1 Centre for Reproductive Medicine and Research Laboratories for Reproductive Medicine, University Hospital and Medical School, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium
2 To whom correspondence should be addressed. e-mail: veerle.frederickx{at}az.vub.ac.be
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Abstract |
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Key words: cryopreservation/mouse/spermatogonia/stem cell/transplantation
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Introduction |
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Since Brinster and Zimmermann (1994) successfully reinitiated spermatogenesis after transferring stem cells from one donor mouse to a sterile recipient mouse, a new field of research has emerged. One day, their testicular stem cell transplantation model may have clinical applications. Prepubertal boys that have to undergo a sterilizing chemotherapy are unable to cryopreserve spermatozoa because of the lack of complete spermatogenesis. They may, however, bank testicular cells, specifically the stem cells, and have them transplanted back into their testes at adult age.
Nevertheless, given the limited number of stem cells in the testes (Tegelenbosch and de Rooij, 1993), an optimized cryopreservation protocol may be a prerequisite for the successful application of this strategy in a clinical study.
Until now, few data have been available in the literature regarding cryopreservation of testicular stem cells as a cell suspension. The first paper on this topic from Avarbock et al. (1996) reports a survival of 30% when testicular cells are cryopreserved by means of an uncontrolled protocol, but that, nevertheless, successful transplantation can be obtained with frozenthawed testicular cell suspensions. This uncontrolled cryopreservation protocol is similar to the one used for somatic cell suspension freezing. Brinster and Nagano (1998
) also reported cryopreservation of testicular cell suspensions by means of a standard procedure as used for somatic cells. In order to evaluate cryopreservation of testicular cell suspensions, in addition to their survival, their progenitor capacity should also be examined. Transplantation of frozenthawed cell suspensions is the method of choice for assessing this functional capacity (Brinster and Nagano, 1998
).
Recently, Izadyar et al. (2002) published a paper in which they show that, in calves, enriched spermatogonia A cell suspensions can be cryopreserved successfully. As a functional assay, in vitro culture and transplantation in recipient mice were applied with success.
This study therefore aimed to evaluate different cryopreservation protocols on the basis of cell survival and recovery in mice. The protocol offering the best survival was evaluated further by germ cell transplantation.
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Materials and methods |
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We used the method as described by Romrell et al. (1976) with a few modifications in order to isolate the seminiferous tubules and to prepare a suspension of spermatogenic cells. The tunica albuginea from the dissected testes was removed and the tubuli seminiferi were transferred into a collagenase type IA solution (1000 IU/ml, Sigma, Belgium). The tubuli were then placed in a shaking warm water bath for 1 h at 37°C. Every 10 min, they were vortexed for a few seconds. After centrifuging for 5 min at 184 g, the supernatant was removed and a 0.025% trypsin (Sigma) solution containing 0.01% DNase (Sigma) was added for 15 min at 37°C. The trypsin reaction was halted by adding 4% fetal bovine serum (FBS; Life Technologies, Belgium) in DMEM/F12. The supernatant was removed after centrifuging for 6 min at 447 g and the pellet was resuspended in DMEM/F12. Cell viability was determined by a trypan blue exclusion test and the concentration of cells was counted in a Neubauer counting chamber. Cell suspensions with a concentration of 7 x 10617.5 x 106 cells/ml were used for further experiments.
Cryopreservation.
We compared two different cryoprotective agents in order to evaluate the survival of a testicular cell suspension and the spermatogonia in particular: 3 mol/l dimethyl sulphoxide (DMSO, Sigma) and 3 mol/ml of the less toxic ethylene glycol (EG, Sigma). These cryoprotective agents were added drop by drop to the cell suspension in a 1:1 (v/v) ratio so that the final concentration in each suspension was 1.5 mol/l cryoprotectant. The total cell suspension was pooled and divided over four vials. Each vial contained on average the cell suspension from two testes in 100 ml of DMEM/F12. The addition of the cryoprotectant was performed within 15 s without any incubation period. Freezing was initiated immediately after adding the cryoprotectant. This method was applied in all protocols.
Three independent series of experiments were performed. In a first series, the uncontrolled cryopreservation protocol as described by Avarbock et al. (1996) was compared with a controlled long cryopreservation protocol. In a second series, a DMSO-based shorter protocol was performed with two different thawing methods: thawing on ice water or thawing in a 37°C warm water bath. Finally, in a third series, this short protocol was performed with a different cryoprotective agent: EG. For all experiments, cell suspensions were frozen in cryotubes with a volume of 1.5 ml (Merck Eurolab, Belgium).
In the first series of experiments, the two different protocols were compared using the same cryoprotective agent, i.e. DMSO.
The cooling rate in the uncontrolled protocol was not mechanically controlled: the cryotube was placed in the freezer, which was equilibrated at 80°C, i.e. a cooling rate of 1°C/min. After 48 h, the cryotube was transferred into liquid nitrogen and stored until further use. For thawing, the cryotubes were plunged into a 37°C water bath, after which DMEM/F12 medium was added drop by drop until the volume was tripled in order to dilute the DMSO. After centrifugation (447 g, 6 min), the supernatant containing the cryoprotective agent was removed and the pellet resuspended in a medium used for transplantation, i.e. DMEM/F12 supplemented with 5% penicillin (Sigma), 5% streptomycin (Sigma) and 10% FBS.
In the controlled long protocol, the cooling rate was regulated by a biofreezer (Planer Kryo 10 series, Planer Products). The cryotubes were placed in the cooling chamber at room temperature. With a cooling rate of 5°C/min, the cryotubes were brought to the seeding temperature of 7°C. The temperature was stabilized for 15 min, during which the seeding occurred. The programme then continued at 0.3°C/min to 80°C, after which the cryotubes were transferred into liquid nitrogen for further storage. In this protocol too, thawing was done in a 37°C water bath and the cryoprotectant was diluted as described above.
The second series of experiments, the shorter protocol, which used DMSO as the cryoprotectant, started like the long protocol but, instead of cooling until 80°C, the slow cooling was halted at 40°C. The cryotubes were then plunged into liquid nitrogen and stored.
For thawing, we compared thawing in a 37°C water bath versus in ice water (rates of 250 or 50°C/min, respectively). The cryoprotectant was diluted as described earlier, after which both concentration and viability of the cell suspension were evaluated.
In a third series of experiments, this protocol was evaluated again, but this time using a different cryoprotectant. As cryoprotective agent, 3 mol/l EG was evaluated (the final concentration of cryoprotective agent being 1.5 mol/l). Thawing, cryoprotectant dilution and evaluation occurred as described earlier.
Transplantation experiments
Animals.
Donor cells were isolated from 6-day-old heterozygote transgenic mice with a C57Bl/6OlaHsd (Harlan, The Netherlands) and a B6129-TgR (Rosa 26) 26 Sor (The Jackson Laboratory, ME) background. We used these mice because they express the Escherichia coli Lac Z reporter gene in all stages of spermatogenesis in a heterozygous way. This means that the donor cells stain blue after incubation with 5-bromo-4-chloro-3-indolyl--D-galactoside (X-gal) (Sigma) (Zambrowicz et al., 1994
). In this way, we can detect the cells that are injected (Lac Z positive) in the donor mice testes, which are Lac Z negative.
In a second series of transplantation experiments, cryptorchid heterozygote ROSA 26 donor mice were used in order to obtain a higher proportion of stem cells in the testicular suspension (Shinohara et al., 2000a). The donor mice were made cryptorchid at the age of 810 weeks. After 24 months post-surgery, they were used as donors in our experiments.
As recipients, we used adult (46 weeks) F1 hybrid males (C57Bl/CbaCa) (Harlan, The Netherlands). In these recipients, endogenous spermatogenesis was suppressed by intraperitoneal busulphan injections (40 mg/kg; ICN Biochemicals) (Bucci and Meistrich, 1987). The recipient mice were 810 weeks old at the time of busulphan treatment. Transplantation was performed within 12 months after this treatment. For transplantation, they were anaesthetized with a mixture of ketamine 100 mg (75 mg/kg) and medetomidin hydrochloride 1 mg/ml (1.0 mg/kg).
The microinjection needles used for injecting the cell suspension into the seminiferous tubules were drawn on a pipette puller (Sutter Instruments, Model P-97, USA) from 30 µl glass micropipettes (Microcaps, Drummond Scientific Company, Broomall, USA). The diameter of the tip of the pipette was set at 60 µm and the tip was sharply bevelled (microbeveller MB3/T, Research Instruments, UK) to an angle of 30°. The injection procedure was based on that described by Ogawa et al. (1997
) with some minor modifications. The upper part of the efferent duct was fixed and the sharp pipette was then introduced manually through the wall of the efferent duct bundle in the direction of the rete testis. Once the pipette was in the correct position, the pressure on the cell suspension in the pipette was raised by means of an air injector (Research Instruments, UK). The cell suspension filled the tubuli seminiferi from the rete testis onwards. Only the tip of the micropipette was filled with 1% trypan blue. This was sufficient to visualize the injection of the cell suspension. In this way, we tried to minimize the toxic effect of trypan blue on the cell suspension. Between 50 and 80% of the testis surface was filled with 10 µl of cell suspension.
Three months after transplantation, the recipient males were killed by cervical dislocation and their testes were fixed for X-gal staining. The tunica was removed and the testes transferred into a 2.5% paraformaldehyde fixative at 4°C for 60 min. After fixation, the testes were washed three times (30 min each time) in a phosphate-buffered saline solution containing 2 mmol/l CaCl2 (Sigma), 2 mmol/l MgCl2 (Sigma), 0.3% glucose (Sigma) and 0.3% fructose (Sigma) (SPM). Then the testes were incubated for 1624 h at 37°C with SPM containing 1 mg/ml X-gal. A second fixation in 10% paraformaldehyde was performed for 24 h. Dark blue coloured tubules were considered positive for X-gal staining and proved that injected stem cells repopulated the tubuli seminiferi of the acceptor mice. At least two different positive tubules had to be observed to consider injection successful. Positive controls (testis from a heterozygote male) were always included in the evaluation. The macroscopic findings were confirmed by histological assessments as described by Brinster and Zimmermann (1994).
Statistics
The cryopreservation results were analysed using a Wilcoxon test (paired results) or a MannWhitney test (unpaired results). Viability and cell concentration before freezing were considered as being 100%, to compare viability and recovery after freezing in all conditions.
The transplantation results were analysed using a 2 test (Yates corrected). The medians of positive tubules were analysed with a MannWhitney test.
P-values below 0.05 were considered significant.
Internal review board approval
The Animal Care and Use Committee at the Brussels Free University approved all experiments in this study.
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Results |
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After freezing with DMSO and thawing in ice water, 87.5% of the original cell concentration was recovered compared with 84.9% (P = NS) when thawing was done in a 37°C water bath. However, the viability of the cells resulting from these two protocols was different: thawing on ice resulted in 48.6% viability, whereas thawing in a 37°C water bath resulted in 57.6% viability (P < 0.05).
Series 3: controlled short EG protocol.
In this third series, we evaluated EG as a cryoprotective agent in the short controlled protocol. We also evaluated two different thawing conditions, i.e. slow and rapid thawing. Thawing occurred either in ice water or in a 37°C water bath.
When EG was used as a cryoprotectant, thawing in ice water resulted in a 95.9% recovery of the original cell suspension and 41.7% viability. After the 37°C thawing, 67.0% of the original cell population was recovered and 67.9% excluded trypan blue and was considered viable (P < 0.05) (both results).
Transplantation
The viability and functional capabilities of the stem cells frozen according to our best protocol were evaluated further through germ cell transplantation of a whole cell suspension containing stem cells to restore fertility in sterile recipient mice.
Normal heterozygote ROSA 26 mice as donor.
In a first series of transplantation experiments in which non-cryptorchid donors were used, three conditions were evaluated. In a first group, the control group, 10 recipients received freshly prepared cell suspension for transplantation. In 13 out of 20 testes, the injection procedure was successful. In 12 out of the 13 successfully injected testes (92%), spermatogenesis could be reinitiated. A total of 57 positive tubules were found in these 12 testes. In a second group, 17 recipients received frozenthawed cell suspensions obtained after the uncontrolled protocol (DMSO group). In 25 testes of these 17 animals, the injection procedure was successful; however, in none of the injected testes was spermatogenesis eventually observed. In a third group, cell suspensions recovered after freezing with the EG protocol were transplanted into the 20 recipient animals. In 28 testes, the injection procedure was successful but again in none of the testis was spermatogenesis eventually observed.
These results are shown in Table II.
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Discussion |
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Since the introduction of germ cell transplantation of frozen testicular cell suspensions (Avarbock et al., 1996), cryobanking of testicular tissue or testicular cell suspensions of prepubertal boys may be a solution to this problem. In this study, we aimed at exploring whether more controlled cryopreservation protocols than the one initially reported by Avarbock et al. (1996
) may improve survival and better preserve the functional capacity of the testicular cell suspensions, including the stem cells.
During controlled freezing, a slower cooling rate is used in order to avoid intracellular ice formation, which is likely to damage the cells. Slow cooling leads to a better cell dehydration (Mazur, 1990), but, on the other hand, this causes an increase of intracellular and extracellular solute concentrations (Mazur, 1970
), stressing the cell membrane by extreme shrinkage (Mazur and Rigopoulos, 1983
). The so-called solution effects are also extremely important in cryo-injury: a decrease in temperature results in a decline of intracellular water volume and eventually higher concentrations of intra- and extracellular solutes and their precipitation. The high concentrations of electrolytes produced by freezing affect membrane lipids so as to make cells leaky. This causes osmotic shock because of the water inflow during thawing (Lovelock, 1953a
,b). The optimal cooling rate for testicular stem cells in particular has not yet been studied but, according to the size of the cell and the nucleus/cytoplasm ratio, these cells compare with lymphocytes, which have an optimal cooling rate of 10°C/min (Thorpe et al., 1976
). After some disappointing preliminary experiments (data not shown), we decided to cool at the slower rate of 0.3°C/min until a temperature of 80°C was reached. This rate is closer to the optimal cooling rate of 1°C/min as for haematopoietic stem cells frozen with 1.25 mol/l glycerol in balanced salt solutions as described by Mazur (1970
). This controlled cooling method provided better results in terms of survival than the uncontrolled method. While viability after uncontrolled freezing was
30%, similar to the viability cited by Avarbock et al. (1996
), this increased to 48% after controlled freezing.
Our results further show that cooling to 80°C is unnecessary and that with a shorter protocol to 40°C a higher proportion of cells were recovered without loss of viability. However, shortening the dehydration phase in the 40°C protocol may induce intracellular ice crystals, too small to cause direct injury to the cells, but which may recrystallize to larger damaging crystals if the thawing rate is too slow (Mazur, 1970). Therefore, in the next experimental series, two different thawing methods were tested. Thawing by plunging the vials in a 37°C water bath yielded better results than thawing in ice water, which corroborates the above hypothesis.
In a subsequent series, we tested an alternative cryoprotectant to DMSO, i.e. EG. DMSO is a widely used and known cryoprotectant, but it is a slow penetrating molecule. EG penetrates the cell much faster and is less toxic than DMSO (Hee-Jun et al., 2002). EG may thus reduce osmotic stress to the cells because a fast-penetrating cryoprotective agent replaces intracellular water more rapidly. We found the recovery of viable cells after cryopreservation with EG to be comparable with that with DMSO in the controlled short protocol.
We then wanted to investigate whether freezing and thawing with EG may also have a favourable effect on the functional capacity of the frozenthawed cells compared with DMSO. In order to examine this functional capacity, frozenthawed testicular cell suspensions were injected into the testes of infertile recipient mice via the rete testis.
After transplantation of frozenthawed cell suspensions, a few recipients exhibited reinitiated spermatogenesis when cryptorchid donors were used. However, the proportion of mice showing spermatogenesis was lower compared with fresh controls. This may indicate that the functional capacity of the stem cells may be seriously damaged by the freezingthawing cycle despite the acceptable viability of the testicular cell suspension as observed after thawing.
These results also stress that a further optimization of our cryoprotocols is mandatory. Other cryoprotectants may be used for this purpose. A review paper by Hovatta (2001) mentions a study by Huhtanen and co-workers in which six different freezing and thawing protocols were compared for both mouse and human testicular cell suspensions. The best cell survival was obtained by slow-programmed freezing and with propanediol and sucrose as cryoprotective agents (65% survival of mouse cells and 60% survival of human cells); however, no detailed methods or results have been published so far and no information on the functional capacity of these cells is available.
Also Brook et al. (2001) used a similar study design in order to evaluate the cryopreservation of both human and murine germ cells. Different cryoprotective agents and different cooling rates were compared. They found that the cryoprotectant used had no effect on the viability of human testicular cells after cryopreservation. They also found that the cooling rate did affect the viability. Cell death increased with cooling rates
0.4°C/min. Again, they did not apply a functional test in order to assess survival of the stem cells.
In a recent elegant publication, Izadyar et al. (2002) have shown that sucrose as a non-permeating cryoprotectant protects stem cells against freezingthawing damage. This protective effect has also been reported in other cell populations, as well as in embryos (Van den Abbeel et al., 1994
).
In their study, Izadyar et al. (2002) cryopreserved isolated bovine type A spermatogonia. They found that cryopreservation in an MEM-based medium supplemented with 10% DMSO, 10% FCS and 0.07 mol/l sucrose and using a non-controlled-rate freezing protocol resulted in 70% cell viability after thawing. The frozenthawed bovine spermatogonia were able to survive in vitro and they retained their ability to proliferate. These authors did apply testicular germ cell transplantation (TGCT) as a functional assay and observed some colonization in mouse testes 23 months after transplantation. Spermatogenesis was not observed because of the limitations of their xenotransplantation model.
The addition of sucrose or another non-permeating sugar may thus be a method by which to improve cryopreservation of testicular cells for transplantation. The exact mechanisms by which these sugars protect cells from cryoinjury are not yet completely understood. The protection may have a colligative basis as proposed by Lovelock (1953a,b) or the sugars may have an effect on the physical state of the external frozen medium as recently proposed by Koshimoto and Mazur (2002
).
Apart from using a suboptimal cryoprotocol, the limited results after TGCT in our experiments may be explained by other factors.
Although overall viability of the testicular cell suspension was acceptable, the proportion of stem cells in these suspensions may still be very low. The absence of any restoration of spermatogenesis when non-cryptorchid donor mice were used may be indicative for this. In the mouse, only 0.03% of testicular cells are stem cells (Tegelenbosch and de Rooij, 1993). However, this proportion may be much higher in the human since the human has relatively more A spermatogonia as compared with the mouse.
We did not evaluate the presence of stem cells by the use of specific markers as was done by Brook et al. (2001) because some controversy still exists on these markers (von Schönfeldt et al., 1999
; Shinohara et al., 2000b
). As proposed by Brinster and Zimmermann (1994
), we rather preferred to assess the presence of functional stem cells by TGCT itself.
Because we used mixed testicular cell suspensions containing different testicular cell types, it may be possible that other damaged cells may have a toxic effect carried over on to the stem cells. Freezing a selected population of testicular stem cells would overcome this hypothetical problem. However, freezing isolated cell populations would probably require huge tissue volumes due to high cell loss during both the freezingthawing procedure and the isolation procedure.
Our results show that reinitiation of spermatogenesis from a frozenthawed testicular cell suspension is feasible. However, the findings of our study also show that although testicular cell suspensions containing stem cells can be frozen using different cryoprotectants, high survival rates do not guarantee preservation of the functionality of these cells. They clearly show that testicular stem cell transplantation should be part of the evaluation of testicular stem cell cryopreservation protocols.
Further optimization of cryopreservation protocols, combined with in vitro culture either before or after cryopreservation, may further improve the feasibility of restoring reproduction in young boys undergoing sterilizing cancer treatment.
In the future, cryopreservation protocols aiming at preserving tissue samples rather than cell suspensions should also be evaluated. It may indeed be attractive to cryopreserve tissue instead of suspensions because in some patients testicular tissue may contain malignant cells, and therefore a considerable risk exists that after transplantation malignancy may reoccur (Jahnukainen et al., 2001). Xenotransplantation of the frozenthawed tissue may one day circumvent this problem (Honaramooz et al., 2002
; Schlatt et al., 2002
; Shinohara et al. (2002
).
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Acknowledgements |
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References |
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Submitted on November 22, 2002; resubmitted on October 14, 2003; accepted on November 11, 2003.