Effects of ginsenoside on survival of human embryonic stem cell-derived cardiac cells after thawing

Jun Beom Ku,Yoon Young Kim,Sun Kyung Oh,Seok Hyun Kim,Young Min Choi,Shin Yong Moon 한국발생생물학회 2012 한국발생생물학회 학술발표대회 Vol.31 No.-

Human embryonic stem cells (hESCs) are promising cell source because of their unique self-renewal and pluripotency. Although hESC-derived cardiac cells are currently generated worldwide, cryopreservation of these cells is still limited due to low rate of post-thaw survival. Cryopreservation of hESC-derived cardiac cells is critical in that their long-term storage can accelerate their use in regenerative medicine. However, to date, there are few reports on efficient cryopreservation and post-thaw survival of hESC-derived cardiac cells. In this study, we evaluated the effects of ginsenoside, which is known to improve survival of rat embryonic cardiomyocytes against myocardial ischemia injury in diabetic rats (Wu et al., 2011), on the survival of hESC-derived cardiac cells after thawing. We induced differentiation into cardiac cells using our previously reported method (Kim et al., 2011). Differentiated, pre-beating stage cardiac cells were cryopreserved using either mass cryopreservation or vitrification. To evaluate the effects of ginsenoside (Re, Rb), we compared three sets: pre- and post-thaw treatment, pre- or post-thaw treatment only. The survival of post-thaw cardiac cells were evaluated using Trypan-blue and Annexin V staining. In addition, the three groups were treated with ROCK inhibitor Y-27632, and compared with non-treatment groups. The effect of ginsenoside was significant in post-thaw treatment group, i.e, thawed cells expressed cardiac specific genes and showed specific functionality such as spontaneous beating. Taken together, we demonstrated favorable effects of ginsenoside on the survival of hESC-derived cardiac cells after cryopreservation and thawing. These results suggest a possible application of well-known cardioprotectant ginsenoside in cell-based tissue engineering using hESC-derived cardiac cells.

Survival Rates with Time Course of Frozen-thawed Pacific Oyster Larvae in Indoor Rearing System

김기태,임한규,장영진 한국발생생물학회 2013 발생과 생식 Vol.17 No.4

Post-thawed larval rearing in Pacific oyster Crassostrea gigas was performed to investigate the survival rate with time course in three kinds of larvae cryopreserved. The highest survival rate and larval activity index (LAI) of post-thawed larvae were obtained from the permeation in 0.2 M sucrose and 2.0 M ethylene glycol (EG) at -1℃/min in freezing speed showing the survival rates just after thawing of 63.8% in trochophore, 84.1% in D-shaped veliger and 56.3% in early umbo veliger. In post-thawed larval rearing with food supply, the larvae lasted their lives until 24 hours in trochophore, 75 hours in D-shaped veliger and 57 hours in early umbo veliger. The results suggested that each larval stage post-thawed revealed no more further development to subsequent respective stage.

Survival Rates with Time Course of Frozen-thawed Pacific Oyster Larvae in Indoor Rearing System

Ki Tae Kim,Young Jin Chang,Han Kyu Lim 한국발생생물학회 2013 발생과 생식 Vol.17 No.4

Post-thawed larval rearing in Pacific oyster Crassostrea gigas was performed to investigate the survival rate with time course in three kinds of larvae cryopreserved. The highest survival rate and larval activity index (LAI) of post-thawed larvae were obtained from the permeation in 0.2 M sucrose and 2.0 M ethylene glycol (EG) at -1℃/min in freezing speed showing the survival rates just after thawing of 63.8% in trochophore, 84.1% in D-shaped veliger and 56.3% in early umbo veliger. In post-thawed larval rearing with food supply, the larvae lasted their lives until 24 hours in trochophore, 75 hours in D-shaped veliger and 57 hours in early umbo veliger. The results suggested that each larval stage post-thawed revealed no more further development to subsequent respective stage.

Survival Rates with Time Course of Frozen-thawed Pacific Oyster Larvae in Indoor Rearing System

Kim, Ki Tae,Lim, Han Kyu,Chang, Young Jin The Korean Society of Developmental Biology 2013 발생과 생식 Vol.17 No.4

Post-thawed larval rearing in Pacific oyster Crassostrea gigas was performed to investigate the survival rate with time course in three kinds of larvae cryopreserved. The highest survival rate and larval activity index (LAI) of post-thawed larvae were obtained from the permeation in 0.2 M sucrose and 2.0 M ethylene glycol (EG) at $-1^{\circ}C/min$ in freezing speed showing the survival rates just after thawing of 63.8% in trochophore, 84.1% in D-shaped veliger and 56.3% in early umbo veliger. In post-thawed larval rearing with food supply, the larvae lasted their lives until 24 hours in trochophore, 75 hours in D-shaped veliger and 57 hours in early umbo veliger. The results suggested that each larval stage post-thawed revealed no more further development to subsequent respective stage.

냉동-해동 적혈구의 제조 및 수혈후 생존능

한규섭,권석운,한복연,김상인,오영철,최범열,김기흥 大韓血液學會 1991 大韓血液學會誌 Vol.26 No.2

γ-LA-Supplementation to IVC for IVP Bovine Embryos

Gaja, A.,Meng, C.L.,Sato, M.,Nakajima, T.,Kubota, Chikara,Kojima, T. Asian Australasian Association of Animal Productio 2010 Animal Bioscience Vol.23 No.1

The present study aimed to examine the effects of ${\gamma}$-linolenic acid (GLA) supplementation to in vitro culture (IVC) medium on in vitro developmental competence, freezability and morphology of in vitro matured and fertilized bovine embryos. In vitro produced (IVP) bovine zygotes were cultured in IVC medium supplemented with 0 (negative control), 15, 31, 62, 125, 250, 500 or 1,000 ppm GLA, 250 ppm linoleic acid albumin (LAA) and without any supplement as a control. Day 6 blastocysts derived from culture control were cultured in IVC medium containing either 62, 250 GLA or 250 LAA for 24 h, and at Day 7 were subjected to freezing or morphological examination by electron microscope. GLA 15 showed a tendency to have a higher cleavage rate at Day 2 (70.3%) than other groups. The hatching rate at Day 9 in LAA (38.2%) was significantly higher than the control and all treatment groups (p<0.05), while the blastocyst rate in LAA (32.4%) did not differ from those of 15 (30.5%), 31 (27.1%), and 62 GLA (33.1%) or the control (35.1%). GLA in concentrations of 125, 250, 500, and 1,000 ppm had significantly detrimental effect on the blastocyst rate compared to 15, 31 and 62 ppm GLA, LAA, and control groups (p<0.05). In contrast, the highest post-thaw survival rate (100%) was observed in the control group (p<0.01). Large lipid droplets were observed in the cytoplasm of trophoblastic cells, even in the control, but were abundant in GLA groups. Taking the results of the study into consideration, the addition of GLA to the culture medium for IVP bovine embryos at the dose of 15 ppm increased the developmental competence of zygotes and enhanced the cleavage rate up to Day 2. However, blastulation rate and post-thaw survival were not increased when GLA was added to the culture media.

참굴 Crassotrea gigas 유생의 냉동과 해동후 경시별 생존율

김기태,장영진,임한규 한국발생생물학회 2012 한국발생생물학회 학술발표대회 Vol.31 No.-

유용양식 대상종인 참굴 Crassostrea gigas은 연안의 환경오염으로 수질환경의 악화로 인해 어미굴의 생리적 활성과 유생의 건강도가 저하로 인한 대량폐사가 빈번히 발생하고 있다. 이에 대한 대책으로서 자연채묘보다는 인공 종묘생산에 의한 양식용 종패생산 기술개발이 필요하며, 무엇보다 유전적으로 우수한 어미와 우량형질을 지닌 유생의 공급이 이루어져야 한다. 그 방법의 일환으로 발생배의 대량 냉동보존기술을 고려해 볼 수 있으며, 기존의 연구에서 생존율 85%의 좋은 결과를 얻은 바 있다. 그러나 이 결과는 해동직후의 생존율로서, 해동 후 경시별 생존율과 추가적인 발생진행을 조사하지 않아 치패생산을 위한 냉동방법으로 활용하기는 어렵다. 따라서 본 연구에서는 참굴의 유생을 이용하여 냉동․해동 후 발생 및 성장에 관한 기초자료를 얻기 위해 적정 CPA의 농도별 냉동실험을 실시하고, 해동유생의 사육을 통한 경시별 생존율, 활성 및 발생 진행 여부를 조사하였다. 충분히 성숙한 암수의 생식소 부위를 절개하여 각각 채란·채정하고 인공수정 후 발생배의 50%가 도달한 담륜자, D형 유생, 초기 각정기 유생을 실험에 사용하였다. 희석액은 0.2 M sucrose와 CPA는 ethylene glycol(EG)를 인공해수에 첨가하여, 최종농도가 각각 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 M 되도록 하였다. 평형시간은 10분씩 설정하였고, CPA에 유생을 침지한 후 0.5 mL straw에 넣어 봉입하여 프로그램냉동기에 의해 -35℃까지 1차 냉동한 다음 -196℃의 액체질소통에 넣어 2차 냉동하였다. 해동에서는 20℃ 담수로 20초간 급속해동하였다. 이어서 해동한 유생의 생존율 및 유생활성지수(larval activity index, LAI)를 산정하였다. 해동유생의 사육시간별 생존율 및 LAI를 알아보기 위해, 담륜자, D형유생, 및 초기 각정기 유생을 여과해수에 담구어 15분간 CPA를 용출시킨 뒤 2 L비커에 수용하여, 최후의 유생이 생존할 때까지 관찰하였다. 사육수는 1일 1회 전량 환수하였고, 해동유생의 먹이로는 Isochrysis galbana를 1일 2회 주되, 매회 20,000 cells/mL씩 공급하였다. 담륜자유생의 해동직후 생존율은 EG 2.0 M에서 63.8±1.3%로 가장 높았으며, LAI 역시 높았다. D형 유생의 생존율은 2.0 M, 2.5 M에서 각각 84.0±1.9%, 84.1±1.5%로 다른 실험구에 비해 높았으며, LAI 또한 3.3±0, 3.4±0으로 다른 농도에 비해 높았다. 초기 각정기 유생의 해동직후 생존율은 2.0 M과 2.5 M에서 각각 56.3±0.6, 55.1±1.4로 다른 실험구들에 비해 높았으며, LAI는 2.0 M에서 2.8±0.1로 높았다. 해동 후 경시별 담륜자의 생존율은 해동직후 63.3%로부터 시간경과에 따라 낮아지다가 24시간째에 전량 폐사하였다. D형 유생은 75시간까지 생존하였다. 초기 각정기 유생의 사육에서도 D형 유생과 비슷한 경향을 보였으며, 해동직후 생존율 55.5%로부터 57시간째에 모두 폐사하였다. 해동 후 생존한 발생배의 경시별 LAI 또한 생존율 변화와 비슷한 경향을 나타내었다. 모든 실험구에서 해동 후 발생 배는 더 이상의 발생 진행이 관찰되지 않았다.