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.     

Isochoric preservation: A novel characterization method

Isochoric (constant volume) preservation is an alternative to traditional cryopreservation methods because it requires less cryoprotectant and is simple to operate. In order to validate that this method automatically minimizes the pressure for a given temperature, pressure and temperature data were collected from a specially designed pressure vessel. This vessel was then used to examine the effect of an isochoric environment on freezing point nucleation in an aqueous antifreeze protein solution, and to generate pressure-temperature phase diagrams for various cryoprotectant solutions. Our results show that the isochoric pressure vessel follows the pressure-temperature phase diagram of water, thereby minimizing the pressure for the given temperature. We also show that the nucleation temperature of the antifreeze protein in an isochoric vessel is lower than that of the isobaric method. Furthermore, the nucleation temperature decreased with increasing concentration in the isochoric vessel while the isobaric nucleation temperature showed no change. These results indicate that the isochoric environment imposes additional constraints on ice formation and warrants further study as these results may change when a different type of cryoprotectant is used. Finally, all of the cryoprotectant phase diagrams exhibited a similar pressure-temperature slope indicating that, regardless of the cryoprotectant used or the mechanism by which it suppresses freezing, isochoric freezing affects the molecules in the same manner. Together, all of these results indicate that the isochoric method of preservation is a valuable tool for characterizing the thermodynamic properties of cryoprotectants and has great potential as a cryopreservation method in the field of cryobiology.     

Analysis of isochoric subcooling

Because ice-I is less dense than water, the formation of an ice nucleus in an isochoric (constant volume) chamber will cause an increase in pressure. This analysis shows that the energy required to overcome such a pressure increase makes homogeneous ice nucleation thermodynamically improbable in an isochoric system at temperatures above -109 degrees C. By suppressing ice nucleation, isochoric cooling is expected to significantly promote vitrification. Because water has a higher freezing temperature and a lower glass-transition temperature than physiological solutions, this analysis represents a scenario for avoiding ice crystallization during the preservation of biological substances. While isochoric cryopreservation has not yet been put into practice, this theoretical, first-order analysis suggests that if attainable it could make organ preservation significantly more effective and practical.     

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.     

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.     

Isochoric conditions enable high subfreezing temperature pancreatic islet preservation without osmotic cryoprotective agents

While biological systems are typically studied under isobaric (constant pressure) conditions, recent reports on the bio-thermodynamics of isochoric (constant volume) systems point to their potential for subfreezing-temperature preservation of biological matter. This preliminary study, in which we report that pancreatic islets can survive multi-day preservation at high subfreezing temperatures in an isochoric chamber without osmotic cryoprotective agents (CPA), highlights the potential of isochoric cryopreservation in an application of clinical value.     

Do physical forces contribute to cryodamage?

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the “two factor theory.” The present study deals with a third, largely ignored component—mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70–110, 250–500, and 1,000–1,250 µm, as a means of altering the surface area with which the cells come in contact. Post‐thaw evaluation included motility at 0 h and after 3 h at 37°C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra‐container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area‐to‐volume ratio, the intra‐container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing. Biotechnol. Bioeng. 2009; 104: 719–728 © 2009 Wiley Periodicals, Inc.     

Thermally defined cryomicroscopy and thermodynamic analysis in lymphocyte freezing

A cryomicroscope is described which provides the possibility of quantifying the volume loss of cells during freezing, detection of intracellular ice formation during cooling and warming, as well as the determination of viability as function of (constant) cooling rates. The basic mechanisms occurring in cryopreservation have been studied with this system using the human lymphocyte suspended in pure saline as a biological model system; experimentally observed exosmosis during freezing is compared to predictions from a thermodynamic model. Cell volume loss during freezing has been determined experimentally for cooling rates of 2.4, 12, 48, and 120 degrees K/min. Exosmosis also was calculated corresponding to various assumptions regarding the concentration dependence of the hydraulic permeability of the cells. Further calculations of exosmosis are performed for determining the effects of the initial cell volume. The temperatures and transition cooling rate ranges of intracellular ice formation have been determined. On the basis of exosmosis and a lethal level of intracellular salt concentration, a hypothetical relative optimum of the cooling rate is theoretically predicted and compared to the experiments.     

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Evaluation of DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of LDL as cryoprotectant

Defining reliable and objective biomarkers of sperm quality is a complex matter, because it does not rely on a particular characteristic of the milt. Susceptibility to cryopreservation varies between ejaculations and throughout the year, and the evaluation of fresh sperm does not always provide accurate information about their fertilization ability after freezing and thawing. DNA is one of the cell components prone to suffering cryodamage and several studies have pointed out the importance of the maintenance of its integrity during sperm cryostorage. The authors analysed sperm from rainbow trout for four weeks during the natural reproductive season. Viability, DNA integrity, and fertilization ability were evaluated. Furthermore, in order to increase membrane and DNA protection during sperm cryopreservation, the authors optimized the use of LDL fraction from egg yolk as a cryoprotectant during the analysed period. Results revealed that the evaluation of DNA damage in fresh sperm reveals subtle cell damage, not evidenced in fresh sperm by the other parameters. DNA fragmentation increased from 8 to 31% during the reproductive season, indicating pre-freezing differences that render the cells more susceptible to cryodamage. Also, the use of 12% LDL (low density lipoprotein) fraction, instead of the commonly used pure egg yolk, improved sperm quality after freezing. When LDL was used, post-thaw quality remained constant throughout the analysed period, providing around 60% of eyed embryos. In contrast, when egg yolk was used, post-thaw quality decreased significantly at the end of the season and the percentage of eyed embryos dropped from 60% to 27%. Results demonstrated that reduction in DNA integrity takes place during the reproductive season affecting susceptibility to cryodamage and that the protective effect of egg yolk is very much improved when only their LDL fraction is added to the cryopreservation extender.     

酒酒球菌液氮超低温保存

【目地】为安全、长期的保藏酒酒球菌,本文研究了菌体生长时间、冷冻方法、解冻温度、菌密度以及保护剂等对酒酒球菌细胞冷冻存活率的影响,找到最优液氮超低温保存方法。【方法】采用平板计数法测定冷冻存活率。【结果】实验结果表明酒酒球菌的最佳保存方法为:首先在稳定期前期离心收集菌体;其次加入保护剂(20 g/L酵母浸提物,40V/V甘油,20 g/L蔗糖,30 g/L谷氨酸钠)稀释菌体,使菌密度为109CFU/mL;然后直接投入液氮冷冻;最后在37℃温水浴中迅速解冻。保存6个月后,其中21株酒酒球菌的冷冻存活率达到99%以上。【结论】初步研究表明酵母浸提物,甘油,蔗糖,谷氨酸钠复合保护剂对酒酒球菌的保护效果较好,液氮超低温保存可用于酒酒球菌的长期保存。     

Improved and reproducible cell viability in the superflash freezing method using an automatic thawing apparatus

Cell cryopreservation stops the biological activity of cells by placing them in the frozen state, and can be used to preserve cells without subculturing, which can cause contamination and genetic drift. However, the freezing process used in cryopreservation can injure or damage the cells due to the cytotoxicity of cryoprotecting agents (CPAs). We have previously reported a CPA-free cryopreservation method based on inkjet technology. In this method, the vitrified cells were exposed to the room temperature atmosphere during the transport of the cells using tweezers, which caused devitrification due to the increased temperature and often lowered the cell viability. In the present study, we developed an automatic thawing apparatus that transports the vitrified cells rapidly into a prewarmed medium using a spring hinge. Observations with a high-speed camera revealed that the spring hinge drops the cells into the prewarmed medium within 20 ms. All heat-transfer simulations for the apparatuses with different designs and rotation speeds showed that the cells remained below the glass-transition temperature during the transport. Finally, the apparatus was evaluated using mouse fibroblast 3T3 cells. The cell viability was improved and its reproducibility was enhanced using this apparatus. The results indicate that the combination of superflash freezing with the rapid thawing process represents a promising approach to circumvent the problems typically associated with the addition of CPAs.     

Combining endocytic and freezing‐induced trehalose uptake for cryopreservation of mammalian cells

Fibroblasts take up trehalose during freezing and thawing, which facilitates cryosurvival of the cells. The aim of this study was to investigate if trehalose uptake via fluid‐phase endocytosis prefreeze increases cryosurvival. To determine endocytic trehalose uptake in attached as well as suspended fibroblasts, intracellular trehalose concentrations were determined during incubation at 37°C using an enzymatically based trehalose assay. In addition, freezing‐induced trehalose uptake of extracellularly added trehalose was determined. Cryosurvival rates were determined via trypan blue staining. Intracellular trehalose contents of attached as well as suspended cells were found to increase linearly with time, consistent with fluid‐phase endocytosis. Furthermore, the intracellular trehalose concentration increased with increasing extracellular trehalose concentration (0–100 mM) in a linear fashion. Prefreeze loading of cells with trehalose via fluid‐phase endocytosis only showed increased cryosurvival rates at extracellular trehalose concentrations lower than 50 mM in the cryopreservation medium. To obtain satisfactory cryosurvival rates after endocytic preloading, extracellular trehalose is needed to prevent efflux of trehalose during freezing and thawing and for freezing‐induced trehalose uptake. At trehalose concentrations greater than 100 mM, cryosurvival rates were similar or slightly higher if cells were not loaded with trehalose prefreeze. Cells that were grown in the presence of trehalose showed a tendency to aggregate after harvesting. It is concluded that it is particularly freezing‐induced trehalose uptake that facilitates cryosurvival when trehalose is used as the sole cryoprotectant for cryopreservation of fibroblasts. Preloading with trehalose does not increase cryosurvival rates if trehalose is also added as extracellular protectant. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:229–230, 2017     

Cryopreservation of Human Oocytes and Ovarian Tissue

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth.     

Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival

Cryopreservation induces partially irreversible damage to equine sperm membranes. Part of this damage occurs due to membrane alterations induced by the membrane changing from the fluid to the gel-state as the temperature is reduced lower than the membrane transition temperature. One way to prevent this damage is to increase the membrane fluidity at low temperatures by adding cholesterol to the membrane. Different concentrations of cholesterol-loaded-cyclodextrins (CLC) were added to stallion sperm to determine the CLC concentration that optimizes cryosurvival. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to control sperm from those same stallions (67% vs. 50%; P<0.05). Addition of CLCs increased the percentages of membrane intact sperm surviving cryopreservation compared to untreated sperm for all stallions (P<0.05). The amount of cholesterol that incorporated into the membranes of the sperm cells increased in a polynomial fashion (R2=0.9978) and incorporated into all sperm membranes. In addition, there was a significant loss of cholesterol from sperm membranes after cryopreservation; however, addition of CLCs to sperm prior to cryopreservation maintained higher cholesterol levels in the sperm after freezing and thawing than untreated sperm (P<0.05). Addition of CLCs also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than control sperm (48 vs. 15; P<0.05). In conclusion, CLCs improved the percentage of post-thaw viability in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLCs to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.     

Osmotic properties of the rabbit corneal endothelium and their relevance to cryopreservation

The process of cryopreservation subjects cells to gross changes in the composition of the solution that surrounds them, changes that cause the cells first to shrink and then to swell by an osmotic mechanism. Empirical methods have been developed that permit many cells to survive freezing and thawing, but the cornea, which is crucially dependent upon the function of its endothelial monolayer, has proved quite refractory. In this paper we explore the osmotic response of the corneal endothelium of the rabbit to solutions ranging in osmolality from 0.25 to 8.6 × isotonic. Boyle van't Hoff behavior was observed between 0.43 and 8.6 × isotonic, and there was an apparent nonosmotic volume of 33.6%. However, ultrastructural damage was observed at the limits of this range, and it appeared that the tolerated range was 0.64–4.4 × isotonic. We show the extent to which dimethyl sulfoxide (Me₂SO) would be expected to moderate changes in volume during freezing and suggest that its initial concentration should be at least 2M to prevent excessive shrinkage. We also show that cell swelling during removal of Me₂SO is especially likely to be hazardous.     

Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.     

Static magnetic field increases survival rate of dental pulp stem cells during DMSO-free cryopreservation

Abstract

Successful and efficient cryopreservation of living cells and organs is a key clinical application of regenerative medicine. Recently, magnetic cryopreservation has been reported for intact tooth banking and cryopreservation of dental tissue. The aim of this study was to assess the cryoprotective effects of static magnetic fields (SMFs) on human dental pulp stem cells (DPSCs) during cryopreservation. Human DPSCs isolated from extracted teeth were frozen with a 0.4-T or 0.8-T SMF and then stored at ?196?°C for 24?h. During freezing, the cells were suspended in freezing media containing with 0, 3 or 10% DMSO. After thawing, the changes in survival rate of the DPSCs were determined by flow cytometry. To understand the possible cryoprotective mechanisms of the SMF, the membrane fluidity of SMF-exposed DPSCs was tested. The results showed that when the freezing medium was DMSO-free, the survival rates of the thawed DPSCs increased 2- or 2.5-fold when the cells were exposed to 0.4-T or 0.8-T SMFs, respectively (p?<?0.01). In addition, after exposure to the 0.4-T SMF, the fluorescence anisotropy of the DPSCs increased significantly (p?<?0.01) in the hydrophilic region. These results show that SMF exposure improved DMSO-free cryopreservation. This phenomenon may be due to the improvement of membrane stability for resisting damage caused by ice crystals during the freezing procedure.     

Evaluation of zebrafish (Danio rerio) PGCs viability and DNA damage using different cryopreservation protocols

Cryopreservation of primordial germ cells (PGCs) is a better alternative for the conservation of the diploid genome in fish until embryo cryopreservation is achieved. A good cryopreservation protocol must guarantee high survival rates but also absence of genetic damage. In this study, a cell toxicity test using several internal and external cryoprotectants was carried out. The best combination of cryoprotectants (DMSO 5 mol/L, ethylene glicol (EG) 1 mol/L, polyvinyl pyrrolidone (PVP) 4%) was used with and without antifreeze proteins (AFPs) at two different concentrations (10 mg/mL and 20 mg/mL) for cryopreservation trials. Different cryopreservation methods were used with single PGCs, genital ridges, and whole zebrafish embryos using cryovials, 0.5 mL straws, microcapsules, and microdrops. All embryos were obtained from the vasa EGFP zf45 transgenic line and viability was evaluated using trypan blue. High cell viability rates after cryopreservation in 0.5 mL straws were obtained (around 90%) and a decrease in viability was only observed when cells were cryopreserved in microcapsules and when AFP at 20 mg/mL was added to the freezing media. Genetic damage was determined by comet assay and was compared in cells cryopreserved in 0.5 mL straws and microcapsules (lowest viability rate). There were significantly more DNA strand breaks after cryopreservation in the cells cryopreserved without cryoprotectants and in those cryopreserved in microcapsules. Genetic damage in the cells cryopreserved with cryoprotectants in 0.5 mL straws was similar to fresh control samples, regardless of the concentration of AFP used. The decrease in PGC viability with the addition of AFP 20 mg/mL did not correlate with an increase in DNA damage. This study reported a successful method for zebrafish PGC cryopreservation that not only guarantees high cell survival but also the absence of DNA damage.     

Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity (Lp), cryoprotectant permeability (Ps), and the reflection coefficient (sigma). Experimental values for Lp and Ps at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (Ea). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.