CN114027295A - Oocyte and embryo vitrification freezing carrier and method - Google Patents
Oocyte and embryo vitrification freezing carrier and method Download PDFInfo
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- CN114027295A CN114027295A CN202111541870.6A CN202111541870A CN114027295A CN 114027295 A CN114027295 A CN 114027295A CN 202111541870 A CN202111541870 A CN 202111541870A CN 114027295 A CN114027295 A CN 114027295A
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- 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
- A01N1/0236—Mechanical aspects
- A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
- A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
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Abstract
The invention provides a vitrifying freezing carrier and a vitrifying freezing method for oocyte and embryo, and belongs to the technical field of animal embryo engineering. The oocyte and animal embryo vitrification freezing carrier comprises a freezing pipe body, one end of the freezing pipe body is provided with a U-shaped bevel opening, and a water absorption material is attached to the inner wall of the U-shaped bevel opening of the freezing pipe body. After the refrigerating fluid containing the oocytes and/or embryos is transferred to the water absorbing material, redundant refrigerating fluid can be automatically absorbed, the volume of the final refrigerating fluid is controlled to be about 0.5 mu l, and the refrigerating efficiency is greatly improved. The recovery rate of the embryo can reach more than 99 percent by adopting the freezing carrier to vitrify and freeze the animal oocyte and/or embryo.
Description
Technical Field
The invention belongs to the technical field of animal embryo engineering, and particularly relates to a oocyte and embryo vitrification freezing carrier and a method.
Background
The vitrification freezing of oocyte and embryo is to dehydrate oocyte and embryo serially and to put the dehydrated oocyte and embryo onto one carrier and fast lower the temperature in liquid nitrogen environment at-196 deg.c to make the liquid inside and outside the cell reach vitrification state. Oocyte and embryo freezing is one of the important methods for preserving fertility and animal genetic resources in women. Vitrification freezing is to treat oocyte and embryo with relatively high concentration and very viscous freezing protectant to freeze the embryo from physiological temperature to-196 deg.c and to freeze the liquid inside and outside the embryo cell into vitrified solid state. Compared with a program freezing method, in the freezing process of the vitrification method, the liquid in the embryo cell can not form ice crystals, thereby avoiding the physical and chemical damages of the ice crystals to the oocyte and the embryo cell and obtaining better freezing effect. The recovery rate of the vitrified frozen embryo is usually more than 95%, if the blastocyst is frozen, the effect is better, the recovery rate can be more than 99%, the recovery rate is obviously higher than that of a program freezing method, and the advantages are obvious, so that more and more centers gradually start to use the vitrified freezing method and become the main method for embryo freezing at the present stage. Likewise, vitrification freezing has become the gold standard for cryopreservation of oocytes.
The center of the prior art discloses a variety of embryo vitrification freezing vehicles, such as open elongated tubules (OPS) and cryotops. However, the survival rate and conception rate of the embryos vitrified frozen by OPS and Cryotop after thawing are not good. This is because the volume of the final freezing fluid affects the cooling and heating speed of the embryo freezing/thawing process, thereby determining the final freezing effect. For example, the final freezing liquid volume of OPS is 2-5 μ l, and the freezing volume is large. The final refrigerating fluid volume of Cryotop can be controlled to be about 1 mul, but the excessive fluid needs to be sucked manually, which needs years of training and working experience of operators to complete the complicated step, so the operation effect is different and unstable.
Disclosure of Invention
In view of the above, the present invention provides a carrier and a method for vitrifying freezing of oocytes and embryos, which can control the volume of the final freezing liquid to be 0.3 to 0.8 μ l, and greatly improve the freezing efficiency.
The invention provides a oocyte and embryo vitrification freezing carrier, which comprises a freezing pipe body, wherein one end of the freezing pipe body is provided with a U-shaped bevel opening.
Preferably, the water absorbent material comprises filter paper.
Preferably, the shape of the water absorbing material comprises a rectangle; the length of the water absorbing material is 18-20 mm, and the width of the water absorbing material is 5-6 mm.
Preferably, the outer edge of the water absorbing material is flush with the outer edge of the U-shaped bevel opening.
Preferably, the angle of the oblique angle of the U-shaped oblique opening is 60 degrees.
Preferably, the length of the U-shaped bevel opening is 0.5-1 cm
Preferably, the freezing pipe body is prepared by beveling one end of a semen freezing pipe; the specification of the semen freezing tube is 0.25 mL.
The invention also provides an oocyte and/or embryo vitrification freezing method based on the oocyte and embryo vitrification freezing carrier, which comprises the following steps:
transferring the refrigerating fluid containing the oocyte and/or embryo to the water absorbing material of the oocyte and embryo vitrification freezing carrier in the scheme, and freezing by liquid nitrogen.
Preferably, the transfer starting time is 5-10 s before the embryo is frozen by refrigerating fluid.
The invention provides a oocyte and embryo vitrification freezing carrier which comprises a freezing pipe body, wherein one end of the freezing pipe body is provided with a U-shaped bevel opening, and a water absorbing material is attached to the inner wall of the U-shaped bevel opening end of the freezing pipe body. After the refrigerating fluid containing the oocyte and/or embryo is transferred to the water absorbing material, redundant refrigerating fluid can be automatically absorbed, so that the volume of the final refrigerating fluid can be controlled to be about 0.5 mu l, the refrigerating efficiency and the recovery rate of the frozen embryo are greatly improved, and the carrier is named as 'Paper Straw' or 'Paper tube'. The freezing carrier of the invention is adopted to carry out vitrification freezing on the oocyte and/or embryo of the animal, and the recovery rate of the embryo is more than 99 percent. In addition, the carrier disclosed by the invention is used for vitrifying and freezing the animal oocyte and/or embryo, the operation is simple, technical personnel can be applied without training, the technical guarantee is provided for commercialization of the vitrifying and freezing technology, and the wide application of the technology in female fertility preservation and livestock breeding is greatly expanded.
Drawings
FIG. 1 is a schematic diagram of the structure of a semen freezing tube, wherein 1-semen freezing tube body, 11-cotton plug;
FIG. 2 is a schematic diagram of a bevel cutting of a semen freezing tube, wherein 1-the semen freezing tube body, 11-a cotton plug, 3-a scalpel, 4-U-shaped bevel length and 5-U-shaped bevel;
FIG. 3 is a schematic view of a water absorbing material inserted into a freezing tube body from a U-shaped bevel, wherein 1 is the semen freezing tube body, 11 is a cotton plug, 2 is the water absorbing material, and 5 is the U-shaped bevel;
FIG. 4 is a schematic diagram of the freezing liquid containing the oocyte and/or embryo added on the water-absorbing material of the oocyte and animal embryo vitrification freezing carrier, wherein, 1-semen freezing tube body, 11-cotton plug, 2-water-absorbing material, 5-U-shaped bevel opening and 6-freezing liquid containing the oocyte and/or embryo.
Detailed Description
The invention provides a oocyte and embryo vitrification freezing carrier, which comprises a freezing pipe body, wherein one end of the freezing pipe body is provided with a U-shaped bevel opening.
The oocyte and embryo vitrification freezing carrier of the present invention is suitable for human and animal.
In the present invention, the water-absorbent material preferably comprises filter paper, which is inexpensive and economical, and the oocyte and embryo vitrification freezing carrier of the present invention can achieve a freezing effect equivalent to Cryotop, but is more economical and lower in cost, the selling price of Cryotop is $ 20, and the embryo vitrification freezing carrier of the present invention costs about $ 0.5. In the present invention, the shape of the water absorbing material preferably includes a rectangle; the length of the water absorbing material is preferably 18-20 mm, and the width of the water absorbing material is preferably 5-6 mm. In the invention, the outer edge of the water absorbing material is flush with the outer edge of the U-shaped bevel opening.
In the invention, the angle of the oblique angle of the U-shaped oblique opening is preferably 60 degrees; the length of the U-shaped bevel opening is 0.5-1 cm.
In the present invention, the method for preparing the oocyte and embryo vitrification freezing carrier preferably comprises the following steps: beveling one end of the semen freezing pipe to form a freezing pipe body with a U-shaped bevel opening; and a water absorbing material is attached to the inner wall of the U-shaped beveled end of the freezing pipe body.
In the present invention, the beveling is preferably performed using a scalpel; the specification of the bevel cutting semen freezing tube is preferably 0.25mL or 0.5mL, the structural schematic diagram of the semen freezing tube is shown in figure 1, and the bevel cutting schematic diagram is shown in figure 2.
In the invention, the water absorbing material is inserted into the freezing pipe body from the U-shaped inclined opening and naturally attached to the inner wall of the freezing pipe body without being adhered by an adhesive. In the present invention, the schematic diagram of the water absorbing material inserted into the freezing pipe body from the U-shaped bevel opening is shown in fig. 3.
The invention also provides an oocyte and/or embryo vitrification freezing method based on the oocyte and embryo vitrification freezing carrier, which comprises the following steps:
transferring the freezing fluid containing the oocyte and/or embryo to the water absorbing material of the vitrification freezing carrier in the scheme, and directly throwing the front end of the freezing fluid into liquid nitrogen for freezing. The frozen vector was then stored in a common liquid nitrogen tank. See figure 4 for a schematic illustration.
The oocyte and embryo vitrification freezing carrier of the invention is preferably sterilized before use; the sterilization mode is preferably gas sterilization, and more preferably ozone sterilization.
In the invention, the transfer time is preferably 5-10 s before the oocyte and the embryo are frozen by refrigerating fluid.
The formula of the refrigerating fluid is not particularly limited, and the conventional vitrification refrigerating fluid and the conventional refrigeration method in the field can be adopted.
After the oocyte and the embryo are subjected to vitrification freezing, the method preferably further comprises thawing; the thawing method comprises the following steps: adding the vitrified frozen carrier into the thawing solution for thawing. The formula of the thawing solution and thawing parameters are not particularly limited, and the vitrification thawing solution commonly used in the field is adopted.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
1) Cutting off the upper part of a thin tube at an angle of 60 degrees at a position of 1.0cm at the front end of a semen freezing tube with the specification of 0.25ml by using a scalpel, so that a U-shaped groove inclined opening is formed at the bottom of the thin tube;
2) inserting chemical filter paper with the length of 20mm and the width of 5mm into a thin tube with an inclined opening of the U-shaped groove, and enabling the front end of the filter paper to be flush with the front end of the thin tube; filling 10-15 paper tubes into a gas sterilization bag, and performing gas sterilization to obtain a sterilized vitrified freezing carrier (paper tube);
3) transferring the refrigerating fluid containing the oocytes and/or embryos onto the water absorbing material of the paper tube obtained in the step 2) 5-10 s before the freezing treatment of the oocytes and embryos by the refrigerating fluid is finished, and transferring the refrigerating fluid into liquid nitrogen for freezing.
And directly putting the paper tube into the thawing solution for thawing.
Example 2
1. Retrieval of oocytes
The method comprises the steps of carrying out intraperitoneal injection on a 10 IU/PMSG (Sigma) mouse (a Swiss mouse and a B6D2 hybridized F1 mother mouse) on a 8-10-week-old mother mouse, and carrying out intraperitoneal injection on 100 IU/HCG after 48-50 h. And (3) carrying out cervical dislocation and killing 18-20 h after HCG (Sigma) injection, taking out the oviduct, placing the oviduct in a culture solution M2(Sigma), dissecting under a microscope, scratching the expanded part of the oviduct by using a syringe needle to see a cumulus complex, transferring the cumulus complex into a hyaluronidase solution (Sigma) containing 80IU/ml for digestion, repeatedly blowing, sucking and degranulating, collecting oocytes with normal shapes and first polar bodies, placing the oocytes into an M16(Sigma) solution, covering, and placing the oocytes in a 5% incubator at 37 ℃ for later use.
2. Embryo acquisition
By using the mouse super-volley scheme, 8-10-week-old female mice (Swiss mice and B6D2 are hybridized to obtain F1 female mice) are subjected to super-volley treatment by intraperitoneal injection of PMSG and HCG, and then are caged with male mice in a ratio of 1: 1. And (3) closing the cages and viewing the suppository, then dislocating and killing the cervical vertebra of the donor mouse, soaking and disinfecting the donor mouse by using alcohol, shearing the abdominal skin in an anatomical disc and expanding the abdominal skin upwards to expose the abdominal wall muscles, shearing the muscles at the front edge of the pubis, poking the internal organs, and exposing the uterine horn. The fat on the uterine horn is stripped off by an ophthalmic scissors. After the cervix is cut, the uterine horn is removed along with the fallopian tube and ovary and placed in a 50mm petri dish. The uterine horn and the fallopian tube junction are opened by a No. 5 thin needle, 1-2 mL of DPBS (Sigma) + 5% BSA solution (37 ℃) is extracted by an injector, a special flat head injection needle is connected, and the uterine horns on two sides enter the uterine body respectively from the uterine body to perform embryo flushing. After the liquid is flushed out and kept stand for a while, the embryo in the well-developed blastocyst stage is picked up by an egg picking needle under a stereomicroscope, and then is put into M16 liquid and is placed in a 5 percent incubator at 37 ℃ for standby.
3. Vitrification freezing procedure
The frozen base Solution (HS) was Calf blood (Fetal Calf Serum, FCS) 10% (v/v) + DPBS (Sigma). Firstly, pre-balancing oocytes or embryos in a basic Solution for 2min at room temperature, then transferring the oocytes or embryos into a balancing Solution (ES), and balancing the basic Solution containing 7.5% (V/V) of dimethyl sulfoxide (DMSO) and 7.5% (V/V) of Ethylene Glycol (EG) for 3 min; finally, in a vitrified refrigerating fluid (VS): equilibrium 90s (oocytes), or 45 (embryos) in a base solution of DMSO 15% (V/V) + EG 15% (V/V) +1.0M sucrose. According to experimental requirements, oocytes or embryos are loaded on different vitrification freezing carriers (OPS, Cryotop and paper tubes) and directly put into liquid nitrogen for freezing.
4. Thawing procedure
Before the recovery of the oocyte or embryo, the thawing solution is heated to room temperature, the freezing carrier is taken out from liquid nitrogen, the front end of the freezing carrier is placed in the thawing solution 1 (base solution +1.0M sucrose) after being exposed in the air for 3s, so that the oocyte or embryo is fallen into the liquid, and after the balancing for 1min, the freezing carrier is sequentially transferred to the thawing solution 2 (base solution +0.5M sucrose), the thawing solution 3 (base solution +0.25M sucrose), and the freezing carrier is respectively 3min in the base solution. Performing in vitro fertilization on the unfrozen oocyte, and observing the fertilization rate and the blastocyst development rate; the thawed embryos will continue to be cultured for survival, development and hatchability.
5. In Vitro Fertilization (IVF) and development of oocytes
The thawed oocytes were transferred to recipient fluid (HTF, Sigma) at 37 deg.C,5％CO2Fertilization was carried out after 2h of subculture. During the period, the male mice of 10-14 weeks are killed by cervical dislocation, the abdomen is cut open to expose the tissues of the male mice testis and epididymis, the epididymis is separated, the semen is taken out after puncture/shear, the obtained mixture is placed in a TYH (Sigma) culture drop and placed at 37 ℃ and 5% CO2And (4) incubating in an incubator to capacitate the sperms for 1-2 h. Observing sperm motility and state under optical microscope, dropping appropriate amount of sperm into the incubated oocyte culture drop according to sperm state, and performing in vitro fertilization (fertilization density of 2 × 10)5Sperm/ml). After about 4h after fertilization, the culture solution is replaced, embryos are washed 3 times with M2, transferred into M6 culture solution and continuously placed in CO2Culturing in an incubator. And (4) counting the recovery rate and the cleavage rate 16-24 hours after fertilization, and counting the blastocyst rate 5 days after fertilization. Hatchability was counted on day 6 after fertilization.
The recovery rate is survival number of the viable oocyte/number of the unfrozen oocyte multiplied by 100 percent;
the cleavage rate is equivalent to fertilized egg number/total number of eggs × 100%.
Blastocyst rate is blastocyst number/total oocytes × 100%.
Hatching rate is the number of hatching blastocysts/total blastocysts multiplied by 100%.
6. Development of embryo
Putting the thawed blastocysts into M16 solution, and placing the blastocysts in a 5% incubator at 37 ℃ for further culture for 48h for observation. Statistical resuscitation and blastocyst rates were recorded at 24 h. Statistical blastocyst hatchability was recorded at 48 h.
The recovery rate is the survival rate of blastula/the number of thawed blastula multiplied by 100 percent;
the blastula rate is the blastula number/total thawed embryo number multiplied by 100 percent;
the blastocyst hatching rate is the number of hatched blastocysts/total blastocysts multiplied by 100%.
Experimental design and results
1. Biometric method
Statistical methods statistical data were performed using SPSS11.5 software, and the counts were tested by χ 2, with the threshold for significance differences set at P < 0.05. Different letters represent significant differences.
2. Vitrification freezing of oocyte
TABLE 1 Effect of different vitrification vectors on oocyte freezing, in vitro fertilization and embryo development
3. Embryo vitrification freezing
TABLE 2 Effect of different vitrification vectors on freezing and its embryonic development
| Experimental group | Number of blastula | Recovery Rate (%) | Percentage of blastocyst (%) | Blastocyst hatching rate (%) |
| Fresh blastocyst | 65 | 58(89％)a | 39(67％)a | |
| OPS | 76 | 64(84％)a | 57(75％)b | 24(42％)b |
| Cryotop | 80 | 68(85％)a | 63(79％)b | 32(51％)c |
| Paper tube | 89 | 79(89％)a | 72(81％)ab | 40(56％)c |
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (9)
1. The utility model provides an oocyte and embryo vitrification carrier that freezes, includes the cryovial body, the one end of cryovial body is the oblique mouth of U type, its characterized in that the inner wall of the oblique mouth end of U type of cryovial body is pasted and is had water absorption material.
2. The oocyte and embryo vitrification freezing vehicle according to claim 1, wherein the water absorbing material includes filter paper.
3. An oocyte and embryo vitrification freezing carrier according to claim 1 or 2, characterized in that the shape of the water absorbing material includes a rectangle; the length of the water absorbing material is 18-20 mm, and the width of the water absorbing material is 5-6 mm.
4. The oocyte and embryo vitrification freezing carrier according to claim 3, wherein the outer edge of the water absorbing material is flush with the outer edge of the U-shaped bevel.
5. The animal embryo oocyte and embryo vitrification freezing carrier according to claim 1, wherein the angle of the oblique angle of the U-shaped bevel is 60 °.
6. The oocyte and embryo vitrification freezing carrier according to claim 1 or 5, wherein the length of the U-shaped bevel is 0.5-1 cm.
7. The oocyte and embryo vitrification freezing carrier according to claim 1, characterized in that the freezing tube body is prepared by beveling one end of a semen freezing tube; the specification of the semen freezing tube is 0.25 mL.
8. A method for vitrification freezing of an oocyte and/or embryo based on the oocyte and embryo vitrification freezing carrier according to any one of claims 1 to 7, comprising the steps of:
transferring a freezing fluid containing the oocyte and/or embryo to a water-absorbing material of the oocyte and embryo vitrification freezing carrier according to any one of claims 1 to 7, and freezing with liquid nitrogen.
9. The vitrification freezing method according to claim 8, wherein the transfer is started 5 to 10 seconds before the embryo is frozen by the freezing liquid.
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| CN202095441U (en) * | 2011-04-25 | 2012-01-04 | 石家庄市第四医院 | Biological material vitrification freezing carrier |
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| CN203555094U (en) * | 2013-11-21 | 2014-04-23 | 上海交通大学医学院附属第九人民医院 | Loading rod system for sealing-type cryopreservation of biological materials |
| US20160057991A1 (en) * | 2013-05-16 | 2016-03-03 | Mitsubishi Paper Mills Limited | Vitrification-cryopreservation implement for cells or tissues |
| CN106459862A (en) * | 2014-06-30 | 2017-02-22 | 三菱制纸株式会社 | Jig for vitrification preservation of cells or tissues |
| CN206949363U (en) * | 2017-06-28 | 2018-02-02 | 中国福利会国际和平妇幼保健院 | It is a kind of to be applied to freezing mankind's rareness sperm and the closed carrier of the double flute of embryo and ovum |
| CN207491934U (en) * | 2017-11-04 | 2018-06-15 | 中国福利会国际和平妇幼保健院 | A kind of closed carrier of single slot for being suitable for freezing mankind's rareness sperm and embryo and ovum |
| CN209882898U (en) * | 2019-01-25 | 2020-01-03 | 千日成 | Vitrification freezing straw |
| CN217429092U (en) * | 2021-12-16 | 2022-09-16 | 草原和牛投资有限公司 | Oocyte and embryo vitrification freezing carrier |
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2021
- 2021-12-16 CN CN202111541870.6A patent/CN114027295A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202095441U (en) * | 2011-04-25 | 2012-01-04 | 石家庄市第四医院 | Biological material vitrification freezing carrier |
| CN202514485U (en) * | 2011-12-29 | 2012-11-07 | 深圳市易瑞生物技术有限公司 | Carrying tool for vitrified cryopreservation of biological materials |
| US20160057991A1 (en) * | 2013-05-16 | 2016-03-03 | Mitsubishi Paper Mills Limited | Vitrification-cryopreservation implement for cells or tissues |
| CN203555094U (en) * | 2013-11-21 | 2014-04-23 | 上海交通大学医学院附属第九人民医院 | Loading rod system for sealing-type cryopreservation of biological materials |
| CN106459862A (en) * | 2014-06-30 | 2017-02-22 | 三菱制纸株式会社 | Jig for vitrification preservation of cells or tissues |
| CN206949363U (en) * | 2017-06-28 | 2018-02-02 | 中国福利会国际和平妇幼保健院 | It is a kind of to be applied to freezing mankind's rareness sperm and the closed carrier of the double flute of embryo and ovum |
| CN207491934U (en) * | 2017-11-04 | 2018-06-15 | 中国福利会国际和平妇幼保健院 | A kind of closed carrier of single slot for being suitable for freezing mankind's rareness sperm and embryo and ovum |
| CN209882898U (en) * | 2019-01-25 | 2020-01-03 | 千日成 | Vitrification freezing straw |
| CN217429092U (en) * | 2021-12-16 | 2022-09-16 | 草原和牛投资有限公司 | Oocyte and embryo vitrification freezing carrier |
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