WO2021238039A1 - Rapid vitrification cryopreservation and recovery method of strongylocentrotus intermedius embryo - Google Patents
Rapid vitrification cryopreservation and recovery method of strongylocentrotus intermedius embryo Download PDFInfo
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- the invention belongs to the field of sea urchin germplasm preservation, and in particular relates to a rapid vitrification cryopreservation and recovery method of sea urchin embryos.
- the middle ball sea urchin also known as Echinodermata, belongs to the Echinodermata, Eleutherozea, and Echinoidea. It is native to the coasts of Hokkaido, Japan and the Far East of Russia. It was introduced from Japan in 1989 and has now formed a certain scale of artificial breeding in Dalian, Shandong and other places. It is the most important economic sea urchin species in the northern coast of my country. In recent years, the market demand and industrial scale of sea urchins have expanded year by year, but the resource quantity and germplasm quality of sea urchins have dropped sharply.
- cryopreservation of fertilized eggs or blastocyst stage embryos obtained after routine artificial induction and insemination of sea urchins during the sea urchin breeding season to recover and hatch.
- Slow cryopreservation is to place sea urchin fertilized eggs or blastocyst stage embryos in dimethyl sulfoxide, equilibrate at -76°C for 10 minutes and then transfer to liquid nitrogen storage (Asahina E, Takahashi T.
- vitrification cryopreservation technology for embryos of fish and crustacean aquatic animals, which is to solidify a high concentration of protective agent (vitrification solution) wrapped with target cells in an ultra-low temperature environment to form irregular vitrification Like solid.
- This technology can make the cell itself and the vitrification fluid appear viscous and not crystallized when frozen, and can greatly improve the survival rate of the cells.
- the existing vitrification solution used in the rapid vitrification cryopreservation technology of fish embryos is based on 100 ml of distilled water, 2.472% sodium chloride, 0.086% potassium chloride, 0.146% calcium chloride dihydrate, 0.0486% magnesium chloride hexahydrate and 0.019% Sodium bicarbonate is mixed, and the vitrification solution used in the rapid vitrification cryopreservation technology of shrimp embryos is composed of 30% glycerin, 6% ethylene glycol, 3mg/mL bovine serum albumin, 10mg/mL polysucrose and 0.5mol /L is a mixture of sucrose.
- the present invention aims to solve the above-mentioned technical problems in the prior art, and provides a rapid vitrification cryopreservation and resuscitation method of sea urchin embryos.
- the technical solution of the present invention is: a rapid vitrification cryopreservation method of sea urchin embryos in the middle ball sea urchin, which is carried out in sequence according to the following steps:
- Embryo collection artificially induce spawning and insemination of sea urchin, and collect embryos that develop to gastrulation stage from fertilized eggs;
- Embryos cryopreservation at ultra-low temperature The embryos that have been equilibrated together with 250 ⁇ l of vitrification solution for the last equilibration are sucked into the straw and sealed by hot pressure, and immediately placed in liquid nitrogen for cryopreservation.
- a method for resuscitating embryos of sea urchin in the middle ball frozen by the above method The frozen straw is taken out of liquid nitrogen and immediately placed in a water bath at 25°C. When the milky white of the vitrified liquid in the straw is about to disappear, the straw is taken out And cut off the seal, eluted with 1 to 2 mL of 0.075M sucrose solution for 5 to 10 minutes, and then added the same volume of filtered seawater every 5 minutes for washing, washing in total 3 times, and incubating with fresh sterile filtered seawater.
- the invention is to add dimethyl sulfoxide, methanol and 1,2 propanediol as vitrification liquid to sterile filtered seawater to realize rapid vitrification and cryopreservation of the embryos of sea urchin in the middle ball, and undergo a special recovery method such as thawing in a water bath at 25°C, so that After thawing, the intact rate of the embryonic gastrulation of the middle ball sea urchin can reach 47.81%, which has the advantages of high intact rate of the embryonic gastrointestinal tract after thawing, easy operation and low cost.
- the method for rapid vitrification and cryopreservation of sea urchin embryos of the present invention is carried out in sequence according to the following steps:
- Embryo collection in the breeding season in October 2019, take out the middle sea urchin (the middle sea urchin cultivated by the Key Laboratory of Maritime Aquaculture of the Ministry of Agriculture and Rural Affairs of Dalian Ocean University) from the water and dry the sea urchin body surface water for use Potassium chloride artificially induced spawning of the sea urchin in the middle ball and placed it upside down in a petri dish. After the sperm (white) or the eggs (orange) are laid, randomly mix 3 sperm and eggs from the sea urchin in a 5L beaker. Continue to observe To the gastrulation stage, collect embryos in the gastrulation stage;
- DMSO dimethyl sulfoxide
- MeOH methanol
- 1,2 propanediol (1,2-PG) 1,2 propanediol
- Embryos cryopreservation at ultra-low temperature suck the equilibrated embryos together with 250 ⁇ l of vitrification solution for the last soaking treatment into a straw with a diameter of 3mm and a length of about 10cm, burn the mouth of the tube with an alcohol lamp, and seal it with tweezers. Throw it into liquid nitrogen (-196°C) and store it in a frozen state;
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Abstract
Disclosed is a rapid vitrification cryopreservation method of a strongylocentrotus intermedius embryo. Vitrification is realized by mixing dimethyl sulfoxide (DMSO), methanol (MeOH) and 1, 2-propylene glycol (1, 2-PG) with filtered and disinfected seawater, precooling for 15 minutes at the temperature of 2℃, and the volume concentrations of the dimethyl sulfoxide, the methanol and the propylene glycol are 1.0%-2.0%, 1.5%-2.0% and 1.5%-2.0% respectively; and the recovery method is realized by means of treatment such as 25℃ water bath thawing. The intact rate of the thawed strongylocentrotus intermedius embryo archenteron can reach 47.81%. The method has the advantages that the intact rate of the thawed embryo archenteron is high, operation is easy and the cost is low.
Description
本发明属于海胆种质保存领域，尤其涉及一种中间球海胆胚胎的快速玻璃化冷冻保存与复苏方法。The invention belongs to the field of sea urchin germplasm preservation, and in particular relates to a rapid vitrification cryopreservation and recovery method of sea urchin embryos.
中间球海胆，又称虾夷马粪海胆，属于棘皮动物门(Echinodermata)，游走亚门(Eleutherozea)，海胆纲(Echinoidea)。原产于日本北海道和俄罗斯远东等地沿海，1989年自日本引入，现已在大连、山东等地形成一定的人工养殖规模，是我国北方沿海最重要的经济海胆种类。近年来，海胆的市场需求以及产业规模逐年扩大，但是海胆的资源量和种质质量却急剧下降。为了维持海胆优良品种的种质质量以及保护海胆资源多样性，目前已有在海胆繁殖季节对海胆进行常规人工催产和授精后得到的受精卵或囊胚期胚胎进行冷冻保存进而复苏孵化的研究，如慢速冷冻保存或程序降温冷冻保存。慢速冷冻保存，是将海胆受精卵或囊胚期胚胎置于二甲基亚砜中，在-76℃中平衡10min后转入液氮保存(Asahina E,Takahashi T.CRYOPRESERVATION OF SEA URCHIN EMBRYOS AND SPERM[J].Development Growth and Differentiation,1979,21(5):423-430.)。程序降温冷冻保存，是将海胆受精卵或囊胚期胚胎置于1.5M二甲基亚砜和0.04M海藻糖(TRE)中，4℃下初始平衡2min，随后在-1℃min至-12℃冷却平衡2min，后以-1℃/min降至-80℃后平衡2min，最后转移到液氮中进行储存(Bellas J,Estefanía Paredes.Advances in the cryopreservation of sea-urchin embryos:Potential application in marine water quality assessment[J].Cryobiology,2011,62(3):0-180.)。但是，以上两种技术所需要的仪器较多且操作比较繁琐，不利于大规模应用。The middle ball sea urchin, also known as Echinodermata, belongs to the Echinodermata, Eleutherozea, and Echinoidea. It is native to the coasts of Hokkaido, Japan and the Far East of Russia. It was introduced from Japan in 1989 and has now formed a certain scale of artificial breeding in Dalian, Shandong and other places. It is the most important economic sea urchin species in the northern coast of my country. In recent years, the market demand and industrial scale of sea urchins have expanded year by year, but the resource quantity and germplasm quality of sea urchins have dropped sharply. In order to maintain the germplasm quality of excellent sea urchin species and protect the diversity of sea urchin resources, there have been researches on cryopreservation of fertilized eggs or blastocyst stage embryos obtained after routine artificial induction and insemination of sea urchins during the sea urchin breeding season to recover and hatch. Such as slow cryopreservation or program cooling and cryopreservation. Slow cryopreservation is to place sea urchin fertilized eggs or blastocyst stage embryos in dimethyl sulfoxide, equilibrate at -76°C for 10 minutes and then transfer to liquid nitrogen storage (Asahina E, Takahashi T. CRYOP RESERVATION OF SEA URCHIN EMBRYOS AND SPERM[J]. Development Growth and Differentiation,1979,21(5):423-430.). The program is cooled and cryopreserved by placing sea urchin fertilized eggs or blastocyst stage embryos in 1.5M dimethyl sulfoxide and 0.04M trehalose (TRE), initial equilibration at 4°C for 2 minutes, and then at -1°C for min to -12 Cool at ℃ for 2min, then reduce to -80℃ at -1℃/min, then equilibrate for 2min, and finally transfer to liquid nitrogen for storage (Bellas J, Estefanía Paredes. Advances in the cryopreservation of sea-urchin embryos: Potential application in marine water quality assessment[J].Cryobiology,2011,62(3):0-180.). However, the above two technologies require more instruments and complicated operations, which are not conducive to large-scale applications.
目前，已有应用于鱼类和甲壳类水产动物的胚胎快速玻璃化冷冻保存技术，是将包裹有目标细胞的高浓度保护剂(玻璃化液)在超低温环境下凝固，形成不规则的玻璃化样固体。该技术可使细胞本身及玻璃化液冷冻时呈现黏稠而不产生结晶的玻璃化状态，可大幅度提高细胞存活率。现有用于鱼类胚胎快速玻璃化冷冻保存技术的玻璃化液是以蒸馏水100毫升、2.472％氯化钠、0.086％氯化钾、0.146％二水氯化钙、0.0486％六水合氯化镁和0.019％碳酸氢钠混合而成，而用于虾类胚胎快速玻璃化冷冻保存技术的玻璃化液是由30％甘油、6％乙二醇、3mg/mL牛血清蛋白、10mg/mL聚蔗糖和0.5mol/L蔗糖混合而成。但是使用这两种玻璃化液对海胆胚胎进行玻璃化冷冻保存，解冻后的海胆胚胎几乎全部破裂，难以找 到完整胚胎，即现有玻璃化液并不适合中间球海胆胚胎的快速玻璃化冷冻保存。At present, there has been a rapid vitrification cryopreservation technology for embryos of fish and crustacean aquatic animals, which is to solidify a high concentration of protective agent (vitrification solution) wrapped with target cells in an ultra-low temperature environment to form irregular vitrification Like solid. This technology can make the cell itself and the vitrification fluid appear viscous and not crystallized when frozen, and can greatly improve the survival rate of the cells. The existing vitrification solution used in the rapid vitrification cryopreservation technology of fish embryos is based on 100 ml of distilled water, 2.472% sodium chloride, 0.086% potassium chloride, 0.146% calcium chloride dihydrate, 0.0486% magnesium chloride hexahydrate and 0.019% Sodium bicarbonate is mixed, and the vitrification solution used in the rapid vitrification cryopreservation technology of shrimp embryos is composed of 30% glycerin, 6% ethylene glycol, 3mg/mL bovine serum albumin, 10mg/mL polysucrose and 0.5mol /L is a mixture of sucrose. However, using these two vitrification fluids to vitrify sea urchin embryos for cryopreservation, almost all of the thawed sea urchin embryos are broken, and it is difficult to find intact embryos, that is, the existing vitrification fluid is not suitable for rapid vitrification cryopreservation of middle ball sea urchin embryos .
发明内容Summary of the invention
本发明是为了解决现有技术所存在的上述技术问题，提供一种中间球海胆胚胎的快速玻璃化冷冻保存与复苏方法。The present invention aims to solve the above-mentioned technical problems in the prior art, and provides a rapid vitrification cryopreservation and resuscitation method of sea urchin embryos.
本发明的技术解决方案是：一种中间球海胆胚胎的快速玻璃化冷冻保存方法，依次按照如下步骤进行：The technical solution of the present invention is: a rapid vitrification cryopreservation method of sea urchin embryos in the middle ball sea urchin, which is carried out in sequence according to the following steps:
a.胚胎的收集：对中间球海胆进行人工催产和授精，收集受精卵发育至原肠期的胚胎；a. Embryo collection: artificially induce spawning and insemination of sea urchin, and collect embryos that develop to gastrulation stage from fertilized eggs;
b.玻璃化液配制：将二甲基亚砜、甲醇及1,2丙二醇与过滤消毒海水混合，置于0～4℃预冷15min，所述二甲基亚砜、甲醇及丙二醇的体积浓度分别为1.0～2.0％、1.5～2.0％和1.5～2.0％；b. Preparation of vitrification solution: mix dimethyl sulfoxide, methanol and 1,2 propylene glycol with filtered and disinfected seawater, and place it at 0～4°C for 15 min. The volume concentration of the dimethyl sulfoxide, methanol and propylene glycol Respectively 1.0～2.0%, 1.5～2.0% and 1.5～2.0%;
c.胚胎冷冻前的平衡处理：将收集的胚胎依次在1/5、1/4、1/3、1/2和1浓度的玻璃化液中各浸泡5～6min；c. Equilibrium treatment before embryo freezing: Soak the collected embryos in vitrification solution of 1/5, 1/4, 1/3, 1/2 and 1 concentration for 5-6 minutes;
d.胚胎超低温冷冻保存：将经过平衡处理的胚胎连同250μl最后一次平衡处理用玻璃化液吸入麦管中并热压封口，立即置入液氮中冷冻保存。d. Embryos cryopreservation at ultra-low temperature: The embryos that have been equilibrated together with 250μl of vitrification solution for the last equilibration are sucked into the straw and sealed by hot pressure, and immediately placed in liquid nitrogen for cryopreservation.
一种以上述方法冷冻后的中间球海胆胚胎复苏方法，将冷冻的麦管从液氮中取出，立即放入25℃水浴中，待麦管中玻璃化液乳白色即将消失时，将麦管取出并剪去封口，用1～2mL的0.075M的蔗糖溶液洗脱5～10min，之后每5min加入同体积过滤海水进行清洗，共清洗3次，用新鲜无菌过滤海水培养。A method for resuscitating embryos of sea urchin in the middle ball frozen by the above method. The frozen straw is taken out of liquid nitrogen and immediately placed in a water bath at 25°C. When the milky white of the vitrified liquid in the straw is about to disappear, the straw is taken out And cut off the seal, eluted with 1 to 2 mL of 0.075M sucrose solution for 5 to 10 minutes, and then added the same volume of filtered seawater every 5 minutes for washing, washing in total 3 times, and incubating with fresh sterile filtered seawater.
本发明是在消毒过滤海水中添加二甲基亚砜、甲醇和1,2丙二醇作为玻璃化液实现对中间球海胆胚胎的快速玻璃化冷冻保存，并经过25℃水浴解冻等专用复苏方法，使得解冻后的中间球海胆胚胎原肠完整率可达到47.81％，具有解冻后胚胎原肠完整率高、易操作及成本低等优点。The invention is to add dimethyl sulfoxide, methanol and 1,2 propanediol as vitrification liquid to sterile filtered seawater to realize rapid vitrification and cryopreservation of the embryos of sea urchin in the middle ball, and undergo a special recovery method such as thawing in a water bath at 25°C, so that After thawing, the intact rate of the embryonic gastrulation of the middle ball sea urchin can reach 47.81%, which has the advantages of high intact rate of the embryonic gastrointestinal tract after thawing, easy operation and low cost.
本发明的一种中间球海胆胚胎的快速玻璃化冷冻保存方法，依次按照如下步骤进行：The method for rapid vitrification and cryopreservation of sea urchin embryos of the present invention is carried out in sequence according to the following steps:
a.胚胎的收集：在2019年10月份繁殖季节将中间球海胆(大连海洋大学农业农村部北方海水增养殖重点实验室培育的中间球海胆)从水中取出并将海胆身体表面海水擦干，利用氯化钾对中间球海胆进行人工催产，倒置于培养皿中，待产精(白色)或产卵(橙黄色)后，随机混合3只以上海胆的***与卵子均匀混合于5L烧杯中，持续观察至原肠期，收集原肠期的胚胎；a. Embryo collection: in the breeding season in October 2019, take out the middle sea urchin (the middle sea urchin cultivated by the Key Laboratory of Maritime Aquaculture of the Ministry of Agriculture and Rural Affairs of Dalian Ocean University) from the water and dry the sea urchin body surface water for use Potassium chloride artificially induced spawning of the sea urchin in the middle ball and placed it upside down in a petri dish. After the sperm (white) or the eggs (orange) are laid, randomly mix 3 sperm and eggs from the sea urchin in a 5L beaker. Continue to observe To the gastrulation stage, collect embryos in the gastrulation stage;
b.玻璃化液配制：将二甲基亚砜(DMSO)、甲醇(MeOH)及1,2丙二醇(1,2-PG)与过滤消毒海水混合，置于2℃预冷15min，所述二甲基亚砜、甲醇及丙二醇的体积浓度分别为1％、2％和2％；b. Preparation of vitrification solution: mix dimethyl sulfoxide (DMSO), methanol (MeOH) and 1,2 propanediol (1,2-PG) with filtered and disinfected seawater, and place it at 2°C for 15 min. The volume concentrations of methyl sulfoxide, methanol and propylene glycol are 1%, 2% and 2% respectively;
c.胚胎冷冻前的平衡处理：将收集的胚胎依次在1/5、1/4、1/3、1/2和1浓度的玻璃化液中在烧杯中各浸泡5min；每一次浸泡时尽量清除上次玻璃化液并用500目筛绢置于烧杯顶部，逐滴加入玻璃化液，利用玻璃化液自然渗透筛绢后落入烧杯，不断滴加玻璃化液使胚胎一直处于玻璃化液中，直至达到平衡时间(5min)，五步共计时25min；c. Equilibrium treatment before embryo freezing: Soak the collected embryos in vitrification solution of 1/5, 1/4, 1/3, 1/2 and 1 concentration in a beaker for 5 minutes; try to soak each time as much as possible Remove the last vitrification liquid and place a 500-mesh sieve on the top of the beaker, add the vitrification liquid drop by drop, use the vitrification liquid to permeate the sieve naturally and then fall into the beaker, continuously add the vitrification liquid to keep the embryo in the vitrification liquid , Until reaching the equilibrium time (5min), a total of 25min in five steps;
d.胚胎超低温冷冻保存：将经过平衡处理的胚胎连同250μl最后一次浸泡处理用玻璃化液吸入直径3mm、长度约为10cm的麦管中，酒精灯灼烧管口，镊子热压封口后，立即丢入液氮(-196℃)中冷冻保存；d. Embryos cryopreservation at ultra-low temperature: suck the equilibrated embryos together with 250μl of vitrification solution for the last soaking treatment into a straw with a diameter of 3mm and a length of about 10cm, burn the mouth of the tube with an alcohol lamp, and seal it with tweezers. Throw it into liquid nitrogen (-196℃) and store it in a frozen state;
需要复苏时，将冷冻的麦管从液氮中取出，立即放入25℃水浴使其快速解冻，待麦管中玻璃化液的乳白色即将消失时，将麦管取出剪去封口，将中间球海胆胚胎置于培养皿中，用1.5mL的0.075M的蔗糖溶液洗脱5min，立即放置于显微镜下进行观察，原肠期胚胎的完整率达47.81％；之后每5min加入5mL过滤海水进行清洗，共清洗3次，最后用新鲜无菌过滤海水培养。When resuscitation is needed, remove the frozen straw from the liquid nitrogen and immediately put it in a 25°C water bath to quickly thaw it. When the milky white of the vitrified liquid in the straw is about to disappear, remove the straw and cut off the seal, and the middle ball The sea urchin embryos were placed in a petri dish, eluted with 1.5 mL of 0.075M sucrose solution for 5 minutes, and immediately placed under a microscope for observation. The intact rate of the gastrulation stage embryos reached 47.81%; after that, 5 mL of filtered sea water was added every 5 minutes for washing. It was washed 3 times, and finally cultured with fresh sterile filtered seawater.
Claims (2)
- 一种中间球海胆胚胎的快速玻璃化冷冻保存方法，其特征在于依次按照如下步骤进行：A rapid vitrification cryopreservation method of sea urchin embryos, characterized in that the following steps are carried out in sequence:a.胚胎的收集：对中间球海胆进行人工催产和授精，收集受精卵发育至原肠期的胚胎；a. Embryo collection: artificially induce spawning and insemination of sea urchin, and collect embryos that develop to gastrulation stage from fertilized eggs;b.玻璃化液配制：将二甲基亚砜、甲醇及1,2丙二醇与过滤消毒海水混合，置于0～4℃预冷15min，所述二甲基亚砜、甲醇及丙二醇的体积浓度分别为1.0～2.0％、1.5～2.0％和1.5～2.0％；b. Preparation of vitrification solution: mix dimethyl sulfoxide, methanol and 1,2 propylene glycol with filtered and disinfected seawater, and place it at 0～4°C for 15 min. The volume concentration of the dimethyl sulfoxide, methanol and propylene glycol Respectively 1.0～2.0%, 1.5～2.0% and 1.5～2.0%;c.胚胎冷冻前的平衡处理：将收集的胚胎依次在1/5、1/4、1/3、1/2和1浓度的玻璃化液中各浸泡5～6min；c. Equilibrium treatment before embryo freezing: Soak the collected embryos in vitrification solution of 1/5, 1/4, 1/3, 1/2 and 1 concentration for 5-6 minutes;d.胚胎超低温冷冻保存：将经过平衡处理的胚胎连同250μl最后一次平衡处理用玻璃化液吸入麦管中并热压封口，立即置入液氮中冷冻保存。d. Embryos cryopreservation at ultra-low temperature: The embryos that have been equilibrated together with 250μl of vitrification solution for the last equilibration are sucked into the straw and sealed by hot pressure, and immediately placed in liquid nitrogen for cryopreservation.
- 一种以权利要求1所述方法冷冻后的中间球海胆胚胎复苏方法，其特征在于：将冷冻的麦管从液氮中取出，立即放入25℃水浴中，待麦管中玻璃化液乳白色即将消失时，将麦管取出并剪去封口，用1～2mL的0.075M的蔗糖溶液洗脱5～10min，之后每5min加入同体积过滤海水进行清洗，共清洗3次，用新鲜无菌过滤海水培养。A method for resuscitating embryos of sea urchin after being frozen by the method of claim 1, wherein the frozen straw is taken out of liquid nitrogen and immediately placed in a water bath at 25°C until the vitrified liquid in the straw is milky white When the straw is about to disappear, take out the straw and cut off the seal, eluting with 1~2mL of 0.075M sucrose solution for 5~10min, then add the same volume of filtered seawater every 5min for washing, wash 3 times in total, and filter with fresh aseptic Sea water culture.
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