Low Concentrations Of Cryoprotectants Research Articles

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200–300 μm) were randomly assigned into four groups. Group1 – Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2– FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2– FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (−196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Cryopreservation: Synergistic Ice Inhibition Effect Enhances Rapid Freezing Cryopreservation with Low Concentration of Cryoprotectants (Adv. Sci. 6/2021)

In article number 2003387, Hai Wang, Li Song, Gang Zhao and co-workers have designed tungsten diselenide (WSe2)-polyvinyl pyrrolidone (PVP) nanoparticles (NPs) for synergetic ice inhibition based on the hydrogen bonds, absorption-inhibition, and photothermal conversation effects. The WSe2-PVP NPs can significantly facilitate the high-efficiency cryopreservation of cell constructs using a rapid freezing method with low concentrations of cryoprotectants.

Synergistic Ice Inhibition Effect Enhances Rapid Freezing Cryopreservation with Low Concentration of Cryoprotectants.

Despite recent advances in controlling ice formation and growth, it remains a challenge to design anti‐icing materials in various fields from atmospheric to biological cryopreservation. Herein, tungsten diselenide (WSe2)‐polyvinyl pyrrolidone (PVP) nanoparticles (NPs) are synthesized through one‐step solvothermal route. The WSe2‐PVP NPs show synergetic ice regulation ability both in the freezing and thawing processes. Molecularly speaking, PVP containing amides group can form hydrogen bonds with water molecules. At a macro level, the WSe2‐PVP NPs show adsorption‐inhibition and photothermal conversation effects to synergistically restrict ice growth. Meanwhile, WSe2‐PVP NPs are for the first time used for the cryopreservation of human umbilical vein endothelial cell (HUVEC)‐laden constructs based on rapid freezing with low concentrations of cryoprotectants (CPAs), the experimental results indicate that a minimal concentration (0.5 mg mL−1) of WSe2‐PVP NPs can increase the viabilities of HUVECs in the constructs post cryopreservation (from 55.8% to 83.4%) and the cryopreserved constructs can also keep good condition in vivo within 7 days. Therefore, this work provides a novel strategy to synergistically suppress the formation and growth of the ice crystalsfor the cryopreservation of cells, tissues, or organs.

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants.

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.

26 Effect of different cryoprotectant concentrations on vitrification of invitro-matured bovine oocytes in paper containers

A great challenge for successful oocyte vitrification is the development of a low-cytotoxic cryoprotectant solution in a safe device allowing ultra-rapid cooling. This study compared different concentrations of cryoprotectants for bovine IVM-oocyte vitrification in a safe paper container device on oocyte survival and cleavage rates. Abattoir ovaries were obtained and cumulus–oocyte complexes (COCs) were recovered by aspirating follicles of 3 to 6mm in diameter. A total of 470 COCs with homogeneous cytoplasm oocytes, surrounded by several layers of cumulus cells were selected, in 5 replicates. Groups of ∼50 COCs were matured in 500µL of semi-defined IVM medium for 22h at 38.8°C in a humidified atmosphere with 5% CO2. After IVM, COCs were allocated to 1 of 3 groups of 20 to 30 COCs, differing only in final concentration of cryoprotectants. A nonvitrified control group (CG) was also tested, totalling 4 groups. Before vitrification, each group was transferred to 500µL of TCM-199 HEPES with 20% fetal bovine serum (FBS) (Base medium, BM) for 5min at 34°C, and COCs were partially denuded by gentle pipetting. Vitrification followed a 3-step protocol at room temperature and groups of 4 to 5 COCs were transferred to BM solution drops containing (1) 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO) for 30 s; (2) 10% EG + 10% DMSO + 0.25M sucrose for 30 s; and (3) vitrification solution (VS), according to each group: high (HG), 20% EG + 20% DMSO + 0.5M sucrose; medium (MG), 15% EG + 15% DMSO + 0.5M sucrose; or low (LG), 10% EG + 10% DMSO + 0.5M sucrose for 30s. Afterwards, COCs were loaded in &amp;lt;1µL of solution and placed in a homemade paper container device, and immediately plunged in liquid nitrogen. Warming was performed placing the paper container in 3mL of 1M sucrose in BM for 2min. After warming, a 3-step protocol was conducted and COCs were transferred to (1) 500µL of 0.5M sucrose in BM for 2 min; (2) 500µL of 0.25M sucrose for 2 min; (3) 500µL of BM for 2min. Then, COCs from each group were transferred to 250µL of semi-defined IVF medium. Motile sperm were recovered by Percoll washing from one bull and added to IVF medium (Day 0) at final concentration of 106 sperm mL−1 for 18h. At Day 1, all presumptive zygotes were cultured in 25µL of SOF medium with 5% FBS under mineral oil at 38.8°C with 5% CO2 and 5% O2. Normal data were subjected to ANOVA and post hoc Tukey test. Cleavage rate was recorded at Day 2 after IVF. Oocyte survival rate was similar (P&amp;gt;0.05) among vitrified groups (HG, 80%; MG, 86%; LG,87%). Cleavage rate differed (P&amp;lt;0.05) in all vitrified groups compared with control (CG, 82%; HG, 10%; MG, 16%; LG, 16%). Although no difference (P&amp;gt;0.05) was observed among vitrified groups, MG and LG showed a slightly increased oocyte survival and cleavage rates compared with HG. In conclusion, the use of either medium or low concentrations of cryoprotectants may be a less toxic alternative for vitrification of IVM bovine oocytes on paper device. This research was funded by CAPES/COFECUB (#88881.142966/2017-01).

Equilibrium vitrification of mouse embryos using low concentrations of cryoprotectants

Previously, we developed a method for vitrification of mouse embryos in a near-equilibrium state using EFS35c, PB1 medium containing 35% (v/v) ethylene glycol, and 0.98 M sucrose. This method has advantages in both slow freezing and vitrification. However, since the vitrification solution in this method contains high concentrations of cryoprotectants and thus has high osmolality, the solution would injure oocytes and embryos with high sensitivity to chemical toxicity and high osmolality. In this study, we examined whether embryos could be vitrified in a near-equilibrium state using a solution containing low concentrations of cryoprotectants and thus with low osmolality. To investigate whether embryos were vitrified in a near-equilibrium state, 2-cell mouse embryos were vitrified with EDFS10/10a, PB1 medium containing 10% (v/v) ethylene glycol, 10% (v/v) DMSO, and 0.4 M sucrose, in liquid nitrogen, stored at −80 °C for 4–28 days, and warmed in water at 25 °C. The viability of the embryos was evaluated by the appearance of embryos after warming and developmental ability. When embryos were vitrified in liquid nitrogen using EDFS10/10a, the survival and developmental ability into blastocysts after storage at −80 °C for 7 days were high, indicating that embryos were vitrified in a near-equilibrium state. A high proportion of embryos vitrified with EDFS10/10a developed to term after transportation with dry ice, re-cooling in liquid nitrogen, and transfer to recipients. Therefore, new equilibrium vitrification developed in this study may be useful for oocytes and embryos that are highly sensitive to the toxicity of cryoprotectants and high osmolality.

Low cryoprotectant concentration rapid vitrification of mouse oocytes and embryos

Vitrification of mammalian oocytes and embryos is typically a two-step procedure involving two solutions of increasing concentrations of cryoprotectants. In the present study, we report a simple vitrification protocol that uses low cryoprotectant concentration and a single medium (LCSM). This medium, along with the traditional high concentration two media (HCTM) protocol, was used to vitrify mouse oocytes, zygotes, and blastocysts using silica capillary, cryotop, cryolock, and 0.25 ml straws. Survival rates, two-cell rates, and blastocyst formation rates were compared for oocytes and zygotes vitrified using both protocols. Results show that the LCSM protocol was as good as or better than the traditional HCTM protocol for vitrifying mouse MII oocytes and zygotes using silica capillary, cryotop, and cryolock. On the other hand, for blastocysts, only silica capillary using LCSM had comparable results with the traditional HCTM protocol while cryolock and cryotop had significantly lower percentages of re-expanded and hatched blastocysts. Collapsing blastocysts prior to vitrification or longer duration for better cryoprotectant distribution in multicellular embryos may improve the outcome. In conclusion, the LCSM protocol, with one medium of much lower cryoprotectant concentrations and shorter equilibration time, reduces exposure to cryoprotectant toxicity while improves efficiency, consistency and reliability for mammalian oocyte and embryo preservation.

Hydrogel Microfiber Encapsulation Enhances Cryopreservation of Human Red Blood Cells with Low Concentrations of Glycerol.

Cryopreservation of red blood cells (RBCs) has been studied as a typical example of cryobiology methodology. To date, a mature and long-term cryopreservation process for RBCs has been developed, which has the weakness of complicated procedures due to high concentrations of glycerol (Gly). Therefore, it is still a research focus to find a new method for cryopreservation of RBCs to reduce the concentrations of cryoprotectants (CPAs). In this study, alginate hydrogels, which have been widely used in preservation research, were selected because of their advantages such as lower cost, cytocompatibility, and crosslinking occurring under mild conditions. With a variety of CPA solutions, the RBC recovery with the cryopreservation of RBCs and RBC-laden hydrogel microfibers was compared in different situations. It was found that hydrogel microfiber encapsulation enhances cryopreservation of human RBCs with low concentrations of CPAs. And Gly can be removed by washing directly with a 0.9% sodium chloride solution. This study validated the feasibility of cryopreservation of RBC-laden hydrogel microfibers. It may provide a new and evolving direction for reducing the concentrations of CPAs used in the preservation of RBCs.

The synergistic effect of trehalose and low concentrations of cryoprotectants can improve post-thaw ram sperm parameters

The objective of this study was to compare the effects of different concentrations of two different cryoprotectants (glycerol, G and ethylene glycol, EG) and trehalose (T), added to the semen extender, on post-thaw ram sperm parameters. Ejaculates, collected from 6 Merino rams, were pooled and evaluated at 37 °C. The pooled samples were divided into six equal aliquots, and diluted in Tris-based extenders containing 5% G, 3% G + 60 Mm T, 1.5% G + 100 Mm T, 5% EG, 3% EG + 60 mM T, and 1.5% EG + 100 Mm T. Subsequently, the samples were cooled to 5 °C, frozen in 0.25-ml French straws, and stored in liquid nitrogen (LN2). Frozen samples were thawed individually, at 37 °C for 25 s in a water bath, for evaluation. Sperm motility was assessed using a phase-contrast microscope with a warm stage maintained at 37 °C. Acrosome integrity (FITC/PNA-PI), sperm viability (SYBR-14/PI), mitochondrial activity (JC-1/PI), DNA damage (COMET assay) and DNA fragmentation (TUNEL test) were determined. The group of samples diluted in an extender containing 5% of glycerol (Group 5% G) displayed higher percentages of subjective motility, viability and mitochondrial activity of sperm, compared to the other groups (P < 0.05). On the other hand, Group 3% G + 60 mM T yielded the second-best results for subjective motility, viability and mitochondrial activity of sperm, when compared to the other groups. The post-thaw sperm parameters of Group 3% G + 60 Mm T did not show any statistically significant difference from those of Group 5% G. There were no statistically significant differences between the groups for acrosome integrity (P > 0.05).The results of the COMET assay showed that the use of low concentrations of cryoprotectants in combination with trehalose decreased sperm DNA damage. Accordingly, Group 1.5% G + 100 mM T and Group 3% EG + 60 mM T benefited from a significantly stronger cryoprotective effect on DNA integrity, in comparison to Group 5% G (P < 0.05). According to the results of the TUNEL test, the combined use of low concentrations of cryoprotectants with trehalose decreased sperm DNA damage, when compared to the use of 5% glycerol, but this difference was statistically insignificant (P > 0.05).In conclusion, G and EG concentrations can be reduced by adding various amounts of T (60 mM, 100 mM) to the semen extender. The addition of 5% of glycerol and 3% G + 60 mM T to the semen extender did not yield statistically different post-thaw sperm parameters, when compared for protection against cryoinjury. Post-thaw sperm parameters can be improved by the supplementation of the semen extender with 3% G + 60 mM T. Thus, we recommend the use of freezing extenders containing low cryoprotectant concentrations (3% G) combined with trehalose to avoid the high level of toxic and osmotic damage caused by 5% G.

29 Time-lapse analysis of bovine embryos derived after invitro fertilization from vitrified and fresh oocytes

Oocyte vitrification enables the long-term conservation of female genetic resources. However, obtaining viable bovine embryos from vitrified oocytes has proven to be difficult. Embryo development is a dynamic process, and critical stages can go unnoticed with the use of traditional morphologic assessments. Therefore, the aim was to evaluate morphokinetic parameters in embryos derived from fresh versus vitrified oocytes. After 22h of IVM, oocytes were divided into (1) oocytes surrounded by multiple layers of cumulus cells (COCs; n=275) and (2) oocytes partially denuded, leaving only corona radiata (CR; n=178). Then, two-thirds of the CR oocytes were subjected to one of two vitrification protocols as follows: high concentration of cryoprotectants (CR-H; n=171; Ortiz-Escribano et al. 2016 Theriogenology 86, 635-641) and low concentration of cryoprotectants (CR-L; n=163; Ishii et al. 2018 J. Reprod. Develop. 6). After warming, vitrified oocytes were incubated ~2h in maturation medium. Invitro fertilization and culture were performed simultaneously for all groups. Four to eight zygotes from each group were assigned randomly to time lapse (Primo Vision; Vitrolife), and group culture was performed in leftovers as a control. Differences between groups in survival (intact zygotes after IVF), cleavage, and blastocyst rates were evaluated by logistic regression. Morphokinetic data (time to reach first (1-2 cells), second (3-4 cells), third (5-8), fourth (9-16 cells), fifth (&amp;gt;16 cells), cleavage, and blastocyst stage, and time in lag phase) were investigated. Survival rates in COCs (98%) and CR (96%) groups were not different; CR-H (87%) showed lower survival than COCs but similar survival to that of CR. Group CR-L (82%) had a lower survival rate than the rest of the groups (P&amp;lt;0.05). Higher cleavage rate (83%) was found in COCs compared with the rest of the groups. The CR and CR-H groups showed similar cleavage rate (65 and 55%, respectively), whereas CR-L had a lower cleavage rate (42%) than other groups (P&amp;lt;0.05). As expected, both vitrified groups showed lower blastocyst rates (4% for CR-H and 10% for CR-L) than fresh COCs (44%; P&amp;lt;0.05). Morphokinetics measures were affected by the treatments: time to reach first cleavage was similar for COCs (35.3h), CR (38.4h), and CR-L (34.8h), whereas CR-H was slower (42.4h). However, the fourth division was reached earlier by CR-H (51.7h), which was significantly faster than for CR-L (88.8 h; P&amp;lt;0.05) but similar for COCs (71.3h) and CR (60.7h). In conclusion, more morphokinetic data are needed to compare different vitrification methods and confirm whether time-lapse analysis can be used to predict blastocyst outcome after oocyte vitrification.

Ultra-fast vitrification of patient-derived circulating tumor cell lines.

Emerging technologies have enabled the isolation and characterization of rare circulating tumor cells (CTCs) from the blood of metastatic cancer patients. CTCs represent a non-invasive opportunity to gain information regarding the primary tumor and recent reports suggest CTCs have value as an indicator of disease status. CTCs are fragile and difficult to expand in vitro, so typically molecular characterization must be performed immediately following isolation. To ease experimental timelines and enable biobanking, cryopreservation methods are needed. However, extensive cellular heterogeneity and the rarity of CTCs complicates the optimization of cryopreservation methods based upon cell type, necessitating a standardized protocol. Here, we optimized a previously reported vitrification protocol to preserve patient-derived CTC cell lines using highly conductive silica microcapillaries to achieve ultra-fast cooling rates with low cryoprotectant concentrations. Using this vitrification protocol, five CTC cell lines were cooled to cryogenic temperatures. Thawed CTCs exhibited high cell viability and expanded under in vitro cell culture conditions. EpCAM biomarker expression was maintained for each CTC cell line. One CTC cell line was selected for molecular characterization, revealing that RNA integrity was maintained after storage. A qPCR panel showed no significant difference in thawed CTCs compared to fresh controls. The data presented here suggests vitrification may enable the standardization of cryopreservation methods for CTCs.

Evaporation heat transfer of liquid nitrogen on microstructured surface at high superheat level

Liquid nitrogen, as a coolant, is generally applied in cell vitrification cryopreservation. It takes heat from the carrier with cell samples through its violent evaporation on the carrier surface. As a result, the temperature of the carrier plunges dramatically. This article focuses on the unsteady evaporation heat transfer characteristics of liquid nitrogen on a microstructured surface etched into the frozen carrier surface at a high superheat level. The heat flux and evaporation heat transfer coefficient of liquid nitrogen were investigated using a lumped capacitance method. The experimental results showed that the cooling rate of the thin film evaporation on the microstructured surface is obviously higher than that of pool boiling, which is currently being used for cell cryopreservation. The heat flux and the evaporation heat transfer coefficient work together to present a parabolic trend with the superheat decreasing during this heat transfer process. Besides, the microstructure of the surface has an important effect on the evaporation heat transfer of liquid nitrogen. The larger the thin film evaporation zone is, the higher the heat transfer coefficient is. The current investigation results in a cell cryopreservation method through vitrification with relatively low concentrations of cryoprotectants.

A study on vitrification of HUVECs at a low concentration of cryoprotectants in a quartz capillary

A study on vitrification of HUVECs at a low concentration of cryoprotectants in a quartz capillary

Cell blebbing upon addition of cryoprotectants: a self-protection mechanism.

In this work, the mechanism of cell bleb formation upon the addition of cryoprotectants (CPAs) was investigated, and the role of cell blebs in protecting cells was determined. The results show that after adding CPAs, the hyperosmotic stress results in the breakage of the cortical cytoskeleton and the detachment of the cell membrane from the cortical cytoskeleton, causing the formation of cell blebs. Multiple blebs decrease the intracellular hydrostatic pressure induced by the extracellular hyperosmotic shock and alleviate the osmotic damage to cells, which reduces the cell mortality rate. In the presence of a low concentration of CPAs, cell blebs can effectively protect cells. In contrast, in the presence of a high concentration of CPAs, the protective effect is limited because of severe disruption in the cortical cytoskeleton. To determine the relationship between blebs and the mortality rate of cells, we defined a bleb index and found that the bleb index of 0.065 can be regarded as a reference value for the safe addition of DMSO to HeLa cells. The bleb index can also explain why the stepwise addition of CPAs is better than the single-step addition of CPAs. Moreover, the mechanism of the autophagy of cells induced by the hyperosmotic stress was studied, and the protective effect associated with the autophagy was compared with the effect of the blebbing. The findings reported here elucidate a self-protection mechanism of cells experiencing the hyperosmotic stress in the presence of CPAs, and they provide significant evidence for cell tolerance in the field of cryopreservation.

C-20: Tissue vitrification

Application of the original tissue vitrification method developed by Fahy and used by my research group for rabbit blood vessels and articular cartilage has evolved over time. The formulation was 2.21 M propylene glycol, 3.10 M formamide and 3.10 M dimethylsulfoxide in EuroCollins solution and it was employed with multiple addition/removal steps to minimize osmotic damage to the cells. Ice-free cryopreservation using VS55, as originally described, is limited to small or thin samples where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides excellent extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using higher concentrations of the same cryoprotectants in EuroCollins solution has several advantages. High viability can be achieved for human and porcine articular cartilage employing an 83% formulation, VS83, in combination with multiple addition/removal steps. Lower cryoprotectant concentrations are not effective for large mammal articular cartilage. VS83 can also be used for tissue matrix preservation with retention of material properties at the expense of cell viability. Addition and removal of the cryoprotectants can be done in a single step followed by washing to minimize residual chemical concentrations. This protocol minimizes tissue cell viability and also reduces tissue immunogenicity suggesting that this vitrification strategy may be an alternative to the decellularization methods being employed for engineering tissues for medical implants. The evolution of these vitrification protocols will be reviewed drawing upon research employing natural and engineered tissues. Disclosure: Author was or is employed by Cell & Tissue Systems, Inc.

Vitrification of Common Carp (Cyprinus Carpio) Spermatozoa, Post-Thaw Sperm Quality, and Fertility

The aim of this investigation was to test a new technology, vitrification, or ultra-rapid freezing of the spermatozoa of common carp, and to study the ability of glucose, BSA, and other cryoprotectants to protect these cells from cryo-injuries. Spermatozoa were isolated and vitrified using 10 different cryoprotectant solutions: (1) Glucose based medium (GBM) + 1% bovine serum albumin (BSA); (2) GBM + 1% BSA + 10% DMSO; (3) GBM + 1% BSA + 20% DMSO; (4) GBM + 1% BSA + 30% DMSO; (5) GBM + 1% BSA + 10% DMA; (6) GBM + 1% BSA + 20% DMA; (7) GBM + 1% BSA + 30% DMA; (8) GBM + 1% BSA + 10% methanol; (9) GBM + 1% BSA + 20% methanol; (10) GBM + 1% BSA + 30% methanol. Fertilization rates for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization rates than the vitrification solutions containing high cryoprotectant concentrations. In conclusion, this study reported successful vitrification of common carp spermatozoa by direct transfer into liquid nitrogen.

034 Survival of mouse oocytes after cooling in lower cryoprotectant concentrations by ultra rapid warming using an IR laser pulse

Seki and Mazur have recently shown [PLoS One. 2012;7(4)] that about 90% of MII mouse oocytes survive vitrification in solutions of EAFS 10/10 that contain only half the usual concentration of solutes, provided that the warming rate is extremely high (117,000 °C/min). They reported that fewer than 10% survived if the concentration of EAFS were further reduced to 33% of normal. We are currently investigating whether high survivals could be obtained in 0.33× and 0.25× EAFS if the warming rates were still higher. For this purpose we have cooled MII oocytes in these media on Cryotops to −196 °C and warmed them 5 times more rapidly at ∼600,000 °C/min by subjecting them to a 13 joule pulse of 15 msec duration from a Nd:YAG laser. Kleinhans et al in a companion paper at this meeting discuss the physics attributes of the laser and the interactions of the pulse with the sample, as well as the jig he developed to permit the manipulation of the Cryotop before and during its exposure to the laser pulse. The osmotic/morphological survival of 31 samples suspended in 0.25× EAFS was 15.2 ± 22% (standard deviation). The survival of two samples in 0.33× EAFS was 30%. The total molality of these solutions is 1.27 and 1.72, respectively. These are in the range used in standard slow-freeze cryopreservation. For comparison, note that the total molality of full strength 1x EAFS is 7.37 molal, 5.8 and 4.3-fold higher. Although these preliminary results are encouraging, we have two caveats. One is that the variability of the former is high as evidenced by the large standard errors. The other is that the survival of controls subjected to the same cooling procedure but warmed at 117,000 °C/min (no laser) was close to the same (13.3% vs. 15.2%). Source of funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. Conflict of interest: None declared. pmazur@utk.edu

Ultra-high cooling rate utilizing thin film evaporation

This research introduces a cell cryopreservation method, which utilizes thin film evaporation and provides an ultra-high cooling rate. The microstructured surface forming the thin film evaporation was fabricated from copper microparticles with an average diameter of 50 μm. Experimental results showed that a cooling rate of approximately 5[Formula: see text]10(4) °C/min was achieved in a temperature range from 10 °C to -187 °C. The current investigation will give birth to a cell cryopreservation method through vitrification with relatively low concentrations of cryoprotectants.

Directional Freezing of Reproductive Cells and Organs

Directional freezing is based on a simple thermodynamic principle where ice crystals are precisely controlled through the sample by regulating the velocity of the sample movement through the predetermined temperature gradient. Directional freezing permits a precise and uniform cooling rate in both small and large volume samples. Directional freezing was used for slow and rapid freezing, as well as for vitrification of oocytes and embryos using the minimum drop size technique. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The method enabled, for the first time, successful freezing of a whole ovary and freeze-drying of mammalian cells followed by thawing and transplantation and rehydration, respectively.

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5min. We evaluated a method reported for human sperm, using small volumes in loops (15μl) or cut standard straws (20μl) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (∼2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol+10% methyl glycol+10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol+10% methyl glycol+10% propanediol (∼50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.