Vitrification Solution Research Articles

Effects of different vitrification solutions on ovarian tissue cryopreservation in prepubertal dogs

Effects of different vitrification solutions on ovarian tissue cryopreservation in prepubertal dogs

Evaluation of cryopreservation efficiency on embryo growth after exposing them in intracellular cryoprotectant ethylene glycol or glycerol and replacing them by trehalose

The present study was conducted to see whether toxicity could be reduced by replacing some of the penetrating cryoprotectant (intracellular cryoprotectant) with a disaccharide as well as finding out the optimum equilibration time in a vitrification solution before rapid cooling. Goat ovaries (1,685) were aspirated and 1,761 culturable oocytes (COC) were recovered. The recovered COCs were matured in maturation medium and evaluated on the basis of cumulus expansion. The matured oocytes were coincubated with fresh semen capacitated in TALP media, for 18 h. The inseminated oocytes were further cultured in mSOF for in vitro embryo development. The cleavage rate was 11.76% and the development rate of embryos to 4–8 and 8–16 cells morulae was 55.6% and 42.4%, respectively. In vitro produced goat embryos (132), 4–16 cell stages, were used for cryopreservation using different protocols. In protocol- 1, 2, 3 and 4, there was no survivability of embryos. In protocol- 5, 6, 7 and 8, the survivability of embryo after freezing was 10, 25, 35.7 and 72.2%, respectively. In protocol- 8, the percentage of live embryos was significantly higher than other protocols. Also it was found that the survivability rate of embryos in protocol 6 and 7 were significantly higher as compared to protocol 1, 2, 3, 4 and 5. However, non-significant differences were found among protocol 1, 2, 3, 4 and 5, and between 6 and 7. From our results, it can be concluded that cryopreservation using 20% ethylene glycol and 0.9 M trehalose for 30 min gives significantly higher post thaw (72.2%) survivability of embryos.

Genetic suppression of cryoprotectant toxicity

We report here a new, unbiased forward genetic method that uses transposon-mediated mutagenesis to enable the identification of mutations that confer cryoprotectant toxicity resistance (CTR). Our method is to select for resistance to the toxic effects of M22, a much-studied whole-organ vitrification solution. We report finding and characterizing six mutants that are resistant to M22. These mutants fall into six independent biochemical pathways not previously linked to cryoprotectant toxicity (CT). The genes associated with the mutations were Gm14005, Myh9, Nrg2, Pura, Fgd2, Pim1, Opa1, Hes1, Hsbp1, and Ywhag. The mechanisms of action of the mutations remain unknown, but two of the mutants involve MYC signaling, which was previously implicated in CT. Several of the mutants may up-regulate cellular stress defense pathways. Several of the M22-resistant mutants were also resistant to dimethyl sulfoxide (Me2SO), and many of the mutants showed significantly improved survival after freezing and thawing in 10% (v/v) Me2SO. This new approach to overcoming CT has many advantages over alternative methods such as transcriptomic profiling. Our method directly identifies specific genetic loci that unequivocally affect CT. More generally, our results provide the first direct evidence that CT can be reduced in mammalian cells by specific molecular interventions. Thus, this approach introduces remarkable new opportunities for pharmacological blockade of CT.

Validation of a Universal Warming/Dilution Protocol for Frozen Embryo Transfers Independent of Vitrification Device/Cryoprotectant

Objective Validate a universal warming/dilution approach for all vitrification (VTF) solutions associated with various device systems. Design Using a 2 × 2 factorial arrangement of treatments, 124 blastocysts derived from 363 research consented slow frozen embryos were vitrified in Glycerol/ethylene glycol (EG) solution (≥7.9M; I.C.E.) or a 15%DMSO/15%EG solution, and subsequently rapidly warmed and diluted by standard operating procedures (SOP) or a universal sucrose step-down dilution procedure (n = 31 embryos/group). Furthermore, we performed a retrospective analysis of our routine success with microSecure VTF (µS-VTF with Glycerol/EG)/warming to applying a universal sucrose dilution protocol, independent of vitrification device/solution, to blastocysts shipped in from outside facilities between 2015-2017. In validating our clinical approach, we contrasted live birth rates achieved in the latter groups (A and B, respectively; see Table) to outcomes reported in the 2015 CDC data, including the national average (C) and four local IVF labs/clinics (D-H). Materials and Methods All research blastocysts were vitrified by µS-VTF using solutions and dilution treatments described above. Conversely, all internal FET cycles implemented a standardized sucrose step-down dilution using I.C.E. diluents (T1 -T4; estimated to possess 1.0M, 0.5M, 0.25M and 0.125M sucrose concentrations; 3 min/step), independent of the VTF device/cryoprotectant, upon rapid warming and blastocyst isolation. All blastocysts underwent isotonic equilibration in Hepes buffered media for 5 min at 37°C before being in-vitro cultured until ET or research developmental assessment. Our live birth pregnancy rates for FETs (A,B; autologous oocytes, Results No statistical differences in survival or sustained development was observed between the combined dilution treatments of vitrified research blastocysts. SOP thawing (93.5%) or standard sucrose dilution (90.3%) outcomes were similar. Mean live births (see Table) following our universal sucrose dilution approach (B) revealed similar or improved outcomes to those reported in the 2015 CDC summary (C-G). The apparent reduced live birth success compared to µS-VTF (A) and a local group (H) is likely due to differences in embryo production (i.e., lab effect), not to a warming-dilution effect post-VTF. Conclusion A universal sucrose, step-down dilution approach has proven to be a simple, cost-effective and highly successful procedure in our treatment for vitrified blastocysts derived from outside IVF programs, thus alleviating the need to purchase and maintain various commercial thaw solutions. Disclosures MCS developed µS-VTF without any commercial interests. Funding None.

Resveratrol supplementation promotes recovery of lower oxidative metabolism after vitrification and warming of in vitro-produced bovine embryos.

Although vitrification is the current method of choice for oocyte and embryo cryopreservation, it may have detrimental effects on reduction-oxidation status and mitochondrial activity. The aim of this study was to evaluate the effect of supplementing invitro culture (IVC) media and/or vitrification solutions with the antioxidant resveratrol on active mitochondria, mitochondrial superoxide production and lipid peroxidation. Abattoir-derived oocytes were matured and fertilised invitro using standard procedures. Following IVF (21h later), zygotes were cultured in IVC medium supplemented with 0 or 0.5µM resveratrol. On Day 7, blastocysts were vitrified using the Cryotech Vitrification Kit (Cryo Tech Laboratory) with or without 0.5µM resveratrol. After warming, active mitochondria, mitochondrial superoxide production and lipid peroxidation were evaluated using Mito Tracker Green FM, MitoSOX Red and BODIPY581/591 C11 staining respectively. The vitrification-warming process significantly increased active mitochondria and mitochondrial superoxide production in bovine embryos (P<0.05, ANOVA). The addition of 0.5µM resveratrol to the IVC medium or vitrification solutions significantly attenuated the increase in active mitochondria (P<0.05), but not in mitochondrial superoxide production, whereas embryos cultured and vitrified with resveratrol showed the highest values for both parameters (P<0.05). Regarding lipid peroxidation, no significant differences were detected between treatments. In conclusion, resveratrol supplementation of IVC medium or vitrification solutions contributes to recovery of an embryo's 'quieter' state (i.e. lower oxidative metabolism) after vitrification. However, supplementation of both solutions with resveratrol seemed to have a pro-oxidant effect.

Effects of cryoprotectants and cryoprotectant combinations on viability and maturation rates of Camelus dromedarius oocytes vitrified at germinal vesicle stage.

Camel fertility faces many problems, which could be solved by assisted reproductive technologies (ARTs). We designed the experiment to explore the effect of different cryoprotectant concentrations and combinations on viability and maturation rates of vitrified/warmed camel oocytes. We collected ovaries directly after slaughtering from local abattoir and transported them to laboratory in a thermo-flask containing normal physiological saline. We aspirated the oocytes from follicles, which is 2-8mm in diameter, washed three times in TCM-199 and then examined under stereo-microscope for selection. We selected morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer. We equilibrated morphologically normal oocytes in equilibration solution (ES), which is half concentration of vitrification one. After equilibration, We transported oocytes to vitrification solution using ethylene glycol (EG, 40%), dimethyl sulphoxide (DMSO, 40%) and EG 40%+DMSO 40%. The obtained results revealed that addition of EG 40%+DMSO 40% resulted in the best quality of vitrified/warmed oocytes, which is demonstrated by higher per cent survival rate (90.16%) and maturation rate (58.95%). While DMSO 40% resulted in 62.79% and 29.54%, respectively, EG 40% reported 86.11% and 53.47%, respectively. We could conclude that vitrification of immature camel oocytes by using 40% EG+40% DMSO is suitable methods to limit drawbacks of vitrification methods, and we need further studies to assess the ability of in vitro-produced blastocyst to develop in vivo and establish pregnancy after embryo transfer.

Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability

Decellularized biological scaffolds hold great promise in cardiovascular surgery. In order to ensure off-the-shelf availability, routine use of decellularized scaffolds requires tissue banking. In this study, the suitability of cryopreservation, vitrification and freeze-drying for the preservation of decellularized bovine pericardial (DBP) scaffolds was evaluated. Cryopreservation was conducted using 10% DMSO and slow-rate freezing. Vitrification was performed using vitrification solution (VS83) and rapid cooling. Freeze-drying was done using a programmable freeze-dryer and sucrose as lyoprotectant. The impact of the preservation methods on the DBP extracellular matrix structure, integrity and composition was assessed using histology, biomechanical testing, spectroscopic and thermal analysis, and biochemistry. In addition, the cytocompatibility of the preserved scaffolds was also assessed. All preservation methods were found to be suitable to preserve the extracellular matrix structure and its components, with no apparent signs of collagen deterioration or denaturation, or loss of elastin and glycosaminoglycans. Biomechanical testing, however, showed that the cryopreserved DBP displayed a loss of extensibility compared to vitrified or freeze-dried scaffolds, which both displayed similar biomechanical behavior compared to non-preserved control scaffolds. In conclusion, cryopreservation altered the biomechanical behavior of the DBP scaffolds, which might lead to graft dysfunction in vivo. In contrast to cryopreservation and vitrification, freeze-drying is performed with non-toxic protective agents and does not require storage at ultra-low temperatures, thus allowing for a cost-effective and easy storage and transport. Due to these advantages, freeze-drying is a preferable method for the preservation of decellularized pericardium. Statement of SignificanceClinical use of DBP scaffolds for surgical reconstructions or substitutions requires development of a preservation technology that does not alter scaffold properties during long-term storage. Conclusive investigation on adverse impacts of the preservation methods on DBP matrix integrity is still missing. This work is aiming to close this gap by studying three potential preservation technologies, cryopreservation, vitrification and freeze-drying, in order to achieve the off-the-shelf availability of DBP patches for clinical application. Furthermore, it provides novel insights for dry-preservation of decellularized xenogeneic scaffolds that can be used in the routine clinical cardiovascular practice, allowing the surgeon the opportunity to choose an ideal implant matching with the needs of each patient.

Shoot tip cryotherapy for efficient eradication of grapevine leafroll‐associated virus‐3 from diseased grapevine in vitro plants

We describe a droplet‐vitrification cryotherapy method for the eradication of grapevine leafroll‐associated virus‐3 (GLRaV‐3) from diseased in vitro shoots of Vitis plants. The procedure involved pre‐culture of 1.0‐mm shoot tips containing five to six leaf primordia (LPs) for 3 days with a pre‐culture medium containing 0.3 M sucrose, 0.16 mM glutathione and 0.14 mM ascorbic acid, treatment of the pre‐cultured shoot tips for 20 min at room temperature with a loading solution composed of 2 M glycerol and 0.4 M sucrose and exposure to plant vitrification solution 2 (PVS2), prior to freezing in liquid nitrogen of dehydrated shoot tips contained in 2.5‐μL PVS2 droplets on aluminium foil strips. Virus localisation showed GLRaV‐3 was not present in apical dome (AD) and LPs 1–4, but it was detected in the basal shoot tip region, approximately 0.5 mm from the AD, as well as in LP 5 and more mature tissues. Histological observations identify that only freezing in liquid nitrogen results in the death of all cells in areas of shoot tips harbouring virus, whereas PVS2 treatment does not. Thus, freezing in liquid nitrogen is a necessary step that eradicates GLRaV‐3. This cryotherapy procedure produced shoot regrowth levels that ranged from 43% to 59%, and all plants recovered after cryotherapy were free of GLRaV‐3 in two wine, one table and one rootstock cultivars. Thus, this procedure can be considered to be efficient and wildly applicable for eradication of GLRaV‐3 from Vitis spp.

Faster, cheaper, defined and efficient vitrification for immature porcine oocytes through modification of exposure time, macromolecule source and temperature

Vitrification reduces the developmental competence of porcine immature oocytes. We investigated the effects of modifying various factors on the viability and development of oocytes after vitrification. These factors included: 1) exposure to the vitrification solution, 2) macromolecule addition (bovine serum albumin (BSA) or polyvinyl pyrrolidone (PVP)), 3) treatment with cytochalasin B, 4) equilibration temperature, and 5) vitrification method (microdrop or Cryotop). Oocytes were equilibrated and vitrified using medium containing ethylene glycol and propylene glycol. After warming, oocytes were subjected to in vitro maturation, stimulated parthenogenetically, and cultured in vitro. Survival rate, nuclear maturation, cleavage, development to the blastocyst stage and their quality were compared between the vitrified groups and the non-vitrified control group. It was found that 1) exposure to the vitrification solution for longer than 30 s was detrimental to embryo development; 2) replacement of BSA with PVP improved embryo development; 3) cytochalasin B treatment reduced the survival rates, but did not affect the blastocyst development rates, 4) equilibration at room temperature (25 °C) was the most beneficial, and 5) the microdrop method improved survival rates. With these adjustments, we were able to establish a simplified and defined cryopreservation system for porcine immature oocytes with improved efficacy.

Development of a vitrification method for preserving human myoblast cell sheets for myocardial regeneration therapy

BackgroundTissue-engineered cardiac constructs have potential in the functional recovery of heart failure; however, the preservation of these constructs is crucial for the development and widespread application of this treatment. We hypothesized that tissue-engineered skeletal myoblast (SMB) constructs may be preserved by vitrification to conserve biological function and structure.ResultsScaffold-free cardiac cell-sheet constructs were prepared from SMBs and immersed in a vitrification solution containing ethylene glycol, sucrose, and carboxyl poly-l-lysine. The cell sheet was wrapped in a thin film and frozen rapidly above liquid nitrogen to achieve vitrification (vitrification group, n = 8); fresh, untreated SMB sheets (fresh group, n = 8) were used as the control. The cryopreserved SMB sheets were thawed at 2 days, 1 week, 1 month, and 3 months after cryopreservation for assessment. Thawed, cryopreserved SMB sheets were transplanted into rat hearts in a myocardial infarction nude rat model, and their effects on cardiac function were evaluated. Cell viability in the cardiac constructs of the vitrification group was comparable to that of the fresh group, independent of the period of cryopreservation (p > 0.05). The structures of the cell-sheet constructs, including cell-cell junctions such as desmosomes, extracellular matrix, and cell membranes, were maintained in the vitrification group for 3 months. The expression of cytokine genes and extracellular matrix proteins (fibronectin, collagen I, N-cadherin, and integrin α5) showed similar levels in the vitrification and fresh groups. Moreover, in an in vivo experiment, the ejection fraction was significantly improved in animals treated with the fresh or cryopreserved constructs as compared to that in the sham-treated group (p < 0.05).ConclusionsOverall, these results show that the vitrification method proposed here preserves the functionality and structure of scaffold-free cardiac cell-sheet constructs using human SMBs after thawing, suggesting the potential clinical application of this method in cell-sheet therapy.

Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing

Grafting of cryopreserved testicular tissue is a promising tool for fertility and testicular function preservation in endangered species, mutant animals, or cancer patients for future use. In this study, we aimed to improve the whole neonatal mouse testicular tissue cryopreservation protocols by comparing cryosurvival, spermatogenesis, and androgen production of grafted testicular tissue after cryopreservation with three different vitrification protocols and an automated computed controlled-rate freezing. Whole neonatal mouse testes were vitrified with various vitrification solutions (V1) 40% EG + 18% Ficoll + 0.35 M Sucrose, (V2) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M PG), or (V3) 15% EG + 15% PG + 0.5 M Sucrose (total solute concentration V1:74.34%, V2:44.0%, and V3:49.22% wt/vol). Alternatively, neonatal testicular tissue was also frozen in 0.7 M DMSO +5% fetal bovine serum using controlled-rate freezing and compared to fresh grafted testicular tissue, sham grafted controls, and the vitrification protocol groups. Fresh (n = 4) and frozen-thawed (n = 4) testes tissues were grafted onto the flank of castrated male NCr Nude recipient mouse. The grafts were harvested after three months. Fresh or frozen-thawed grafts with controlled-rate freezing had the highest rate of tissue survival compared to other vitrified protocols after harvesting (p < 0.05). Both controlled-rate freezing and V1 protocol groups displayed the most advanced stages of spermatogenesis with elongated spermatids and spermatozoa in 17.6 ± 1.3% and 16.3 ± 1.9% of seminiferous tubules based on histopathological evaluation, respectively. Hosts of the testicular graft from controlled-rate freezing had higher levels of serum testosterone compared to all other vitrified-thawed graft groups (p < 0.05). This study shows that completed spermatogenesis from whole neonatal mouse testes were obtained when frozen with controlled-rate freezing and V1 vitrification solution and that testicular cryopreservation efficacy vary with the protocol and vitrification technique.

Three-dimensional culture of fresh and vitrified ovarian tissue in a novel tyramine substituted hyaluronan gel

Follicle culture in matrices that allow 3-D growth may better mimic in vivo conditions by preserving follicle architecture and critical granulosa-oocyte interactions. This study explores methodology for culturing fresh and vitrified ovarian tissue. We further describe the application of a relatively new biomaterial for in vitro follicle maturation and the production of functional metaphase II oocytes. Mouse pre-antral follicle model for IVM and testing of a hyaluronan matrix. Ovaries from 12 to 14 day B6D2F1 pups were removed. Ovaries were vitrified using an ethylene glycol (EG)/DMSO protocol. Ovaries were equilibrated in chilled vitrification solutions (VS) containing 5, 10, 15 and finally 20% EG/DMSO in L-15 with 20% SSS. Ovaries were loaded on a nylon mesh and plunged in to a specially vented cryovial filled with LN2. Pre-antral follicles (FL) and ovarian follicle clusters were collected from fresh and vitrified-warmed ovaries either by mechanical isolation using needles or by enzymatic digestion in collagenase. The HA hydrogel (3 mg/ml) cross-linking was initiated by mixing with 0.03% hydrogen peroxide in a 25:1 ratio. HA gel was then quickly pipetted into the culture dish. Isolated preantral follicles or intact clusters with 4-6 follicles were rapidly seeded into the HA before gelling was completed. Follicle culture was performed in α-MEM supplemented with 5% FBS, 100 mIU/ml FSH, and ITS. Dishes were incubated at 37°C with 6% CO2. Final maturation was triggered with hCG (1.5 IU/ml) and EGF (5 ng/ml) after 12 days of growth. Outcome parameters monitored were follicle morphology, survival in culture, germinal vesicle breakdown, oocyte maturation and meiotic spindle retardance using Oosight Imaging system. Vitrified-warmed ovarian tissue was easily dissected into clusters with 4-5 intact follicles without exposure to enzyme. In contrast, enzymatic follicle isolation was more efficient and preferable for fresh tissue. With fresh ovaries, maturation rate in FL-clusters was similar to that observed with isolated follicles. Meiotic spindle retardance in ovulated oocytes from both culture types did not differ. The overall percent oocyte maturation to metaphase II was lower with vitrified tissue. HA is a natural matrix and supported follicle growth and oocyte maturation. The 3-D culture of FL-clusters provided a method to further mimic in vivo growth by preserving interfollicle connections and architecture. Functional competence of oocytes needs further assessment. Enzymatic treatment of vitrified-warmed ovaries for follicle isolation may not be the best approach due to tissue fragility.Table 1IVM summary data for fresh and vitrified tissueFrozenaFreshbFollicle - ClusterFollicle - IsolatedFollicle - ClusterFollicles observed during IVC69130154Antrum formationna51% (66/130)53% (81/154)Ovulated complexes after hCG93% (64/69)71% (92/130)66% (101/154)GVBD (MI)52% (33/64)30% (28/92)28% (28/101)Maturation to MII34% (22/64)59% (54/92)55% (56/101)Spindle Retardance (Range)na1.34- 3.641.62- 3.51aMechanically isolated follicle cluster. bEnzymatic isolation. Open table in a new tab

Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis.

BackgroundThe domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family.MethodsImmature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC–MS/MS.ResultsA total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented.ConclusionsThe identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Cryosurvival after exposure of IVF-derived Nellore embryos to different cryoprotectants and exposure times during vitrification

We evaluated the effects of the vitrification solution (i.e., ethylene glycol (EG) + dimethyl sulfoxide (DMSO) with or without propylene glycol (PG)) and of exposure time on the re-expansion and hatching rates of vitrified Bos indicus embryos. In vitro produced embryos (n = 1050) were divided into seven groups: control group (non-vitrified embryos) and six vitrification groups with different cryoprotectant concentrations and exposure times. After vitrification, embryos were cultured for determination of re-expansion and hatching rates. Vitrification with 25% DMSO +25% EG (exposure for 1 min and 20 s) resulted in the highest re-expansion (65.2%) and hatching (68.2%) rates. The lowest re-expansion and hatching rates were observed in vitrification with 12.5% DMSO + 25% EG + 12.5% PG with both tested exposure times (i.e., 3 min + 1 min and 1 min + 20 s). A combination of DMSO + EG is efficient to preserve blastocysts, especially following a short exposure time.

Droplet-vitrification cryopreservation of in vitro-grown shoot tips of grapevine (Vitis spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.

Cryopreservation of an endangered Hladnikia pastinacifolia Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.

Critical vitrification steps towards survival of Garcinia hombroniana (Clusiaceae) shoot tips after cryopreservation

A cryopreservation protocol was developed for in vitro shoot tips of Garcinia hombroniana using the vitrification technique. Four critical steps in the technique were investigated, namely preculture, loading, dehydration with Plant Vitrification Solution 2 (PVS2), and unloading. Shoot tips precultured for 48 hr gave significantly higher survival (75 %) compared to 24 hr preculture (50 %) after cryopreservation. Treatment with 1 M glycerol plus 0.4 M sucrose as a loading solution gave higher survival (45.83 %) compared to the other treatments (0.4 M sucrose + 2 M glycerol; 0.4 M sucrose). Shoot tips dehydrated with PVS2 for 25 min gave the highest survival after immersion in liquid nitrogen. Stepwise PVS2 treatment for 15 min with 50 % PVS2 followed by 10 min with 100 % PVS2 solution improved survival of the shoot tips after cryopreservation (41.67 %). Murashige and Skoog medium with 0.4 M sucrose gave significantly higher survival (66.67 %) than MS with 1.2 M sucrose (25 %) as an unloading solution. Water content was shown to decrease throughout the whole vitrification steps from 6.83 ± 1.66 g g-1 dw for fresh shoot tips down to 2.93 ± 0.28 g g-1 dw after PVS2 treatment. Further study on each step including recovery medium is required to improve the survival. Nevertheless, the present study showed the potential of using the vitrification technique for cryopreservation of G. hombroniana.

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Effect of Catalase or Alpha Lipoic Acid Supplementation in the Vitrification Solution of Ovine Ovarian Tissue.

The present study evaluates the effect of different concentrations of antioxidants (catalase - CAT and alpha lipoic acid - ALA) on the follicular activation and morphology, DNA damage, ROS production, and mitochondrial activity in vitrified sheep ovarian tissue. This experiment was divided into two steps. First, ovarian fragments were distributed into the following treatments: fresh tissue or control (CTR), incubation (INC), vitrification without antioxidant (VWA), with CAT (10, 20, or 40 IU mL-1) or ALA (25, 50, or 100 μM mL-1). After vitrification/warming, the fragments were additionally incubated for 24 hours and evaluated for morphology and follicular activation, as well as reactive oxygen species (ROS) levels in the culture medium. For the second step, other ovarian fragments were submitted to CTR, VWA, CAT40, and ALA100. After vitrification/warming, the fragments were incubated for 24 hours and evaluated by cell density of ovarian stroma, DNA damage, and mitochondrial and intracellular ROS levels. The percentage of morphologically normal follicles in vitrified ovarian tissue in the presence of ALA in all concentrations did not differ (p > 0.05) from fresh tissue or CTRs. The percentage of activated follicles was higher in ALA100 μM mL-1 than those observed for the treatments INC, CAT (40 IU mL-1), or ALA (25 or 50 μM mL-1). The use of CAT affected (p < 0.05) the density of stromal cells (40 IU mL-1), ROS levels (10 and 20 IU mL-1), as well as DNA damage revealed by ©H2AX (40 IU mL-1). Although 100 μM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.

In vitro and in vivo survival of mouse morulas and blastocysts following vitrification in 45% glycerol

The toxicity of cryoprotectant agents is one of the critical factors for the successful vitrification of mammalian embryos, which depends on the concentration, time and temperature of exposure to the cryoprotectant. Moreover, embryos from different species or stages of development have distinct levels of tolerance to cryoprotectant agents. This study aimed to evaluate the in vitro and in vivo survivals of mouse embryos after distinct times of exposure to two cryoprotectant concentrations prior to vitrification. In Experiment 1, compact morulas, blastocysts and expanded blastocysts were exposed to 10% glycerol for 10 min (group 1) or to 25% glycerol for 10, 5 and 2.5 min (groups 2, 3, and 4, respectively) prior to their exposure to the vitrification solution containing 45% glycerol for 1 min at 20°C before immersion in liquid nitrogen. Embryos were thawed in a water bath at 20°C for 20 sec, and the cryoprotectant was diluted in 1 M sucrose for 10 min. Then, embryos were morphologically evaluated following in vitro culture for 1 and 48 h. In vitro survivals of compact morulas and blastocysts were not affected by glycerol concentration and/or equilibration time prior to vitrification. However, expanded blastocysts demonstrated a lower survivability to vitrification. In Experiment 2, fresh and vitrified (according to procedures in group 1, Experiment 1) compact morulas and blastocysts were transferred to recipients following in vitro culture for 1 h at room temperature. Pregnancy rates, based on the proportion of viable fetuses, were similar between vitrified and fresh compact morulas (27% and 33%, respectively). However, vitrified blastocysts demonstrated a lower in vivo survival than controls (9% vs. 52%, respectively).