• 한국해양생명과학회지
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• 제1권1호
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• pp.70-78
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• 2016

Microalgae are of significant importance for future biotechnological applications. Many microalgae banks or laboratories attempt to maintain various microalgae for further research purposes. Cryopreservation has been preferred to reduce a labor-intensive and costly routine sub-culturing. Cryopreservation can also diminish the genetic drift risk. However, cryopreservation as a long term storage of microalgae method are still in developing progress because it cannot be generalized for all microalgae. Microalgae types, cryoprotectant agents (CPAs) types, freezing and thawing methods are the most important factors that should be considered for cryopreservation. In this short review the basic principles and the current advanced of microalgae cryopreservation methods are discussed with a suggested starting parameters for microalgae cryopreservation.

• Journal of Plant Biotechnology
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• 제45권4호
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• pp.400-408
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• 2018

21세기 들어 전 세계는 환경파괴에 따른 지구 온난화로 인하여 생물 다양성이 지속적으로 감소하였다. 생물다양성의 감소는 생태계의 자정 및 복원능력 등을 악화 시켜 환경오염이 더욱 심화 되었고 나아가 생물의 생존을 위협하고 있다. 우리나라는 현재 나고야 의정서로 인해 국내 유전자원을 효율적으로 발굴 보호해야 하는 중요한 시점에 있다. 본 연구에서 사용하는 미세조류는 유용 생물자원으로 활용도가 높으나 동결보존하는 방법이 개발되어 있지 않아 장기보존에 어려움이 있다. 대부분의 원핵생물이나 균류 등의 미생물은 종에 관계없이 glycerol 을 이용하여 저온동결보존이 가능하나 미세조류는 동결보존제의 종류와 농도, 유리화 과정 및 해동방법 등에 따라 세포 재생률에 차이가 있기 때문에 구체적인 연구가 필요하다. 미세조류 중 규조류는 전 세계적으로 발견되며 규산질로 구성된 cell wall 은 이미 산업적으로 많이 이용되고 있다. 본 연구에서는 해양 규조류 Nizschia frustulum과 Nitzschia amabilis를 이용하여 장기 동결보존을 위한 기초연구를 수행하였다. 동결보존제로는 세포막 침투성 보존제인 glycerol, DMSO, methanol을 각각 5, 10, 15% 농도로 제조하여 실험하였고 메탄올의 경우 시약 특성상 5, 10, 12% 농도로 실험하였으며 미세조류는 N. frustulum과 N. amabilis 두 종을 각각 $10^2$, $10^3$, $10^4cells\;ml^{-1}$로 희석하여 사용하였다. 실험에 사용한 미세조류 두 종 모두 메탄올 12%에서 가장 높은 생존율을 보였으며 N. frustulum은 $6.94{\pm}0.31%$, N. amabilis는 $8.85{\pm}0.16%$로 나타났다. 동결 후 해동한 미세조류를 3주간 재배양한 결과에서는 N. frustulum이 재배양 초기 농도보다 약10배 증가하였고 N. amabilis는 약12배 증가한 결과를 나타내었다. 본 연구에서는 유용생물자원인 미세조류 Nizschia sp.에 한하여 적합한 동결보존제를 탐색하였으며 이 자료를 바탕으로 더 많은 동결보존제에 대한 효과와 다양한 미세조류 종에 적합한 동결보존 기법 연구가 필요한 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권3호
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• pp.328-335
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• 2009

This study was undertaken to investigate the influence of exposure and removal of four different cryoprotectants (CPAs) on the ATP content of cumulus cell-enclosed (COs) and cumulus cell-denuded (DOs) immature porcine oocytes. The in vitro nuclear maturation of the COs, exposed to and removed from the CPAs was also assessed. Both COs and DOs were exposed to 1.5 M concentrations of each CPA (ethylene glycol (EG); propylene glycol (PG); dimethyl-sulfoxide (DMSO); and glycerol (G)) for durations of 5, 15, and 30 minutes at room temperature ($23.5{\pm}1.5^{\circ}C$), and immersed in physiological saline supplemented with 20% (v/v) fetal bovine serum for 5 minutes ($39^{\circ}C$) to remove each CPA. Before, during and after exposure to each CPA, the ATP content of both the COs and the DOs was measured. After removal from each CPA an aliquot of the COs was matured for 44${\pm}$2 h, and their nuclear maturation rates were measured up to the metaphase stage of the second meiotic division (the M-II stage). The maturation rates up to the M-II stage were not significantly different between all the groups that were exposed to each CPA for 5 minutes. For 15 and 30 minute exposures, the maturation rates of the COs exposed to PG, DMSO and EG were almost the same as those of the control groups; however, the rates of G group exposed for 15 and 30 minutes were significantly lower (p<0.05) than the control group. These groups were also found to have a decrease in the ATP content of COs and DOs during and after exposure for the same periods (p<0.05). In addition, although the ATP contents of the COs after exposure to EG for any period were the same as the controls, the ATP content of the DOs exposed to EG for any period were significantly lower (p<0.05) than those of the controls. When the ATP content of the COs and DOs of each CPA were compared, the DOs exposed to PG were found to have a significantly greater level (p<0.05) than DOs exposed to G for any duration. In addition, the ATP content of DOs exposed to PG for 30 min and removal was also higher (p<0.05) than when exposed to DMSO for the same period. These findings indicate that PG may be a useful CPA for the cryopreservation of immature porcine oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제38권1호
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• pp.24-30
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• 2011

Objective: The objectives of this study were to analyze efficacy of immature and mature mouse oocytes after vitrification and warming by applying various combinations of cryoprotectants (CPAs) and/or super-rapid cooling using slush nitrogen ($SN_2$). Methods: Four-week old ICR female mice were superovulated for GV- and MII-stage oocytes. Experimental groups were divided into two groups. Ethylene glycol (EG) only group: pre-equilibrated with 1.5 M EG for 2.5 minutes and then equilibrated with 5.5 M EG and 1.0 M sucrose for 20 seconds. EG+dimethylsulfoxide (DMSO) group: pre-equilibrated with 1.3 M EG+1.1 M DMSO for 2.5 minutes and equilibrated with 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 20 seconds. The oocytes were loaded onto grids and plunged into $SN_2$or liquid nitrogen ($LN_2$). Stored oocytes were warmed by a five-step method, and then their survival, maturation, cleavage, and developmental rates were observed. Results: The EG only and EG+DMSO groups showed no significant difference in survival of immature oocytes vitrified after warming. However, maturation and cleavage rates after conventional insemination were greater in the EG only group than in the EG+DMSO group. In mature oocytes, survival, cleavage, and blastocyst formation rates after warming showed no significant difference when EG only or EG+DMSO was applied. Furthermore, cleavage and blastocyst formation rates of MII oocytes vitrified using $SN_2$ were increased in both the EG only and EG+DMSO groups. Conclusion: A combination of CPAs in oocyte cryopreservation could be formulated according to the oocyte stage. In addition, $SN_2$ may improve the efficiency of vitrification by reducing cryoinjury.

• 약학회지
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• 제37권2호
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• pp.143-148
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• 1993

The objective of this study was to evaluate the sedative activity of four aporphine alkaloids (APA) and nine cyclopeptide alkaloids(CPA), which had been isolated from the seeds (sanjoin) of Zizyphus vulgaris var. spinosus, and the fruits and stem bark of Zizyphus jujuba var. inermis. The assessment of sedative activity was carried out, employing a hexobarbital-induced sleeping time method in mice. When the relative sedative potency of sanjoinine-A(CPA) was given as one unit, those of nuciferine (APA), lysicamine (APA), chlorpromazine (positive control), and sanjoinine -Ahl (an epimer of sanjoinine-A) were 13, 6.5, 5, and 3, respectively. The sedatvie activities of other CPAs were much lower than those of sanjoinine-A and -Ahl, and other APAs were not active. On heat treatment, nuciferine and lysicamine were degraded into some artifacts which exhibited no sedative activity, while sanjoinine-A was converted into sanjoinine-Ahl which showed more potent sedative activity. These results suggested that nuciferine and sanjoinine-A were major sedative components of native sanjoin, and that sanjoinine-A and its epimeric artifact, sanjoinineAhl were the active principles of roasted sanjoin. It provides a scientific basis for heat-processing (roasting) of this Oriental medicine.

• 대한조선학회논문집
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• 제56권4호
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• pp.352-360
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• 2019

International Maritime Organization (IMO) regulates an emission of greenhouse gases by creating an Energy Efficiency Design Index (EEDI) to reduce environmental pollution. In propulsion system field, studies are under way on Energy Saving Device (ESD), which can improve propulsion efficiency with the propeller, to reduce the EEDI. Among the studies, the study of Pre-Swirl Stator (PSS) has been actively conducted from long time ago. Recently the variable pith angle type pre-swirl stator has been studied to improve the propulsion efficiency in non-uniform flow fields of the Stern. However, for traditional design methods, no specific design method has been established on the blade or location of radius. In this study, proper design method is proposed for each blade or location for radius according to hydrodynamic pitch angle.

• 한국수산과학회지
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• 제47권1호
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• pp.39-44
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• 2014

This study aimed to find an optimal diluent and cryoprotective agent (CPA) during cryopreservation of common Korean bitterling Acheilognathus signifier sperm. The bitterling is an endangered species in Korea, and this study will enable conservation and further technical development of artificial seed production. We tested the effects of cryopreservation and toxicity by the type of diluent and CPA. The optimal combination of diluent and CPA for cryopreservation was 300 mM glucose+10% methanol, resulting in a survival rate and sperm activity index (SAI) of $96.3{\pm}1.5%$, and $3.0{\pm}0.0$ respectively, and no significant difference compared to fresh sperm. The survival rate and SAI of post-thawed sperm was $9.7{\pm}1.5%$, $0.8{\pm}0.3$ respectively, which was significantly higher than had been achieved with other diluents and CPAs.

• 한국수산과학회지
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• 제44권6호
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• pp.665-670
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• 2011

The rotifer Brachionus plicatilis is one of the most important food organisms in aquaculture. The resting eggs produced by mictic female rotifers are easily stored and hatched, making them useful as the starter for the mass culture of rotifers in marine larval culture. This study examined the optimum preservation method for resting eggs to ensure a high hatching rate. To produce resting eggs, the marine rotifer B. plicatilis was cultured with Nannochloris oculata (KMMCC 16). The resting eggs were harvested and cryopreserved using 5% and 10% methanol (MeOH), dimethylsulfoxide (DMSO), and glycerol as cryoprotectant agents (CPAs). The cryopreservation comprised slow or rapid freezing and the resting eggs were stored for one month in liquid nitrogen ($-196^{\circ}C$). The resting eggs were also dried at different temperatures (30, 40, and $50^{\circ}C$) and for different times (1, 2, and 3 h). In general, the hatching rates of the resting eggs preserved with CPA were higher than those without CPA and the slow freezing method was better than the rapid freezing method. However, the optimum CPA concentration for the hatching rate of the resting eggs varied with the freezing method and kind of CPA, and the CPA also affected the viability of the resting eggs. Dried resting eggs had a high, rapid hatching rate over 80%. The moisture content of the resting eggs cryopreserved in liquid nitrogen affected the hatching rate. Drying at $30^{\circ}C$ for 1 hour resulted in a high hatching rate of the resting eggs. In conclusion, drying at $30^{\circ}C$ for 1 hour and preservation in liquid nitrogen with the slow freezing method, without CPA, is recommended for a high hatching rate (ca. 95%) of rotifer resting eggs.

• 융합정보논문지
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• 제11권1호
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• pp.128-135
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• 2021

바지락 발생배의 냉동보존을 위한 동해방지제의 효과를 규명하기 위하여4종의 보존액, dimethyl sulfoxide, ethylene glycol, glycerol 및 1,2-propanediol을 사용하여 발생배의 생존율을 조사한 결과로, 생존율의 범위는 0-64.3%였고, 대조구의 생존율은 82.3%을 보였다. 발생배의 냉동보존을 위한 프로그래 동결 과정은 최초 25℃에서 10분 동안 유지하다가 자동 냉동보존 프로그램 장치에 의하여 -12℃까지 분당 -1℃씩 조정하였으며 -35℃까지는 분당 -2℃씩 하강시켜 액체 질소통에 스트로우를 넣어 해동 후 관찰한 결과, 바지락 생존율은 DMSO(dimethyl sulfoxide) 2.0M 실험구에서 64.3%로 가장 좋은 결과를 보였다.

• Clinical and Experimental Reproductive Medicine
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• 제37권4호
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• pp.307-319
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• 2010

목적: 유리화 동결액의 조성조절과 냉각속도 증진을 통한 동결보호제의 농도를 낮추는 전략을 통해 세포에 미치는 독성을 감소시켜 유리화 동결 및 융해 후 생쥐 배아의 생존율 및 발생률을 증진시키고, 궁극적으로 배아의 유리화 동결법을 개선하고자 하였다. 연구방법: 생쥐 배아와 포배기를 그리드를 이용한 유리화 동결법을 이용하여 동결/융해하였다. 동결액 내 ethylene glycol와 dimethylsulphoxide (DMSO)의 농도와 당의 농도를 조절하여 생쥐 배아의 융해 후 생존율과 발생률을 관찰하였고, 냉각속도의 증가와 동결억제제의 농도와의 상관관계를 포배기의 융해 후 생존율과 발생률에 따라 비교하였다. 또한 융해 후 배아를 대리모에 이식하여 산자를 생산함으로 냉각속도의 효율성을 알아보았다. 결과: EG를 단독으로 사용한 동결보존액 보다는 DMSO와 혼합된 동결보존액의 사용이 보다 유리하다는 결과를 얻을 수 있었다. 슬러시 질소에 의한 냉각속도의 증가가 동결보존의 대상의 상해를 줄여 유리화 동결의 효율성을 증진시키는 것으로 생각된다. 결론: 혼합된 동결보호제를 사용하였을 때 생쥐 배아의 유리화 동결 후 생존과 발생률이 증진되었다. 슬러시 질소를 이용한 유리화 동결의 도입은 냉각속도의 증가를 통해 기존 유리화 동결방법의 효율을 증진시켜 생존율과 융해후 발생률, 임신율 증진에 기여하였다. 또한 냉각속도의 증진은 유리화 동결의 필수요건인 고농도의 동결억제제에 대한 노출을 감소시킬 수 있었다. 이러한 노력은 생식력의 보전을 위한 유리화 동결법의 효율 향상에 기여할 것이다.