CN114874976A - Sodium hyaluronate double-phase gel and application thereof - Google Patents
Sodium hyaluronate double-phase gel and application thereof Download PDFInfo
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- 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
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
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- C—CHEMISTRY; METALLURGY
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- C—CHEMISTRY; METALLURGY
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
- C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
- C08K5/00—Use of organic ingredients
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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
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Abstract
The invention provides a sodium hyaluronate dual-phase gel, which consists of cross-linked sodium hyaluronate and free sodium hyaluronate. The invention also provides application of the sodium hyaluronate dual-phase gel in culture of the zona pellucida-free fertilized eggs or embryos, and a culture method of the zona pellucida-free fertilized eggs or embryos of mammals. The sodium hyaluronate double-phase gel provided by the invention can effectively maintain development potential and stability of the fertilized eggs or embryos without the zona pellucida, so that the fertilized eggs or embryos without the zona pellucida can normally develop. The culture method has the advantages of simpler operation, effectiveness and convenience.
Description
Technical Field
The invention belongs to the technical field of embryo culture, and particularly relates to a sodium hyaluronate dual-phase gel and application thereof.
Background
The development of the egg cells takes place in the follicles, and when the first layer of follicular cells completely envelops the egg cells, an acellular membrane, called zona pellucida, begins to form outside the egg cells. In the continuous process of cleavage of fertilized eggs, the maintenance of the development potential of the fertilized eggs needs to ensure that the blastomeres keep mutual connection and a certain spatial structure. The zona pellucida plays a very critical role in maintaining the inter-cleavage interconnection and spatial structure. In the implementation process of the assisted reproduction technology, the phenomenon of the deletion or the abnormality of the zona pellucida can exist; in addition, the structure of the transparent belt also falls off in the in vitro operation process. Fertilized eggs lacking the zona pellucida can cause the blastomeres to grow flat after cell division by using the conventional embryo culture method, thereby influencing the later development potential of the embryos.
For the embryos without zona pellucida, there are also cases reported in the literature that culture was successfully performed by using methods such as the pendant drop method and agarose gel. However, the method has certain defects in use: the pendant drop method is complex to operate, and the amount of culture solution is very small, so that the stability of the embryo culture environment is difficult to ensure, and the available embryo efficiency obtained finally is still low; although agarose or other gel-like materials can be used to maintain the spatial structure of embryo development to some extent, the stability of the embryo development effect is poor due to the impermeability and difficulty in controlling the strength, and the adverse effects of agarose or other gel components on the embryo development and the late implantation are difficult to evaluate.
Therefore, there is an urgent need to develop a product and culture method that can effectively maintain the development potential and stability of the zona pellucida-free fertilized egg or embryo.
Disclosure of Invention
Based on the above, the present invention aims to provide a sodium hyaluronate dual-phase gel, which can effectively maintain development potential and stability of the zona pellucida-free fertilized eggs or embryos, so that the zona pellucida-free fertilized eggs or embryos can normally develop during the culture process.
The specific technical scheme is as follows.
A sodium hyaluronate bi-phase gel consisting of cross-linked sodium hyaluronate and free sodium hyaluronate.
In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (1.2-6).
In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (1.2-4).
In a preferred embodiment, the weight ratio of the cross-linked sodium hyaluronate and the free sodium hyaluronate in the sodium hyaluronate dual-phase gel is 1: (1.5-3.5).
In a more preferred embodiment, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (2-3.5).
In some embodiments, the cross-linked sodium hyaluronate is prepared from a cross-linking agent selected from at least one of butanediol glycidyl ether, divinyl sulfone, bis-sulfosuccinimidyl suberate, 3 '-dithiobis-sulfosuccinimidyl propionate, and 4, 4' -dithiobis-sulfosuccinimidyl butyrate.
In a preferred embodiment, the cross-linking agent is selected from butanediol glycidyl ether.
The invention also provides application of the sodium hyaluronate double-phase gel in culture of fertilized eggs or embryos without zona pellucida.
The invention also provides a method for culturing the mammal fertilized eggs without the zona pellucida or the embryos, which comprises the following steps: (1) adding the sodium hyaluronate double-phase gel into a culture dish, and then sequentially covering a culture solution and mineral oil on the sodium hyaluronate double-phase gel; (2) placing the culture dish in an incubator to ensure that the pH value of the sodium hyaluronate dual-phase gel is the same as that of the culture solution; (3) and taking out the culture dish, embedding the mammal non-zona pellucida oosperm or embryo into the sodium hyaluronate diphasic gel by using a thinned embryo transfer tube, and then putting the culture dish into an incubator for continuous culture.
In some embodiments, the culture fluid is selected from at least one of an embryo culture fluid, a fertilization fluid culture fluid, a cleavage embryo culture fluid, a blastocyst culture fluid, and an in vitro manipulation culture fluid.
In a preferred embodiment, the culture fluid is selected from embryo culture fluids.
In some embodiments, the mammal is a mouse, rat, rabbit, pig, monkey, cow, sheep, dog, or horse.
The invention provides a sodium hyaluronate double-phase gel capable of effectively maintaining development potential and stability of fertilized eggs or embryos without zona pellucida, which can be used as a 'bracket' for development of the fertilized eggs or embryos without zona pellucida to replace the zona pellucida to form a restriction effect on the fertilized eggs or embryos, so that the division process can be carried out in a normal cell arrangement mode, and the problem of poor development potential of the embryos due to incapability of maintaining the spatial structure of the fertilized eggs or embryos without zona pellucida in the development process is effectively solved, thereby enabling the fertilized eggs or embryos without zona pellucida to normally develop. Further, the inventor also finds that the ratio of the cross-linked sodium hyaluronate and the free sodium hyaluronate in the sodium hyaluronate dual-phase gel has obvious influence on the culture effect, and the proper ratio of the cross-linked sodium hyaluronate and the free sodium hyaluronate is obtained through optimization, so that the sodium hyaluronate dual-phase gel can meet the constraint effect on fertilized eggs or embryos without zona pellucida, and can meet the requirement on nutrient exchange, thereby obtaining a good culture effect.
The invention also provides a method for culturing mammal non-zona pellucida oosperms or embryos by using the sodium hyaluronate dual-phase gel, and the method has the advantages of simplicity, effectiveness and convenience in operation.
Drawings
FIG. 1 is a diagram showing the developmental state of fertilized egg of normal mouse cultured in comparative example 4.
FIG. 2 is the development state of mouse zona pellucida-free fertilized egg cultured by sodium hyaluronate biphase gel according to example 1 of the present invention.
FIG. 3 is a view showing the development state of a zona pellucida-free fertilized egg of a mouse cultured by the conventional method in comparative example 3.
FIG. 4 shows the development status of mouse zona pellucida-free fertilized egg cultured by sodium hyaluronate biphase gel according to example 4 of the present invention.
FIG. 5 shows the development status of zona pellucida-free fertilized eggs of mice cultured by the sodium hyaluronate duplex gel of the present invention in example 5.
FIG. 6 is a view showing the development state of a zona pellucida-free fertilized egg of a mouse cultured in comparative example 1.
FIG. 7 is a view showing the development state of a zona pellucida-free fertilized egg of a mouse cultured in comparative example 2.
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The invention provides a sodium hyaluronate dual-phase gel, which consists of cross-linked sodium hyaluronate and free sodium hyaluronate.
The sodium hyaluronate state comprises a liquid phase and a solid phase. The uncrosslinked sodium hyaluronate is liquid, and free sodium hyaluronate is connected by a crosslinking agent to form a network structure, so that the physical property of the sodium hyaluronate is changed to form a jelly-like solid state, namely the crosslinked sodium hyaluronate. Gels containing both cross-linked sodium hyaluronate and sodium hyaluronate in the free state are known as sodium hyaluronate bi-phase gels. The cross-linked sodium hyaluronate has certain hardness and structure, and the sodium hyaluronate is a main component of extracellular matrix, so that the sodium hyaluronate has no negative effect on the embryo development. The cross-linking agent used to prepare the cross-linked sodium hyaluronate may be selected from, but is not limited to, the following cross-linking agents: butanediol glycidyl ether (BDDE), divinyl sulfone (DVS), disulfosuccinimidyl suberate (BS3), 3 '-dithiobis-sulfosuccinimidyl propionate (DTSSP), 4' -dithiobis-sulfosuccinimidyl butyrate (DTSSB). The cross-linked sodium hyaluronate can be directly purchased from the market to obtain finished products, and can also be prepared according to the conventional technical means in the field.
In a preferred embodiment, the crosslinking agent is butanediol glycidyl ether (BDDE).
In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (1.2-6).
In some embodiments, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (1.2-4).
Further, the weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate in the sodium hyaluronate dual-phase gel is 1: (1.5-3.5).
In a preferred embodiment, the weight ratio of the cross-linked sodium hyaluronate and the free sodium hyaluronate in the sodium hyaluronate dual-phase gel is 1: (2-3.5).
In a more preferred embodiment, the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: 3.
the preparation method of the sodium hyaluronate double-phase gel comprises the following steps: and crushing the cross-linked sodium hyaluronate, and mixing the crushed cross-linked sodium hyaluronate with the free sodium hyaluronate according to a proportion to obtain the sodium hyaluronate double-phase gel.
The sodium hyaluronate double-phase gel can be used for culturing fertilized eggs or embryos without zona pellucida. It can be used as a 'bracket' for the development of fertilized eggs or embryos without zona pellucida to replace zona pellucida to form a restriction effect on the fertilized eggs or embryos.
The sodium hyaluronate dual-phase gel disclosed by the invention is used for culturing the mammalian zona pellucida oosperms or embryos, so that a 'bracket' can be provided for the development of the zona pellucida oosperms or embryos, and the exchange requirement of nutrient substances can be met, thereby effectively maintaining the development potential and stability of the zona pellucida oosperms or embryos.
In some embodiments, the method for culturing the mammalian zona pellucida zygote or embryo comprises the following steps: (1) adding the sodium hyaluronate double-phase gel into a culture dish, and then sequentially covering a culture solution and mineral oil on the sodium hyaluronate double-phase gel; (2) placing the culture dish in an incubator to ensure that the pH value of the sodium hyaluronate dual-phase gel is the same as that of the culture solution; (3) and taking out the culture dish, embedding the mammal non-zona pellucida oosperm or embryo into the sodium hyaluronate diphasic gel by using a thinned embryo transfer tube, and then putting the culture dish into an incubator for continuous culture.
In some embodiments, the culture dish is placed in the incubator for at least 6h in step (2).
In some embodiments, the culture fluid is selected from at least one of an embryo culture fluid, a fertilization fluid culture fluid, a cleavage embryo culture fluid, a blastocyst culture fluid, and an in vitro manipulation culture fluid.
In a preferred embodiment, the culture fluid is selected from embryo culture fluids.
In some embodiments, the mammal is an oviparous mammal, including but not limited to a mouse, rat, rabbit, pig, monkey, cow, sheep, dog, or horse.
In some embodiments, the culture dish may be a common cell culture dish, a 96-well plate, a 48-well plate, a 24-well plate, a 12-well plate, a 4-well dish, and other culture dishes containing fixed-well wells.
In some embodiments, the incubator is a carbon dioxide incubator or a three gas incubator.
The present invention is further illustrated by the following specific examples.
The cross-linked sodium hyaluronate used in the examples below was purchased from china huaxi organism; the embryo culture solution comprises a cleavage stage culture solution and a blastocyst stage culture solution, which are purchased from Vitrolife, Sweden, and the product number of the cleavage stage culture solution is 10128, and the product number of the blastocyst stage culture solution is 10132.
Example 1
The present example provides a sodium hyaluronate dual phase gel, which is composed of sodium hyaluronate (cross-linked sodium hyaluronate) cross-linked by butanediol glycidyl ether and free sodium hyaluronate, and a weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate is 1: 3.
example 2
The present embodiment provides a sodium hyaluronate dual-phase gel, which is composed of sodium hyaluronate (cross-linked sodium hyaluronate) cross-linked with butanediol glycidyl ether and free sodium hyaluronate, and a weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate is 1: 1.5.
example 3
The present example provides a sodium hyaluronate dual phase gel, which is composed of sodium hyaluronate (cross-linked sodium hyaluronate) cross-linked by butanediol glycidyl ether and free sodium hyaluronate, and a weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate is 1: 5.
example 4
The present example provides a sodium hyaluronate dual phase gel, which is composed of sodium hyaluronate (cross-linked sodium hyaluronate) cross-linked by butanediol glycidyl ether and free sodium hyaluronate, and a weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate is 1: 1.
example 5
The present example provides a sodium hyaluronate dual phase gel, which is composed of sodium hyaluronate (cross-linked sodium hyaluronate) cross-linked by butanediol glycidyl ether and free sodium hyaluronate, and a weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate is 1: 6.5.
the sodium hyaluronate double-phase gel of the above embodiments 1 to 5 is used for culturing mouse fertilized eggs without zona pellucida, and the culture effect is tested.
Meanwhile, the following comparative examples were set up during the experiment:
comparative example 1: culturing mouse fertilized eggs without zona pellucida by using butanediol glycidyl ether crosslinked sodium hyaluronate (crosslinked sodium hyaluronate);
comparative example 2: culturing mouse fertilized eggs without zona pellucida by using free sodium hyaluronate;
comparative example 3: performing contrast culture on the mouse fertilized eggs without the zona pellucida by using a conventional culture method;
comparative example 4: the normal mouse zygotes were cultured using a conventional culture method.
The culture method of the invention comprises the following steps: (1) adding the sodium hyaluronate double-phase gel of the embodiment 1-5 into a culture dish respectively to serve as a 'bracket', and then sequentially covering a proper amount of a culture solution in a cleavage stage and mineral oil on the sodium hyaluronate double-phase gel; (2) placing the culture dish in 6% CO 2 Balancing in a cell incubator for 8 hours to ensure that the sodium hyaluronate double-phase gel and the culture solution in the cleavage stage reach the same pH value; (3) taking out the culture dish, sucking the mammal fertilized eggs without the transparent belts by using a thinned embryo transfer tube and embedding the fertilized eggs into the sodium hyaluronate double-phase gel bracket of the culture dish obtained in the step (2), so that the nontransparent fertilized eggs are fixed in the gel bracket; then the culture dish is put into a cell culture box for continuous culture. The continuous culture process is consistent with the conventional embryo culture, and the blastocyst stage culture solution is replaced when the embryo develops to the fusion embryo stage or the mulberry embryo stage.
The culture method of comparative examples 1 to 2: the culture method of the present invention was performed in accordance with the present invention except that the cross-linked sodium hyaluronate and the free sodium hyaluronate were added to the culture dish in step (1).
The conventional culture method in comparative examples 3 to 4 was as follows: (1) adding a proper amount of a culture solution in a cleavage stage into a culture dish, and covering mineral oil above the culture solution; (2) placing the culture dish in 6% CO 2 Balancing in a cell incubator for 8 hours; (3) sucking the mouse zona pellucida-free fertilized eggs by using a thinned embryo transfer tube and putting the fertilized eggs into a culture solution; and then putting the culture dish into an incubator for continuous culture, and replacing the culture solution in the blastocyst stage when the embryo develops to the fusion embryo stage or the mulberry embryo stage.
The fertilized eggs without the zona pellucida cultured by the sodium hyaluronate double-phase gel disclosed by the embodiments 1-3 of the invention have good development state and can be normally expanded into blastocysts. Among them, the best effect was obtained by culturing in example 1, and high-quality blastocysts were obtained (FIG. 2). The development state of normal mouse fertilized eggs (comparative example 4) cultured by the conventional method is shown in FIG. 1, and high-quality blastocysts can be cultured. The developmental state of the fertilized eggs of the mouse with no zona pellucida cultured by the conventional method (comparative example 3) is shown in FIG. 3. It is understood that the culturing effect of the zona pellucida-free fertilized eggs cultured in example 1 is not much different from that of normal fertilized eggs, and high-quality blastocysts can be obtained, whereas high-quality blastocysts cannot be cultured by the conventional method.
The effect of the sodium hyaluronate double-phase gel of examples 4 to 5 on culturing of mammal fertilized eggs without zona pellucida is shown in fig. 4 to 5, wherein blastocysts can be generated, but cannot be expanded by self-force after the blastocysts develop, and high-quality embryos are difficult to form. From the culture results of the sodium hyaluronate double-phase gel in examples 1 to 5 on fertilized eggs without zona pellucida, it can be seen that the weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate in the sodium hyaluronate double-phase gel affects the culture effect, and when the weight ratio of the cross-linked sodium hyaluronate to the free sodium hyaluronate is 1: (1.2-6), not only can the binding effect on the fertilized eggs without the zona pellucida be met, but also the requirement on nutrient exchange can be met; especially, the weight ratio of the two is 1: 3 (example 1), the culture effect was better (FIG. 2). When the weight ratio of the two is too high (for example, 1: 1 in example 4), the binding force is too strong, which in turn affects the exchange of nutrients and makes it difficult to form a good quality embryo (FIG. 4). When the weight ratio of the two is too low (for example, 1: 6.5 in example 5), the binding effect on the fertilized eggs without zona pellucida cannot be effectively satisfied, the cell connection between blastomeres is loose, and a high-quality blastocyst cannot be formed (fig. 5).
When only the cross-linked sodium hyaluronate was used for culture (comparative example 1), the cross-linked hyaluronic acid with high concentration could not exchange culture solution effectively, resulting in gradual apoptosis of blastomeres during development and failure to develop to blastocyst stage (fig. 6); when only free sodium hyaluronate was used for the culture (comparative example 2), the free hyaluronic acid was hardly bound to the culture medium, resulting in failure to achieve tight cell connection between blastomeres, dispersion of the distribution, and finally failure to develop to the blastocyst stage (fig. 7).
In conclusion, the sodium hyaluronate double-phase gel and the culture method can effectively maintain development potential and stability of the fertilized eggs or embryos without the zona pellucida, so that the fertilized eggs or embryos without the zona pellucida can normally develop.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The sodium hyaluronate dual-phase gel is characterized by consisting of cross-linked sodium hyaluronate and free sodium hyaluronate.
2. The sodium hyaluronate bi-phase gel of claim 1, wherein the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (1.2-6).
3. The sodium hyaluronate bi-phase gel of claim 1, wherein the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (1.2-4).
4. The sodium hyaluronate bi-phase gel of claim 1, wherein the weight ratio of cross-linked sodium hyaluronate and free sodium hyaluronate in the sodium hyaluronate bi-phase gel is 1: (1.5 to 3.5), more preferably 1: (2-3.5).
5. The sodium hyaluronate biphel gel of any one of claims 1 to 4, wherein the cross-linked sodium hyaluronate is prepared from at least one cross-linking agent selected from butanediol glycidyl ether, divinyl sulfone, bis-sulfosuccinimidyl suberate, 3 '-dithiobis-sulfosuccinimidyl propionate, and 4, 4' -dithiobis-sulfosuccinimidyl butyrate.
6. The sodium hyaluronate bi-phase gel of claim 5, wherein the crosslinking agent is selected from butanediol glycidyl ether.
7. The use of the sodium hyaluronate bi-phase gel according to any one of claims 1 to 6 in the culture of fertilized eggs or embryos without zona pellucida.
8. A method for culturing mammal fertilized eggs or embryos without zona pellucida is characterized by comprising the following steps: (1) adding the sodium hyaluronate bi-phase gel of any one of claims 1 to 6 to a culture dish, and then sequentially covering a culture solution and mineral oil on the sodium hyaluronate bi-phase gel; (2) placing the culture dish in an incubator to ensure that the pH value of the sodium hyaluronate dual-phase gel is the same as that of the culture solution; (3) and taking out the culture dish, embedding the mammal non-zona pellucida oosperm or embryo into the sodium hyaluronate diphasic gel by using a thinned embryo transfer tube, and then putting the culture dish into an incubator for continuous culture.
9. The method of culturing a mammalian zona pellucida fertilized egg or embryo according to claim 8, wherein the culture medium is at least one selected from the group consisting of an embryo culture medium, a fertilization medium, a blastocyst culture medium, and an in vitro manipulation culture medium.
10. The method for culturing the mammal zona pellucida-free fertilized egg or embryo according to claim 8 or 9, wherein the mammal is a mouse, rat, rabbit, pig, monkey, cow, sheep, dog or horse.
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| US20190160202A1 (en) * | 2016-05-04 | 2019-05-30 | ETH Zürich | Transglutaminase mediated high molecular weight hyaluronan hydrogels |
| CN111269877A (en) * | 2020-02-21 | 2020-06-12 | 南京市江宁医院 | Method for polymerization and in-vitro culture of embryo without zona pellucida before implantation |
| CH717854A2 (en) * | 2020-09-10 | 2022-03-15 | Hyamed Biotechnology Zhuhai Co Ltd | Process for the preparation of sodium hyaluronate gel. |
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| WO1999021415A1 (en) * | 1997-10-28 | 1999-05-06 | Stem Cell Sciences Pty. Ltd. | Nuclear transfer for production of transgenic animal embryo |
| US20190160202A1 (en) * | 2016-05-04 | 2019-05-30 | ETH Zürich | Transglutaminase mediated high molecular weight hyaluronan hydrogels |
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