CN118421551A - Freezing and recovering method of gonad and chimeric embryo - Google Patents
Freezing and recovering method of gonad and chimeric embryo Download PDFInfo
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Abstract
The invention discloses a freezing and recovering method of gonads and a chimeric embryo, wherein the freezing and recovering method comprises the following steps: s100, placing gonads of the embryo in cell cryopreservation liquid for cryopreservation; s200, taking out the frozen gonads, removing cell frozen stock solution, adding a germ cell culture medium into the cell frozen stock solution for cultivation until the concentration of gonad germ cells in the culture medium is not lower than 1X 10 5 cells/mL; wherein the germ cell culture medium in step S200 comprises at least 0.1% to 0.3% by volume of serum, and the serum is derived from the biological species corresponding to the embryo. The invention is based on direct cryopreservation of gonads, can directly culture germ cells after recovery and is applied to subsequent breeding, greatly reduces the seed protection cost, and provides a new thought and direction for seed protection of wild rare or endangered birds.
Description
Technical Field
The invention relates to the field of genetics, in particular to a freezing and recovering method of gonads and a chimeric embryo.
Background
Aiming at the problem of preservation of the biological diversity of poultry germplasm, poultry species are generally raised in multiple species as live flocks to maintain a continuation of the overall breed. However, the development of climate change and urbanization processes is shrinking habitat of local chickens and other avian species, and avian influenza and other epidemic diseases endanger avian species. In addition, the cost of maintaining these lines in vivo is high in the case of the specialized chickens raised by the research facility, which also makes part of the breeds gradually disappear (Fulton and Delany, 2003).
Sperm cryopreservation is an effective method for preserving genetic diversity in avian species, while artificial insemination is a non-invasive hen fertilization method. However, artificial insemination using frozen and thawed cock sperm is not efficient. Furthermore, because the cock is a homogamete (ZZ), the W sex chromosome is carried only by females (WZ, hart-Johnson and Mankelow,2022; whyte et al, 2016), the use of frozen and thawed sperm does not restore the complete genetic background, and the hatching egg hatching rate of an artificially inseminated hen is generally low, also giving great uncertainty in the preservation of the breed.
Primordial Germ Cells (PGCs) are embryonic undifferentiated progenitor cells that develop into functional gametes and transfer the parent's genetic information to the next generation. Normalization of avian primordial germ cells relies on autonomous preformation of cells with maternally deposited germplasm (Tsunekawa et al, 2000; naito, 2001). Unlike other vertebrates, avian PGCs enter blood vessels early in the embryo and migrate along the blood circulation until reaching the lateral mesoderm. From there, they colonize gonadal primordia and proliferate during the embryonic stage, forming a resident germ cell population (Nieuwkoop and Sutasurya, 1979). This feature provides an excellent opportunity for manipulation of avian germ cell lines, either by retrieving embryonic blood for in vitro PGC culture or by introducing exogenous PGCs into the vascular system of a surrogate host to produce chimeric birds (Woodcock et al, 2017).
Current studies have shown that cultured PGCs isolated from embryonic blood or reproductive crescent can be infinitely expanded in vitro, providing a more efficient method for the biological pool of chicken species (Nandi et al, 2016). However, the use of breeds of local chickens to obtain embryos at the correct developmental stage is cost prohibitive because of the small feeding scale of local chickens, low egg production, and the large amount of technical resources required for this technology to isolate and culture cells. Cryopreservation of reproductive tissues (ovaries and testes) of newly hatched chicks has proven to be a viable method for germ line transmission by chimeric receptor hosts in master and slave frozen donor tissues. However, these methods are technically demanding, since the avian reproductive organs are located in the abdominal cavity, the implantation of frozen tissues into the recipient requires special surgery, and the use of immunosuppressive drugs is often required to reduce rejection of the transplanted tissues (Blackburn et al 2023).
Cryopreservation of embryonic gonads is an alternative (Tajima et al, 1998). Germ cells isolated from embryonic gonads have demonstrated the ability to transfer germ line (Nakamura et al, 2013). In addition, frozen and thawed Gonadal Germ Cells (GGCs) isolated from late-development (day 9-10 embryo) embryos are introduced directly into sterile hosts, and can produce large numbers of homozygous offspring containing multiple genotypes (Hu et al, 2022). However, this approach is to achieve stable production in modern pregnant hens, requiring a number of female donor gonads.
Disclosure of Invention
Aiming at the prior art, the invention aims to overcome the problems of high seed preservation cost, complex seed preservation mode and the like of poultry seed preservation problems in the prior art, and provides a method for freezing and recovering gonads and chimeric embryos, which are based on direct freezing and storing gonads, can directly culture germ cells after recovery and are applied to subsequent breeding, greatly reduce seed preservation cost and provide a new idea and direction for seed preservation of wild rare or endangered birds.
In order to achieve the above object, the present invention provides a method for freezing and resuscitating gonads, comprising:
S100, placing gonads of the embryo in cell cryopreservation liquid for cryopreservation;
S200, taking out the frozen gonads, removing cell frozen stock solution, adding a germ cell culture medium into the cell frozen stock solution for cultivation until the concentration of gonad germ cells in the culture medium is not lower than 1X 10 5 cells/mL; wherein,
The germ cell culture medium in step S200 comprises at least 0.1% to 0.3% by volume of serum, and the serum is derived from the biological species corresponding to the embryo.
Preferably, in step S100, the embryo body is a chicken embryo. The embryo body can be obtained by a reasonable mode which can be understood and operated by a person skilled in the art, such as hatching fertilized eggs to obtain chick embryos, wherein the temperature of the embryo body is 37.2-38 ℃ and the humidity of the embryo body is 55-70% during cultivation, and the egg turning angle is 40-50 degrees.
Preferably, the freezing process in step S100 specifically includes: and cooling the gonad in the cell freezing solution to not higher than-75 ℃ at a speed of 1 ℃/min-2 ℃/min through a procedural cooling box, pre-freezing for 10-15 hours at the temperature of-85 ℃ to-75 ℃, and then freezing in a liquid nitrogen environment for a long time.
Preferably, the germ cell medium further comprises: b-27 supplement, glutaMax, nonessential amino acids, embryoMax nucleosides, beta-mercaptoethanol, ovalbumin, pyruvic acid, caCl 2, heparin sodium and growth factors.
Preferably, the growth factor is selected from the group consisting of human activin a and human FGF2.
Preferably, in step S300, the process of removing the cell cryopreservation solution specifically includes: thawing gonads frozen in cell frozen stock solution at 35-37 ℃ for 8-15 s, transferring into a centrifuge tube, and balancing on ice for 25-35 min; and then placing the centrifuge tube in a condition of 1800-2200 r/min for 3-5 s, and removing the cell frozen stock solution.
Preferably, in step S200, the incubation process specifically includes:
S201, directly placing gonads of the cell-removed frozen stock solution in a germ cell culture medium to cultivate for 18-22 hours, and then flushing the gonads with the germ cell culture medium to obtain a primary culture mixed solution;
S202, sucking a germ cell culture medium in the primary culture mixed solution, and continuously culturing the sucked germ cell culture medium;
S203, discarding 1/4 volume part to 1/5 volume part of the cultured germ cell culture medium every 36h to 48h, and supplementing the germ cell culture medium which is not involved in the culture until the cell concentration in the cultured germ cell culture medium is not lower than 1 multiplied by 10 5 cells/mL.
The invention also provides a chimeric embryo obtained by injecting resuscitating gonadal germ cells into a recipient embryo, wherein the resuscitating gonadal germ cells are obtained by adopting the cryopreservation and resuscitating method of gonads.
The gonads hatched for a plurality of days are directly frozen, gonad tissues are directly placed in an improved gonad germ cell special culture medium without digestion after recovery, and a large number of gonad germ cells can be obtained through specific cell culture operation. The invention greatly simplifies the culture operation of germ cells, and compared with the seed preservation by using semen, the invention overcomes the defect of incomplete genetic material; compared with the use of early embryo for seed preservation, the invention is simpler, more convenient and feasible; the present invention allows for a reduced amount of hatching eggs (about 4 chick embryos per successful blood PGC cell line, and only 1 chick embryo per successful gonadal germ cell line) compared to methods using chick embryo blood to isolate PGCs (primordial germ cells). The biological seed protection cost is greatly reduced, and simultaneously, a new thought and direction are provided for the seed protection of wild rare birds and endangered birds.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 is a graph showing the climbing of resuscitated gonadal germ cells from the gonads in example 2;
FIG. 2 is a graph of immunofluorescence results of the ability of resuscitating gonadal germ cells to proliferate in test example 2;
FIG. 3 is a line graph of the ability of the resuscitating gonadal germ cells of example 2 to proliferate;
FIG. 4 is a graph of the results of the re-colonisation ability of the resuscitating gonadal germ cells of example 3;
FIG. 5 is a flow chart of the cryopreservation and resuscitation method provided by the present invention.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The method of the invention can be applied to proper species adaptively, and a person skilled in the art can design proper specific proportion of the culture medium according to the needs. However, based on the genetic characteristics of birds, which are characteristic of avian germ cell lines, in vitro PGC culture can be performed by retrieving embryo blood, or exogenous PGCs can be introduced into the vascular system of a surrogate host to generate chimeric birds, the gonads of birds are more suitable for cryopreservation and resuscitation and application to the construction of subsequent chimeric embryos.
However, since the hatching conditions and the germ cell culture medium adopted for different birds are greatly different, in a more specific embodiment of the present invention, the breeding objects mainly include chickens, and the chickens are taken as specific objects to better describe the technical scheme of the present invention.
Wherein, the B-27 supplement, glutaMax, nonessential amino acids, beta-mercaptoethanol, pyruvic acid and custom basal medium (12.0mM glucose,and CaCl 2 -free) are conventional commercial products or custom products of ThermoFisher Scientific (Sieimer's technology); embryoMax nucleosides is a conventional commercial product of Merck Millipore (Merck milbo); ovalbumin and heparin sodium are conventional commercial products from Sigma company; human activin a is a conventional commercial product of Peprotech (paripetak); human FGF2 is a conventional commercial product from R & D Biosystems; chicken serum Biosera is a conventional commercial product of company.
The shallow flower Su Saisi chicken used in the specific examples is a conventional commercial shallow flower Su Saisi chicken offered by the company of the Wisha breeding in France (China).
A specific flow chart of the invention is shown in fig. 5, which is described in detail below in connection with incubation. The embryo body may be obtained in a reasonable manner as will be appreciated by those skilled in the art and as will be appreciated by those skilled in the art, for example, in the specific embodiment of the present invention, the fertilized egg is hatched to obtain a chick embryo. The present invention is not limited to this specific acquisition mode and will not be described in detail herein.
1. Hatching chicken embryo:
Eggs were incubated in an incubator at 37.8C with a humidity of 60% and an egg turning angle of 45℃for 10 days. It should be noted that, the person skilled in the art can adjust the cultivation conditions in a specific cultivation process according to actual needs, the cultivation conditions may also be selected within a wide range, for example, more specifically, the temperature of the cultivated embryo body may be within a range of 37.2-38 ℃, the humidity of the cultivated embryo body may be within a range of 55% -70%, and the egg turning angle of the cultivated embryo body may be within a range of 40 ° -50 °.
2. Gonad acquisition and cryopreservation:
The chick embryo is dissected by using an ophthalmic scissors and an ophthalmic forceps, the gonad is taken out, and after PBS cleaning, the gonad is directly transferred to a freezing tube containing 150 mu L of freezing solution for subsequent freezing preservation. Judging sex according to gonad morphology and marking on a freezing tube. The freezing tube is placed in a program cooling box, placed in a refrigerator at the temperature of minus 80 ℃ for 12 hours, and then transferred into liquid nitrogen for long-term freezing. The selection of the frozen solution can be performed by a person skilled in the art according to the actual situation, for example, STEM-CELLBANKER frozen solution can be selected, and it should be noted that the specific frozen solution is only taken as an example, and it is not meant that only the frozen solution can be used.
3. Gonadal germ cell culture medium preparation:
The formulation of the chicken primordial germ cell culture medium is as follows: 1 XB-27 supplement (Thermo FISHER SCIENTIFIC), 2.0mM GlutaMax (Thermo FISHER SCIENTIFIC), 1 Xnonessential amino acids (Thermo FISHER SCIENTIFIC), 1X EmbryoMax nucleosides (Merck Millipore), 0.1mM beta-mercaptoethanol (Thermo FISHER SCIENTIFIC), 0.2% ovalbumin (Sigma), 1.2mM pyruvic acid (Thermo FISHER SCIENTIFIC), 0.15mM CaCl 2, 0.01% heparin sodium (Sigma) in DMEM, custom basal medium (12.0 mM glucose, and CaCl 2 -free; thermoFisher Scientific). The following growth factors were added prior to use: 25ng/mL human activin A (Peprotech), 4ng/mL human FGF2 (R & D Biosystems). The special culture medium for gonadal germ cells needs to be additionally added with 0.2 percent chicken serum (Biosera). Meanwhile, it should be further pointed out that the amount of the above specific reagent may be selected within a wide range, and those skilled in the art can appropriately adjust according to the actual situation; the type of reagent is not limited thereto, and those skilled in the art can use some germ cell culture media known in the art, and use the inventive scheme on the basis of such germ cell culture media conventionally used, and add serum as the gonadal germ cell dedicated culture medium of the present application. It is still worth mentioning that the use of the specific agents described above and their formulation according to the application can better improve the survival rate of later resuscitation.
4. Gonadal resuscitating:
The frozen vials were removed as needed, thawed at 37℃for 10 seconds, and the gonadal tissue was transferred to a 1.5mL centrifuge tube and equilibrated on ice for 30 minutes. The frozen stock was removed by brief centrifugation at 2000rpm for 4 seconds, then 1mL of medium was added and transferred to a sterile petri dish along with the gonads.
5. Gonadal germ cell acquisition and culture:
Gonadal tissue was not digested with digestive enzymes and gonadal was picked using a sterile syringe needle directly into 48 well plates pre-loaded with 300 μl of medium. After 20 hours of culture, the culture broth was gently aspirated using a pipette (1000 mL) to purge the gonads, and the culture medium except for the gonads was aspirated to another 48-well plate for continuous culture. One third of the gonadal germ cell medium was changed every 2 days until the cells grew to 1X 10 5 cells/mL.
6. Gonadal germ cell identification and proliferation potency assay:
Immunofluorescence was used to detect the germ cell marker gene SSEA-1 to identify germ cells. Cell counts were performed on days 4, 6, 8 of culture to determine the growth rate of cells by inoculating 3X 10 4 cells into 500mL of medium in 24 well plates.
7. Gonadal germ cell function verification:
marking the gonadal germ cells identified in step 6 with green fluorescent dye, injecting the successfully marked germ cells into normal receptor embryos hatched for 2.5 days, continuing to dissect the gonads after 8 th day of hatching and detecting the re-colonisation ability of the germ cells cultured by the method of the invention by observing GFP signals.
The following is a detailed description of a specific example using a Light Sussex (Light Sussex) as a Light flower Su Saisi.
Preparation example 1, hematoblast medium:
Preparing a reagent and a blood germ cell culture medium with the following contents: 1 XB-27 supplement, 2.0mM GlutaMax,1 Xnonessential amino acids, 1X EmbryoMax nucleosides,0.1mM beta-mercaptoethanol, 0.2% ovalbumin, 1.2mM pyruvic acid, 0.15mM CaCl 2, 0.01% heparin sodium in DMEM, custom basal medium (12.0mM glucose,and CaCl 2 -free), and 25ng/mL human activin A and 4ng/mL human FGF2 that need to be added prior to use are blood germ cell culture medium A1.
Preparation example 2 gonadal germ cell culture medium:
On the basis of preparation example 1, 0.2% of chicken serum is added, namely gonadal germ cell culture medium A2.
Preparation example 3, reconstituted hematopoiesis cell culture medium:
On the basis of preparation example 1, 5% of chicken serum was added, namely, the reconstituted blood germ cell medium A3.
Example 1, cryopreservation of the gonads of shallow-flowered Su Saisi chickens:
hatching is carried out by using 60 hatching eggs of the Su Saisi chickens with shallow flowers, and the hatching is carried out for 10 days in an incubator with the temperature of 37.8 ℃ and the humidity of 60 percent and the egg turning angle of 45 degrees.
And (5) egg candling is carried out on the hatching eggs hatched for 10 days, and 48 fertilized eggs are obtained. After breaking the eggshell of the fertilized egg and removing the shell membrane, the embryo was de-headed and placed in a 100 mm petri dish, neck tissue, wings and legs were removed, the abdominal cavity was opened, and the embryo was rinsed three times with PBS. The chick embryos were dissected using an ophthalmic scissors and an ophthalmic forceps and the gonads were removed, and after PBS washing, the gonads were transferred to a cryopreservation tube containing 150. Mu.L STEM-CELLBANKER cryopreservation solution. Judging sex according to gonad morphology and marking on a freezing tube. The male embryo gonadal 48 tubes (24 tubes for each of left and right side gonads) and the female embryo gonadal 48 tubes (24 tubes for each of left and right side gonads) were obtained together. The freezing tube is placed in a program cooling box, placed in a refrigerator at the temperature of minus 80 ℃ for 12 hours, and then transferred into liquid nitrogen for long-term freezing.
Example 2 resuscitation of cryopreserved gonads and cultivation of gonadal germ cells:
After 9 months of the gonadal cryopreservation, the gonadal cryopreservation tube was removed, thawed in a 37 ℃ metal bath for 10 seconds until ice was lost, and gonadal tissue was transferred to a 1.5mL screw cap Eppendorf tube and equilibrated on ice for 30 minutes. The frozen stock solution was removed by brief centrifugation at 2000rpm for 4 seconds, and then 1mL of the gonadal germ cell medium A2 prepared in preparation 2 was added and transferred to a sterile petri dish together with the gonads using a P1000Eppendorf pipette.
Under a stereoscopic microscope in a biosafety cover, one gonad was picked up with a sterile hypodermic needle and then transferred to one well of a 48-well plate, which was pre-filled with 300 μl of gonadal germ cell medium A2 prepared in preparation example 2. After 20 hours of incubation, gonadal tissue was gently washed several times using medium A2 and P1000 Eppendorf pipettes in the wells to remove germ cells attached to the tissue mass. The cell suspension (but not including the tissue mass) was then carefully aspirated and transferred to a new well on a 48-well plate. One-third of the culture medium A2 in the new wells was changed every 2 days until the number of cells therein (i.e., resuscitated gonadal germ cells) exceeded 1X 10 5 cells/mL (critical value for successful reproduction).
The climbing of germ cells from gonads is shown in FIG. 1. As can be seen from FIG. 1, when frozen gonadal tissue is placed in culture medium A2 for 20 hours, germ cells spontaneously shed into the culture medium, loosely adhere to somatic cells and the bottom of the culture well, and then transition to suspension cells.
Example 3 preparation of chimeric embryo:
The resuscitated gonadal germ cells incubated in example 2 were labeled using Live Cell Labeling kit Green Fluorescence-Cytopainter (ab 187964). Resuscitating gonadal germ cells with GFP markers were injected into normal recipient embryos incubated for 2.5 days.
Comparative example 1
The procedure of example 2 was followed except that gonadal germ cell medium A2 was replaced with blood germ cell medium A1 prepared in preparation example 1.
Comparative example 2
The procedure of example 2 was followed except that gonadal germ cell medium A2 was replaced with the reconstituted blood germ cell medium A3 prepared in preparation example 3.
Detection example 1
The proliferation success rate, differentiation rate, and average cell number of the germ cells cultured in example 2, comparative example 1, and comparative example 2 were examined, and the results obtained are shown in table 1.
TABLE 1 culture proliferation of cryopreserved gonadal germ cells
In general, for fresh gonads, over two to three weeks of culture, each gonad can produce more than 1X10 5 germ cells. As can be seen from Table 1, when "gonadal germ cell culture medium A2" was used for gonads of Light susex chick embryos frozen for 9 months, success rate of gonadal cell proliferation was male 63%, female was left side gonad 88%, right side gonad 50%, and cell differentiation rate was 0; when the 'blood germ cell culture medium A1' is used, the success rate of gonadal cell proliferation is 19% for male, 25% for female, 13% for right and 0 for cell differentiation; when "reproducing blood germ cell culture medium A3" was used, the success rate of gonadal cell proliferation was 56% for male, 75% for female, 63% for right, 13% for male, 25% for female, and 38% for right. Although the success rate of gonadal cell proliferation was also relatively high after the use of the reconstituted blood germ cell medium A3, the germ cell differentiation rate was also relatively high. However, it is known that the germ cells, after differentiation, exhibit adherent growth, and the differentiated cells lose germ cell stem properties and cannot be used for subsequent multiple seed. Furthermore, it should be noted that, since differentiated cells cannot be used for multiple seed later, the presence of cell differentiation means that the corresponding culture well cannot be used for continuous culture, and further means that the culture well is not used for culture.
Detection example 2
Marker gene identification was performed on resuscitated gonadal germ cells cultured in example 2 using immunofluorescence techniques. Cells were fixed in 4% PFA for 20min and then treated with 1% Triton X-100 for 15 min. After 30 minutes of treatment in 2% goat serum, cells were incubated with DAZL antibody (1:250 dilution, abcam) and SSEA-1 antibody (1:250 dilution, R & D system) for 1 hour, then secondary antibodies were labeled with AF488 and AF546 for 30 minutes to identify the target signal, and immunofluorescence results are shown in fig. 2. As can be seen from fig. 2, the cultured resuscitating gonadal cells express gonadal markers SSEA1 and DAZL (fig. 2), indicating that the resuscitating gonadal cells obtained still have germ cell characteristics.
In one well of a 24-well plate, 3×10 4 cells were inoculated into 500mL of gonadal germ cell medium A2 prepared in preparation example 2, and the cell growth rate was analyzed. A total of 8 germ cell lines from Light susex female embryos were analyzed. First, on day 4 of culture, all cells were re-inoculated into the same well with fresh medium after counting, and the cell numbers were counted again in the same manner on days 6 and 8. The results are shown in FIG. 3. Germ cells extracted from frozen gonads were 6-fold increased 4 days before culture, then slightly slowed down, 1.9-fold increased 4-6 days, 1.5-fold increased 6-8 days, and cell doubling time was 1.94 days.
Detection example 3
The chimeric embryos from example 3 were continued to be hatched, the gonads were dissected after day 8 and the re-colonisation ability of the resuscitated gonadal germ cells cultured by the method of the invention was examined by observing GFP signals. The results are shown in FIG. 4, in which donor cells labeled green successfully migrate to the host gonads and colonize, indicating that germ cells obtained by the present method are functional.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (8)
1. A method for cryopreserving and resuscitating gonads, comprising:
S100, placing gonads of the embryo in cell cryopreservation liquid for cryopreservation;
S200, taking out the frozen gonads, removing cell frozen stock solution, adding a germ cell culture medium into the cell frozen stock solution for cultivation until the concentration of gonad germ cells in the culture medium is not lower than 1X 10 5 cells/mL; wherein,
The germ cell culture medium in step S200 comprises at least 0.1% to 0.3% by volume of serum, and the serum is derived from the biological species corresponding to the embryo.
2. The method according to claim 1, wherein in step S100, the embryo is a chicken embryo.
3. The method for freezing and resuscitating gonads according to claim 1 or 2, wherein the freezing process in step S100 specifically comprises: and cooling the gonad in the cell freezing solution to not higher than-75 ℃ at a speed of 1 ℃/min-2 ℃/min through a procedural cooling box, pre-freezing for 10-15 hours at the temperature of-85 ℃ to-75 ℃, and then freezing in a liquid nitrogen environment for a long time.
4. A method of cryopreserving and resuscitating a gonad according to claim 1 or 2, wherein the germ cell culture medium further comprises: b-27 supplement, glutaMax, nonessential amino acids, embryoMax nucleosides, beta-mercaptoethanol, ovalbumin, pyruvic acid, caCl 2, heparin sodium and growth factors.
5. The method of claim 4, wherein the growth factor is selected from the group consisting of human activin A and human FGF2.
6. The method according to claim 1 or 2, wherein in step S200, the process of removing the cell cryopreservation solution comprises: thawing gonads frozen in cell frozen stock solution at 35-37 ℃ for 8-15 s, transferring into a centrifuge tube, and balancing on ice for 25-35 min; and then placing the centrifuge tube in a condition of 1800-2200 r/min for 3-5 s, and removing the cell frozen stock solution.
7. The method for cryopreservation and resuscitating gonads according to claim 1 or 2, wherein in step S200, the incubation process specifically comprises:
S201, directly placing gonads of the cell-removed frozen stock solution in a germ cell culture medium to cultivate for 18-22 hours, and then flushing the gonads with the germ cell culture medium to obtain a primary culture mixed solution;
S202, sucking a germ cell culture medium in the primary culture mixed solution, and continuously culturing the sucked germ cell culture medium;
S203, discarding 1/4 volume part to 1/5 volume part of the cultured germ cell culture medium every 36h to 48h, and supplementing the germ cell culture medium which is not involved in the culture until the cell concentration in the cultured germ cell culture medium is not lower than 1 multiplied by 10 5 cells/mL.
8. A chimeric embryo obtained by injecting resuscitating gonadal cells into a recipient embryo, wherein said resuscitating gonadal cells are obtained by the cryopreservation and resuscitating method of gonads according to any one of claims 1 to 7.
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