Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods

We examined possible genotype effects on the survival of 8- to 16-cell mouse embryos isolated from four inbred strains (C57BL/6N, BALB/cAnN, DBA/2N, and C3H/HeN), a outbred stock (ICR), and various crosses after cryopreservation by vitrification or conventional slow freezing using glycerol solutions. The rates of in vitro development of C57BL/6N, BALB/cAnN, C3H/HeN, and ICR embryos to expanded blastocysts ranged from 86% to 94% after slow freezing and 85% to 97% after vitrification. The cryopreservation method did not significantly influence in vitro embryo survival after thawing (P >0.05). Although genotype significantly influenced the in vitro survival of embryos (P = 0.008), this presumably resulted from an increased difficulty in assessing the quality grade of C3H/HeN embryos prior to cryopreservation. The rates in vivo development of C57BL/6N, BALB/cAnN, C3H/HeN, DBA/2N, and ICR embryos to normal day 18–19 fetuses ranged from 19% to 64% after slow freezing and from 18% to 63% after vitrification. The in vivo development of cryopreserved embryos was significantly influenced by cryopreservation method and genotype (P = 0.01 and P = 0.001, respectively). Vitrification yielded significantly higher rates of in vivo development than that after slow freezing (P > 0.05). In vivo development rates of DBA/2N and ICR♀ X B6D2F1 ♂ embryos after cryopreservation were significantly higher than that of embryos from BALB/cAnN and C3H/HeN mice (P < 0.05). These results indicate that parental genotype exerts little or no effect on the ability of embryos to develop in vitro after vitrification or slow freezing. Differences in the ability of cryopreserved embryos to develop normally in vivo may reflect inherent genotype related differences in their post-implantation developmental potential and not their sensitivity to cryoinjury. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Evaluation of sheep ovarian tissue cryopreservation with slow freezing or vitrification after chick embryo chorioallantoic membrane transplantation

The aim of our investigations was to compare the effectiveness of two methods for cryopreservation of sheep ovarian tissue, slow freezing and vitrification. The quality of cryopreserved tissues was evaluated after 5 days of thawing and chorioallantoic membrane (CAM) transplantation. Follicular structure, stromal integrity and neovascularization were assessed. The areas of fibrosis and necrosis were measured using MICROVISIBLE software, and proliferation was assessed with Ki-67 immunostaning. After 5 days of culture, the proportion of primordial follicles decreased, whereas the primary and intermediary follicles increased insignificantly (p > .05). Only necrosis in the vitrified culture group increased significantly (p < .05). It was established also that 5 days CAM culture was not suitable methodology for detection of folliculogenesis. Follicular quality decreased after culture, but was better in fresh and slow frozen tissues than after vitrification (p < .05). Cellular proliferative activity fell, but it preserved to some extent in all groups. In conclusion, follicles was preserved better in grafted tissue after slow freezing than vitrification and stroma was more susceptible to ischemia in vitrified rather than conventional freezing in this view. Vitrification may not be a suitable alternative to the slow freezing.     

Parasite cryopreservation by vitrification

Parasitic protozoa and helminths and parasitic/vector insects each have distinct requirements for cryopreservation. Most parasitic protozoa respond to cryopreservation stresses similarly to other single cell suspensions, but few species are currently routinely cryopreserved by protocols specifically designed for vitrification. With slow equilibrium cooling, some protozoa osmotically dehydrated by solutes concentrated in the residual unfrozen fraction will survive by vitrifying. Several species of helminths, together with insect embryos cannot be cryopreserved by slow cooling protocols and have an absolute requirement for vitrification. Studies incorporating slow cooling and stepped cooling of both protozoa and helminths, particularly the intraerythrocytic stages of malaria and the schistosomula larvae of Schistosoma mansoni have aided in the design of vitrification protocols for parasites. For helminths, the most widely used cryopreservation protocol, originally successful for cryopreserving S. mansoni schistosomula, consists of the addition of ethanediol in two steps, followed by rapid cooling (approximately 5100 degrees C min(-1)) to -196 degrees C. This technique exploits the temperature-dependent differential in permeability of the cryoprotectant additive (CPA) to first permeate into the organism at 37 degrees C followed by a dehydration-mediated internal CPA increase in concentration resulting from incubation in a second higher CPA concentration at 0 degree C. Samples are rapidly warmed/diluted (approximately 14,000 degrees C min(-1)) to recover the organisms from liquid nitrogen storage. Variations on this technique have also been successful in cryopreserving the larvae and adult worms of filariae, muscle stage larvae of Trichinella spp., the infective stages of gastro-intestinal nematode parasites and insect embryos. Other protocols where the dehydration step precedes CPA addition have been used to cryopreserve entomogenous nematode larvae by vitrification. Techniques that utilize high concentrations of CPA cocktails and slower cooling, developed for the vitrification of mammalian embryos, have been applied to the cryopreservation of parasitic protozoa, but with limited success to date. Where cryopreservation by classical slow cooling methods is possible, vitrification has enhanced the levels of survival obtained. And vitrification has enabled the successful cryopreservation of those parasitic species not able to be cryopreserved by traditional methods. Since a limited number of parasitic organisms has been cryopreserved using vitrification protocols, there is considerable scope for further improvement in the cryopreservation techniques used for many parasitic species.     

A simple vitrification technique for sheep and goat embryo cryopreservation

The aim of this study was to evaluate pregnancy and embryo survival rate of vitrified in vivo produced Merino sheep and Criolla goat (morulae and blastocysts) embryos, using the plastic tips of micropipettes, as containers (Cryo-tips). The embryos were exposed, at room temperature, to two successive equilibration solutions for a period of 5 min and then to a vitrification solution (VS) for 30 s. Then embryos were then loaded in 1 μl VS, into a plastic micropipette tip, and plunged into liquid nitrogen. On thawing, the embryos were warmed (37 °C) and placed into cryoprotectant dilutions (three-step-process). In the ovine, the morula and blastocyst pregnancy rates (47.1% vs 50%) and embryo survival rates (41.2% vs 50%) recorded were similar for both embryonic stages. Unlike the sheep, no pregnancies were recorded in goat vitrified/thawed morulae embryos, following transfer. However, in contrast, goats receiving blastocysts recorded high rates of pregnancy and embryo survival (64% and 64%, respectively). This technique allows for easy handling of cryopreserved embryos, is simple and efficient in both ovine embryo stages and also for goat vitrified blastocysts. The technique has definite potential application.     

Field trial to compare pregnancy rates of bovine embryo cryopreservation methods: vitrification and one-step dilution versus slow freezing and three-step dilution

We designed and conducted a field trial to obtain accurate pregnancy rates of Day 7 bovine embryos after vitrification in PB1 containing 6.5 M glycerol and 6% BSA (w/v) and one-step dilution in 1 M sucrose compared with controlled slow freezing in 1.5 M glycerol and three-step dilution. Embryos were collected from superovulated donor cows, and Grade 1 and 2 morulae and blastocysts were randomly assigned to each cryopreservation treatment group. Dutch farmers were solicited to participate in the field trial by an advertisement that offered cryopreserved embryos at subsidized cost. Within a period of 11 wk, one of six technicians visited 150 farms. Standard nonsurgical methods were used to transfer a total of 728 cryopreserved embryos. Recipient cows, mainly multiparous and of various breeds, the so-called "bottom-end" of the national herd, received embryos either 6, 7 or 8 d after standing estrus during natural estrous cycles. We compiled a database on 22 factors that may influence establishment of pregnancy in order to check randomization of each factor over cryopreservation treatment groups and embryo transfer technicians and to perform the statistical tests. Overall pregnancy rates were 44.5% (n = 393) for vitrified embryos and 45.1% (n = 335) for slowly frozen embryos. Pregnancy rates were not significantly different (ANOVA, P = 0.79 or Chi- square analysis, P = 0.88). The registered data confirm that all factors were randomly distributed over cryopreservation methods and technicians. Technician was not a significant source of variation in pregnancy rate (analysis of variance, P = 0.79). Although three technicians performed better with the one-step procedure and the other three performed better using the three-step method, the interaction between the technician and cryopreservation method was not significant (Tukey's test for nonadditivity, P = 0.13). Our results indicate that 1) vitrification and one-step dilution can be successfully used in the field without significant reduction in the pregnancy rate and 2) the pregnancy rate obtained using the "bottom-end" of the herd is satisfactory for practical application.     

Successful cryopreservation of mouse ovaries by vitrification

We developed a new method of cryopreservation of whole ovaries by vitrification using DAP213 (2 M dimethyl sulfoxide, 1 M acetamide, and M propylene glycol) as a cryoprotectant. Four-week-old C57BL/6 mice that underwent partial ovariectomy were orthotopically transplanted with cryopreserved or fresh ovaries (experimental or control group) isolated from 10-day-old green fluorescent protein (GFP)-transgenic mice (+/+). GFP-positive pups were similarly obtained from both groups by natural mating or in vitro fertilization (IVF) followed by embryo transfer, indicating that the cryopreserved ovaries by vitrification retain their fecundity. However, a statistically significant difference (P < 0.05) was found between both groups with respect to the following parameters: the number of GFP-positive pups born by natural mating/grafted ovary (0.8 +/- 0.3 for the experimental group versus 2.0 +/- 0.7 for the control group, mean +/- SEM), the number of collected oocytes by superovulation per mouse (7.0 +/- 1.7 for the experimental group versus 22.7 +/- 3.2 for the control group), the percentage of two-cell embryos obtained from GFP-positive oocytes by IVF (38.5% for the experimental group versus 90.0% for the control group). Histologically, normal development of follicles and formation of corpora lutea were observed in frozen-thawed grafts. However, estimated number of follicles decreased in frozen-thawed ovaries compared with fresh ovaries. Taken together, cryopreservation of the ovary by vitrification seems a promising method to preserve ovarian function, but further studies are required to overcome the possible inhibitory effects of this method on the growth of the ovarian graft.     

Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezing

The cryopreservation of oocytes is valuable for the preservation of women's fertility and might also be an interesting tool to preserve animal genetic biodiversity but it is not often used because of the very poor fertility recovered after thawing, especially in rabbit species. The objective of our study was to evaluate the effect of slow-freezing and vitrification on the structural integrity of ovulated rabbit oocytes, their ATP contents, and their developmental competence. Results show that, whatever the method is used, cryopreservation has a dramatic effect on the metabolic integrity, the structural integrity, and the developmental ability of the oocytes. Vitrification and slow freezing both impair the rabbit oocytes viability after thawing but the processes act differently. Further studies are needed to improve the cryopreservation techniques in rabbit species. Moreover, we underlined that morphology and maintenance of the structural integrity of the oocytes are not suitable enough to assess the potential for further development of cryopreserved M_II oocytes. The assessment of ATP metabolism allows efficient evaluation of the viability of the frozen or vitrified oocytes. It should be used in addition to parthenogenesis to better assess the potential for further development.     

Cryopreservation effects on canine sperm morphometric variables and ultrastructure: Comparison between vitrification and conventional freezing

Semen cryopreservation is an increasingly demanded technique in canids, particularly in order to preserve and spread high genetic value material. Sperm vitrification may represent an interesting alternative to costly and time consuming conventional freezing. The objective of this study was to evaluate the effect of sperm vitrification on sperm morphometry and ultrastructure compared to conventional freezing. Pools of nine beagle dogs were both frozen and vitrified. Computerized morphological parameters (length, wide, area and perimeter) and sperm ultrastructure, using scanning and transmission microscopy, were analysed in both fresh and in thawed/warmed samples. There were no differences (p > 0.05) between post-thaw and fresh morphometric variables of the sperm heads. However, cluster analysis revealed that sperm-heads turned out to be smaller after thawing (p < 0.05) in two of the four subpopulations. Vitrification-warming process led to an overall increase in sperm-head size. Furthermore, the sperm head size increased after warming in two subpopulations (p < 0.05). In conclusion, the variations in the sperm head area depended on the cryopreservation procedure (conventional freezing or vitrification). Conventional freezing tended to decrease the head dimensions, at least in some subpopulations, and vitrification led to an overall increase in the sperm head size. Decondensation of chromatin and plasma membrane blebbing in the head region was observed by transmission electron microscopy in several vitrified sperm, which might explain the increase of head dimensions detected by CASA-Morph system.     

Ovarian tissue cryopreservation by stepped vitrification and monitored by X-ray computed tomography

Ovarian tissue cryopreservation is, in most cases, the only fertility preservation option available for female patients soon to undergo gonadotoxic treatment. To date, cryopreservation of ovarian tissue has been carried out by both traditional slow freezing method and vitrification, but even with the best techniques, there is still a considerable loss of follicle viability. In this report, we investigated a stepped cryopreservation procedure which combines features of slow cooling and vitrification (hereafter called stepped vitrification). Bovine ovarian tissue was used as a tissue model. Stepwise increments of the Me₂SO concentration coupled with stepwise drops-in temperature in a device specifically designed for this purpose and X-ray computed tomography were combined to investigate loading times at each step, by monitoring the attenuation of the radiation proportional to Me₂SO permeation. Viability analysis was performed in warmed tissues by immunohistochemistry. Although further viability tests should be conducted after transplantation, preliminary results are very promising. Four protocols were explored. Two of them showed a poor permeation of the vitrification solution (P1 and P2). The other two (P3 and P4), with higher permeation, were studied in deeper detail. Out of these two protocols, P4, with a longer permeation time at −40 °C, showed the same histological integrity after warming as fresh controls.     

Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability

Human spermatozoa can be successfully cryopreserved avoiding the use of cryoprotectants through vitrification at very high cooling rates (up to 7.2 x 10(5) degrees C/min). This is achieved by directly plunging a copper cryoloop loaded with a sperm suspension into liquid nitrogen. After storage, vitrified spermatozoa are instantly thawed by melting in an agitated, warm medium. The goal of the present study was to compare the quality of spermatozoa cryopreserved using this rapid vitrification method with that of spermatozoa cooled relatively slowly by preexposure of the loaded cryoloop to liquid nitrogen vapor (-160 degrees C) with speed in the range 150-250 degrees C/min) before immersion into liquid nitrogen. Both cooling modes led to comparable results in terms of the motility, fertilization ability, and DNA integrity of the warmed spermatozoa. In both cases, instant thawing by melting in a warm medium was essential for successful cryopreservation. Our findings suggest that optimal regimes for the cryoprotectant-free cryopreservation of spermatozoa need not be restricted to very fast cooling before storage in liquid nitrogen, a wide range of cooling rates being acceptable. Herein, we discuss the implications of this finding in the light of the physics of extra- and intracellular vitrification.     

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6 M concentrations compared to only 1 M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.     

Improved cryopreservation by diluted vitrification solution with supercooling-facilitating flavonol glycoside

The effect of kaempferol-7-O-glucoside (KF7G), one of the supercooling-facilitating flavonol glycosides which was originally found in deep supercooling xylem parenchyma cells of the katsura tree and was found to exhibit the highest level of supercooling-facilitating activity among reported substances, was examined for successful cryopreservation by vitrification procedures, with the aim of determining the possibility of using diluted vitrification solution (VS) to reduce cryoprotectant toxicity and also to inhibit nucleation at practical cooling and rewarming by the effect of supplemental KF7G. Examination was performed using shoot apices of cranberry and plant vitrification solution 2 (PVS2) with dilution. Vitrification procedures using the original concentration (100%) of PVS2 caused serious injury during treatment with PVS2 and resulted in no regrowth after cooling and rewarming (cryopreservation). Dilution of the concentration of PVS2 to 75% or 50% (with the same proportions of constituents) significantly reduced injury by PVS2 treatment, but regrowth was poor after cryopreservation. It is thought that dilution of PVS2 reduced injury by cryoprotectant toxicity, but such dilution caused nucleation during cooling and/or rewarming, resulting in poor survival. On the other hand, addition of 0.5 mg/ml (0.05% w/v) KF7G to the diluted PVS2 resulted in significantly (p < 0.05) higher regrowth rates after cryopreservation. It is thought that addition of supercooling-facilitating KF7G induced vitrification even in diluted PVS2 probably due to inhibition of ice nucleation during cooling and rewarming and consequently resulted in higher regrowth. The results of the present study indicate the possibility that concentrations of routinely used VSs can be reduced by adding supercooling-facilitating KF7G, by which more successful cryopreservation might be achieved for a wide variety of biological materials.     

Effective surface-based cryopreservation of human embryonic stem cells by vitrification

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing.Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.     

Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification

We cryopreserved infantile rat ovaries by vitrification and assessed their viability by autotransplantation. Hemilateral ovarian transplantation was performed on rats on postnatal Days 10 to 12. The left ovary of each rat was dissected out, cryopreserved by vitrification using a modified vitrification solution (VS1), and then autotransplanted under the capsule of the right kidney. The right ovary of each rat was removed. For the control, the left ovary was dissected out from each rat and was immediately transplanted by the same procedure, without cryopreservation. Rats were nursed until weaning, and then the day of vaginal opening, estrous cyclicity from the day of vaginal opening until postnatal Day 84, and histology of ovarian grafts at postnatal Day 84 were examined. The time course of development of endocrine function of cryopreserved grafts was similar to that of fresh grafts. In ovarian transplants recovered on postnatal Day 84, antral follicles and corpora lutea (CL) were observed in addition to small follicles, although the number of antral follicles in cryopreserved grafts was smaller than in the fresh grafts. These results indicate that cryopreservation of ovarian tissue by vitrification can be used for the preservation of fertility and endocrine function of ovaries.     

Improved cryopreservation by diluted vitrification solution with supercooling-facilitating flavonol glycoside

The effect of kaempferol-7-O-glucoside (KF7G), one of the supercooling-facilitating flavonol glycosides which was originally found in deep supercooling xylem parenchyma cells of the katsura tree and was found to exhibit the highest level of supercooling-facilitating activity among reported substances, was examined for successful cryopreservation by vitrification procedures, with the aim of determining the possibility of using diluted vitrification solution (VS) to reduce cryoprotectant toxicity and also to inhibit nucleation at practical cooling and rewarming by the effect of supplemental KF7G. Examination was performed using shoot apices of cranberry and plant vitrification solution 2 (PVS2) with dilution. Vitrification procedures using the original concentration (100%) of PVS2 caused serious injury during treatment with PVS2 and resulted in no regrowth after cooling and rewarming (cryopreservation). Dilution of the concentration of PVS2 to 75% or 50% (with the same proportions of constituents) significantly reduced injury by PVS2 treatment, but regrowth was poor after cryopreservation. It is thought that dilution of PVS2 reduced injury by cryoprotectant toxicity, but such dilution caused nucleation during cooling and/or rewarming, resulting in poor survival. On the other hand, addition of 0.5 mg/ml (0.05% w/v) KF7G to the diluted PVS2 resulted in significantly (p < 0.05) higher regrowth rates after cryopreservation. It is thought that addition of supercooling-facilitating KF7G induced vitrification even in diluted PVS2 probably due to inhibition of ice nucleation during cooling and rewarming and consequently resulted in higher regrowth. The results of the present study indicate the possibility that concentrations of routinely used VSs can be reduced by adding supercooling-facilitating KF7G, by which more successful cryopreservation might be achieved for a wide variety of biological materials.     

Effect of conventional controlled-rate freezing and vitrification on morphology and metabolism of bovine blastocysts produced in vitro

This study compares the effects of conventional controlled-rate freezing and vitrification on the morphology and metabolism of in vitro-produced bovine blastocysts. Day 7 expanded blastocysts cultured in synthetic oviduct fluid with 5% fetal calf serum were frozen in 1.36 M glycerol, 0.25 M sucrose or vitrified in 25% glycerol, 25% ethylene glycol. Cell alterations and in vitro development were evaluated immediately after thawing or after 72 h. The effect of cryopreservation on inner cell mass and trophectoderm (TE) cell number as well as glucose, pyruvate, and oxygen uptakes, and lactate release by blastocysts were evaluated. Immediately after thawing, blastocysts showed equivalent cell membrane permeabilization after both cryopreservation procedures, while alterations in nuclear staining were more frequent in vitrified embryos. After culture, similar survival and hatching rates were observed. Both procedures decreased cell number immediately after thawing and after 72 h. However, the number of TE cells was lower in frozen embryos than in vitrified ones. In relation to this, frozen blastocysts showed a decrease in glucose, pyruvate, and oxygen uptake, although those parameters were not altered in vitrified embryos. An increased glycolytic activity was also observed in frozen embryos, indicating a stress response to this procedure.