Construction method of tilapia ventral fin cell line

A construction method and technology of tilapia, applied in the field of cell biology, can solve the problems that the construction of tilapia pelvic fin cell lines has not been reported yet, and achieve the effect of strong cell reproduction ability

Pending Publication Date: 2022-01-18

GUANGDONG OCEAN UNIVERSITY

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Problems solved by technology

[0004] Through the above analysis, the existing problems and defects of the prior art are: ...

Abstract

The invention belongs to the technical field of cell biology, and discloses a construction method of a tilapia ventral fin cell line. The construction method of the tilapia ventral fin cell line comprises the following steps of preparing a cell culture medium; pretreating tilapia ventral fin cell blocks; carrying out primary culture on the pelvic fin cells of the tilapia; and carrying out subculture on the pelvic fin cells of the tilapia. According to the construction method of the tilapia ventral fin cell line, the culture medium suitable for growth and proliferation of the cells and suitable culture conditions are optimized according to the characteristics of the tilapia ventral fin cells, the tilapia ventral fin cell line is successfully constructed, and the cells are high in reproductive capacity, the method can be applied to important research on related functional genes of cytobiology, molecular biology, virology, toxicology, oncology, genetics, immunology and the like, and provides a basis for developing fish virus infection ways and infection mechanisms on the molecular cell level and developing viral vaccines and the like.

Application Domain

Cell dissociation methodsCulture process +4

Technology Topic

Molecular biologyBiophysics +14

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[0034] In order to make the objects, technical solutions and advantages of the present invention, the present invention will be described in further detail below with reference to the embodiments. It is to be understood that the specific embodiments described herein are intended to explain the present invention and is not intended to limit the invention.

[0035] For the presence of the prior art, the present invention provides a method of constructing cell lines in ventral tilapia, the present invention in conjunction with the following drawings in detail below.

[0036] like figure 1 , The construction method according to an embodiment of the present invention tilapia ventral cell lines include the steps of:

[0037] S101, configuration cell culture medium;

[0038] S102, tilapia pelvic pretreated cell mass;

[0039] S103, tilapia pelvic primary culture cells;

[0040] S104, tilapia pelvic subcultured cells.

[0041] like figure 2 Shown embodiment the present invention provides the step S101, the configuration of the cell culture medium, comprising:

[0042] S201, M199 medium powder taken using sterile deionized water after dissolution, aliquots were filtered off with suction and;

[0043] S202, after taking a small amount of the culture solution was aseptically test, placed under storage conditions of 4 ℃, basal cell culture medium to obtain;

[0044] S203, fetal calf serum were added into the base medium, vitamin C, sodium bicarbonate, and the double anti-epidermal growth factor, mixed, and sterilized to adjust the pH, to obtain complete cell culture medium.

[0045]The fetal bovine serum provided by the embodiment of the present invention accounted for 10 to 20% of the total volume of the cell culture medium, the concentration of the vitamin C of 1 to 50 μg / ml, and the concentration of sodium bicarbonate is 1 to 100 μg / L, the The concentration of the epidermal growth factor was 5 to 100 ng / ml.

[0046] The sterilization treatment provided by the embodiment of the present invention includes: filtration by filtration of a filter film having a diameter of 0.2 μm diameter micropores.

[0047] Double-resistant bipodies provided by embodiments of the present invention include penicillin 150 to 200 U / mL, streptomycin 0.05 ~ 0.2 mg / ml.

[0048] The pH of the complete cell culture medium according to the embodiment of the present invention is 7.5 to 8.

[0049] like image 3 As shown, in step S102 of the embodiment of the present invention, the pretreatment of the tilapia fin cell block, including:

[0050] S301, take healthy tilapia, measurement, weigh, immersion in 75% ethanol, and disinfect and anesthesia, immediately anatomy;

[0051] S302, cut the tissue of the squid, soaked, disinfect, rinse in penicillin-streptomycin double anti-solution, cut the tilapia belly fin cell block into 0.5 ~ 1.5mm under sterile conditions 3 Organization block;

[0052] S303, soaking the tissue block at 0.25% trypsin and 0.02% EDTA digestion 10 ~ 25min, then diges 0.5 to 2.5 h by 0.5% hyaluronase, which is pre-treated tilapous fin cells. piece.

[0053] The preparation method of penicillin-streptomycin double anti-solution provided in the embodiment of the present invention includes: mixing penicillin, streptomycin with a quantity of three vapor, to obtain the bluemycin-streptomycin dual anti-solution.

[0054] The embodiment of the present invention provides soaking, disinfection, rinsing, including: tissue tilapous fins into a concentration of 1000 qu / ml penicillin-streptomycin bishydrate, disinfecting treatment with 75% alcohol, using The D-PBS rinsing 3 to 5 times of amphotericin B of 2.5 ug / ml concentration.

[0055] like Figure 4 As shown, in step S103 of the embodiment of the present invention, the primary culture of the tilapia fin cells, including:

[0056] S401, transplanting the pre-treated tilapia belly fin cell block to 25cm equipped with base cell culture medium 2 In the cell culture flask, maintained at 26 ° C;

[0057] S402, removes the base cell culture medium, adding 2 ml of trypsin for digestion treatment, terminating digestion, filtration, centrifugation, resuscitation;

[0058] S403, transplanted to another 25cm 2 In the cell culture flask, complete cell culture medium is supplemented, and the medium is placed in a 28 ° C cell incubator, and the medium is replaced once a day.

[0059] The digestion process provided by the embodiment of the present invention has a time of 15 to 30 min.

[0060] The centrifugation of the embodiments of the present invention is: centrifugation for 10 to 15 min under conditions of 1000 to 1500 rpm.

[0061] The complementary complete cell culture medium provided in the embodiment of the present invention, comprising: a complete cell culture medium 2 to 5 ml of a complete cell culture medium was added to the graft, and 3 to 6 ml of complete cell culture medium was added to 24h.

[0062] like Figure 5 As shown, in step S104 of the embodiment of the present invention, the passage culture of the tilapia fin cells, including:

[0063] S501, after the original cultured cells have long formed by a single layer, the medium is absorbed, and PBS rinsing cells are added, and 0.25% of the trypsin containing 0.25% EDTA is added to stand;

[0064] S502, the microscope is observed, and the cytotransmondracence is subjected to the cytometry, and fresh and complete cell cultured cell suspensions are added;

[0065] S503, inoculated into two 25 cm 2 cell culture flasks in the ratio of 1: 3, in the 28 ° C incubator, the biochemical culture is carried out, and 50 generations of 50 generations of cermic fin cell lines were cultured.

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