University of Münster, D-48149 Münster;
Institute of Molecular Medicine, University of Düsseldorf, D-40225 Düsseldorf; and
Institute of Transfusion Medicine, University of Münster, D-48149 Münster, Germany

³Correspondence: Institute of Experimental Dermatology, University of Münster, Röntgenstrasse 21, D-48149 Münster, Germany. E-mail: los{at}uni-muenster.de

SPECIFIC AIMS

Cryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering, but their storage and thawing are associated with severe damage, suggesting the need for better cryopreservation methods. We analyzed whether apoptosis is involved in cryoinjury, whether caspases are activated during the freeze-thaw process, and whether inhibition of caspases improves the recovery and survival of several cryopreserved cell types and hematopoietic progenitor cells.

PRINCIPAL FINDINGS

1. Caspase inhibition improves recovery from cryopreservation
Proteases of the caspase family are the key enzymes responsible for apoptotic cell death. To determine the involvement of components of the apoptotic machinery in cryoinjury, we investigated the effect of caspase inhibition during thawing of several cryopreserved cell lines that were frozen in liquid nitrogen for > 5 years. Jurkat T cells, HeLa cervix, and 293 kidney carcinoma cells grew to a markedly higher density when they were thawed and taken into culture in the presence of the irreversible caspase inhibitor zVAD-fmk vs. controls that had not obtained zVAD-fmk (data not shown). This indicated that inhibition of caspases strongly improved cell survival. The enhanced survival conferred by zVAD-fmk showed a clear dose dependency. Maximal effects were obtained with doses of 20–40 µM; higher concentrations of zVAD-fmk were less effective.

Additional evidence for a cryoprotective effect of caspase inhibition was provided by Rat-1-MycER cells transfected with the caspase inhibitor CrmA. Rat-1-MycER-CrmA cells showed on average a threefold higher confluency 4 days after thawing than Rat-1-MycER cells not expressing CrmA (data not shown). This underscores the positive effect of caspase inhibition on the survival of cryopreserved cells. Further supplementation of the growth medium with zVAD-fmk led to an improvement in the recovery of both cell lines, but was less effective in the CrmA-expressing cells.

2. Inhibition of caspase-3, which is activated during freeze and postfreeze process, exerts cryoprotective effects
To answer the question of whether cells are preferentially damaged during freezing or during the thawing process, we compared the effect of caspase inhibition in cells that had been either frozen, thawed, or frozen, and thawed in the presence of zVAD-fmk. The monocytic cell line MonoMac-6 and murine splenocytes were frozen in medium with or without zVAD-fmk and stored for 14 months in liquid nitrogen or at -70°C. When transferred into culture, again cells that had been frozen and stored in the presence of zVAD-fmk displayed a higher viability than the controls as assessed by trypan blue exclusion 2 h after thawing (Fig. 1 A). The protection conferred by the caspase inhibitor was stronger in cells kept at -70°C, a condition that fosters cryoinjury more than preservation in liquid nitrogen. A further decrease in the number of dead cells was noticed when zVAD-fmk was added to the growth medium during thawing. Remarkably, the combined use of zVAD-fmk in both the freeze and the growth medium of cells stored at -70°C suppressed cell death to an extent obtained otherwise under optimal conditions in liquid nitrogen without zVAD-fmk (Fig. 1B ).

View larger version (45K): [in this window] [in a new window]   Figure 1. Differential effect of zVAD-fmk on cell survival and caspase activation during the freeze/thaw process. A, B) The monocytic cell line MonoMac-6 and murine splenocytes were frozen in standard freeze medium in the presence or absence of 30 µM zVAD-fmk. Cells were stored in liquid nitrogen (A) or at -70°C (B). 14 months after freezing, cells were thawed in culture medium in the presence and absence of 30 µM zVAD-fmk. Two hours post-thawing, cell death was assessed by the uptake of trypan blue. Results are given as mean ± SD from two thawed vials measured in tetraplicate cultures. C) Activation of caspase-3 in cultures of cells frozen and thawed in the presence and absence of zVAD-fmk. Jurkat cells were frozen with or without zVAD-fmk. After 3 wk of storage in liquid nitrogen, cells were thawed in the presence or absence of the caspase inhibitor. Six hours later cells were lysed and subjected to the DEVDase assay (upper panel) or immunoblotting (lower panel) using a caspase-3-specific antibody. The immunoblot shows the 32 kDa precursor of caspase-3, the inactive p21 intermediate fragment, and the p17 and p12 active subunits.

To confirm that the effects were indeed due to inhibition of caspase activation, Jurkat cells were frozen in liquid nitrogen in the absence or presence of different zVAD-fmk concentrations. After 3 wk, cells were thawed and transferred into growth medium with or without zVAD-fmk. Six hours later the cells were lysed, and caspase-3-like DEVDase activity and caspase-3 processing were determined by fluorogenic substrate assays and immunoblot analyses, respectively (Fig. 1C ). In cells that were frozen and thawed in the absence of zVAD-fmk, a strong DEVDase activity was measured. This was in accordance with the appearance of the active p17 and p12 subunits of caspase-3. This indicated that the freeze-thaw process did indeed induce caspase activation. Addition of zVAD-fmk solely to the freeze medium led to a marked decrease in caspase-3 activation. An even more pronounced inhibition was observed when zVAD-fmk was added to the growth medium during the thaw cycle. Using Jurkat cell clones that overexpressed the anti-apoptotic protein Bcl-2 or lacked the death receptor-associated protein FADD, we found that Bcl-2 but not FADD deficiency conferred protection against cryoinjury. Moreover, unlike zVAD-fmk, inhibitors of calpain and cathepsins were unable to protect the cells (data not shown). This suggests that cryoinjury-induced apoptosis is mediated by a death receptor-independent mitochondrial caspase pathway that does not involve other noncaspase proteases.

3. Caspase inhibition greatly improves the colony-forming activity of hematopoietic progenitor cells
Cryopreservation is widely used for the storage and subsequent transplantation of hematopoietic progenitor cells (HPC) in the treatment of cancer and other diseases. Clinical use of HPC requires efficient cryopreservation and recovery protocols. To assess the effect of caspase inhibition on the recovery of HPC, we used colony assays with peripheral blood-derived progenitor cells. Cryopreserved HPC from different patients were thawed and cultured on methylcellulose. Two weeks later, the number of burst-forming units representing erythroid colonies (BFU-E) and colony-forming units representing granulocyte-macrophage progenitors (CFU-GM) were counted. Figure 2 A shows representative data with HPC derived from one patient. Preincubation of HPC with 30 µM zVAD-fmk for 2 h before plating caused a two- to threefold increase in colony numbers. However, addition of the caspase inhibitor to the methylcellulose medium and continued culture in the presence of zVAD-fmk slightly inhibited colony formation, indicating that, in contrast to short-term inhibition, prolonged inhibition of caspases might impair cell survival. We also examined whether caspase inhibition changed the colony composition and cellular differentiation of HPC by comparing the BFU-E/CFU-GM ratio. No significant differences in overall colony composition were detected after short- or long-term culture with zVAD-fmk (Fig. 2B ).

View larger version (30K): [in this window] [in a new window]   Figure 2. Inhibition of caspase activity strongly improves clonogenicity of cryopreserved hematopoietic stem cells. A) HPC were frozen in liquid nitrogen and thawed in culture medium in the presence and absence of 30 µM zVAD-fmk. Left panel: experiments with cells preincubated for 2 h with or without the caspase inhibitor, before the cells were further incubated on methylcellulose in the absence of zVAD-fmk. Right panel: results with cells continuously incubated in methylcellulose medium containing 30 µM zVAD-fmk for 14 days. Two weeks after the thaw process, the number of erythroid progenitor-derived colonies (BFU-E) and granulocyte-macrophage progenitor-derived colonies (GM-CFU) was determined. Results with HPC cultures from one representative donor measured in duplicate cultures. B) Calculation of the BFU-E/GM-CFU ratio revealed that incubation with zVAD-fmk for 2 h or 14 days did not strongly affect the cellular composition of HPC cultures.

The question of whether zVAD-fmk application for cryopreservation markedly influences cell physiology was investigated in an experimental system requiring the interplay of multiple gene products. We used Rat-1-MycER cells in which activation of the c-Myc oncogene is triggered by incubation with ß-estradiol. In the presence of the hormone, Rat-1-MycER cells underwent apoptosis after serum deprivation. Rat-1-MycER cells treated with zVAD-fmk during the first 24 h after thawing were as sensitive to serum withdrawal after c-Myc induction as their untreated counterparts (data not shown). Since c-Myc induced apoptosis is cell cycle dependent and requires de novo protein synthesis as well as the activation of several signaling pathways, this indicates that zVAD-fmk does not have broad unspecific effects.

CONCLUSIONS AND SIGNIFICANCE

The present results clearly demonstrate the successful cryopreservation and recovery of cells by using a caspase inhibitor as a cryoprotective agent. We document for the first time that the freeze-thaw process is associated with caspase activation, suggesting that apoptosis plays an important role in the cryoinjury of cells. Consequently, we demonstrate that inhibition of caspases, either by overexpression of CrmA and Bcl-2 or by addition of the pharmacological inhibitor zVAD-fmk, prevents caspase activation and enhances the recovery of cryopreserved cells. The caspase inhibitor was protective when added to both the freeze solution and the culture medium during the thaw process. Furthermore, addition of zVAD-fmk in the freeze and postfreeze process prevented the activation of caspase-3. The application of zVAD-fmk proved to be beneficial not only for the survival of cells kept in liquid nitrogen, but even more strongly for cells stored at -70°C. This observation might relevant for conditions when storage or transportation facilities in liquid nitrogen are not available.

An important finding was the observation that addition of the caspase inhibitor significantly improved the recovery of HPC. We were able to increase the colony-forming activity of cryopreserved HPC up to threefold by brief treatment with zVAD-fmk after thawing. HPC are currently of high interest for their use in bone marrow transplantation and other applications. Therefore, our finding may provide a perspective with which to improve the rate of successful cell engraftments.

Moreover, we found no significant differences in the colony composition of HPC after zVAD-fmk treatment. However, our data also suggest that short-term inhibition of caspases is superior to the use of zVAD-fmk in prolonged cell culture. The inhibitory effect of long-term exposure or higher concentrations of zVAD-fmk might indicate that zVAD-fmk itself exerts some toxicity or that caspases play a role in processes unrelated to cell death.

Our data suggest the short-term usage of zVAD-fmk or other pharmacological caspase inhibitors as cryoprotective agents (Fig. 3 ). Caspase inhibitors might not only be beneficial for stem cell transplantation, but also for other applications that require preservation of biological materials. It is conceivable that apoptosis might be involved in the damage of organs and tissues that are stored by cooling or freezing during transport before transplantation. Finally, the increasing use of cryopreserved germ cells and embryonic stem cells requires better cryopreservation methods. Because apoptosis is involved in cryoinjury, caspase inhibitors in combination with other cryoprotectant agents might be important for the long-term storage of living cells and critical for the success of tissue engineering, cell, and tissue transplantation.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram indicating the role of caspases and caspase inhibition in modulation of cryoinjury-induced death.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0034fje; to cite this article, use FASEB J. (August 7, 2002) 10.1096/fj.02-0034fje

² Both authors share senior authorship.

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