• Title, Summary, Keyword: Vitrification

Search Result 377, Processing Time 0.048 seconds

Comparison of Vitrification and Slow Freezing-thawing Method on 1-cell Zygotes (생쥐 1-세포기 수정란의 동결방법에 있어서 초자화동결과 완만동결의 비교)

  • Lee, Ji-Hyang;Han, Hyuck-Dong;Koo, Hye-Young
    • Clinical and Experimental Reproductive Medicine
    • /
    • v.28 no.3
    • /
    • pp.191-198
    • /
    • 2001
  • Objective : This study was conducted to examine the effect of vitrification on the survival and in vitro development of mice 1-cell zygotes. Method: Effects of exposure to vitrification solution and vitrification, with different concentrations of the cryoprotectant solution, were examined. The 1-cell zygotes were also subjected to a slow freezing-thawing method to compare with vitrification method. Solution composed of ethylene glycol (6.0 M, 5.0 M, 4.0 M) and sucrose (1.0 M) were used as cryopropectant. The experiments employed the method loading the embryos on electron microscope grids. Results: I. The effects of exposure in vitrification solution. 1-cell zygotes were non-toxic at all concentrations of the vitrification solution showing the survival rate between 88.1% and 97.5%. Development into 2-cell was more successful in the higher concentrations of the vitrification solution. Therefore, higher concentrations of the vitirification solution do not seem to cause any problems in vitrification procedure. II. The effects of vitrification method. 1-cell zygotes showed the survival rate between 78.8% and 92.4%. The lowest and the highest survival rate was observed in the 6.0 M and 4.0 M vitrification solution, respectively. 2-cell development rates varied from 77.6% to 91.3%. Blastocyst development rate was shown highest in 5.0 M and the lowest in 4.0 M solution. Therefore, the highest 2-cell and blastocyst development rate was observed in 5.0 M solution. III. Comparison of vitrification and slow freezing-thawing method on 1-cell zygotes. This experiment showed that 1-cell zygotes had the highest survival and development rates in 5.0 M vitrification solution. Vitrified group of 1-cell zygotes, in the 5.0 M vitrification solution, were compared with the group processed in slow freezing-thawing method. The development rate into 2-cell and blastocyst as well as the survival rate were higher in the vitrified group than in the slowly freezed group. Conclusion: 1. The results demonstrate that the best cryoprotectant is a 5.0 M vitrification solution for 1-cell zygotes. 2. Vitrification method significantly increases the survival rate of the 1-cell zygote and its development into 2-cell and blastocyst. Equilibration and exposure time during the vitrification was remarkerbly short in this experiment. Total time, from the exposure to vitirification solution to storage in the liquid nitrogen, was taken only 90 seconds. In contrast, the slow freezing-thawing method have taken more than four hours. Taken together, we presume that the overall time used for the procedure contributes to the results as an important parameter. 3. The loading of 1-cell zygotes on the EM grid is technically more simple and takes less time than the straw or cryo vial method.

  • PDF

Vitrification of Mouse Morulae (마우스 상실배의 Vitrification에 관한 연구)

  • ;;M. Kasai
    • Korean Journal of Animal Reproduction
    • /
    • v.15 no.3
    • /
    • pp.173-177
    • /
    • 1991
  • In vitro survival of the mouse morulae frozen by vitrification method(Kasai et al., 1990) was investigated in the present study. The embryos were plunged into LN2 directly after exposure to the vitrification solutions(EFS, GFS and DFS). The results were obtained as follows. The viability of morulae after freezing and thawing was high in EFS(96.7∼100.0%) and GFS vitrification solution(93.3∼96.7%), and the lowest in DFS vitrification solution(0.00∼0.03%).

  • PDF

Serum or serum albumin in a vitrification solution and EGF or FGF affect in vitro viability of frozen-thawed bovine blastocysts after vitrification (동결액에 첨가된 macromolecule 및 EGF, FGF가 vitrification 법으로 동결한 소 수정란의 체외생존성에 미치는 영향)

  • Lee, Eun-song;Fukui, Yutaka
    • Korean Journal of Veterinary Research
    • /
    • v.38 no.2
    • /
    • pp.394-400
    • /
    • 1998
  • Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

  • PDF

Studies on In Vitro Fertilization after Vitrification Freezing of Immatured Canine Oocytes (개 미숙난자의 Vitrification 동결 후 체외수정에 관한 연구)

  • 박상훈;박종민;김상근
    • Journal of Embryo Transfer
    • /
    • v.17 no.2
    • /
    • pp.117-121
    • /
    • 2002
  • This study was carried out to investigate the in vitro fertilization rate of canine immature oocytes cryopreserved by vitrification freezing. The vitrification solutions of EPS and EDS were consisted of 40% ethylene glycol 18% Ficoll and 0.3M sucrose, and 20% ethylene glycol, 16.5% DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10% FCS, respectively. The oocytes were exposed The developmental rate of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 3.8%, 10.7%, 46%, respectively. to EFS or EDS at $25^{\circ}C$ and loaded into straw fer 30 sec. The straws was slowly immersed into L$N_2$. Fertilization and survival rate was defined as development rate on in vitro culture or FDA-test. 1. The fertilization rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was very low(5.3%~31.4%) than the unfrozen oocyte(60.0%). And the fertilization rate after vitrification freezing of immature oocytes was very higher than that of mature oocytes. 2. The survival rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was 55.0%, 40.0%, 28.6% and 17.1%, respectively. And the survival rate after vitrification freezing of immature oocytes was slightly higher than that of mature oocytes.

Evaluation of vitrification for cryopreservation of teeth

  • Dissanayake, Surangi C.;Che, Zhong-Min;Choi, Seong-Ho;Lee, Seung-Jong;Kim, Jin
    • Journal of Periodontal and Implant Science
    • /
    • v.40 no.3
    • /
    • pp.111-118
    • /
    • 2010
  • Purpose: The aim of this study was to investigate whether vitrification in the cryopreservation of periodontal ligament (PDL) cells could be useful for tooth banking. Methods: In step 1, primary cultured human PDL cells were cryopreserved in 100% conventional cryopreservation media and 100% vitrification media (ESF40 media) in different temperatures for 2 weeks. In step 2, a series of modified vitrification formulae named T1 (75% vitrification media + 25% F-media), T2 (50% vitrification media + 50% F-media) and T3 (25% vitrification media + 75% F-media) were used to store PDL cells for 2 weeks and 4 weeks in liquid nitrogen. MTT assay was performed to examine the viability of PDL cells. Results: Maximum cell viability was achieved in cells stored in 100% conventional cryopreservation media at $-196^{\circ}C$ (positive control group) in step 1. Compared to the positive control group, viability of the cells stored in 100% vitrification media was very low as 10% in all test conditions. In step 2, as the percentage of vitrification media decreased, the cell viability increased in cells stored for 2 weeks. In 4-week storage of cells in step 2, higher cell viability was observed in the T2 group than the other vitrification formulae while the positive control group had the highest viability. There was no statistically significant difference in the cell viability of 2-week and 4-week stored cells in the T2 group. Conclusions: These observations indicate 100% vitrification media is not successful in PDL cell cryopreservation. Conventional cryopreservation media is currently the most appropriate media type for this purpose while T2 media would be interesting to test for long-term storage of PDL cells.

Closed vitrification of mouse oocytes using the CryoLogic vitrification method: A modification that improves developmental competence

  • Jo, Jun Woo;Jee, Byung Chul;Suh, Chang Suk;Kim, Seok Hyun
    • Clinical and Experimental Reproductive Medicine
    • /
    • v.40 no.4
    • /
    • pp.148-154
    • /
    • 2013
  • Objective: To compare the mouse oocyte vitrification outcomes of the CryoLogic vitrification method (CVM) and the conventional open method using a Cryotop. Two CVM methods (original CVM and modified CVM) were tested. Methods: Mature oocytes obtained from female BDF-1 mice were vitrified by two-step exposure to equilibrium and vitrification solutions. Three vitrification protocols were tested on three groups: the CVM-kit, modified CVM, and Cryotop groups. After exposure to the two solutions, the oocytes were vitrified. After warming, the oocytes were fertilized in vitro, and the embryo development was assessed. Blastomeres positive for caspase were counted using an in situ assay kit. The spindle morphology and chromosome configurations of warmed vitrified oocytes were also assessed. Results: The modified CVM and Cryotop groups showed similar developmental capacities, and similar proportions of cells with intact spindles and chromosome configurations. The modified CVM protocol was superior to the original CVM protocol for developmental competence and intact spindle preservation. However, the CVM group showed a relatively higher number of apoptotic cells in blastocysts. Conclusion: Closed vitrification using the modified CVM protocol may be used as an alternative to the conventional open method, but strategies to decrease apoptosis in the blastomere need to be investigated.

Post-thaw Embryo Development following Vitrification or Slow Freezing of Rabbit Normal and Nuclear Transplant Embryos (토끼의 정상 및 핵이식배의 유리화 및 완만동결에 따른 융해 후 발달율)

  • 강다원;최창용;하란조;강태영;심보웅;최상용;이효종;박충생
    • Journal of Embryo Transfer
    • /
    • v.13 no.1
    • /
    • pp.1-9
    • /
    • 1998
  • In order to improve the cryopreservation by vitrification or slow freezing of nuclear transplant rabbit embryos, the effects of factors affecting embryo cryopreservation such as cryoprotectants, equilibration, cooling rate and post-thaw dilution on post-thaw survial and development were determined using intact embryos of morular stage. And the post-thaw development of nuclear transplanted embryos cryopreserved under the optimal conditions examined was compared between vitrification and slow freezing. The cryoprotectant solution used was ethyleneglycol-ficoll-sucrose (EFS) or ethyleneglycol-poly-vinylpyrrolidone-galactose- I (EPG- I ) for vitrification, and EPG- II for slow freezing. To examine the viability of frozen-thawed embryos, the nuclear transplanted embryos were co-cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) for 24 hrs and the intact morulae were co-cultured with BOEC for 5 days and 3 days to hatching blastocyst stage in 39 ˚C 5% $CO_2$ incubator. The results obtained were as follows: Following vitrification with EFS, the post-thaw development of rabbit morulae to hatching blastocyst was significantly(P<0.05) higher in compacted stage(82.4%) than in early morular stage(60.0%). The post-thaw development of compacted morulae to hatching blastocyst was similarly high in vitrification with EFS(82.4%), EPG- I (85.0%) and in slow freezing with EPG- II (83.3%). Following vitrification with EPG- I, the post-thaw development of intact rabbit morulae to hatching blastocyst was similar as 78.0% and 85.0% in 1-step and 2-step post-thaw dilution, respectively. The post-thaw development of nuclear transplanted rabbit embryos of compacted morulae stage to hatching blastocyst was similarly 43.6% and 40.0% in vitrification with EPG- Iand slow freezing with EPG- II, respectively. These results indicated that the rabbit nuclear transplant and intact embryos of morulae stage could be well cryopreserved with either vitrification or slow freezing procedure.

  • PDF

Comparison of the Efficiency between Slow Freezing and Vitrification Method for Cryopreservation of Human Embryos (인간 수정란의 완만 동결과 유리화 동결의 비교)

  • Kim, Eun-Kuk;Kim, Mi-Yeon;Son, Sun-Mi;Kim, Dong-Won
    • Journal of Embryo Transfer
    • /
    • v.23 no.1
    • /
    • pp.19-24
    • /
    • 2008
  • The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.

Glass Property Models, Constraints, and Formulation Approaches for Vitrification of High-Level Nuclear Wastes at the US Hanford Site

  • Kim, Dongsang
    • Journal of the Korean Ceramic Society
    • /
    • v.52 no.2
    • /
    • pp.92-102
    • /
    • 2015
  • Current plans for legacy nuclear wastes stored in underground tanks at the U.S. Department of Energy's Hanford Site in Washington are that they will be separated into high-level waste and low-activity waste fractions that will be vitrified separately. Formulating optimized glass compositions that maximize the waste loading in glass is critical for successful and economical treatment and immobilization of these nuclear wastes. Glass property-composition models have been developed and applied to formulate glass compositions for various objectives for the past several decades. Property models with associated uncertainties combined with composition and property constraints have been used to develop preliminary glass formulation algorithms designed for vitrification process control and waste-form qualification at the planned waste vitrification plant. This paper provides an overview of the current status of glass property-composition models, constraints applicable to Hanford waste vitrification, and glass formulation approaches that have been developed for vitrification of hazardous and highly radioactive wastes stored at the Hanford Site.

The Effect of Cryopreservation Condition on Developmental Rate of Pronuclear Stage Embryos and Vitrification of Mouse Oocytes (생쥐 난자의 유리화 동결과 전핵기 배아의 동결 조건이 배아의 발달에 미치는 영향)

  • Kim, Ji-Chul;Park, Sung-Baek;Nam, Yoon-Sung;Seo, Byoung-Boo;Kim, Jae-Myeoung;Song, Hai-Bum
    • Journal of Embryo Transfer
    • /
    • v.26 no.3
    • /
    • pp.201-207
    • /
    • 2011
  • The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.