CN115141796A - Culture medium for rockfish reproductive stem cells and long-term culture method thereof - Google Patents
Culture medium for rockfish reproductive stem cells and long-term culture method thereof Download PDFInfo
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Abstract
The application belongs to the technical field of in vitro culture of reproductive stem cells, and particularly relates to a culture medium for the reproductive stem cells of grouper and a long-term culture method thereof; the application provides a culture medium of rockfish germ stem cells, which comprises the following components: the fetal bovine serum, antibiotics, beta-mercaptoethanol, glutaMAX, cytokines, medaka embryo extract and Advanced DMEM/F12 culture medium prolong the time of in vitro culture of the rockfish spermatogonial stem cells to 20 months, prolong the time of in vitro culture of the oogonial stem cells to 5 months, and can simultaneously meet the long-term in vitro culture requirements of the rockfish spermatogonial stem cells and the rockfish oogonial stem cells, thereby solving the technical problem that the culture medium required by in vitro long-term culture of rockfish reproductive stem cells in the prior art is lacked.
Description
Technical Field
The application belongs to the technical field of reproductive stem cell in-vitro culture, and particularly relates to a culture medium for rockfish reproductive stem cells and a long-term culture method thereof.
Background
At present, the research on the self-renewal mechanism of the germ stem cells is concentrated in mammals such as mice and human beings, and a long-term culture system of the germ stem cells of a plurality of mammals such as mice and human beings is successfully established based on the research; however, the research on the self-renewal mechanism of the fish germ stem cells is less, and the fish germ stem cells are concentrated on the fish spermatogonial stem cells, because the nutrient medium required for maintaining the long-term self-renewal of the fish germ stem cells is not clear, a proper culture medium is lacked to culture the fish germ stem cells in vitro for a long time, most of the fish spermatogonial stem cells can survive in the culture medium for only 1 to 3 months, but the survival time of the fish oogonial stem cells is shorter, for example, the zebra fish oogonial stem cells can survive in the culture medium for only about 6 weeks, because of the physiological characteristic difference of the fish spermatogonial stem cells and the oogonial stem cells, the fish spermatogonial stem cell culture medium cannot be directly used for culturing the oogonial stem cells, and the culture medium cannot be used universally when the fish spermatogonial stem cells and the fish oogonial stem cells are cultured in vitro for a long time.
Meanwhile, the number of species of fishes is extremely large, the physiological characteristics are different, and the nutrient substrates required for maintaining the long-term self-renewal of the reproductive stem cells of different fishes are different, for example, different from most fishes, the grouper is hermaphrodite, namely the grouper is female in the initial maturity, and after the grouper participates in reproduction as the female, the female fish starts to be turned into the male fish in a sexual reversion way, so that the culture medium of the reproductive stem cells of other fishes cannot be commonly used with the grouper due to the great difference of the physiological characteristics of the grouper and other fishes, the grouper is a marine fish with the quality, delicious taste, rich nutrition, high economic value and important production and application values, the physiological characteristics of hermaphrodite different from most fishes also have important scientific research values, however, the related research of the culture medium of the reproductive stem cells of the grouper in the prior art is lacked, and the requirement of the long-term culture of the reproductive stem cells of the grouper cannot be met.
Disclosure of Invention
In view of the above, the present application provides a culture medium for grouper germ stem cells and a long-term culture method thereof, which are used for solving the technical problem that the culture medium required for the in vitro long-term culture of grouper germ stem cells is lacking in the prior art.
The first aspect of the application provides a culture medium for rockfish germ stem cells, which comprises the following components: basal medium and nutrient components;
the nutrient components comprise: fetal bovine serum, antibiotics, beta-mercaptoethanol, glutaMAX, cytokines and pre-treated medaka embryos;
the cytokines include: bFGF, GDNF, LIF and EGF.
The basic culture medium is an Advanced DMEM/F12 culture medium or a DMEM/F12 culture medium.
Preferably, in the culture medium of the rockfish germ stem cells, the volume ratio of the fetal bovine serum to the basic culture medium is 5-20: 100, respectively;
the antibiotics are penicillin and streptomycin:
the concentration of the penicillin is 100Units/mL;
the concentration of the streptomycin is 100 mug/mL;
the concentration of the beta-mercaptoethanol in the basic culture medium is 55uM/mL;
the concentration of the GlutaMAX in the basal medium is 2mM/L;
the concentration of the pretreated medaka embryos in the basal medium is 2 grains/mL;
the concentrations of bFGF, GDNF, LIF and EGF in the basal medium are all 20ng/mL.
Preferably, the medaka embryo is a pretreated medaka day 7 embryo.
The collected medaka day 7 embryos are washed by PBS, then ground by a tissue disruptor for 20 minutes, repeatedly frozen and thawed three times in liquid nitrogen and 37 ℃ water bath, and finally centrifuged at 3500g for 30 minutes at 4 ℃ under the centrifugal force to obtain medaka embryos as pretreated medaka day 7 embryos.
The pre-treated medaka embryos can be added with PBS to adjust to 400 embryos per ml and then stored in a refrigerator at-20 ℃, and then taken out when necessary and added into a basal medium.
The second aspect of the application provides a long-term culture method of rockfish germ stem cells, which utilizes a culture medium of rockfish germ stem cells to carry out long-term in-vitro culture and comprises the following steps:
step 1, cutting the sex gland of the grouper into pieces to obtain a sex gland tissue block of the grouper;
step 2, dispersing the grouper gonad tissue blocks in a culture container for tissue block culture to obtain adherent grouper gonad tissue blocks;
step 3, performing first tissue block culture on the gonad tissue blocks of the adherent groupers to obtain first adherent cells;
step 4, scraping off somatic cells in the first adherent cells by using a cell scraper, cleaning by using double-resistant PBS (phosphate buffered saline), and culturing a second tissue block to obtain second adherent cells;
step 5, scraping somatic cells in the second adherent cells by using a cell scraper to obtain third adherent cells;
step 6, infiltrating the obtained third adherent cells with pancreatin, removing the pancreatin, and washing with PBS to obtain purified germ stem cells;
and 7, adding a culture medium of the grouper germ stem cells into the purified germ stem cells for in vitro culture.
It should be noted that, the traditional magnetic bead immune sorting method and the cell flow sorting method are expensive, and are generally only used for separating and purifying germ stem cells of model animals such as mice and the like, and are not suitable for large-size and non-model animals such as groupers; the existing separation and purification method of fish stem cells utilizes the principle that spermatogonial stem cells are attached slowly compared with somatic cells, firstly, sperms are digested into a single cell state, then, non-attached spermatogonial stem cells are obtained by a differential wall attaching method, but the differential wall attaching method is easy to introduce somatic cells into the non-attached spermatogonial stem cells, the proliferation speed of the somatic cells is extremely high, so that when the non-attached spermatogonial stem cells are cultured in vitro, the somatic cells can be excessively proliferated to cause that germ stem cells lack growth space and nutritional conditions and are finally lost, long-term in-vitro culture cannot be realized, the sperms are directly digested into the single cell state, the damage to the germ stem cells and the microenvironment is large, the germ stem cells are not beneficial to the long-term in-vitro culture, meanwhile, the damage to the cells and the microenvironment is large in the process of separating the cells by the gradient centrifugation method, and the introduced somatic cells are not easy to separate; in the long-term culture method of the rockfish germ stem cells, the destruction of the tissue block culture method to the germ stem cells and the microenvironment thereof is small, the primary culture of the rockfish germ stem cells can be easily started, the original physiological properties of the rockfish germ stem cells are maintained, then in the process of tissue block culture of the rockfish gonad tissue blocks, the adherent cells migrated from the rockfish gonad tissue blocks are not subjected to pancreatin and are inoculated into a new culture container for subculture, but in the process of tissue block culture of the rockfish gonad tissue blocks, only the culture medium is replaced, so that the adherent cells are subjected to non-subculture in the culture container, and in the non-subculture process, because the culture medium is compared with adherent cells, the reproductive stem cell clusters in the cell clusters are very firm in adherent wall and not easy to fall off, adherent somatic cells are excessively proliferated and crowded in a culture container along with long-term non-passage, and gradually fall off to expose the bottom of a blank culture bottle, and the reproductive stem cells of the groupers gradually migrate to the exposed bottom of the culture bottle to obtain the reproductive stem cells of the adherent groupers, after the reproductive stem cells of the groupers grow to be large enough, the remaining somatic cells are infiltrated and digested by pancreatin, and the purified reproductive stem cells of the groupers without somatic cells are obtained after the washing by using double-antibody PBS (phosphate buffer solution). According to the method, a tissue block culture method and non-passage culture are combined in the process of separating the somatic cells in the gonads of the groupers, so that the purified reproductive stem cells without somatic cells are obtained; in the process of separating somatic cells in the gonad of the grouper, adherent somatic cells can be scraped off by cell scraping, so that the separation of the somatic cells in the gonad of the grouper is accelerated, and the purified germ stem cells of the grouper without the somatic cells are obtained; and then placing the purified grouper germ stem cells in a culture medium of the grouper germ stem cells, so that the grouper germ stem cells can be cultured for more than 20 months for a long time, and the in-vitro culture time of the grouper germ stem cells is greatly prolonged.
Preferably, in step 1, the volume of the rockfish gonad tissue block is 1mm 3 。
Preferably, in steps 2, 3 and 4, the culture medium used in the tissue block culture, the first tissue block culture and the second tissue block culture is the culture medium of the grouper germ stem cells.
It should be noted that, in the tissue mass culture process, compared with other commercially available or self-made culture media, the culture medium for the grouper germ stem cells provided by the application can meet the nutrient medium required by the grouper germ stem cells for self-renewal, is beneficial to improving the self-renewal and proliferation rate of the germ stem cells, reducing the death and differentiation conditions of the grouper germ stem cells, and accelerating the separation of somatic cells in the grouper gonads.
Preferably, in step 2, the tissue block culture specifically comprises: subjecting the mass of rockfish gonadal tissue to a temperature of 28 ℃ in an atmosphere of 5% CO 2 Culturing the lower tissue block for 10 days;
in step 3, the first tissue block culture specifically comprises: adding the gonadal tissue blocks of Armillaridae into the mixture at 28 deg.C under 5% CO 2 Culturing the lower tissue block for 30 days;
in step 4, the second tissue block culture specifically comprises: subjecting the first adherent cells to a temperature of 28 deg.C under an atmosphere of 5% 2 The lower tissue mass was cultured for 50 days.
The culture method provided by the present application is characterized in that the cultured rockfish gonad tissue mass, the cultured adherent rockfish gonad tissue mass and the first adherent cells are all placed in a medium containing 5% CO 2 The culture in the atmosphere is that the basic culture medium in the culture medium of the rockfish reproductive stem cells is NaHCO 3 Buffer system for rendering the germ stem cells of grouper 5% CO 2 Growth under conditions with simultaneous CO 2 The culture in the atmosphere can obviously reduce the oxidation of the rockfish reproductive stem cells, slow down the aging and poisoning phenomena of the rockfish reproductive stem cells and improve the growth and proliferation rate of the rockfish.
Preferably, in steps 2, 3 and 4, the volume of the medium used in the tissue piece culturing, the first tissue piece culturing and the second tissue piece culturing is 3 to 5ml.
Preferably, in steps 2, 3 and 4, the culture medium is replaced every 2 to 3 days during the tissue block culture, the first tissue block culture and the second tissue block culture.
Preferably, in step 6, the pancreatin is used in a volume of 2 to 3ml;
the pancreatin infiltration time is 1-1.5 min;
the number of times of PBS washing was 2 to 3.
Preferably, in step 6, before obtaining the purified germ stem cells after infiltrating the obtained third parietal cells with pancreatin, removing pancreatin, washing with PBS, further comprising:
step 6.1, adding 5 to 6ml of the culture medium of the rockfish germ stem cells at a temperature of 28 ℃ under an atmosphere of 5% 2 Culturing for 5 days to obtain germ stem cells;
and 6.2, repeating the step 6-step 6.1 for 3-5 times to obtain the purified germ stem cells.
Preferably, the sex gland of the grouper is the sex gland of male Epinephelus coioides or the sex gland of female Epinephelus coioides.
In summary, the present application provides a culture medium for rockfish germ stem cells and a long-term culture method thereof, wherein the culture medium comprises: the method is characterized in that fetal calf serum, antibiotics, beta-mercaptoethanol, glutaMAX, cell factors, medaka embryo extract and an Advanced DMEM/F12 culture medium are added into the culture medium, nutrient components contained in medaka embryos greatly prolong the self-renewal and proliferation time of rockfish germ stem cells, compared with single cell factors, the four cell factors including bFGF, GDNF, LIF and EGF are cooperated with one another to meet the regulation and control requirements of the self-renewal and proliferation of rockfish germ stem cells, so that the death and differentiation conditions of rockfish germ stem cells are reduced, the self-renewal and proliferation of rockfish germ stem cells can be maintained for a long time, meanwhile, glutaMAX replacing L-glutamine in the culture medium not only provides energy and participates in protein synthesis and nucleic acid metabolism, the cell ammonolysis is avoided, the introduction of pathogens is avoided being unfavorable for the long-term culture of rockfish germ stem cells by using commercial bovine fetal serum, the culture medium is added into the rockfish stem cells to achieve the purpose of prolonging the in-vitro culture time of rockfish germ stem cells to 20 months, and the problem that the rockfish germ stem cells lack of the in-vitro culture technology for rockfish germ cells can be provided, and the rockfish germ cells can be solved.
Drawings
In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings needed to be used in the detailed description of the present application or the prior art description will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a microscope photograph of adherent tissue blocks of the gonad of a rockfish after 10 days of tissue block culture using the tissue blocks of rockfish in example 2 of the present application;
FIG. 2 is a microscope photograph of the bottom germ stem cell clusters in adherent cells after 30 days of first tissue mass culture of the gonadal tissue mass of adherent grouper in example 2 of the present application;
FIG. 3 is a microscopic image of the in vitro cultured Epinephelus coioides reproductive stem cells obtained by the method for long-term culture of Epinephelus coioides provided in example 2 of the present application;
FIG. 4 is a specific marker gene expression profile of the in vitro cultured grouper germ stem cells by the method for long-term culture of grouper germ stem cells provided in example 2 of the present application;
FIG. 5 is a flow chart of the method for long-term culture of the germ stem cells of grouper provided in example 2 of the present application;
wherein, FIG. 1A is a microscope picture of an adherent grouper ovary tissue block, FIG. 1B is a microscope picture of an adherent grouper testis tissue block, FIG. 2A is a microscope picture of a grouper oogonial stem cell cluster, and FIG. 2B is a microscope picture of a grouper spermatogonial stem cell cluster; 3A is a microscopic image of rockfish oogonial stem cells, and 3B is a microscopic image of rockfish spermatogonial stem cells; 4A is the expression map of the specific marker gene of the grouper oogonial stem cell, and 4B is the expression map of the specific marker gene of the grouper spermatogonial stem cell.
Detailed Description
The application provides a culture medium for rockfish germ stem cells and a long-term culture method thereof, which are used for solving the technical problem that the culture medium required by the in-vitro long-term culture of rockfish germ stem cells is lacked in the prior art.
The technical solutions of the present application will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1
The embodiment 1 of the present application provides a culture medium for rockfish germ stem cells, which comprises the following components as shown in table 1:
| fetal bovine serum | 15％ |
| Penicillin and streptomycin | 1X |
| Beta-mercaptoethanol | 55uM/mL |
| GlutaMAX | 2mM |
| Pre-treatment of medaka day 7 embryos | 2 grains/mL |
| bFGF (basic fibroblast growth factor) | 20ng/mL |
| GDNF (glial cell line-derived neurotrophic factor) | 20ng/mL |
| LIF (leukemia inhibitory factor) | 20ng/mL |
| EGF (epidermal growth factor) | 20ng/mL |
| Advanced DMEM/F12 medium |
TABLE 1
The preparation method of the pretreated medaka day 7 embryos comprises the following steps:
step 1, incubating fertilized medaka embryos at the water temperature of 28 ℃, and collecting medaka embryos which grow normally on the 7 th day;
step 2, washing the embryo with PBS (phosphate buffer solution) for three times, and grinding the embryo with a tissue disruptor for 20 minutes;
step 3, repeatedly freezing and thawing for three times in liquid nitrogen and 37 ℃ water bath;
and 4, centrifuging the mixture for 30 minutes at 4 ℃ by using a centrifugal force of 3500g, taking the supernatant, centrifuging the mixture for 30 minutes at 4 ℃ by using a centrifugal force of 18000g, taking the supernatant, adding PBS (phosphate buffer solution) to adjust the volume of the supernatant to 400 embryos per milliliter, and storing the mixture in a refrigerator at-20 ℃.
Example 2
The application embodiment 2 provides a long-term culture method of rockfish germ stem cells, which utilizes the rockfish germ stem cells provided in embodiment 1 to perform in-vitro long-term culture and comprises the following steps:
step 1, pretreating the gonads of the groupers
Removing tissue components such as blood vessels, blood, membranes, fat, sperms or ova and the like from the gonads of the groupers as far as possible, and repeatedly cleaning the groupers by using double-antibody PBS (phosphate buffer solution);
step 2, preparing the grouper gonad tissue block
Transferring the gonads washed by the double-antibody PBS in the step 1 to a 1.5mL sterile centrifuge tube to avoid the gonads from being polluted, and shearing the gonads in the sterile centrifuge tube into 1mm pieces by using small scissors 3 Small fragments are obtained to obtain the rockfish gonad tissue block;
step 3, preprocessing the grouper gonad tissue block
Standing or centrifuging the aseptic centrifuge tube containing the rockfish gonad tissue block in the step 2 at a low speed, removing supernatant in the aseptic centrifuge tube, and then adding 1ml of the culture medium prepared in the example 1 into the aseptic centrifuge tube for resuspension to obtain uniformly dispersed rockfish gonad tissue blocks;
step 4, culturing the tissue blocks of the gonad of the groupers
Transferring the rockfish gonad tissue block obtained in step 3 to a new culture flask, adding 5ml of the culture medium provided in example 1, culturing the tissue block for 50 days at 28 deg.C and 5% CO 2 An incubator, wherein a primary culture medium is replaced for three days in the culture process; the culture process is divided into three stages, the first stage is cultured for 10 days, the tissue block of the Epinephelus malabaricus adheres to the wall, somatic cells (such as fiber-like cells and epidermoid cells) in the tissue block of the adherent Epinephelus malabaricus firstly migrate to the bottom of a culture bottle (as shown in figure 1), reproductive stem cells subsequently migrate, and the second stage is cultured for 30 days, because the reproductive stem cell clusters adhere to the wall very firmly and are not easy to fall off compared with the somatic cells, and the somatic cells migrating in the tissue block of the adherent Epinephelus malabaricus are not passaged, over-proliferated and over-proliferated for a long timeCrowding, gradual and natural shedding, finally exposing the bottom of the blank culture flask, and gradually migrating the germ stem cells from the tissue block to the exposed bottom of the culture flask, wherein the germ stem cells grow in clusters (as shown in figure 2), and the diameter of the germ stem cell cluster is about 2 to 5mm at this stage 2 The area of the germ stem cell cluster can be marked by circles, then somatic cells in the non-circled area are scraped off by a cell scraper, and then the somatic cells are cleaned for 2 times by double-antibody PBS (phosphate buffered saline), so that the separation of the somatic cells is accelerated; culturing for 50 days in the third stage, marking the area of the germ stem cell cluster by drawing circles again, scraping off somatic cells in a non-circled area by using a cell scraper, cleaning for 2 times by using double-resistant PBS (phosphate buffered saline), accelerating to separate the somatic cells, scraping off or naturally dropping off most of the somatic cells in the stage, adding 2mL of pancreatin into a culture bottle when the number of the germ stem cell clusters in the culture bottle is large and the area is large, gently shaking the culture bottle to soak all the cells by using pancreatin digestive juice, allowing the somatic cells with weak adherence to be easily digested by using the pancreatin, not blowing off the pancreatin digestive juice in the pancreatin digestive process to avoid the germ stem cell cluster from dropping and being digested, observing under a microscope, digesting for about 1 minute, dropping off most of the somatic cells, sucking away the pancreatin, cleaning for two times by using the PBS, adding the culture medium prepared in the step 1, culturing for 5 days, and repeating the pancreatin digestive step for 3-5 times to obtain the purified rockfish germ stem cells without the somatic cells;
step 5, culturing the reproductive stem cells of the purified grouper in vitro
Adding the culture medium of the cultured Mylopharyngodon Piceus germ stem cells described in example 1 to the purified Mylopharyngodon Piceus germ stem cells prepared in step 4, and culturing in vitro at 28 deg.C, 5% CO 2 Replacing a culture medium once for three days in the culture process of the incubator, culturing the rockfish spermatogonial stem cells in vitro for 20 months, culturing the rockfish spermatogonial stem cells in the culture bottle after 5 months of oogonial stem cells, wherein a microscopic picture is shown as figure 3, the rockfish spermatogonial stem cells and the oogonial stem cells in the culture bottle can be seen from the cell morphology shown as figure 3, no somatic cells are present, further performing specific marker gene expression analysis on the rockfish spermatogonial stem cells and the oogonial stem cells in the culture bottle, and the result is shown as figure 4, which indicates that the rockfish spermatogonial stem cells and the oogonial stem cells are preserved and analyzedThe method maintains the original physiological characteristics and can differentiate into adult stem cells for generating sperms and adult stem cells for generating ova, so that the long-term culture system for the groupers greatly prolongs the in-vitro culture time of the grouper germ stem cells, avoids the problem that the grouper germ stem cells are finally died due to lack of growth space and nutritional conditions along with the extension of the culture time because the somatic cells are introduced and have high proliferation rate and excessive proliferation and low proliferation speed in the prior art, and cannot realize the long-term in-vitro culture of the grouper germ stem cells.
It will be further noted that the gonads of the groupers in step 1 of this example 1 include adult female coir rockfish gonad tissues and adult female coir rockfish gonad tissues; in step 2, preparing 1mm gonad by cutting 3 The rockfish gonad tissue block is convenient for cells in the middle of the gonad tissue block to fully contact with a culture medium, and is beneficial to cell proliferation; in the step 3, the gonad tissue blocks of the groupers are uniformly dispersed, so that the phenomenon of contact inhibition of cells is avoided; in the step 4, the obtained purified germ stem cells are rockfish spermatogonial stem cells and rockfish oogonial stem cells, in the step 4, due to individual differences of rockfish gonads, the germ stem cells migrate from tissue blocks of the rockfish gonads to the bottom of an exposed culture flask, the time for the germ stem cells to grow in a clustering manner is possibly longer, the time of the first tissue block culture stage and the second tissue block culture stage can be prolonged to two months, and the tissue block culture stage in the third stage can be prolonged.
The method for pretreating the gonads of the groupers comprises the following steps: firstly, transferring prepared adult male Epinephelus coioides with the body length of about 70cm at the age of 4 and adult female Epinephelus coioides with the body length of about 35cm at the age of 1 to a big water bucket, and adding eugenol into the water to anaesthetize the Epinephelus coioides; dissecting the fish belly with scissors, tweezers, etc., completely removing the whole gonad, removing excessive tissue as much as possible, putting the gonad into a bottle filled with double-resistant PBS, quickly screwing down a cover, and semi-burying the bottle in ice, wherein the whole process is as fast as possible and pollution is reduced as much as possible; and then bringing the gonadal sample back to the laboratory as soon as possible, repeatedly cleaning the whole gonadal by using double-antibody PBS on a clean bench, shearing a small part of the gonadal, and finishing the pretreatment of the gonadal of the grouper.
The above embodiments are only used for illustrating the technical solutions of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present application.
Claims (10)
1. A culture medium for rockfish germ stem cells is characterized by comprising the following components: a basal medium and nutrients;
the nutrient components comprise: fetal bovine serum, antibiotics, beta-mercaptoethanol, glutaMAX, cytokines and pre-treated medaka embryos;
the cytokines include: bFGF, GDNF, LIF and EGF.
2. The culture medium for the rockfish germ stem cells according to claim 1, wherein the volume ratio of the fetal bovine serum to the basal medium is 5-20: 100, respectively;
the antibiotics are penicillin and streptomycin:
the concentration of the penicillin is 100Units/mL;
the concentration of the streptomycin is 100 mug/mL;
the concentration of the beta-mercaptoethanol in the basic culture medium is 55uM/mL;
the concentration of the GlutaMAX in the basal medium is 2mM/L;
the concentration of the pretreated medaka embryos in the basal medium is 2 grains/mL;
the concentrations of bFGF, GDNF, LIF and EGF in the basal medium are all 20ng/mL.
3. A long-term culture method of rockfish germ stem cells is characterized by comprising the following steps:
step 1, cutting the sex gland of the grouper into pieces to obtain a sex gland tissue block of the grouper;
step 2, dispersing the grouper gonad tissue blocks in a culture container for tissue block culture to obtain adherent grouper gonad tissue blocks;
step 3, performing first tissue block culture on the gonad tissue blocks of the adherent groupers to obtain first adherent cells;
step 4, scraping off somatic cells in the first adherent cells by using a cell scraper, cleaning by using double-resistant PBS (phosphate buffered saline), and culturing a second tissue block to obtain second adherent cells;
step 5, scraping somatic cells in the second adherent cells by using a cell scraper to obtain third adherent cells;
step 6, infiltrating the obtained third cell with pancreatin, removing the pancreatin, and cleaning with PBS to obtain a purified germ stem cell;
and 7, adding a culture medium of the grouper germ stem cells in the claim 1 into the purified germ stem cells for in vitro culture.
4. The method for long-term culture of rockfish germ stem cells according to claim 3, wherein the culture medium used in the tissue mass culture, the first tissue mass culture and the second tissue mass culture in steps 2, 3 and 4 is the culture medium of rockfish germ stem cells according to claim 1.
5. The method for long-term culture of the reproductive stem cells of the grouper as claimed in claim 3, wherein in the step 2, the tissue mass culture comprises: subjecting the mass of rockfish gonadal tissue to a temperature of 28 ℃ in an atmosphere of 5% CO 2 Culturing the lower tissue block for 10 days;
in step 3, the first tissue block culture specifically comprises: the tissue mass of the gonadal gland of the adherent grouper is treated at a temperature of 28 ℃ under an atmosphere of 5% CO 2 Culturing the lower tissue block for 30 days;
in step 4, the second tissue piece culture specifically comprises: will be provided withFirst adherent cells at a temperature of 28 ℃ under an atmosphere of 5% CO 2 The lower tissue mass was cultured for 50 days.
6. The method for long-term culture of rockfish germ stem cells according to claim 3, wherein the volume of the culture medium used in the tissue mass culture, the first tissue mass culture and the second tissue mass culture in steps 2, 3 and 4 is 3-5 ml.
7. The method for long-term culture of the reproductive stem cells of grouper as claimed in claim 3, wherein in steps 2, 3 and 4, the culture medium is replaced every 2 to 3 days during the culture of the tissue mass, the culture of the first tissue mass and the culture of the second tissue mass.
8. The method for long-term culture of the reproductive stem cells of grouper as claimed in claim 3, wherein in step 6, the pancreatin is used in a volume of 2 to 3ml;
the pancreatin infiltration time is 1-1.5 min;
the number of PBS washes was 2-3.
9. The method for long-term culture of the reproductive stem cells of grouper as claimed in claim 3, wherein step 6, before obtaining the purified reproductive stem cells after the step of soaking the obtained third parietal cells with pancreatin, removing pancreatin and washing with PBS, further comprises the steps of:
step 6.1, adding 5 to 6ml of the culture medium for the rockfish germ stem cells according to claim 1 at a temperature of 28 ℃ under an atmosphere of 5% CO 2 Culturing for 5 days to obtain germ stem cells;
and 6.2, repeating the step 6-6.1 for 3 to 5 times to obtain the purified germ stem cells.
10. The method for long-term culture of the rockfish germ stem cells according to claim 3, wherein the rockfish gonads are male Epinephelus coioides gonads or female Epinephelus coioides gonads.
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