Primary culture medium for gills of crassostrea gigas and application of primary culture medium

Abstract

The invention belongs to the technical field of biology, particularly relates to a marine mollusk cell culture technology, and particularly relates to a primary culture medium for gills of crassostrea gigas and application of the primary culture medium. The culture medium comprises a basic culture medium and an additive, and the volume ratio of the basic culture medium to the additive is (65.895-70.895): (29.105-34.105); the additive is a cell growth component and a cell proliferation promoting component. The culture medium can be used for in-vitro culture of primary cells of gills of crassostrea gigas, and survival and growth of the gills of the crassostrea gigas cultured in vitro can be better maintained; crassostrea gigas serum with a specific proportion is added into the culture medium, and when the culture medium with the proportion is used for culturing primary cells of gills of the crassostrea gigas, bacteria can be effectively inhibited; the invention also provides an application method of the culture medium, the method is simple to operate, and the cultured gills of crassostrea gigas are high in cell density, good in form and strong in antibacterial ability.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly focuses on marine mollusk cell culture technology, in particular to a primary culture medium for long oyster gill cells and an application thereof. Background Art

[0002] The giant oyster (Crassostrea gigas) belongs to the phylum Mollusca, class Bivalvia, family Ostreidae, genus Crassostreae in the animal kingdom. As a species with significant economic value, the giant oyster occupies an important position in China's marine aquaculture industry. With the completion of the whole genome sequencing of the giant oyster, marine shellfish such as the giant oyster have made significant progress in the verification of functional genes. However, the lack of giant oyster cell lines has limited the in-depth study and exploration of the gene functions and regulatory mechanisms of the giant oyster, as well as the innovation of shellfish aquaculture technology, which has become a major bottleneck in research and application development.

[0003] In the cell culture process of marine mollusks, bacterial contamination is one of the most common and difficult problems to solve. Therefore, it is crucial to use antibacterial substances to reduce the risk of bacterial contamination. Although certain progress has been made in the primary cell culture technology of long oysters, there is still room for improvement in the antibacterial treatment of tissues (mantle, gills, digestive glands and gonads) and cell separation and preparation technology. In particular, the gills, as the main line of defense of long oysters against pathogenic microorganisms, are more difficult to inhibit bacteria in the cell culture process due to the large variety and number of microorganisms in the gill tissues. At present, research on cell culture and functional verification using long oyster gill cells is still relatively limited. Given the abundance of long oyster resources, it has great potential and economic value in the application of cell culture technology. Summary of the invention

[0004] In view of the prior art, the present invention provides a primary culture medium for gill cells of an oyster and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A primary culture medium for gill cells of an oyster comprises a basal culture medium and an additive, wherein the volume ratio of the basal culture medium to the additive is 65.895-70.895:29.105-34.105; and the additive comprises a cell growth component and a cell proliferation promoting component.

[0007] The volume of the cell growth component accounts for 28% to 33% of the volume of the primary culture medium of the long oyster gill cells, and the components thereof are long oyster serum, FBS, antibiotics, mixed salts (CaCl2, NaCl, KCl, MgCl2), and sulfate (MgSO4).

[0008] The volume of the cell proliferation promoting components accounts for 1.105% of the volume of the primary culture medium of the long oyster gill cells, and the components include Poly-D-lysine, HEPES buffer, EGF and beta-mercaptoethanol.

[0009] The volume ratio of the long oyster serum to FBS is 0.5-1.

[0010] The long oyster serum is obtained by centrifuging the long oyster hemolymph to obtain the supernatant, filtering and sterilizing with a 0.22 μm filter membrane; the centrifugation conditions are: centrifugation temperature 4° C., centrifugation speed 800×g, and centrifugation for 15 minutes.

[0011] The antibiotics include the following components: 10 kU / mL penicillin, 10 mg / mL streptomycin, and 5 mg / mL gentamicin.

[0012] The mixed salt includes the following ingredients: 0.6 g CaCl2, 20.2 g NaCl, 0.54 g KCl, 3.9 g MgCl2, and pure water is used to make the volume to 100 mL.

[0013] The sulfate is MgSO4, and the preparation concentration is 50g / L.

[0014] The basic culture medium is L-15 culture medium.

[0015] An application of the primary culture medium for long oyster gill cells, and application of the culture medium in primary culture of long oyster gill cells.

[0016] A method for primary culture of oyster gill cells:

[0017] (1) Sterilize the gill tissue of the oyster with a disinfectant and then cut it into pieces;

[0018] (2) preparing a suspension of gill cells of oysters by using a tissue lysate from the shredded tissue in step (1);

[0019] (3) diluting the oyster gill cell suspension obtained in step (2) using the primary culture medium described in claim 1, and then adding the dilution to a culture bottle for culture.

[0020] The tissue lysis solution in step (2) is a mixture of trypsin, papain, collagenase and EDTA; the final concentrations of the components of the lysis solution in the long oyster gill cell suspension are: 2.5 mg / mL trypsin, 5 mg / mL papain, 0.5 mg / mL collagenase, 0.2 mg / mL EDTA; wherein the volume ratio between the chopped tissue pieces and the tissue lysis solution is 1:1.

[0021] The enzymatic hydrolysis time of the tissue lysate is 15 min, and the enzymatic hydrolysis temperature is 37°C.

[0022] The culture conditions in step (3) are: 18° C., static culture.

[0023] The culture flask is treated with a Poly-D-lysine solution. After 0.1 mg / mL Poly-D-lysine is spread overnight, excess Poly-D-lysine solution is sucked away, and the flask is dried in a super-clean room for 1 hour for standby use.

[0024] The disinfectants in step (1) are 75% alcohol disinfectant, chlorhexidine disinfectant and antibiotic disinfectant. The action time of each disinfectant is: 75% alcohol disinfectant soaks for 10-15 seconds, chlorhexidine disinfectant soaks for 10 minutes, and antibiotic disinfectant soaks for 15 minutes. After use, the disinfectant needs to be rinsed with PBS.

[0025] The antibiotic disinfectant comprises the following components: 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL gentamicin, 10 μg / mL kanamycin, 2 μg / mL amphotericin B and 100 μg / mL chloramphenicol, and the solvent is pure water.

[0026] The primary culture medium of the invention adds nutrients for cell growth and substances for promoting cell proliferation to the basic culture medium, and the two substances together ensure the survival and growth of the oyster gill cells cultured in vitro.

[0027] Among them, the long oyster serum in the cell growth component provides the sample with a growth environment for the long oyster gill cells, and also provides essential nutrients such as amino acids, trace elements, ions, hormones, etc. It also has antibacterial effects, and cooperates with antibiotics to prevent bacteria from contaminating the culture medium. At the same time, other ingredients simulate seawater salinity and restore the seawater salinity in the long oyster semi-open system.

[0028] Poly-D-lysine in the cell proliferation promoting substance is an auxiliary adhesion factor, which is coated in the cell culture bottle, promoting cell growth and promoting cell adhesion and reproduction; the buffer in the ingredients maintains the pH of the culture medium, and EGF activates the tyrosine kinase in the cytoplasmic domain of the receptor, thereby initiating signal transduction of DNA synthesis and cell proliferation; β-mercaptoethanol neutralizes the oxygen free radicals accumulated in the cell culture medium to improve the environment around the cells.

[0029] The invention cultured the long oyster gill cells in a culture medium. The method is simple to operate, and the cultured long oyster gill cells have high cell density and good morphology.

[0030] Advantages of the present invention include:

[0031] The culture medium of the present invention can be used for in vitro culture of primary oyster gill cells, and can better maintain the survival and growth of in vitro cultured oyster gill cells; a specific proportion of oyster serum is added to the culture medium, and the culture medium of the proportion can effectively inhibit bacteria when culturing primary oyster gill cells; the application also provides an application method of the culture medium, which is simple to operate, and the cultured oyster gill cells have a large cell density, good morphology, and strong antibacterial ability; specifically:

[0032] 1. The oyster gill cell culture medium of the present application has a stronger antibacterial effect by adding a specific proportion of oyster serum, and can better maintain the growth and metabolism of primary cultured oyster gill cells in vitro;

[0033] 2. The application of the oyster gill cell culture medium in the present application is used to cultivate oyster gill cells. The method is simple to operate, and the cultured oyster gill primary cells have strong antibacterial ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The antibacterial effect of the long oyster serum provided by the embodiment of the present invention, Figure 1 a depicts the growth of E. coli on LB solid culture plates, and Figure 1 b shows the growth of Vibrio splendens on 2216E solid culture plates. In these two sub-figures, numbers 1 to 4 represent four different treatment combinations: (1) complete medium with long oyster serum, (2) complete medium without long oyster serum, (3) long oyster serum alone, and (4) phosphate buffered saline (PBS). The application area of ​​each treatment is marked with a blue circle in the figure to facilitate observation and comparison of the differences in the effects of different treatment combinations on bacterial growth.

[0035] Figure 2 is the effect of the medium containing oyster serum of the present invention on cell viability. Cg represents the medium containing oyster serum, Cg — Denotes medium without long oyster serum.

[0036] Figure 3It is the effect of different proportions of oyster serum against Vibrio splendens provided in the embodiment of the present invention. The complete culture medium components of the Blank group include 10% FBS, 100U / mL penicillin, 100μg / mL streptomycin, 50μg / mL gentamicin, and L-15 culture medium; 0% represents a complete culture medium for gill cells without oyster serum, and compared with the Blank group, the following components are added to the culture medium: 0.6g / L CaCl2, 20.2g / L NaCl, 0.54g / L KCl, 3.9g / L MgCl2, 50g / L MgSO4, 10mM HEPES buffer, 10ng / mL EGF, and 0.05mM β-mercaptoethanol; 5%, 10% and 25% represent the volume ratios containing oyster serum, and the remaining components are the same as the complete culture medium for gill cells without oyster serum.

[0037] Figure 4 The image analysis software Image J was used to measure Figure 3 The radius of the inhibition zone on each plate. DETAILED DESCRIPTION

[0038] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0039] The oyster gill cell culture medium of the invention comprises a basic culture medium, a cell growth component and a cell proliferation promoting component. The culture medium can be used for in vitro culture of oyster gill cells and can better maintain the survival and growth of in vitro cultured oyster gill cells.

[0040] Example 1: Antibacterial ability of long oyster serum

[0041] 1. Experimental materials: Escherichia coli (purchased commercially and preserved in the laboratory strain bank for resuscitation), Vibrio splendens (purchased commercially and preserved in the laboratory strain bank for resuscitation), 2216E solid culture medium provided by Qingdao Haibo Biological Co., Ltd., LB solid culture medium from Shenggong Co., Ltd., long oyster serum, and PBS.

[0042] Complete medium for gill cells of oysters containing serum and complete medium for gill cells of oysters without serum (the ingredients lack oyster serum, and the other ingredients are the same as the complete medium for gill cells of oysters containing serum); wherein, the complete medium for gill cells of oysters containing oyster serum is 10mL of FBS, 10mL of oyster serum, 1mL of antibiotics, 10mL of mixed salts, 2mL of sulfate, 1mL of HEPES buffer, 5μL of 1M β-mercaptoethanol and 0.1mL of 10μg / mL EGF, and the volume of L-15 basic medium is fixed to 100mL.

[0043] The antibiotics include 10 kU / mL penicillin, 10 mg / mL streptomycin, and 5 mg / mL gentamicin.

[0044] The mixed salt includes the following ingredients: 0.6 g CaCl2, 20.2 g NaCl, 0.54 g KCl, 3.9 g MgCl2, and pure water is used to make the volume to 100 mL.

[0045] The sulfate is MgSO4, and the preparation concentration is 50g / L.

[0046] 2. Experimental method: Shake the bacteria to resuscitate Escherichia coli and Vibrio splendens, obtain the culture fluid of Escherichia coli and Vibrio splendens, and evenly apply it to LB solid medium and 2216E solid medium respectively. Each plate is divided into four equal parts, and the four substances of serum-containing long oyster gill cell complete medium, serum-free long oyster gill cell complete medium, long oyster serum, and PBS are applied respectively, and the growth of colonies in each application area and the surrounding areas is observed. The amount of material applied in each area is controlled at 10μL. The culture temperature of LB plate is 37℃, and the culture temperature of 2216E plate is 16℃.

[0047] 3. Experimental results: Observe the changes in the number of colonies on the plate after 24 hours of culture. The results are shown in Figure 1 , the number of colonies in each area in the statistical chart, the data are as follows:

[0048]

[0049] Complete medium (Cg + ) had a significant antibacterial effect. In contrast, the complete medium (Cg - ) and only applying the long oyster serum had a weaker antibacterial effect. It is speculated that the long oyster serum cooperates with certain substances in the culture medium to obtain a more obvious antibacterial effect; the long oyster serum group was compared with the PBS group and found that the long oyster serum had a certain antibacterial effect. This shows that the long oyster serum provided by the present application can be added to the culture medium to more effectively prevent bacterial infection.

[0050] Example 2: Inhibition of different proportions of serum against Vibrio splendidus

[0051] 1. Experimental materials: 2216E solid culture medium provided by Qingdao Haibo Biological Company, Vibrio splendens (purchased commercially and preserved in the laboratory strain bank for strain recovery), and complete culture medium containing different long oyster sera;

[0052] The complete medium containing different long oyster serum is L-15 medium and additives, and the additives are cell growth components and cell proliferation promoting components;

[0053] The volume of the cell growth component accounts for 28% to 33% of the volume of the primary culture medium of the long oyster gill cells, and the components thereof are long oyster serum, FBS, antibiotics, mixed salts (CaCl2, NaCl, KCl, MgCl2), and sulfate (MgSO4).

[0054] The volume of the cell proliferation promoting components accounts for 1.105% of the volume of the primary culture medium of the long oyster gill cells, and the components include Poly-D-lysine, HEPES buffer, EGF and beta-mercaptoethanol.

[0055] The antibiotic components include: 10 kU / mL penicillin, 10 mg / mL streptomycin, and 5 mg / mL gentamicin.

[0056] The mixed salt includes the following ingredients: 0.6 g CaCl2, 20.2 g NaCl, 0.54 g KCl, 3.9 g MgCl2, and pure water is used to make the volume to 100 mL.

[0057] The sulfate is MgSO4, and the preparation concentration is 50g / L.

[0058] The final concentrations of the components in the culture medium are 100U / mL penicillin, 100μg / mL streptomycin, 50μg / mL gentamicin, 0.6g / L CaCl2, 20.2g / L NaCl, 0.54g / L KCl, 3.9g / LMgCl2, 50g / L MgSO4, the concentration of HEPES buffer is 10mM, the concentration of EGF is 10ng / mL, and the final concentration of β-mercaptoethanol is 0.05mM.

[0059] The volume of the final culture medium contained oyster serum at 0%, 5%, 10% and 25% respectively, and the volume of the final culture medium contained FBS at 10%.

[0060] 2. Experimental method: Prepare the plate used for the inhibition zone experiment, prepare 2216E solid culture medium, pour a thin layer of 2216E culture medium when pouring the plate, air dry in the ultra-clean room, put the Oxford cup in the center of the plate, pour 2216E solid culture medium for the second time, air dry and solidify in the ultra-clean room. Prepare complete culture medium containing different long oyster serums. The specific concentrations of each component are shown in the experimental materials. The Blank group only contains FBS, 100U / mL penicillin, 100μg / mL streptomycin, and 50μg / mL gentamicin, which account for 10% of the complete culture medium. Shake the bacteria at 16℃ for 24h to revive Vibrio splendens, and take 100μL of the bacterial solution and evenly spread it on the 2216E solid culture medium. After the coating is completed, add the complete culture medium containing different proportions of long oyster serum into the Oxford cup, add 200μL of complete culture medium to each plate, and then put the 2216E plate into a 16℃ constant temperature incubator for culture, and observe the changes in the inhibition zone every day.

[0061] 3. Experimental results: Comparing the inhibition degree of Vibrio splendens with different serum proportions, the experimental results show that when the serum proportion is 5%, the complete medium produces the largest inhibition zone and has the best antibacterial effect on Vibrio splendens ( Figure 3 ). The image analysis software Image J was used to measure the radius of the inhibition zone on each plate. The results are as follows Figure 4 As shown, the radius of the inhibition zone produced in the complete medium containing 5% serum was the largest.

[0062] Example 3: Effect of oyster serum on the activity of oyster gill cells

[0063] 1. Experimental materials: The primary culture medium of the gill cells of the oyster is the basic culture medium and additives, wherein the additives are cell growth components and components promoting cell proliferation.

[0064] Taking 100 mL of primary culture medium for long oyster gill cells as an example, the required volumes of each component in the additive are as follows: 10 mL of FBS, 5 mL of long oyster serum, 1 mL of antibiotics, 10 mL of mixed salts, 2 mL of sulfate, 1 mL of HEPES buffer, 5 μL of 1 M β-mercaptoethanol and 0.1 mL of 10 μg / mL EGF.

[0065] The antibiotic components include: 10 kU / mL penicillin, 10 mg / mL streptomycin, and 5 mg / mL gentamicin.

[0066] The mixed salt includes the following ingredients: 0.6 g CaCl2, 20.2 g NaCl, 0.54 g KCl, 3.9 g MgCl2, and pure water is used to make the volume to 100 mL.

[0067] Poly-D-lysine solution is used to coat cell culture flasks.

[0068] The description of each reagent is as follows:

[0069]

[0070] 2. Experimental methods:

[0071] (1) Treat the cell culture flask with Poly-D-lysine solution. After overnight spreading with 0.1 mg / mL Poly-D-lysine, remove the excess Poly-D-lysine solution and dry in a clean room for 1 hour.

[0072] (2) Wash the gill tissue of the long oyster with PBS to remove the mucus on the surface of the tissue, then sterilize and disinfect it with 75% alcohol, chlorhexidine disinfectant, and antibiotic disinfectant, and use an ophthalmic scalpel to repeatedly shear the tissue to make tissue paste;

[0073] (3) preparing a suspension of gill cells of oysters by using a tissue lysate from the shredded tissue in step (2);

[0074] (4) Dilute the oyster gill cell tissue suspension prepared in step (3) using the prepared primary culture medium, and assemble it into the cell culture bottle that was completely dried in step (1), and culture it at 18° C. Then, the CCK-8 cell viability was detected every day by an ELISA instrument.

[0075] At the same time, the primary culture medium without adding oyster serum was used as the control group. 1 is the primary culture medium; 2 is the primary culture medium without adding oyster serum; 3 is oyster serum; 4 is PBS as the blank control group.

[0076] The tissue lysis solution in step (3) is a mixture of trypsin, papain, collagenase and EDTA; the final concentrations of the components of the lysis solution in the long oyster gill cell suspension are: 2.5 mg / mL trypsin, 5 mg / mL papain, 0.5 mg / mL collagenase, 0.2 mg / mL EDTA; wherein the volume ratio between the tissue paste and the tissue lysis solution is 1:1.

[0077] The enzymatic hydrolysis time of the tissue lysate is 15 min, and the enzymatic hydrolysis temperature is 37°C.

[0078] The disinfectants in step (1) are 75% alcohol disinfectant, chlorhexidine disinfectant and antibiotic disinfectant. The action time of each disinfectant is: 75% alcohol disinfectant soaks for 10-15 seconds, chlorhexidine disinfectant soaks for 10 minutes, and antibiotic disinfectant soaks for 15 minutes. After use, the disinfectant needs to be rinsed with PBS.

[0079] The antibiotic disinfectant comprises the following components: 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / mL gentamicin, 10 μg / mL kanamycin, 2 μg / mL amphotericin B and 100 μg / mL chloramphenicol, and the solvent is pure water.

[0080] 3. Experimental results: In the first 3 days of the experiment (0-3 days), the viability of the cells showed a gradual downward trend. Especially on the 3rd day, the cells in the culture medium containing long oyster serum showed higher viability compared with the gill cell culture medium without serum. However, on the 9th day, bacterial contamination was observed in the culture medium, and the increase in the number of bacteria at this time affected the determination of cell viability. At this stage, the measured cell viability actually reflects the degree of bacterial contamination. Through CCK-8 viability analysis of cell viability in two different culture media (with and without serum), it was found that the degree of bacterial contamination in the group with the addition of long oyster serum was relatively light. Therefore, it can be concluded that long oyster serum can significantly reduce the degree of bacterial contamination, and this result is in Figure 2 It is shown in .

[0081] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A primary culture medium for long oyster gill cells, It is characterized in that The culture medium comprises a basic culture medium and additives, wherein the volume ratio of the basic culture medium to the additives is 65.895-70.895:29.105-34.105; the additives comprise cell growth components and components promoting cell proliferation.

2. The primary culture medium of long oyster gill cells according to claim 1, It is characterized in that The volume of the cell growth component accounts for 28% to 33% of the volume of the primary culture medium of the long oyster gill cells, and its components are long oyster serum, FBS, antibiotics, mixed salt (CaCl 2 、NaCl、KCl、MgCl 2 ), sulfate (MgSO 4 ); The volume of the cell proliferation promoting components accounts for 1.105% of the volume of the primary culture medium of the long oyster gill cells, and the components include Poly-D-lysine, HEPES buffer, EGF and beta-mercaptoethanol.

3. The primary culture medium of long oyster gill cells according to claim 2, It is characterized in that The volume ratio of the long oyster serum to FBS is 0.5-1.

4. The primary culture medium of long oyster gill cells according to claim 3, It is characterized in that The long oyster serum is obtained by centrifuging the long oyster hemolymph to obtain the supernatant, filtering and sterilizing with a 0.22 μm filter membrane; the centrifugation conditions are: centrifugation temperature 4° C., centrifugation speed 800×g, and centrifugation for 15 minutes.

5. The primary culture medium of long oyster gill cells according to claim 1, It is characterized in that The basic culture medium is L-15 culture medium.

6. Use of the primary culture medium of long oyster gill cells according to claim 1, Features: The culture medium is used in primary culture of long oyster gill cells.

7. A method for primary culture of oyster gill cells, Features: (1) Sterilize the gill tissue of the oyster with a disinfectant and then cut it into pieces; (2) preparing a suspension of gill cells of oysters by using a tissue lysate to obtain the shredded tissue blocks in step (1); (3) Using the primary culture medium described in claim 1, dilute the oyster gill cell suspension obtained in step (2) to 1-4×10 7 / mL, and then added to the culture bottle for culture.

8. The method according to claim 7, Features: The tissue lysis solution in step (2) is a mixture of trypsin, papain, collagenase and EDTA; the final concentrations of the components of the lysis solution in the long oyster gill cell suspension are: 2.5 mg / mL trypsin, 5 mg / mL papain, 0.5 mg / mL collagenase, 0.2 mg / mL EDTA; wherein the volume ratio between the chopped tissue pieces and the tissue lysis solution is 1:

1.

9. The method according to claim 7, Features: The culture conditions in step (3) are: 18° C., static culture.

10. The method according to claim 7, Features: The culture flask is treated with a Poly-D-lysine solution. After 0.1 mg / mL Poly-D-lysine is spread overnight, excess Poly-D-lysine solution is sucked away, and the flask is dried in a super-clean room for 1 hour for standby use.

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