Vitrification Protocol Research Articles

Development of sperm vitrification protocols for freshwater fish (Eurasian perch, Perca fluviatilis) and marine fish (European eel, Anguilla anguilla)

Vitrification was successfully applied to the sperm of two fish species, the freshwater Eurasian perch (Perca fluviatilis) and marine European eel (Anguilla anguilla). Sperm was collected, diluted in species-specific non-activating media and cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). Additional sperm quality parameters such as sperm head morphometry parameters (in case of European eel) and fertilizing capacity (in case of Eurasian perch) were carried out to test the effectiveness of vitrification. The vitrification method for Eurasian perch sperm resulting the highest post-thaw motility (14±1.6%) was as follows: 1:5 dilution ratio, Tanaka extender, 30% cryoprotectant (15% methanol+15% propylene-glycol), cooling device: Cryotop, 2μl droplets, and for European eel sperm: dilution ratio 1:1, with 40% cryoprotectant (20% MeOH and 20% PG), and 10% FBS, cooling device: Cryotop, with 2μl of sperm suspension. Viable embryos were produced by fertilization with vitrified Eurasian perch sperm (neurulation: 2.54±1.67%). According to the ASMA analysis, no significant decrease in head area and perimeter of vitrified European eel spermatozoa were found when compared to fresh spermatozoa.

Assessment of the effect of different vitrification solutions on human ovarian tissue after short-term xenotransplantation onto the chick embryo chorioallantoic membrane.

Chemotherapy can lead to the loss of fertility and premature ovarian failure in young women who suffer from malignant diseases. Freezing ovarian tissue by vitrification allows for the preservation of a large number of follicles prior to treatment, yet no established protocols have been optimized with respect to the vitrification solution. The aim of the present study was to evaluate the early stage of human ovarian tissue xenotransplantated onto the chick embryo chorioallantoic membrane after vitrification, and to determine the effect of different vitrification solutions on ovarian tissue quality-as defined by morphology and viability of follicles, neovascularization, cell proliferation, and apoptosis. Each vitrification protocol had a different impact on ovarian tissue at the early transplantation stage; one process using the lowest concentrations of ethylene glycol and dimethylsulfoxide, plus sucrose, demonstrated a moderate advantage compared to the other protocols. We also demonstrated that the chorioallantoic membrane model can be a useful alternative for short-term xenotransplantation studies of angiogenesis into human ovarian cortical tissue.

Étude prospective comparant l’efficacité de la vitrification ovocytaire en système fermé ou en système ouvert

ObjectivesOocyte vitrification using an open device is thought to be a source of microbiological and chemical contaminations that can be avoided using a closed device. The principal purpose of this study was to compare the two vitrification protocols: closed and open system. The secondary aim was to study the effects of the storage in the vapor phase of nitrogen (VPN) on oocytes vitrified using an open system and to compare it to those of a storage in liquid nitrogen (LN). MethodsForty-four patients have been included in our study between November 2014 and May 2015. Two hundred and fourteen oocytes have been vitrified at germinal vesicle (GV), metaphase I (0PB) and metaphase II (1PB) stages. We vitrified 96 oocytes (59 GV/37 0PB) using a closed vitrification device and 118 oocytes (57 GV/31 0PB/30 1PB) using an open device. The vitrified oocytes were then stored either in LN or in VPN. The main outcome measures were the survival rate after warming (SR), meiosis resumption rate (MRR) and maturation rate (MR). ResultsThe global post-thaw SR was significantly higher for oocytes vitrified using an open system (93.2%) compared to those vitrified using a closed one (64.5%; P<0.001). On the contrary, there was no significant difference in terms of global MRR and MR (82.1% vs. 87.5% and 60.7% vs. 61.2% using closed and open system respectively). The SR, MRR and the MR were not significantly different when vitrified oocytes were stored in VPN or LN (91.6, 83.8, 64.5% vs. 93.9, 89.8, 59.1% respectively). ConclusionTaking into account the limits of our protocol, the open vitrification system remains the more efficient system. The use of sterile liquid nitrogen for oocyte vitrification and the subsequent storage in vapor phase of nitrogen could minimize the hypothetical risks of biological and chemical contaminations.

High survival of mouse oocytes using an optimized vitrification protocol.

The method of vitrification has been widely used for cryopreservation. However, the effectiveness of this method for mammalian oocytes could be improved by optimizing each step of the process. In the present study, we tested the effects of varying several key parameters to determine the most effective protocol for mouse oocyte vitrification. We found that cryoprotectant containing ethylene glycol and dimethylsulfoxide plus 20% fetal calf serum produced the highest rates of oocyte survival, fertilization, and blastocyst formation. The duration and temperature of oocyte exposure to vitrification and thawing solutions influenced survival rate. The presence of cumulus cells surrounding oocytes and the incubation of thawed oocytes in Toyoda-Yokoyama-Hosoki medium also increased oocyte survival. Open pulled straw and nylon loop methods were more effective than the mini-drop method. Finally, the combination of these improved methods resulted in better spindle morphology when compared to the unimproved methods. These results demonstrate that the outcomes of mouse oocyte vitrification can be improved by a suitable combination of cryopreservation methods, which could be applied to future clinical research with human oocytes.

Integration of tissue culture and cryopreservation methods for propagation and conservation of the fern Osmunda regalis L.

A simple and reliable technique for in vitro multiplication and long-term preservation using liquid nitrogen was developed for gametophytes of Osmunda regalis. The effect of Knop’s medium and various concentrations (1/2, 1/4, 1/8) of mineral salts provided in Murashige and Skoog (MS) basal medium, together in the presence or absence of both ammonium nitrate and a full complement of vitamins on gametophyte proliferation and sporophyte production was determined. Moreover, the effectiveness of gametophyte cryopreservation by vitrification and encapsulation–vitrification techniques was assessed. Maximum gametophyte proliferation (89 %) occurred on the ammonium nitrate- and vitamin-free [(-NH4NO3)(-vit)] MS medium with 1/2 or 1/4 strength mineral salts. The maximum production of sporophytes (30 plantlets per gametophyte clump) required 1/8MS (-NH4NO3)(-vit) medium. The flow cytometric analysis revealed that the sporophytes contained twofold more pg DNA than gametophytes. This confirmed that the sporophytes were obtained by sexual reproduction. The vitrification protocol and PVS2 solution were ineffective for cryopreservation. The greatest survival rate (81.6 %) following cryo-exposure occurred following treatment of encapsulated gametophytes with PVS3 solution for 3 h. This protocol allowed the recovery of gametophyte cultures following 6 weeks after rewarming. Finally, 100 % of sporophytes produced in vitro were successfully acclimated to ex vitro conditions. Application of the in vitro and cryopreservation methods made it possible to improve the number and time of O. regalis sporophyte production. Whole system of micropropagation can be completed in approximately 1 year. The protocols open new avenues for the mass propagation, germplasm conservation and resource management of the species.

Addition of sucrose on cryoprotectant solution of vitrification enhances the quality of Dorper sheep embryos produced in vivo

This study aimed to evaluate the efficacy of adding sucrose in vitrification solution of ovine embryos produced in vivo. Forty Dorper ewes were selected and superovulated. Immediately prior to the embryo collection by laparotomy, a laparoscopy was performed to verify the superovulatory response. The recovered flushing was followed by embryo evaluation and embryos were divided in two experimental groups where embryos from Control group were submitted to a traditional vitrification protocol and embryos from Sucrose group to a modified vitrification protocol with sucrose. After warming, embryos were again divided regarding cryoprotectant removal (Indirect) or not (Direct). The embryo quality was classified as embryos of degrees I (excellent or good), II (regular), III (poor) and IV (dead or degenerate). It was also verified the homogeneity of mass, occurrence of embryonic mass retraction and rupture of pellucid zone. The results were expressed as percentages and were subjected to Chi-square test with P &lt; 0.05. The embryos vitrified in the presence of sucrose had lower proportions of lower-quality embryos after warming (22.20 vs. 44.50%), higher percentages of homogeneous embryos after warming (63.89 vs. 38.89 %) while concerning other parameters there was no difference between these groups. It can be concluded that the addition of 0.4 M sucrose during vitrification improves the embryo quality.

Addition of sucrose on cryoprotectant solution of vitrification enhances the quality of Dorper sheep embryos produced in vivo

&lt;p&gt;This study aimed to evaluate the efficacy of adding sucrose in vitrification solution of ovine embryos produced &lt;em&gt;in vivo&lt;/em&gt;. Forty Dorper ewes were selected and superovulated. Immediately prior to the embryo collection by laparotomy, a laparoscopy was performed to verify the superovulatory response. The recovered flushing was followed by embryo evaluation and embryos were divided in two experimental groups where embryos from Control group were submitted to a traditional vitrification protocol and embryos from Sucrose group to a modified vitrification protocol with sucrose. After warming, embryos were again divided regarding cryoprotectant removal (Indirect) or not (Direct). The embryo quality was classified as embryos of degrees I (excellent or good), II (regular), III (poor) and IV (dead or degenerate). It was also verified the homogeneity of mass, occurrence of embryonic mass retraction and rupture of pellucid zone. The results were expressed as percentages and were subjected to Chi-square test with P &amp;lt; 0.05. The embryos vitrified in the presence of sucrose had lower proportions of lower-quality embryos after warming (22.20 vs. 44.50%), higher percentages of homogeneous embryos after warming (63.89 vs. 38.89 %) while concerning other parameters there was no difference between these groups. It can be concluded that the addition of 0.4 M sucrose during vitrification improves the embryo quality.&lt;/p&gt;

51 OPTIMIZATION OF VITRIFICATION PARAMETERS FOR RHESUS MACAQUE BLASTOCYSTS

The cryobiological characteristics of rhesus macaque embryos are not well defined, and only a few studies report limited success rates after recovery from slow-freezing cryopreservation. Vitrification is the method currently in use by many infertility clinics with 85 to 96% embryos surviving post-thaw. However, success of thawing rhesus macaque embryos after vitrification is not well documented. Therefore, multiple vitrification parameters were evaluated on rhesus expanded blastocysts by comparing culture medium, vitrification method, and embryo integrity. Embryos (n = 204) were produced by standard IVF and cultured in either Global (single-step; LifeGlobal Group, Guilford, CT, USA) or Quinns Advantage (sequential; SAGE, Trumbull, CT, USA) in a humidified tri-gas mixture (6% CO2, 5% O2, 89% N2) at 37°C until blastocyst expansion was observed after 7 to 9 days of culture. Embryos were then vitrified in straws using the Global Fast Freeze Vitrification protocol or in cryotops using the Kitazato method (VT 601-Top-Kitazato, Japan). Additional embryos were biopsied with an objective-mounted laser to remove ~10 to 20 trophectoderm cells before vitrification with Global Fast Freeze in straws. Embryos were thawed at 30°C and cultured at 37°C for an additional 48 h or until arrested. Morphology was graded as good (re-expanded), poor (shrunken cytoplasm), or lysed, following thaw, and success of good or hatched embryos was analysed by Fisher’s exact test with P &lt; 0.05. Because our goal was to provide good quality embryos for embryo transfer, the ability to hatch was our standard for determining success. There was no significant difference in embryo quality between culture medium, vitrification method, or embryo integrity. Overall, 90% of the embryos were scored as morphologically good regardless of culture medium based on re-expansion post-thaw &lt;24 h (55/61) but had a relatively low hatching rate of 25% (15/61), demonstrating that not all re-expanded embryos will go on to achieve the hatched state. Initial trials vitrifying with the Global protocol were associated with decreased recovery rates as many embryos were lost. Cryotops were able to increase our recovery rate to 100% (n = 22) but with only 5% surviving post-thaw (1/22). Decreasing the amount of media used to recover embryos from straws enabled a re-evaluation of the cryotop technique with an improved recovery of 92% (60/65). Biopsied embryos tended to have a higher hatch rate (48%; 10/21) compared with intact embryos frozen at the same stage (26%; 9/35). The trophectoderm biopsy likely allows for better permeation of the cryoprotectant before vitrification in embryos due to a collapsed blastocoel. Taken together, these data suggest that vitrification is a viable method of preserving embryos of genetic value and those that undergo manipulation, including trophectoderm.

46 VITRIFICATION AT THE GERMINAL VESICLE STAGE TRIGGERS PRECOCIOUS MEIOTIC RESUMPTION BUT DOES NOT AFFECT CYTOPLASMIC MATURATION IN CUMULUS-ENCLOSED PORCINE OOCYTES DURING IN VITRO MATURATION

Previously we have reported a vitrification protocol that allows preservation of immature porcine oocytes in large numbers (Somfai et al. 2014 PLoS One 9, e97731). However, despite high survival rates, embryo development rates have remained low. The aim of our current research is to reveal factors potentially responsible for reduced developmental competence of vitrified oocytes. As a first step, we investigated the effects of vitrification at the germinal vesicle stage on subsequent nuclear progression and the normality of cytoplasmic functions during in vitro maturation (IVM). Cumulus-enclosed porcine oocytes were vitrified in microdrops, stored, and then warmed by our method (Somfai et al. 2015 Reprod. Fertil. Dev. 27, 124). Then the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cyclic adenosine monophosphate, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG. The following 24 h of IVM was performed in porcine oocyte medium without any supplementation. We compared vitrified/warmed oocytes (vitrified group) with freshly collected immature oocytes (control group) in terms of (1) nuclear progression, (2) intracellular glutathione (GSH), and (3) adenosine triphosphate levels throughout IVM. Each experiment was replicated at least 3 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. A total of 510 oocytes were vitrified of which 422 (82.3%) survived. Only live oocytes were subjected to subsequent assays. Orcein staining revealed that after 22 h of IVM, a significantly higher percentage (P &lt; 0.05) of vitrified oocytes showed germinal vesicle breakdown compared with the control group (22.0 v. 0.9%, respectively). In a similar fashion, after 30 h IVM, a significantly higher (P &lt; 0.05) percentage of oocytes reached the metaphase-II (MII) stage in the vitrified group than in the control group (21.8 v. 0%, respectively). After 46 h of IVM, there was no difference between the vitrified and control groups in terms of the percentage of MII stage oocytes (93.9 and 86.3%, respectively). Analysis of GSH levels in oocytes by the 5,5′-dithio-bis-2-nitrobenzoic acid-glutathione disulfide reductase recycling assay showed no significant difference between the vitrified and control groups at 0 h (6.7 and 7.0 pmol, respectively), 22 h (5.5 and 5.5 pmol, respectively), and 46 h (6.9 and 7.9 pmol, respectively) of IVM. Adenosine triphosphate assay (FL-ASC; Sigma-Aldrich Co., St. Louis, MO) revealed similar adenosine triphosphate contents in the oocytes of the vitrified and control groups at 0 h (1.53 and 1.61 pmol, respectively), 22 h (1.67 and 1.70 pmol, respectively), and 46 h (1.65 and 1.83 pmol, respectively) of IVM. In conclusion, vitrification triggered precocious nuclear maturation even in the presence of dibutyryl cyclic adenosine monophosphate; however, it did not affect GSH levels and overall metabolism. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS.

Cryopreservation of Persian sturgeon (Acipenser persicus) embryos by DMSO-based vitrificant solutions

Vitrification could provide a promising tool for the cryopreservation of fish embryos. To achieve successful cryopreservation, several parameters should be taken into account in the design of a vitrification protocol. In the present study, some relevant factors were investigated (choice of a proper vitrificant solutions and temperature for thawing) using neurulation-stage embryos. Six DMSO-based vitrificant solutions (V1–V6) were tested using a 6-step incorporation protocol. DMSO-based vitrificant solutions contained DMSO + permeable cryoprotectants + nonpermeable cryoprotectants. Embryos were immersed in vitrificant solutions for 7 minutes and directly plunged into liquid nitrogen. After vitrification (−196 °C for 10 minutes), the thawing was performed in a water bath at 0 or 20 °C and then embryos incubated until hatched. Our results demonstrated that some embryos vitrified in 5 of 6 vitrification solutions survived and hatched out, but none survived after vitrification in V2. The highest survival rate (45.45%) was observed in samples frozen with the best vitrificant solution (V6) and thawing combination (20 °C). These results establish that cryopreservation of Persian sturgeon (Acipenser persicus) embryos by DMSO-based vitrificant solutions is possible.

Efficient automatable and aseptic vitrification of human pluripotent stem cells using bio-safe and chemically defined media

Human pluripotent stem (hPS) cells, mainly represented by human embryonic stem (hES) and human induced pluripotent stem (hiPS) cells, are considered as a virtually unlimited material for biomedical research and cell-based therapies. However, their use in the clinics requires efficient and bio-safe handling. Cryopreservation is an obligate key step of storage and transportation, during which the cells undergo extreme physical and chemical conditions prone to alter their viability as well as their biological properties. Human PS cells cryopreservation routinely consists in conventional slow-freezing resulting in poor survival rates mainly due to cell damages due to excessive dehydration and water crystallization. In order to prevent this, several groups have adapted vitrification protocols to hPS cells. Vitrification is a cryopreservation method based on the conversion of a liquid into a glass-like state by an infinite enhancement of its viscosity and without formation of ice crystals. It has been shown that vitrification protocols are more effective than slow freezing (1–3). However, they cannot provide any insurance of biological safety since cells are stored in containers that are predisposed to leakage when plunged into liquid nitrogen (i.e. non aseptic containers). This study presents our newly developed hPS cells cryopreservation method.

Cryopreservation of Day 8 equine embryos after blastocyst micromanipulation and vitrification

Pregnancy rates after cryopreservation of large equine blastocyst stage embryos have remained lower than other domesticated livestock species. It is generally accepted that the embryonic capsule is the primary barrier to cryoprotectant entry into the embryo proper and techniques need to be developed to circumvent this obstacle. Therefore, the objective of this study was to develop an efficient Day 8 equine embryo cryopreservation protocol through blastocyst micromanipulation and vitrification. Grade 1 and 2 embryos recovered from mares (n = 15) 8 days after ovulation were used in these experiments. In experiment 1, the effect of either one- or two-puncture treatments before aspiration of blastocoel fluid and exposure to vitrification solutions was evaluated. No difference was detected in mean embryo volume across treatment groups after exposure to vitrification solutions or after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increase at 48 and 72 hours during in vitro culture were 100%, 83%, and 75% compared with 93%, 67%, and 50% for one- and two-puncture treatment groups, respectively. Capsule loss was 25% for one-puncture and 50% for two-puncture treatment groups. In experiment 2, no difference was detected in mean embryo volume for indirect introduction (aspiration of blastocoel fluid + equilibration) and direct introduction (injection of cryoprotectant into blastocoel cavity) treatment groups, after exposure to dilution solution or to culture medium. There was no difference in mean embryo volume for the indirect and direct introduction treatment groups after 1, 24, 48, and 72 hours of culture. Percent of embryos re-expanding at 24 hours and percent of embryos showing diameter increases at 48 and 72 hours during in vitro culture were 100%, 76.9%, and 69.2%, respectively, for both treatment groups. Those embryos subjected to the direct introduction treatment had a higher (P = 0.05) percent capsule loss (70%) compared with the indirect introduction treatment group (31%). The pregnancy rate after transfer of vitrified expanded Grade 1 blastocysts using the indirect introduction method was 83% (5/6). Three pregnancies were allowed to continue to term and resulted in the birth of three healthy foals. The vitrification protocol used in this study has the potential to become a key tool for the successful cryopreservation of equine expanded blastocysts.

Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel (Camelus dromedarius)

The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me2SO) for equilibration, and cooling in 16% EG + 16% Me2SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ∼24 °C/15 min and body 37 °C/3 min), and the replacement of Me2SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo–derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and re-expansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37 °C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me2SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (P < 0.05). The modified protocol of loading EG at 37 °C for 3 minutes has increased the embryo survival of the original protocol from 67% to 91% and the developmental rate from 57% to 83% at 5-day culture. These results were comparable to or better than those reported in human or other species, indicating that this optimized method is well suited to any commercial embryo transfer program in the dromedary camel.

Development of Cryopreservation Techniques for Gorgonian (Junceella juncea) Oocytes through Vitrification.

Gorgonian corals are slowly declining due to human interaction and environmental impacts. Cryopreservation of gorgonian corals is an ex-situ method of conservation, ensuring future reproduction. The present study assessed the vitrification properties of cryoprotectant (CPT) mixtures using the cryotop, cryoloop and open pulled straw (OPS) cryopereservation methods prior to experimentation on gorgonian (Junceella juncea) oocytes. Investigations of the equilibration and vitrification solutions’ (ES and VS) effect on oocytes throughout different incubation periods were conducted. The cryotop method was found to be the most successful in ensuring vitrification. The most favourable VS was composed of propylene glycol (PG), ethylene glycol (EG) and methanol with concentrations of 3.5M, 1.5M and 2M respectively. Experiments were performed using the cryotop method to cryopreserve Junceella juncea oocytes using VS2, the solution had the least impact on oocytes at 5°C rather than at 26°C. The success of the vitrification procedures was determined by adenosine triphosphate (ATP) levels in cooled-thaw oocytes and the highest viability obtained from the present study was 76.6 ± 6.2%. This study provides information regarding gorgonian corals’ tolerance and viability throughout vitrification to further advance the vitrification protocol on whip corals.

Effect of different rehydration temperatures on the survival of human vitrified-warmed oocytes.

To evaluate the effect of different exposure temperatures during the dilution process on the survival rate of vitrified oocytes and following development. Patients were divided at random into two groups for different dilution temperature (20-22 °C, RT group; 37 °C,37 °C group) according to computer-generated random numbers on the day of oocyte warming. The survival and fertilization rates of vitrified oocytes as well as the implantation and clinical pregnancy rates of the resulting embryos were recorded. A total of 662 and 676 oocytes were warmed in the 37 °C group and RT group, respectively, and significant difference was observed in the survival rate between 37 °C group (88.37%) and RT group (79.88%) (P = 0.0000). There was significant difference between the survival rate of 37 °C group (87.27%) and RT group (75.64%) in nondonor patients (P = 0.0001). Multiple linear regression analysis showed that dilution temperature (β = 0.079, P = 0.017) and clinical outcomes of fresh cycles (β = 0.063, P = 0.001) were significantly and independently associated with survival rate. No significant difference was found between the 37 °C group and RT group in: fertilization rate (66.67 versus 65.37%), implantation rate (20.0 versus 19.46%), clinical pregnancy rate (37.5 versus 35.0%). In conclusion, the results of this study give supportive evidence of the application of 37 °C in the dilution process, especially for oocytes of poor quality. Further studies with well-controlled experimental groups are needed to optimize protocols for human oocyte vitrification.

DEVELOPING REGENERATION SYSTEM FOR CRYOPRESERVATION IN SUGARCANE (SACCHARUM OFFICINARUM L.)

A regeneration system for cryopreservation of sugarcane (Saccharum officinarum L.), commercial cultivar ‘U-Thong 5’, has been established. Somatic embryogenesis has been done using immature inflorescence segments as explants. The highest induction percentage of the initial embryogenic callus was obtained in Murashige and Skoog media supplemented with 10-15 µM 2,4-dichlorophenoxy-acetic acid, 50 ml/L coconut water, 500 mg/L casein hydrolysate, and 6% (w/v) sucrose in dark condition. Embryogenic callus developed to somatic embryos and plantlets on MS media containing 50 ml/L coconut water, 0.5 gm/L orchid fertilizer (21-21-21) and 6% (w/v) sucrose. Shoots were able to root on these media. For cryopreservation, a vitrification protocol using different plant vitrification solutions was employed, PVS2, PVS3, and variants of PVS3 (various concentrations of glycerol and sucrose). Somatic embryos in the early stages have been precultured on MS media containing 50 ml/L coconut water, 2 µM abscisic acid, 0.3 M sucrose for 24 h. After 24 h, the somatic embryos have been cultured on the same media with 0.5 M for 24 h. Subsequently, the somatic embryos have been treated with loading solution comprising of 0.5 M sucrose and 2 M glycerol in MS media for 30 min. Then, somatic embryos were dehydrated with different types of PVSs for 30 min at 5°C prior to being immersed in liquid nitrogen for 7 days. After freezing in liquid nitrogen, regeneration percentages of the somatic embryos treated with PVS3 and variants of PVS3 were 34-42%. In contrast using PVS 2 during dehydration step, lower survival and regeneration percentages of the somatic embryos were obtained, when compared to those of PVS3.

Effects of Cryopreservation on the Molecular Integrity of Ovarian Grafts

INTRODUCTION: The objective of this study was to compare the effects of slow freezing and vitrification on the molecular integrity of ovarian grafts. METHODS: Fresh bovine ovaries were processed and divided into three groups (fresh control, slow freezing, and vitrification) before transplantation. For slow freezing and vitrification, 1.5 M dimethylsulfoxide-based cryoprotectant and a two-step vitrification method were used, respectively. Two pieces of ovarian tissue were transplanted onto the back muscle of the severe combined immunodeficient mouse (six animals for each group). Ovarian grafts were recovered on day 7 and day 28 after transplantation to assess DNA damage, apoptosis, and autophagy by histology, immunohistochemistry, and Western blot analysis. RESULTS: The slow freezing group showed significantly higher normal follicle count compared with the vitrification group. There was no significant difference in apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, caspase 3) among three groups; however, autophagy (LC3B) was decreased in both slow freezing and vitrification groups. On day 28, a significant decrease in LC3B in the vitrification group was noted compared with the slow freezing group. There was no difference in expression of DNA damage and repair markers (γH2AX, Rad51, PCNA) and vascular density makers (CD31). CONCLUSION: The expression pattern of biochemical markers for DNA damage and repair and apoptosis in ovarian grafts was similar between three groups. There is not yet strong evidence that vitrification is better than slow freezing. Therefore, further improved vitrification protocols are needed.

Improved low-CPA vitrification of mouse oocytes using quartz microcapillary

Cryopreservation by low-cryoprotectant (CPA) vitrification has the potential to combine all the advantages of the conventional high-CPA vitrification and slow-freezing approaches while avoiding their drawbacks. However, current low-CPA vitrification protocol for cryopreservation of oocytes requires a lengthy and multi-step procedure for unloading CPAs. In this study, we report a much-simplified procedure of using quartz microcapillary (QMC) for low-CPA vitrification of mouse oocytes with only one step for unloading CPAs. The immediate viability of oocytes after the improved low-CPA vitrification was determined to be more than 90%. Moreover, no significant difference was observed in terms of embryonic development from the two-cell to blastocyst stages between the fresh and vitrified oocytes after in vitro fertilization (IVF). This improved low-CPA vitrification technology has the potential for efficient cryopreservation of oocytes to preserve the fertility of mammals including humans for assisted reproductive medicine, maintenance of animal resource and endangered species, and livestock management.

"Universal" vitrification of cells by ultra-fast cooling.

Long-term preservation of live cells is critical for a broad range of clinical and research applications. With the increasing diversity of cells that need to be preserved (e.g. oocytes, stem and other primary cells, genetically modified cells), careful optimization of preservation protocols becomes tedious and poses significant limitations for all but the most expert users. To address the challenge of long-term storage of critical, heterogeneous cell types, we propose a universal protocol for cell vitrification that is independent of cell phenotype and uses only low concentrations of cryoprotectant (1.5 M PROH and 0.5 M trehalose). We employed industrial grade microcapillaries made of highly conductive fused silica, which are commonly used for analytical chemistry applications. The minimal mass and thermal inertia of the microcapillaries enabled us to achieve ultrafast cooling rates up to 4,000 K/s. Using the same low, non-toxic concentration of cryoprotectant, we demonstrate high recovery and viability rates after vitrification for human mammary epithelial cells, rat hepatocytes, tumor cells from pleural effusions, and multiple cancer cell lines.

Effective cryopreservation of golden Syrian hamster embryos by open pulled straw vitrification.

Golden Syrian hamster embryos are difficult to cryopreserve due to their high sensitivity to cryoprotectants and in vitro handling. The objective of this study is to develop a robust open pulled straw (OPS) vitrification technique for cryopreserving hamster embryos at various developmental stages. We first systematically tested the concentrations of cryoprotectants and the exposure times of two-cell embryos to various vitrification solutions. We identified pretreatment of two-cell embryos with 10% (v/v) ethylene glycol (EG) + 10% (v/v) dimethylsulfoxide (DMSO) for 30 s followed by exposure in the vitrification solution, EDFS30 (containing 15% EG + 15% DMSO), for 30 s before plunging into liquid nitrogen (two-step exposure method) as the optimal OPS vitrification protocol. We then investigated the resourcefulness of this protocol for vitrifying hamster embryos at different developmental stages. The results showed that high blastocyst rates from embryos vitrified at two-cell, four-cell, eight-cell, or morula stage (62%, 78%, 80%, or 72%, respectively), but not those verified at pronuclear (0%) or blastocyst stage (24%; P < 0.05), were achieved by this protocol. When embryos vitrified at the two-cell stage were recovered and then directly transferred to recipient females, 29% of them developed to term, a development rate not significantly different (P > 0.05) from the 40% birth rate of the unvitrified controls. In conclusion, we have developed an effective two-step OPS vitrification protocol for hamster embryos.