CN115141796B - Culture medium for grouper germ stem cells and long-term culture method thereof - Google Patents
Culture medium for grouper germ 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 germ stem cells, and particularly relates to a culture medium of germ stem cells of groupers and a long-term culture method thereof; the culture medium for the grouper germ stem cells provided by the application comprises the following components: the method comprises the steps of culturing fetal calf serum, antibiotics, beta-mercaptoethanol, glutaMAX, cytokines, medaka embryo extract and Advanced DMEM/F12 culture medium, so that the in-vitro culture time of the grouper primordial stem cells is prolonged to 20 months, the in-vitro culture time of the grouper primordial stem cells is prolonged to 5 months, and the long-term in-vitro culture requirements of the grouper primordial stem cells and the grouper primordial stem cells can be met simultaneously, thereby solving the technical problem that the culture medium required by the in-vitro long-term culture of the grouper reproduction stem cells is lacking in the prior art.
Description
Technical Field
The application belongs to the technical field of in-vitro culture of germ stem cells, and particularly relates to a culture medium of germ stem cells of groupers and a long-term culture method thereof.
Background
At present, research on self-renewal mechanism of germ stem cells is concentrated in mammals such as mice, human and the like, and a long-term culture system of germ stem cells of a plurality of mammals such as mice, human and the like 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 research is concentrated on the fish germ stem cells, because the nutrient medium required for maintaining the long-term self-renewal of the fish germ stem cells is not clear, the fish germ stem cells are cultured for a long time in vitro due to the lack of a proper culture medium, most of the fish germ stem cells can only survive for 1 to 3 months in the culture medium, and the survival time of the fish germ stem cells is shorter, for example, zebra fish germ stem cells can only survive for about 6 weeks in the culture medium, because of the physiological characteristic difference of the fish germ stem cells and the germ stem cells, the culture medium of the fish germ stem cells cannot be directly used for culturing the germ stem cells, and the culture medium for the long-term in vitro culture of the fish germ stem cells and the fish germ stem cells cannot be used universally.
Meanwhile, the species number of fishes is very large, physiological characteristics are different, nutrient matrixes required for maintaining long-term self-renewal of germ stem cells of different fishes are also different, for example, the fishes are hermaphrodite fishes, namely female fishes are in primary sexual maturity, female fishes begin to be sex-reversed into male fishes after one year to a plurality of years after the fishes participate in reproduction as female fishes, therefore, the physiological characteristics of the fishes are very different, the culture conditions required for in-vitro culture of the germ stem cells of the fishes are very different from those of the other fishes, the culture medium of the germ stem cells of the other fishes cannot be used for being universal for the fishes, the fishes are famous and precious marine fishes, the meat quality is delicious, the nutrition is rich, the economic value is very high, the important production and application value is realized, meanwhile, the physiological characteristics of the hermaphrodite fishes different from most fishes also have important scientific research value, however, the related research of the germ stem cell culture medium of the fishes is lacking in the prior art, and the requirement for in-vitro culture of the germ stem cells of the fishes cannot be met.
Disclosure of Invention
In view of the above, the application provides a culture medium for the germ stem cells of the groupers 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 the germ stem cells of the groupers is lacking in the prior art.
The first aspect of the application provides a culture medium for germ stem cells of groupers, which comprises the following components: a basal medium and nutrients;
the nutritional ingredients include: fetal bovine serum, antibiotics, beta-mercaptoethanol, glutaMAX, cytokines, and pre-treatment of medaka embryos;
the cytokines include: bFGF, GDNF, LIF and EGF.
The basal medium is an advanced DMEM/F12 medium or a DMEM/F12 medium.
Preferably, in the culture medium of the grouper germ stem cells, the volume ratio of the fetal calf serum to the basal culture medium is 5-20: 100;
the antibiotics are penicillin and streptomycin:
the concentration of penicillin is 100Units/mL;
the concentration of the streptomycin is 100 mug/mL;
the concentration of the beta-mercaptoethanol in the basal medium is 55 mu M;
the concentration of GlutaMAX in the basal medium is 2mM;
the concentration of the pretreated medaka embryo in the basal medium is 2 grains/mL;
the concentrations of bFGF, GDNF, LIF and EGF in the basal medium were 20ng/mL.
Preferably, the medaka embryo is a day 7 embryo of a pretreated medaka.
The collected medaka embryos were washed with PBS, ground for 20 minutes with a tissue breaker, repeatedly freeze-thawed three times in a water bath at 37℃in liquid nitrogen, and centrifuged at 3500g for 30 minutes at 4℃to obtain medaka embryos as pretreated medaka embryos on day 7.
The pretreated medaka embryos can be adjusted to 400 embryos per milliliter by adding PBS, stored at-20 ℃ in a refrigerator, taken out when needed, and added to a basal medium.
The second aspect of the application provides a long-term culture method of the grouper germ stem cells, which utilizes a culture medium of the grouper germ stem cells for long-term in vitro culture and comprises the following steps:
step 1, shearing gonads of the groupers to obtain gonad tissue blocks of the groupers;
step 2, dispersing the grouper gonad tissue blocks in a culture container for tissue block culture to obtain the attached grouper gonad tissue blocks;
step 3, culturing the gonad tissue blocks of the garrupa to obtain first adherent cells;
step 4, scraping somatic cells in the first adherent cells by using cell scraping, cleaning by using double-antibody PBS, and culturing a second tissue block to obtain second adherent cells;
step 5, scraping somatic cells in the second adherent cells by using cell scraping 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 cell flow sorting method are only used for separating and purifying germ stem cells of model animals such as mice generally due to high price, and are not applicable to large and non-model animals such as groupers; the existing separation and purification method of fish stem cells utilizes the principle that the spermatogonial stem cells are slower than the somatic cells in adherence, the spermary is digested into a single cell state firstly, then the non-adherent spermatogonial stem cells are obtained through a differential adherence method, however, the somatic cells are easily introduced into the non-adherent spermatogonial stem cells through the differential adherence method, the proliferation speed of the somatic cells is extremely high, so that when the non-adherent spermatogonial stem cells are subjected to in vitro culture, the somatic cells excessively proliferate to cause the germ stem cells to lack of growth space and nutrition conditions and finally die, long-term in vitro culture cannot be realized, the spermary is directly digested into the single cell state, the germ stem cells and the microenvironment thereof are greatly destroyed, the long-term in vitro culture of the germ stem cells is not facilitated, and meanwhile, the cells and the microenvironment thereof are also greatly destroyed in the process of separating the cells by a gradient centrifugation method, and the introduced somatic cells are not easy to separate; in the long-term culture method of the grouper germ stem cells, the tissue block culture method has less damage to the germ stem cells and the microenvironment thereof, the primary culture of the grouper germ stem cells can be easily started, the original physiological properties of the grouper germ stem cells are maintained, then in the process of tissue block culture of the grouper germ tissue blocks, the adherent cells migrated from the grouper germ tissue blocks are not subjected to pancreatin and inoculated into a new culture container for subculture, but in the process of tissue block culture of the grouper germ tissue blocks, only the culture medium is replaced, so that the adherent cells are subjected to non-passage culture in the culture container, and in the non-passage culture process, as compared with the adherent cells, the germ stem cell clusters in the cell clusters are very firm and are not easy to fall off, the adherent cells are not passaged for a long time, and are excessively proliferated and crowded in the culture container, the bottom of a blank culture bottle is gradually fallen off, the grouper germ cells are gradually migrated to the bottom of the exposed bottle, and the adherent germ cells are subjected to the subculture, and the germ cells of the grouper germ cells are not infiltrated by the PBS (poly-styrene) to the germ cell clusters, and the germ cell clusters of the grouper germ cells are not purified after the germ cells are subjected to the passage culture, and the germ cell clusters of the germ cells are not subjected to the non-passage culture; in the process of separating somatic cells in the gonads of the groupers, the adherent somatic cells can be scraped off by cell scraping, so that the somatic cells in the gonads of the groupers are separated quickly, and purified germ stem cells of the groupers without 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 a long time of more than 20 months, and the in-vitro culture time of the grouper germ stem cells is greatly prolonged.
Preferably, in the step 1, the volume of the grouper gonad tissue block is 1mm 3 。
Preferably, in the steps 2, 3 and 4, the culture medium used in the process of culturing the tissue mass, the first tissue mass and the second tissue mass is the culture medium of the grouper germ stem cells.
In the tissue block culture process, compared with other commercial or self-made culture mediums, the culture medium for the grouper germ stem cells can meet the nutrition substrate required by the self-renewal of the grouper germ stem cells, is favorable for improving the self-renewal and proliferation rate of the germ stem cells, reduces the death and differentiation of the grouper germ stem cells and quickens the separation of somatic cells in the gonads of the groupers.
Preferably, in step 2, the tissue block culture is specifically: the gonad tissue block of the grouper is heated to 28 ℃ and the atmosphere is 5 percent CO 2 Culturing the lower tissue block for 10 days;
in step 3, the first tissue block culture is specifically: the gonad tissue block of the garrupa is subjected to the atmosphere of 5 percent CO at the temperature of 28 DEG C 2 Culturing the lower tissue block for 30 days;
in step 4, the second tissue block culture is specifically: the first adherent cells were subjected to an atmosphere of 5% CO at a temperature of 28 ℃C 2 The lower tissue mass was cultured for 50 days.
The method for culturing the grouper gonad tissue block, the grouper gonad tissue block and the first adherent cells are all placed in the medium containing 5% CO 2 The culture in the atmosphere of (a) is due to the fact that the basal medium in the culture medium of the germ stem cells of the garrupa is NaHCO 3 Buffer system for enabling the germ stem cells of the garrupa to be in 5 percent CO 2 Under conditions of growth in CO 2 The culture in the atmosphere can obviously reduce the oxidation of the germ stem cells of the groupers, slow down the aging and poisoning phenomena of the germ stem cells of the groupers and improve the growth and proliferation rate of the groupers.
Preferably, in the steps 2, 3 and 4, the volume of the culture medium used in the tissue mass culture, the first tissue mass culture and the second tissue mass culture is 3 to 5ml.
Preferably, in the steps 2, 3 and 4, the culture medium is replaced every 2 to 3 days during the tissue mass culture, the first tissue mass culture and the second tissue mass culture.
Preferably, in the step 6, the use volume of the pancreatin is 2-3 ml;
the time of the pancreatin infiltration is 1-1.5 min;
the PBS cleaning times are 2-3 times.
Preferably, in step 6, the step of infiltrating the obtained third adherent cells with pancreatin, removing pancreatin, and after washing with PBS, further comprises, before obtaining purified germ stem cells:
step 6.1, adding 5-6 ml of culture medium of the grouper germ stem cells at the temperature of 28 ℃ and under the atmosphere of 5% CO 2 Culturing for 5 days to obtain germ stem cells;
and step 6.2, repeating the steps 6 to 6.1 for 3 to 5 times to obtain the purified germ stem cells.
Preferably, the garrupa gonad is male garrupa gonad or female garrupa gonad.
In summary, the application provides a culture medium for germ stem cells of groupers and a long-term culture method thereof, wherein the culture medium comprises the following components: the application provides a method for culturing the germ stem cells of the groupers, which comprises the steps of preparing a culture medium of the groupers, carrying out the culture of the germ stem cells of the groupers, carrying out the culture of the germ cells of the groupers, and carrying out the culture of the germ cells of the groupers.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a microscopic view showing a gonad tissue block of an adherent grouper after tissue block culture for 10 days using a grouper tissue block in example 2 of the present application;
FIG. 2 is a microscopic view of germ cell clusters at the bottom of the adherent cells after the first tissue mass culture for 30 days with the garrupa gonad tissue mass of example 2 of the present application;
fig. 3 is a microscopic view of the germ stem cells of groupers after culturing the germ stem cells of groupers in vitro by the long-term culturing method of the germ stem cells of groupers provided in the embodiment 2 of the application;
FIG. 4 is a graph showing the expression of specific marker genes of the germ stem cells of groupers cultured in vitro according to the long-term culturing method of germ stem cells of groupers provided in example 2 of the present application;
fig. 5 is a flowchart of a long-term culture method of grouper germ stem cells provided in embodiment 2 of the present application;
wherein, fig. 1A is a microscopic view of an ovary tissue block of an adherent grouper, fig. 1B is a microscopic view of a testis tissue block of an adherent grouper, fig. 2A is a microscopic view of an oosperm stem cell cluster of an grouper, and fig. 2B is a microscopic view of an sperm stem cell cluster of an grouper; 3A is a microscopic image of the garrupa oosperm stem cells, and FIG. 3B is a microscopic image of the garrupa oosperm stem cells; 4A is the expression pattern of the specific marker gene of the garrupa oosperm stem cell, and FIG. 4B is the expression pattern of the specific marker gene of the garrupa seminal stem cell.
Detailed Description
The application provides a culture medium of grouper 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 grouper germ stem cells is lacking in the prior art.
The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Example 1
The embodiment 1 of the application provides a culture medium for germ stem cells of groupers, which comprises the following components
Table 1 shows:
| fetal bovine serum | 15％ |
| Penicillin, streptomycin | 1X |
| Beta-mercaptoethanol | 55μM |
| GlutaMAX | 2mM |
| Pretreatment of medaka day 7 embryo | 2 grains/mL |
| bFGF (basic fibroblast growth factor) | 20ng/mL |
| GDNF (glial cell derived neurotrophic factor) | 20ng/mL |
| LIF (leukemia inhibitory factor) | 20ng/mL |
| EGF (epidermal growth factor) | 20ng/mL |
| Advanced DMEM/F12 medium |
TABLE 1
Wherein, the preparation method of the pretreated medaka day 7 embryo comprises the following steps:
step 1, incubating fertilized medaka embryos at a water temperature of 28 ℃, and collecting medaka embryos which are on the 7 th day and are normal in development;
step 2, washing the embryo three times by using PBS (phosphate buffer solution), and grinding the embryo for 20 minutes by using a tissue breaker;
step 3, repeatedly freezing and thawing for three times in liquid nitrogen and 37 ℃ water bath;
step 4, centrifuging at the temperature of 4 ℃ for 30 minutes by using a centrifugal force of 3500g, taking the supernatant, centrifuging at the temperature of 4 ℃ for 30 minutes by using a centrifugal force of 18000g, taking the supernatant, adding PBS (phosphate buffer solution) to adjust to 400 embryos per milliliter, and storing in a refrigerator at the temperature of minus 20 ℃.
Example 2
The embodiment 2 of the application provides a long-term culture method of grouper germ stem cells, which utilizes the grouper germ stem cells provided in the embodiment 1 to carry out in vitro long-term culture, and comprises the following steps:
step 1, pretreatment of gonads of groupers
Removing blood vessels, blood, films, fat, sperms or ovum and other tissue components of the gonads of the groupers as much as possible, and repeatedly cleaning by using double-antibody PBS;
step 2, preparing gonad tissue blocks of groupers
Transferring the gonads washed by the double-antibody PBS in the step 1 to a 1.5mL sterile centrifuge tube, avoiding the gonads from being polluted, and then shearing the gonads in the sterile centrifuge tube into 1mm by using small scissors 3 Small fragments to obtain gonad tissue blocks of groupers;
step 3, preprocessing the gonad tissue block of the grouper
Standing or centrifuging the sterile centrifuge tube containing the grouper gonad tissue blocks in the step 2 at a low speed, removing supernatant in the sterile centrifuge tube, and then adding 1ml of the culture medium prepared in the example 1 into the sterile centrifuge tube for resuspension to obtain the grouper gonad tissue blocks with uniform dispersion;
step 4, performing tissue block culture on the gonad tissue blocks of the groupers
Transferring the grouper gonad tissue block in the step 3 into a new culture bottle, and adding 5ml of the culture medium provided in the example 1 to perform tissue block culture for 50 days, wherein the environment of tissue block culture is 28 ℃ and 5% CO 2 The incubator is used for replacing the culture medium once in three days in the culture process; the culturing process is divided into three stages, wherein the first stage is used for culturing for 10 days, somatic cells (such as fiber-like cells and epidermic-like cells) in the tissue blocks of the garrupa are firstly migrated to the bottom of a culture flask (shown in figure 1), germ stem cells are subsequently migrated, and the second stage is used for culturing for 30 days, and the germ stem cell clusters are very firm and difficult to fall off compared with the somatic cells, and the somatic cells migrated by the tissue blocks of the garrupa gradually naturally fall off due to long-term non-passage, excessive proliferation and crowding, finally the bottom of a blank culture flask is exposed, and the germ stem cells gradually migrate from the tissue blocks to the bottom of the exposed culture flask and grow in a clustered shape (shown in figure 2), and the diameter of the germ stem cell clusters is about 2-5 mm in the stage 2 The germ stem cell cluster region can be circled and labeled, then somatic cells in the non-circled region are scraped off by cell scraping, and then washed 2 times with double-antibody PBSQuickening the separation of somatic cells; culturing for 50 days in the third stage, marking the area of the germ stem cell cluster in a circling way again, scraping off somatic cells in a non-circling area by using cell scraping, then cleaning for 2 times by using double-antibody PBS (phosphate buffer solution), accelerating the separation of the somatic cells, scraping off or naturally shedding most of the somatic cells in the stage, adding 2mL of pancreatin into a culture flask when the germ stem cell cluster in the culture flask is more in number and larger in area, gently shaking the culture flask to enable pancreatin digestive juice to infiltrate all the cells, enabling the somatic cells which are not firmly attached to pass through pancreatin to be easily digested, avoiding the shedding digestion of the germ stem cell cluster in the pancreatin digestion process, observing under a microscope, digesting for about 1 minute, shedding most of the somatic cells, sucking away the pancreatin, cleaning twice by using the PBS, and culturing for 5 days by adding the culture medium prepared in the step 1, and repeating the pancreatin digestion step for 3-5 times to obtain purified grouper germ stem cell without somatic cells;
step 5, culturing the purified grouper germ stem cells in vitro
Adding the culture medium of the grouper germ stem cells described in the example 1 into the purified grouper germ stem cells prepared in the step 4 for long-term in vitro culture, wherein the in vitro culture environment is 28 ℃ and 5% CO 2 The culture process is to replace a culture medium for three days, the culture process is to culture the grouper spermatogonial stem cells in vitro for 20 months, and the grouper spermatogonial stem cells in the culture bottle after 5 months, the microscopic image is shown as shown in figure 3, the cell morphology shown in figure 3 shows that the grouper spermatogonial stem cells and the grouper spermatogonial stem cells are in the culture bottle, no somatic cells exist, the specific marker gene expression analysis is further carried out on the grouper spermatogonial stem cells and the grouper spermatogonial stem cells in the culture bottle, the result is shown as figure 4, which shows that the grouper spermatogonial stem cells and the grouper spermatogonial stem cells keep the original physiological characteristics, and can be differentiated into the adult stem cells for producing sperms and the adult stem cells for producing the ova, therefore, the long-term culture system of the grouper provided by the application greatly prolongs the in vitro culture time of the grouper germ stem cells, avoids the growth rate of the grouper germ cells being fast and excessive proliferation, the proliferation rate of the grouper germ cells is very slow, and the proliferation rate of the germ cells is prolonged along with the culture time in the prior art,the germ stem cells of the groupers lack of growth space and nutrition conditions to finally die, and long-term in vitro culture of the germ stem cells of the groupers cannot be realized.
It will also be noted that the garrupa gonad in step 1 of this example 1 includes adult male garrupa gonad tissue and adult female garrupa gonad tissue; in step 2, the gonads are sheared to prepare 1mm 3 The gonad tissue block of the grouper is convenient for the cells in the middle of the gonad tissue block to fully contact with the culture medium, thereby being 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 the grouper sperm stem cells and the grouper egg stem cells, in the step 4, the germ stem cells migrate from the grouper gonad tissue blocks to the bottom of an exposed culture bottle due to the individual difference of the grouper gonad, the time for clustered growth can be 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 also be prolonged.
The pretreatment of the gonads of the groupers comprises the following steps: firstly, transferring prepared adult male Epinephelus coioides with the age of 4 and the length of about 70cm and adult female Epinephelus coioides with the age of 1 and the length of about 35cm to a big water bucket, and then adding eugenol into water to anesthetize the fish; then using tools such as scissors tweezers to dissect the fish belly, completely taking out the whole gonad, removing redundant tissues as much as possible without damaging the coated membrane, putting the gonad into a bottle with double-antibody PBS, quickly screwing a cover, and half burying the bottle in ice, wherein the whole process is as fast as possible, and pollution is reduced as much as possible; and then the gonad sample is brought back to a laboratory as soon as possible, the whole gonad is repeatedly cleaned by using double-antibody PBS on an ultra-clean workbench, and a small part of gonad is sheared, so that the pretreatment of the grouper gonad is completed.
The above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (7)
1. The application of a culture medium of the grouper germ stem cells in culturing the grouper germ stem cells is characterized by comprising the following components: a basal medium and nutrients;
the basic culture medium is an Advanced DMEM/F12 culture medium;
the nutritional ingredients include: fetal bovine serum, antibiotics, beta-mercaptoethanol, glutaMAX, cytokines, and pre-treatment of medaka embryos;
the cytokine consists of bFGF, GDNF, LIF and EGF;
the volume ratio of the fetal calf serum to the basic culture medium is 5-20: 100;
the antibiotics are penicillin and streptomycin:
the concentration of penicillin is 100Units/mL;
the concentration of the streptomycin is 100 mug/mL;
the concentration of the beta-mercaptoethanol in the basal medium is 55 mu M;
the concentration of GlutaMAX in the basal medium is 2mM;
the concentration of the pretreated medaka embryo in the basal medium is 2 grains/mL;
the concentrations of bFGF, GDNF, LIF and EGF in the basal medium were 20ng/mL.
2. A long-term culture method of grouper germ stem cells, which is characterized by comprising the following steps:
step 1, shearing gonads of the groupers to obtain gonad tissue blocks of the groupers;
step 2, dispersing the grouper gonad tissue blocks in a culture container for tissue block culture to obtain the grouper gonad tissue blocks, wherein a culture medium used for the tissue block culture is a culture medium of the grouper germ stem cells in claim 1;
step 3, culturing the garrupa gonad tissue block to obtain a first garrupa gonad tissue block, wherein a culture medium used for culturing the first tissue block is a culture medium of the garrupa germ stem cell in claim 1;
scraping somatic cells in the first adherent cells by using cell scraping, cleaning by using double-antibody PBS, and culturing a second tissue block to obtain second adherent cells, wherein a culture medium used for culturing the second tissue block is a culture medium of the grouper germ stem cells in claim 1;
step 5, scraping somatic cells in the second adherent cells by using cell scraping 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 the culture medium of the grouper germ stem cells in claim 1 into the purified germ stem cells for in vitro culture.
3. The method for long-term culture of germ stem cells of grouper according to claim 2, wherein in step 2, the tissue mass culture is specifically: the gonad tissue block of the grouper is heated to 28 ℃ and the atmosphere is 5 percent CO 2 Culturing the lower tissue block for 10 days;
in step 3, the first tissue block culture is specifically: the gonad tissue block of the garrupa is subjected to the atmosphere of 5 percent CO at the temperature of 28 DEG C 2 Culturing the lower tissue block for 30 days;
in step 4, the second tissue block culture is specifically: the first adherent cells were subjected to an atmosphere of 5% CO at a temperature of 28 ℃C 2 The lower tissue mass was cultured for 50 days.
4. The long-term culture method of the grouper germ stem cells according to claim 2, wherein in the step 2, the volume of a culture medium used in the tissue mass culture process is 3-5 ml;
in the step 3, the volume of the culture medium used in the first tissue block culture process is 3-5 ml;
in the step 4, the volume of the culture medium used in the second tissue block culture process is 3-5 ml.
5. The method for long-term culture of grouper germ cells according to claim 3, wherein in the step 2, the culture medium is replaced every 2-3 days in the tissue mass culture process;
in the step 3, the culture medium is replaced every 2-3 days in the first tissue block culture process;
in the step 4, the culture medium is replaced every 2-3 days in the second tissue block culture process.
6. The long-term culture method of the grouper germ stem cells according to claim 2, wherein in the step 6, the use volume of pancreatin is 2-3 ml;
the pancreatin infiltration time is 1-1.5 min;
the PBS cleaning times are 2-3 times.
7. The method for long-term culture of grouper germ cells according to claim 2, wherein the gonad of grouper is male garrupa gonad or female garrupa gonad.
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