CN117535233A - Thawing method for ovum or embryo - Google Patents
Thawing method for ovum or embryo Download PDFInfo
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- CN117535233A CN117535233A CN202210918016.5A CN202210918016A CN117535233A CN 117535233 A CN117535233 A CN 117535233A CN 202210918016 A CN202210918016 A CN 202210918016A CN 117535233 A CN117535233 A CN 117535233A
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- Prior art keywords
- embryo
- ovum
- thawing
- oil
- liquid
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- 210000001161 mammalian embryo Anatomy 0.000 title claims abstract description 88
- 210000004681 ovum Anatomy 0.000 title claims abstract description 73
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 70
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 70
- 238000010257 thawing Methods 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 51
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000003085 diluting agent Substances 0.000 claims abstract description 17
- 238000004140 cleaning Methods 0.000 claims abstract description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 15
- 238000005406 washing Methods 0.000 claims abstract description 7
- 239000003921 oil Substances 0.000 claims description 45
- 235000019198 oils Nutrition 0.000 claims description 45
- 210000002257 embryonic structure Anatomy 0.000 claims description 15
- 235000010446 mineral oil Nutrition 0.000 claims description 13
- 239000002480 mineral oil Substances 0.000 claims description 13
- 239000008157 edible vegetable oil Substances 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 6
- 229920002545 silicone oil Polymers 0.000 claims description 6
- 235000015112 vegetable and seed oil Nutrition 0.000 claims description 6
- 239000008158 vegetable oil Substances 0.000 claims description 6
- 230000003204 osmotic effect Effects 0.000 claims description 4
- 235000013601 eggs Nutrition 0.000 claims 11
- 239000012531 culture fluid Substances 0.000 claims 1
- 230000000630 rising effect Effects 0.000 abstract description 4
- 239000007791 liquid phase Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 17
- 238000010438 heat treatment Methods 0.000 description 8
- 238000004017 vitrification Methods 0.000 description 7
- 239000013078 crystal Substances 0.000 description 5
- 238000001816 cooling Methods 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 3
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a defrosting method of ovum or embryo, comprising the following steps: respectively filling the preheated oil, the diluent, the cleaning liquid and the culture solution into different containers; taking out the carrier filled with ovum or embryo from liquid nitrogen, and immersing ovum or embryo into container filled with oil for thawing; sucking out the thawed ovum or embryo from the carrier, and transferring into a container filled with diluent; sucking ovum or embryo out of the container containing diluent, and transferring into the container containing cleaning solution; the ovum or embryo is sucked out of the vessel containing the washing liquid and moved into the vessel containing the culture liquid. When the embryo thawing device is used for thawing the embryo, the embryo thawing device has the advantages of having the temperature rising speed and safety similar to those of the traditional thawing liquid phase, avoiding the embryo from leaving the carrier, increasing the convenience of operation, along with simple equipment structure, easy implementation, low cost and the like.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for thawing an ovum or embryo.
Background
The vitrification freezing technology relies on high-speed cooling and heating speed to avoid ice crystal generation, so that the stored biological sample is prevented from being damaged by ice crystal, and the survival rate after thawing is improved. Wherein, the high-speed cooling is realized by directly soaking liquid nitrogen, and the high-speed heating is realized by directly soaking thawing liquid.
Prior vitrification freezing techniques, the ovum and embryo are placed on a carrier in the final processing step. The embryo or ovum has vitrification frozen liquid around it. The vitrified refrigerating fluid contains high-concentration cryoprotectant, and the cryoprotectant can avoid the generation of ice crystals by being matched with a high-speed cooling speed. When thawing is required, the operator removes the carrier from the liquid nitrogen and immediately immerses a portion of the egg or embryo in the thawing solution to raise the temperature. The rate of thawing and warming is a critical factor in determining whether an ovum or embryo can be resuscitated (additional critical factors include the rate of freezing and cooling). If the rate of heating is too slow, ice crystals may develop around or within the embryo, resulting in the death of the ovum or embryo. In order to accelerate the temperature rising speed, the prior art is to take out the carrier filled with ovum or embryo from liquid nitrogen, and then instantly insert the carrier into the thawing liquid for temperature rising and thawing. This method is much faster than placing the carrier in air. However, there is a serious problem in the prior art: the ovum or embryo on the carrier floats out of the carrier when the ovum or embryo is soaked in the thawing solution. When an ovum or embryo leaves the carrier and floats freely in the thawing solution, it is difficult for the operator to find the ovum or embryo. Due to the high osmotic pressure in the thawing solution, the ovum or embryo can only be soaked in it for a short time, and when the embryo floats outside the carrier, it often takes a long time to search through a microscope, resulting in the death or loss of the ovum or embryo.
Disclosure of Invention
In view of the above, the present invention aims to solve at least one of the drawbacks (shortcomings) of the prior art, and provides a method for thawing an ovum or embryo, which is characterized in that the method comprises the following steps:
the carrier containing the ovum or embryo is removed from the liquid nitrogen and the ovum or embryo is immersed in a container containing oil for thawing.
The temperature of the oil is 30-40 ℃.
The temperature of the oil was 37 ℃.
The oil is one of vegetable oil, edible oil, mineral oil and silicone oil.
The oil is mineral oil.
The method comprises the following steps:
a. respectively filling the preheated oil, the diluent, the cleaning liquid and the culture solution into different containers;
b. taking out the carrier filled with ovum or embryo from liquid nitrogen, and immersing ovum or embryo into container filled with oil for thawing;
c. sucking out the thawed ovum or embryo from the carrier, and transferring into a container filled with diluent to reduce osmotic pressure around the ovum or embryo;
d. sucking out ovum or embryo from the container filled with diluent, and moving into the container filled with cleaning solution to wash the ovum or embryo;
e. sucking ovum or embryo out of the container with cleaning liquid, and transferring into the container with culture liquid to wash ovum or embryo for the second time.
In step a, the temperature of the preheated oil is in the range of 30 to 40 ℃.
The temperature of the preheated oil was 37 ℃.
The oil is one of vegetable oil, edible oil, mineral oil and silicone oil.
The oil is mineral oil.
In steps c, d, e the ovum or embryo is aspirated through a glass capillary.
After step e, the vessel containing the culture solution is moved into an incubator for culture recovery.
In step b, the carrier is withdrawn from the liquid nitrogen to be immersed in the oil for less than one second.
In step c, the ovum or embryo is transferred to a container containing a diluent, and then transferred to a container containing a washing liquid after waiting three minutes.
In step d, the ovum or embryo is transferred to the vessel containing the washing liquid, and then transferred to the vessel containing the culture liquid after waiting for five minutes.
By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that: the invention uses mineral oil as thawing liquid, and has the following advantages: 1) The thawing and heating speed is much faster than that of the thawing and heating method when the thawing and heating method is directly placed in the air, and the thawing and heating effect is the same as that of the traditional thawing liquid. 2) The adopted thawing liquid is oil, which is different from the traditional thawing liquid, embryos or ova can be always limited in the water phase of the carrier by the oil and cannot float away from the carrier, so that the embryo or ovum thawing liquid is convenient for operators to find and operate. In addition, when the embryo is thawed, the temperature rising speed and the safety which are the same as those of the traditional thawing liquid are achieved, meanwhile, the embryo is prevented from leaving the carrier, and the convenience of operation is improved. Effectively reduces the operation times of the microscope, and ensures that the automatic vitrification freezing and automatic unfreezing are connected more smoothly and conveniently. The invention also has the characteristics of simple equipment structure, easy implementation, low cost and the like.
Detailed Description
The following examples are further illustrative and supplementary of the present invention and are not intended to limit the invention in any way.
The invention comprises a method for thawing an ovum or embryo, which comprises the following steps:
the carrier containing the ovum or embryo is removed from the liquid nitrogen and the ovum or embryo is immersed in a container containing oil for thawing.
Further, the temperature of the oil is 30-40 ℃.
Preferably, the temperature of the oil is 37 ℃.
Further, the oil is one of vegetable oil, edible oil, mineral oil and silicone oil.
Preferably, the oil is a mineral oil.
The invention also comprises a method for thawing the ovum or embryo, which comprises the following steps:
a. respectively filling the preheated oil, the diluent, the cleaning liquid and the culture solution into different containers;
b. taking out the carrier filled with ovum or embryo from liquid nitrogen, and immersing ovum or embryo into container filled with oil for thawing;
c. sucking out the thawed ovum or embryo from the carrier, and transferring into a container filled with diluent to reduce osmotic pressure around the ovum or embryo;
d. sucking out ovum or embryo from the container filled with diluent, and moving into the container filled with cleaning solution to wash the ovum or embryo;
e. sucking ovum or embryo out of the container with cleaning liquid, and transferring into the container with culture liquid to wash ovum or embryo for the second time.
Further, in step a, the temperature of the preheated oil is in the range of 30 to 40 ℃.
Preferably, the temperature of the preheated oil is 37 ℃.
Further, the oil is one of vegetable oil, edible oil, mineral oil and silicone oil.
Preferably, the oil is a mineral oil.
In steps c, d, e the ovum or embryo is aspirated through a glass capillary.
After step e, the vessel containing the culture solution is moved into an incubator for culture recovery.
In step b, the carrier is withdrawn from the liquid nitrogen to be immersed in the oil for less than one second.
In step c, the ovum or embryo is transferred to a container containing a diluent, and then transferred to a container containing a washing liquid after waiting three minutes.
In step d, the ovum or embryo is transferred to the vessel containing the washing liquid, and then transferred to the vessel containing the culture liquid after waiting for five minutes.
The detailed operation steps of the invention are as follows:
the operator first places the oil in a container, in this example a petri dish, and preheats the oil to 37 degrees. In addition, two containers and a glass capillary tube for moving embryos were prepared, and the two containers were filled with a dilution liquid and a cleaning liquid, respectively. The capillary tube is filled with a conventional thawing or diluting solution. The operator places the pre-heated oil under a microscope and then removes the carrier from the liquid nitrogen, immediately immersing a portion of the embryo-containing oil.
Generally, to avoid slow heating of embryos or ova in air, the time from carrier leaving liquid nitrogen to immersion in oil is controlled to be within about one second. Embryos or ova in the oil and nearby vitrification frozen liquid will heat up rapidly to avoid ice crystal generation. Generally, the temperature rise process takes about 2 seconds. The operator then observes under a microscope the portion of the carrier containing the embryo or ovum, and since the embryo or ovum and vitrification frozen solution belong to the aqueous phase and do not mix with the oil nor fly out of the carrier, the operator only needs to use a glass capillary to suck the embryo off the carrier and remove it to the next solution for subsequent thawing steps, e.g. in a dilution.
If the vitrification frozen liquid on the carrier is too little to directly suck out the embryo, the traditional thawing liquid or diluent which is filled in the glass capillary at a point near the embryo or ovum can be blown out, and then the solution is sucked away, and the solution drives the embryo to be sucked into the capillary together. After transferring the embryo to the diluent, the embryo stays for 3 minutes, then transfers to the cleaning solution for 5 minutes, finally washes the embryo clean by the cleaning solution, and transfers to the culture dish. The operator can use the culture droplets in the culture dish to wash the embryo again, remove the residual cleaning liquid, and transfer the embryo to a clean incubator for continuous culture recovery.
Although the present invention has been disclosed by the above embodiments, the scope of the present invention is not limited thereto, and each of the above components may be replaced with similar or equivalent elements known to those skilled in the art without departing from the spirit of the present invention.
Claims (15)
1. A method for thawing an ovum or embryo, comprising the steps of:
the carrier containing the ovum or embryo is removed from the liquid nitrogen and the ovum or embryo is immersed in a container containing oil for thawing.
2. The method for thawing an ovum or embryo according to claim 1, wherein the temperature of the oil is 30-40 ℃.
3. The method of thawing eggs or embryos according to claim 2, wherein the temperature of the oil is 37 ℃.
4. A method of thawing an ovum or embryo according to any of claims 1-3 wherein the oil is one of vegetable oil, edible oil, mineral oil, silicone oil.
5. The method of thawing eggs or embryos according to claim 4, wherein the oil is mineral oil.
6. A method for thawing an ovum or embryo, comprising the steps of:
a. respectively filling the preheated oil, the diluent, the cleaning liquid and the culture solution into different containers;
b. taking out the carrier filled with ovum or embryo from liquid nitrogen, and immersing ovum or embryo into container filled with oil for thawing;
c. sucking out the thawed ovum or embryo from the carrier, and transferring into a container filled with diluent to reduce osmotic pressure around the ovum or embryo;
d. sucking out ovum or embryo from the container filled with diluent, and moving into the container filled with cleaning solution to wash the ovum or embryo;
e. sucking ovum or embryo out of the container with cleaning liquid, and transferring into the container with culture liquid to wash ovum or embryo for the second time.
7. The method of thawing eggs or embryos according to claim 6, wherein in step a, the preheated oil has a temperature ranging from 30 to 40 ℃.
8. The method of thawing eggs or embryos according to claim 7, wherein the preheated oil has a temperature of 37 ℃.
9. The method for thawing an ovum or embryo according to any one of claims 6 to 8, wherein the oil is one of vegetable oil, edible oil, mineral oil, silicone oil.
10. A method of thawing an ovum or embryo according to claim 9, wherein the oil is mineral oil.
11. The method of thawing eggs or embryos according to claim 6, wherein in steps c, d, e the eggs or embryos are aspirated through glass capillaries.
12. The method of thawing eggs or embryos according to claim 6, wherein after step e, the container containing the culture fluid is moved to an incubator for resuscitative culture.
13. The method of thawing an ovum or embryo according to claim 6 wherein in step b the carrier is removed from the liquid nitrogen to be immersed in the oil for less than one second.
14. The method of thawing eggs or embryos according to claim 6, wherein in step c, the eggs or embryos are transferred to a container containing a diluent, and then transferred to a container containing a washing liquid after waiting three minutes.
15. The method of thawing eggs or embryos according to claim 6, wherein in step d, the eggs or embryos are transferred to the vessel containing the washing liquid after waiting five minutes after being transferred to the vessel containing the culture liquid.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210918016.5A CN117535233A (en) | 2022-08-01 | 2022-08-01 | Thawing method for ovum or embryo |
| PCT/CN2023/109157 WO2024027532A1 (en) | 2022-08-01 | 2023-07-25 | Method for thawing ovum or embryo |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210918016.5A CN117535233A (en) | 2022-08-01 | 2022-08-01 | Thawing method for ovum or embryo |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117535233A true CN117535233A (en) | 2024-02-09 |
Family
ID=89790522
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210918016.5A Pending CN117535233A (en) | 2022-08-01 | 2022-08-01 | Thawing method for ovum or embryo |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN117535233A (en) |
| WO (1) | WO2024027532A1 (en) |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004081103A (en) * | 2002-08-27 | 2004-03-18 | Yamaguchi Technology Licensing Organization Ltd | New method of ex vivo culture of feline embryo |
| WO2006059626A1 (en) * | 2004-12-03 | 2006-06-08 | Nipro Corporation | Device for biosample storage |
| CN104663649B (en) * | 2015-02-13 | 2016-08-24 | 安徽医科大学 | Human oocytes cryoprotective agent |
| CN104823966A (en) * | 2015-05-19 | 2015-08-12 | 上海交通大学医学院附属第九人民医院 | Trace sperm cryopreservation carrier and corresponding freezing and thawing methods thereof |
| CN109673623A (en) * | 2019-02-21 | 2019-04-26 | 白晓红 | A kind of glass freezing reagent and vitrifying defrosting reagent and its application and application method |
| CN111226909B (en) * | 2020-03-09 | 2022-03-25 | 佛山辅康生物科技有限公司 | Vitrification thawing solution and thawing method for ovum and cleavage stage embryo |
| CN114836291A (en) * | 2021-02-01 | 2022-08-02 | 深圳拜尔洛克生物技术有限公司 | System and method for thawing and recovering biological material |
-
2022
- 2022-08-01 CN CN202210918016.5A patent/CN117535233A/en active Pending
-
2023
- 2023-07-25 WO PCT/CN2023/109157 patent/WO2024027532A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024027532A1 (en) | 2024-02-08 |
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