Needle Immersed Vitrification Research Articles

Cryopreservation of African painted dog (Lycaon pictus) ovarian tissue.

Development of techniques for the preservation and use of gonadal tissues are increasingly needed for the genetic management of the endangered African painted dog (Lycaon pictus). Here we evaluated two cryopreservation techniques for ovarian tissue (2 × 2 × 1 mm3 fragments, n = 11 individuals): needle immersed vitrification (NIV), with equilibration in a 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG) solution, and vitrification in a 15% DMSO, 15% EG, and 0.5 M sucrose solution, and slow freezing in cryovials with either the equilibration (SF-E) or vitrification (SF-V) solutions. Following warming, tissues were either fixed and embedded for evaluation of density of morphologically normal follicles, semi-quantitative scoring of stromal cell preservation, and apoptotic index (TUNEL stain), and/or flash-frozen for expression of proliferation (PCNA), apoptosis (CASP3, BCL2), or oxidative stress (GPX3, SOD1, SOD2) pathway genes (n = 4). Needle immersed vitrification maintained higher density of morphologically normal follicles compared to the slow freezing protocols applied (p < 0.05), with no significant changes in expression of select genes among treatment groups. A slight increase in apoptotic index was observed in all cryopreservation groups, but only reached significance in SF-E compared with fresh tissue controls (p < 0.05). Future research should be dedicated to developing improved methods for ovarian tissue culture in the species, both as a means to evaluate the efficacy of tissue cryopreservation techniques and for the production of viable oocytes from banked ovarian tissue in the endangered African painted dog.

Vitrification of the ovarian tissue in sturgeons

The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me2SO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M Me2SO in the ES, and 1.5 M MeOH and 5.5 M Me2SO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M Me2SO in the ES, and 3 M PG and 3 M Me2SO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons.

A novel strategy for conservation of European eel (Anguilla anguilla) genetic resources: Cryopreservation of ovarian stem cells

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me2SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN2 at −80 °C displayed the highest OSC viability. Different cooling rates ranging from −1 to −40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me2SO) with VS3 (3 M PG and 3 M Me2SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species.

Preservation of zebrafish genetic resources through testis cryopreservation and spermatogonia transplantation

Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

Cryopreservation and transplantation of common carp spermatogonia.

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5–10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

Cryovial monolayer vitrification for ovarian tissue cryopreservation.

The cryopreservation of ovarian tissue has been proved to be effective for fertility preservation. To find a better cryopreservation method, we tested the efficacy of cryovial monolayer vitrification method in comparison with that of needle immersed vitrification (NIV). Ovaries from 10 female kunming mice aged 6-8weeks were cut into pieces and allocated into group A (cryovial monolayer vitrification method), group B (NIV method) and group C (fresh control). In group A, pieces of ovarian tissue were layered around the inner wall of cryovial as a monolayer; and in group B, pieces of ovarian tissue were pierced with a needle. Other than the difference in the carrier for ovarian tissue, the cryoprotectants and the protocols in group A and B were the same. The viability and in vitro growth potential of the follicles after warming in groups A and B were evaluated respectively and compared with those of fresh control. The results showed that the viabilities of the follicles were not statistically different among three groups. The average diameter of follicles did not show statistically significant difference among three groups before culture and between group A and group B after culture (p>0.05), but demonstrated statistically significant difference between group A and group C (p<0.01), group B and group C (p<0.01), respectively. In the procedure of loading ovarian tissue onto carriers, group A took less time compared with group B. In addition, the small pieces and debris which were harder or impossible to be pierced with needle in group B could be easily layered onto the inner wall of the cryovial in group A. Hence the follicles existed within the small pieces and debris of the ovarian tissue could also be cryopreserved. It is concluded that, in cryopreserving both viability and growth potential of follicles, the cryovial monolayer vitrification method is comparable with NIV method but with higher efficiency.

Cryopreservation of human ovarian tissue using the silver closed vitrification system.

The aim of this study is to evaluate the feasibility of using a hand-made silver container for the cryopreservation of human ovarian cortex. Human ovarian cortex tissues were vitrified using an open vitrification system (OVS) of needle immersed vitrification (NIV) and two closed vitrification systems (CVS) of a plastic vial (plastic CVS) and a silver container (silver CVS). Outcomes of vitrification were evaluated morphologically and histologically by in vitro culture and xenotransplantation. The apoptosis of primordial follicles was assessed by TUNEL staining. The production of E2 and P4 was examined by a chemiluminescent immunoassay. Blood vessels were visualized with CD31 staining. Compared with the fresh ovarian cortex tissue, ovarian cortex tissues that were vitrified using the three different carriers and then warmed showed significantly reduced percentages of normal primordial follicles, viability of primordial follicles, E2 and P4 levels during in vitro culture and decreased amounts of blood vessels. However, much better outcomes were obtained with NIV and silver CVS than with plastic CVS, based on the better morphology and viability of primordial follicles, higher E2 and P4 production during an in vitro culture, and greater numbers of blood vessels after xenografting. Importantly, the outcomes of ovarian cortex cryopreservation with silver CVS were similar and comparable to those with NIV. The hand-made silver container as a CVS is a promising carrier for the cryopreservation of the human ovarian cortex.

First successful vitrification of salmonid ovarian tissue

Due to a lack of cryopreservation protocols for fish eggs and embryos, alternative techniques which will enable storage of female genetic resources are crucial for future development of reproduction management in conservation biology and aquaculture. Experiments were conducted to develop an optimal vitrification protocol for cryopreservation of brown trout Salmo trutta juvenile ovarian tissue. Needle immersed vitrification (NIV) method was used where ovaries were pinned on an acupuncture needle, passaged through equilibration and vitrification solutions containing different combinations and concentrations of methanol (MeOH), propylene glycol (PG) and dimethyl sulfoxide (Me2SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me2SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively unaltered perinucleolar follicles. The method developed for brown trout could be applied to the conservation of female genetic resources of other salmonid species, including endangered and endemic species or populations.

Erratum to: Attempts to improve human ovarian transplantation outcomes of needle immersed vitrification and slow-freezing by host and graft treatments.

Purpose To investigate if needle-immersed vitrification or slow-freezing yields better implantation results for human ovarian tissue and which method benefits more when combined with the “improvement protocol” of host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A.

Attempts to improve human ovarian transplantation outcomes of needle-immersed vitrification and slow-freezing by host and graft treatments.

To investigate if needle-immersed vitrification or slow-freezing yields better implantation results for human ovarian tissue and which method benefits more when combined with the "improvement protocol" of host melatonin treatment and graft incubation with biological glue + vitamin E + vascular endothelial growth factor-A. Human ovarian tissue was preserved by needle-immersed vitrification or slow-freezing and transplanted into immunodeficient mice, either untreated (groups A and C, respectively) or treated with the improvement protocol (groups B and D, respectively). Grafted and ungrafted slices were evaluated by follicle counts, apoptosis assay and immunohistochemistry for Ki67 and platelet endothelial cell adhesion molecule (PECAM). Follicle number in the recovered grafts was limited. The number of atretic follicles was significantly higher after vitrification with/without the improvement protocol and slow-freezing than that after slow-freezing + the improvement protocol. Stroma cell apoptosis was the lowest in the group D. PECAM staining showed a peripheral and diffuse pattern in the group D (mostly normal follicular morphology) and a diffuse pattern in all other groups (few follicles, mostly atretic), with significantly higher diffuse levels in the vitrification groups. Ki67 staining was identified in all normal follicles. Follicles did not survive transplantation in the vitrification groups. Ovarian sample preparation with slow-freezing + the improvement protocol appears to yield better implantation outcomes than needle-immersed vitrification with/without the improvement protocol. The real quality of frozen tissue can be assessed only after grafting and not after thawing/warming.

Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica)

Cryopreservation of ovarian tissue has been the only effective way of ex situ conservation of female germplasm in avian species. A novel needle-in-straw (NIS) vitrification method was developed to store tissue in straws instead of cryovials. Fragments of ovarian tissue from one-week old Japanese quail were transfixed on an acupuncture needle. They were immersed in equilibration and vitrification solutions containing dimethyl sulphoxide, ethylene glycol and sucrose. A layer of tin foil was rolled over the tissue fragments and the tin foil package was plunged into liquid nitrogen and inserted into a pre-cooled, 0.5-ml straw which was stored in liquid nitrogen. Tissue was also preserved using a needle immersed vitrification (NIV) method, in which tissue fragments transfixed on needles without tin foil and were stored in cryovials filled with liquid nitrogen. Cryopreserved tissue was warmed at room temperature (RT) or 37°C and the ratio of normal follicles to total visible follicles was determined by histological methods. In addition, cryopreserved and warmed tissue was cultured on the chorioallantoic membranes of fertilized chicken eggs for 5–6 days. The viability and vascularization of the grafts were evaluated. The tissue cryopreserved by NIS and warmed at RT showed comparable follicle morphology to fresh tissue and to that preserved by NIV and warmed at RT. No significant impairment on the viability or vascularization of the grafted tissue was observed. The NIS method allows tissue to be stored and transported safely and efficiently and can be used instead of cryovials in tissue cryobanking.

Needle immersed vitrification can lower the concentration of cryoprotectant in human ovarian tissue cryopreservation

To investigate whether needle immersed vitrification (NIV) can further lower the concentration of cryoprotective agent (CPA). Experimental cross-sectional controlled in vitro study. University teaching hospital. Human ovarian biopsy tissues were obtained from ten women undergoing gynecology operations. Ovarian cortical tissues were cryopreserved using slow freezing or vitrification. The vitrification solutions used were as follows: group A: 2.69 mol/L ethylene glycol (EG)+2.11 mol/L dimethylsulfoxide (DMSO)+0.5 mol/L sucrose; group B: 2.42 mol/L EG+1.90 mol/L DMSO+0.5 mol/L sucrose; group C: 2.15 mol/L EG+1.69 mol/L DMSO+0.5 mol/L sucrose; and group D: 1.88 mol/L EG+1.48 mol/L DMSO+0.5 mol/L sucrose. Histologic evaluations were performed using light and electron microscopy. Apoptosis was assessed by TUNEL staining. Tissue damage after cryopreservation was measured by the levels of lactate dehydrogenase (LDH) in culture. The proportion of normal ultrastructure of granulosa cells and stromal cells in groups B and C was higher than that in group A. The proportion of TUNEL-positive primordial follicles and stromal cells in the NIV groups decreased with reduction of concentration. Additionally, LDH levels in groups B and C were lower than in group A. The NIV method could further lower the concentration of CPA. Therefore, we can use the CPA of group C as an optimal concentration for NIV.