• 한국수정란이식학회지
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• 제9권1호
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• pp.65-71
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• 1994

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$^{\circ}C$ 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$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 3$0^{\circ}C$ and 35$^{\circ}C$. 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.).

• 생명과학회지
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• 제24권11호
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• pp.1252-1257
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• 2014

DMSO는 배양포유류세포 동결보존의 동결보호제로써 일반적으로 사용 되어져 왔지만, DNA 메틸화 및 히스톤의 수식에 의해 일부 세포에서는 분화를 일으키는 것으로도 알려져 있다. 동결보존시의 배양세포의 안정된 분화형질유지에는 메틸화를 일으키는 DMSO 이외의 동결보호제의 사용이 필요하다. 세포독성이 낮고, 동물정자동결보존에 효과적인 것으로 알려진 아미도 화합물이 동일하게 포유류의 배양세포의 동결보존에서 동결보호작용이 있는지를(8종류의 아미드 화합물) 배양 마우스 혈관내피세포를 이용해 조사했다. 조사한 아미드 화합물 중에 아세트아미드와 락트아미드의 2종류가 배양세포에 대해서 동결보호작용이 있고, 가장 효과적인 것은 농도가 1.5 M의 락트아미드이다. 배양세포의 동결보존에 관해서는 삼투압 스트레스를 받지 않을 필요가 있기 때문에, 1.5 M 락트아미드 용액을 제작 시, 용매를 각 희석율의 PBS로 하고, 삼투압을 바꾼 동결 보존액에 동결세포의 생존율을 조사했다. 그 결과, 0.4배 농도의 PBS가 삼투압 스트레스를 가장 낮고 생존율이 가장 높음을 확인했다. 동결보존배지에 고 분자량재료를 첨가하면 세포생존율이 개선되는 것이 알려져 있기 때문에 BSA, HES, 데키스트란의 효과를 조사했다. 그 결과, 락트아미드를 이용한 동결보존배지는 $0.4{\times}PBS$를 이용한 1.5 M 락트아미드용액에 1%의 BSA를 첨가한 경우, DMSO의 동결보호작용에 필적하는 동결보호작용을 나타내는 것을 확인했다.

• KSBB Journal
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• 제27권6호
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• pp.347-351
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• 2012

This study was conducted to optim ize the composition of CEM (cabbage extract medium) and cryoprotectants on the growth of Lactobacillus plantarum, a probiotics growing in plant and milk. For this, we analyzed the growth characteristics of Lb. plantarum in CEM and subsequently optimized the medium composition by addition of carbon, nitrogen sources and buffering agents. Among carbon sources, glucose showed the best result to increase the cell density after dilution of CEM. When 0.5% yeast extract and 1% soy peptone were supplemented in the diluted CEM, Lb. plantarum grew up to the maximum cell density. Addition of buffering agents in CEM was not significantly effective to increase the cell density. Meanwhile, addition of 12% skim milk, 5% sucrose and 0.5% glycerol showed a cryoprotective effect against cell damage of Lb. plantarum during freeze drying process showing high survival rate after 150 days. This optimized CEM can be used for economical production of bacterial cells particularly originated from a plant-related ecosystem.

• 한국가금학회지
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• 제40권3호
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• pp.197-205
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• 2013

동결 닭 원시생식세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화하기 위해서는, 닭 원시생식세포의 동결보존기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시생식세포는 배양 5.5일령의 닭 원시생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시생식세포를 분리했다. 15% 각각의 EG를 동결보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 10% EG 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 상업용 닭(한협3호)에서도 오계와 비슷한 경향의 결과를 확인하였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였다.

• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.59-65
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• 2004

Objectives: The aim of this study was to assess toxicities of cryoprotectants. Methods: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1, 2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. Results: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH ($75.9{\pm}27.0$) or the control ($99.0{\pm}18.3$) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO ($14.2{\pm}1.5$) and PROH ($11.2{\pm}1.4$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.2{\pm}0.9$, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). Conclusions: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

• 한국가금학회지
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• 제41권4호
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• pp.249-259
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• 2014

동결 닭 원시 생식 세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화 하기 위해서는, 닭 원시 생식 세포의 동결 보존 기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시 생식 세포는 배양 5.5일령의 닭 원시 생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시 생식 세포를 분리했다. 15% 각각의 EG를 동결 보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 동결 보호제로 10% EG를 이용한 유리화 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 10% EG를 이용한 완만 동결 처리군에서 66.14%로 동일한 농도의 PG 처리군(50.11%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였고, 이는 한국재래닭(오계)의 원시 생식 세포의 동결 보존의 실용화가 보다 더 향상될 수 있는 또 하나의 방법이 될 수 있음을 시사한다.

• KSBB Journal
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• 제30권3호
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• pp.109-113
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• 2015

Current drying and encapsulation methods for probiotics manufacturing are complicate and cost-burdened processes. The aim of this study was to develop a simple ingredient mixture to make probiotic granules via one-step process, providing not only a cryoprotective effect during freezing and drying but also high survival ratio in gastrointestinal tract. As cryoprotectans, commercially available ingredients including skim milk, monosaccharide (trehalose or glycerin), maltodextrins (with low or high degree of equivalents) were used. Their cryoprotective effect during lyophilization and survival ratios in artificial gastric juice and bile salt were measured against 3 strains of lactic acid bacteria (LAB) (Lactobacillus plantarum, Lb. brevis, and Lactococcus lactis). As results, 3 mixtures with different compositions showed a cryprotective effect on LAB tested and the best compostion was dependant upon LAB; skim milk 10%, trehalose 15%, glycerin 0.5%, and NaCl 1% was for Lb. plantarum and Lc. lactis, and maltodextrin 10% instead of skim milk was for Lb. brevis. In addition, those mixtures showed similar survival effect on LAB tested. These results demonstrate that skim milk or maltodextrins with trehalose, glycerin, and NACl can be effectively used for onestep lyophilization of LAB as an alternative method of encapsulation.

• ALGAE
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• 제24권4호
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• pp.257-265
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• 2009

Marine microalgae are a key diet component in finfish and shellfish aquaculture. Cryopreservation of the microalgae is suggested by many other studies as the best method for long-term storage. To test cryopreservation efficacy, 19 taxas of marine microalgal species were examined. In the first experiment we compared dimethylsulfoxide ($Me_2SO$) and glycerol, which are most widely used as cryoprotectant agents (CPAs). The cryopreservation comprised two freezing procedures. Firstly, the samples containing the CPAs were kept at $4^{\circ}C$ for 10 min before being plunged into liquid nitrogen ($-196^{\circ}C$). Secondly, samples containing CPAs were pre-cooled ($-1^{\circ}C$ $min^{-1}$ to $-80^{\circ}C$ before being plunged into liquid nitrogen. Most of the species were successfully cryopreserved using $Me_2SO$, whereas the Prasinophyceae (T. striata and T. suecica) were successfully cryopreserved using glycerol. In general, the cooling method had no influence on the survival of the microalgae except in the case of the Tetraselmis species. In the second experiment, the cultured solution was divided before cryopreservation into concentrated and non-concentrated groups to identify the effect of cell density during cryopreservation. After 12 months of storage, the samples were again divided into centrifugation and non-centrifugation groups to learn the effect of $Me_2SO$ on the culture. Viability and growth of the microalgae were not influenced by cell density or the centrifugal removal of the $Me_2SO$ after thawing.

• 한국임상수의학회지
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• 제29권1호
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• pp.27-32
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• 2012

The purpose of this study was to evaluate survival rates of vitrified mouse blastocysts in various vitrification solutions (cryoprotectants) and apparatuses. The mouse blastocysts were harvested from culture of mouse 2 cell embryo and were divided into three group (i) untreated (control); (ii) exposed to cryoprotectant agents; or (iii) cryopreserved by various vitrification apparatuses. Vitrification solutions are 40% ethylene glycol (EG) + 5.8 mg/mL ficoll + 0.5M sucrose (EFS solution), 3M glycerol + 3M EG (ES solution), 20% EG + 20% dimethyl sulfoxide (ED solution), 3M EG + 1.0 m sucrose (ES solution). Vitrification apparatuses consisted of 5 groups ; closed plastic straw (CPS), electron microscope (EM) grid, cryoloop, open pulled straw (OPS), and glass micropippete in plastic straw (GPS). The survival rates of control were 88.0%. The survival rates of exposed blastocysts in EFS, GE, ED, and ES solutions were 70.8%, 43.5% (P<0.01), 83.3% and 65.2%, respectively. The survival rates of vitrified blastocysts in CPS, EM grid, cryoloop, OPS and GPS were 56.5% (P< 0.01), 72.7%, 83.3%, 60.9% (P<0.05) and 54.2% (P<0.01), respectively. Among the vitrification solutions, the highest survival rate was seen in blastocysts vitrified in EG + DMSO (83.3%). The survival rate was not significantly different from that of the control (88%). Blastocysts cryopreserved with glycerol in all groups had an overall low survival rate of 43.5%. Survival rate of mouse blastocysts between vitrification apparatuses showed higher in cryoloop.

• 한국수정란이식학회지
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• 제4권1호
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• pp.1-13
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• 1989

This paper reviews the most important steps that have generated consistent progress in principles and developmental progress of embryo cryopreservation, and also study on freezing procedure and its application by conventional method and current improved method for freezing procedure and its appilcation of embryo cryopreservation in farm animals. Four were of particular interest: 1.The transport of water across the ccli membrane (zona pellucida) during freezing and thawing accordinglyplays a role in determing whether the celi survives. This movement of water is controlied mainly by extracellular phase changes and by the nature and concentration of any cryoprotective agent present. Therates of cooling, freezing and warming, and the intervals over which they are applied are further decisi've factors in determining whether a cryopreservation procedure allows survival after thawing. 2.The first successful deep freezing experiments with sheep morula and blastocysts during the seventies were based on the early procedures used for mouse embryos.Current research during the eighties is developed with the aim of simplifying and improving current procedures such as one-step dilution and rapid or ultra-rapid cooling by using the model of laboratory animals. 3.The conventional method for the embryo cryopreservation is described. An alternative to this method which may result in high survival and also in reducing of the freezing and thawing time is done by combing a permeable cryoprotectant such as glycerol, DMSO or propanediol and a non-permeable compound such as sucrose, trehalose, raffinose or lactose. 4.Finally a different approach to the preservation of embryos, named vitrification, is introduced. This procedure depends upon the ability of concentrated solutions of cryoprotective agents such as glycerol and propanediol to supercool to very low temperature (-196$^{\circ}C$) during rapid cooling before solidifying without formation of ice. However, more complete data are necessary for successful vitrification of blastocysts.