CN116676257A - In-vitro culture method of scylla paramamosain spermatogonial stem cells - Google Patents

Abstract

The invention relates to a method for in vitro culture, induced differentiation and application of scylla paramamosain spermatogonium stem cells, which is an invertebrate cell culture method, belongs to the field of cell biology, and can be used for in vitro culture of scylla paramamosain spermatogonium stem cells for 395 days and continuous culture for 15-20 generations. The scylla paramamosain spermatogonia stem cells obtained by the method have the stem property and proliferation activity, and can be differentiated into somatic cells such as bone cells, fat cells and the like through induction. The method can provide basic experimental materials for culturing the crustacean cells and carrying out proliferation and differentiation and other molecular biology research on the cell level.

Description

A kind of in vitro culture method of spermatogonia stem cells

technical field

The invention relates to the field of cell engineering, in particular to a method for culturing spermatogonia stem cells in vitro.

Background technique

Cell culture technology is also called cell cloning technology. Cell culture is used in cytogenetics, biochemical and molecular laboratory research and toxicology research.

By artificially controlling cell culture conditions, the effects of cell metabolism, physical, chemical, and biological factors can be studied. Compared with animal experiments, cell culture is less expensive and economical, and can be cultivated in large quantities, which is beneficial to the production of biological products. At present, many cell lines have been successfully established, including normal cells, stem cells, tumor cells and defective cells of humans and model animals, but crustacean cell culture is still difficult. So far, there has been no report on a stable method for the application of crustacean cell culture technology, and there is no report on the application of crustacean cells for cell-level related experiments.

Crustacean cells are very sensitive to the growth conditions of the external environment. At present, the commercial media that can be purchased are basal culture and specific media for other species. The use of basal media alone is not enough to meet the normal survival requirements of crustacean cells in vitro. need. In the existing reports on the initial attempts to culture crustacean cells, whether using TC199, M199, L-15 or other media, they cannot keep the cells active for a long time, and the cell proliferation rate is far lower than the death rate, and it is impossible to carry out Subculture. Therefore, there has been no breakthrough in cell culture technology in crustaceans.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis and are crucial to the transmission of biological genetic information. Crustacean spermatogonial stem cells have a high degree of self-renewal and differentiation capabilities. Under appropriate culture conditions, they have the ability to form embryoid bodies and have the potential to differentiate into various types of somatic cells. However, the culture technology of crab spermatogonial stem cells is not perfect, and there are no reports about the in vitro culture of crab SSCs. Whether crabs can obtain stable spermatogonial stem cell lines and whether crab SSCs can differentiate into different types of somatic cells remains to be further proved.

Exploring the application of crab SSCs in the fields of germplasm resource protection and germ cell transplantation, and obtaining stable cultured spermatogonial stem cell lines are of great significance for in-depth research on the differentiation potential of crustacean nutrition metabolism and cell physiology. The present invention obtains the optimal culture conditions of the crustacean Scylla pseudocavea spermatogonial stem cells through multi-level, multi-element, and multi-faceted conditions, and designs a medium for the first time to provide an in vitro culture of the Scylla spermatogonial stem cell line. The method also provides a method for inducing spermatogonial stem cells to differentiate into somatic cells, and inducing lentivirus infection of spermatogonial stem cells to express foreign genes.

Contents of the invention

The purpose of the present invention is to break through the current situation that crustacean cells cannot be cultured and communicated in vitro for a long time, and provide a method for culturing spermatogonial stem cells in vitro for the first time in Scylla syringae, filling the gap in the experimental technology of crustacean cells, and providing the above-mentioned A method for inducing spermatogonial stem cells to differentiate into somatic cells, and inducing lentivirus infection of spermatogonial stem cells to express foreign genes.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

An in vitro culture method for spermatogonial stem cells of Scylla pseudocavei, comprising the steps of:

A, prepare culture medium, save for subsequent use;

B, add described culture medium in centrifuge tube, standby;

C, add described culture medium in the culture hole of petri dish or hole plate, standby;

D. Take the tissue and put it into the centrifuge tube, cut the tissue into pieces, blow and beat the tissue, then insert the centrifuge tube into the ice and let it stand, then gently aspirate and discard the supernatant, mix the cells by blow and beat and count , transferring a suspension of a certain number of cells to the culture dish or the culture well of the well plate;

E. Move the culture wells of the culture dish or the well plate into an incubator for culture, then slowly suck and discard the medium in the upper layer along the edge of the culture well of the culture dish or the well plate, and add an appropriate amount of corresponding culture solution , do not float the cells, and move them into the incubator to continue culturing;

F, change the medium regularly, slowly absorb and discard the old described medium stock solution when changing the medium, and add new described medium;

G. After the primary cells cover the bottom of the culture dish or the culture well of the well plate, passage or further screening is carried out.

Preferably, in step A, the culture medium includes the following components: DMEM high-glucose medium as the base medium, adding fetal bovine serum, mud crab serum, penicillin-streptomycin-amphotericin B mixed antibiotics, L-glutamic acid, non-essential amino acids, pyruvic acid, basic fibroblast growth factor, and β-mercaptoethanol; the preparation method of the mud crab serum is: drawing blood on the mud crab, and anticoagulant Mix the reagents, centrifuge, collect the supernatant, filter and sterilize to obtain the mud crab serum.

Adding a certain amount of mud crab serum can alleviate the incompatibility of the cells in vitro culture, and can provide nutrients that other animal serum does not have for the spermatogonial stem cells of the mud crab during the culture process, so that the spermatogonial stem cells of the mud crab can The growth state of primary stem cells remained stable in vitro.

Preferably, in step A, the configuration of the medium is: add 200mL fetal bovine serum, 20mL mud crab serum, 10mL penicillin-streptomycin-amphotericin B mixed antibiotics, 2mL L - glutamic acid, 4mL non-essential amino acids, 2mL sodium pyruvate, 200ng basic fibroblast growth factor and 10mL β-mercaptoethanol; Blood was drawn at the place, then mixed with an equal volume of the anticoagulant, centrifuged at 2000×g for 20min at 4°C,

The supernatant was collected and sterilized by filtration with a 0.22 μm filter membrane to obtain the mud crab serum.

Preferably, in step A, after the preparation of the culture medium is completed, store it at low temperature for later use; in step B, insert the centrifuge tube into ice for later use; in step D, use ophthalmic scissors to cut the tissue, and then use a pipette Blow and blow the tissue, then insert the centrifuge tube into ice and let it stand for 1 h, the number of cells in the suspension is 1×10 ⁸ cells/ml; in step E, culture the culture dish or the well plate Move the wells into an incubator and incubate at 26-29°C for 24 hours, discard the culture medium in the upper layer and add the culture solution to continue the culture; in step F, the regular frequency of changing the liquid is once every 1-2 days; in step G, The cells were continuously passaged at a ratio of 1:2 each time the monolayer was confluent.

Preferably, in step A, after the preparation of the culture medium is completed, store it at 4°C for later use; in step D, soak the ophthalmic scissors in 75% ethanol before use, put them in an ultra-clean bench, sterilize them with ultraviolet light for 30 minutes, and then take them out and put them in a super clean table. Dry in a clean bench; in step E, the culture temperature is 28°C.

An application of the above-mentioned in vitro culture method of Scylla pseudospermum stem cells for differentiating the Scylla pseudospermia stem cells into somatic cells of Scylla pseudospermum.

Preferably, the somatic cells include one or more of bone cells and fat cells.

Preferably, the induction condition for differentiating the spermatogonial stem cells of Scylla pseudocarpus into osteocytes is: adding 10 mmol/L sodium β-glycerophosphate, 50 mg/L vitamin C and 10 ^-5 mmol/L dexamethasone to the medium Mixed into an osteogenic induction medium, placed the cells in a 28°C incubator for induction culture, replaced the osteoinductive medium every 3 days, and continuously induced for 9 days; the induction conditions for differentiating the spermatogonial stem cells of Scylla pseudomiterus into adipocytes For: add 0.05mmol/L 3-isobutyl-1-methylxanthine, 0.2mmol/L indomethacin, 10mg/L insulin and 10-3mmol/L dexamethasone in the culture medium as the composition Lipid-inducing medium, cells were placed in a 28°C incubator to induce cells to produce lipid droplets. After induction and culture for 24 hours, the current state of cells and the size of lipid droplets were maintained for 48 hours with the medium added with 10 mg/L insulin, and then replaced. The above-mentioned adipogenic induction medium continued to induce the increase of lipid droplets, and this was repeated for 3 cycles.

An application of the above-mentioned in vitro culture method of Scylla pseudospermis stem cells, which is used for screening and cultivating cells with specific and stable expression.

Scylla pseudospermum stem cells can stably express specific genes, and can be used as an excellent carrier for in vitro cell biology research of Scylla pseudocarpus.

Preferably, the method of screening and cultivating the specific stably expressed cells is as follows: Infect 1× ¹⁰⁵ cells/ml of Scylla pseudospermoid stem cells with 100moi of lentivirus containing green fluorescent protein (GFP) plasmid, and pass through a fluorescence microscope after 48h Detect the efficiency of GFP-positive cells transfected spermatogonial stem cells in vitro. After 8 days, the cells that do not express green fluorescent protein (GFP) are basically killed, and the surviving cells all express protein stably, and can continue to be cultured for 25 days to obtain the specific stably expressing cells.

Compared with the prior art, implementing the present invention has the following beneficial effects:

The present invention establishes a method for culturing spermatogonial stem cells in vitro. Among them, the Scylla pseudocavei spermatogonial stem cells can be cultured for more than 395 days, and can be continuously cultured for 15-20 generations; the Scylla pseudocavena spermatogonial stem cells obtained by the method of the present invention have stemness and proliferative activity; Primitive stem cells can be induced to differentiate into somatic cells, such as bone cells and fat cells.

The testis cells of Scylla pseudocarpus cultured by the method of the present invention are observed and identified by cell morphology, and the morphology is observed under an optical microscope, and the specific markers of spermatogonia are detected by immunocytochemistry, and it is determined to be Scylla pseudocarpus. Crab spermatogonial stem cells. The cells express stem cell marker molecules and germ cell marker molecules; express Nanog proliferation activity and germ cell marker protein DAZL; cell cycle analysis shows diploid and tetraploid; they can still recover after cryopreservation and still have the ability to proliferate.

The resulting spermatogonial stem cells of Scylla pseudocarpus can stably express GFP after lentivirus infection, and can continue to be cultured for 25 days.

To sum up, the method of the present invention obtains a continuously passaged Scylla pseudomae spermatogonial stem cells, which have stem cell properties, can be differentiated into somatic cells, and can function well as tool cells.

Description of drawings

Figure 1 is a picture of the spermatogonial stem cells cultured in Scylla pseudocavena, in which: A is the spermatogonial stem cells cultured for 24 hours, B is the growth state of the 48 spermatogonial stem cells, C is the growth state of the spermatogonial stem cells after recovery, D is the HE staining of the testicular tissue, E HE staining of P4 spermatogonial stem cells, F is HE staining of spermatogonial stem cells after thawing.

Figure 2 shows the apoptosis and proliferation of spermatogonial stem cells of Scylla pseudocarpus in culture for 14 passages, in which: A is the staining of living and dead cells, B is the proportion of living and dead cells, and C is the detection of CCK8. The results showed that the proportion of living cells was higher than 99%, and the cells were in a state of continuous proliferation 24h and 48h after passage.

Fig. 3 is the result of chromosome ploidy analysis of the spermatogonial stem cells of Scylla pseudosyrpa. The results show that the cells have haploid, diploid and tetraploid karyotypes.

Figure 4 shows the results of indirect immunofluorescent staining of the spermatogonial stem cells of Scylla pseudosyrpa, in which: the spermatogonial stem cells of Scylla pseudocarpus expressed A as the germ cell marker protein DAZL, B as the germ cell marker protein piwi, and C as the stem cell marker molecule Nanog. The results showed that the spermatogonial stem cells of Scylla pseudosyrpa expressed germ cell marker proteins DAZL and piwi (A, B) and stem cell marker molecule Nanog (C).

Figure 5 Scylla pseudomaculata spermatogonial stem cells differentiated into different types of somatic cells in vitro, in which: A and B are differentiated and cultured osteoblasts, and C and D are differentiated and cultured adipocytes.

Fig. 6 is the Scylla macula spermatogonial stem cells stably expressing green fluorescent protein (GFP) after lentivirus infection.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings.

Experimental Materials

The preparation of the medium of Scylla pseudomae spermatogonial stem cells: 13.37g of DMEM, 4.5g of glucose and 0.11g/L of sodium pyruvate (pH 7.7) were added to every 1L of sterilized ddH ₂ O to obtain DMEM high-glucose medium. Add the following components in turn to each 1L of basal medium DMEM: 200mL fetal bovine serum (FBS), 20mL mud crab serum (mix the mud crab hemolymph with an equal volume of anticoagulant, centrifuge at 2000×g for 20min at 4°C, collect Supernatant, 0.22μm filter sterilization-20 ℃ storage), 10mL penicillin-streptomycin-amphotericin B mixed antibiotics (100×), 2mL L-glutamic acid (100×, 2mM), 4mL non-essential amino acids ( 100×, 1 mM), 2 mL sodium pyruvate (100×, 1 mM), 200 ng basic fibroblast growth factor (0.1 mg/mL) and 10 mL β-mercaptoethanol (50 mM). The prepared complete medium was sterilized by 0.22 μm filter and stored at 4°C. Adding a certain amount of mud crab serum can alleviate the incompatibility of the cells in vitro culture, and can provide nutrients that other animal serum does not have for the spermatogonial stem cells of the mud crab during the culture process, so that the spermatogonial stem cells of the mud crab can The growth state of primary stem cells remained stable in vitro.

During the stage of induction of Scylla macae spermatogonial stem cells and lentivirus infection, basic fibroblast growth factor and β-mercaptoethanol were not added to the medium.

All instruments used in the experiment were sterilized by high temperature and high pressure.

Example 1: Cultivation of spermatogonial stem cells of Scylla pseudocavena

1. Isolation of Testicular Cells from Scylla pseudocarpus

Take about 300g of blue crabs, soak the crab body in PBS containing 1% double antibody for 30min, spray with 75% alcohol and dissect. Carefully clip the testis with tweezers and place it in a 50ml centrifuge tube filled with medium, cut the tissue with ophthalmic scissors, then blow the tissue with a pipette gun, insert the centrifuge tube into ice and let it stand for 1 hour to wait for the cells to release in the medium. Discard the supernatant, mix the cells by pipetting and count. Add 1× ¹⁰⁸ cells/ml cell suspension into the culture medium in the culture well of the culture dish or well plate, blow and mix well, and culture at 28°C.

2. Cultivation and passage of testicular cells of Scylla pseudocarpus

After culturing for 24 hours, slowly discard the upper layer of medium along the edge of the culture dish or well plate, add an appropriate amount of medium to prevent the cells from floating, and move it into the incubator to continue culturing. Change the medium once every 2 days. Subculture after the primary cells cover the bottom of the culture dish or well plate. First, suck out the medium in the well, wash it with DPBS for 3 times, add 200 μL DPBS and put it in a refrigerator at 4 °C for 5 minutes, suck out the DPBS, add 2 mL of medium to resuspend, and suck out 1 mL into a new well according to the ratio of 1:2. In the well, continue to culture at 28°C. After the cells covered the monolayer, they were passaged again for subsequent identification and other experiments.

Microscopic observation shows that the testis cell suspension of Scylla pseudocavea contains a large amount of cells, and a small amount of cells adhere to the wall after 24 hours of culture. After changing the medium for 3 days, obvious cell proliferation can be observed, and the cells are round or irregular in shape. After about a week Larger cell colonies are formed, the cells in the middle accumulate and grow, and the bottom is full of cells and then subcultured. Add new medium to resuspend, and pipette the cells evenly into the culture wells of the new well plate. Each time the monolayer was covered with cells, the ratio of 1:2 was continuously passaged, as shown in Figure 1, A is the growth state of spermatogonial stem cells for 24 hours, B is the growth state of 48 spermatogonial stem cells, and C is the growth state of spermatogonial stem cells after recovery).

Example 2: Identification of spermatogonial stem cells of Scylla pseudocavena

1. Hematoxylin and eosin (HE) staining of the testicular cells of Scylla pseudocarpus

Adjust the cell density to 1×10 ⁵ /well, inoculate the cells into a 12-well plate, and culture at 28°C. When the cells grew to 60% confluence, they were washed twice with DPBS, fixed with 4% paraformaldehyde for 30 minutes, washed twice with DPBS, stained with hematoxylin staining solution for 10 minutes, washed twice with DPBS, and stained with eosin staining solution for 1 minute. Washed twice with 70% ethanol, observed under a microscope, the nucleus was blue, and the cytoplasm was pink or red.

Hematoxylin and eosin (HE) staining is shown in Figure 1E and F, the cells are pink, which is consistent with the results of hematoxylin and eosin (HE) staining in the testis tissue sections of Scylla pseudocarpus (Figure 1D), and there are spermatozoa of various stages The distribution of cells indicates that the cultured cells are spermatogonia of Scylla syringae.

2. Cryopreservation and recovery of spermatogonial stem cells from Scylla pseudocavena

Take the monolayer cells in a 6-well plate as an example. After washing 3 times with DPBS, add 200 μL DPBS and put it in a refrigerator at 4°C for 5 minutes, suck out the DPBS, and add 500 μL of general freezing medium to resuspend the cells. Collect into a 2mL cryopreservation tube, place the cryopreservation tube in a freezer box, carry out gradient cooling at -80°C, and transfer to liquid nitrogen after 24 hours. After 125 days of frozen storage, take out the cryopreservation tube and dissolve it quickly in a 37°C water bath, add 2ml of medium to resuspend the cells, transfer to a 10ml centrifuge tube, centrifuge at 800×g for 5min, discard the supernatant, add 1mL of medium, and pipette well Mix the cells, transfer them to a culture dish or a culture plate for culture, and culture and passage according to the above method. After the cells covered the monolayer, some cells were taken out and placed in a 12-well plate, and stained with hematoxylin and eosin (HE) according to the above method.

The spermatogonial stem cells of Scylla pseudocavena can still be revived after cryopreservation. As shown in Figure 1C, the 10th generation cells that have been cryopreserved for 125 days grow well and still have the corresponding cell shape.

3. Cell staining, cck8 identification of spermatogonial stem cell apoptosis and proliferation

1) Put the circular 24-well cell slide in 75% alcohol for 6 hours, take out the slide with tweezers and put it on the tinfoil. Put the spermatogonial stem cells on slides, wait until the cells grow to 70% confluence, discard the medium, carefully wash the cells twice with DPBS, add 4% paraformaldehyde to fix for 30min, wash the cells twice with DPBS before use. Add 1μL LiveDye and 1μL NucleiDye to 1mL Assay Buffer to prepare StainingSolution. Take the prepared cell slides into a new 24-well plate, with the cells facing up, add 0.5mL of Stainingsolution, incubate at 37°C in the dark for 30min, wash the cells twice with DPBS, and cover with the coverslip onto glass slides and analyze cells by fluorescence microscopy using appropriate filters as soon as possible. The results showed that as shown in Figures 2A and 2B, the proportion of living cells in the spermatogonial stem cells of Scylla pseudocarpus was higher than 99%, and the proportion of dead cells was low. It shows that the spermatogonial stem cells of Scylla pseudocarpus have cell activity.

2) Inoculate the suspension of spermatogonial stem cells (1×10 ⁴ cells/well) of Scylla pseudocarpus in a 96-well plate, and after culturing for 12h, 24h and 48h, add 10 μL of CCK-8 solution to each well of the 96-well plate , taking care not to introduce air bubbles into the holes. Gently pat to mix and incubate in the incubator for 4h, and measure the absorbance at 450nm with a microplate reader. The results showed that as shown in Figure 2C, the Scylla pseudomaculata spermatogonial stem cells had the largest proliferation at 24h and 48h, indicating that the Scylla pseudomaculata spermatogonial stem cells had a strong proliferation ability, and the cells were in a state of continuous proliferation at 24h and 48h after passage.

4. Karyotyping

Take the spermatogonial stem cells in good growth state and discard the medium, resuspend the cells with pre-cooled DPBS, transfer to a 10ml centrifuge tube, centrifuge at 500×g for 6min, and discard the supernatant. Resuspend with pre-cooled DPBS, centrifuge at 500×g for 6 min, and discard the supernatant. Now configure 70% ethanol to pre-cool at 4°C. Take 1 mL of 70% ethanol to mix by pipetting, and fix overnight at 4°C. Centrifuge at 500×g for 6 min, discard the supernatant. Add RNase A (200 μg/ml) and propidium iodide (20 μg/ml) to PBS, take 1 ml of resuspended cells and incubate at 37°C for 15 minutes for DNA staining, transfer to a flow tube, and place on ice for flow detection .

The karyotype analysis is shown in Figure 3, which shows that in addition to the diploid and tetraploid peaks of the spermatogonial stem cells of Scylla pseudocavena, there is a higher haploid peak, that is, there are haploid, diploid and tetraploid nuclei type.

5. Indirect Immunofluorescence

In order to identify the cultured Scylla pseudospermis testis cells as spermatogonia stem cells, the germ cell marker proteins DAZL, piwi and stem cell marker Nanog were detected by immunofluorescence.

Place 5 slices of cell slides prepared from Scylla pseudospermia stem cells into a new 24-well plate with the cell side up, wash once with DPBS, add 200 μL of 1% Triton-X 100 to each well for 10 min to permeate , washed twice with DPBS, added 200 μL of 5% goat serum to each well to block for 60 min, and washed once with DPBS. Add 200 μL DAZL rabbit anti-antibody (1:200) to one well, add 200 μL piwi mouse anti-antibody (1:200) to one well, add 200 μL Nanog rabbit anti-antibody (1:200) to the other well, and incubate overnight at 4°C. Serum and mouse serum served as negative controls. After equilibrating at room temperature for 20 minutes, wash 3 times with DPBST, add 200 μL FITC-labeled goat anti-rabbit secondary antibody (1:250) and 200 μL Cy3-labeled goat anti-mouse secondary antibody (1:250), incubate at room temperature for 2 hours in the dark, and wash 3 times with DPBS , add 200 μL DAPI (1:500) to each well, incubate in the dark for 10 minutes, wash once with DPBS, carefully remove the slide with tweezers, dry at room temperature for 5 minutes, add 6 μL of anti-fluorescence quenching mounting medium on the slide, and place the slide Put it upside down on a glass slide and observe under a fluorescent microscope.

The results of indirect immunofluorescence staining are shown in Figure 4, showing that the spermatogonial stem cells of Scylla pseudocarpus express the germ cell marker proteins DAZL and piwi (Figure 4A, Figure 4B), and have the characteristics of Nanog (Figure 4C) stem cells.

Example 3: Inducing the differentiation of spermatogonia stem cells into somatic cells

1. Differentiation of spermatogonia stem cells into osteoblasts

10mmol/L sodium β-glycerophosphate, 50mg/L vitamin C and 10-5mmol/L dexamethasone were added to the medium and mixed to form an osteogenic induction medium. Inoculate 5× ¹⁰⁵ spermatogonial stem cells of Scylla pseudocavena into a 24-well plate and culture at 28°C. When the cells are fused to 70%, remove the medium, add osteogenic induction medium, and place the cells at 28°C. Induced at ℃, the medium was replaced every 3 days, and the induction was continued for 9 days. Alizarin red staining was performed to observe the differentiation of Scylla pseudomaculata spermatogonial stem cells under a microscope.

The results are shown in Figure 5A, after being induced with osteogenic induction medium, the Scylla pseudomae spermatogonial stem cells could differentiate into osteoblasts.

2. Differentiation of spermatogonia stem cells into adipocytes

Add 0.05mmol/L 3-isobutyl-1-methylxanthine, 0.2mmol/L indomethacin, 10mg/L insulin and 10-3mmol/L dexamethasone to the medium as adipogenic induction medium . Inoculate 5× ¹⁰⁵ spermatogonial stem cells of Scylla pseudocavena into a 24-well plate and culture in a 28°C incubator. When the cells are fused to 70%, remove the medium and add adipogenic induction medium to induce the cells to produce lipids. After 24 hours of induction culture, use the spermatogonial stem cell medium added with 10 mg/L insulin to maintain the current state of the cells and the size of lipid droplets for 48 hours, and then change to the lipid induction medium to continue to induce the increase of lipid droplets, and so on for 3 times For each cycle, oil red O staining was performed to observe the differentiation of Scylla pseudosperm stem cells under a microscope.

The results are shown in Fig. 5B, after being induced with adipogenic induction medium, Scylla pseudomaculata spermatogonial stem cells could differentiate into adipocytes.

Example 4: Infection and expression of spermatogonial stem cells from Scylla pseudocavena

According to the virus titer, the added amount of the virus solution was calculated, and the virus solution and polybrene were added dropwise to the spermatogonial stem cells. Seal the cell culture plate with a parafilm, and centrifuge at 3000×g for 1 hour in a well-plate centrifuge to make the virus evenly attached to the cell surface. Remove the sealing film, wipe around the orifice plate with alcohol to avoid contamination, and then put it back into the incubator to continue culturing. After 24 hours of virus infection, the cells were replaced with fresh medium to continue culturing. After 48 hours, the infection of the cells was observed with a fluorescence microscope.

The expression of green fluorescent protein was observed under a fluorescence microscope to judge the transfection efficiency, and the results are shown in Figure 6. After about 8 days, the cells that do not express green fluorescent protein are basically killed, leaving the cells that stably express green fluorescent protein in the spermatogonial stem of Scylla pseudocarpus, and the cells that stably express can continue to be cultured for 25 days.

Comparative example 1: 2×L15 culture medium for culturing spermatogonial stem cells of mud crab

In the previous experiments, we tried to use conventional 2×L15 medium (purchased from Thermo Fisher Scientific (China) Co., Ltd., Gibco ^TM ) to cultivate Scylla cryptae spermatogonial stem cells. The experimental steps are as follows:

Take about 300g of blue crabs, soak the crab body in PBS containing 1% double antibody for 30min, spray with 75% alcohol and dissect. Carefully clip the testis with tweezers and place it in a 50ml centrifuge tube filled with 2×L15 medium, cut the tissue with ophthalmic scissors, blow the tissue with a pipette gun, insert the centrifuge tube into ice and let it stand for 1 hour to wait for the cells to release at 2×L15 in L15 medium. Discard the supernatant, mix the cells by pipetting and count. Add 1× ¹⁰⁸ cells/ml cell suspension into the culture wells of the 6-well plate, mix well by pipetting, and culture at 28°C. Observed under a microscope, after 24 hours, a large number of cells floated. After 48 hours, only a few cells adhered to the wall. After 72 hours, the field of view was full of cell fragments, which proved that the cells were completely apoptotic, and subsequent culture and passage experiments could not be carried out.

Comparative example 2: Schneider Drosophila culture medium for culturing spermatogonial stem cells of Scylla pseudophylla

The previous experiments tried to use the conventional Schneider Drosophila medium (purchased from Thermo Fisher Scientific (China) Co., Ltd., Gibco ^TM ) to cultivate Scylla pseudomaculata spermatogonial stem cells. The experimental steps are as follows:

Take about 300g of blue crabs, soak the crab body in PBS containing 1% double antibody for 30min, spray with 75% alcohol and dissect. Carefully clamp out the testis with tweezers and place it in a 50ml centrifuge tube containing Schneider Drosophila medium, cut the tissue with ophthalmic scissors, blow the tissue with a pipette gun, insert the centrifuge tube into ice and let it stand for 1 hour to wait for the cells to release in the Schneider fruit fly in the fly medium. Discard the supernatant, mix the cells by pipetting and count. Add 1× ¹⁰⁸ cells/ml cell suspension into the culture wells of the 6-well plate, mix well by pipetting, and culture at 28°C. Observed under a microscope, after 24 hours, the culture medium was very turbid, and the field of view was full of cell debris, which proved that the cells were completely apoptotic, and subsequent culture and passage experiments could not be carried out.

Comparative example 3: Grace insect culture medium for culturing spermatogonial stem cells of Scylla pseudocarpus

The previous experiment tried to use the conventional Grace insect medium (purchased from Thermo Fisher Scientific (China) Co., Ltd., Gibco ^TM ) to cultivate the spermatogonial stem cells of the mud crab. The experimental steps are as follows:

Take about 300g of blue crabs, soak the crab body in PBS containing 1% double antibody for 30min, spray with 75% alcohol and dissect. Use tweezers to carefully remove the testis and place it in a 50ml centrifuge tube filled with Grace insect medium, cut the tissue with ophthalmic scissors, blow the tissue with a pipette gun, insert the centrifuge tube into ice and let it stand for 1 hour to wait for the cells to be released in Grace insect culture Base. Discard the supernatant, mix the cells by pipetting and count. Add 1× ¹⁰⁸ cells/ml cell suspension into the culture wells of the 6-well plate, mix well by pipetting, and culture at 28°C. Observed under a microscope, after 24 hours, the culture medium was very turbid, and the field of view was full of cell debris, which proved that the cells were completely apoptotic, and subsequent culture and passage experiments could not be carried out.

Comparative example 4: RPMI-1640 medium for culturing spermatogonial stem cells of mud crab

In the previous experiment, we tried to use conventional RPMI-1640 medium (purchased from Thermo Fisher Scientific (China) Co., Ltd., Gibco ^TM ) to cultivate Scylla pseudomaculata spermatogonial stem cells. The experimental steps are as follows:

Take about 300g of blue crabs, soak the crab body in PBS containing 1% double antibody for 30min, spray with 75% alcohol and dissect. Carefully clip the testis with tweezers and place it in a 50ml centrifuge tube containing RPMI-1640 medium, cut the tissue with ophthalmic scissors, blow the tissue with a pipette gun, insert the centrifuge tube into ice and let it stand for 1 hour to wait for the cells to be released in RPMI-1640. 1640 medium. Discard the supernatant, mix the cells by pipetting and count. Add 1× ¹⁰⁸ cells/ml cell suspension into the culture wells of the 6-well plate, mix well by pipetting, and culture at 28°C. Observed under a microscope, 24 hours later, the cell culture medium was relatively turbid, and a small amount of cells adhered to the wall. After 48 hours, the field of view was full of cell fragments, which proved that the cells were completely apoptotic, and subsequent culture and passage experiments could not be carried out.

Effect Example 1

From the comparison of Example 1 and Comparative Examples 1 to 4, it can be seen that it is difficult for conventional stem cell culture methods to make crustacean stem cells survive in vitro for more than 3 days, but the present invention overcomes the current difficult problem of crustacean in vitro cell culture and successfully For example, crustaceans can be continuously cultured and conveyed in vitro. Among them, the spermatogonial stem cells of the mud crab can be cultured for more than 395 days, and can be continuously cultured for 15-20 generations, which greatly guarantees the spermatogenesis of the mud crab. The proliferation ability and survival rate of stem cells in vitro make the establishment of primary cell culture system a high success rate, lay a research foundation for further construction of cell lines, and provide a reference for the development of crustacean in vitro culture technology.

The above disclosures are only preferred embodiments of the present invention, and should not be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention still fall within the scope of the present invention.

Claims (10)

1. a method for culturing in vitro of the spermatogonial stem cells of the pseudo-cave crab, is characterized in that, comprises the steps: A, prepare culture medium, save for subsequent use; B, add described culture medium in centrifuge tube, standby; C, add described culture medium in the culture hole of petri dish or hole plate, standby; D. Take the tissue and put it into the centrifuge tube, cut the tissue into pieces, blow and beat the tissue, then insert the centrifuge tube into the ice and let it stand, then gently aspirate and discard the supernatant, mix the cells by blow and beat and count , transferring a suspension of a certain number of cells to the culture dish or the culture well of the well plate; E. Move the culture well of the culture dish or the well plate into an incubator for cultivation, then slowly suck and discard the old upper layer of the culture medium along the edge of the culture well of the culture dish or the well plate, and add an appropriate amount of the corresponding For the new medium, do not make the cells float, and move it into the incubator to continue culturing; F, change the medium regularly, slowly absorb and discard the old described medium stock solution when changing the medium, and add new described medium; G. After the primary cells cover the bottom of the culture dish or the culture well of the well plate, passage or further screening is carried out. 2. the method for culturing in vitro of Scylla pseudocaveus spermatogonia stem cells as claimed in claim 1, characterized in that, in step A, the medium comprises the following components: DMEM high-glucose medium is the base medium, adding fetal bovine Serum, blue crab serum, penicillin-streptomycin-amphotericin B mixed antibiotics, L-glutamic acid, non-essential amino acids, pyruvate, basic fibroblast growth factor and β-mercaptoethanol; The preparation method of the mud crab serum is as follows: blood is drawn from the body of the mud crab, mixed with an anticoagulant, centrifuged, the supernatant is collected, and the mud crab serum is obtained after filtering and sterilizing. 3. The in vitro culture method of Scylla pseudocaveus spermatogonial stem cells as claimed in claim 2, characterized in that, in step A, the configuration of the culture medium is: add 200mL fetal bovine serum, 20mL quasi- Blue crab serum, 10mL penicillin-streptomycin-amphotericin B mixed antibiotics, 2mL L-glutamic acid, 4mL non-essential amino acids, 2mL sodium pyruvate, 200ng basic fibroblast growth factor and 10mL β-mercaptoethanol; The preparation method of the mud crab serum is as follows: blood is drawn from the joint membrane of the adult mud crab, then mixed with an equal volume of the anticoagulant, centrifuged at 2000×g for 20 min at 4°C, and the supernatant is collected , using a 0.22 μm filter membrane to filter and sterilize to obtain the serum of the mud crab. 4. The in vitro culture method of Scylla pseudocaveus spermatogonial stem cells as claimed in claim 1, characterized in that, in step A, after the preparation of the culture medium is completed, it is stored at low temperature for subsequent use; in step B, the centrifuge tube is inserted into the ice In step C, the orifice plate is a 6-well plate; in step D, use ophthalmic scissors to cut the tissue, then use a pipette gun to blow the tissue, and then insert the centrifuge tube into ice and let it stand 1h, the number of cells in the suspension is 1× ¹⁰⁸ cells/ml; in step E, move the culture dish or the culture well of the well plate into an incubator and incubate at 26-29°C for 24h, aspirate and discard the old Add the culture medium in the upper layer and add new culture medium to continue the culture; in step F, the frequency of regular liquid change is 1 to 2 days; in step G, the ratio of 1:2 for each cell confluence Serial passage. 5. The in vitro culture method of Scylla pseudocavena spermatogonial stem cells according to claim 4, characterized in that in step A, after the preparation of the medium is completed, it is stored at 4°C for later use; in step D, the ophthalmic scissors are used Before soaking to 75% ethanol, put it in the ultra-clean bench, sterilize it by ultraviolet light for 30 minutes, then take it out and put it in the ultra-clean bench to dry; in step E, the culture temperature is 28°C. 6. The application of the in vitro culture method of Scylla pseudomaculata spermatogonial stem cells as claimed in claim 1, characterized in that it is used to differentiate the Scylla pseudomaculata spermatogonial stem cells into somatic cells of Scylla pseudomaculata. 7. The application according to claim 6, wherein the somatic cells include one or more of bone cells and fat cells. 8. The application according to claim 6, characterized in that, the induction condition for the differentiation of Scylla pseudomaculata spermatogonial stem cells into osteocytes is: adding 10mmol/L sodium β-glycerophosphate, 50mg/L Vitamin C and ^10-5 mmol/L dexamethasone were mixed into an osteogenic induction medium, and the cells were placed in a 28°C incubator for induction culture, and the osteoinduction medium was replaced every 3 days for 9 consecutive days of induction; The induction condition for the differentiation of Scylla spermatogonial stem cells into adipocytes is: add 0.05mmol/L 3-isobutyl-1-methylxanthine, 0.2mmol/L indomethacin, 10mg/L L insulin and 10-3mmol/L dexamethasone were used as the adipogenic induction medium, and the cells were placed in a 28°C incubator to induce the cells to produce lipid droplets. After induction and culture for 24 hours, they were maintained with the medium added with 10 mg/L insulin The current state of the cells and the size of the lipid droplet were changed for 48 hours, and then the adipogenic induction medium was changed to continue to induce the increase of the lipid droplet, and this was repeated for 3 cycles. 9. The application of an in vitro culture method of Scylla pseudospermia spermatogonia stem cells as claimed in claim 1, characterized in that it is used for screening and cultivating cells with specific stable expression. 10. The application according to claim 9, characterized in that the screening and culturing method of the cells with specific and stable expression is: infecting 1×10 ⁵ /ml blue crabs with 100 moi of lentiviruses containing green fluorescent protein particles For spermatogonial stem cells, the efficiency of transfecting spermatogonial stem cells with GFP-positive cells in vitro was detected by fluorescence microscope after 48 hours. After 8 days, the cells that did not express green fluorescent protein were basically killed, and the surviving cells continued to be cultured to obtain the specific stable expression. cell.