Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system.

STUDY QUESTION: What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? SUMMARY ANSWER: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. WHAT IS KNOWN ALREADY: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers. STUDY DESIGN, SIZE, DURATION: Non-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm(3)) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. MAIN RESULTS AND THE ROLE OF CHANCE: Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P < 0.05) by Week 3, but diameters in vitrified follicles were smaller (P < 0.05) by Week 6 (566 ± 27 µm) than those of the fresh follicles (757 ± 26 µm). Antrum formation rates were lower (P < 0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture. LIMITATIONS, REASONS FOR CAUTION: Only in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. WIDER IMPLICATIONS OF THE FINDINGS: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. STUDY FUNDING/COMPETING INTEREST(S): National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep.

Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called "ultra-rapid cooling", and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species.

Deep supercooling, vitrification and limited survival to -100{degrees}C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae.

Larvae of the freeze-avoiding beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) in Alaska have mean supercooling points in winter of -35 to -42 degrees C, with the lowest supercooling point recorded for an individual of -58 degrees C. We previously noted that some larvae did not freeze when cooled to -80 degrees C, and we speculated that these larvae vitrified. Here we present evidence through differential scanning calorimetry that C. c. puniceus larvae transition into a glass-like state at temperatures<-58 degrees C and can avoid freezing to at least -150 degrees C. This novel finding adds vitrification to the list of insect overwintering strategies. While overwintering beneath the bark of fallen trees, C. c. puniceus larvae may experience low ambient temperatures of around -40 degrees C (and lower) when microhabitat is un-insulated because of low snow cover. Decreasing temperatures in winter are correlated with loss of body water from summer high levels near 2.0 to winter lows near 0.4 mg mg(-1) dry mass and concomitant increases in glycerol concentrations (4-6 mol l(-1)) and thermal hysteresis. Finally, we provide direct evidence that Cucujus from Wiseman, Alaska, survive temperatures to -100 degrees C.

Normothermic blood perfusion of isolated rabbit kidneys. II. In vitro evaluation of renal function followed by orthotopic transplantation.

This study describes the use of a blood perfusion apparatus to assess the renal function of isolated kidneys. Eight fresh kidneys were obtained from healthy rabbits and perfused with blood at 36 degrees C for 2 hours. Rabbit blood was drawn and diluted to a hematocrit of 25%. The kidneys were evaluated for their capacity to support life in an autograft model. Blood and urine samples were taken at regular time intervals during kidney perfusion. Serum creatinine was measured in surviving rabbits after transplantation. Over the course of the perfusion, arterial pressure was maintained at 87.2 +/- 5.5 mm Hg. The renal blood flow (3.7 +/- 1.0 ml/min per g) and urine output (0.11 +/- 0.04 ml/min per g) were continuously monitored. Glomerular filtration rate (0.29 +/- 0.02 ml/min per g) and fractional reabsorption (FR) of sodium and glucose indicated appreciable tubular function (FR(Na) = 67.9 +/- 8.5%, FR(Glu) = 91.2 +/- 5.8%). Protein was excluded from urine at 99.8% +/- 0.1%. After transplantation, the peak creatinine was 6.8 +/- 3.2 mg/dl at 1.90 +/- 0.92 days for the seven surviving rabbits and was above 16 mg/dl for the only rabbit that died 4 days after operation. The level of free hemoglobin generated at the end of the perfusion (2.6% +/- 2.8%) was correlated with the postoperative peak creatinine (r2 = 0.84). Perfusion of seven additional kidneys by using the roller pump lead to a final hemolysis of only 0.34 +/- 0.14%. Kidneys transplanted after 2 hours of blood perfusion were able to resume normal function and support life. Hemolysis was a measurable stress factor causing delayed function of the kidney after transplantation. Introduction of a roller pump significantly reduced the hemolysis.

Permanent life support by kidneys perfused with a vitrifiable (7.5 molar) cryoprotectant solution.

BACKGROUND: Vitrification (glass formation) is a potential method for indefinite term organ preservation that eliminates all of the conventional problems of freezing and thawing. A 7.5 M mixture of cryoprotectants known as VS4 is sufficiently concentrated, in combination with applied pressure, to preclude ice formation entirely during cooling to below the glass transition temperature (about -125 degrees C), at which point vitrification takes place, arresting further changes over time. METHODS: Rabbit kidneys were perfused with VS4 according to three different protocols. The kidneys were evaluated using an autograft model with immediate contralateral nephrectomy. RESULTS: All three methods permitted long-term survival, but the best results were obtained when the highest concentrations were perfused at about -3 degrees C. Using the latter protocol, the survival rate was 10/10, serum creatinine returned to a normal baseline after transient elevation, other clinical chemistry results normalized, and no histological damage was apparent 3 weeks after autografting. CONCLUSIONS: The results described provide the strongest evidence to date that it may be possible to bank kidneys for unlimited periods in the absence of ice for later transplantation.

Vitrification enhancement by synthetic ice blocking agents.

Small concentrations of the synthetic polymer polyvinyl alcohol (PVA) were found to inhibit formation of ice in water/cryoprotectant solutions. Ice inhibition improved with decreasing molecular weight. A PVA copolymer of molecular weight 2 kDa consisting of 20% vinyl acetate was found to be particularly effective. PVA copolymer concentrations of 0.001, 0.01, 0.1, and 1% w/w decreased the concentration of glycerol required to vitrify in a 10-ml volume by 1, 3, 4, and 5% w/w, respectively. Dimethyl sulfoxide concentrations required for vitrification were also reduced by 1, 2, 2, and 3% w/w, respectively. Crystallization of ice on borosilicate glass in contact with cryoprotectant solutions was inhibited by only 1 ppm of PVA copolymer. Devitrification of ethylene glycol solutions was also strongly inhibited by PVA copolymer. Visual observation and differential scanning calorimeter data suggest that PVA blocks ice primarily by inhibition of heterogeneous nucleation. PVA thus appears to preferentially bind and inactivate heterogeneous nucleators and/or nascent ice crystals in a manner similar to that of natural antifreeze proteins found in cold-hardy fish and insects. Synthetic PVA-derived ice blocking agents can be produced much less expensively than antifreeze proteins, offering new opportunities for improving cryopreservation by vitrification.

Cryopreservation of the mammalian kidney. II. Demonstration of immediate ex vivo function after introduction and removal of 7.5 M cryoprotectant.

The objective of the present study was to determine whether rabbit kidneys could be perfused with a 7.5 M vitrification solution (VS4, which vitrifies under applied pressure) without loss of function. To answer this question, kidneys were perfused with VS4 using a computer-based machine to gradually raise and lower concentration and then attached to the aorta and vena cava of a perfusor rabbit using an apparatus that permitted renal blood flow and renal function to be measured. About half (6/13) of the kidneys so evaluated resumed substantial immediate function after a transient period of severely reduced blood flow. Loss of function did not occur if cryoprotectant concentration was limited to 3.8 M. The loss of function produced by VS4 could be partially reproduced by artificially limiting blood reflow in control kidneys to simulate the transiently low flows caused by VS4 exposure. These results provide the first evidence that both the parenchyma and the vascular system of a sensitive mammalian organ can survive exposure to a vitrifiable concentration of cryoprotectant.

Organ perfusion equipment for the introduction and removal of cryoprotectants.

A new system for perfusing organs with cryoprotective agents or other solutions is described. It represents the first system that combines the sophistication and versatility required for successful organ cryopreservation protocols with the experimental ease and reproducibility required for sustaining a long-term research or organ-processing effort with a high throughput of successfully perfused organs. Operation is simple, fully documented, and readily checked by visual means, and maintenance is not difficult. It appears that this system could be adapted for use with a wide variety of organ perfusion protocols and could lead to improved results and greater patient safety with less human effort.

Cryopreservation of the mammalian kidney. I. Transplantation of rabbit kidneys perfused with EC and RPS-2 at 2-4 degrees C.

The requirements of organ cryopreservation differ from those of conventional organ preservation. The encouraging results of Karow's group with dog kidneys transplanted after perfusion with more than 4 M dimethyl sulfoxide were based on an RPS-2 (renal preservation solution 2) vehicle solution, but transplantation of rabbit kidneys after perfusion with RPS-2 has not been reported. We evaluated RPS-2 in comparison to Euro-Collins solution (EC) using a modified technique for rabbit kidney autotransplantation and a computer-based organ perfusion machine designed for the introduction and removal of cryoprotective agents. Consistent success in rabbit kidney transplantation was found to depend on the anesthetic used, the hydration volumes administered, and direct ureter-to-ureter anastomosis. RPS-2 was found to be equivalent to EC for short-term (about 5 h) preservation by either perfusion or simple cold storage. However, good results with EC were associated with perfusion at 4 degrees C, recovery being significantly worse at 2 degrees C. In addition, we found that the solitary rabbit kidney is not able to fully compensate for the loss of the contralateral kidney, the result being persistent (to 3 weeks) mild elevation of serum creatinine, potassium, and calcium and persistent moderate reduction of serum phosphate. These results establish perfusates, perfusion conditions, transplantation techniques, computer-based perfusion control techniques, and a general clinical baseline that are permissive of further direct experiments on cryoprotectant introduction and removal.

Molecular nanotechnology.

Molecular nanotechnology involves the ability to manufacture objects to precise atomic specifications. A central postulate is that any structure that can be specified and that does not violate physical law can be built. Three pathways to molecular nanotechnology are proximate probe technology (the use of improvements of the scanning tunneling microscope, STM), biotechnology, and supramolecular chemistry. Combinations of these technologies appear particularly powerful. The biotechnological approach should make it possible to use in vitro translation systems to manufacture polymers containing at least 10 times as many different artificial monomers as there are natural amino acids. These polymers could further adsorb various other molecular devices, and the use of STMs should enable the complexes to be arranged into sophisticated machines, including molecular computers. The implications include pocket superautomated analyzers and the ability to base medical therapy on the biochemical individuality of specific patients.

The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats.

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1-1.5 degrees C/minute down to -40 degrees C at which point the vials were plunged into liquid nitrogen at -196 degrees C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival.

Short-term and long-term possibilities for interventive gerontology.

Worldwide demographic trends, including the aging of the human population and the steadily declining fertility rate in developed nations, are creating enormous economic pressures due to the ever-increasing demand for health care services for the elderly and the ever-decreasing ability of the young to pay for these services. An attractive cost-containment strategy is accelerated research on basic molecular mechanisms of aging and rapid clinical application of the results. This approach should result in maintenance of health and productivity over a longer fraction of the lifespan and thereby to a reduction in the ratio of health care expenditures to lifelong earnings. This strategy should lead to many important improvements in the human condition. Sooner or later, our armamentarium will be supplemented by powerful and useful tools from the field of molecular engineering. With proper care, both these developments may help us to live longer, healthier, happier lives.

Deuterium oxide, dimethylsulfoxide and heat shock confer protection against hydrostatic pressure damage in yeast.

Deuterium oxide, dimethylsulfoxide (Me2SO) and heat shock treatment were all significantly effective at baro-injury as measured by plating efficiency after decompression. The content of unfreezable cell water was observed to increase during heat treatment, and this increase was associated with increase in viability.

Rapid and uniform electromagnetic heating of aqueous cryoprotectant solutions from cryogenic temperatures.

Devitrification (ice formation during warming) is one of the primary obstacles to successful organ vitrification (solidification without ice formation). The only feasible approach to overcoming either devitrification or its damaging effects in a large organ appears at present to be the use of some form of electromagnetic heating (EH) to achieve the required high heating rates. One complication of EH in this application is the need for warming within a steel pressure vessel. We have previously reported that resonant radiofrequency (RF) helical coils provide very uniform heating at ambient temperatures and low heating rates and can be modified for coaxial power transmission, which is necessary if only one cable is to penetrate through the wall of the pressure vessel. We now report our initial studies using a modified helical coil, high RF input power, and cryogenic aqueous cryoprotectant solutions [60% (w/v) solution of 4.37 M dimethylsulfoxide and 4.37 M acetamide in water and 50% (w/w) 1,2-propanediol]. We also describe the electronic equipment required for this type of research. Temperatures were monitored during high-power conditions with Luxtron fiberoptic probes. Thermometry was complicated by the use of catheters needed for probe insertion and guidance. The highest heating rates we observed using catheters occurred at temperatures ranging from about -70 to -40 degrees C, the temperature zone where devitrification usually appears in unstable solutions during slow warming. We find that in this range we can achieve measured heating rates of approximately 300 degrees C/min in 30- to 130-ml samples using 200 to 700 W of RF power without overheating the sample at any point. However, energy conservation calculations imply that our measured peak heating rates may be considerably higher than the true heating rates occurring in the bulk of our solutions. We were able to estimate the overall true heating rates, obtaining an average value of about 20 degrees C/min/100 W/100 ml, which implies a heating efficiency close to 100%. It appears that it should be possible to warm vitrified rabbit kidneys rapidly enough under high-pressure conditions to protect them from devitrification.

Physical problems with the vitrification of large biological systems.

Vitrification is an attractive potential pathway to the successful cryopreservation of mature mammalian organs, but modern cryobiological research on vitrification to date has been devoted mostly to experiments with solutions and with biological systems ranging in diameter from about 6 through about 100 microns. The present paper focuses on concerns which are particularly relevant to large biological systems, i.e., those systems ranging in size from approximately 10 ml to approximately 1.5 liters. New qualitative data are provided on the effect of sample size on the probability of nucleation and the ultimate size of the resulting ice crystals as well as on the probability of fracture at or below Tg. Nucleation, crystal growth, and fracture depend on cooling velocity and the magnitude of thermal gradients in the sample, which in turn depend on sample size, geometry, and cooling technique (environmental thermal history and thermal uniformity). Quantitative data on thermal gradients, cooling rates, and fracture temperatures are provided as a function of sample size. The main conclusions are as follows. First, cooling rate (from about 0.2 to about 2.5 degrees C/min) has a profound influence on the temperature-dependent processes of nucleation and crystal growth in 47-50% (w/w) solutions of propylene glycol. Second, fracturing depends strongly on cooling rate and thermal uniformity and can be postponed to about 25 degrees C below Tg for a 482-ml sample if cooling is slow and uniform. Third, the presence of a carrier solution reduces the concentration of cryoprotectant needed for vitrification (CV). However, the CV of samples larger than about 10 ml is significantly higher than the CV of smaller samples whether a carrier solution is present or not.

Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

Cryoprotectant toxicity is a fundamental obstacle to the full potential of artificial cryoprotection, yet it remains in general a poorly understood phenomenon. Unfortunately, most relevant biochemical studies to date have not met the basic criteria required for demonstrating mechanisms of toxicity. A model biochemical study of cryoprotectant toxicity was that of Baxter and Lathe, which demonstrated that alteration of a specific enzyme (fructose diphosphatase, or FDPase) was the cause of impaired glycolysis after treatment with and removal of dimethyl sulfoxide (D). FDPase alteration by D was reported to be preventable by the simultaneous presence of amides. This protection could be due to a "counteracting solute" effect similar to that employed by nature, but we find no meaningful correlation between the general protein stabilizing or destabilizing tendency of the cryoprotectant medium and its toxicity. Baxter and Lathe postulated that the effect of D arises from hydrogen bonding between D and the epsilon amino groups of surface lysine residues on FDPase, and it was found that molecules which resembled this group could block the alteration induced by D, presumably by competing with lysine residues for association with D. However, we find that the interaction between D and lysine in the presence of water is actually thermochemically repulsive, and that the presence of formamide does not affect the interaction between D and lysine, implying no useful complex formation between formamide and D. We were also unable to demonstrate that the blocking compounds consistently reduce toxicity when added to D rather than substituting for D, contrary to predictions based on complex formation between blocking compounds and D. In summary, it seems that present concepts of cryoprotectant toxicity are in need of serious revision.

Some emerging principles underlying the physical properties, biological actions, and utility of vitrification solutions.

Vitrification solutions are aqueous cryoprotectant solutions which do not freeze when cooled at moderate rates to very low temperatures. Vitrification solutions have been used with great success for the cryopreservation of some biological systems but have been less successful or unsuccessful with other systems, and more fundamental knowledge about vitrification solutions is required. The purpose of the present survey is to show that a general understanding of the physical behavior and biological effects of vitrification solutions, as well as an understanding of the conditions under which vitrification solutions are required, is gradually emerging. Detailed nonequilibrium phase diagram information in combination with specific information on the tolerance of biological systems to ice and to cryoprotectant at subzero temperatures provides a quantitative theoretical basis for choosing between vitrification and freezing. The vitrification behavior of mixtures of cryoprotective agents during cooling is predictable from the behavior of the individual agents, and the behavior of individual agents is gradually becoming predictable from the details of their molecular structures. Progress is continuing concerning the elucidation of mechanisms and cellular sites of toxicity and mechanisms for the reduction of toxicity. Finally, important new information is rapidly emerging concerning the crystallization of previously vitrified cryoprotectant solutions during warming. It appears that vitrification tendency, toxicity, and devitrification all depend on subtle variations in the organization of water around dissolved substances.

The relevance of cryoprotectant "toxicity" to cryobiology.

Cryoprotective agents are essential for the cryopreservation of almost all biological systems. These additives, however, do not usually permit 100% survival after freezing and thawing, though from a theoretical point of view they should be able to fully suppress all known types of freezing injury. In view of the known biological and physicochemical effects of cryoprotectants, it is suggested that the toxicity of these agents is a key limiting factor in cryobiology. Not only does this toxicity prevent the use of fully protective levels of additive, but it may also be manifested in the form of cryoinjury over and beyond the cryoinjury due to classical causes. Evidence for this extra injury ("cryoprotectant-associated freezing injury") is reviewed. It is suggested that better suppression of toxicity is possible and will lead to advances in cryopreservation.

Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification.

The failure of complex mammalian organs, such as the kidney, to function following freezing to low temperatures is thought to be due largely to mechanical disruption of the intercellular architecture by the formation of extracellular ice. Classical approaches to the avoidance of ice formation through the imposition of ultra-rapid cooling and warming rates or by gradual depression of the equilibrium freezing point during cooling to -80 degrees C have not been adequate. An alternative approach relies on the ability of highly concentrated aqueous solutions of cryoprotective agents to supercool to very low temperatures. At sufficiently low temperatures, these solutions become so viscous that they solidify without the formation of ice, a process termed vitrification. When embryo suspensions are cryopreserved using conventional procedures, this supercooling behaviour allows intracellular vitrification, even in the presence of extracellular ice. We have therefore used mouse embryos to examine the feasibility of obtaining high survival following vitrification of both the intra- and extracellular solutions and report here that in properly controlled conditions embryos seem to survive in high proportions after cryopreservation in the absence of ice.