A culture medium for marine invertebrate cells

Abstract

The present invention relates to the technical field of cell culture media, and relates to a cell culture medium for marine invertebrates, which comprises the following components in parts by weight: 0.5-1 part by weight of a composite supplement, 1-4 parts by weight of glucose, 0.3-0.5 part by weight of L-glutamine, 0.01-0.05 part by weight of vitamins, 10-15 parts by weight of fetal bovine serum, 0.02-0.04 part by weight of penicillin, 0.01-0.03 part by weight of trehalose, 0.3-0.7 part by weight of a sodium chloride osmotic pressure regulator, 0.5-1 part by weight of inorganic salts, and 10-25 parts by weight of a sodium bicarbonate buffer; wherein, the composite supplement is prepared by mixing ostracods, diatoms and disodium cyclohexanediaminetetraacetate in a mass ratio of 1:0.2-0.6:0.1-0.5. By adopting the composite supplement, the applicable range of the culture medium is improved. After enzymolyzing ostracods and diatoms and adding them to the cell culture medium, it more meets the requirements of marine invertebrate cells compared with chemically synthesized culture media, thereby effectively promoting cell growth and achieving the purpose of rapid growth and stable passage.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture media, and specifically, to a cell culture medium for marine invertebrates. Background Art

[0002] Marine invertebrates, as one of the oldest and most biodiverse groups of organisms on Earth, occupy an important position in the marine ecosystem. They are extremely diverse in species, ranging from tiny plankton to large starfish, corals, and giant squids, forming 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, and carrying out material metabolism and energy conversion. During the long process of evolution, these cells have gradually adapted to the particularity of the marine environment, such as extreme conditions like high salinity, low temperature, high pressure, and insufficient light. However, traditional cell culture media for marine invertebrates have a narrow application range and cannot achieve rapid growth and stable passage. In view of this, a cell culture medium for marine invertebrates is provided. Summary of the Invention

[0003] The purpose of the present invention is to provide a cell culture medium for marine invertebrates to solve the problems of narrow application range and inability to achieve rapid growth and stable passage of the cell culture medium for marine invertebrates as proposed in the above background art.

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

[0005] As a further improvement of this technical solution, the specific preparation method of the composite supplement is as follows:

[0006] Clean and dry ostracods and diatoms, crush them using a crusher with a sieve, mix them thoroughly, then add the composite enzyme to a sodium bicarbonate buffer to prepare an enzyme solution. Add the mixed ostracod and diatom powder to the enzyme solution, and carry out an enzymatic hydrolysis reaction under the condition of a constant temperature water bath at 35 - 40 °C for 2 - 3 h. After the reaction ends, heat it at 80 - 100 °C for 3 - 5 min to terminate the enzyme activity, then add cyclohexanediaminetetraacetic acid disodium, continuously stir for 15 - 20 min, centrifuge at 3000 - 5000 rpm / min for 5 - 10 min using a centrifuge, and then filter to remove the remaining solid particles. After sterilization, a composite supplement is obtained.

[0007] Ostracods are a type of small crustaceans, and diatoms are single-celled or multi-celled microalgae. After enzymatically hydrolyzing ostracods and diatoms, rich nutrients such as amino acids, lipids, and vitamins can be released, which not only expands the scope of application of the culture medium, but also, since natural biological materials as nutrient sources are more natural than chemically synthesized culture medium components and are more in line with the needs of marine invertebrate cells, these components can effectively promote cell growth. Thus, marine invertebrate cell lines can fully utilize the rich nutrients contained in ostracods and diatoms as food sources to achieve the purpose of rapid growth and stable subculture.

[0008] However, as aquatic organisms, ostracods and diatoms can absorb heavy metals from the environment, such as lead and mercury, because their body surfaces or cell structures have a certain ability to adsorb or uptake heavy metal ions in the environment. When heavy metals enter the culture medium, they will have a toxic effect on cells, reducing the survival rate and proliferation ability of cells. Therefore, cyclohexanediaminetetraacetic acid disodium is introduced, and cyclohexanediaminetetraacetic acid disodium can form stable complexes with heavy metals, thereby reducing the toxicity of heavy metals.

[0009] As a further improvement of this technical solution, the inorganic salts are composed of 3 - 5 parts by weight of potassium chloride, 1 - 3 parts by weight of calcium chloride, 0.5 - 1 part by weight of magnesium sulfate, and 0.4 - 0.6 part by weight of sodium dihydrogen phosphate.

[0010] As a further improvement of this technical solution, the vitamins include vitamin B and vitamin C necessary for cell growth and metabolism.

[0011] As a further improvement of this technical solution, the aperture size of the sieve of the crusher is 80 - 100 mesh.

[0012] As a further improvement of this technical solution, the composite enzyme is composed of protease, lipase, chitinase, and cellulase, and the mass ratio is 4:1.5 - 2.5:2.4 - 3.4:0.8 - 1.2.

[0013] As a further improvement of the technical solution, the mass ratio of the mixed substrate of ostracods and diatoms to the composite enzyme is 1:1 to 3.

[0014] As a further improvement of the technical solution, the specific preparation method of the animal cell culture medium is as follows:

[0015] Add glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer to deionized water in sequence, stir and dissolve thoroughly with a magnetic stirrer. Under sterile conditions, add vitamins and penicillin, continue to stir and mix, then add a composite supplement, filter and sterilize the culture medium with a sterile filter, and finally add fetal bovine serum to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0016] 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.

[0017] As a further improvement of the technical solution, the pore size of the filter membrane of the sterile filter is 0.1 - 0.22 μm.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] In this marine invertebrate cell culture medium, a composite supplement is used to prepare a marine invertebrate cell culture medium. The use of ostracods and diatoms broadens the scope of application of the culture medium. After enzymolysis of ostracods and diatoms, rich nutrients such as amino acids, lipids, and vitamins are added to the cell culture medium. Since natural biological materials as nutrient sources are more natural than chemically synthesized culture medium components and are also more in line with the needs of marine invertebrate cells, it effectively promotes cell growth and achieves the purpose of rapid growth and stable passage. Specific Embodiments

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] A marine invertebrate cell culture medium, comprising the following components in parts by weight: 0.5 - 1 part by weight of a composite supplement, 1 - 4 parts by weight of glucose, 0.3 - 0.5 part by weight of L-glutamine, 0.01 - 0.05 part by weight of vitamins, 10 - 15 parts by weight of fetal bovine serum, 0.02 - 0.04 part by weight of penicillin, 0.01 - 0.03 part by weight of trehalose, 0.3 - 0.7 part by weight of a sodium chloride osmotic pressure regulator, 0.5 - 1 part by weight of inorganic salts, and 10 - 25 parts by weight of a sodium bicarbonate buffer; wherein, the composite supplement is prepared by mixing ostracods, diatoms, and disodium cyclohexanediaminetetraacetate in a mass ratio of 1:0.2 - 0.6:0.1 - 0.5; the inorganic salts consist of 3 - 5 parts by weight of potassium chloride, 1 - 3 parts by weight of calcium chloride, 0.5 - 1 part by weight of magnesium sulfate, and 0.4 - 0.6 part by weight of sodium dihydrogen phosphate; the vitamins include vitamins B and C essential for cell growth and metabolism; the composite 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.

[0022] Example 1: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows:

[0023] Prepare the following components: 1 part by weight of a composite supplement, 4 parts by weight of glucose, 0.5 part by weight of L-glutamine, 0.05 part by weight of vitamins, 15 parts by weight of fetal bovine serum, 0.04 part by weight of penicillin, 0.03 part by weight of trehalose, 0.7 part by weight of a sodium chloride osmotic pressure regulator, 1 part by weight of inorganic salts, and 25 parts by weight of a sodium bicarbonate buffer; wherein, the composite supplement is prepared by mixing ostracods, diatoms, and disodium cyclohexanediaminetetraacetate in a mass ratio of 1:0.2:0.1; the inorganic salts consist 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 part by weight of sodium dihydrogen phosphate; the vitamins include vitamins B and C essential for cell growth and metabolism; the composite enzyme consists of protease, lipase, chitinase, and cellulase, with 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;

[0024] Wash and dry the ostracods and diatoms, crush them using a pulverizer with a sieve, mix them thoroughly, then add the composite enzyme to the sodium bicarbonate buffer to prepare an enzyme solution, add the mixed ostracod and diatom powder to the enzyme solution, carry out an enzymatic hydrolysis reaction under the condition of a 35°C constant temperature water bath for 3 h, after the reaction ends, heat at 100°C for 5 min to terminate the activity of the enzyme, then add disodium cyclohexanediaminetetraacetate, stir continuously for 15 min, centrifuge at 3000 rpm / min for 5 min using a centrifuge, and then filter to remove the remaining solid particles, and obtain the composite supplement after sterilization;

[0025] Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer were successively added to deionized water, and dissolved thoroughly with a magnetic stirrer at a stirring speed of 300 rpm / min for 10 min. Under sterile conditions, vitamins and penicillin were added, and stirring and mixing were continued. Then, a composite supplement was added, and the culture medium was filtered and sterilized using a 0.22 μm sterile filter. Finally, fetal bovine serum was added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0026] Example 2: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows:

[0027] Prepare the following components: 1 part by weight of composite supplement, 4 parts by weight of glucose, 0.5 part by weight of L-glutamine, 0.05 part by weight of vitamins, 15 parts by weight of fetal bovine serum, 0.04 part by weight of penicillin, 0.03 part by weight of trehalose, 0.7 part by weight of sodium chloride osmotic pressure regulator, 1 part by weight of inorganic salts and 25 parts by weight of sodium bicarbonate buffer; among them, the composite supplement is prepared by mixing ostracods, diatoms and disodium cyclohexanediaminetetraacetate in a mass ratio of 1:0.4:0.3; the inorganic salts consist 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 part by weight of sodium dihydrogen phosphate; the vitamins include vitamins B and C necessary for cell growth and metabolism; the composite enzyme consists of protease, lipase, chitinase and cellulase, with 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;

[0028] The ostracods and diatoms were cleaned and dried, crushed using a pulverizer with a sieve, and mixed thoroughly. Then, the composite enzyme was added to the sodium bicarbonate buffer to prepare an enzyme solution. The mixed ostracod and diatom powder was added to the enzyme solution, and an enzymatic hydrolysis reaction was carried out under the condition of a 35°C constant temperature water bath for 3 h. After the reaction ended, the enzyme activity was terminated by heating at 100°C for 5 min. Then, disodium cyclohexanediaminetetraacetate was added, and stirring was continued for 15 min. Centrifugation was carried out at 3000 rpm / min for 5 min using a centrifuge, and the remaining solid particles were removed by filtration. After sterilization, the composite supplement was obtained;

[0029] 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.

[0030] Example 3: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows:

[0031] 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;

[0032] 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;

[0033] Add glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer to deionized water in sequence, and fully stir and dissolve them using a magnetic stirrer. The stirring speed of the magnetic stirrer is 300 rpm / min, and the stirring and dissolving time is 10 min. Under sterile conditions, add vitamins and penicillin, continue to stir and mix, then add the composite supplement, filter and sterilize the culture medium using a 0.22 μm sterile filter, and finally add fetal bovine serum to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0034] Example 4: In this example, the specific preparation method of the marine invertebrate cell culture medium is as follows:

[0035] Prepare the following components: 1 part by weight of 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 part by weight of penicillin, 0.01 part by weight of trehalose, 0.3 part by weight of sodium chloride osmotic pressure regulator, 0.5 part by weight of inorganic salts and 10 parts by weight of sodium bicarbonate buffer; wherein, the composite supplement is prepared by mixing ostracods, diatoms and disodium cyclohexanediaminetetraacetate in a mass ratio of 1:0.4:0.3; the inorganic salts are 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 vitamins B and C necessary for cell growth and metabolism; the composite enzyme is composed of protease, lipase, chitinase and cellulase, and the mass ratio is 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;

[0036] Clean and dry the ostracods and diatoms, crush them using a pulverizer with a sieve, mix them evenly, then add the composite enzyme to the sodium bicarbonate buffer to prepare an enzyme solution, add the mixed ostracod and diatom powder to the enzyme solution, carry out an enzymatic hydrolysis reaction under the condition of a 35°C constant temperature water bath for 3 h. After the reaction ends, heat at 100°C for 5 min to terminate the enzyme activity, then add disodium cyclohexanediaminetetraacetate, continuously stir for 15 min, centrifuge at 3000 rpm / min for 5 min using a centrifuge, and then filter to remove the remaining solid particles. After sterilization, obtain the composite supplement;

[0037] Glucose, L-glutamine, inorganic salts, trehalose, sodium chloride osmotic pressure regulator and sodium bicarbonate buffer were successively added to deionized water, and dissolved by sufficient stirring with a magnetic stirrer. The stirring speed of the magnetic stirrer was 300 rpm / min, and the stirring and dissolving time was 10 min. Under sterile conditions, vitamins and penicillin were added, and stirring and mixing were continued. Then, a composite supplement was added. The culture medium was filtered and sterilized using a 0.22 μm sterile filter. Finally, fetal bovine serum was added to the filtered and sterilized culture medium to obtain a marine invertebrate cell culture medium.

[0038] Comparative Example 1: The method of Example 2 was adopted, and the addition amount of the composite supplement was 0.01 part by weight.

[0039] Comparative Example 2: The method of Example 2 was adopted, and the composite supplement was only prepared by enzymatic hydrolysis of ostracods and no diatoms were added.

[0040] Comparative Example 3: The method of Example 2 was adopted, and the composite supplement was only prepared by enzymatic hydrolysis of diatoms and no ostracods were added.

[0041] The present invention has good performance in the application of the technical field of cell culture media by preparing a marine invertebrate cell culture medium. The specific detection is shown in the following table:

[0042] Marine invertebrate cells were inoculated into a 96-well plate, about 5000 cells per well, and cultured at 37 °C and 5% CO 2 under standard culture conditions. At the designated time point of 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added to each well, and the culture was continued for 4 hours. Since the MTT reagent can be metabolized by living cells into a colored substance, the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) reader at specific wavelengths of 450 nm and 570 nm. The absorbance was measured every 30 min, and the net absorbance of Examples 1-4 and Examples 1-3 was calculated. The net absorbance was calculated as ΔA = A 450 −A 570 . The slope of the change in net absorbance over time was calculated using linear regression analysis. The larger the slope, the higher the cell metabolic activity and the better the cell proliferation. The cells were inoculated into a culture dish and subcultured once a week. The cultured cells were collected, diluted 1:3 for subculture, transferred to a new culture dish after dilution, and after 10 subcultures, the absorbance was measured at 450 nm. The cell survival rate was the ratio of the absorbance of the experiment to the absorbance of the control blank experiment. The control blank experiment was the culture medium prepared without adding the composite supplement. The larger the cell survival rate, the better the cell stable proliferation, which was used to indirectly evaluate the stable passage ability of the cells.

[0043] The obtained data are shown in Table 1

[0044] Table 1 Performance data of Examples 1-4 and Comparative Examples 1-3

[0045]

[0046] Comparing Examples 1-4 with Comparative Examples 1-3, it can be seen that in a marine invertebrate cell culture medium, the composite supplement has a significant impact on the growth of animal cells.

[0047] According to Examples 1-3, as the component ratio in the composite supplement changes continuously, 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. Amino acids and fatty acids produced after enzymatic hydrolysis can provide necessary nutrients for cells. Amino acids produced by protein enzymatic hydrolysis are important raw materials for cell protein synthesis, while fatty acids are important components of cell membranes. As phytoplankton, diatoms are rich in vitamins, minerals, and carbohydrates. In particular, sugars produced after enzymatic hydrolysis of carbohydrates can serve as the energy source for cells. Diatoms also contain antioxidant substances, which help reduce oxidative stress and protect cells from damage. An appropriate ratio of ostracods and diatoms can provide more balanced nutrition, enabling cells to grow and divide better. Excess or deficiency of any one component will lead to nutritional imbalance. Excessive ostracods will result in amino acid surplus, while excessive diatoms will result in carbohydrate surplus, both of which are not conducive to cell growth and thus affect cell growth.

[0048] According to Examples 2 and 4, as the content of other components in a marine invertebrate cell culture medium changes continuously, 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. Changes in other components have little impact on cell growth within a certain range.

[0049] According to the above test experiments, Example 2 is used as the optimal example and compared with Comparative Examples 1-3 respectively.

[0050] By comparing Example 1 and Comparative Example 1, it can be seen that the lower the addition amount of the composite supplement is 0.01 part by weight, the worse the cell growth effect in a marine invertebrate cell culture medium. This is because the composite supplement is obtained by enzymatically hydrolyzing ostracods and diatoms and is rich in various nutrients such as amino acids, fatty acids, vitamins, and minerals. When the addition amount of the composite supplement is too low, sufficient nutritional support cannot be obtained, and the metabolic process of cells will be restricted, resulting in slow growth or stagnation, thus affecting their growth and division. In addition, when a very small amount of the composite supplement is added, almost all components of the culture medium are chemically synthesized, and there is no natural biological material as a nutrient source to meet the needs of marine invertebrate cells. Therefore, the growth of animal cells is slower.

[0051] By comparing Example 1 and Comparative Example 2, it can be seen that the composite supplement is only prepared by enzymatically hydrolyzing ostracods without adding diatoms, and the worse the cell growth effect in a marine invertebrate cell culture medium. This is because diatoms, as phytoplankton, contain rich vitamins, minerals, and antioxidants, and can provide more comprehensive nutritional support. Diatoms contain rich antioxidants, and the lack of these components will cause cells to be more vulnerable to oxidative damage, affecting the health and growth of cells.

[0052] By comparing Example 1 and Comparative Example 3, it can be seen that the composite supplement is only prepared by enzymatically hydrolyzing diatoms without adding ostracods, and the worse the stability of the active components in a marine invertebrate cell culture medium. This is because the nutrients produced after enzymatically hydrolyzing ostracods are mainly proteins, fats, and other trace elements. Although diatoms contain rich vitamins and minerals, their amino acid and fatty acid contents are relatively low. Therefore, the lack of these key nutrients will result in poor stability of the active components of cells.

[0053] 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. The above embodiments and the descriptions in the specification are only preferred examples of the present invention and are not used to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

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

2. 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.

3. 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.

4. A marine invertebrate cell culture medium according to claim 1, characterized in that: The sieve aperture of the pulverizer is 80-100 meshes.

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

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