A cell culture medium for the mantle of Mytilus coruscus and its application

Abstract

The invention relates to the field of cell culture and discloses a culture medium for thick-shelled mussel mantle cells and an application thereof. The culture medium comprises a basal culture medium and additives, wherein the additives comprise thick-shelled mussel serum, proanthocyanidins, bovine insulin, growth factors, antibiotics, defensins, mussel mucin and a buffer. The culture medium can be used for in vitro culture of thick-shelled mussel mantle cells and can better maintain the survival and growth of in vitro cultured thick-shelled mussel mantle cells. Defensins in a specific proportion are added to the culture medium, and the best growth and reproduction state can be achieved when the thick-shelled mussel mantle cells are cultured using the culture medium in the specific proportion. The application also provides an application method of the culture medium, which is simple to operate and can produce cultured thick-shelled mussel mantle cells with high cell density, good morphology and high wall adhesion.

Description

Technical Field

[0001] The invention relates to the field of cell culture, in particular to a culture medium for mantle cells of thick-shelled mussels and applications thereof. Background Art

[0002] Thick-shelled mussels are mainly distributed along the coasts of the Yellow Sea, Bohai Sea and East China Sea in my country, with the largest resource volume along the Zhejiang coast. Thick-shelled mussels have the advantages of delicious meat, rich nutrition, high protein content, fast growth and reproduction, strong disease resistance, easy artificial breeding, and rapid detection of environmental water quality. They are a cultured shellfish with important economic value in Zhoushan, Zhejiang. Currently, the mantle cells of shellfish are often used for experiments at the cellular level such as RNA interference and cell attack. Common shellfish include cuming clams, Hepu pearl shells, etc., such as a shellfish disclosed in Publication No. CN107043741A. The preparation of a pearl oyster mantle epithelial cell line and the application of the cell line and cells. The method for preparing the cell line is to culture the mantle epithelial cells of pearl oysters, i.e. river clams or sea clams, in vitro for 15 months and pass them to the 50th generation; the pearl oyster mantle epithelial cell line maintains the morphological, growth and proliferation characteristics of the pearl oyster mantle epithelial cells. The culture method uses the most commonly used culture medium and performs primary cell screening and culture under simple conditions, ensuring that the cells finally obtained can proliferate without special reagents and conditions, providing better space for their future applications.

[0003] Another example is Publication No. CN113462634A, which discloses an in vitro culture medium, preparation method, and use method for mantle cells of the Pinctada maxima (Pinctada maxima). The medium comprises a basal cell culture medium, a Radix Isatidis extract, a Flos Lonicerae extract, fetal bovine serum, bovine fluids, kanamycin, a balanced salt solution, and glucose. This method utilizes the addition of the Chinese medicinal herbs Radix Isatidis and Flos Lonicerae, bovine fluids, and other components to the basal cell culture medium, optimizing the proportions of each component to achieve in vitro mantle cell culture. The added Radix Isatidis extract and Flos Lonicerae extract inhibit harmful bacteria in the external environment throughout the culture process, while also extending the cell lifespan.

[0004] Currently, there are few technologies that use the mantle cells of thick-shelled mussels at the cellular level. However, thick-shelled mussels are abundant in resources and grow and reproduce extremely quickly. Applying them to cell culture technology has extremely high value and economic advantages. At the same time, the existing technology has not yet disclosed relevant content about the culture medium of thick-shelled mussel mantle cells. Summary of the Invention

[0005] In view of the fact that relevant content of the thick-shelled mussel mantle cell culture medium has not been disclosed in the prior art, the present application provides a thick-shelled mussel mantle cell culture medium and its application. The thick-shelled mussel mantle cell culture medium provided in this application includes a basal culture medium, component a and component b. The culture medium can be used for in vitro culture of thick-shelled mussel mantle cells and can better maintain the survival and growth of in vitro cultured thick-shelled mussel mantle cells.

[0006] The specific technical solutions of the present invention are:

[0007] A culture medium for mantle cells of thick-shelled mussels, comprising a basic culture medium and an additive, wherein the additive comprises a component and a component b.

[0008] The component a comprises thick-shelled mussel serum, proanthocyanidins, bovine insulin, growth factors, antibiotics, defensins, mussel mucin and buffer, and the component b comprises proanthocyanidins, bovine insulin, mussel mucin and growth factors;

[0009] The defensins include Mytilus thunbergii defensin ALD-1 and Mytilus thunbergii defensin ALD-2 in a mass ratio of 1:2.

[0010] The present application provides a culture medium for mantle cells of thick-shelled mussels. The culture medium is based on L-15 medium, wherein components A and B are additives. Component A primarily provides nutrients required for cell growth, and component B primarily promotes cell proliferation. Together, components A and B ensure the survival and growth of thick-shelled mussel mantle cells cultured in vitro. The minimal medium provides basic nutrients. The thick-shelled mussel serum in component a simulates the growth environment of thick-shelled mussel mantle cells and provides some essential nutrients, such as amino acids, trace elements, ions, and required hormones. Growth factors include attachment factors and expansion factors, which promote cell growth and promote cell adherence and reproduction. Proanthocyanidins scavenge free radicals and reduce oxidative damage. Antibiotics include penicillin, streptomycin, gentamicin, and amphotericin B, which are antibacterial and prevent bacterial contamination of the culture medium. Defensins also have antibacterial effects and can also reduce damage to thick-shelled mussel mantle cells caused by toxic and harmful substances produced by metabolism during the culture process. The mussel mucin in component b promotes cell adhesion and adhesion. The buffer maintains the pH of the culture medium. In addition, a specific ratio of defensins (thick-shelled mussel defensin ALD-1 and thick-shelled mussel defensin ALD-2 in a mass ratio of 1:2) is added to the culture medium. Only when the culture medium with this ratio is used to culture the thick-shelled mussel mantle cells can the optimal growth and reproduction state be achieved.

[0011] Preferably, the final concentrations of the components in the additive are: thick-shelled mussel serum 15%, proanthocyanidins 20,000 μg / mL, bovine insulin 12 μg / mL, growth factor 0.008 μg / mL, defensin 0.06 μg / mL, and mussel mucin 0.08 μg / mL.

[0012] Preferably, the antibiotics include the following components: 60 μg / ml penicillin, 100 μg / ml streptomycin, 40 μg / mL gentamicin, and 0.1 μg / ml amphotericin B.

[0013] Preferably, the defensins include Mytilus thunbergii defensin ALD-1 and Mytilus thunbergii defensin ALD-2 in a mass ratio of 1:2.

[0014] Preferably, the basal culture medium is L-15 culture medium, the buffer is HEPES buffer, and the pH of the thick-shelled mussel mantle cell culture medium is 7.2-7.3.

[0015] Preferably, the method for preparing the thick-shelled mussel serum comprises: centrifuging the thick-shelled mussel blood, taking the supernatant, filtering and sterilizing the supernatant, and the centrifugation conditions are: centrifugation temperature 4°C, centrifugation speed 12000 rpm, and centrifugation time 10 min.

[0016] A method for culturing mantle cells of Mytilus thunbergii using a culture medium for mantle cells of Mytilus thunbergii comprises the following steps:

[0017] (1) Cutting the mantle tissue of thick-shelled mussel to make tissue paste;

[0018] (2) enzymatically hydrolyzing the tissue paste in step (1) to prepare a thick-shelled mussel mantle cell suspension;

[0019] (3) Adding component b and component a to L-15 culture medium and mixing them evenly to prepare a culture medium for the mantle cells of thick-shelled mussels;

[0020] (4) Using the mantle cell culture medium prepared in step (3), the thick-shelled mussel mantle cell suspension prepared in step (2) is diluted and assembled for culture.

[0021] The present application also provides a method for applying the above-mentioned thick-shelled mussel mantle cell culture in culturing thick-shelled mussel mantle cells. The method is simple to operate, and the cultured thick-shelled mussel mantle cells have high cell density, good morphology, and high wall adhesion.

[0022] Preferably, the enzyme used in the enzymatic hydrolysis in step (2) is trypsin.

[0023] Preferably, the dilution factor in step (4) is 1.5×10 4 pcs / ml.

[0024] Preferably, the conditions for the bottling culture in step (4) are: CO2 content of 5%, temperature of 37°C, and static culture.

[0025] Compared with the existing technology, this application has the following technical effects:

[0026] (1) The present application provides a culture medium for mantle cells of thick-shelled mussels, which can be used for in vitro culture of mantle cells of thick-shelled mussels and can better maintain the survival and growth of mantle cells of thick-shelled mussels cultured in vitro;

[0027] (2) The culture medium in this application contains a specific ratio of defensins (Mussel mussellin ALD-1 and Mussel mussellin ALD-2 in a mass ratio of 1:2). The optimal growth and reproduction state of Mussel mussellin mantle cells can be achieved when cultured using the culture medium in this ratio.

[0028] (3) The present application also provides a method for using a thick-shelled mussel mantle cell culture medium in culturing thick-shelled mussel mantle cells. The method is simple to operate, and the cultured thick-shelled mussel mantle cells have a high cell density, good morphology, and high wall adhesion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the morphological development of the mantle cells of the thick-shelled mussel of the present invention.

[0030] Figure 2 It is a schematic diagram of the flow cytometer detection results of the thick-shelled mussel mantle cells of the present invention. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the embodiments.

[0032] Example 1:

[0033] A culture medium for mantle cells of thick-shelled mussels comprises a basal culture medium and an additive. The final concentrations of the additive components are as follows: 15% thick-shelled mussel serum, 20,000 μg / mL proanthocyanidins, 12 μg / mL bovine insulin, 0.008 μg / mL growth factor, 0.06 μg / mL defensin, and 0.08 μg / mL mussel mucin. The antibiotics comprise the following components: 60 μg / mL penicillin, 100 μg / mL streptomycin, 40 μg / mL gentamicin, and 0.1 μg / mL amphotericin B. The defensins comprise thick-shelled mussel defensin ALD-1 and thick-shelled mussel defensin ALD-2 at a mass ratio of 1:2. The basal culture medium is L-15 medium, the buffer is a HEPES buffer, and the pH of the thick-shelled mussel mantle cell culture medium is 7.2-7.3.

[0034] Example 2:

[0035] An application of a culture medium for mantle cells of thick-shelled mussels comprises the following steps:

[0036] (1) Use PBS to wash the mantle tissue of thick-shelled mussels, remove the mucus on the surface of the tissue, cut the mantle tissue into several small pieces with a scalpel, add PBS to the small pieces, and use ophthalmic scissors to repeatedly cut the tissue to make tissue paste;

[0037] (2) adding trypsin to the tissue paste in step (1) for enzymatic digestion into micro-tissue particles, and then blowing the micro-tissue particles into a thick-shelled mussel mantle cell suspension;

[0038] (3) Adding component b and component a in Example 1 to L-15 culture medium and mixing them evenly to prepare a culture medium for the mantle cells of thick-shelled mussels;

[0039] (4) Using the mantle cell culture medium prepared in step (3), dilute the thick-shelled mussel mantle cell suspension prepared in step (2) (the dilution concentration is 1.5×10 4 The culture conditions were as follows: placing the cells in a CO2 incubator with a CO2 content of 5% and culturing at 37°C.

[0040] Comparative Example 1

[0041] Compared with Example 1, the culture medium in step (3) is L-15 culture medium (5% fetal bovine serum), and the other conditions are the same as those in Example 2.

[0042] Comparative Example 2

[0043] Compared with Example 2, the mass ratio of thick-shelled mussel defensin ALD-1 to thick-shelled mussel defensin ALD-2 in step (3) is 1:1, and the other conditions are the same as those in Example 2.

[0044] Comparative Example 3

[0045] Compared with Example 2, the mass ratio of thick-shelled mussel defensin ALD-1 to thick-shelled mussel defensin ALD-2 in step (3) is 1:3, and the other conditions are the same as those in Example 2.

[0046] Comparative Example 4

[0047] Compared with Example 2, the mass ratio of thick-shelled mussel defensin ALD-1 to thick-shelled mussel defensin ALD-2 in step (3) is 1:4, and the other conditions are the same as those in Example 2.

[0048] Test example:

[0049] The growth state of the mantle cells of the thick-shelled mussels after culturing for 24 hours in Example 2 and Comparative Examples 1 to 4 was observed. Figure 1 ;

[0050] like Figure 1 As shown, compared with Comparative Example 1, Example 1 has a higher cell density, is closely arranged, and is firmly attached to the inner wall of the culture bottle; compared with Comparative Example 1, Comparative Examples 2, 3, and 4 have slightly higher cell densities and better morphology; compared with Comparative Examples 2, 3, and 4, Example 1 has a higher cell density, is closely arranged, and is better attached. This shows that the culture medium provided by the present application can well maintain the survival and growth of thick-shelled mussel mantle cells cultured in vitro, and that the culture medium containing a specific ratio of defensins (thick-shelled mussel defensin ALD-1 and thick-shelled mussel defensin ALD-2 at a mass ratio of 1:2) can achieve the best growth and reproduction state when culturing thick-shelled mussel mantle cells.

[0051] The culture medium application method of Example 2 and Comparative Examples 1 to 4 was used to culture the mantle cells of thick-shelled mussels, and the cell density was detected and recorded using flow cytometry (once every 6 hours). The test results are as follows: Figure 2 ;

[0052] like Figure 2 As shown, Example 2 compared with Comparative Example 1 demonstrates that the culture medium of Example 2, when supplemented with mussel serum and defensin ALD, is more effective in accelerating the growth and proliferation of mussel mantle cells and shortening the culture time compared with fetal bovine serum. ALD also better protects mantle cells from environmental damage and promotes cell-to-cell adhesion. Comparison of Example 2 with Comparative Examples 2, 3, and 4 demonstrates that the specific ratio of ALD-1 and ALD-2 in the culture medium of Example 2 is more conducive to the proliferation of mussel mantle cells.

[0053] 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 fall within the scope of protection of the technical solution of the present invention.

Claims

1. A culture medium for mantle cells of thick-shelled mussels, characterized in that: The method comprises L-15 culture medium and additives, wherein the additives are as follows: 15% thick-shelled mussel serum, 20,000 μg / mL proanthocyanidins, 12 μg / mL bovine insulin, 0.008 μg / mL growth factor, 0.06 μg / mL defensin, 0.08 μg / mL mucin, antibiotics and HEPES buffer, wherein the defensins include thick-shelled mussel defensin ALD-1 and thick-shelled mussel defensin ALD-2 in a mass ratio of 1:2, and the antibiotics are: 60 μg / ml penicillin, 100 μg / ml streptomycin, 40 μg / mL gentamicin and 0.1 μg / ml amphotericin B.

2. A thick-shelled mussel mantle cell culture medium according to claim 1, characterized in that: The pH of the thick-shelled mussel mantle cell culture medium is 7.2-7.

3.

3. A thick-shelled mussel mantle cell culture medium according to claim 1, characterized in that: The thick-shelled mussel serum is prepared by centrifuging thick-shelled mussel blood to obtain the supernatant and filtering and sterilizing it.

4. A thick-shelled mussel mantle cell culture medium according to claim 3, characterized in that: The centrifugation temperature was 4°C.

5. A thick-shelled mussel mantle cell culture medium according to claim 3, characterized in that: The centrifugal speed was 12000 rpm.

6. A thick-shelled mussel mantle cell culture medium according to claim 3, characterized in that: The centrifugation time is 10 minutes.

7. Use of the thick-shelled mussel mantle cell culture medium according to any one of claims 1 to 6 in culturing thick-shelled mussel mantle cells, characterized in that: The following steps are involved: (1) Cutting the mantle tissue of thick-shelled mussel to make tissue paste; (2) enzymatically hydrolyzing the tissue paste in step (1) to prepare a thick-shelled mussel mantle cell suspension; (3) adding component b and component a into L-15 culture medium and mixing evenly to prepare a culture medium for the mantle cells of thick-shelled mussels; (4) Using the mantle cell culture medium prepared in step (3), dilute the thick-shelled mussel mantle cell suspension prepared in step (2), and culture in bottles.

8. The use according to claim 7, characterized in that: The enzyme used in the enzymatic hydrolysis in step (2) is trypsin.

9. The use according to claim 7, characterized in that: The dilution factor in step (4) is 1.5×10 4 pcs / ml.

10. The use according to claim 7, characterized in that: The conditions for the bottling culture in step (4) are: CO2 content of 5%, temperature of 37°C, and static culture.

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