• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 춘계학술대회
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• pp.80-80
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• 2019

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of (Fragaria x ananassa DUCH. cvs. 'Derunoka' and 'Jumbo pure berry'. The shoot tips of strawberry were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7M). Precultured explants were treated with loading solution (LS, C4) containing glycerol 17.5% and sucrose 17.5% for 40 min and exposed to dehydration solution (B1) containing 50% of glycerol and 50% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M Sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on MS medium for 4 weeks was 77.8% and 60.0% for 'Derunoka' and 'Jumbo pure berry', respectively. This result shows droplet-vitrification would be a promising method for cryobanking strawberry germplasm.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2023년도 임시총회 및 춘계학술대회
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• pp.33-33
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• 2023

Potatoes are the world's 4th major food crop after maize, rice, and wheat and also are a staple food for 1.3 billion people. Due to their wide adaptability to various environmental conditions, their yeild capacity, and high commercial value, potatoes have contributed to global food security. Many potato germplasms are commonly preserved as whole plants in fields or in storage to maintain their particular genetic combinations. However, field maintenance is expensive and has the risk of potential losses from diseases, pests, plant ageing and climate change. Over the past four decades, meaningful efforts have been made toward the safe long-term conservation of potatoes through cryopreservation methods such as droplet-vitrification. In this study, we tested 4 Korean potato varieties('Golden Egg', 'Golden Ball', 'Ja-Young' and 'Ha-Ryeong') with the modified potato droplet -vitrification protocol. Potato shoot tips are precultured in a sucrose-enriched medium(0.3 and 0.7M for 7 and 17hrs, respectively) and submitted to a loading step with C4 solution for osmoprotection. The treated explants were dehydrated with Plant Vitrification Solution(PVS)2 which is 80% A3 solution in ice for 30 minutes. Thawing and unloading steps were performed with 0.8M sucrose solution for 30 sec(40℃) followed by 30min(25℃, room temperature). In a potato post-culture medium(MS+0.1 mg·L^-1 GA₃+0.1 mg·L^-1 kinetin), we obtained a survival rates of post-thawed explants ranging 16.1-82.2%. The results suggest that modified and optimized protocols are required dependinig on every cultivar, genetic and ecological types. To achieve higher survival and regeneration rates, each step within the cryoprocedure must be carefully optimized.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.3-7
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• 2002

In vitro embryo culture techniques provide significant contributions not only for a basic research of fertilization and early embryogenesis, but also for a low cost mass production of bovine embryos for transfer, embryo diagnosis, nuclear cloning and the production of transgenic cows. This presentation introduces newly developed serum-free media (IVD101 and IVMD101) that are effective far high yields of transferable embryos of excellent quality from in vitro-matured and fertilized oocytes. Both serum-free media are superior to a conventional serum-containing medium on the increased rates of blastocyst formation, post-thaw embryo viability, and pregnancy after transfer. Furthermore, reduced risks of calf mortality and large calf syndrome are also observed for the serum-free-derived embryos. Serum-derived embryos contain a large number of lipid droplets and immature mitochondria in their cytoplasm that may account for the lower production of transferable embryos and poor embryo quality.

• Restorative Dentistry and Endodontics
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• 제31권3호
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• pp.192-202
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• 2006

본 연구의 목적은 흰쥐 상악 대구치를 발거 한 후 급속 냉동보존을 통해 치아를 보관하였을 때 in vivo MTT 검색법을 이용하여 치주인대세포의 활성도를 측정하고자 하였다. 실험 방법은 74마리 4주령의 암컷 Sprague-Dawley계의 흰쥐를 사용하여 각군당 10마리의 쥐에서 상악 좌우 제1, 2 대구치를 발거하여 모두 40개의 치아를 사용하였다. 대조군은 즉시 발치군이며 냉동군은 F medium에 5% Dimethylsulfoxide (DMSO) 6% Hydroxyethyl starch (HES)를 포함한 군(1군), 10% DMSO를 포함한 군(2군) 그리고 $Viaspan^(R)$에 5% DMSO 6% HES(3군), 10% DMSO를 포함한 군 (4군)으로 나누어 1주일간 액체질소에 냉동한 뒤 해동하였다. 냉장군은 F medium (5군)와 $Viaspan^(R)$ (6군)에 넣어 1주간 $4^{\circ}C$ 냉장고에서 보관하였다. 냉동 및 냉장보존한 후에는 in vivo MTT검색법을 시행하였다. 개개 치아의 치근면 단위면적으로 표준화하기 위해 치근을 1% eosin Y 용액에 12시간 담근 후 1% acid alcohol로 용해시켜 530 nm에서 측정한 흡광도 값을 in vivo MTT 측정값으로 나누었다. 통계 분석을 위해 Two way ANOVA와 Duncan's Multiple Range Test를 95% 신뢰 구간에서 시행하였다. 그리고 각 군당 2개의 치아를 같은 방법으로 처리한 후 $10{\mu}m$ 두께로 냉동 절단하여 광학 현미경과 편광 현미경하에서 관찰하였다. 1, 2군은 3, 4군보다. 높은 흡광도를 나타내었으며 6군은 5군보다. 높은 흡광도를 나타내었다(p<0.05). 광학 현미경 관찰에서 MTT 결정은 파란색을, 편광현미경에서는 밝은 오렌지색을 나타내었고 전체적으로 in vivo MTT검색법의 결과와 같은 양상을 나타내었다. 본 연구의 결과 5%, 10% DMSO를 사용한 F medium 배지로 냉동보존한 경우가 $Viaspan^(R)$을 배지로 냉동하였을 때나 냉장보존 하였을 때보다 통계적으로 유의차 있게 높은 세포 활성도를 나타내었다(p<0.05).

• 한국수정란이식학회지
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• 제23권3호
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• pp.223-227
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• 2008

The cryopreservation of Hanwoo embryos has become an integral part of assisted reproduction in animal. The objective of this study was to assess the effect of The objectives of this study were: (1) to evaluate the influence of bovine embryo developmental stage on in vitro embryo development after freezing, (2) to study the efficiency compared with conventional freezed embryos at different embryo source. For conventional slow-freezing, day 7 or 8 expanded blastocysts were collected. The standard freezing medium was 1.8 M ethylene glycol (EG). Embryos were equilibrated in 1.8 Methylene glycol(EG) with 0.1 M sucrose in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25 ml-straw and placed directly into cooling chamber of programmable freezer precooled to $-7^{\circ}C$, after 2 min, the straw was seeded, maintained at $-7^{\circ}C$ for 8 min, and then cooled to $-35^{\circ}C$ at $0.3^{\circ}C$/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 see and exposed to $37^{\circ}C$ water for 20 sec. Straws were then removed from $37^{\circ}C$ water. Rates of blastocyst survive and hatched were evaluated at 12 to 48h post-warming. The re-expansion and hatched rates of morula embryos were significantly lower than those obtained for blastocysts and expansion blastocysts (31.6%, 10.5% vs, 68.9%, 22.2% vs, 73.7%, 53.6%, respectively). No differences in re-expansion rates were found between in vivo and in vitro blastocysts. whereas hatched rates was significantly higher (51.2%) in vivo compared with in vitro embryos (18.6%). in conclusion, demonstrate that conventional freezing can be used successfully in cryopreservation of in vitro and in vivo bovine embryos, and that it might be considered for use in commercial programs and embryo preservation.

• 한국수정란이식학회지
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• 제23권4호
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• Clinical and Experimental Reproductive Medicine
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• 제28권2호
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• pp.121-129
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• 2001

Objective: The aim of this study is to compare the efficiency of a method for the cryopreservation of mouse blastocyst.. Methods: Mouse embryos were obtained at 2-cell stage and cultured to blastocyst stage in T6 medium supplemented with 10% fetal bovine serum. Morphologically normal blastocysts were collected and randomly divided to one control and four experimental groups. In control group, blastocysts were cultured in vitro continuously for additional two days. In group 2, blastocysts were exposed to vitrification solution (ethylene glycol) only without cryopreservation (exposure only group). In group 3, 4 and 5, blastocysts were cryopreserved by slow-freezing procedure with glycerol (slow-fteezing group) or by vitrification procedure using EM grids (EM grids group) and cryoloop (cryoloop group), respectively. Frozen blastocysts were thawed and cultured for additional two days. Twenty four hours after thawing, some blastocysts were fixed and stained with Hoechst 33342 (bisbenzimide) and the number of nuclei in each blastocysts were counted to confirm the survival of bias to cysts in experimental groups. Results: Survival rate and hatching rate of the blastocysts in slow-freezing group (24 h: 72.4% and 66.0%, 48 h: 63.2% and 64.6%) and EM grids group (24 h: survival rate 77.3%, 48 h: 70.1% and 71.4%) were significantly lower ($X^2$-test p<0.05) than those of control group (24 h: 93.4% and 86.0%, 48 h: 88.5% and 90.7%). In contrast, the survival rate and hatching rate of the blastocysts in cryoloop group (24 h: 84.1% and 84.1%,48 h 79.3% and 87.7%) is well compared with those in the control group. The mean (${\pm}SD$) cell number of blastocyst in the exposure only ($89.2{\pm}11.5$), EM grids ($85.0{\pm}10.3$) and cryoloop ($89.0{\pm}11.0$) groups, except slow-freezing group ($79.0{\pm}10.0$), were not significantly different from that of control group ($93.1{\pm}13.9$) 24 h after thawing (Student's t-test). Conclusion: This study demonstrates that higher survival rate of vitrified-thawed mouse blastocyst can be obtained using cryoloop as the embryo container at freezing rather than slow-freezing or vitrification using EM grids. The results of this study suggest that vitrification using cryoloop (with ethylene glycol) may be a preferable procedure for mouse blastocyst cryopreservation and could be applied to the human blastocyst cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Clinical and Experimental Reproductive Medicine
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• 제35권3호
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• pp.203-212
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• 2008

목 적: 정자의 동결 과정에서 생길 수 있는 급격한 온도 차에 의한 동결 충격이나 동결 상해등에 의한 세포막의 손상, 세포의 기능 장애 등은 정자의 수정능에 영향을 미칠 수 있다. 본 연구에서는 인간 정자를 동결 보존하는 과정에서 콜레스테롤 전처리가 정자의 운동성 및 기능보존에 미치는 영향을 알아보고자 하였다. 연구방법: 본원을 내원한 14명 남성의 정자를 대상으로 콜레스테롤을 첨가하지 않은 대조군 (control)과 여러 농도의 콜레스테롤을 동결보존액에 첨가한 실험군에서 정자의 동결-융해 후 상태를 다음 3가지 방법으로 비교, 분석하였다. 1) 정자 분석, 2) calcium ionophore로 유도된 첨체 반응 검사, 3) 정자 염색질 구조 분석 (sperm chromatin structure assay). 결 과: 첫째로 인간 정자의 운동성은 $0.5{\mu}g$ 농도의 콜레스테롤을 첨가한 동결보존액에서 동결-해동하였을 경우, 콜레스테롤을 첨가하지 않은 군에 비해 유의적 차이를 보이며 증가하는 것을 확인하였다 ($33.46{\pm}1.48%$ vs. $30.10{\pm}1.07%$, p<0.05). 다음으로 동된 정자의 첨체 반응 검사에서도 콜레스테롤을 첨가한 동결보존액에서의 첨체 반응이 일어나는 정자의 비율이 첨가하지 않은 군에 비해 유의하게 높게 관찰되었다 ($53.60{\pm}1.60%$ vs. $47.40{\pm}1.86%$, p<0.05). 마지막으로 정자 염색질 구조 분석에서는 콜레스테롤을 첨가한 군이 첨가하지 않은 군에 비해 정자의 DNA손상이 적게 나타남을 확인하였다. 결 론: 본 실험은 동결보존액을 통한 정자 원형질막 내 콜레스테롤 함유량의 증가가 동결-융해 후 정자의 운동성과 수정능(capacitation status)을 증가시키고 DNA 손상을 방지하는 역할을 한다는 결과를 보여주었다. 이러한 결과를 통해 동결보존액 내 콜레스테롤의 첨가는 인간 정자의 동결보존 동안 발생할 수 있는 동결 상해를 줄여줄 수 있는 유용한 방법으로 사료된다.

• 한국수정란이식학회지
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• 제23권4호
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• pp.269-274
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• 2008

The purpose of this study was to determine the effects of Taxol pre-treatment to in vitro matured bovine oocytes, and sucrose and trehalose added to vitrification solution on spindle morphology and embryonic development following cryopreservation. Bovine oocytes were collected from ovaries and matured in tissue culture medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS), 0.05ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone and $1{\mu}g/ml$ estradiol for 22h in $39^{\circ}C$, 5% $CO_2$, TCM 199-HEPES containing 20% FBS was used as basic medium (BM) to prepare vitrification solution. Oocytes were pre-treated with $1\;{\mu}M$ Taxol in maturation medium for 15 min prior to vitrification. Oocytes were exposed to 1.6 M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM and then were exposed to 3.2 M EG, 2.6 M DMSO and 0.5 M sucrose in BM or 3.2 M EG, 2.6 M DMSO and 0.5 M trehalose in BM. Oocytes with cumulus cells and oocytes without cumulus cells were considered as control 1 and control 2, respectively and held in TCM 199-HEPES at $39^{\circ}C$. Oocytes were frozen using modified solid surface vitrification and were stored in cryotubes in liquid nitrogen for more than 1 week. Frozen oocytes were thawed in TCM 199-HEPES containing 0.5 M, 0.25 M and 0.1 M sucrose in BM for 2 min, respectively or 0.5 M, 0.25 M and 0.1 M trehalose in BM for 2 min, respectively. Immunoflurorescence staining of oocytes was performed to assess spindle morphology and chromosome configuration of oocytes. The rates of cleavage and blastocyst were examined following in vitro fertilization. Normal spindle morphology rate of oocytes pre-treated with Taxol prior to vitrification was not higher than that of other vitrified groups. Taxol pre-treatment did not increase cleavage and blastocyst formation rates, although control groups showed significantly higher rates (p<0.05). Percentages of normal spindle and embryonic development were not significantly different among vitrified groups regardless of type of sugar. In conclusion, Taxol pre-treatment of oocytes before cryopreservation did not reduce the damage induced by vitrification and subsequently did not improve embryonic development following vitrification. Trehalose may be used as an alternative non-permeating cryoprotectant in vitrification solution.