Penetrating Cryoprotectants Research Articles

Liposome-based Freezing Medium Improves the Outcome of Mouse Prepubertal Testicular Tissue Cryopreservation

AbstractCryopreservation of testicular tissue holds an important role in the field of fertility preservation, particularly for prepubertal boys diagnosed with cancer. However, prepubertal testicular tissue cryopreservation is still considered to be in the experimental stage necessitating the refinement of cryopreservation protocol. Considering the fact that loss of membrane lipids is the primary cause of freeze–thaw-induced loss of testicular cell functions, in this study, we explored the beneficial properties of exogenous supplementation of membrane lipids in the form of liposomes in enhancing the cryosurvival of prepubertal testicular tissue. The freezing medium supplemented with liposomes (prepared from soy lecithin, phosphatidylethanolamine, phosphatidylserine, and cholesterol) was used for the experiments. Prepubertal testicular tissues from Swiss albino mice were cryopreserved in a liposome-containing freezing medium (LFM) composed of 0.25 mg/mL liposomes, 5% DMSO, and 30% FCS in the DMEM/F12 medium using a slow freezing protocol. The tissues were thawed and assessed for various testicular cell functions. Freezing in LFM mitigated the loss of viability, decreased malondialdehyde level (p < 0.05), and reduced apoptosis (p < 0.05) in the testicular cells compared to the testicular tissue cryopreserved in the control freezing medium (CFM). Further, DMSO (5%) appears to be the ideal penetrating cryoprotectant for prepubertal testicular tissue cryopreservation with liposome-based freezing medium. Similar enhancement in cryosurvival of cells was observed in adult human testicular tissue frozen with LFM. These findings highlight the translational value of liposome-based freezing medium in the cryopreservation of testicular tissue of prepubertal boys undergoing chemotherapy.

Freezing Hu ram semen: influence of different penetrating cryoprotectants and egg yolk level on the post-thaw quality of sperm.

The Hu sheep is a renowned breed known for its high reproductive rate. It is in estrus all year round, and its breeding population is gradually expanding. However, the current techniques for cryopreserving semen have limited effectiveness, which hinders the continuous development of this species. The purpose of this study is to explore the effects of different penetrating cryoprotectants (CPAs) and egg yolk (EY) concentrations on the cryopreservation of Hu ram semen to determine the most effective combination. In this study, the effects of glycerol (GLY), ethylene glycol (EG), dimethylacetamide, dimethyl sulfoxide, different proportions of GLY and EG, EY on sperm quality after thawing were investigated by detecting sperm total motility (TM), progressive motility (PM), straight-line velocity, curvilinear velocity, average path velocity, amplitude of lateral head displacement, wobble movement coefficient, average motion degree, functional integrity (plasma membrane integrity, acrosome integrity) and reactive oxygen species (ROS) level. When GLY and EG were added together, compared to other concentration groups, 6% GLY significantly (p<0.05) increased TM, PM, plasma membrane integrity, and acrosome integrity of thawed sperm. Additionally, it significantly (p<0.05) decreased the ROS level of sperm. In this study, the TM, PM, and membrane integrity of the 6% EG were significantly (p<0.05) higher than those of the control, 1% GLY+5% EG and 6% GLY+6% EG groups. Compared to other concentration groups, 20% EY significantly (p<0.05) improved the TM, PM, and plasma membrane integrity of thawed sperm. However, the integrity of the acrosome increased with the higher concentration of EY. In conclusion, the post-thawed Hu ram semen diluted with a diluent containing 6% GLY and 20% EY exhibited higher quality compared to the other groups.

PROTECTIVE ENVIRONMENTS FOR ANIMAL SPERM: EVOLUTION OF METHODS AND TOPICAL ASPECTS (REVIEW ARTICLE)

A retrospective review of domestic and foreign sources of literature is presented, as well as data of published own research on cryopreservation of animal sperm. The main historical stages of the creation of protective environments for deep freezing of sperm are given. In the 30s of the last century, a phenomenon characterized by the death of spermatozoa upon sharp cooling in the range of positive temperatures was discovered. It is called temperature shock of sperm. To prevent it, it is proposed to add substances containing phospholipids to the composition of diluents. Such environments can contain both simple components - native chicken egg yolk or milk, and high-tech - lipoproteins, isolated phospholipids of various origins. To stabilize protein-lipid complexes of plasma membranes and acrosomes of sperm during the cooling process, carbohydrates are added to the diluents. Sugars are components of energy supply for sperm and, along with salts, they are the main osmotic regulators. A combination of two or three carbohydrates in the medium was traditionally considered necessary. However, the Kharkiv school of reproductive specialists has proven the possibility of creating effective protective environments using only one sugar - sucrose or lactose - based on considerable practical experience. The effectiveness of germ cell freezing is shown depending on the cryoprotectants used. Glycerin is the first known endocellular cryoprotectant, which is still unsurpassed in sperm cryopreservation. Our own experimental data on the effect of combinations of glycerol with substances from the amide group on the main biological indicators of sperm after deconservation are presented. Cryoprotectants dimethylacetamide (DMAC) and dimethylformamide (DMF) were tested in own experiments on stallion semen. The experiments studied the effect of different concentrations of the above-mentioned penetrating cryoprotectants both on the main physiological characteristics of stallion sperm (motility, survival), and on the degree of damage to the membrane apparatus of sperm. The effectiveness of certain combinations of these substances has been proven. Methods of preventing the negative impact of oxygen and the development of lipid peroxidation processes in sperm during cryopreservation are presented. The concept of using additional hormonal components in diluents, in particular prostaglandin F2a, is revealed. The materials related to the effect on the quality of reproductive cells of healing preparations are displayed. Keywords: artificial insemination, environments, semen, animals, bulls, stallions, cryoprotectants, freezing.

Cryopreservation did not affect sperm DNA methylation levels of genes related to fertilization and embryonic development of cynomolgus macaque (Macaca fascicularis)

DNA methylation alters gene expression in numerous biological processes, including embryonic development. It is little known about the effect of cryopreservation on sperm DNA methylation. The present study has investigated whether cryopreservation causes abnormal DNA methylation in cynomolgus macaque sperm for five critical genes that includes the maternally imprinted gene (SNRPN), genes associated with male infertility (HSPA1L, MTHFR) and genes involved in embryonic development (TET3, LZTR1). Our results showed that sperm motility, the percentage of acrosomal integrity, DNA integrity and mitochondrial membrane potential were decreased after cryopreservation either being frozen with penetrating cryoprotectant, glycerol (Gly) or ethylene glycol (EG), compared to fresh sperm (p = 0.000), but the methylation patterns of the five target genes from cynomolgus macaque sperm samples were not affected after cryopreservation as evaluated by the Bisulfite Sequencing PCR (BSP) method. The data indicates that the current protocol for sperm cryopreservation of cynomolgus macaque is safe in terms of DNA methylation levels in these genes related to critical sperm functions.

Influence of helium, xenon, and other noble gases on cryopreservation of Hela and l929 cell lines

Any biological material contains dissolved gases that affect physical and biological processes associated with cooling and freezing. However, in the cryobiology literature, little attention has been paid to the effect of gasses on cryopreservation. We studied the influence of helium, neon, krypton, xenon, argon, nitrogen, and sulfur hexafluoride on the survivability of HeLa and L929 cell lines during cryopreservation. Saturation of a cell suspension with helium, neon, and sulfur hexafluoride enhanced survival of HeLa and L929 cells after cryopreservation. Helium exerted the most significant effect. For a range of noble gases, the efficiency of the positive effect decreased as the molecular mass of the gas increased. This paper discusses possible mechanisms for the influence of gases on the cryopreservation of biological material. The most probable mechanism is the disruption of the frozen solution structure with gas-filled microbubbles produced during water crystallization. Ultimately, it was concluded that helium and neon can be used to improve methods for cryopreservation of cell suspensions with a low concentration of conventional penetrating cryoprotectants or even without them.

Induction of vacuolization in spermatozoa from men with oligoasthenoteratozoospermia after cryopreservation with glycerol and polyvinylpyrrolidone

Background. The two–step method using penetrating cryoprotectant glycerol is a routine for cryopreservation of spermatozoa from men with normal spermatogenesis. However, the use of this freezing method in the case of oligoasthenoteratozoospermia (OAT) leads to additional cells damage and there is a need to search for new effective cryopreservation method. The sperm vacuolization inherent in male gametes plays a negative role, reducing the fertilizing ability of cells. Objective. The aim of the study was to assess the degree of vacuolization induced in human OAT sperm after cryopreservation by a two–stage method using glycerol and polyvinylpyrrolidone (PVP), molecular mass 360000. Methods. The isolated sperm fraction was divided into 3 groups: group I – native spermatozoa, II – spermatozoa cryopreserved by the two–stage method with 10% glycerol, group III – spermatozoa cryopreserved by the two–stage method with 10% PVP. Results. About 5% of spermatozoa contained vacuoles in group I. The number of spermatozoa with one small vacuole increased to (19.68 ± 2.27)% in group II. And in group III the number of cells without vacuoles was comparable with native spermatozoa: 95.59 ± 1.59 and (96.09 ± 2.02)%, respectively. Conclusion. Thus, the use of a penetrating cryoprotectant of glycerol at 10% concentration for two–stage method cryopreservation of human spermatozoa initiates the vacuoles formation while the use of non–penetrating PVP can prevent vacuolization.

Protocol for Cryopreservation of Endothelial Monolayers.

One of the major challenges in the preservation of complex tissues is the cryosensitivity of the endothelium, the single layer of cells lining blood vessels, corneas, and other tissues. The increasing importance of endothelial monolayers in tissue-engineered constructs for transplantation and research warrants the need to develop protocols for the successful cryopreservation of cells in monolayers. In this chapter, we describe a recently published cryopreservation protocol that we developed based on examination of various factors that influence the post-thaw recovery of endothelial monolayers. To efficiently investigate cryopreservation protocol parameters, we employed an interrupted slow-cooling procedure (graded freezing) that allows dissecting loss of cell viability into contributions from slow-cooling injury and rapid-cooling injury. Our optimized protocol involves culturing cells on Rinzl plastic coverslips, using a combination of a penetrating cryoprotectant (5% dimethyl sulfoxide) and a non-penetrating cryoprotectant (6% hydroxyethyl starch), addition of 2% chondroitin sulfate, controlled cooling at 0.2°C/min or 1°C/min, and removal of cryoprotectant immediately after thaw. The protocol has been validated for human umbilical vein and porcine corneal endothelial cell monolayers.

KrioBlast TM as a New Technology of Hyper-fast Cryopreservation of Cells and Tissues. Part I. Thermodynamic Aspects and Potential Applications in Reproductive and Regenerative Medicine.

Kinetic (dynamic) vitrification is a promising trend in cryopreservation of biological materials because it allows avoiding the formation of lethal intracellular ice and minimizes harmful effects of highly toxic penetrating cryoprotectants. A uniform cooling protocol and the same instruments can be used for practically all types of cells. In modern technologies, the rate of cooling is essentially limited by the Leidenfrost effect. We describe a novel platform for kinetic vitrification of biological materials KrioBlast TM that realizes hyper-fast cooling and allows overcoming the Leidenfrost effect. This opens prospects for creation of a novel technology of cell cryopreservation for reproductive and regenerative medicine.

Влияние инкубации с криопротекторами на количество эпитопов Gal-α-1,3-Gal в клетках линии PK-15

Transplantation of bioprostheses derived from bovine and porcine tissues is an advanced method of correcting defects in blood vessels and valves, however when using it a hyperacute immune response associated with the presence of Gal-alpha- 1,3-Gal (I±-Gal epitope) on the surface of cell membranes can develop. We have studied the effect of penetrating and nonpenetrating cryoprotectants on the amount of I±-Gal epitopes on membranes of PK-15 cell line of porcine origin. Cell labeling with BSIB4 lectin conjugated with FITC and flow cytometry revealed a significant increase in the number of fluorescent cells after incubation with various concentrations of dimethyl sulfoxide and polyethylene glycol with MW of 1500 (PEG-1500). The revealed effect may be associated with an additional exposure of I±-Gal epitopes on cell membrane, locally deformed due to the action of cryoprotectants.Probl Cryobiol Cryomed 2016; 26(4): 331–339

Влияние инкапсуляции в альгинатные микросферы на жизнеспособность мезенхимальных стромальных клеток после экспозиции с проникающими криопротекторами

The effect of encapsulation into alginate microspheres (AMS) on viability of mesenchymal stromal cells (MSCs) after exposure to the solutions of penetrating cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG) and 1,2-propane diol (1,2‑PD) with concentrations varying from 1.5 to 9 M during 1.5 and 5 min was studied. It was shown that the viability of MSCs both as a suspension and encapsulated in AMS decreased with a rise in cryoprotectant concentration and exposure time. Encapsulation into AMS significantly protected the cells from cytotoxic damage of the cryoprotectants, e. g . if the 5-min exposure to 9 M DMSO, EG and 1,2-PD resulted in the death of quite all the cells in the suspension, the viability of encapsulated MSCs after exposure under similar conditions made 60, 80 and 52% respectively. It was found that the studied penetrating cryoprotectants had a various cytotoxicity to MSCs, and it decreased in the row DMSO > 1,2-PD > EG. The findings can be used to develop a multi-component vitrifying solution. Probl Cryobiol Cryomed 2016; 26(3): 213–220

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

029 Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

Cholesterol to phospholipid ratio is used as a representation for membrane fluidity, and predictor of cryopreservation success but results are not consistent across species and ignore the impact of membrane proteins. Therefore, this research explored the modulation of membrane fluidity and protein organization of rooster sperm during cooling and cryopreservation using the fluorescent stains Merocyanine 540 (phospholipid order) and Rhodamine 640 (protein order). Rooster semen samples (3 roosters and breeds per pool; 4 pools total) were diluted in Lakes Low Temperature (LLT) and Sasaki diluent and cooled to 5 °C over 30 min (Step 1). The samples were then diluted with the penetrating cryoprotectants (CPA) glycerol (LLT) or methylacetamide (Sasaki), equilibrated for 30 min (Step 2), loaded into straws and cryopreserved. The cryopreservation and thawing (Step 3) was performed using the optimized procedures for each diluent. Computer automated semen analysis (CASA) was used to analyze motility characteristics and flow cytometry was used to analyze plasma membrane phospholipid, protein organization and plasma membrane integrity (PMI) at each step. Percent values from CASA and flow cytometry fluorescent medians (FM) were transformed using arcsine and Box–Cox, respectively. The GLM ANOVA was used to detect differences in motility characteristics and membrane organization and included the effects of cryopreservation step and rooster semen pool. In the total motility analyses cryopreservation diluent/CPA was a significant ( P P P P P Source of funding: USDA-ARS-NCGRP National Animal Germplasm Program. Conflict of interest: None declared. phil.purdy@ars.usda.gov

090 The influence of cryopreservation on marine microalgal cells

The development of cryopreservation methods for microalgae opens great prospects for marine biotechnology and aims to establish a bank of cryopreserved cultures. Nine of eleven marine microalgal species used in this study (the diatoms, green, red, and golden algae), including five previously untested species, were successfully recovered after freezing to ultra-low temperatures (−196 °C) using penetrating (Me 2 SO, glycerol, and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants. The chief factor for the successful preservation of microalgal cells during freeze-thawing was shown to be the cooling rate. The survival rate for many algal species did not exceed 15%, but recovery of the initial cell concentration usually took 6–12 days after thawing. The pigment composition and content were evaluated as additional indicators of the algal functional activity. The pigment content tended to decrease after thawing as compared with unfrozen cells and increase during cell recovery. Three diatom species ( Attheya arenicola , A. longicornis , and A. ussurensis ) were successfully cryopreserved for the first time. For these weakly silicified marine diatoms, step freezing created the possibility of gradual cryoprotectant penetration inside the cells and a stepwise release of water out of the cells. None of the tested protocols gave a positive result for the diatoms of the Pseudo-nitzschia genus. The failure in cryopreservation of the Pseudo-nitzschia species may be explained by peculiarities in the cell wall composition: the Pseudo-nitzschia cell wall is composed of more silica and fewer organic components than those of the other tested diatoms, which makes the wall much more rigid and, probably, not permeable enough for penetrating protectants, even if the step freezing method is used. In our experiments with the Pseudo-nitzschia species, we observed that most of the thawed cells were dead but had almost undamaged cell walls. In contrast, the high tolerance to a wide range of cryopreservation conditions of the tiny green algae may be explained by its low water content, small cell size and thin cell wall. Cryosensitivity of marine algal cells was shown to depend on the differences in natural intrinsic characteristics rather than their taxonomic position. The mechanisms by which some algae withstand freezing better than others are not yet clear, but we suggest that the biochemical and physical properties of algal cell walls determine the water flow inside and outside the cell and enable the high flexibility needed for adaptation to different environments. Our results are a step in the development of a cryopreservation technology for some marine microalgae and the search for the nature of their cryoresistance. Source of funding: Grant 1 12-I-P6-07 of the Far Eastern Branch of Russian Academy of Sciences, grant 1 12-04-31974 of the Russian Foundation for Basic Research, grant 1 12-04-13006-12 of the Russian Foundation for Basic Research-Far Eastern Federal University, and grant 1 11 G34.31.0010 of Program at the Far Eastern Federal University. Conflict of interest: None declared. borodandy@gmail.com

Optimization of Ethylene Glycol Concentrations, Freezing Rates and Holding Times in Liquid Nitrogen Vapor for Cryopreservation of Rhesus Macaque (Macaca mulatta) Sperm

Ethylene glycol (EG) has been speculated to be the most appropriate penetrating cryoprotectant for cryopreservation of rhesus macaque sperm due to its higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN(2)) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.18, 0.35, 0.7, 1.4 and 2.1 M), 0.7 M EG showed the most effective cryoprotection (P<0.05). Sperm frozen with 0.7 M EG at -183°C/min showed higher post-thaw motility than sperm frozen at -10, -67 or -435°C/min (P<0.05). Sperm frozen in LN(2) vapor at -183°C/min with 0.7 M EG and a holding time of 10 min showed higher post-thaw motility compared with a holding time of 5 or 15 min (P<0.05). The function of sperm cryopreserved at the optimized EG concentration, freezing rate and holding time was further evaluated by in vitro fertilization. Of the 36 oocytes collected from gonadotropin-stimulated rhesus macaques, 61.1% were fertilized, and 61.1, 44.4 and 36.1% of the oocytes developed to 2 cells, morulae and blastocysts, respectively. Our findings provide an alternative penetrating cryoprotectant and optimal protocol for genetic preservation purposes in this important species.

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75–90% motility and 14–16 μm s −1 swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 °C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 °C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40–45% viability and 30–35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5–6 × 10 6/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 °C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.

Slow cooling and temperature-controlled protein crystallography

In cryocrystallography, rapid sample cooling is generally deemed essential to prevent solvent crystallization and associated sample damage. We show that by carefully and completely removing all external solvent, many protein crystals can be successfully cooled to T = 100 K at only 0.1 K/s without additional penetrating cryoprotectants. Slow cooling provides an alternative when flash cooling fails, and enables diffraction studies of protein structure and function at all temperatures between T = 300 K and T = 100 K.

Cryopreservation of mammalian oocytes by using sugars: Intra- and extracellular raffinose with small amounts of dimethylsulfoxide yields high cryosurvival, fertilization, and development rates

Accumulation of intra- and extracellular sugars such as trehalose, glucose, and raffinose is central to survival strategies of a variety of organisms coping with extreme conditions including freezing and almost complete drying. The objective of the present study was to investigate the potential application of intra- and extracellular raffinose in combination with low concentrations of dimethylsulfoxide (Me2SO) to mammalian oocyte cryopreservation. To this end, the fertilization and embryonic development of cryopreserved metaphase II (M II) mouse oocytes were studied in comparison to unfrozen controls. For cryopreservation, M II oocytes were microinjected with 0.1M raffinose, and then cooled to −196°C in the presence of either 0.3M raffinose and 0.5M Me2SO (cryopreservation group 1) or 0.3M raffinose and 1.0M Me2SO (cryopreservation group 2). The control groups included untreated oocytes (untreated control) and oocytes microinjected with raffinose, but not frozen (injection control). The post-thaw survival rates were 83.9% and 80.6% for the cryopreservation group 1 and 2, respectively. The fertilization and blastocyst rates in the cryopreservation group 1 (90.0% and 77.8%, respectively) and 2 (94.6% and 72.5%, respectively) were also high and similar to the ones of the injection controls (97.8% and 78.5%, respectively) and untreated controls (98.8% and 83.6%, respectively). These results are consistent with the findings of our earlier studies and support the use of sugars as intra- and extracellular cryoprotectants. Furthermore, the results of the present study indicate that the presence of intra- and extracellular sugars alleviates high concentrations of conventional penetrating cryoprotectants, and thus minimizes their toxicity.

Successful Cryopreservation of Mouse Oocytes by Using Low Concentrations of Trehalose and Dimethylsulfoxide1

Sugars such as trehalose, sucrose, and glucose are effectively used by a variety of animals (e.g., brine shrimp, tardigrades, some frogs, and insects), as well as by bacteria, yeasts, and plant seeds to survive freezing and extreme drying. The objective of this study was to examine the potential application of sugars to mammalian oocyte cryopreservation. To this end, we used trehalose, a nonreducing disaccharide, and mouse metaphase II oocytes as models. Our experiments show that extracellular trehalose alone affords some protection at high subzero temperatures (e.g., -15 degrees C), which diminishes with further cooling of the oocytes to -30 degrees C and below. When present both intracellularly and extracellularly, trehalose dramatically improves the cryosurvival with increasing extracellular concentrations to 0.5 M, even after cooling to -196 degrees C. Furthermore, the combination of intracellular and extracellular trehalose with small amounts of a conventional penetrating cryoprotectant (i.e., 0.5 M dimethylsulfoxide) provide high survival, fertilization, and embryonic development rates statistically similar to untreated controls. When transferred to foster mothers, cryopreserved oocytes give rise to healthy offspring showing the proof of principle. Our experiments with differential scanning calorimetry indicate that when cooled using the same cryopreservation protocol, the mixture of 0.5 M trehalose and cryopreservation medium undergoes glass transition at high subzero temperatures, which further substantiates the use of sugars as intracellular and extracellular cryoprotectants. Taken together, our results are in agreement with the survival schemes in nature and demonstrate the successful use of sugars in cryopreservation of mammalian oocytes.

Vitrification Successfully Preserves Hepatocyte Spheroids

This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196 degrees C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 +/- 14.39 to 70.38 +/- 11.29 microg/10(6) cells and from 56.84 +/- 14.48 to 71.79 +/- 16.65 microg/10(6) cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.

Stallion semen cryopreservation using different cryoprotective agents on the skim milk trehalosa extender

Cryoprotective agents (CPAs), protect the sperm during cryopreservation. There are two general classes of CPAs, at first penetrating cryoprotectants, these pass through the sperm membrane and act both intracellular and extracellularly, and the second non-penetrating cryoprotectants, these act only extracellularly. The objective of this study was to evaluate the addition of different CPAs namely glycerol (G), combination of ethylene glycol with glycerol (EG) and dimethilformamide (DMF) using skim milk trehalosa extender. The semen collected from 3 stallions using artificial vagina twice aweeks. The semen was evaluated, centrifugated and diluted in skim milk extender supplemented with 50 mM trehalose and three different CPAs with the concentration of sperm were 200x10 6 ml -1 . Extended semen was then packed into minitub 0.3 ml and equilibrated at 4 o C for 2 hours, freeze in the liquid nitrogen vapor for 10 minutes and stored in liquid nitrogen container -196 o C. After 24 hours, the semen was thawed at 37 o C for 30 second. Results of this experiment indicated that the percentage of motile and viable sperm in skim trehalosa extender using DMF and glycerol better than combination of ethylene glycol and glycerol. Key Words : Cryopreservation, Stallion Sperm, Cryoprotectant