Abstract: Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol. In fact, both damaging mechanisms are important, their relative contributions depending on cell type, cooling rate, and warming rate. A consensus has developed that intracellular freezing is dangerous, whereas extracellular ice is harmless. If the water permeability of the cell membrane is known it is possible to predict the effect of cooling rate on cell survival and the optimum rate will be a tradeoff between the risk of intracellular freezing and effects of the concentrated solutes. However, extracellular ice is not always innocuous: densely packed cells are more likely to be damaged by mechanical stresses within the channels where they are sequestered and with complex multicellular systems it is imperative not only to secure cell survival but also to avoid damage to the extracellular structure. Ice can be avoided by vitrification--the production of a glassy state that is defined by the viscosity reaching a sufficiently high value (approximatly 10(13) poises) to behave like a solid, but without any crystallization. Toxicity is the major problem in the use of vitrification methods. Whether freezing is permitted (conventional cryopreservation) or prevented (vitrification), the cryoprotectant has to gain access to all parts of the system. However, there are numerous barriers to the free diffusion of solutes (membranes), and these can result in transient, and sometimes equilibrium, changes in compartment volumes and these can be damaging. Hence, the processes of diffusion and osmosis have important effects during the introduction of cryoprotectants, the removal of cryoprotectants, the freezing process, and during thawing. These phenomena are amenable to experiment and analysis, and this has made it possible to develop effective methods for the preservation of a very wide range of cells and some tissues; these methods have found widespread applications in biology and medicine.

Abstract: This paper discusses the importance of the successive steps of the vitrification technique and reviews the current development and use of vitrification and of the two derived protocols, encapsulation-vitrification and droplet-vitrification Vitrification refers to the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate Samples are thus cryopreserved without detrimental intracellular ice formation In a standard vitrification protocol, excised explants are precultured on medium enriched with sucrose, treated (loaded) with a loading solution composed of 2 M glycerol + 04 M sucrose, dehydrated with a highly concentrated vitrification solution [eg the PVS2 vitrification solution, which contains 30 percent (w/v) glycerol, 15 percent (w/v) ethylene glycol and 15 percent (w/v) DMSO and 04 M sucrose], frozen and rewarmed rapidly, unloaded with basal culture medium supplemented with 12 M sucrose, and then transferred to standard culture conditions In the encapsulation-vitrification technique, the explants are encapsulated in alginate beads, loaded and dehydrated with a vitrification solution before rapid immersion in liquid nitrogen In the droplet-freezing technique, excised explants are loaded, treated with the vitrification solution and frozen in individual microdroplets of vitrification solution placed on aluminium foils, which are immersed rapidly in liquid nitrogen These three techniques have been applied to different tissues of over 100 plant species from temperate and tropical origins and the number of cases where they are being tested on a large scale or applied routinely is increasing

Abstract: A glassy state of matter results if crystallization is avoided upon cooling or increasing density. However, the physical factors controlling the ease of vitrification and nature of the glass transition remain elusive. Using numerical simulations of polydisperse hard disks, we find a direct relation between medium-range crystalline ordering and the slow dynamics which characterizes the glass transition. This suggests an intriguing scenario that the strength of frustration controls both the ease of vitrification and nature of the glass transition. Vitrification may be a process of hidden crystalline ordering under frustration, at least in our system.

Abstract: Cryopreservation of human oocytes and embryos is a necessary tool in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing costs. Vitrification is a cryopreservation technique that leads to a glass-like solidification, with rapid cooling of cells or tissues. Nowadays vitrification is claimed to be the future of cryopreservation of human embryos due to improved survival rates and clinical outcomes. This study was conducted at a university clinic to assess the safety and efficiency of vitrification of human zygotes as a routine procedure. A total of 849 pronuclear-stage (PN) zygotes were vitrified between March 2004 and July 2006. During this period, 103 cycles of cryopreserved embryo transfer were completed. In total, 339 PN zygotes were thawed resulting in an 89% survival rate (302 PN zygotes). The mean number of embryos per transfer was 2.2. The pregnancy rate obtained was three times higher (36.9%) than that obtained with the slow-rate freezing method (10.2%) used previously in the same centre. In conclusion, vitrification of human zygotes at the pronuclear stage seems to be a successful and reliable method with favourable outcomes and can be recommended as a routine technique for cryopreservation of human embryos.

Abstract: Vitrification appears to be a viable method for the cryopreservation of human metaphase II (MII) oocytes, but concerns regarding the concentration of cryoprotectants used during vitrification have been raised. In an attempt to circumvent this potential problem, the majority of protocols are carried out at room temperature. Exposing oocytes to temperatures below 37°C, however, leads to rapid microtubule depolymerization. Polarized light microscopy was used to measure meiotic spindle retardance following exposure to cryoprotectants and vitrification in human and mouse oocytes. To quantify the extent of depolymerization, spindle retardance was determined before and after each treatment. Exposure to vitrification and warming solutions at room temperature (21–22°C) caused the spindle of mouse MII oocytes to depolymerize. In contrast, no measurable changes in the meiotic spindle were detected by maintaining the temperature at 37°C during the exposure regimen. By carrying out the entire vitrification and warming procedure at 37°C, the spindle was also unaffected. Comparable results were obtained with vitrification of human MII oocytes at 37°C. Analysis of sibling human oocytes demonstrated that slow freezing, in contrast to vitrification, was unable to preserve the meiotic spindle. Using a vitrification protocol employing 37°C impacts negligibly on the meiotic spindle. Thus, fertilization can proceed without having to await spindle reformation.

Abstract: The capability to encapsulate single cells in droplets while retaining high cell viability (>90%) has great impact on tissue engineering, high-throughput screening, as well as clinical diagnostics and therapeutics. We demonstrate a novel method to vitrify a small number of cells using cell-encapsulating droplets. The method allows vitrification at low cryoprotectant concentration (1.5 M propanediol and 0.5 M trehalose), similar to that used in slow freezing protocols. The method was successfully applied to five different mammalian cell types: AML-12 hepatocytes, NIH-3T3 fibroblasts, HL-1 cardiomyocytes, mouse embryonic stem cells, and RAJI cells.

Abstract: Vitrification technology has shown great promise for cryopreservation of human embryos. The majority of this work has been with blastocyst-stage embryos. This report describes initial clinical results following vitrification of human embryos on day 3 of culture at the 6- to 8-cell stage. A total of 236 embryos were cryopreserved on cryoloops using a vitrification protocol. Warmed embryos were cultured until day 5 before transfer to the patient. The post-warming survival rate was 85%. The clinical pregnancy rate was 44% (34/77), and the implantation rate was 20% (40/201). In transfers where at least one warmed embryo reached the blastocyst stage by the day of transfer, the clinical pregnancy rate was 58% (28/48). The cryoloop was an excellent vessel for ultra-rapid cryopreservation of embryos. This study supports the effectiveness of a dimethylsulphoxide/ethylene glycol cryoprotectant combination for vitrification of human embryos at the 6- to 8-cell stage.

Abstract: Background The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method. Methods HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing. Results Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency. Conclusions This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.

Abstract: We put forward a new strategy for cryopreservation, namely vitrification or ice-free preservation, of cell–biomaterial constructs for tissue-engineering applications. In this study, for a period of...

Abstract: Vitrification of laser treated human blastocysts using reduced concentrations of permeable cryoprotectants was carried out by submerging cut standard straws (CSS) into liquid nitrogen. The CSS were made by cutting a standard 0.25 ml straw at an angle of � 45. After laser assisted hatching, 6 day blastocysts (n = 250) were loaded into droplets of � 0.75 ll in the CSS and were either plunged directly into liquid nitrogen or first encased in a standard 0.5 ml straw (aseptic technique) before being vitrified. Permeable cryoprotectants (ethylene glycol + Me2SO) at concentrations of 15% and 20% v:v were tested for their effect on post warming re-expansion and post transfer pregnancy rates. Our results indicate that the use of reduced concentrations of cryoprotectants and aseptic packaging of blastocysts did not have any statistically significant impact on the study outcomes. 2007 Elsevier Inc. All rights reserved.

Abstract: The purpose of the present study was to investigate the effects of two vitrification procedures on developmental capacity and ultrastructural changes of matured swamp buffalo oocytes. In vitro-matured oocytes were vitrified by using 35 and 40% ethylene glycol as vitrification solution for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified-warmed oocytes, evaluated on the basis of ooplasm homogeneity, oolemma integrity and zona pellucida intactness, as well as parthenogenetic blastocyst rates of vitrified-warmed oocytes were significantly higher with SSV (89.3 and 13.6%, respectively) than ISV (81.8 and 5.5%, respectively). However, they were still significantly lower than that of control oocytes (100 and 34.2%, respectively). For examining the ultrastructural changes, fresh, VS-exposed (ISV and SSV), and vitrified-warmed (ISV and SSV) oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified-warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. The present study demonstrated the feasibility of vitrification of in vitro-matured swamp buffalo oocytes.

Abstract: Vitrification of nuclear wastes is attractive because of its flexibility, the large number of elements which can be incorporated in the glass, its high corrosion durability and the reduced volume of the resulting waste form. Vitrification is a mature technology and has been used for high level nuclear waste (HLW) immobilisation for more than 40 years in France, Germany and Belgium, Russia, UK, Japan and the USA. Vitrification involves melting of waste materials with glass-forming additives so that the final vitreous product incorporates the waste contaminants in its macro- and micro-structure. Hazardous waste constituents are immobilised either by direct incorporation into the glass structure or by encapsulation when the final glassy material can be in form of a glass composite material (GCM). Both borosilicate and phosphate glasses are currently used to immobilise nuclear wastes, moreover in addition to relatively homogeneous glasses novel GCM are used to immobilise problematic waste streams. The spectrum of wastes which are currently vitrified increases from HLW to low and intermediate wastes (LILW) such as legacy wastes in Hanford, USA and nuclear power plant operational wastes in Russia and Korea.

Abstract: Glycerol alone or in combination with other additives is one of the most widely used and successful cryoprotectants for human sperm. The glycerol method requires rigorous post thaw sample washing for use in ART and this may lead to low sperm yield from oligospermic samples. In this study the feasibility of the use of sucrose in sperm cryopreservation was explored. Sucrose as cryoprotectant was combined with direct plunging of sample into liquid nitrogen (vitrification) as a freezing method. Sucrose treated sperm from normozoospermic and severly oligozoospermic samples underwent rapid freeze and thaw. Motility and viability were evaluated before freezing (after sucrose equilibration) as well as post freezing (after thaw). The 100 mM concentration of sucrose showed better cryoprotectant features compared to that of higher concentrations (200-1000 mM). Sucrose (100 mM)treated sperm maintained low but acceptable motility (30%) and satisfactory viability (60%) after freezing and thawing. The cryoprotectant capacity of sucrose for normozoospermic and oligozoospermic samples were identical. The sucrose method utilizes rapid freezing of a micro volume of sample and thus quickly freezes, thaws, and maximizes recovery of the sperm from the sample.

Abstract: We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 06 M sucrose) or that do not (37% v/v) EG, 06 M sucrose and 30% (v/v) EG, 06 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs To protect against contamination a straw-in-straw technique was employed Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (>96%), multipotency and karyotype Rapid cooling in 37% (v/v) EG, 06 M sucrose afforded good viability but did not preserve structural integrity Rapid cooling in 30% (v/v) EG, 06 M sucrose additionally reduced cell viability to 77% Slow-cooling reduced cell viability to 65% and damaged the neurospheres This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures

Abstract: OBJECTIVE To compare the effects of vitrification with slow-freezing on the developmental ability of day 3 cleavage stage embryos. METHODS Patients who had no less than 4 high quality embryos were included in this study. These embryos were cryopreserved using the methods of vitrification or slow-freezing. In the cryopreserved embryo transfer cycles, the embryos which were cryopreserved using one of the methods were chosen randomly. The developmental ability of embryos was compared between these two groups. RESULTS A total of 80 patients were included in this study with 160 embryos. In the group of slow-freezing, 73 (91%) embryos were survived and achieved 15 (38%) clinical pregnancies. Among these, 3 were twins and the implantation rate was 25% (18/73). In the group of vitrification, 71 (89%) embryos were survived and achieved 19 (48%) clinical pregnancies. Among these, 9 were twins and the implantation rate was 39% (28/71), which was significantly higher than the slow-freezing group (P < 0.05). Otherwise, the clinical pregnant rate and multiple pregnant rate was higher in the group of vitrification than the slow-freezing group, but had no significance. CONCLUSION Vitrification is more benefit for the developmental ability of the thawed embryos and more suitable for the cryopreservation of day 3 cleavage stage embryos.

Abstract: This study was designed to investigate the effect of vitrification and post-thaw survival and chromosomal aberrations caused by vitrification of vitrified 8-cell mouse embryos in comparison with a control group. To this purpose the survival rate and the frequency of chromosomal aberrations were assessed in frozen-thawed 8-cell mouse embryos after various storage durations in the presence of ethylene glycol as cryoprotectant. eight-cell mouse embryos were obtained from NMRI mice 3 days after mating. Retrieved embryos were transferred to vitrification solution containing ethylene glycol as cryoprotectant, then transferred into a vitrification straw using standard technique, and vitrified in liquid nitrogen. Six groups of embryos according to storage duration (24 hours, 1 and 2 weeks, 1-6 months) were frozen. After appropriate storage periods embryos were thawed and studied for their viability 4-6 hours after thawing and intact embryos were transferred to fresh medium containing colcemid. After 48 hours, the embryos were fixed and studied for their chromosome abnormalities using Tarkowsky's drying technique. Results indicate that freezing affects the viability and chromosome structure of embryos when compared with the control group. Furthermore increasing the storage duration reduces the viability and increases the chromosome aberrations of embryos (such as aneuploidy and polyploidy). This result might indicate that the effects of vitrification on the cytoskeleton or other cellular organelle might produce chromosomal alterations leading to cell death.

Abstract: The present study aims to develop a reliable method for in vitro micropropagation and long-term storage of shoot tips in Prunus avium using vitrification and encapsulation dehydration methods. For micropropagation, maximum number of shoots per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L BA (benzyl amino purine). Several pretreatments prior to cryogenic storage were conducted to enhance shoot tips recovery after cryostorage. Using vitrification method, two different preculture treatments were tested to enhance regrowth after both dehydration and cryopreservation. Further accumulation of sucrose and glycerol was tested. The highest regrowth using this method was (up to 77.8%) when shoot tips were exposed to 60 min PVS2 at 0oC. Growth recovery of cryopreserved shoot tips using encapsulation dehydration was (up to 76.3%) obtained when encapsulated shoot tips were pregrown for 3 d in liquid medium containing 0.75 M sucrose, desiccated to 20-22% moisture content and immersed in liquid nitrogen. Recovery of whole plantlets from cryopreserved shoot tips took place directly without transitory callus formation. Both methods are promising techniques for in vitro propagation and germplasm preservation of P. avium shoot tips.

Abstract: Aim: This study was designed to compare the survival rates, follicular growth rates, and ovulation rates of vitrified preantral follicles (PF) from ovaries with those isolated from a vitrified ovarian cortical strip. Methods: Mouse ovaries were divided into three groups: those not treated by vitrification of the PF (control), those treated by vitrification of the PF isolated from the ovaries (group I), and those treated by vitrification of ovarian tissue followed by PF isolation (group II). The group I samples were exposed to equilibration solution (EG-20) for 5.0 min plus vitrification solution (EFS-40) for 0.5 min, while the group II samples were exposed to EG-20 for 10.0 min plus EFS-40 for 2.0 min, before vitrification. They were subsequently placed on an electron microscope grid, and submerged immediately in liquid nitrogen. After thawing, the survival rate and the growth rate of the follicles were evaluated every 2 days. Results: In the in vitro condition, the follicles grew and developed into antral follicles in groups I and II. The survival rate of the group I samples was higher than that of the group II samples during the in vitro culture (P < 0.05). The growth rates of the follicles in group I were higher than those in group II after day 6 (P < 0.05). The ovulation rate of the samples in group I was higher than that of group II (P < 0.05). Conclusion: These results demonstrated that direct PF vitrification appeared to be better than vitrification of the PF isolated from ovarian tissue.