Ethylene Glycol이 포유류 초기배자의 생존성에 미치는 독성 효과 분석

김현,유대중,최창용,성환후 한국동물번식학회 2015 Reproductive & developmental biology Vol.39 No.3

본 연구에서 배아의 생식세포 동결에 가장 흔히 쓰이고 있는 두 가지 동결 보호제, 즉 DMSO와 EG의 독성을 비교하고자 생쥐 수정란 모델을 이용한 실험을 하였다. 생후 6주령의 암컷 생쥐 F1 hybrid mice에 10 IU의 PMSG를 복강 주사하여 과배란을 유도하고, 2-세포기 배아를 획득하고 DMSO와 EG 각각 노출시킨 후, 배양을 하였다. 배반포의 전체 세포수는 2-세포기 단계에서 DMSO(68.1±24.1)로 EG(81.2±27.0) 혹은 control(99.0±18.3)(p<0.001) 처리구에 비해서 유의적으로 낮았다. DMSO 처리구가 EG 처리구에 비해 세포수가 적었다. DMSO(15.4±1.5)와 EG(10.2±1.4) 두 처리구는 대조구(6.1±0.9, p<0.0001)와 비교해서 배반포에서 세포사 비율이 더 높음을 확인했다. 또한, DMSO 처리구는 EG 처리구(p<0.001)보다 더 많은 세포사멸된 세포가 확인되었다. DMSO 또는 EG 처리군과 대조군 사이에는 배아 부화율에 있어서 차이가 있었으며, 이는 배아에 대한 동결 보호제의 잠재적인 독성을 확인한 결과였다. 이번 연구에서 장기간 처리했을 때 EG 처리군보다 DMSO 처리군에서 배아발달과 세포수가 저하된 것은 DMSO의 독성이 더 높을 것으로 사료된다. This study was carried out to evaluate the effects of embryonic stage and toxicities of cryoprotectant on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology. Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and ethylene glycol (EG) 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 EG. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO (68.1±24.1) at the 2-cell stage was significantly lower than that were treated with EG (81.2±27.0) or the control (99.0±18.3) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the EG treated group (p<0.05). Both DMSO (15.4±1.5) and EG (10.2±1.4) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.1±0.9, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the EG treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or EG at room temperature. When comparing two cryoprotective agents, EG appeared to be less toxic than DMSO at least in a murine embryo model.

GC-OTC/FID에서 Dead Time 결정을 위한 새로운 방법 개발에 대한 연구

오도석,김성화,고은아,전형우 대한화학회 2019 대한화학회지 Vol.63 No.4

GC-OTC/FID(Gas chromatography-Open Tubular Column/Flame Ionization Detector) 계에서 극성 용매(Alcohols)를 분 리 하기 위하여 DMSO(Dimethyl sulfoxide)를 사용하였다. 이 계에서는 극성 용매들 보다 DMSO가 늦게 용출이 된다. 이런 계 에서 크로마토그래픽 인자인 조정된 머무름 시간(tR'=tR-tO)과 용량 인자{k'=(tR-tO)/tO} 및 분리 인자{α=(tR2-tO)/(tR1-tO)}를 구하 기 위하여 불감시간(tO)이 필요하다. 그러나 이런 계에서 tO 를 구하기 위한 보고가 현재까지 된 바가 없기 때문에, 본 연구에 서는 tO 를 구하는 방법을 개발하고자 하였다. tO 를 계산하기 위하여 DMSO의 머무름 시간(DMSO tR)을 상용로그로 전환하 였다(f(x)=log tR(DMSO)→tO, tO=log 9.551=0.980). 개발된 방법의 적합 여부를 확인하기 위하여 CH4의 tR과 ln tR(DMSO)를 log tR(DMSO)와 비교하였다. 세 가지 방법 중 CH4 tR 과 ln tR(DMSO)는 k' 과 α를 계산하는데 적합하지 않았다. 본 연구에서 개발한 방 법인 log tR(DMSO)는 일반적인 기준인 k'(1<k'<10)과 α(1<α<2)를 만족하였다. 본 연구에서 개발한 계산방법은 쉽고 편리하기 때 문에, 이와 유사한 계에서도 활용될 것으로 기대된다. In the system of GC-OTC/FID (Gas chromatography-Open Tubular Column/Flame Ionization Detector), DMSO (Dimethyl sulfide) solvent was used to separate the polar solvents (Alcohols). In this system DMSO was eluted later than the separated polar solvents. At this system to calculate chromatographic factors [adjusted retention time (tR'=tR-tO), capacity factor{ k'=(tR-tO)/tO} and separation factor {α=(tR2-tO)/(tR1-tO)}], dead time(tO) is necessary, but the method to calculate it has not been reported yet. Therefore, we have tried to develop tO. To calculate tO, we conversed DMSO retention time (DMSO tR) to logarithm (f(x)=log tR(DMSO)→tO, tO=log 9.551=0.980). To confirm the optimization of the developed method, we compared with CH4 tR and ln tR(DMSO). Both of the values calculated by CH4 tR and ln tR(DMSO) were not suitable in the calculation k' and α. The developed method in this study{log tR(DMSO)} has satisfied both of the values k' criteria (1<k'<10) and α (1<α<2). The developed calculation method in this study was easy and convenient, therefore it can be expected to be applied to these similar systems.

사람의 제대혈관 장력에 미치는 DMSO(Dimethyl Sulfoxide)의 효과

김상재,강석한,정인덕,김기순 한양대학교 의과대학 1998 한양의대 학술지 Vol.18 No.2

Starting from 1960s through to 1980s, dimethyl sulfoxide (DMSO) earned fame as a valuable agent that could be used for many purposes including patient therapy. DMSO is dipolar aprotic compound endowed with unique physical and chemical properties. It is a potent solvent for a broad spectrum of solutes. DMSO has been known to exert varions biological effects such as diuretic, vasodilatory, analgesic, anti-inflammatory and cardiovascular effects. Also DMSO has been reported to reduce intracranial pressure and improve cerebral blood flow in experimentally induced ischemia and vasospasm. Some studies presented a possibility that may alter vascular smooth muscle tension. On the other hand, human umbilical vessels are ideal for the study of vasoactive substances because of poor innervation of autonomic nerves to umbilical vessels. This study was undertaken to investigate effects of DMSO on the vascular tension of human umbilical vein (HUV) and artery (HUA). DMSO(1-10%) relaxed HUV and HUA preparations precontracted with KCl (3×10^-2M) in a concentration-dependent manner. At all concentrations, vasodilating effect of DMSO was greater in HUV than HUA. Pretreatment with methylene blue (5×10^-6M), soluble guanylate cyclase inhibitor and L-NAME (10^-4M), NO synthase inhibitor, did not alter relaxing action of DMSO. After pretreament with indomethacin, cyclooxygenase inhibiotor, the DMSO-induced relaxing response was partially suppressed only in HUA (41 ± 5.7%). Following chemical denudation with saponin (0.03mg/ml, 45min), the DMSO-induced relaxing responses of both umbilical vessels were not changed. These results suggest that the DMSO induces an endothelium-independent relaxation both in the human umbilical artery and vein, and that the DMSO-induced relaxing response of HUA results, at least in part, from the mediation of prostanoids.

Evaluation of Fertilizing Ability using Frozen Thawed Sperm in the Longtooth Grouper, Epinephelus bruneus

오성립,이치훈,Hyeong-Cheol Kang,송영보,김형배,이영돈 한국발생생물학회 2013 발생과 생식 Vol.17 No.4

This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: 99.5±0.8%, DMSO 7.5%: 99.5±0.7%, and DMSO 10.0%: 99.6±0.6%). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: 96.2±2.3%, DMSO 7.5%: 95.3±3.6%, 10.0%: 96.6±1.8%) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm (98.4%±0.5) and DMSO concentration level of 5.0% (97.8±0.1%) were not significantly different. However, with 7.5% (97.2±0.6%) and 10.0% DMSO concentrations (95.9±0.2%) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; 66.8±1.8%, HR: 82.0±12.9%) and 2 years (FR; 78.5±14.8%, HR: 79.3±0.6%) cryopreserved sperm were lower than control (FR; 100%, HR: 91.1±3.6%) and 2 days cryopreserved sperm (FR; 99.6±0.6%, HR: 96.6±1.8%). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.

Evaluation of Fertilizing Ability using Frozen Thawed Sperm in the Longtooth Grouper, Epinephelus bruneus

Oh, Seong-Rip,Lee, Chi-Hoon,Kang, Hyeong-Cheol,Song, Young-Bo,Kim, Hyung-Bae,Lee, Young-Don The Korean Society of Developmental Biology 2013 발생과 생식 Vol.17 No.4

This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: $99.5{\pm}0.8%$, DMSO 7.5%: $99.5{\pm}0.7%$, and DMSO 10.0%: $99.6{\pm}0.6%$). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: $96.2{\pm}2.3%$, DMSO 7.5%: $95.3{\pm}3.6%$, 10.0%: $96.6{\pm}1.8%$) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm ($98.4%{\pm}0.5$) and DMSO concentration level of 5.0% ($97.8{\pm}0.1%$) were not significantly different. However, with 7.5% ($97.2{\pm}0.6%$) and 10.0% DMSO concentrations ($95.9{\pm}0.2%$) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; $66.8{\pm}1.8%$, HR: $82.0{\pm}12.9%$) and 2 years (FR; $78.5{\pm}14.8%$, HR: $79.3{\pm}0.6%$) cryopreserved sperm were lower than control (FR; 100%, HR: $91.1{\pm}3.6%$) and 2 days cryopreserved sperm (FR; $99.6{\pm}0.6%$, HR: $96.6{\pm}1.8%$). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.

포유류 초기 배아의 효율적인 동결 보존 방법에 관한 연구

김현,조영무,고응규,김성우,성환후,야마노우치 케이타로 한국수정란이식학회 2014 한국동물생명공학회지 Vol.29 No.3

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. This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Two to eight cell embryos were obtained from oviducts of mated F1 hybrid female mice superovulated by PMSG and hCG. Two-step EG, DMSO and 4-step EG, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in EG and DMSO was significantly higher than 4~8 cell (65.4% versus 61.2%, 81.1% versus 72.5%) (p<0.01, p<0.01), but the development rates of 4~8 cell embryos in EG and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4~8 cell embryos in EG were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in EG(77.0% versus 64.4%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was sinificantly higher in EG than in DMSO during 48 hr (p<0.01). The survival rate of 4~8 cell embryo was 62.5% in EG and 73.3% in DMSO. The development rates of embryo in EG were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. The survival rate of embryo in 2 cell stage was higher than in 4~8 cell stage, and EG appears more effective cryoprotectant than DMSO because EG showed better development rates of embryos in 2 and 4~8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

The effects of Dimethyl sulfoxide on mouse embryonic development and methylation

Na Eun Cho,Myoung Ok Kim 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Mouse embryonic stem cells (mESCs) undergo self-renewal in the presence of the cytokine leukemia inhibitory factor (LIF). LIF is added to the mouse stem cells culture medium, and removal results in rapid differentiation. Dimethyl sulfoxide (DMSO) is one of the most commonly used solvents in drug test. We exposed 4-day mESC cultures to different concentrations of DMSO (0.1%, 0.5%, 1.0%, and 2.0%) to identify the safest dose exhibiting efficacy as a solvent. In the absence of LIF, mESCs grown under common possibility conditions were treated with DMSO. In addition, as a control for differentiation, mESCs were grown in the absence of LIF. DMSO upregulated the mRNA expression level of pluripotency markers. Furthermore, DMSO reduced the mRNA expression levels of mesodermal marker (Hand1), ectodermal marker (β-tubulin3) and endodermal markers (Foxa2 and Sox17) in mESCs. These results indicate that DMSO treatment enhances the pluripotency and interrupts the differentiation of mESCs. We also show that members of the Tet oncogene family are critical to inhibiting the differentiation and methylation of mESCs. DMSO is appropriate for maintaining the multiple possibilities of mESCs in the absence of LIF, and mESCs can be maintained in an undifferentiated state using DMSO. Therefore, DMSO may function in part as a substitute for LIF.

생쥐배아의 동결보존에 관한 실험적 연구

이여일,권영숙,박현정,Lee, Yu-Il,Kwon, Young-Sook,Park, Hyun-Jeong 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.1

Objectives: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Materials and Methods: Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. Results: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1 %,79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The developmen1 rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. Conclusions: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

Purification of 5-hydroxymethyl-2-furfural from DMSO by extraction method

박석규,이경원,이관영,조진구 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

5-Hydroxymethyl-2-furfural (HMF) is an essential platform to be transformed to a variety of bio-based chemicals but mass production of HMF has not been achieved in industry by now for the reason of difficulty in isolating HMF. Representatively, although DMSO is well known as the best solvent for selective dehydration of fructose into HMF, separation of HMF from DMSO still acts as a hurdle in mass production of HMF. Liquid-liquid extraction is used as a typical procedure to remove DMSO solvent in synthetic chemistry but there are few profound studies on separation of HMF from DMSO. In this study, 9 Kinds of organic solvents were screened for efficient extraction of HMF with minimizing the extraction of DMSO. Finally, 90% of HMF in 99% of purity could be separated from DMSO using MIBK/HX (9:1).

Development of DMSO-free Cryopreservation Media for Cells Producing Biopharmaceuticals

Beom Jin KIM,Do Hee LEE,Duk Jae OH 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Many studies have shown that cells are likely to experience genetic or phenotypic variations over generations. The only way to prevent this is to keep the cells frozen at extremely low temperatures ( -80℃ to -196℃). However, this cryopreservation method can have a negative effect on cells because it provides cells with an extreme environment (high osmotic pressure, physical and chemical stress, etc.). To reduce this negative effect as much as possible, CPAs (cryoprotective agents) are used. The most representative CPA is DMSO (dimethyl sulfoxide). However, the use of DMSO is somewhat controversial as studies have shown that it may reduce cell viability or affect gene expression systems. Therefore, we conducted research to develop preservatives using substitute substances other than DMSO. Similar substances were evaluated to select two CPAs as highly permeable substances such as DMSO were considered key candidates. These can prevent ice formation and inhibit physical damage to cells by removing water from the cells. However, recently, the freeze protection effect of antioxidants was confirmed as it has been argued that not only the physical protection of cells but also the chemical damage to ROS outbreaks should be controlled. In conclusion, we identified cell-permeable substances that have the key effects to replace DMSO, and additionally designed cryopreservation compositions by adding functional additives. Like this, we aim to eliminate controversial DMSO when freeze-preserving cells for biopharmaceutical production, and to develop products with equal or better freeze-preservation capabilities without using DMSO.