Vitrification of human monocytes

Human monocytes purified from peripheral blood by counterflow centrifugal elutriation were cryopreserved in a vitreous state at 1 atm pressure. The vitrification solution was Hanks' balanced salt solution (HBSS) containing (w/v) 20.5% Me2SO, 15.5% acetamide, 10% propylene glycol, and 6% polyethylene glycol. Fifteen milliliters of this solution was added dropwise to 1 ml of a concentrated monocyte suspension at 0 degrees C. Of this, 0.8 ml was drawn into silicone tubing and rapidly cooled to liquid nitrogen temperature, stored for various periods, and rapidly warmed in an ice bath. The vitrification solution was removed by slow addition of HBSS containing 20% fetal calf serum. The numerical cell recovery was about 92% and most of these retained normal phagocytic and chemotactic ability. Differential scanning calorimeter records of the solution show a glass transition at -115 degrees C during cooling and warming, but no evidence of ice formation during cooling. Devitrification occurs at about -70 degrees C during warming at rates as rapid as 80 degrees C/min. The amount of devitrification is dependent upon the warming rate. Freeze-fracture freeze-etch electron microscope observations revealed no ice either intra- or extracellularly in samples rapidly cooled to liquid nitrogen temperatures except for small amounts in some cellular organelles. However, if these cell suspensions were warmed rapidly to -70 degrees C and then held for 5 min, allowing devitrification to occur, the preparation contained significant amounts of both intra- and extracellular ice. Biological data showed that this devitrification was associated with severe loss of cell function.     

Vitrification of rabbit tissues with propylene glycol and trehalose

A previous study had suggested the use of a mixture of propanediol and trehalose for the preservation of tissues by vitrification. In this paper, we describe experiments in which stepwise procedures were developed for adding these cryoprotectants to high final concentrations in two rabbit tissues—carotid artery and cornea. The tissue concentration of the additives was measured at the end of each step so that the temperature of the next step could be chosen to reduce toxicity but avoid freezing. This process was arrested when a concentration had been reached that should permit vitrification if the tissues were cooled rapidly to −175 °C. They were stored at that temperature; warmed rapidly by conduction; the cryoprotectants removed by stepwise dilution; and appropriate active functions measured. These were contraction and relaxation for arteries and endothelial integrity and ability to control stromal swelling for the corneas. In control experiments the exposure and functional assays were carried out without vitrification. It was shown that the tissue concentration of propanediol was 33%w/w in artery and 30% in cornea. These permitted cooling to −175 °C without freezing but devitrification occurred during the warming of the arteries, though not of the corneas, despite the lower tissue concentration reached in the cornea. The function of the vitrified arteries was severely reduced but the endothelium of the corneas was substantially intact although we were unable to demonstrate any ability to control stromal swelling during normothermic perfusion. It appears that concentrations of cryoprotectants sufficient to prevent freezing in these tissues during cooling were well tolerated so long as appropriate stepwise means of addition and removal were used. Devitrification during warming remained a major problem with arteries, but not with corneas. We suggest that the composition of the aqueous phase in the tissue with respect to components other than the vitrifying agents may be crucial here and that the search for agents that will suppress devitrification is an important avenue for further study.     

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.     

Glass transition behavior of the vitrification solutions containing propanediol, dimethyl sulfoxide and polyvinyl alcohol

Knowledge of the glass transition behavior of vitrification solutions is important for research and planning of the cryopreservation of biological materials by vitrification. This brief communication shows the analysis for the glass transition and glass stability of the multi-component vitrification solutions containing propanediol (PE), dimethyl sulfoxide (Me₂SO) and polyvinyl alcohol (PVA) by using differential scanning calorimetry (DSC) during the cooling and subsequent warming between 25 and −150 °C. The glass formation of the solutions was enhanced by introduction of PVA. Partial glass formed during cooling and the fractions of free water in the partial glass matrix increased with the increasing of PVA concentration, which caused slight decline of glass transition temperature, T_g. Exothermic peaks of devitrification were delayed and broadened, which may result from the inhibition of ice nucleation or recrystallization of PVA.     

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage,access and transport

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me₂SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me₂SO and EG, respectively. Concentration changes in EG/Me₂SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.     

Thermodynamic aspects of vitrification

Vitrification is a process in which a liquid begins to behave as a solid during cooling without any substantial change in molecular arrangement or thermodynamic state variables. The physical phenomenon of vitrification is relevant to both cryopreservation by freezing, in which cells survive in glass between ice crystals, and cryopreservation by vitrification in which a whole sample is vitrified. The change from liquid to solid behavior is called the glass transition. It is coincident with liquid viscosity reaching 10¹³ Poise during cooling, which corresponds to a shear stress relaxation time of several minutes. The glass transition can be understood on a molecular level as a loss of rotational and translational degrees of freedom over a particular measurement timescale, leaving only bond vibration within a fixed molecular structure. Reduced freedom of molecular movement results in decreased heat capacity and thermal expansivity in glass relative to the liquid state. In cryoprotectant solutions, the change from liquid to solid properties happens over a ∼10 °C temperature interval centered on a glass transition temperature, typically near −120 °C (±10 °C) for solutions used for vitrification. Loss of freedom to quickly rearrange molecular position causes liquids to depart from thermodynamic equilibrium as they turn into a glass during vitrification. Residual molecular mobility below the glass transition temperature allows glass to very slowly contract, release heat, and decrease entropy during relaxation toward equilibrium. Although diffusion is practically non-existent below the glass transition temperature, small local movements of molecules related to relaxation have consequences for cryobiology. In particular, ice nucleation in supercooled vitrification solutions occurs at remarkable speed until at least 15 °C below the glass transition temperature.     

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

Summary In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.Abbreviations BA 6-benzylaminopurine - DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige and Skoog medium (1962) - PVS2 vitrification solution     

Effective vitrification of human induced pluripotent stem cells using carboxylated ε-poly-l-lysine

Derivation of human induced pluripotent stem (iPS) cells could enable their widespread application in future. Establishment of highly efficient and reliable methods for their preservation is a prerequisite for these applications. In this study, we developed a vitrification solution comprising ethylene glycol (EG) and sucrose as well as carboxylated ε-poly-l-lysine (PLL); this solution inhibited devitrification. Human iPS cells were vitrified in 200-μL vitrification solutions comprised 6.5 M EG, 0.75 M sucrose and 0 or 10% w/v carboxylated PLL with 65 mol% of the amino groups converted to carboxyl groups [PLL (0.65)] in a cryovial by directly immersing in liquid nitrogen. After warming, attached colony and recovery rates of human iPS cells vitrified by adding PLL (0.65) were significantly higher than those for cells without PLL (0.65) and vitrification solution (DAP213: 2 M dimethyl sulfoxide, 1 M acetamide and 3 M propylene glycol). Furthermore, even after warming at room temperature, attached colony and recovery rates of iPS cells vitrified with PLL (0.65) were reduced to a lesser extent than those vitrified with either DAP213 or EG and sucrose without PLL (0.65). This could be attributed to inhibition of devitrification by PLL (0.65), as differential scanning calorimetry indicated less damage after vitrification with PLL (0.65). In addition, human iPS cells vitrified in the solution with PLL (0.65) had normal karyotypes and maintained undifferentiated states and pluripotency as determined by immunohistochemistry and teratoma formation. Addition of PLL (0.65) successfully vitrified human iPS cells with high efficiency. We believe that this method could aid future applications and increase utility of human iPS cells.     

Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification

Cultured cells and somatic embryos derived from the mesophyll tissue of asparagus (Asparagus officinalis L.) were cryopreserved by vitrification. The vitrification solution (PVS) contains (w/v) 22% glycerol, 15% ethylene glycol, 15% propylene glycol and 7% DMSO in Murashige-Skoog medium enriched with 0.5M sorbitol. After initial cryoprotection with sorbitol supplemented MS medium containing 12% ethylene glycol, cells or embryos were exposed stepwise to 85% PVS at 0°C. They were loaded into 0.5 ml transparent straws, and were then plunged directly into liquid nitrogen. After rapid warming, PVS was removed and diluted stepwise. The highest survivals of vitrified cells and embryos were about 65 and 50%, respectively. Surviving embryos developed into plantlets.Abbreviations DMSO dimetyl sulfoxide - PVS vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - MS Murashige-Skoog salt medium - NAA naphthalene acetic acid - BA 6-benzyladenine     

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196 °C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250 °C/min to −196 °C and for each cooling rate, subjecting them to five warming rates back above 0 °C at rates ranging from 610 to 118,000 °C/min. In samples warmed at the highest rate (118,000 °C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610 °C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000 °C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.     

Biotransformation activity in vitrified human liver slices

In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (-196 degrees C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,2-propanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37 degrees C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7-ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 microM 7-EC and its primary metabolite 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans.     

Developmental competence and gene expression of immature oocytes following liquid helium vitrification in bovine

The objective of this study was to develop an effective ultra-rapid vitrification method and evaluate its effect on maturation, developmental competence and development-related gene expression in bovine immature oocytes. Bovine cumulus oocyte complexes were randomly allocated into three groups: (1) controls, (2) liquid nitrogen vitrification, and (3) liquid helium vitrification. Oocytes were vitrified and then warmed, the percentage of morphologically normal oocytes in liquid helium group (89.0%) was significantly higher (P < 0.05) than that of the liquid nitrogen group (81.1%). When the vitrified–thawed oocytes were matured in vitro for 24 h, the maturation rate in liquid helium group (50.6%) was higher (P < 0.05) than liquid nitrogen group (42.6%). Oocytes of liquid helium vitrification had higher cleavage and blastocyst rates (41.1% and 10.0%) than that of liquid nitrogen vitrification (33.0% and 4.5%; P < 0.05) after in vitro fertilization. Moreover, the expression of GDF9 (growth/differentiation factor-9), BAX (apoptosis factor) and ZAR1 (zygote arrest 1) was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) when the vitrified–thawed oocytes were matured 24 h. The expression of these genes was altered after vitrification. Expression of GDF9 and BAX in the liquid helium vitrification group was not significantly different from that of the control, however there were significant differences between the liquid nitrogen vitrification group and control. In conclusion, it was feasible to use liquid helium for vitrifying bovine immature oocytes. There existed an association between the compromised developmental competence and the altered expression levels of these genes for the vitrified oocytes.     

Comparison between the temperatures of intracellular ice formation in fresh mouse oocytes and embryos and those previously subjected to a vitrification procedure

When cells that have been subjected to supposedly innocuous freezing or vitrification procedures are used as the source material for subsequent experiments, it is important that they possess or exhibit the same relevant properties as fresh cells. In this study, we compared the temperatures of intracellular ice formation (IIF) in previously vitrified mouse oocytes/embryos with those in fresh intact ones. In the case of MII oocytes, 2-cell embryos, 4-6-cell embryos, and morulae, there are no significant differences (p > 0.05); namely, -33.3 °C (fresh) vs. -35.4 °C (vitrified) with MII oocytes, -40.6 °C (fresh) vs. -38.7 °C (vitrified) with 2-cell embryos, -38.0 °C (fresh) vs. -39.4 °C (vitrified) with 4-6-cell embryos, -24.5 °C (fresh) vs. -24.2 °C (vitrified) with morulae. But, in 8-cell embryos, there is a significant difference (p < 0.05) between fresh (−37.9 °C) and vitrified (−32.9 °C). If we include this significant difference, the overall IIF temperature of fresh cells is 0.74 °C lower than that of previously vitrified cells. If we exclude it, the IIF temperature for fresh cells is 0.32 °C higher than that for previously vitrified cells. Our conclusion then is that there is no difference between the IIF temperatures of fresh and previously vitrified cells.     

Rapid and uniform electromagnetic heating of aqueous cryoprotectant solutions from cryogenic temperatures

Devitrification (ice formation during warming) is one of the primary obstacles to successful organ vitrification (solidification without ice formation). The only feasible approach to overcoming either devitrification or its damaging effects in a large organ appears at present to be the use of some form of electromagnetic heating (EH) to achieve the required high heating rates. One complication of EH in this application is the need for warming within a steel pressure vessel. We have previously reported that resonant radiofrequency (RF) helical coils provide very uniform heating at ambient temperatures and low heating rates and can be modified for coaxial power transmission, which is necessary if only one cable is to penetrate through the wall of the pressure vessel. We now report our initial studies using a modified helical coil, high RF input power, and cryogenic aqueous cryoprotectant solutions [60% (w/v) solution of 4.37 M dimethylsulfoxide and 4.37 M acetamide in water and 50% (w/w) 1,2-propanediol]. We also describe the electronic equipment required for this type of research. Temperatures were monitored during high-power conditions with Luxtron fiberoptic probes. Thermometry was complicated by the use of catheters needed for probe insertion and guidance. The highest heating rates we observed using catheters occurred at temperatures ranging from about -70 to -40 degrees C, the temperature zone where devitrification usually appears in unstable solutions during slow warming. We find that in this range we can achieve measured heating rates of approximately 300 degrees C/min in 30- to 130-ml samples using 200 to 700 W of RF power without overheating the sample at any point. However, energy conservation calculations imply that our measured peak heating rates may be considerably higher than the true heating rates occurring in the bulk of our solutions. We were able to estimate the overall true heating rates, obtaining an average value of about 20 degrees C/min/100 W/100 ml, which implies a heating efficiency close to 100%. It appears that it should be possible to warm vitrified rabbit kidneys rapidly enough under high-pressure conditions to protect them from devitrification.     

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, −10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.     

Relationship between bovine oocytes developmental competence and mRNA expression of apoptotic and mitochondrial genes following the change of vitrification temperatures and cryoprotectant concentrations

The present study analyzed the relationship between bovine oocytes developmental competence and mRNA expression of apoptotic and mitochondrial genes following the change of vitrification temperatures (VTs) and cryoprotectant agent concentrations (CPAs). Cumulus oocyte complexes were randomly divided into five groups: control, vitrified in liquid nitrogen (LN; −196 °C) with 5.6 M CPAs (LN 5.6 M), LN with 6.6 M CPAs (LN 6.6 M), liquid helium (LHe; −269 °C) with 5.6 M CPAs (LHe 5.6 M), and LHe with 6.6 M CPAs (LHe 6.6 M). After vitrification and warming, oocytes of vitrified and control groups were subjected to in vitro maturation (IVM), in vitro fertilization and in vitro culture. The blastocyst rate in LHe 5.6 M group was the highest among the four vitrified groups (13.7% vs. 9.4%, 1.3%, and 8.4%; P < 0.05). The mRNA expression level of 8 apoptotic- and 12 mitochondria-related genes were detected through qRT-PCR after IVM. Lower VT (LHe, −269 °C) positively affected the mRNA expression levels of apoptotic genes (BAD, BID, BTK, TP53, and TP53I3) and mitochondrial genes (COX6B1, DERA, FIS1, NDUFA1, NDUFA4, PRDX2, SLC25A5, TFB1M, and UQCRB), and reduced oxidative stress from freezing. Decreased CPAs (5.6 M) positively affected mRNA expression levels of apoptotic genes (BAD, BCL2A1, BID, and CASP3) in LHe vitrification but negatively affected apoptotic genes (BAD, BAX, BID, BTK, and BCL2A1) in LN vitrification. In conclusion, decreased VTs and CPAs in LHe vitrification may increase the blastocyst rate by changing the mRNA expression levels of these apoptotic and mitochondrial genes for the vitrified oocytes.     

New device for the vitrification and in-straw warming of in vitro produced bovine embryos

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0 M sucrose; protocol W3) or directly in 0.5 M (protocol W1/0.5) or 0 M sucrose (protocol W1/0). Similar survival rates were recorded 24 h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24 h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5 M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70 °C) were tested. When warming solution passed through the new vitrification/warming device at 45 °C, 61.5% of blastocysts were fully re-expanded or hatched at 24 h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24 h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45 °C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.     

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.Abbreviations DMSO dimethyl sulfoxide - PVS2 vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - BA 6-benzylaminopurine - MT Murashige-Tucker basal medium - INAA naphthaleneacetic acid     

The anti-oxidant roles of Taurine and Hypotaurine on acrosome integrity,HBA and HSPA2 of the human sperm during vitrification and post warming in two different temperature

Despite advances in vitrification techniques for sperm cryopreservation, cryo-damages of sperm caused by generation of reactive oxygen species (ROS) continue to impede implementation of this technique. This study analyses the effects of taurine and hypotaurine as anti-oxidants during vitrification of human sperms. The study was performed in two steps. In the first step, 20 normospermic semen samples were vitrified in the presence of varying concentrations of taurine and hypotaurine, and their effects as anti-oxidant agents on classical sperm parameters, hyaluronan-binding assay (HBA), lipid peroxidation (LPO) and acrosome reaction (AR) were studied. Statistical analyses showed that the sperm parameters in all vitrified groups decreased significantly (P < 0.05) compared to the fresh group. However, HBA and acrosome integrity in vitrified groups containing taurine and 50 mM of hypotaurine were better than in the control group (P < 0.05). The morphology of the vitrified group was good only in the group that contained 50 mM of hypotaurine (P < 0.05).Based on the results from the first step, 50 mM of hypotaurine was considered the ideal anti-oxidant formulation and further tests were carried out on 10 normospermic semen samples with this protecting agent. In addition to the mentioned parameters, the expression of heat shock proteinA2 (HSPA2) was studied in the vitrified group with 50 mM hypotaurine, warmed under two different warming temperatures 37 and 42 °C. 50 mM Hypotaurine was found to equally improve motility, morphology, HBA, and AR after warming at 37 °C and 42 °C (P < 0.05). However, at both warming temperatures, the expression of HSPA2 was reduced in all vitrified groups comparing to the fresh group (P < 0.05). In conclusion, taurine and hypotaurine antioxidants, especially 50 mM hypotaurine, are able to reduce deleterious cryo-injuries on morphology, acrosome and HBA and improve sperm recovery at both warming temperatures (37 and 42 °C). However, they do not have any protective action on expression of HSPA2.     

Physical aspects of vitrification in aqueous solutions

Vitrification is the process by which a liquid solidifies at temperatures usually far below the normal freezing point, but without the formation of any crystalline phase. The liquid has formed a glass. Occasionally, when the liquid consists of a solution, one of the components freezes to form a crystalline phase during cooling, but the remainder of the solution vitrifies. The product is then a partially crystallized glass. Glass formation, as a feature of aqueous solutions, either of the whole solution or of the remaining fraction after crystallization of ice, is discussed. We focus on the general principles involved in glass formation and also discuss in detail the effect of pressure on the nucleation and vitrification of the solution. In particular we look at the physical processes involved as well as the chemical aspects of the solutes which can be used in vitrifiable aqueous solutions. In any application of vitrification of aqueous solutions the material properties of the resultant glass are also important; these are also briefly considered. Recent work concerning the nature of the devitrification (crystallization during warming) event at high pressures is detailed.