Directional freezing is based on a simple thermodynamic principle where ice crystals are precisely controlled through the sample by regulating the velocity of the sample movement through the predetermined temperature gradient. Directional freezing permits a precise and uniform cooling rate in both small and large volume samples. Directional freezing was used for slow and rapid freezing, as well as for vitrification of oocytes and embryos using the minimum drop size technique. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The method enabled, for the first time, successful freezing of a whole ovary and freeze-drying of mammalian cells followed by thawing and transplantation and rehydration, respectively.
The multithermal gradient (MTG) technique is based on the principle of directional freezing (Fig. 1), which allows control over ice crystal propagation through a predetermined thermal gradient, by regulating the velocity of the sample movement through this temperature gradient. This method also enables incorporation of controlled seeding into the freezing process. When any liquid is cooled below its freezing point, it remains as a liquid, in an unstable super-cooled state, until freezing starts randomly at distributed nucleation sites and spreads throughout the entire volume of the liquid. When using the conventional method of freezing, ice grows with uncontrolled velocity and morphology and may disrupt and kill the cells in the samples. Ideally, ice crystal propagation should be such that it does not disrupt the cells or tissue. The laterally varying gradient used in our technology allows cooling to proceed at differing rates under varied temperature regimes, thereby facilitating full control over nucleation and ice crystal morphology. This technique allows very precise control of the cooling rate (0.01–1000°C/min) within a large volume. The MTG freezing apparatus can control ice crystal propagation by changing the thermal gradient (G) or the liquid-ice interface velocity (V), thereby optimizing the ice crystal morphology during the freezing process (Fig. 2) (Arav 1999). Thus, maximizing the survival rate of cells subjected to freezing and thawing requires careful control of the freezing process, that is, interface velocity. Using cryo-microscopy observation, we found that survival of sperm had a biphasic curve, where at a very slow velocity, ice will grow in a planar form, which will kill all cells. At higher velocities, ice crystals will form secondary branches and survival will increase; also, at 300 μm/s, ice will start to form ‘needle-like’ ice crystals, which will increase post-thaw motility and will permit very high survival depending on the space between ice crystals (Arav 1999; Arav et al. 2002a,b). Finally, at >3000 μm/s, directional solidification will not occur and survival will decrease. We used directional freezing for freezing and vitrification of many gametes, embryos, somatic cells, tissue and organs such as sperm of domestic and wild animals, oocytes and embryos vitrification, stem cells, ovarian tissue and whole ovaries (Saragusty and Arav 2011).
Directional Freezing of Semen from Domestic Species
In a field study, (Arav et al. 2002a,b; Saragusty et al. 2009a) using the directional freezing for bull semen, we demonstrated that it is possible to perform two freeze/thawing cycles of the same sample without compromising their fertilization and conception rate. This experiment was repeated with rabbit semen (Si et al. 2006). Double freezing of bull semen has been used for progeny tests and cattle breeding and also for sorting semen by sex (X, Y sorting) after freezing and thawing (Rudriguez-Martinez 2012). Directional freezing of sex-sorted sperm (dolphins) showed very good results (O’Brien and Robeck 2006). This concept is commercially applied today as a regular basis for sex sorting of bulls semen in the UK and Japan (Cogent UK). The advantages of large volume freezing are apparent when one has to consider the storage space and cost of large number of samples under liquid nitrogen over an extended interval. We then used the MTG where insemination with large volume is required, such as with stallions semen, which showed that ejaculates frozen by MTG had a post-thaw progressive linear motility (PLM) of 50 ± 2%, compared with 37 ± 1% for conventional freezing PLM (Saragusty et al. 2007). Also with pigs (Arav et al. 2002a,b), high post-thaw motilities of 45% were obtained using the MTG with a large volume sample. We were also applied the technique on avian and ram sperm (Arav et al. 2002a,b) with excellent post-thaw motility. Freezing large volumes of pulled buck semen showed a very high post-thaw motility and more than 40% pregnancy rates following cervical insemination (Gacitua and Arav 2005).
Directional Freezing of Semen from Wild Animals
One of the first comparative studies on wild animals spermatozoa were on dolphin sperm. Post-thaw results showed that within the same sperm type, directional freezing was superior (p < 0.05) to conventional techniques for maintaining motility and in immediate post-thaw viabilities (O’Brien and Robeck 2006). Another comparative study was carried out on sperm from killer whales between conventional freezing (using straws) and directional freezing (using hollow tubes) (Robeck et al. 2011). The post-thaw results showed that directional freezing was superior to conventional freezing in all parameters of motility and viability. For example, best total motility achieved post-thawing in conventional freezing was 54.1%, whereas with directional freezing, it was 77.7%. The same authors performed also an important study on beluga dolphins. They showed that when directional freezing and trehalose were used, the post-thaw results were statistically better than those achieved with conventional freezing (O’Brien and Robeck 2010). The directional freezing technology developed for beluga semen was used for artificial insemination (AI) on seven beluga females, which resulted with a pregnancy and a delivery of twin beluga calves with one surviving (Robeck et al. 2010). This was the first beluga ‘baby’ born from frozen-thawed semen. We performed a study on semen collected post-mortem from three species of gazelle (Saragusty et al. 2006). The semen was frozen in 8 ml samples using the hollow tubes and the directional freezing device. The post-thaw results showed that the time of semen collection after death can be also crucial. In fact, semen collected from two Gazelles gazelle 7 and 24 h after death, respectively, had a post-thaw motility rate of 63 ± 3.21 vs 45.55 ± 1.97%, respectively. For the Gazelle dorcas, post-thaw motility was 57.25 ± 4.77% and semen was frozen 6 h after death. In addition, better sperm morphologies and intact acrosome rates were also observed. Conversely, frozen semen collected from Gazelle acaiae showed low post-thaw motility of only 15%, which might be due to a long interval between time of death and sperm cryopreservation. Artificial insemination is very important for reducing inbreeding in elephants. To date, artificial insemination in elephants was only performed with fresh-chilled sperm. Most likely the fact that no cryopreserved sperm was reported being used for AI is because of the low post-thaw semen quality. Freezing of sperm was carried out using directional freezing at a large volume (Saragusty et al., 2007). The results showed post-thaw motility of 57.2% and acrosome integrity was 57.1%, and 52% of the cells exhibited normal morphology. The same group also performed a study on hippopotamus (Saragusty et al. 2010), freezing sperm using directional freezing in hollow tubes of 2.5 ml (for post-thaw evaluations) and 8 ml samples (for storage). Sperm were evaluated after dilution, after chilling and after freeze–thawing. Post-thaw motility was between 8.5 ± 3.13 and 18.86 ± 7.96%, depending on the extender used. More than 75% of sperm showed intact acrosome after thawing, with 50–66% of the cells showing intact morphology. Semen from European brown hare was collected via electroejaculation. Semen was cryopreserved using directional freezing, 2 ml samples frozen in hollow tubes (Hildebrandt et al. 2009). Fresh and thawed semen were both used for AI. Conception rate, pregnancies delivered and fertility rate were not different between the two groups, even though the post-thaw motility of the frozen semen was lower (46.9 ± 5.8%) compared with that of fresh semen (91.6 ± 2.4%). Another study was performed to compare the sperm from rhesus macaque cryopreserved with conventional freezing and directional freezing (Si et al. 2010). Sperm was assessed upon thawing for motility and acrosome integrity as well as used for in vitro fertilization. The results between the freezing methods were not statistically different, although the directional freezing frozen-thawed sperm yielded higher blastocysts formation. Finally, the most important result using this technique was the birth of young Rhino born after AI with sperm cryopreserved by directional freezing (Hermes et al. 2009). Sperm was collected from 35- to 36-year-old rhinoceros in the UK, frozen by directional freezing system and shipped to Hungary. Two AI attempts were performed using different concentrations. The first dose contained 135 million motile cells did not give result in pregnancy, whereas the second of 500 million motile cells resulted in a live offspring. This technology could allow the cryopreservation of sperm worldwide for future use in AI.
Directional Vitrification of Oocytes and Embryos – the Minimum Drop Size Technique
The device for directional vitrification was described 20 years ago (Arav 1992) and is based on the cryomicroscope developed by Rubinsky (Rubinsky et al. 1991). The cryomicroscope stage with a glass slide consists of two bases with the temperatures being set to −180°C to +40°C, which create a temperature gradient (G) of 220°C/mm between the two bases. The slide is pushed at a constant velocity (V) from the warm to the cold base. The cooling rate (B) is calculated as a product of the multiplication of G and V. The oocytes and embryos were placed with minimum drop size (MDS) of 0.07 μl on the slide and cooled rapidly to −180°C. In this device, a relatively high cooling rate can be achieved, for example, when G = 220°C/mm and V = 1mm/s, the calculated cooling rate is CR = 13 200°C/min. The MDS and the directional cooling stage were used successfully for vitrification with low cryoprotectant concentrations (17.5% (v/v) propylene glycol, 20% (v/v) foetal calf serum, 0.1 m sucrose and 40 mg/ml anti-freeze proteins) of bovine and ovine embryos (Arav 1992; Arav et al. 1994).
Whole Ovary Freezing
For many years, attempts to cryopreserve large organs have been unsuccessful because of the problems associated with heat transfer (Arav et al. 2005) and the non-homogeneous rate of cooling between the core and the periphery of the organ. To overcome these problems, two methodologies have been proposed: the directional freezing (Arav et al. 2005) and the vitrification methodology (Courbiere et al. 2009). Vitrification, which has been proposed for the first time for whole organs such as kidneys, has many drawbacks: chemical toxicity and osmotic shock following exposure to high concentration of cryoprotectants (>50%). Fractures of the organ could be caused by vitrification and warming procedures (Fahy et al. 1990), and devitrification if the storage temperature is above the glass transition temperature. The directional freezing methodology has solved the heat transfer problem by maintaining a uniform cooling rate through the entire organ. The uniform cooling rate was made possible by building our freezing device based on two principles: (i) large mass of conductive material that enables rapid removal of latent heat being released owing to crystallization in the freezing front, and (ii) as the sample is moving through the temperature gradient at a controlled velocity, we are able to create a very precise freezing front, which results with a uniform cooling rate throughout the sample (Gavish et al. 2008). The alternative for the whole ovary freezing is the cryopreservation of cortical slices by slow freezing or vitrification (Huang et al. 2008). However, these methods have major technical limitation because upon thawing and grafting of the cortical tissue, there is a vast loss of follicles during the period of ischaemia and before the tissue becomes revascularized (Arav and Natan 2009), compromising ovarian function. The survival for a long time (6 years) of frozen transplanted whole ovary in a large animal successfully demonstrates the potential application of this technique to preserve human fertility (Arav et al. 2010). The use of sheep ovaries as a model for humans is very relevant, because the sheep ovaries have strong similarities with ovaries of young women, displaying a high cortical primordial follicles density. This is the first time that long-term follow-up of transplanted whole ovary where a large number of follicles were observed. Cryopreservation of intact human ovaries with its vascular pedicle is not associated with any signs of apoptosis or ultrastructural alterations in any cell types, confirming that whole organ ovary transplantation may be a viable option in the future (Arav and Natan 2009).
For many years, scientists have been aspiring for the lyophilization of cells for the simplicity it allows in the storage and transportation of dried materials. In a recent publication, we reported that ovine freeze-dried granulosa cells and lymphocytes, stored for 3 years at room temperature, maintained their DNA integrity and ability to produce normal embryos after nuclear transfer (Loi et al. 2008). Additionally, we have performed freeze–thawing and freeze-drying experiments with mononuclear cells derived from mice bone marrow, which were transfused into sub-lethally irradiated mice. We have seen increased survival following injection of freeze-dried and rehydrated cells. The freeze–thawing experiments were performed on blood from male mice that were injected into irradiated female mice. One month after the injection of the frozen-thawed cells, blood was taken from the female mice and PCR was performed, showing the presence of Y chromosome. These preliminary findings suggest that the cells can incorporate into the bone marrow and form new white blood cells. We have then continued on improving the viability and functionality of the dried cells after rehydration by adding an antioxidant to the Lyophilization solution and by changing the freezing and drying parameters. This was carried out on mononuclear cells collected from human umbilical cord blood units, where we have achieved a viability rate of 85% after freeze-drying and rehydration with distilled water. Furthermore, when colony forming unit assay was carried out, there was no difference between the numbers of colonies formed before freezing and those formed after freeze-drying and rehydration (Natan et al. 2009). This assay has shown that the hematopoietic stem cells have maintained their capability to differentiate into different blood cells following freeze-drying. This was the first report that shows how cells that were subjected to complete lyophilization followed by rehydration have maintained not only their viability, but also their functionality.
Conflicts of interest
The authors have no conflicts of interest to declare.