Isochoric vitrification: An experimental study to establish proof of concept

Ice-free vitreous cryopreservation (vitrification) is regarded as the principal method for avoiding ice crystallization damage in cryopreserved tissues and organs. We previously established the fundamental thermodynamics of isochoric (constant volume) systems for cryopreservation, and now extend this novel approach to vitrification in an isochoric system. This was achieved by measuring pressure changes in a 2 ml isochoric chamber containing a variety of aqueous solutions of the ubiquitous cryoprotective additives (CPA), dimethyl sulfoxide (Me₂SO) and Propane-diol. The CPAs, ranging in concentrations from 0 to 49%(w/v), were prepared in a proprietary preservation solution (Unisol^®) in anticipation of future applications to tissue and organ banking. Pressures developed in the system were monitored as a function of CPA concentration and cooling rate when the isochoric chamber was cooled to cryogenic temperature (−160 °C). This study corroborated our previous findings that pressure increases in accordance with the thermodynamics of partially frozen systems of low concentrations of CPAs. A key finding of this study was that in an isochoric system of higher concentrations of CPA, which vitrifies, there is no increase in pressure. In fact, an increase in pressure is a measure of failure to vitrify and a measure of devitrification. Comparison with results from the literature show that the concentration of CPAs needed for vitrification in an isochoric chamber is substantially lower than that needed for vitrification in isobaric systems at 1 atm and hyperbaric systems at 1000 atm. In addition, isochoric chambers are much more effective in promoting vitrification than hyperbaric pressure chambers, and are less expensive, easier to design, and implement.     

The effect of undissolved air on isochoric freezing

This study evaluates the effect of undissolved air on isochoric freezing of aqueous solutions. Isochoric freezing is concerned with freezing in a constant volume thermodynamic system. A possible advantage of the process is that it substantially reduces the percentage of ice in the system at every subzero temperature, relative to atmospheric freezing. At the pressures generated by isochoric freezing, or high pressure isobaric freezing, air cannot be considered an incompressible substance and the presence of undissolved air substantially increases the amount of ice that forms at any subfreezing temperature. This effect is measurable at air volumes as low as 1%. Therefore eliminating the undissolved air, or any separate gaseous phase, from the system is essential for retaining the properties of isochoric freezing.     

The thermodynamic principles of isochoric cryopreservation

The goal of this study is to introduce the fundamental thermodynamic principles of isochoric (constant volume) cryopreservation for low temperature preservation of biological materials. Traditionally, cryopreservation is performed in an isobaric process (constant pressure) at 1 atm, because this is our natural environment and it is most convenient experimentally. More than half a century of studies on cryopreservation shows that the major mechanism of damage during isobaric cryopreservation is the increase in intracellular ionic concentration during freezing, which presumably causes chemical damage to the components of cells. Cryoprotectants as well as hyperbaric pressures have been developed as methods to reduce the extent of chemical damage during freezing. The theoretical studies in this paper show that in isochoric cryopreservation, the increase in solution concentration during freezing is lower at each temperature by almost an order of magnitude from that in isobaric cryopreservation. This suggests that isochoric cryopreservation could be a preferential alternative to isobaric cryopreservation. The technology for isochoric cryopreservation is very simple; freezing in a constant volume chamber. Using a simple isochoric cryopreservation device, we confirm the theoretical thermodynamic predictions.     

Escherichia coli viability in an isochoric system at subfreezing temperatures

In comparison with isobaric (constant pressure) freezing, isochoric (constant volume) freezing reduces potential mechanical damage from ice crystals and exposes stored biological matter to a lower extracellular concentration, at the price of increased hydrostatic pressure. This study evaluates the effects of isochoric freezing to low temperatures and high pressures on Escherichia coli (E. coli) survival. The viability of E. coli was examined after freezing to final temperatures between −5 °C and −20 °C for periods from 0.5 h to 12 h, with recovery periods from 0 h to 24 h. Freezing for up to two hours to −10 °C and −15 °C had little effect on the percentage of viable E. coli, relative to the controls. However, after two hours of exposure at −20 °C, when left to recover for 24 h, a 75% reduction in survival is observed. Furthermore, after 12 h of isochoric freezing at −15 °C and −20 °C, E. coli population is reduced by 2.5 logs while freezing to these temperatures in conventional isobaric atmospheric conditions reduces population by only one log. This suggests that the combination of low temperature and high pressure experienced during isochoric freezing close to the triple point may be more detrimental to biological matter survival than the combination of elevated concentration, low temperature, and ice crystallization experienced during conventional freezing, and that this effect may be related to the time of exposure to these conditions.     

草莓愈伤组织超低温贮藏及植株再生研究

本文介绍了草莓愈伤组织的诱导,超低温贮藏及不同解融方法对其细胞存活率和芽分化的影响。     

拟南芥悬浮细胞系的玻璃化法超低温保存

悬浮培养细胞系是植物生理生化研究的好材料之一。为了保持细胞系的遗传稳定性,需要采用超低温保存技术。玻璃化法是一种不用程序降温仪的超低温保存技术。本文报道了从模式植物拟南芥建立悬浮细胞系并对其进行玻璃化法超低温研究。细胞经过合理的预培养处理和保护剂处理,直接投入液氮贮存。复温后的细胞能恢复生长,恢复生长的细胞保持着植株再生能力。国外,拟南芥悬浮细胞系的程序降温法保存和包埋脱水法保存已经报道,玻璃化法保存尚未见报道。     

Effects of cryopreservation on the survival rate of the seven-band grouper (Epinephelus septemfasciatus) embryos

The effects of cryopreservation and the vitrification solution on the embryo hatchability of the seven-band grouper Epinephelus septemfasciatus were evaluated in this study. Six small molecule cryoprotectants (PG, MeOH, Gly, DMF, DMSO and EG) and four macromolecular cryoprotectants (glucose, fructose, sucrose and trehalose) were used to determine the embryo toxicity levels. Results showed that the embryo survival rate was higher when the PM (24% PG + 16% MeOH):Gly ratios were 3:1 and 4:1. Further experiments showed that the embryo survival rates in PMG3S (35% PMG3 + 5% sucrose) and PMG3T (35% PMG3 + 5% trehalose) were relatively higher, which are 29.24 ± 10.81% and 27.01 ± 3.39%, respectively. When treated with PMG3S and PMG3T by using 5-step method, embryos at somite stage and tail-bud stage shrank in the first 6 min and gradually recovered in volume to the original. This indicated the successful permeation of the vitrification solutions into cells. Then, embryos at the embryoid body formation stage, the somite stage and the tail-bud stage were cryopreserved with PMG3S and PMG3T. In total, 82 floating embryos were obtained, 14 of which developed further, with 8 embryos at the tail-bud stage developing to the heartbeat stage, 4 embryos at the body formation stage development to the somite stage, and 2 embryos at the somite stage hatched to larval fish.     

Tolerance to vitrification of rat embryos at various developmental stages

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.     

Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures

Sperm vitrification is a low cost and simple technique that does not require special equipment and may represent an attractive alternative to the costly and time consuming conventional dog spermatozoa cryopreservation techniques. The objective of this study was to evaluate different cryoprotectants and warming temperatures on the vitrification of dog spermatozoa. Pooled semen samples from 10 beagle dogs were vitrified with four extenders, based on Tris, citric acid and glucose, 20% egg yolk (TCG-20% EY) and different combinations of sucrose and/or trehalose: 250 mM sucrose; 250 mM trehalose; 125 mM sucrose + 125 mM trehalose; 250 mM sucrose + 250 mM trehalose. Samples were vitrified by dropping 50 μL of sperm suspension directly into liquid nitrogen. After vitrification, warming was done either fast (at 65 °C for 2–5 s) or slow (at 37 °C for one minute). Motility was assayed using a computer-aided sperm analysis (CASA) system; membrane integrity and acrosomal status were analyzed by fluorescence microscopy. For comparison, samples were also conventionally frozen in liquid nitrogen vapor using a TCG-20% egg yolk extender plus 5% glycerol. Frozen straws were thawed in a water bath at 37 °C for 30 s. Poorer motility results (P < 0.05) but similar viability were obtained when vitrification was performed, compared to conventional freezing (P > 0.05). When vitrification was used, cryoprotectants containing either 250 mM sucrose or 250 mM trehalose and warmed at 37 °C returned the best sperm quality variables.     

Cryogenic protection of oocytes with antifreeze proteins

Proteins belonging to a family of compounds known as “antifreeze proteins” interact with occytes and protect the oolemma from damage at cryogenic temperatures. Experiments were performed with pig oocytes rapidly cooled to cryogenic temperatures in vitrifying solutions with and without antifreeze proteins. Four different types of antifreeze polypeptides and glycoproteins were tested. The integrity of the oolemma was examined with Fluoroscein Diacetate (FDA) staining and morphological examinations. Results show that the pig oocyte oolemma is a primary site of injury during exposure to low temperatures and that all the different proteins have a similar ability to interact with and protect the oolemma. Our results may be important in developing solutions for long-term preservation of oocytes at cryogenic temperatures (cryopreservation). © 1993 Wiley-Liss, Inc.     

香蕉茎尖超低温保存过程中的细胞超微结构观察(简报)

超低温保存(Cryopreservation)通常称为液氮保存或LN(-196℃)保存,是目前植物种质资源长期稳定保存的理想方法,已经成功应用于多种植物种质资源保存。玻璃化法(Vitrification)超低温保存植物种质资源始于20世纪80年代末,Uagami等首次     

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.     

Further work on the cryopreservation of articular cartilage with particular reference to the liquidus tracking (LT) method

The cryopreservation of articular cartilage with survival of living cells has been a difficult problem. We have provided evidence that this is due to the formation of ice crystals in the chondrons. We have developed a method in which the concentration of the cryoprotectant dimethyl sulphoxide (Me(2)SO) is increased progressively, in steps, as cooling proceeds so that ice is never allowed to form, but the very high concentrations of Me(2)SO required at low temperatures are reached only at those low temperatures. In this paper, we describe some new experiments with discs of ovine articular cartilage similar to those used in our previous studies and we show that continuous stirring throughout the process resulted in a significant increase in the rate of (35)S sulphate incorporation into glycosoaminoglycans (GAGs), now reaching 87% of the corresponding fresh control values. We confirmed that the method is also effective for human knee joint cartilage, which gave 70% of fresh control ability to synthesise GAGs; continuous stirring was also used in this experiment. We then extended the method to ovine knee joint osteochondral dowels and showed that, again with continuous stirring, the method produced tissue concentrations of Me(2)SO that were sufficient to prevent freezing in dowels too, and to permit cell function at 60% of control. The most important mechanical property (instantaneous compressive modulus) was unaffected by the process. Finally, we experimented with some technical variations to facilitate clinical use-a more rapid process for warming and removal of Me(2)SO was developed and a method of short-term storage before or after cryopreservation was developed. Finally, pilot experiments were carried out to provide proof of principle for a closed, continuous flow method in which both temperature and Me(2)SO concentration were computer-controlled.     

Vitrification of porcine articular cartilage

The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. A vitrification solution containing a 55% cryoprotectant formulation, VS55, previously studied using rabbit articular cartilage, was evaluated using porcine articular cartilage. Specimens ranging from 2 to 6 mm in thickness were obtained from 6 mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The results of post-rewarming viability assessments employing alamarBlue demonstrated a large decrease (p < 0.001) in viability in all three sizes of cartilage specimen vitrified with VS55. This is in marked contrast with prior experience with full thickness, 0.6 mm rabbit cartilage. Microscopic examination following cryosubstitution confirmed ice formation in the chondrocytes of porcine cartilage vitrified using VS55. Experiments using a more concentrated vitrification formulation (83%), VS83, showed a significant treatment benefit for larger segments of articular cartilage. Differences between the VS55 and the VS83 treatment groups were significant at p < 0.001 for 2 mm and 4 mm plugs, and at p < 0.01 for full thickness, 6 mm plugs. The percentage viability in fresh controls, compared to VS55 and VS83, was 24.7% and 80.7% in the 2 mm size group, 18.2% and 55.5% in the 4 mm size group, and 5.2% and 43.6% in the 6 mm group, respectively. The results of this study continue to indicate that vitrification is superior to conventional cryopreservation with low concentrations of dimethyl sulfoxide by freezing for cartilage. The vitrification technology presented here may, with further process development, enable the long-term storage and transportation of living cartilage for repair of human articular surfaces.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Cryopreservation of Luciola praeusta Kiesenwetter (Coleoptera: Lampyridae) embryos by vitrification

There is an urgent need to preserve the ever-decreasing number of different species of fireflies all over the world. We sought to develop a vitrification procedure to cryopreserve the firefly embryos. The late stages of Luciola praeusta Kiesenwetter embryos were collected. Several impermeable and permeable protectants with various concentrations in different mediums (TNM-FH insect medium, Grace's medium, Dulbecco's Modification of Eagle's Medium (DMEM) and Dulbecco's Phosphate-Buffered Saline (DPBS)) were used. Embryos culturing in TNM-FH medium yielded the highest survival rate of 75.3 ± 3.6%. One-step, two-step and three-step methods were used in equilibrium procedure respectively. The highest survival rate (73.7% ±3.2%) occurred in embryos treated by three-step method ((1.5 M ethylene glycol (EG) + 2 M EG plus 8% polyvinylpyrrolidone (PVP) + 3 M EG, 8% PVP and 15% trehalose). Additionally, embryos exposed to 0.5 M trehalose presented a significantly higher survival rate (71.8 ± 2.7%) than embryos preserved in 0.5 M sucrose.     

In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN₂) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN₂ has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.     

Vitrification of posterior corneal lamellae

Cryopreservation of corneas has not yet been established as a routine method. Unsatisfactory experimental results with conventional techniques prompted us to explore the possibilities of vitrification. The aim of the present study was to optimize the heat exchange between the corneal tissue and cooling medium by reducing the corneal tissue volume and using a suitable sample container. A further objective was to promote vitrification by developing a new device for rapid cooling to -140 degrees C, just below the vitrification temperature of the cryopreservation medium. Experiments were done using posterior lamellar discs from pig corneas with a diameter of 7.5 mm and a thickness of 250-350 microm. The volume of tissue to be vitrified was 88% lower with posterior corneal lamellae than with the previously used corneoscleral discs. A very thin-walled (0.05 mm), teflon-coated bag served as the sample container. Immersed in only 0.1 ml of the vitrification solution VS41a, the lamellae were cooled to a final storage temperature of -196 degrees C. After warming and organ-culturing for 24h, the endothelium was stained with trypan blue and alizarin red, to determine cell viability. Vitrification of corneal lamellae without apparent ice formation or cracking of the specimen was achieved. Despite the successful vitrification, only a maximum of 10% of the endothelial cells was vital after warming. Thus, the toxicity of the cryoprotective agents and the devitrification that occurred during the heating process require further optimization of the method.