WO2024027532A1 - Method for thawing ovum or embryo - Google Patents

Method for thawing ovum or embryo Download PDF

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WO2024027532A1
WO2024027532A1 PCT/CN2023/109157 CN2023109157W WO2024027532A1 WO 2024027532 A1 WO2024027532 A1 WO 2024027532A1 CN 2023109157 W CN2023109157 W CN 2023109157W WO 2024027532 A1 WO2024027532 A1 WO 2024027532A1
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oil
embryos
eggs
embryo
egg
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PCT/CN2023/109157
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French (fr)
Chinese (zh)
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陈昊楠
舒怡玮
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深圳拜尔洛克生物技术有限公司
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Publication of WO2024027532A1 publication Critical patent/WO2024027532A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor

Definitions

  • the present invention relates to the field of bioengineering technology, and in particular to a method for thawing eggs or embryos.
  • Vitrification technology relies on high-speed cooling and heating to avoid the generation of ice crystals, so that preserved biological samples can avoid damage by ice crystals and improve the survival rate after thawing.
  • the high-speed cooling is achieved by direct immersion in liquid nitrogen, while the high-speed heating is achieved by direct immersion in thawing liquid.
  • Vitrification liquid contains high-concentration cryoprotectants. These cryoprotectants combined with high cooling speed can avoid the formation of ice crystals.
  • the operator takes the carrier out of the liquid nitrogen and immediately immerses the part containing the eggs or embryos in the thawing solution to heat it up.
  • the speed of thawing and warming is a key factor that determines whether eggs or embryos can be revived (another key factor also includes the speed of freezing and cooling). If the temperature rises too slowly, ice crystals will form around or inside the embryo, causing the death of the egg or embryo.
  • the existing technology is to take out the carrier containing the eggs or embryos from the liquid nitrogen, and then insert the carrier into the thawing solution instantly to heat up and thaw. This method heats up much faster than placing the carrier in air.
  • the existing technology there is also a serious problem in the existing technology: when the eggs or embryos on the carrier are soaked in thawing liquid, they will float out of the carrier. When the eggs or embryos leave the carrier and float freely in the thawing fluid, it is difficult for the operator to find the eggs or embryos. Due to the high osmotic pressure in the thawing solution, eggs or embryos can only be immersed in it for a short time. When the embryos float out of the carrier, it often takes a long time to search through a microscope, leading to the death of the eggs or embryos. Or lost.
  • the present invention aims to solve at least one defect (shortcoming) described in the above-mentioned prior art and provide a method for thawing eggs or embryos, which is characterized in that the method includes the following steps:
  • the carrier containing eggs or embryos is removed from the liquid nitrogen, and the eggs or embryos are immersed in a container containing oil to thaw.
  • the temperature of the oil is 30 to 40°C.
  • the temperature of the oil is 37°C.
  • the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
  • the oil is mineral oil.
  • the method includes the following steps:
  • step a the temperature of the preheated oil ranges from 30 to 40°C.
  • the temperature of the preheated oil is 37°C.
  • the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
  • the oil is mineral oil.
  • steps c, d, and e the eggs or embryos are aspirated through the glass capillary tube.
  • step e move the container containing the culture medium to the incubator for culture and recovery.
  • step b the time from taking the carrier out of liquid nitrogen to immersing it in oil is less than one second.
  • step c after the eggs or embryos are moved to the container containing the diluent, wait three minutes before transferring them to the container containing the cleaning solution.
  • step d after the eggs or embryos are moved to a container containing cleaning fluid, wait for five minutes before transferring them to a container containing culture fluid.
  • the present invention uses mineral oil as the thawing liquid, which has the following advantages: 1) The speed of thawing and heating is much faster than directly placing it in the air, and the effect of thawing and heating is the same as that of traditional thawing liquid. 2)
  • the thawing liquid used is oil, which is different from traditional thawing liquid. The embryos or eggs will always be limited by the oil in the water phase of the carrier and will not float away from the carrier, making it easier for operators to find and operate.
  • thawing embryos when thawing embryos, it has the same heating speed and safety as traditional thawing solutions, while preventing the embryos from leaving the carrier, increasing the convenience of operation. It effectively reduces the number of microscope operations and makes the connection between automated vitrification and automated thawing smoother and more convenient.
  • the invention also has the characteristics of simple equipment structure, easy implementation and low cost.
  • the invention includes a method for thawing eggs or embryos, which includes the following steps:
  • the carrier containing eggs or embryos is removed from the liquid nitrogen, and the eggs or embryos are immersed in a container containing oil to thaw.
  • the temperature of the oil is 30 to 40°C.
  • the temperature of the oil is 37°C.
  • the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
  • the oil is mineral oil.
  • the invention also includes a method for thawing eggs or embryos, which includes the following steps:
  • step a the temperature range of the preheated oil is 30 to 40°C.
  • the temperature of the preheated oil is 37°C.
  • the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
  • the oil is mineral oil.
  • steps c, d, and e the eggs or embryos are aspirated through the glass capillary tube.
  • step e move the container containing the culture medium to the incubator for culture and recovery.
  • step b the time from taking the carrier out of liquid nitrogen to immersing it in oil is less than one second.
  • step c after the eggs or embryos are moved to the container containing the diluent, wait three minutes before transferring them to the container containing the cleaning solution.
  • step d after the eggs or embryos are moved to a container containing cleaning fluid, wait for five minutes before transferring them to a container containing culture fluid.
  • the operator first places the oil in a container, which is a petri dish in this embodiment, and preheats the oil to 37 degrees.
  • the two containers are loaded with diluent and cleaning solution respectively.
  • the capillary tube is filled with traditional thawing solution or diluent.
  • the operator places the preheated oil under the microscope, then takes the carrier out of the liquid nitrogen and immediately immerses the part containing the embryos in the oil.
  • the time from the carrier leaving the liquid nitrogen to being immersed in the oil should be controlled within one second.
  • the embryo or egg within the oil and the nearby vitrification fluid heat up rapidly to avoid the formation of ice crystals.
  • the heating process takes about 2 seconds.
  • the operator observes the part of the carrier containing the embryo or egg under the microscope. Since the embryo or egg and the vitrification liquid belong to the water phase, they will not mix with the oil or float out of the carrier, so the operator only needs Aspiration of the embryos using a glass capillary allows the embryos to be removed from the carrier and moved to the next solution for subsequent thawing steps, such as a diluent.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Developmental Biology & Embryology (AREA)
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Abstract

Disclosed herein is a method for thawing an ovum or embryo. The method comprises the following steps: respectively loading a preheated oil, a diluent, a cleaning liquid and a culture solution into different containers; taking out a carrier loaded with the ovum or embryo from liquid nitrogen, and immersing the ovum or embryo into the container loaded with the oil for thawing; sucking out the thawed ovum or embryo from the carrier and moving same into the container loaded with the diluent; sucking out the ovum or embryo from the container loaded with the diluent and moving same into the container loaded with the cleaning liquid; and sucking out the ovum or embryo from the container loaded with cleaning liquid and moving same into the container loaded with the culture solution. According to the present invention, when the embryo is thawed, the embryo is prevented from leaving the carrier while a heating speed and safety comparable to those of traditional thawing liquids are achieved, thereby improving the operation convenience. In addition, the present invention is characterized by a simple device structure, ease of implementation, low cost, and the like.

Description

一种卵子或胚胎的解冻方法A method of thawing eggs or embryos 技术领域Technical field
本发明涉及生物工程技术领域，特别是涉及一种卵子或胚胎的解冻方法。The present invention relates to the field of bioengineering technology, and in particular to a method for thawing eggs or embryos.
背景技术Background technique
玻璃化冷冻技术依靠高速的降温和升温速度来避免冰晶的产生，使保存的生物样本避免冰晶的伤害，提高了解冻后的存活率。其中的高速降温由直接浸泡液氮得以实现，而高速升温则通过直接浸泡解冻液得以实现。Vitrification technology relies on high-speed cooling and heating to avoid the generation of ice crystals, so that preserved biological samples can avoid damage by ice crystals and improve the survival rate after thawing. The high-speed cooling is achieved by direct immersion in liquid nitrogen, while the high-speed heating is achieved by direct immersion in thawing liquid.
现有的玻璃化冷冻技术，卵子和胚胎在最后的处理步骤会被放置在一个载体上。胚胎或卵子的周围具有玻璃化冷冻液。玻璃化冷冻液含有高浓度冷冻保护剂，这些冷冻保护剂配合高速的降温速度可以避免冰晶的产生。当需要解冻的时候，操作人员将载体从液氮中取出，并立刻将装有卵子或胚胎的一部分浸入解冻液内进行升温。解冻升温的速度是决定卵子或胚胎能否复苏的关键因素(另外的关键因素还包括冷冻降温的速度)。如果升温速度过慢，冰晶就会在胚胎周围或者胚胎内部产生，导致卵子或胚胎死亡。为了加快升温速度，现有技术是将装有卵子或胚胎的载体由液氮中取出后，瞬间将载体插入解冻液中进行升温解冻。此方法比将载体放置在空气中的升温速度快很多。但是现有技术也存在一个严重的问题：载体上的卵子或胚胎当泡在解冻液的时候会由载体中向外漂出。当卵子或胚胎离开载体并在解冻液里自由漂浮的时候，操作员很难找到卵子或胚胎。由于解冻液中的渗透压很高，卵子或胚胎只能在其中浸泡很短时间，当胚胎漂出载体外部时，往往需要通过显微镜花费很长的时间进行寻找，从而导致卵子或胚胎的死亡，或者丢失。With existing vitrification technology, eggs and embryos are placed on a carrier in the final processing step. The embryo or egg is surrounded by vitrification fluid. Vitrification liquid contains high-concentration cryoprotectants. These cryoprotectants combined with high cooling speed can avoid the formation of ice crystals. When it is time to thaw, the operator takes the carrier out of the liquid nitrogen and immediately immerses the part containing the eggs or embryos in the thawing solution to heat it up. The speed of thawing and warming is a key factor that determines whether eggs or embryos can be revived (another key factor also includes the speed of freezing and cooling). If the temperature rises too slowly, ice crystals will form around or inside the embryo, causing the death of the egg or embryo. In order to speed up the temperature rise, the existing technology is to take out the carrier containing the eggs or embryos from the liquid nitrogen, and then insert the carrier into the thawing solution instantly to heat up and thaw. This method heats up much faster than placing the carrier in air. However, there is also a serious problem in the existing technology: when the eggs or embryos on the carrier are soaked in thawing liquid, they will float out of the carrier. When the eggs or embryos leave the carrier and float freely in the thawing fluid, it is difficult for the operator to find the eggs or embryos. Due to the high osmotic pressure in the thawing solution, eggs or embryos can only be immersed in it for a short time. When the embryos float out of the carrier, it often takes a long time to search through a microscope, leading to the death of the eggs or embryos. Or lost.
发明内容Contents of the invention
综上所述，本发明旨在解决上述现有技术所述的至少一种缺陷(不足)，而提供一种卵子或胚胎的解冻方法，其特征在于，该方法包括如下步骤：To sum up, the present invention aims to solve at least one defect (shortcoming) described in the above-mentioned prior art and provide a method for thawing eggs or embryos, which is characterized in that the method includes the following steps:
将装有卵子或胚胎的载体由液氮中取出，并将卵子或胚胎浸入装有油的容器内解冻。 The carrier containing eggs or embryos is removed from the liquid nitrogen, and the eggs or embryos are immersed in a container containing oil to thaw.
油的温度为30至40℃。The temperature of the oil is 30 to 40°C.
油的温度为37℃。The temperature of the oil is 37°C.
所述油为植物油、食用油、矿物油、硅油中的一种。The oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
所述油为矿物油。The oil is mineral oil.
该方法包括如下步骤：The method includes the following steps:
a、分别将预热后的油、稀释液、清洗液和培养液装入不同的容器中；a. Put the preheated oil, diluent, cleaning solution and culture solution into different containers respectively;
b、将装有卵子或胚胎的载体由液氮中取出，并将卵子或胚胎浸入装有油的容器内解冻；b. Remove the carrier containing eggs or embryos from the liquid nitrogen, and immerse the eggs or embryos in a container containing oil to thaw;
c、将解冻后的卵子或胚胎由载体上吸出，并移动至装有稀释液的容器中，以降低卵子或胚胎周围的渗透压；c. Aspirate the thawed eggs or embryos from the carrier and move them to a container containing diluent to reduce the osmotic pressure around the eggs or embryos;
d、将卵子或胚胎由装有稀释液的容器中吸出，并移动至装有清洗液的容器中对卵子或胚胎进行冲洗；d. Aspirate the eggs or embryos from the container containing the diluent and move them to the container containing the cleaning liquid to rinse the eggs or embryos;
e、将卵子或胚胎由装有清洗液的容器中吸出，并移动至装有培养液的容器中，对卵子或胚胎进行二次冲洗。e. Aspirate the eggs or embryos from the container containing the cleaning solution, move them to the container containing the culture solution, and rinse the eggs or embryos twice.
在步骤a中，预热后的油的温度范围为30至40℃。In step a, the temperature of the preheated oil ranges from 30 to 40°C.
预热后的油的温度为37℃。The temperature of the preheated oil is 37°C.
所述油为植物油、食用油、矿物油、硅油中的一种。The oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
所述油为矿物油。The oil is mineral oil.
在步骤c、d、e中卵子或胚胎通过玻璃毛细管吸出。In steps c, d, and e, the eggs or embryos are aspirated through the glass capillary tube.
在步骤e之后，将装有培养液的容器移动至培养箱内培养复苏。After step e, move the container containing the culture medium to the incubator for culture and recovery.
在步骤b中，载体由液氮取出到浸入油中的时间小于一秒。In step b, the time from taking the carrier out of liquid nitrogen to immersing it in oil is less than one second.
在步骤c中，卵子或胚胎被移动至装有稀释液的容器中后，等待三分钟后再转移至装有清洗液的容器中。In step c, after the eggs or embryos are moved to the container containing the diluent, wait three minutes before transferring them to the container containing the cleaning solution.
在步骤d中，卵子或胚胎被移动至装有清洗液的容器中后，等待五分钟后再转移至装有培养液的容器中。In step d, after the eggs or embryos are moved to a container containing cleaning fluid, wait for five minutes before transferring them to a container containing culture fluid.
采用上述技术方案，与现有技术相比，本发明所产生的有益效果在于： 本发明使用矿物油作为解冻液，具有如下优点：1)解冻升温的速度比直接放置在空气中快很多，同时解冻升温的效果与传统的解冻液相同。2)采用的解冻液为油，这点与传统解冻液不同，胚胎或卵子会一直被油限位在载体的水相内，不会飘离载体，方便操作人员寻找和操作。此外，在胚胎解冻的时候，拥有跟传统解冻液相同的升温速度和安全性的同时，避免胚胎离开载体，增加了操作的便利性。有效减少了显微镜的操作次数，使自动化玻璃化冷冻和自动化解冻衔接更加顺畅和方便。本发明还具有设备结构简单，易于实施，成本低廉等特点。By adopting the above technical solution, compared with the existing technology, the beneficial effects produced by the present invention are: The present invention uses mineral oil as the thawing liquid, which has the following advantages: 1) The speed of thawing and heating is much faster than directly placing it in the air, and the effect of thawing and heating is the same as that of traditional thawing liquid. 2) The thawing liquid used is oil, which is different from traditional thawing liquid. The embryos or eggs will always be limited by the oil in the water phase of the carrier and will not float away from the carrier, making it easier for operators to find and operate. In addition, when thawing embryos, it has the same heating speed and safety as traditional thawing solutions, while preventing the embryos from leaving the carrier, increasing the convenience of operation. It effectively reduces the number of microscope operations and makes the connection between automated vitrification and automated thawing smoother and more convenient. The invention also has the characteristics of simple equipment structure, easy implementation and low cost.
具体实施方式Detailed ways
下列实施例是对本发明的进一步解释和补充，对本发明不会构成任何限制。The following examples further explain and supplement the present invention and do not constitute any limitation to the present invention.
本发明包括一种卵子或胚胎的解冻方法，包括如下步骤：The invention includes a method for thawing eggs or embryos, which includes the following steps:
将装有卵子或胚胎的载体由液氮中取出，并将卵子或胚胎浸入装有油的容器内解冻。The carrier containing eggs or embryos is removed from the liquid nitrogen, and the eggs or embryos are immersed in a container containing oil to thaw.
进一步地，油的温度为30至40℃。Further, the temperature of the oil is 30 to 40°C.
优选地，油的温度为37℃。Preferably, the temperature of the oil is 37°C.
进一步地，所述油为植物油、食用油、矿物油、硅油中的一种。Further, the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
优选地，所述油为矿物油。Preferably, the oil is mineral oil.
本发明还包括一种卵子或胚胎的解冻方法，包括如下步骤：The invention also includes a method for thawing eggs or embryos, which includes the following steps:
a、分别将预热后的油、稀释液、清洗液和培养液装入不同的容器中；a. Put the preheated oil, diluent, cleaning solution and culture solution into different containers respectively;
b、将装有卵子或胚胎的载体由液氮中取出，并将卵子或胚胎浸入装有油的容器内解冻；b. Remove the carrier containing eggs or embryos from the liquid nitrogen, and immerse the eggs or embryos in a container containing oil to thaw;
c、将解冻后的卵子或胚胎由载体上吸出，并移动至装有稀释液的容器中，以降低卵子或胚胎周围的渗透压；c. Aspirate the thawed eggs or embryos from the carrier and move them to a container containing diluent to reduce the osmotic pressure around the eggs or embryos;
d、将卵子或胚胎由装有稀释液的容器中吸出，并移动至装有清洗液的容器中对卵子或胚胎进行冲洗； d. Aspirate the eggs or embryos from the container containing the diluent and move them to the container containing the cleaning liquid to rinse the eggs or embryos;
e、将卵子或胚胎由装有清洗液的容器中吸出，并移动至装有培养液的容器中，对卵子或胚胎进行二次冲洗。e. Aspirate the eggs or embryos from the container containing the cleaning solution, move them to the container containing the culture solution, and rinse the eggs or embryos twice.
进一步地，在步骤a中，预热后的油的温度范围为30至40℃。Further, in step a, the temperature range of the preheated oil is 30 to 40°C.
优选地，预热后的油的温度为37℃。Preferably, the temperature of the preheated oil is 37°C.
进一步地，所述油为植物油、食用油、矿物油、硅油中的一种。Further, the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
优选地，所述油为矿物油。Preferably, the oil is mineral oil.
在步骤c、d、e中卵子或胚胎通过玻璃毛细管吸出。In steps c, d, and e, the eggs or embryos are aspirated through the glass capillary tube.
在步骤e之后，将装有培养液的容器移动至培养箱内培养复苏。After step e, move the container containing the culture medium to the incubator for culture and recovery.
在步骤b中，载体由液氮取出到浸入油中的时间小于一秒。In step b, the time from taking the carrier out of liquid nitrogen to immersing it in oil is less than one second.
在步骤c中，卵子或胚胎被移动至装有稀释液的容器中后，等待三分钟后再转移至装有清洗液的容器中。In step c, after the eggs or embryos are moved to the container containing the diluent, wait three minutes before transferring them to the container containing the cleaning solution.
在步骤d中，卵子或胚胎被移动至装有清洗液的容器中后，等待五分钟后再转移至装有培养液的容器中。In step d, after the eggs or embryos are moved to a container containing cleaning fluid, wait for five minutes before transferring them to a container containing culture fluid.
本发明的详细操作步骤如下：The detailed operating steps of the present invention are as follows:
操作人员首先将油放在一个容器内，在本实施例中容器为培养皿，并将油预热到37度。此外准备两个容器和移动胚胎的玻璃毛细管，两个容器中分别装载稀释液和清洗液。毛细管内填充传统的解冻液或者稀释液。操作人员将已经预热好的油放置到显微镜下，然后将载体由液氮中取出，立刻将装有胚胎的一部分浸入油内。The operator first places the oil in a container, which is a petri dish in this embodiment, and preheats the oil to 37 degrees. In addition, prepare two containers and glass capillary tubes for moving embryos. The two containers are loaded with diluent and cleaning solution respectively. The capillary tube is filled with traditional thawing solution or diluent. The operator places the preheated oil under the microscope, then takes the carrier out of the liquid nitrogen and immediately immerses the part containing the embryos in the oil.
一般来说，为了避免胚胎或卵子在空气中缓慢升温，从载体离开液氮到浸入油的时间大概会控制在一秒以内。油内的胚胎或卵子以及附近的玻璃化冷冻液会快速升温，以避免冰晶的产生。一般来说，升温的过程需要2秒左右。然后操作人员在显微镜下观察载体装有胚胎或卵子的部分，由于胚胎或卵子和玻璃化冷冻液属于水相，并不会与油混合在一起，也不会飘出载体，因此操作人员只需要使用玻璃毛细管将胚胎吸走则可以将胚胎由载体上取出，并移动到下一个溶液中以进行后续的解冻步骤，例如稀释液中。 Generally speaking, in order to prevent the embryo or egg from slowly heating up in the air, the time from the carrier leaving the liquid nitrogen to being immersed in the oil should be controlled within one second. The embryo or egg within the oil and the nearby vitrification fluid heat up rapidly to avoid the formation of ice crystals. Generally speaking, the heating process takes about 2 seconds. The operator then observes the part of the carrier containing the embryo or egg under the microscope. Since the embryo or egg and the vitrification liquid belong to the water phase, they will not mix with the oil or float out of the carrier, so the operator only needs Aspiration of the embryos using a glass capillary allows the embryos to be removed from the carrier and moved to the next solution for subsequent thawing steps, such as a diluent.
如果载体上的玻璃化冷冻液过少而导致不能直接吸出胚胎，也可以先稍微在胚胎或卵子附近吹出一点已经在玻璃毛细管内填充好的传统解冻液或者稀释液，再将溶液吸走，溶液则会带动胚胎一同吸入到毛细管内。胚胎被转移到稀释液后，停留3分钟，然后再转移到清洗液内再停留5分钟，最后通过清洗液将胚胎冲洗干净，再转移到培养皿内。操作员可以使用培养皿内的培养微滴，再次冲洗胚胎，将残余的清洗液清除后，转移到干净的培养箱中继续培养复苏。If there is too little vitrification solution on the carrier and the embryo cannot be sucked out directly, you can also blow out a little traditional thawing solution or diluent that has been filled in the glass capillary near the embryo or egg, and then suck the solution away. The embryo will be sucked into the capillary together. After the embryos are transferred to the diluent, stay for 3 minutes, then transfer to the cleaning solution and stay for another 5 minutes. Finally, rinse the embryos through the cleaning solution, and then transfer to the culture dish. The operator can use the culture droplets in the petri dish to rinse the embryos again, remove the remaining cleaning fluid, and then transfer them to a clean incubator to continue culture and recovery.
尽管通过以上实施例对本发明进行了揭示，但是本发明的范围并不局限于此，在不偏离本发明构思的条件下，以上各构件可用所属技术领域人员了解的相似或等同元件来替换。 Although the present invention has been disclosed through the above embodiments, the scope of the present invention is not limited thereto. Without departing from the concept of the present invention, each of the above components can be replaced with similar or equivalent components understood by those skilled in the art.

Claims (15)

  1. 一种卵子或胚胎的解冻方法，其特征在于，该方法包括如下步骤：A method for thawing eggs or embryos, characterized in that the method includes the following steps:
    将装有卵子或胚胎的载体由液氮中取出，并将卵子或胚胎浸入装有油的容器内解冻。The carrier containing eggs or embryos is removed from the liquid nitrogen, and the eggs or embryos are immersed in a container containing oil to thaw.
  2. 根据权利要求1所述的卵子或胚胎的解冻方法，其特征在于，油的温度为30至40℃。The egg or embryo thawing method according to claim 1, characterized in that the temperature of the oil is 30 to 40°C.
  3. 根据权利要求2所述的卵子或胚胎的解冻方法，其特征在于，油的温度为37℃。The egg or embryo thawing method according to claim 2, characterized in that the temperature of the oil is 37°C.
  4. 根据权利要求1至3中任一项所述的卵子或胚胎的解冻方法，其特征在于，所述油为植物油、食用油、矿物油、硅油中的一种。The egg or embryo thawing method according to any one of claims 1 to 3, characterized in that the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
  5. 根据权利要求4所述的卵子或胚胎的解冻方法，其特征在于，所述油为矿物油。The egg or embryo thawing method according to claim 4, wherein the oil is mineral oil.
  6. 一种卵子或胚胎的解冻方法，其特征在于，该方法包括如下步骤：A method for thawing eggs or embryos, characterized in that the method includes the following steps:
    a、分别将预热后的油、稀释液、清洗液和培养液装入不同的容器中；a. Put the preheated oil, diluent, cleaning solution and culture solution into different containers respectively;
    b、将装有卵子或胚胎的载体由液氮中取出，并将卵子或胚胎浸入装有油的容器内解冻；b. Remove the carrier containing eggs or embryos from the liquid nitrogen, and immerse the eggs or embryos in a container containing oil to thaw;
    c、将解冻后的卵子或胚胎由载体上吸出，并移动至装有稀释液的容器中，以降低卵子或胚胎周围的渗透压；c. Aspirate the thawed eggs or embryos from the carrier and move them to a container containing diluent to reduce the osmotic pressure around the eggs or embryos;
    d、将卵子或胚胎由装有稀释液的容器中吸出，并移动至装有清洗液的容器中对卵子或胚胎进行冲洗；d. Aspirate the eggs or embryos from the container containing the diluent and move them to the container containing the cleaning liquid to rinse the eggs or embryos;
    e、将卵子或胚胎由装有清洗液的容器中吸出，并移动至装有培养液的容器中，对卵子或胚胎进行二次冲洗。e. Aspirate the eggs or embryos from the container containing the cleaning solution, move them to the container containing the culture solution, and rinse the eggs or embryos twice.
  7. 根据权利要求6所述的卵子或胚胎的解冻方法，其特征在于，在步 骤a中，预热后的油的温度范围为30至40℃。The egg or embryo thawing method according to claim 6, characterized in that in the step In step a, the temperature of the preheated oil ranges from 30 to 40°C.
  8. 根据权利要求7所述的卵子或胚胎的解冻方法，其特征在于，预热后的油的温度为37℃。The egg or embryo thawing method according to claim 7, characterized in that the temperature of the preheated oil is 37°C.
  9. 根据权利要求6至8中任一项所述的卵子或胚胎的解冻方法，其特征在于，所述油为植物油、食用油、矿物油、硅油中的一种。The egg or embryo thawing method according to any one of claims 6 to 8, wherein the oil is one of vegetable oil, edible oil, mineral oil, and silicone oil.
  10. 根据权利要求9所述的卵子或胚胎的解冻方法，其特征在于，所述油为矿物油。The egg or embryo thawing method according to claim 9, wherein the oil is mineral oil.
  11. 根据权利要求6所述的卵子或胚胎的解冻方法，其特征在于，在步骤c、d、e中卵子或胚胎通过玻璃毛细管吸出。The thawing method of eggs or embryos according to claim 6, characterized in that in steps c, d, and e, the eggs or embryos are sucked out through a glass capillary tube.
  12. 根据权利要求6所述的卵子或胚胎的解冻方法，其特征在于，在步骤e之后，将装有培养液的容器移动至培养箱内进行培养复苏。The egg or embryo thawing method according to claim 6, characterized in that after step e, the container containing the culture medium is moved to an incubator for culture and recovery.
  13. 根据权利要求6所述的卵子或胚胎的解冻方法，其特征在于，在步骤b中，载体由液氮取出到浸入油中的时间小于一秒。The egg or embryo thawing method according to claim 6, characterized in that in step b, the time for the carrier to be taken out from liquid nitrogen and immersed in oil is less than one second.
  14. 根据权利要求6所述的卵子或胚胎的解冻方法，其特征在于，在步骤c中，卵子或胚胎被移动至装有稀释液的容器中后，等待三分钟后再转移至装有清洗液的容器中。The egg or embryo thawing method according to claim 6, characterized in that, in step c, after the egg or embryo is moved to a container filled with diluent, wait for three minutes and then transfer to a container filled with cleaning liquid. in the container.
  15. 根据权利要求6所述的卵子或胚胎的解冻方法，其特征在于，在步骤d中，卵子或胚胎被移动至装有清洗液的容器中后，等待五分钟后再转移至装有培养液的容器中。 The thawing method of eggs or embryos according to claim 6, characterized in that, in step d, after the eggs or embryos are moved to a container filled with cleaning fluid, wait for five minutes and then transfer to a container filled with culture fluid. in the container.
PCT/CN2023/109157 2022-08-01 2023-07-25 Method for thawing ovum or embryo WO2024027532A1 (en)

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