본 연구에서 생쥐 초기 배아를 이용하여 동결 보존된 배아의 생존율 또는 발달율에 영향을 미치는 요인들의 상관관계를 알아본 결과, 배아의 발달 단계에 따른 동결 - 해동 후 생존율에 있어서는 2세포기 배아가 4~8세포기 배아보다 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 4~8세포기 배아가 유의하게(p＜0.01), 높아 생존율과 배 발달율과는 상관관계가 없는 것으로 사료된다. 또한, 동결 보호제에 따른 배아의 생존율에 있어서는 2, 4~8세포기 배아 모두 DMSO에서 유의하게 높았으나(p＜0.01), 배 발달율에 있어서는 EG가 DMSO에 비해 더 유의하게 높은 성적을 나타내어(p＜0.01, p＜0.05) 동해로 인한 상해가 적은 것으로 생각되어 2, 4~8세포기 배아에서 EG가 더 효과적인 동결 보호제로 사료되며, 동결 프로그램으로는 완만 동결 - 급속 해동법이 더 우수한 프로그램으로 보인다.

본 실험은 국화의 바이로이드 제거에 이용되는 초저온처리 시 국화 품종 'White ND'을 적합한 처리조건을 확립하기 위해 초저온처리의 단계별 요인을 실험하였다. 그 결과 생장점의 크기는 1 mm(엽원기 2~3매 포함)에서 높은 생존율과 신초 재생율을 나타내었고, vitirification 처리시 PVS3가 효과적이었으며, 처리 시간은 60분 처리 하였을 때 높은 생존율 및 정 상 신초 재생율을 보였다. 또한 vitrification을 위한 전처리 조건은 sucrose 농도를 88 mM 24시간, sucrose 0.3 M 16시간, sucrose 0.5 M 6시간, sucrose 0.7 M 3시간으로 처리하는 것이 초저온 처리 후 생존율 및 신초 재생율을 높이는데 효과적이었으며, 재생된 정상 식물체는 모본과 비교하여 ploidy level이 동일한 것으로 보아 식물체의 유전적 변이가 일어나지 않았다.

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at — 7℃ to — 35℃ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen- thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/ 0.5 ml, and 43.6 % frozen at 20embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at —7 ℃ to —35 ℃ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen-thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/ 0.5ml, and 43.6 % frozen at 20 embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

The aim of this study was to evaluate the post-thawed characteristics of leopard cat semen. In this experiment, semen was collected from two leopard cats (A and B) at wild animal center in Seoul Grand Park in Korea. After collection, the sperms were washed with D-PBS and diluted by the freezing medium (Irvine science, USA) and stored in liquid nitrogen. The post-thawed concentration was for A and for B. The viability of post-thawed sperm from A and B individual was 24.0% and 19.0%, respectively. Pre-freezing motility of A and B individual semen was 68.54% and 56.65. Leopard cat A had more normal sperm than that of B (69.5% vs. 54.5%). Acrosome integrity analysis detected live (14.5% vs. 9.0%), damage (39.0% vs. 44.0%) and dead (46.0% vs. 47.0%) in leopard cat A and B, respectively. The present results concluded that leopard cat semen can be collected successfully by electro-ejaculation method and cryopreserved successfullyfor future use in different assisted reproductive technologies. The cryopreservation protocol needs to be modified for increasing post-thawed viability of leopard cat spermatozoa.

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.

This study were carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen bovine embryos. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3O water. Survival rate was defined by FDA test. The results are summarized as follows : 1. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various kinds of cryoprotective agents added 0.25M and O.50M sucrose were 28.6% and 25.0%, 35.1% and 31.6%, 32.4% and 24.4%, 34.2% and 28.2%, 18.9% and 17.6%, 14.7.% and 21.6%, respectively. 2. The survival rate of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol, i.5M and 2.OM DMSO and 1.5M and 2.OM propanediol were 22.9~37.8%, 2O.7~31. 3%, 19.2~30.0% and 17.2~25.0%, respectively. 3. The temperature thawed at 2 after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 3 and 35. 4. The equilibration time on the survival rate of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (1O~20 min.).

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: ‘Frost Eureka limon’ and ‘Cook Eureka limon’, using a droplet-vitrification method. The shoot tips that were excised from in vitro grown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at 25℃ and incubated in a loading solution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at 25℃. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at 0℃ or PVS3 at 25℃. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in ½ MS for 30 min at 25℃. Shoot tips were post-cultured overnight on survival medium and then micrografted onto ‘trifoliate orange’ (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% for cryopreserved shoot tips of ‘Frost Eureka limon’ and ‘Cook Eureka limon’, respectively, when pre-cultured in 0.3 M and 0.5 M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at 0℃. We also investigated whether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoot tips. The viability of cooled samples, following culturing on woody plant media (WPM) containing ¼ ammonium nitrate overnight before micrografting, was the highest (70.3%) in ‘Frost Eureka limon’. The study described here is a cost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruit plants and for breeding disease resistance.

Sweet potato (Ipomoea batatas L.) shoot tips grown in vitro were successfully cryopreserved by encapsulationvitrification. Encapsulated explants are very easily manipulated, due to the relatively large size of the alginate beads, and a large number of samples can be treated simultaneously. In this study, the effects of sucrose preculture, cryoprotectant preculture, and post-warm recovery media on regrowth, following liquid nitrogen (LN) exposure, were investigated to establish an efficient encapsulation-vitrification protocol for sweet potato. Shoot tips of plants grown in vitro were precultured in 0.3 M sucrose for 2 d before encapsulation. Encapsulated shoot tips were pre-incubated in liquid MS (Murashige and Skoog) medium containing 0.5 M sucrose for 16 h, before preculturing in sucrose-enriched medium (0.7 M sucrose) for 8 h. Shoot tips were osmoprotected with 35% plant vitrification solution 3 (PVS3) for 3 h, before being dehydrated with PVS3 for 2 h at 25°C. The encapsulated and dehydrated shoot tips were transferred to 2 mL cryotubes, suspended in 0.5 mL PVS3, and plunged directly into liquid N. High levels of shoot formation were obtained for the cv. Yeulmi (65.7%) and Yeonwhangmi (80.3%). The regrowth rates of cryopreserved samples in Yeulmi (78.9%) and Yeonwhangmi (91.3%), following culture on ammonium-free MS medium for 5 d, were much higher than those cultured on standard MS medium (65.7% and 80.3%, respectively). This encapsulation-vitrification is a promising method for the long-term preservation of sweet potato.

Cryopreservation has been known as an efficient method for long-term preservation of clonally propagated plants, and several cryopreservation methods have been developed. Among them, a droplet-vitrification method for potato using axillary shoot tips in vitro has been established previously. In this study, we have optimized the procedure in which explants were submitted to a step-wise pre-culture in liquid sucrose-enriched medium (0.3 and 0.7 M for 7 and 17 h, respectively). The pre-cultured explants were dehydrated with PVS3 (w/v, 50% glycerol + 50% sucrose) for 90 min or modified PVS2 vitrification solution (w/v, 37.5% glycerol + 15% DMSO + 15.0% ethylene glycol + 22.5% sucrose) for 30 min. This two dehydration solutions produced post-cryopreservation regeneration percentages of 57.2% and 80.9%, respectively. We also compared a new post-culture medium (0.1 mg L ･ -1 GA3, 0.1 mg L ･ -1 kinetin) with the conventional one (0.15 mg L ･ -1 IAA, 0.2 mg L ･ -1 zeatin, 0.05 mg L ･ -1 GA3); the shooting initiation rates were 80.9% and 43.5%, respectively. The results suggest that the modified droplet-vitrification protocol described in this study is more effective, easier to implement, and more economical than the droplet-vitrification protocols currently used for potato.

Human embryonic stem cells (hESCs) have the potential for use in regenerative medicine and in the field of basic research. Therefore, effective cryopreservation and storage of hESCs are important for preservation of newly established cell line for various purposes. Despite poor survival and slow recovery after thawing, the conventional slow freezing method is most commonly used for cryopreservation of hESCs due to its simplicity and ease of use for freezing a large number of hESCs appropriate to clinical applications. Here we controlled the clump size (Group Ⅰ; 400～450 ㎛, Group Ⅱ; 800～900 ㎛, and Group Ⅲ; 1500～1700 ㎛) of hESCs at 5 days after plating using a glass pipette during cryopreservation in order to obtain a larger amount of hESCs after thawing. Attachment rates differed significantly (P<0.05) in each of the three groups and the average of attachment rate of GroupⅡ was highest in SNUhES4 and H1. In particular, the attachment rate of Group Ⅱ in SNUhES3 showed a significant improvement with ROCK inhibitor Y-27632. These results indicate that clump size and cell-cell adhesions of GroupⅡ are appropriate for cryopreservation compared to the Group Ⅰ and Group Ⅲ. This method increased cell viability and reduced the recovery time leading to various experiments, and therefore has an advantage for use with hESCs like newly established in particular. We demonstrated that use of this effective cryopreservation method with control of the clump size of hESCs can effectively improve the attachment rate and survival of post-thaw hESCs with and without Y-27632.

Recently, human embryonic stem (hES) cells have become very important resources for ES cell basic research, cell replacement therapy, and other medical applications; thus, efficient cryopreservation methods for these cells are needed. This study examined whether a newly developed minimum volume cooling (MVC) vitrification method, which was tested through cryopreservation of sensitive bovine oocytes, can be used for freezing hES cells. Feeder-free cultured hES cell (MB03) colonies were mechanically dissected into several small clumps following enzymatic treatment. We compared the freezing efficiency of a slow-cooling method using a cryo-module (0.4-0.6C/min, 20-30 clumps/vial) and MVC vitrification using a modified 0.5-ml French mini-straw designated as a MVC straw (>/min, 10 clumps/straw) After thawing, in vitro survival of hES cell clumps was higher for MVC-vitrified cells (80.8%, 97/120) than for slow-cooled cells (38.2%, 39/102). Further, the proliferation rate of surviving MVC-vitrified cells was similar to that of control hES cells from 2 weeks after thawing. In addition, vitrified-thawed hES cells demonstrated a normal karyotype, were positively immunostained for surface marker antibodies (AP, SSEA-4 and TRA-1-60) and the Oct-4 antibody, and could differentiate into all three embryonic germ layer cells in vitro. This result demonstrates that hES cell clumps can be successfully cryopreserved by a newly developed MVC vitrification method without loss of human cell characteristics.

본 연구에서는 보다 효율적인 동결 보존법을 수립하기 위하여 현재 사용되는 동결 보존액과 동결방법을 정자의 운동성 측면에서 비교해 보았다. 즉, 세 종류의 조성이 다른 동결보존액인 TYB, dithiothreitol을 첨가한 TYB+DTT, KS II 등이 동결보존 전후에 있어 운동성에 미치는 영향을 조사하였으며, 또한 vapor freezing 방법과 computerized freezer를 사용한 동결방법이 정자 운동성에 미치는 영향을 알아보았다. 정자