CN110923194A

CN110923194A - Kit for primary cell culture and application thereof

Info

Publication number: CN110923194A
Application number: CN201911337507.5A
Authority: CN
Inventors: 黄诚坤; 钟国亮; 劳海金
Original assignee: According To Guangdong Biological Technology Co Ltd
Current assignee: According To Guangdong Biological Technology Co Ltd
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2020-03-27
Anticipated expiration: 2039-12-23
Also published as: CN110923194B

Abstract

The invention provides a kit for primary cell culture, comprising: culture solution A, fetal bovine serum, GlutaMAX^TMThe preparation method comprises the following steps of (1) supplementing liquid, a penicillin-streptomycin-neomycin solution, EGF, bFGF, collagenase IV, DNase I and a penicillin-streptomycin-amphotericin B mixed solution; the culture solution A is added with inorganic salt, amino acid and vitamin. The reagent provided by the kit for primary cell culture provided by the invention can be used for preparing a solution required for shearing a tissue sample, digesting the tissue sample and realizing cell culture proliferation in the process of primary cell culture, and the enzyme level, the trace element level, the amino acid level and the vitamin level in the prepared solution can adaptively meet the nutritional requirements of the primary cells in each culture stage.

Description

Kit for primary cell culture and application thereof

Technical Field

The invention belongs to the field of animal cell culture, and particularly relates to a kit for primary cell culture and application thereof.

Background

Primary culture, also called primary culture, refers to the first culture of cells, tissues and organs taken directly from the body, and strictly speaking, primary culture refers to the culture before successful passage, where the cells retain the basic properties of the original cells, and if normal cells, the diploid number is still retained. However, in practice, the cultured cells within the first to tenth generations are generally collectively referred to as primary cell culture.

The primary culture cell has biological characteristics which do not change greatly because the tissue is just isolated, the hereditary property of the original diploid is still kept, the gene retention is over 90 percent, the primary culture cell is suitable for drug sensitivity tests and mechanism research related tests, the data of the primary culture cell is more convincing, and meanwhile, a corresponding pathological change cell line or strain is established through the primary culture technology, the pathological change, molecular heredity, transfer evolution mechanism and the like of the cell are researched, and the primary culture cell plays a very important role in researching and treating various diseases. The primary cells are used as important tool cells in molecular biology research, and along with the rapid development of biological science research, the primary cells are widely used in biological experiments, and the demand of the primary cells is in a continuous increasing trend along with the rapid development of the biological science research. Therefore, there is a need to develop a method capable of rapidly culturing primary generation cells to meet the current scientific development needs.

Disclosure of Invention

The invention aims to provide a kit for primary cell culture and application thereof, so as to rapidly obtain a large amount of primary cells.

According to one aspect of the invention, there is provided a kit for primary cell culture, comprising: culture solution A, fetal bovine serum, GlutaMAX^TMThe preparation method comprises the following steps of (1) supplementing liquid, a penicillin-streptomycin-neomycin solution, EGF, bFGF, collagenase IV, DNase I and a penicillin-streptomycin-amphotericin B mixed solution; inorganic salt, amino acid and vitamin are added into the culture solution A; the inorganic salt comprises at least one of calcium chloride, copper sulfate, ferric nitrate, ferrous sulfate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, monosodium phosphate, disodium phosphate and zinc sulfate; the amino acids include L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride monohydrate, L-cystine dihydrochloride, L-glutamic acid, L-glutamine, glycine, and L-histidine hydrochloride monohydrateAt least one of L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate and L-valine; the vitamins include at least one of vitamin H, choline chloride, folic acid, inositol, nicotinamide, vitamin B5, vitamin B6, vitamin B2, vitamin B1, and vitamin B12.

Through a plurality of improvements and attempts, the inventor finds that primary cells cultured in a culture medium containing EGF or bFGF show excellent proliferation potential, provides a reagent set comprising EGF and bFGF in the kit for culturing the primary cells, and can culture a large amount of primary cells in a short time and high efficiency by culturing the primary cells by using the provided reagent set.

Preferably, the inorganic salts comprise the following species and their corresponding concentrations in broth a: 0.1-0.15 g/L of calcium chloride, 0.5-1 μ g/L of copper sulfate, 10-80 μ g/L of ferric nitrate, 0.2-0.5mg/L of ferrous sulfate, 0.05-1 g/L of magnesium sulfate, 0.2-0.5 g/L of potassium chloride, 1-1.5 g/L of sodium bicarbonate, 5-10 g/L of sodium chloride, 0.05-0.1 g/L of monosodium phosphate, 0.05-0.1 g/L of disodium phosphate and 0.3-0.7 mg/L of zinc sulfate.

Preferably, the amino acids comprise the following species and their corresponding concentrations in broth a: 3-4 mg/L of L-alanine, 0.1-0.2 g/L of L-arginine hydrochloride, 5-10 mg/L of L-asparagine monohydrate, 5-8 mg/L of L-aspartic acid, 0.01-0.02 g/L of L-cysteine hydrochloride monohydrate, 0.02-0.05g/L of L-cystine dihydrochloride, 5-10 m g/L of L-glutamic acid, 0.2-0.5 g/L of L-glutamine, 0.01-0.03 g/L of glycine, 0.02-0.05g/L of L-histidine hydrochloride monohydrate, 0.03-0.07 g/L of L-isoleucine, 0.03-0.07 g/L of L-leucine, 0.07-0.12 g/L of L-lysine hydrochloride, 15-20 m g/L of L-methionine, 0.02-0.05g/L of L-phenylalanine, 0.01-0.03 g/L of L-proline, 0.02-0.05g/L of L-serine, 0.04-0.06 g/L of L-threonine, 8-10 mg/L of L-tryptophan, 0.04-0.06 g/L of L-tyrosine disodium salt dihydrate and 0.04-0.06 g/L of L-valine.

Preferably, the vitamins include the following species and their corresponding concentrations in broth a: vitamin H2-5 mu g/L, choline chloride 7-10 mg/L, folic acid 1.5-3 mg/L, inositol 0.1-0.2 g/L, nicotinamide 1-4 mg/L, vitamin B51-4 mg/L, vitamin B61-4 mg/L, vitamin B20.1-0.3 mg/L, vitamin B11-3 mg/L and vitamin B120.5-1 mg/L.

Aiming at the nutrition requirement of primary cell proliferation, the kit for primary cell culture provides a culture solution A meeting the nutrition required by primary cell proliferation, and the nutrition provided by the culture solution A is balanced by limiting the types and contents of amino acid, inorganic salt and vitamin contained in the culture solution A, thereby being beneficial to the absorption and transformation of the primary cells.

Preferably, other additives are added into the culture solution A, and the other additives comprise at least one of D-glucose, hydroxyethylpiperazineethiosulfonic acid, hypoxanthine, linoleic acid, phenol red, putrescine, sodium pyruvate, DL-lipoic acid and thymidine.

Preferably, the other additives include DL-lipoic acid; in the culture solution A, the concentration of DL-lipoic acid is 0.05-0.15 mg/L. In the process of primary cell proliferation and growth, the consumption speed of vitamins, especially vitamin C and vitamin E is high, while the content of vitamin C and vitamin E in a culture medium is low and is not enough to meet the utilization requirement of primary cells, and DL-lipoic acid can be used as a temporary supplement.

Preferably, the other additives include hydroxyethylpiperazineethiosulfonic acid and putrescine; in the culture solution A, the concentration of the hydroxyethylpiperazineethiosulfonic acid is 3-5 g/L, and the concentration of the putrescine is 50-100 mu g/L.

In the process of primary cell proliferation, strict requirements are imposed on the pH value level of a culture medium, in a culture solution A, the pH value level of the culture medium is synergistically regulated by hydroxyethyl piperazinethiosulfonic acid and putrescine, so that the pH value of the culture medium is stabilized within a certain range, and the drastic change of the pH value level of the culture medium caused by the consumption of nutrient substances in the culture medium is avoided. In addition, the inventor also finds that the addition of putrescine can also remarkably improve the proliferation speed of primary cells, and the reason is probably that the putrescine can be combined with polyamine regulatory sites of NMDA receptors to strengthen the generation of NMDA induction and promote the absorption and utilization of trace elements by organisms, but detailed action mechanisms are still to be researched.

Preferably, the other additives include hypoxanthine and thymidine; in the culture solution A, the concentration of hypoxanthine is 2-4 mg/L, and the concentration of thymidine is 0.2-0.5 mg/L.

According to the DNA synthesis pathway in cells, in the proliferation process of primary cells, hypoxanthine and thymidine are used as raw materials, and the two are catalyzed by enzyme to generate corresponding nucleotides, so that the synthesis pathway of nucleotides from amino acids and other small molecular compounds is a supplementary pathway of the main nucleotide production pathway. This ensures that the primary cells are sufficiently supplied with amino acids during their growth and proliferation.

According to another aspect of the present invention, there is provided a method of culturing primary cells using the above-described kit for primary cell culture, characterized by comprising the steps of: s1, preparing a culture solution B and a dissociation solution: the culture solution B comprises culture solution A + 5-20% fetal bovine serum 1X GlutaMAX^TMThe supplementary solution +1X penicillin-streptomycin-penicillin solution + 70-120 ng/mL EGF + 70-120 ng/mL bFGF, and the dissociation solution comprises the following components: the kit comprises a culture solution A, fetal calf serum, collagenase IV, DNase I and a mixed solution of penicillin-streptomycin-amphotericin B, wherein the volume ratio of the dosage of the culture solution A to the volume of the mixed solution of fetal calf serum, DNase I to the volume of the mixed solution of penicillin-streptomycin-amphotericin B is as follows: culture solution A: fetal bovine serum: DNA enzyme I: mixed solution of penicillin-streptomycin-amphotericin B35-4: 6-15: 2-3: 0.1-1, the concentration of collagenase IV in the dissociation liquid is 0.4-0.5 g/L; s2, shearing a tissue sample in the culture solution A; s3, placing the tissue sample treated in the S2 into dissociation liquid for digestion, and collecting cells after digestion; and S4, placing the cells obtained in the step S3 in a culture solution B for culture.

Preferably, S1 further includes the steps of: preparing a preservation solution, wherein the preservation solution comprises the following components: preparing a cleaning solution from a culture solution A, 5-20% fetal calf serum and 1X penicillin-streptomycin-neomycin solution, wherein the cleaning solution comprises the following components: normal saline and 0.5-1.2% penicillin-streptomycin-amphotericin B mixed solution; the specific operation of S2 is: the obtained tissue sample is preserved in preservation solution and brought to a cell culture place, the tissue sample is placed in culture solution A, blood clots and fat on the surface of the tissue sample are removed from the culture solution A, the tissue sample is cleaned by cleaning solution, the tissue sample is placed in the culture solution A again, and the tissue sample is cut into pieces in the culture solution A.

According to the method, the reagents provided by the kit for culturing the primary cells are prepared into the solutions required by each stage, and the solutions are applied to the culture of the primary cells, so that the efficiency of the primary cell culture can be effectively improved.

Drawings

FIG. 1 is a statistical plot of cell numbers plotted against the data provided in Table 1.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1

1. Assembly of the kit

The reagents required to assemble the kit for primary cell culture of this example were as follows: culture solution A, Fetal Bovine Serum (FBS), GlutaMAX^TMReplenisher, penicillin-streptomycin-neomycin solution, EGF (epidermal growth factor, bFGF (basic fibroblast growth factor), collagenase IV, DNase I, penicillin-streptomycin-amphotericin B mixed solution, and normal saline, wherein GlutaMAX is a natural product of natural plant origin, and natural plant origin^TMThe major component of the supplement, L-alanine-L-glutamine dipeptide, was purchased from ThermoFisher scientific. The composition of culture solution A is shown in Table 1.

TABLE 1 composition of the particular constituents of culture A referred to in this example

The reagent group composed of the above reagents is the core component of the kit for primary cell culture provided in this embodiment.

2. Tissue sample sources for primary cell culture

The tissue sample used in this example was lung tissue collected from general thoracic surgery at general hospital of medical university and surgically excised for lung cancer.

3. Primary cell culture experiment

3.1 preparation of the desired reagents

At normal temperature, preparing a culture solution B, a preservation solution, a dissociation solution and a cleaning solution according to the following components, wherein the culture solution B needs to be prepared before each use, and if the dissociation solution is prepared for later use, the dissociation solution is frozen below 0 ℃.

Culture solution B culture solution A + 10% FBS +1X GlutaMAX^TMMake-up +1 Xpenicillin-streptomycin-penicillin solution +100ng/mL EGF +100ng/mL bFGF.

Preservation solution: culture solution A + 10% FBS +1X penicillin-streptomycin-neomycin solution.

Dissociation liquid: 40mL of the culture solution A +10mL of FBS +0.5mL of the mixed solution of penicillin-streptomycin-amphotericin B (triple antibody) +25mg of collagenase IV +2.5mL of DNase I (2000U/mg) (10 mL of DMEM was added per bottle for dissolution).

Cleaning solution: saline + 1% triple antibody.

3.2 pretreatment of reagents and devices

3.2.1 Sterilization: sterilizing and drying experimental equipment such as scissors, tweezers, blades, needle holders, culture bottles, culture dishes and the like under high pressure.

3.2.2 bottle laying: one flask was filled with prepared 0.1% gelatin and placed in the incubator for 1 hour before use.

3.2.3 preheating: before the experiment, the culture solution A, the culture solution B and the preservation solution are preheated in a water bath kettle at 37 ℃, and the dissociation solution is subjected to warm melting.

3.3 treatment of tissue samples

3.3.1 tissue samples obtained by surgery are placed in a preservation solution and placed in an ice box to be brought back to the cell culture laboratory.

3.3.2 in the culture solution A, removing the tissues such as blood clots, fat and the like on the surface of the tissue sample.

3.3.3 the tissue sample is rinsed in a centrifuge tube filled with a cleaning solution 3-4 times, then the tissue sample is transferred to a culture dish and cleaned twice by the cleaning solution.

3.3.4 placing the washed tissue sample into a clean petri dish, adding a small amount of culture solution A, and shearing the tissue sample with scissors and forceps until no obvious large tissue blocks exist.

3.3.5 freshly prepared dissociation solution 2 mL-5 mL (in other examples, dissociation solution is added according to the size of the tissue sample, standard 1 cm)³5mL) was placed in a 37 ℃ incubator and digested for 30 minutes, during which time digestion was continuously observed.

3.3.6 after digestion, the tissue samples were transferred to a 15mL centrifuge tube, the tube was placed in a centrifuge and centrifuged at 1000rpm for 8 minutes with the cells in the lower pellet, and the supernatant was discarded to collect the cells.

3.3.7 washing the cells collected in the previous step with 200g of preservation solution twice for 5 min each time, centrifuging the mixed solution containing the cells after each washing, and discarding the supernatant to collect the cells.

3.3.8 adding the cells collected in the previous step into the culture solution B, mixing, spreading the cell suspension in a culture bottle paved with 0.1% gelatin, and transferring the culture bottle to a 37 ℃ incubator.

3.4 culture of tissue samples

3.4.1 setting three days before entering the formal culture period as a pre-culture period, in the pre-culture period, carrying out half liquid change on cell suspension to be cultured every day, and preheating a culture solution B for liquid change to 37 ℃ before liquid change.

3.4.2 entering the main culture period the next day after the pre-culture period, collecting the cell suspension supernatant after the pre-culture period after the main culture period, collecting the supernatant into a 15mL centrifuge tube, and washing the flask with culture solution A twice.

3.4.3 the supernatant from the previous step was centrifuged at 1000rpm for 5 minutes and the supernatant from the centrifugation was discarded.

3.4.4 adding fresh culture solution B into the precipitate collected in the previous step, mixing, adding into the original culture flask, observing under microscope, and culturing in incubator.

3.4.5 in the culture process, the whole culture solution is changed every 2-3 days, and the culture solution B for changing the culture solution is preheated to 37 ℃.

Comparative example 1

As a comparative example of example 1, this example used the kit for primary cell culture provided in example 1 to perform a cell culture experiment, which was different from example 1 in that: this example prepared medium B with reagents provided in the kit just before the start of the cell culture experiment, and prepared medium B before the experiment was performed during the cell culture experiment, without preparing medium B before the start of each step involving medium B. In addition to the above differences, the operation and parameter settings of the various steps of the cell culture experiments performed in this example were strictly consistent with those of example 1.

Comparative example 2

As a comparative example to example 1, this example carried out the culture experiment of primary cells using commercial DMEM/F-12 medium (purchased from ThermoFisher Scientific) and RPMI Medium 1640 (purchased from ThermoFisher Scientific).

In this example, the composition of each reagent is as follows:

culture solution C: DMEM/F-12 culture medium added with 10% FBS, and the washing solution is RPMI culture medium 1640;

digestion solution: DMEM/F-12 medium supplemented with trypsin;

washing liquid: RPMI medium 1640.

The specific process of primary cell culture is as follows:

1. pretreatment of reagents and devices

1.1, sterilization: sterilizing and drying experimental equipment such as scissors, tweezers, blades, needle holders, culture bottles, culture dishes and the like under high pressure.

1.2 bottle laying: one flask was filled with prepared 0.1% gelatin and placed in the incubator for 1 hour before use.

2 treatment of tissue samples

2.1 tissue samples obtained by surgery were placed in culture medium C and returned to the cell culture laboratory in an ice box.

2.2 in the culture solution C, the tissue such as blood clot and fat on the surface of the tissue sample is removed.

And 2.3, rinsing the tissue sample in a centrifuge tube filled with a washing solution for 3-4 times, transferring the tissue sample to a culture dish, and washing twice by using the washing solution.

2.4 placing the washed tissue sample into a clean culture dish, adding a small amount of culture solution C, and shearing the tissue sample by scissors and tweezers until no obvious large tissue blocks exist.

2.5 add digestion solution 2-5 mL (in other examples, add dissociation solution according to tissue sample size, standard 1cm3/5mL), put into incubator to digest for 30 minutes, during which time the digestion is continuously observed.

2.6 after digestion, the tissue samples were transferred to a 15mL centrifuge tube, the centrifuge tube was placed in a centrifuge and centrifuged at 1000rpm for 8 minutes with the cells in the lower pellet, and the supernatant was discarded to collect the cells.

2.7 washing the cells collected in the previous step with 200g of culture medium C twice for 5 minutes each time, centrifuging the mixed solution containing the cells after each washing, and discarding the supernatant to collect the cells.

2.8 adding the cells collected in the previous step into the culture solution C, uniformly mixing, paving the cell suspension in a culture bottle paved with 0.1% gelatin, and transferring the culture bottle to an incubator.

3 culture of tissue samples

3.1 setting three days before entering the formal culture period as a pre-culture period, and in the pre-culture period, adopting the culture solution C to perform half liquid change on the cell suspension to be cultured every day.

3.2 entering a formal culture period on the next day after the pre-culture period, collecting the cell suspension clear liquid after the pre-culture period after the formal culture period, collecting the clear liquid into a 15mL centrifuge tube, and cleaning the culture bottle twice by adopting the culture solution C.

3.3 centrifuging the supernatant obtained in the previous step at 1000rpm for 5 minutes, and discarding the supernatant obtained after centrifugation.

3.4 adding fresh culture solution C into the precipitate collected in the previous step, mixing, adding into the original culture flask, observing under microscope, and culturing in incubator.

3.5 in the culture process, the culture solution C is adopted to carry out total solution change every 2 to 3 days.

Example 2

This example is a test example, and in the experiment process of example 1, comparative example 1 and comparative example 2, the cells of the primary culture were counted, and the counting results are shown in table 2 and fig. 1, so it can be seen that the efficiency of the primary cell culture can be significantly improved by performing the primary cell culture experiment using the reagent provided by the kit for primary cell culture of example 1, compared to the existing commercial culture medium. Example 1, comparative example 1 and comparative example 2 the primary cell culture method provided in example 1 is most beneficial for the propagation of primary cells.

TABLE 2 cell culture number (ten thousand) of each example after entering the official culture period

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.

Claims

1. A kit for primary cell culture, comprising: culture solution A, fetal bovine serum, GlutaMAX^TMThe preparation method comprises the following steps of (1) supplementing liquid, a penicillin-streptomycin-neomycin solution, EGF, bFGF, collagenase IV, DNase I and a penicillin-streptomycin-amphotericin B mixed solution;

inorganic salt, amino acid and vitamin are added into the culture solution A;

the inorganic salt comprises at least one of calcium chloride, copper sulfate, ferric nitrate, ferrous sulfate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, monosodium phosphate, disodium phosphate and zinc sulfate;

the amino acid comprises at least one of L-alanine, L-arginine hydrochloride, L-asparagine monohydrate, L-aspartic acid, L-cysteine hydrochloride monohydrate, L-cystine dihydrochloride, L-glutamic acid, L-glutamine, glycine, L-histidine hydrochloride monohydrate, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate and L-valine;

the vitamins include at least one of vitamin H, choline chloride, folic acid, inositol, nicotinamide, vitamin B5, vitamin B6, vitamin B2, vitamin B1, and vitamin B12.

2. The kit for primary cell culture according to claim 1, wherein the inorganic salts comprise the following species and their corresponding concentrations in the culture broth a:

0.1-0.15 g/L of calcium chloride, 0.5-1 μ g/L of copper sulfate, 10-80 μ g/L of ferric nitrate, 0.2-0.5mg/L of ferrous sulfate, 0.05-1 g/L of magnesium sulfate, 0.2-0.5 g/L of potassium chloride, 1-1.5 g/L of sodium bicarbonate, 5-10 g/L of sodium chloride, 0.05-0.1 g/L of monosodium phosphate, 0.05-0.1 g/L of disodium phosphate and 0.3-0.7 mg/L of zinc sulfate.

3. The kit for primary cell culture according to claim 1, wherein the amino acids comprise the following species and their corresponding concentrations in the culture broth a:

3-4 mg/L of L-alanine, 0.1-0.2 g/L of L-arginine hydrochloride, 5-10 mg/L of L-asparagine monohydrate, 5-8 mg/L of L-aspartic acid, 0.01-0.02 g/L of L-cysteine hydrochloride monohydrate, 0.02-0.05g/L of L-cystine dihydrochloride, 5-10 mg/L of L-glutamic acid, 0.2-0.5 g/L of L-glutamine, 0.01-0.03 g/L of glycine, 0.02-0.05g/L of L-histidine hydrochloride monohydrate, 0.03-0.07 g/L of L-isoleucine, 0.03-0.07 g/L of L-leucine, 0.07-0.12 g/L of L-lysine hydrochloride, 15-20 mg/L of L-methionine, 0.02-0.05g/L of L-phenylalanine, 0.01-0.03 g/L of L-proline, 0.02-0.05g/L of L-serine, 0.04-0.06 g/L of L-threonine, 8-10 mg/L of L-tryptophan, 0.04-0.06 g/L of L-tyrosine disodium salt dihydrate and 0.04-0.06 g/L of L-valine.

4. The kit for primary cell culture according to claim 1, wherein the vitamins comprise the following species and their corresponding concentrations in the culture broth a:

vitamin H2-5 mu g/L, choline chloride 7-10 mg/L, folic acid 1.5-3 mg/L, inositol 0.1-0.2 g/L, nicotinamide 1-4 mg/L, vitamin B51-4 mg/L, vitamin B61-4 mg/L, vitamin B20.1-0.3 mg/L, vitamin B11-3 mg/L and vitamin B120.5-1 mg/L.

5. The kit for primary cell culture according to claim 1, wherein the culture solution a further comprises other additives, the other additives comprising at least one of D-glucose, isethionic acid, hypoxanthine, linoleic acid, phenol red, putrescine, sodium pyruvate, DL-lipoic acid, and thymidine.

6. Kit for primary cell culture according to claim 5, characterized in that: the other additives include DL-lipoic acid; in the culture solution A, the concentration of DL-lipoic acid is 0.05-0.15 mg/L.

7. Kit for primary cell culture according to claim 5, characterized in that: the other additives comprise hydroxyethyl piperazinethionamic acid and putrescine; in the culture solution A, the concentration of the hydroxyethylpiperazineethiosulfonic acid is 3-5 g/L, and the concentration of the putrescine is 50-100 mu g/L.

8. Kit for primary cell culture according to claim 5, characterized in that: the other additives comprise hypoxanthine and thymidine; in the culture solution A, the concentration of hypoxanthine is 2-4 mg/L, and the concentration of thymidine is 0.2-0.5 mg/L.

9. A method of culturing primary cells using a kit for primary cell culture according to any of claims 1-8, comprising the steps of:

s1, preparing a culture solution B and a dissociation solution:

the culture solution B comprises the following components of the culture solution A + 5-20% of fetal bovine serum 1X and GlutaMAX^TMMake-up solution +1X said penicillin-streptomycin-penicillin solution + 70-120 ng/mL said EGF + 70-120 ng/mL said bFGF,

the dissociation liquid comprises the following components: the culture solution A, the fetal calf serum, the collagenase IV, the DNase I and the mixed solution of the penicillin-streptomycin-amphotericin B are mixed, wherein the volume ratio of the dosage of the culture solution A to the volume ratio of the fetal calf serum to the mixed solution of the DNase I to the mixed solution of the penicillin-streptomycin-amphotericin B is as follows: the culture solution A: the fetal bovine serum: the DNA enzyme I: the mixed solution of penicillin-streptomycin-amphotericin B is 35-4: 6-15: 2-3: 0.1-1, the concentration of the collagenase IV in the dissociation liquid is 0.4-0.5 g/L;

s2, shearing a tissue sample in the culture solution A;

s3, placing the tissue sample treated in the step S2 in the dissociation liquid for digestion, and collecting cells after digestion;

s4, placing the cells obtained in the S3 into the culture solution B for culture.

10. A method of culturing primary cells using a kit for primary cell culture according to claim 9, wherein:

the S1 further includes the steps of:

preparing a preservation solution, wherein the preservation solution comprises the following components: the culture solution A + 5-20% of fetal bovine serum +1X of the penicillin-streptomycin-neomycin solution,

preparing a cleaning solution, wherein the cleaning solution comprises the following components: normal saline and 0.5-1.2% penicillin-streptomycin-amphotericin B mixed solution;

the specific operation of S2 is:

and preserving the obtained tissue sample in the preservation solution to a cell culture place, placing the tissue sample in the culture solution A, removing blood clots and fat on the surface of the tissue sample in the culture solution A, cleaning the tissue sample by using the cleaning solution, placing the tissue sample in the culture solution A again, and shearing the tissue sample in the culture solution A.