• Journal of Veterinary Clinics
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• v.28 no.4
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• pp.394-398
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• 2011

Mesenchymal stem cells (MSC) are pluripotent cells that can be found in umbilical cord blood from new borne babies as well as placenta, bone marrow, adipose tissue, amniotic fluid, muscle, et al. MSC are capable of renewing themselves without differentiation in long-term culture, also can be differentiated into various tissues under specific condition. Formulating a cryopreservation protocol for the MSC is required because these cells cannot survive for long periods under in vitro culture conditions and a new formulation of harmless cryoprotectant is needed for the direct injection of MSC into patients. The undifferentiated MSC were frozen with a vitrification solution of 40% ethylene glycol, 20% Ficoll-70 and 0.3M sucrose. The survival rate after thawing and their proliferation rate were examined and compared with slow rate cooling methods using dimethylsulfoxide (DMSO). The vitrification method showed high survival rate after thawing and proliferation capacity comparable to DMSO. It can be suggested that ultra-rapid cooling method by vitrification is reliable methods for long term preservation of MSC and the vitrification solution with ethylene glycol, Ficoll-70 and sucrose will be more beneficially used for direct transplantation of MSC into patients than DMSO solution.

• Korean Journal of Poultry Science
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• v.28 no.1
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• pp.69-76
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• 2001

Avian primordial germ cells (PGCs) originate from the epiblast and appear in the germinal crescent. These PGCs enter the developing blood vessels during stage 10∼12 (H&H), circulate in the blood stream, migrate into the developing gonadal anlage and differentiate into germ cells. However, it is not clear until when the migratory ability of PGC is maintained. This study was conducted to examine whether migratory ability is present in PGCs from the gonad at later embryonic developmental stages. In the present study, gonads were dissected from 5-, 6- and 10-day old quail embryos and treated with trypsin-EDTA. Gonadal PGCs (gPGCs) were purified by Ficoll-density-gradient-centrifugation and labeled with PKH26 fluorescent dye. The PKH26-labeled gPGCs were microinjected into the blood vessel of the recipient quail embryo. Manipulated recipients were incubated for 3 days, embedded in paraffin and sdctioned. The foreign gPGCs were detected by fluorescent and confocal laser microscopy. As a result, quail gPGCs, from 10, 6 and 5 day old embryos could migrate through the recipient blood stream at early stage and settle in the gonads. Thus, results suggest that gPGCs from upto 10-day old embryos keep properties seen in circulating PGC. Therefore, the PGCs of 10-day old embryonic gonads can be used for the tools of genetic manipulation.

• Korean Journal of Microbiology
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• v.22 no.4
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• pp.207-214
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• 1984

Improved methods for the isolation and purification of conidial protoplast were investigated and conidial proplast from auxotrophic mutants were fused. The reaction time for isolation of protoplasts from the swollen condiospores preincubated in liquid minimal medium supplimented with 2-deoxy-D-glucose was shorten by reaction with mixture of 2% driselase and 2% ${\beta}-glucuronidase$ (1:1). The conidial protoplast could be highly purified by using 5% Ficoll 400 as a centrifugation medium. Nucleus of the conidial protoplast was stained with Giemsa stain and the conidial protoplast had one nucleus. It was also confirmed that the conidial protoplast was true protoplast with no cell wall remnant at the outside of plasma menbrane. Fusion frequencies of the conidial protoplast from auxotrophic mutants ranged from $3.4{\times}10^{-1}\;to\;4.9{\times}10^{-1}$. These values were higher than those of mycelial protoplast by a factor of 5 to 28.

• Journal of Embryo Transfer
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• v.8 no.1
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• pp.31-36
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• 1993

The post-thaw survival of mouse embryos of the various developmental stages was determined after cryopreservation by vitrification in a solution containing ethylene glycol, Ficoll and sucrose (EFS). All the embryos were equilibrated for 2 minutes just prior to freezing. The number of blastomeres during in vitro development was counted by nuclei higher rates of post-thaw survival were obtained from the embryos of 2-cell(92.2%), 8-cell(77.2%) or morula stage(90.0%) than those of blastocyst stage(62.7%). The number of blastomeres per embryo following in vitro culture for 24 hours was significantly(P<0.05) smaller as 66.0f22.3 in vitrified and thawed morulae than fresh morulae(91.7$\pm$12.2).

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.251-256
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• 1995

Two experiments were conducted to study the production of in vitro fertilized bovine embryos and the viability of blastocysts cryopreserved by vitrification. In experiment 1, production rate of in vitro matured bovine oocytes after fertilization in medium containing bovine oviduct epithelial cells (BOEC), cumulus cells and granulosa cells to blastocysts were 18.4, 14.6 and 13.1%, respectively. Developmental percentages of blastocysts produced at day 6, 7 and 8 were 8.5, 10.6 and 15.2% respectively. Hatching rate of bovine embryos produced was 60.0%. In experiment 2, post-thawed surviving embryos in a vitrification solution consisting of 7.15M ethylene glycol, 2.5 mM ficoll and 0.3 M sucrose were 36.4% (56/154). Also, survival rate of bovine embryos after exposed to vitrification solution at 1, 2, 3, 4 and 5 min were 84.0, 88.0, 71.0, 48.0 and 24.0% respectively.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.131-137
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• 1997

This study was carried out to investigate the in vivo development rates of vitrified-thawed mouse expanding, hatching and hatched blastoc ysts(BL). In vitro fertilization produced blastocysts were vitrified in EFS40(40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). Expanding a and hatching blastocysts were equilibrated in 20% ethylene glyco](EG) for 5 min. before exposure to EFS40 at 25°C for 1 min., they were then vitrified in liquid nitrogen. Hatched blastocysts which cultured in m-CR1 medium supple mented 0.4% bovine serum albumin on day 5. were equilibrated in 10% EG for 5 min. and then vitrified in EFS40 for 30 sec. After thawing, re-expanding blastocysts were transferred to recipients(3 day of pseudopregnant) on one or both uterine horns(6-8 embryos per a horn). Preg¬n nancy rates of recipients and implantation were a assccessed by autopsy on 15 gestation. The res¬u ults obtained in these experiments were summar¬1 ized as follows; 1) The pregnancy and live fetus rates, for vitrified expanding BL(77.8 and 25.0%) and hatching BL(77.8 and 26.4%)n vitro were not significantly difference in those of control BL (66.7 and 42.9%: 83.3 and 40.4%), respectively, 2) in vitro development of vitrified hatched BL was 34.0%. and 3) in vivo developmental rate of vitrified hatched BL was only 33.3%. These results suggested that proposed rapid vitrification p procedures used EFS40 cryoprotectant can be effectively performed in mouse expanding Ihatching blastocysts and that mouse blastocysts a after being hatched from zona pellucida can be successfully cryopreserved.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.173-179
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• 2004

This study was conducted to investigate the toxi-cological effects of different vitrification solution on development of immature porcine oocytes in vitro. Oocytes were exposed to EFS solution [40% ethylene glycol (EG) + 18% Ficoll + 0.3M sucrose], ES solution (5.5M EG + 1.0M sucrose) or GE solution [10% glycol (G) + 20% EG], and these oocytes were transferred to sucrose solution directly. Maturation rates were significantly (P<0.05) higher in the ES solution (44.5%) and control (57.6%) than in the EFS solution (38.8%) and GE solution (22.4%). No differences among three solution were found in fertilization rates. Cleavage rates was significantly (P<0.05) higher in the ES solution (47.1%) and control (65.9%) than in the EFS solution (21.9%) and GE solution (19.0%), but no difference among three solutions was found in the blastocyst formation rates. These results indicate that combination of EG and sucrose solutions had effects on development of immature porcine oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.287-292
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• 1998

The objective of this study was to confirm whether the vitrification method using EFS35 has detrimental effect for cytoskeleton and chromosome constitution of the mouse oocytes by indirect immunocytochemistry and chromosome analysis. Mouse oocytes were vitrified using EFS35 which consisted of 35% ethylene glycol, 18% ficoll, 0.3 M sucrose and 10% FBS in M2 medium. The results obtained in this experiment were summarized as follows: When the survival rates after being exposed or vitrified in EFS35 were examined, there were not different between two groups (97.7 and 89.3%). Also, when the microtubule morphology and microfilament distribution in vitrified oocytes were examined, normal percentage of two cytoskeleton in vitrified group (95.5 and 100%) was not different from that in control (97.5 and 100%) and exposed group (92.3 and 100%). In addition, the rate of oocytes containing a normal chromosome number in vitrified group (73.5%) after IVF was not different from that in control (79.5%) and exposed group (78.7%). These results indicated that the cytoskeletal morphology and chromosome constitution of mouse oocytes were not affected by cryoprotectant (EFS35) or freezing apd that vitrification methods using EFS35 was suitable for cryopreservation of mouse oocytes.

• Journal of Genetic Medicine
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• v.5 no.1
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• pp.55-60
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• 2008

Purpose : The ability to perform chromosome analysis of cryopreserved cord blood mononuclear cells is important for future retrospective studies. We compared the karyotypes of cryopreserved cells with cells before cryopreservation. Methods : One cord blood (CB) sample was obtained from normal healthy volunteer. Karyotype analysis was performed before cryopreservation. After mononuclear cell separation with Ficoll-Hypaque, the mononuclear cells were cryopreserved by programmed controlled-rate freezer and then transferred into the liquid nitrogen ($-196^{\circ}C$) for 3 days. After rapid thawing, cytogenetic analysis was performed as the same method for each sample by different conditions. The samples were divided by three groups. The first group was no culture before cryopreservation, the second group was 72 hours culture before cryopreservation, but no 24 hours culture after thawing and the third group was 72 hours culture before cryopreservation and 24 hours culture after thawing. Results : The chromosome analysis was successful in the second and third groups of CB sample. Conclusion : The successful result from CB samples may suggest the usefulness of long-term cryopreservation for retrospective study in various clinical settings including hematologic malignancies.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.171-176
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• 1998

This study was carried out to confirm whether the in vivo developmental potential of mouse hatched blastocysts (HBs) can be obtained by vitrification method using the cryoprotectant EFS35. The HBs ($\theta$ 130$\mu\textrm{m}$) were cultured in vitro until day 5 from zygotes produced in vivo and were equilibrated in 10% ethylene glycol(EG) for 5 min, and then were exposed or vitrified in EFS35 (35% EG, 18% Ficoll and 0.3M sucrose). After 30 min thawing, re-expanding HBs were transferred into one or both uterine horns of pseudopregnant recipients on day 3 (4~6 embryos /horn). Pregnancy rates of recipients and implantation were assessed by autopsy on day 15 of gestation. The results obtained in these experiments were summarized as follows : After thaw-ing, in vitro survival of HBs was not significantly different between exposed (65.5%) and vitrified(54.5%) group. Also, when the in vivo development potential was examined, total implantation was not different between control (58.5% ) and vitrified (41.0%) group, although the live fetus formation of vitrified group (24.0%) was significantly lower than that of control (58.3%) group (p< 0.05). These results suggested that vitrification freezing method of mouse HBs using EFS35 can be used to make wide the utility of embryo transfer of the more embryos produced in vitro.