Application of Vacuum Infiltration in Cryopreservation of Isolated Grape Buds

Preserving the gene pool of grapes, referred to the vegetatively propagated plants is a complicated task, which can be also solved by cryopreservation of the buds. To saturate such bulk and heterogeneous samples with cryoprotectants the novel methods are required. The effectiveness of vacuum infiltration and 60 min standard passive saturation (soaking) of isolated grape buds of the Russian Concord variety with a cryoprotective solution PVS 2 were compared in this research. To saturate by vacuum infiltration the buds were incubated in cryoprotective solution for 15 min at 40 kPa, afterwards, the pressure was gradually increased to atmospheric level. The efficiency of bud saturation was evaluated with low-temperature differential scanning calorimetry by changing the enthalpies and temperatures of phase transitions as well as the intensity of heat capacity jump at glass transition. The use of vacuum in the saturation of isolated grape buds were found to lead to a strong rise in cryoprotectant concentration in them and a significant decrease in the amount of free water crystallized during cooling compared to passive soaking in a PVS 2 vitrification solution.

Efficiency of cryoprotectors for cryopreservation of two orchid species from Americas

The objective of this study was to evaluate the efficiency of cryoprotective solution (PVS2) combined with phloroglucinol for the cryopreservation of seeds of two orchid species, Encyclia cordigera and Epidendrum ciliare. Seeds of Encyclia cordigera had 91.03% initial viability and 91.99% germination. The treatment of the seeds with PVS2 at 0 °C with 1% phloroglucinol for 60 min returned 93.79% viability and 91.01% germination after recovery from LN, consequently resulting in faster development of protocorms. For Epidendrum ciliare, seed viability was 85.65% and germination was 85.90%. Seed exposure to the PVS2 at 0 °C with 1% phloroglucinol for 180 min showed viability of 39.23% and germination of 37.88%. Despite lower germination, 78.90% of the protocorms reached stage P3 of development, when evaluated 45 days after sowing, not significantly different from the control 1, and showed normal development. These results indicate that PVS2 cryoprotective solution is efficient when combined with phloroglucinol for the cryopreservation and successful recovery of seeds of Encyclia cordigera and Epidendrum ciliare. The present study also indicates that response to cryopreservation and success of recovery after cold storage is species-specific and requires adjustments in exposure time to PVS2 at 0 °C prior to immersion in LN.

Effect of Vitamins and Amino Acids as Cryoprotectant Solution Supplementation for the Cryopreservation of Tambaqui Semen (Colossoma macropomum)

Background: The addition of antioxidant substances to a cryoprotective solution can increase its protective capacity, shielding spermatozoa from the oxidative stress caused by the seminal cryopreservation process. However, there is no record of a seminal cryopreservation protocol of tambaqui (Colossoma macropomum) using antioxidants as a supplement to the cryoprotective solution. Thus, the objective of this study was to evaluate the effects of adding vitamin C, vitamin E, cysteine, and/or taurine to the seminal cryopreservation of tambaqui.Materials, Methods & Results: Pools of semen (n = 10) were diluted in cryoprotective solutions supplemented with: vitamin C (T1), vitamin E (T2), vitamin C + vitamin E (T3), cysteine (T4), taurine (T5), and taurine + cysteine (T6). The control treatment (T7) was not supplemented. Diluted semen was loaded in 0.5 mL straws, frozen in a dry-shipper, stored in a cryogenic cylinder, and then thawed in a water bath (45ºC for eight seconds). The quality of fresh and cryopreserved semen was evaluated by measuring total motility, progressive motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity, and straightness using a computerized system of sperm analysis. Sperm membrane integrity parameters were analyzed using eosin-nigrosin staining, sperm morphology was assessed using pink bengal staining, and motility duration was measured by a digital timer. Data were analyzed using the statistical program SAS (2002) and the results were expressed as means ± standard error of the mean. The results showed that, in general, there was no significant increase in seminal quality when antioxidants were added to the cryoprotective solution. The T5 treatment promoted an increase (P < 0.05) in progressive motility when compared to T1 (6.33 ± 1.14% and 2.98 ± 0.88%, respectively). However, it did not differ significantly (P > 0.05) from the other treatments. Treatments T2 and T5 presented the highest values of VCL (34.74 ± 2.58 and 33.60 ± 1.81 μm.s-1, respectively). These were higher (P < 0.05) than T1 (26.31 ± 1.64 μm.s-1) but not different (P > 0.05) from the T7 control (30.87 ± 1.49 μm.s-1). The VSL and VAP results showed that T1 presented the lowest velocity (9.89 ± 1.75 and 15.06 ± 1.92 μm.s-1, respectively) compared to the other treatments (P < 0.05) that did not differ from each other. Combining the two vitamins (T3) or the two amino acids (T6) was not advantageous in relation to the use of only one of these antioxidants.Discussion: The present study reports, for the first time, results of the addition of antioxidants to the tambaqui seminal freezing medium. The addition of taurine and vitamin E, although not significantly different from the control treatment, resulted in a tendency to increase sperm kinetics. This effect may be due to the action of taurine as a regulator of Ca2+ transporters, which is necessary to trigger sperm activation, and to the ability of vitamin E to scavenge reactive oxygen species produced during lipid peroxidation. On the other hand, the reduced sperm quality observed when vitamin C was used may have been related to the toxicity caused by a high concentration of this vitamin. In addition, once the safe dose of antioxidants has been exceeded, the normal physiological functions of reactive oxygen species can be inhibited. Thus, it is concluded that the use of vitamin E and taurine promotes promising results of curvilinear velocity after thawing of sperm. Therefore, these treatments are recommended, as well as more tests to determine their optimal concentrations.

An Ultrafast Vitrification Method for Cell Cryopreservation

Ultrafast cooling is the key to successful cell vitrification cryopreservation of lower concentration cryoprotective solution. This research develops a cell cryopreservation methodology which utilizes thin film evaporation and achieves vitrification of relatively low concentration cryoprotectant with an ultrafast cooling rate. Experimental results show that the average cooling rate of dimethylsulfoxide (DMSO) cryoprotective solution reaches 150,000 °C/min in a temperature range from 10 °C to −180 °C. The ultrafast cooling rate can remarkably improve the vitrification tendencies of the cryoprotective solution. This methodology opens the possibility for more successful cell vitrification cryopreservation.

Criopreservação de sementes de rainha do abismo (Sinningia leucotricha)

The objective was to evaluate the use of cryogenic solutions in cryopreservation Sinningia leucotricha seeds by the vitrification method in liquid nitrogen. The treatments were: T1 - control: without cryoprotective solution; T2 - PVS1; T3 - modified PVS1; T4 - PVS2; T5 - modified PVS2; T6 - PVS3; T7 - PVS3 modified; T8 - PVS2 + 1% phloroglucinol. After 15 days of immersion of seeds in LN, the cryotubes were removed and rapidly reheated to a temperature of 40 °C water bath for 1.5 minutes. Then, the seeds were washed with wash solution for 20 min. Later they were submitted to the germination test which was conducted on blotting paper moistened with distilled water packaged in crystal polystyrene boxes kept in a growth chamber at 25 ± 2 °C and 16 hours photoperiod. Was evaluated the germination percentage the germination speed index (GSI), and at the end of the experiment will determine the length of shoot (LS) and root (LR), and dry mass of seedlings (DMS). We used a completely randomized design with eight treatments and five replications, consisting of 100 seeds. The direct submission of the queen of the abyss seeds in liquid nitrogen provided the highest germination values, GSI, LS, LR and DMS. The queen of the abyss seeds can be cryopreserved in liquid nitrogen directly without the need to cryoprotectant solutions.

An Ultra Fast Cooling Method for Cell Vitrification Cryopreservation Utilizing Thin Film Evaporation

Ultra-fast cooling is the key to successful cell vitrification cryopreservation of lower concentration cryoprotective solution. This research develops a cell cryopreservation methodology which utilizes thin film evaporation and achieves vitrification of relatively low concentration cryoprotectant with an ultra-fast cooling rate. Experimental results show that the average cooling rate of dimenthylsulphoxide cryoprotective solution reaches 150,000°C/min in a temperature range from 10°C to −180°C. The ultra-fast cooling rate can remarkably improve the vitrification tendencies of the cryoprotective solution. This methodology opens the possibility for more successful cell vitrification cryopreservation.

Comparision Of Different Cryoprotective Solution - Albumin Vs Autologous Plasma - Its Effect On Cell Recovery and Clonogenic Potential Of Cryopreserved Peripheral Blood Hematopoietic Stem and Progenitor Cells

Background Peripheral blood is a preferable source of hematopoietic stem and progenitor cells (HSPCs) used for autologous transplantation. HSPCs are mobilized to peripheral blood and collected by leukapheresis. Prior to cryopreservation the cells need to be processed including the addition of cryoprotective mixture as dimethyl sulfoxide (DMSO) prediluted in human serum albumin solution (HSAS). In Europe there is no commercially available albumin manufactured in packs with tubing which would enable the use of sterile tubing welder. Alternatively cryoprotective solution can be prepared using autologous plasma (AP) obtained during the same leukapheresis, allowing for the preparation of HSPCs in a completely closed system and hence to reduce the risk of contamination. The goal of our study was to test if the HSAS may be replaced by autologous plasma without negative impact on cell recovery and clonogenicity. Methods Samples were prospectively collected from 18 patients with multiple myeloma (n=13) and lymphomas (n=5) mobilized with chemotherapy combined with G-CSF. Small volumes (1.5 ml) of cell suspensions obtained from the leukapheresis products were divided into 2 parts (0,5ml) placed in separate small vials, each containing different cryoprotective mixture - 5% HSAS or AP with a final 7.5% DMSO concentration. The final volume of cell suspensions equaling 1 ml, the cell concentration (0.7–1.5 × 108 /ml). The cells were frozen in IceCube, using a computer controlled cooling program and stored in liquid nitrogen for 2 - 4 months. Concentration of total protein and individual electrophoretic fractions of plasma proteins were measured. The quality of cryoprotective mixtures was evaluated by cell recovery and clonogenic potential. The recovery was determined by comparing number of living cells before and after cryopreservation, using trypan blue staining. Clonogenic potential was carried out by colony forming unit (CFU) assays. Depending on CD34+ percentage, 5-40 × 103 living cells were plated (in triplicates) in MethoCult medium and cultured for 14 days. Results The median recovery of nucleated cells for AP was 68.3% (range 40.6-96.1) and was similar to HSAS 68.5%, (41.7-100); (p=0.3; Wilcoxon matched pairs test). The number of CFUs calculated per 100 000 cryopreserved cells did not differ significantly between tested cryoprotective mixture: 187.3 (11.3-806.3) for albumin, 130.5 (15-924.2) for autologous plasma (p=0.5; Wilcoxon matched pairs test). No significant differences were observed when the number of specific types of CFUs were compared. Neither total protein nor albumin concentration of plasma correlated with the clonogenic potential of the leukapheresis product cryopreserved in AP when samples from patients with higher concentration than median were compared with the other (Mann-Whitney U test). Between January and July 2013 more than 50 successful transplants of autologous HSPCs cryopreserved with 7.5% DMSO prediluted in autologous plasma were performed in our Department. Conclusions Commercially available human serum albumin can be replaced by autologous plasma in procedure of HSPCs cryopreservation. The use of autologous plasma for cryoprotective mixture preparation does not appear to negatively affect cell recovery and clonogenic potential of leukapheresis product. The advantage of such solution is possibility of HSPCs preparation in closed system to reduce risk of auto-HSCT product contamination to the minimum. Disclosures: No relevant conflicts of interest to declare.

Vitrification of cleavage stage mouse embryos by the cryoloop procedure

By decreasing the volume of the cryoprotective solution it is possible to increase dramatically the freezing speed and — at the same time — reduce the toxicity and osmotic side effects of cryoprotectants (CPA). The objective of our study was to vitrify Day-3 cleavage stage mouse embryos (n = 229) with the cryoloop technology using a new composition of vitrification media. Embryos were exposed to a 2-step loading of CPA, ethylene glycol (EG) and propylene glycol (PG), before being placed on the surface of a thin filmy layer formed from the vitrification solution in a small nylon loop, then they were rapidly submerged into liquid nitrogen. After warming, the CPA was diluted out from the embryos by a 3-step procedure. Survival of embryos was based on morphological appearance after thawing and continued development to expanded blastocysts upon subsequent 48-hour culture. Embryos of the two control groups were either treated likewise except that they were not vitrified, or cultured in vitro without any treatment. Our data show that a high percentage of embryos survived (92.7%) vitrification in the mixture of EG and PG combined with cryoloop carrier and developed normally (89.1%) in vitro after thawing. To our knowledge this is the first report of the successful vitrification of cleavage stage mouse embryos using VitroLoop vitrification procedure.

68 VITRIFICATION OF CLEAVAGE-STAGE MOUSE EMBRYOS BY THE VITROLOOP™ (CRYOLOOP) PROCEDURE

By decreasing the volume of the cryoprotective solution, we were able to dramatically increase the freezing speed and decrease the toxicity and osmotic side effects of the cryoprotectants (CP). Several carriers have been developed successfully (Vajta G et al. 1998 Mol. Reprod. Dev. 51, 53–58; Liebermann J et al. 2002 Reprod. Biomed. Online 4, 146–150; Park SP et al. 1999 Hum. Reprod. 15, 1787–1790; Chung HM et al. 2000 Fertil. Steril. 73, 545–551; Kuwayama M and Kato O 2000 Fertil. Steril. 74(Suppl. 3), S49 O-127; Matsumoto H et al. 2001 Cryobiology 42, 139–144; Lane M et al. 1999 Fertil. Steril. 72, 1073–1078; Dinnyés A et al. 2000 Biol. Reprod. 63, 513–518). The objective of our study was to vitrify Day 3 cleavage stage mouse embryos with the Vitroloop™ (Vitrolife, Kungsbacka, Sweden) cryopreservation technology. Vitrification was carried out in RapidVit™ Cleave (Vitrolife) solutions (holding, equilibration, and vitrification medium). Embryos were exposed to a 2-step loading of CP, ethylene glycol (EG), and propylene glycol (PG), before being placed in a small loop attached to the lid of a cryo-vial and rapidly submerged into liquid nitrogen (LN). First, the embryos were transferred from the G-MOPS holding medium to the equilibration medium containing 8% EG for 2 min. Then, embryos were transferred into a 20-μL drop of vitrification medium containing 16% EG, 16% PG, 10 mg mL–1 Ficoll 400, and 0.65 m sucrose for 30 s. After that, the embryos (maximum of 2 at a time) were transferred onto the loop, which was quickly sealed in a cryo-vial of LN and stored. After storage in LN, embryos were warmed by a 3-step dilution of the CP with sucrose (RapidWarmCleave™, Vitrolife) carried out at 37°C. First, the loop with the embryos was quickly immersed in 37°C warming medium 1 (0.65 m sucrose) for 20 s. Then, the embryos were transferred into warming medium 2 (0.25 m sucrose) for 1 min, then into warming medium 3 (0.125 m sucrose) for 2 min, and finally into warming medium 4 (G-MOPS medium) for 5 min. Following warming, embryos were cultured in G1 medium (Vitrolife) at 37°C with 6.5% CO2 and maximum humidity in air. Embryo viability was assessed by 48 h in vitro culture; the survival of embryos was based on morphological appearance in vitro after thawing and continued development to expanded blastocysts upon subsequent culture. The control embryos were treated likewise except that they were not vitrified. A total of 229 cleavage-stage embryos were vitrified and warmed; out of these, 11 were lost (11/229; 4.8%). Of the remaining 218 embryo, 202 survived vitrification (202/218; 92.7%) and 180 developed further to expanded blastocysts during in vitro culture (180/202; 82.6%). In the control group, 91.4% of the embryos developed to expanded blastocysts (75/82) indicating that the solutions used were not toxic. Our data show that a high percentage of cleavage-stage mouse embryos survived vitrification in the mixture of EG and PG combined with the use of cryoloop and developed normally in vitro after thawing. To our knowledge, this is the first report of the successful use of the Vitroloop™ vitrification procedure with cleavage-stage mouse embryos. The authors thank Vitrolife Ltd. (Kungsbacka, Sweden) and FertiCad Ltd. (Budapest, Hungary) for providing the solutions. The 3-month fellowship for Phillip Klambauer was provided by CEEPUS.

83 DEVELOPMENT OF BOVINE IN VITRO-PRODUCED BLASTOCYSTS AFTER CRYOPRESERVATION IN CHEMICALLY DEFINED SOLUTIONS AND ONE-STEP DILUTION

Efficient embryo cryopreservartion using a protein-free defined solution would be beneficial for hygienic commercial embryo transport. In addition, one-step rehydration of frozen–thawed embryos could allow direct transfer of cryopreserved embryos after thawing. The objective of this study was to assess the viability in vitro of bovine in vitro-produced (IVP) embryos after freezing and thawing in a base cryoprotective solution [modified synthetic oviduct fluid (mSOF) medium containing 1.8 m ethylene glycol and 0.05 m trehalose] supplemented with various concentrations of polyvinyl alcohol (PVA). The methods used for in vitro embryo production and embryo cryopreservation were modified from those described previously (Murakami et al. 1998 Cryobiology 36, 206–212). Briefly, oocytes collected from cow ovaries were matured, fertilized in vitro for 5 h, and cultured in mSOF containing 0.4% bovine serum albumin (BSA) at 38.5�C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 (reduced-O2 atmosphere; Day 0). On Day 3, only cleaved embryos were co-cultured with bovine cumulus cells in mSOF containing 5% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 in air. On Day 7, embryos that reached the blastocyst stage were selected and immersed directly into the base cryoprotective solution supplemented with 0.4% BSA (0.4BSA group, the protein-containing control group) or 1.5, 3.0, or 6.0% PVA (1.5PVA, 3PVA, and 6PVA groups, respectively) at room temperature. After exposure to solution, embryo–cryoprotectant solutions were loaded into 0.25-mL plastic straws and cryopreserved by the standard slow freezing method (about 60 to 100 embryos in each group). The frozen straws were placed in air for 10 s, and plunged into a 38.5�C water bath for 10 s; the contents were expelled into a plastic dish. The embryos were then transferred directly to pre-incubated minimum essential medium alpha medium containing 5% FBS and MEM nonessential amino acids solution (mαMEM) for one-step rehydration. These frozen.thawed embryos were washed well and cultured in fresh mαMEM in a reduced-O2 atmosphere for 3 days to examine the developmental potential in vitro. Data were analyzed by ANOVA. The percentage of re-expanded embryos after freezing and thawing was significantly higher (P < 0.05) in 0.4BSA and 1.5PVA groups than in the 3PVA and 6PVA groups (95.4% in 0.4BSA and 90.9% in 1.5PVA v. 73.4% in 3PVA and 57.7% in 6PVA). In addition, there was no significant difference between 0.4BSA and 1.5PVA groups in the percentage of embryos developed into hatched blastocysts (82.0 v. 80.2%), but the percentage decreased with increasing PVA concentration (51.1% in 3PVA and 29.3% in 6PVA, respectively). These results suggest that the biological product BSA added in our standard cryoprotective solution can be replaced with 1.5% PVA to support similar viability of frozen.thawed bovine IVP embryos after one-step dilution. Further studies, including direct transfer of these frozen embryos after thawing, are needed to substantiate these results.