Marine invertebrate cell culture medium

Abstract

The invention relates to the technical field of cell culture media, in particular to a marine invertebrate cell culture medium. Comprising the following components in parts by weight: 0.5 to 1 part of a compound supplement, 1 to 4 parts of glucose, 0.3 to 0.5 part of L-glutamine, 0.01 to 0.05 part of vitamin, 10 to 15 parts of fetal calf serum, 0.02 to 0.04 part of penicillin, 0.01 to 0.03 part of trehalose, 0.3 to 0.7 part of a sodium chloride osmotic pressure regulator, 0.5 to 1 part of inorganic salt and 10 to 25 parts of a sodium bicarbonate buffer agent, wherein the compound supplement is prepared by mixing meson, diatom and disodium cyclohexane diamine tetraacetate according to the mass ratio of 1: (0.2-0.6): (0.1-0.5), the application range of the culture medium is widened by adopting the compound supplement, the meson and the diatom are added into the cell culture medium after enzymolysis, and compared with a chemically synthesized culture medium, the culture medium better meets the requirements of marine invertebrate cells; therefore, cell growth is effectively promoted, and the purposes of rapid growth and stable passage are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture medium, in particular to a marine invertebrate cell culture medium. Background Art

[0002] Marine invertebrates, as one of the oldest and most biodiverse biological groups on Earth, occupy an important position in the marine ecosystem. They are of various species, ranging from tiny plankton to huge starfish, corals and giant squids, constituting an indispensable part of the marine ecosystem. The cells of marine invertebrates, as the basic units of life activities, undertake key tasks such as maintaining the life activities of organisms, transmitting genetic information, material metabolism and energy conversion. These cells have gradually adapted to the particularity of the marine environment during the long-term evolution process, such as extreme conditions such as high salinity, low temperature, high pressure and insufficient light. However, traditional marine invertebrate cell culture media have a narrow application range and cannot achieve rapid growth and stable propagation. In view of this, a marine invertebrate cell culture medium is provided. Summary of the invention

[0003] The object of the present invention is to provide a marine invertebrate cell culture medium to solve the problem that the marine invertebrate cell culture medium mentioned in the above background technology has a narrow application range and cannot achieve rapid growth and stable passage.

[0004] To achieve the above object, the present invention provides a marine invertebrate cell culture medium, comprising the following components in parts by weight: 0.5-1 parts by weight of a composite supplement, 1-4 parts by weight of glucose, 0.3-0.5 parts by weight of L-glutamine, 0.01-0.05 parts by weight of vitamins, 10-15 parts by weight of fetal bovine serum, 0.02-0.04 parts by weight of penicillin, 0.01-0.03 parts by weight of trehalose, 0.3-0.7 parts by weight of a sodium chloride osmotic pressure regulator, 0.5-1 parts by weight of an inorganic salt and 10-25 parts by weight of a sodium bicarbonate buffer; wherein the composite supplement is prepared by mixing ostracods, diatoms and disodium cyclohexanediaminetetraacetic acid in a mass ratio of 1:0.2-0.6:0.1-0.5.

[0005] As a further improvement of the present technical solution, the specific preparation method of the composite supplement is as follows: The ostracods and diatoms are cleaned and dried, crushed using a grinder with a screen, and mixed thoroughly, then the complex enzyme is added to a sodium bicarbonate buffer to prepare an enzyme solution, the mixed ostracod and diatom powders are added to the enzyme solution, and the enzymatic hydrolysis reaction is carried out in a constant temperature water bath at 35-40°C for 2-3 hours. After the reaction is completed, heating is performed at 80-100°C for 3-5 minutes to terminate the activity of the enzyme, and then disodium cyclohexanediaminetetraacetic acid is added, stirring is continued for 15-20 minutes, and centrifugation is performed at 3000-5000rpm / min in a centrifuge for 5-10 minutes. The remaining solid particles are removed by filtration, and the complex supplement is obtained after sterilization.

[0006] Ostracods are a type of small crustaceans, and diatoms are single-cell or multi-cell microalgae. After enzymatic hydrolysis of ostracods and diatoms, rich nutrients such as amino acids, lipids, vitamins, etc. can be released, which not only expands the scope of application of the culture medium, but also because natural biomaterials as nutrient sources are more natural than chemically synthesized culture medium components and are more in line with the needs of marine invertebrate cells. Therefore, these ingredients can effectively promote cell growth, so that marine invertebrate cell lines can make full use of the rich nutrients contained in ostracods and diatoms as food sources to achieve the purpose of rapid growth and stable subculture.

[0007] However, as aquatic organisms, ostracods and diatoms can absorb heavy metals such as lead and mercury from the environment. This is because their body surface or cell structure has a certain adsorption or uptake ability for heavy metal ions in the environment. When heavy metals enter the culture medium, they will have toxic effects on cells, reducing cell survival rate and proliferation ability. Therefore, disodium cyclohexanediaminetetraacetic acid is introduced. Disodium cyclohexanediaminetetraacetic acid can form stable complexes with heavy metals, thereby reducing the toxicity of heavy metals.

[0008] As a further improvement of the technical solution, the inorganic salt consists of 3-5 parts by weight of potassium chloride, 1-3 parts by weight of calcium chloride, 0.5-1 parts by weight of magnesium sulfate and 0.4-0.6 parts by weight of sodium dihydrogen phosphate.

[0009] As a further improvement of the present technical solution, the vitamins include vitamin B and vitamin C which are necessary for cell growth and metabolism.

[0010] As a further improvement of the technical solution, the mesh size of the pulverizer is 80-100 meshes.

[0011] As a further improvement of the technical solution, the complex enzyme consists of protease, lipase, chitinase and cellulase, with a mass ratio of 4:1.5-2.5:2.4-3.4:0.8-1.2.

[0012] As a further improvement of the technical solution, the mass ratio of the mixed substrate of ostracods and diatoms to the complex enzyme is 1:1-3.

[0013] As a further improvement of the present technical solution, the specific preparation method of the animal cell culture medium is as follows: Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer are sequentially added to deionized water, and a magnetic stirrer is used to fully stir and dissolve. Under sterile conditions, vitamins and penicillin are added, and stirring and mixing are continued. Then, a composite supplement is added, and the culture medium is filtered and sterilized using a sterile filter. Finally, fetal bovine serum is added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0014] As a further improvement of the technical solution, the stirring speed of the magnetic stirrer is 200-300 rpm / min, and the stirring and dissolving time is 5-10 min.

[0015] As a further improvement of the technical solution, the pore size of the sterile filter membrane is 0.1-0.22 μm.

[0016] Compared with the prior art, the present invention has the following beneficial effects: In the marine invertebrate cell culture medium, a composite supplement is used to prepare a marine invertebrate cell culture medium, ostracods and diatoms are used to improve the application range of the culture medium, and rich nutrients such as amino acids, lipids, vitamins, etc. produced by enzymatic hydrolysis of ostracods and diatoms are added to the cell culture medium. Since natural biological materials are used as nutrient sources, they are more natural than chemically synthesized culture medium components and are more in line with the needs of marine invertebrate cells, thereby effectively promoting cell growth and achieving the purpose of rapid growth and stable passage. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0018] A marine invertebrate cell culture medium comprises the following components in parts by weight: 0.5-1 parts by weight of a composite supplement, 1-4 parts by weight of glucose, 0.3-0.5 parts by weight of L-glutamine, 0.01-0.05 parts by weight of vitamins, 10-15 parts by weight of fetal bovine serum, 0.02-0.04 parts by weight of penicillin, 0.01-0.03 parts by weight of trehalose, 0.3-0.7 parts by weight of a sodium chloride osmotic pressure regulator, 0.5-1 parts by weight of an inorganic salt and 10-25 parts by weight of a sodium bicarbonate buffer; wherein the composite supplement The filler is prepared by mixing ostracods, diatoms and disodium cyclohexanediaminetetraacetic acid in a mass ratio of 1:0.2-0.6:0.1-0.5; the inorganic salt consists of 3-5 weight parts of potassium chloride, 1-3 weight parts of calcium chloride, 0.5-1 weight part of magnesium sulfate and 0.4-0.6 weight part of sodium dihydrogen phosphate; the vitamins include vitamin B and vitamin C necessary for cell growth and metabolism; the complex enzyme consists of protease, lipase, chitinase and cellulase in a mass ratio of 4:1.5-2.5:2.4-3.4:0.8-1.2.

[0019] Example 1: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows: The following components are prepared: 1 part by weight of a composite supplement, 4 parts by weight of glucose, 0.5 parts by weight of L-glutamine, 0.05 parts by weight of vitamins, 15 parts by weight of fetal bovine serum, 0.04 parts by weight of penicillin, 0.03 parts by weight of trehalose, 0.7 parts by weight of a sodium chloride osmotic pressure regulator, 1 part by weight of an inorganic salt, and 25 parts by weight of a sodium bicarbonate buffer; wherein the composite supplement is prepared by mixing ostracods, diatoms, and disodium cyclohexanediaminetetraacetic acid in a mass ratio of 1:0.2:0.1; the inorganic salt is composed of 5 parts by weight of potassium chloride, 3 parts by weight of calcium chloride, 1 part by weight of magnesium sulfate, and 0.6 parts by weight of sodium dihydrogen phosphate; the vitamins include vitamin B and vitamin C necessary for cell growth and metabolism; the composite enzyme is composed of protease, lipase, chitinase, and cellulase in a mass ratio of 4:2.5:3.4:1.2; the mass ratio of the mixed substrate of ostracods and diatoms to the composite enzyme is 1:3; The ostracods and diatoms are cleaned and dried, crushed with a grinder equipped with a screen, and mixed thoroughly, and then the composite enzyme is added to a sodium bicarbonate buffer to prepare an enzyme solution, and the mixed ostracod and diatom powders are added to the enzyme solution, and an enzymatic hydrolysis reaction is carried out in a constant temperature water bath at 35°C for 3 hours. After the reaction is completed, the enzyme activity is terminated by heating at 100°C for 5 minutes, and then disodium cyclohexanediaminetetraacetic acid is added, and stirring is continued for 15 minutes, and centrifuged at 3000 rpm / min for 5 minutes. The remaining solid particles are removed by filtration, and the composite supplement is obtained after sterilization; Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer are added to deionized water in sequence, and a magnetic stirrer is used to fully stir and dissolve. The stirring speed of the magnetic stirrer is 300 rpm / min, and the stirring and dissolving time is 10 minutes. Under sterile conditions, vitamins and penicillin are added, and stirring and mixing are continued. Then, a composite supplement is added, and the culture medium is filtered and sterilized using a 0.22 μm sterile filter. Finally, fetal bovine serum is added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0020] Example 2: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows: The following components are prepared: 1 part by weight of a composite supplement, 4 parts by weight of glucose, 0.5 parts by weight of L-glutamine, 0.05 parts by weight of vitamins, 15 parts by weight of fetal bovine serum, 0.04 parts by weight of penicillin, 0.03 parts by weight of trehalose, 0.7 parts by weight of a sodium chloride osmotic pressure regulator, 1 part by weight of an inorganic salt, and 25 parts by weight of a sodium bicarbonate buffer; wherein the composite supplement is prepared by mixing ostracods, diatoms, and disodium cyclohexanediaminetetraacetic acid in a mass ratio of 1:0.4:0.3; the inorganic salt is composed of 5 parts by weight of potassium chloride, 3 parts by weight of calcium chloride, 1 part by weight of magnesium sulfate, and 0.6 parts by weight of sodium dihydrogen phosphate; the vitamins include vitamin B and vitamin C necessary for cell growth and metabolism; the composite enzyme is composed of protease, lipase, chitinase, and cellulase in a mass ratio of 4:2.5:3.4:1.2; the mass ratio of the mixed substrate of ostracods and diatoms to the composite enzyme is 1:3; The ostracods and diatoms are cleaned and dried, crushed using a grinder with a screen, and mixed thoroughly, and then the composite enzyme is added to a sodium bicarbonate buffer to prepare an enzyme solution, and the mixed ostracod and diatom powders are added to the enzyme solution, and an enzymatic hydrolysis reaction is carried out in a constant temperature water bath at 35°C for 3 hours. After the reaction is completed, the enzyme activity is terminated by heating at 100°C for 5 minutes, and then disodium cyclohexanediaminetetraacetic acid is added, and the mixture is stirred for 15 minutes, and centrifuged at 3000 rpm / min for 5 minutes. The remaining solid particles are removed by filtration, and the composite supplement is obtained after sterilization; Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer are added to deionized water in sequence, and a magnetic stirrer is used to fully stir and dissolve. The stirring speed of the magnetic stirrer is 300 rpm / min, and the stirring and dissolving time is 10 minutes. Under sterile conditions, vitamins and penicillin are added, and stirring and mixing are continued. Then, a composite supplement is added, and the culture medium is filtered and sterilized using a 0.22 μm sterile filter. Finally, fetal bovine serum is added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0021] Example 3: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows: The following components are prepared: 1 part by weight of a composite supplement, 4 parts by weight of glucose, 0.5 parts by weight of L-glutamine, 0.05 parts by weight of vitamins, 15 parts by weight of fetal bovine serum, 0.04 parts by weight of penicillin, 0.03 parts by weight of trehalose, 0.7 parts by weight of a sodium chloride osmotic pressure regulator, 1 part by weight of an inorganic salt, and 25 parts by weight of a sodium bicarbonate buffer; wherein the composite supplement is prepared by mixing ostracods, diatoms, and disodium cyclohexanediaminetetraacetic acid in a mass ratio of 1:0.6:0.5; the inorganic salt is composed of 5 parts by weight of potassium chloride, 3 parts by weight of calcium chloride, 1 part by weight of magnesium sulfate, and 0.6 parts by weight of sodium dihydrogen phosphate; the vitamins include vitamin B and vitamin C necessary for cell growth and metabolism; the composite enzyme is composed of protease, lipase, chitinase, and cellulase in a mass ratio of 4:2.5:3.4:1.2; the mass ratio of the mixed substrate of ostracods and diatoms to the composite enzyme is 1:3; The ostracods and diatoms are cleaned and dried, crushed using a grinder with a screen, and mixed thoroughly, and then the composite enzyme is added to a sodium bicarbonate buffer to prepare an enzyme solution, and the mixed ostracod and diatom powders are added to the enzyme solution, and an enzymatic hydrolysis reaction is carried out in a constant temperature water bath at 35°C for 3 hours. After the reaction is completed, the enzyme activity is terminated by heating at 100°C for 5 minutes, and then disodium cyclohexanediaminetetraacetic acid is added, and the mixture is stirred for 15 minutes, and centrifuged at 3000 rpm / min for 5 minutes. The remaining solid particles are removed by filtration, and the composite supplement is obtained after sterilization; Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer are added to deionized water in sequence, and a magnetic stirrer is used to fully stir and dissolve. The stirring speed of the magnetic stirrer is 300 rpm / min, and the stirring and dissolving time is 10 minutes. Under sterile conditions, vitamins and penicillin are added, and stirring and mixing are continued. Then, a composite supplement is added, and the culture medium is filtered and sterilized using a 0.22 μm sterile filter. Finally, fetal bovine serum is added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0022] Example 4: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows: The following components are prepared: 1 part by weight of a composite supplement, 1 part by weight of glucose, 0.3 part by weight of L-glutamine, 0.01 part by weight of vitamins, 10 parts by weight of fetal bovine serum, 0.02 parts by weight of penicillin, 0.01 part by weight of trehalose, 0.3 part by weight of a sodium chloride osmotic pressure regulator, 0.5 part by weight of an inorganic salt, and 10 parts by weight of a sodium bicarbonate buffer; wherein the composite supplement is prepared by mixing ostracods, diatoms, and disodium cyclohexanediaminetetraacetic acid in a mass ratio of 1:0.4:0.3; the inorganic salt is composed of 3 parts by weight of potassium chloride, 1 part by weight of calcium chloride, 0.5 part by weight of magnesium sulfate, and 0.4 part by weight of sodium dihydrogen phosphate; the vitamins include vitamin B and vitamin C necessary for cell growth and metabolism; the composite enzyme is composed of protease, lipase, chitinase, and cellulase in a mass ratio of 4:1.5:2.4:0.8; the mass ratio of the mixed substrate of ostracods and diatoms to the composite enzyme is 1:1; The ostracods and diatoms are cleaned and dried, crushed using a grinder with a screen, and mixed thoroughly, and then the composite enzyme is added to a sodium bicarbonate buffer to prepare an enzyme solution, and the mixed ostracod and diatom powders are added to the enzyme solution, and an enzymatic hydrolysis reaction is carried out in a constant temperature water bath at 35°C for 3 hours. After the reaction is completed, the enzyme activity is terminated by heating at 100°C for 5 minutes, and then disodium cyclohexanediaminetetraacetic acid is added, and the mixture is stirred for 15 minutes, and centrifuged at 3000 rpm / min for 5 minutes. The remaining solid particles are removed by filtration, and the composite supplement is obtained after sterilization; Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer are added to deionized water in sequence, and a magnetic stirrer is used to fully stir and dissolve. The stirring speed of the magnetic stirrer is 300 rpm / min, and the stirring and dissolving time is 10 minutes. Under sterile conditions, vitamins and penicillin are added, and stirring and mixing are continued. Then, a composite supplement is added, and the culture medium is filtered and sterilized using a 0.22 μm sterile filter. Finally, fetal bovine serum is added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0023] Comparative Example 1: The method of Example 2 was adopted, and the added amount of the composite supplement was 0.01 parts by weight.

[0024] Comparative Example 2: The method of Example 2 was adopted, and the composite supplement was prepared only by enzymatic hydrolysis of ostracods, without adding diatoms.

[0025] Comparative Example 3: The method of Example 2 was adopted, and the composite supplement was prepared only by enzymatic hydrolysis of diatoms, without adding ostracods.

[0026] The present invention prepares a marine invertebrate cell culture medium, which has good performance in the application of the cell culture medium technology field. Specific detection is shown in the following table: Marine invertebrate cells were inoculated into a 96-well plate, with about 5000 cells per well. The cells were cultured under standard culture conditions at 37°C and 5% CO2. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added to each well at a specified time point of 48 h, and the culture was continued for 4 hours. Since the MTT reagent can be metabolized by living cells into colored substances, the absorbance was measured at specific wavelengths of 450 nm and 570 nm using an ELISA reader. The absorbance was measured every 30 minutes, and the net absorbance of Examples 1-4 and 1-3 was calculated. The net absorbance calculation ΔA=A 450 −A 570 , linear regression analysis was used to calculate the slope of the net absorbance changing with time. The larger the slope, the higher the cell metabolic activity and the better the cell proliferation. The cells were inoculated into culture dishes and subcultured once a week. The cultured cells were collected and subcultured at a dilution of 1:3. After dilution, they were transferred to new culture dishes. After 10 subcultures, the absorbance was measured at 450nm and recorded simultaneously. The cell survival rate was the ratio of the absorbance of the experiment to the absorbance of the comparative blank test. Among them, the comparative blank test was a culture medium prepared without adding compound supplements. The larger the cell survival rate, the better the stable cell proliferation, which was used to indirectly evaluate the stable subculture ability of the cells.

[0027] The obtained data are shown in Table 1 Table 1 Performance data of Examples 1-4 and Comparative Examples 1-3 By comparing Example 1-4 with Comparative Example 1-3, it can be seen that in a marine invertebrate cell culture medium, the composite supplement has a significant effect on the growth of animal cells.

[0028] According to Examples 1-3, as the proportion of components in the composite supplement continues to change, the cell growth effect in a marine invertebrate cell culture medium first increases and then decreases. This is because ostracods are rich in protein, fat and other trace elements, and the amino acids and fatty acids produced after enzymatic hydrolysis can provide necessary nutrition for cells. The amino acids produced by protein enzymatic hydrolysis are important raw materials for cell protein synthesis, and fatty acids are important components of cell membranes. Diatoms, as phytoplankton, are rich in vitamins, minerals and carbohydrates, especially carbohydrates produced after enzymatic hydrolysis. Sugars can be used as an energy source for cells. Diatoms also contain antioxidants, which help reduce oxidative stress and protect cells from damage. The appropriate ratio of ostracods and diatoms can provide more balanced nutrition, allowing cells to grow and divide better. Excess or insufficient any one component will lead to nutritional imbalance. Too many ostracods will lead to excess amino acids, and too many diatoms will lead to excess carbohydrates, both of which are not conducive to cell growth, thereby affecting cell growth.

[0029] According to Example 2 and Example 4, as the contents of other components in a marine invertebrate cell culture medium continue to change, the cell growth effect in a marine invertebrate cell culture medium does not change significantly. This is because the composite supplement is one of the key components in the culture medium, providing the nutrients required for cell growth, and changes in other components have little effect on cell growth within a certain range.

[0030] According to the above test experiments, Example 2 is used as the optimal example and compared with Comparative Examples 1-3 respectively.

[0031] By comparing Example 1 and Comparative Example 1, it can be seen that the added amount of the composite supplement is 0.01 parts by weight, and the cell growth effect in a marine invertebrate cell culture medium is worse. This is because the composite supplement is obtained by enzymatic hydrolysis of ostracods and diatoms, and is rich in various nutrients, such as amino acids, fatty acids, vitamins and minerals. When the added amount of the composite supplement is too low, sufficient nutritional support cannot be obtained, and the metabolic process of the cells will be restricted, resulting in slow growth or stagnation, thereby affecting its growth and division. In addition, a small amount of composite supplement is added. The culture medium components are almost all chemically synthesized, and there is no natural biological material as a nutrient source to meet the needs of marine invertebrate cells. Therefore, animal cells grow more slowly.

[0032] By comparing Example 1 and Comparative Example 2, it can be seen that the composite supplement is prepared only by enzymatic hydrolysis of ostracods without adding diatoms. The worse the cell growth effect in a marine invertebrate cell culture medium, this is because diatoms, as phytoplankton, are rich in vitamins, minerals and antioxidants, and can provide more comprehensive nutritional support. Diatoms are rich in antioxidants. The lack of these ingredients will make cells more susceptible to oxidative damage, affecting cell health and growth.

[0033] By comparing Example 1 and Comparative Example 3, it can be seen that the composite supplement is prepared only by enzymatic hydrolysis of diatoms without adding ostracods, and the stability of the active components in the marine invertebrate cell culture medium is poor. This is because the nutrients produced after enzymatic hydrolysis of ostracods are mainly proteins, fats and other trace elements. Although diatoms are rich in vitamins and minerals, their amino acid and fatty acid content is relatively low. Therefore, the lack of these key nutrients will lead to poor stability of the cell active components.

[0034] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only preferred examples of the present invention, and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A marine invertebrate cell culture medium, characterized in that: The invention comprises the following components in parts by weight: 0.5-1 parts by weight of a composite supplement, 1-4 parts by weight of glucose, 0.3-0.5 parts by weight of L-glutamine, 0.01-0.05 parts by weight of vitamins, 10-15 parts by weight of fetal bovine serum, 0.02-0.04 parts by weight of penicillin, 0.01-0.03 parts by weight of trehalose, 0.3-0.7 parts by weight of a sodium chloride osmotic pressure regulator, 0.5-1 parts by weight of an inorganic salt and 10-25 parts by weight of a sodium bicarbonate buffer; The composite supplement is prepared by mixing ostracods, diatoms and disodium cyclohexanediaminetetraacetic acid in a mass ratio of 1:0.2-0.6:0.1-0.

5.

2. A marine invertebrate cell culture medium according to claim 1, characterized in that: The specific preparation method of the composite supplement is as follows: The ostracods and diatoms are cleaned and dried, crushed using a grinder with a screen, and mixed thoroughly, then the complex enzyme is added to a sodium bicarbonate buffer to prepare an enzyme solution, the mixed ostracod and diatom powders are added to the enzyme solution, and the enzymatic hydrolysis reaction is carried out in a constant temperature water bath at 35-40°C for 2-3 hours. After the reaction is completed, heating is performed at 80-100°C for 3-5 minutes to terminate the activity of the enzyme, and then disodium cyclohexanediaminetetraacetic acid is added, stirring is continued for 15-20 minutes, and centrifugation is performed at 3000-5000rpm / min in a centrifuge for 5-10 minutes. The remaining solid particles are removed by filtration, and the complex supplement is obtained after sterilization.

3. A marine invertebrate cell culture medium according to claim 1, characterized in that: The inorganic salt is composed of 3-5 parts by weight of potassium chloride, 1-3 parts by weight of calcium chloride, 0.5-1 parts by weight of magnesium sulfate and 0.4-0.6 parts by weight of sodium dihydrogen phosphate.

4. A marine invertebrate cell culture medium according to claim 1, characterized in that: The vitamins include vitamin B and vitamin C, which are necessary for cell growth and metabolism.

5. A marine invertebrate cell culture medium according to claim 2, characterized in that: The sieve aperture of the pulverizer is 80-100 meshes.

6. A marine invertebrate cell culture medium according to claim 2, characterized in that: The complex enzyme consists of protease, lipase, chitinase and cellulase, and the mass ratio is 4:1.5-2.5:2.4-3.4:0.8-1.

2.

7. A marine invertebrate cell culture medium according to claim 2, characterized in that: The mass ratio of the mixed substrate of ostracods and diatoms to the complex enzyme is 1:1-3.

8. The marine invertebrate cell culture medium according to claim 1, characterized in that: The specific preparation method of the animal cell culture medium is as follows: Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer are sequentially added to deionized water, and a magnetic stirrer is used to fully stir and dissolve. Under sterile conditions, vitamins and penicillin are added, and stirring and mixing are continued. Then, a composite supplement is added, and the culture medium is filtered and sterilized using a sterile filter. Finally, fetal bovine serum is added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

9. The marine invertebrate cell culture medium according to claim 6, characterized in that: The stirring speed of the magnetic stirrer is 200-300 rpm / min, and the stirring and dissolving time is 5-10 min.

10. The marine invertebrate cell culture medium according to claim 8, characterized in that: The pore size of the sterile filter membrane is 0.1-0.22 μm.

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