CN111808816A

CN111808816A - Culture medium for culturing primary cells of gastric cancer solid tumors

Info

Publication number: CN111808816A
Application number: CN201910289074.4A
Authority: CN
Inventors: 尹申意; 张函槊
Original assignee: Beijing Genex Health Technology Co ltd
Current assignee: Beijing Genex Health Technology Co ltd
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2020-10-23

Abstract

The invention discloses a culture medium for culturing primary cells of gastric cancer solid tumors. The invention provides a method for culturing primary cells of gastric cancer solid tumor and a matched reagent, wherein a special serum-free culture medium is prepared, and a suspension culture system is used for culturing tumor cells derived from gastric cancer solid tumor in vitro, so that the interference of normal cells is eliminated to the maximum extent while the normal amplification of the cancer cells is ensured. The gastric cancer primary cell culture obtained by the method can be used for in-vitro experiments, next generation sequencing, animal model construction, cell line construction and the like of various cell levels. It is expected that the culture method and the special culture medium provided by the invention have wide application prospects in the fields of research and clinical diagnosis and treatment of gastric cancer.

Description

Culture medium for culturing primary cells of gastric cancer solid tumors

Technical Field

The invention relates to the technical field of biology, in particular to a culture medium for culturing gastric cancer solid tumor primary cells.

Background

Gastric cancer is one of the most common health malignancies that severely threaten humans. China is a country with high incidence of gastric cancer, and the incidence number and the death number of the gastric cancer respectively account for 42.6 percent and 45 percent of the incidence number and the death number of the stomach cancer in the world. The incidence rate of gastric cancer in China is 11.8%, and the gastric cancer accounts for the fourth place in all malignant tumors. The mortality rate of gastric cancer is 22.0%, and the cancer accounts for the fifth place in all malignant tumors. With the development of economy, the improvement of living standard and the change of life style, the incidence rate of gastric cancer is on the rising trend. In addition, the risk of recurrence and metastasis of gastric cancer is high, and more than 50% of gastric cancer patients have different degrees of recurrence and metastasis within months to years after radical treatment.

Although research into the etiology and development of gastric cancer by scientific and medical institutions in various countries of the world has been heavily invested, human beings are still poorly aware of the disease. Gastric cancer is a complex disease, the occurrence and development of which are dynamic processes, and the interaction of a plurality of signal molecules is involved, so that a complex molecular regulation network is formed and is influenced by external environmental factors. The etiology and development process of gastric cancer are highly variable among individuals and cannot be determined in a short time. Therefore, the trend of individual accurate research by using the primary cell culture of the gastric cancer solid tumor as a model is the research field of gastric cancer and even the diagnosis and treatment field of gastric cancer.

The existing primary tumor cell culture technology mainly comprises 2D culture, 3D culture, reprogramming culture and the like, and the methods all face the problems of extremely long culture period, low culture success rate, difficult removal of mixed cells and the like in different degrees.

Disclosure of Invention

In order to effectively solve the technical problems, the invention provides a novel gastric cancer solid tumor primary cell culture technology and a matched reagent.

In a first aspect, the invention claims a medium for culturing primary cells of gastric carcinoma solid tumors.

The culture medium for culturing the primary cells of the gastric cancer solid tumor consists of antibacterial antifungal agents of three antibiotics (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein R-spondin, human recombinant protein Wnt-3a, human recombinant protein Noggin, SB202190(4- (4-fluorophenyl) -2- (4-hydroxyphenyl) -5- (4-pyridyl) -1H-imidazole), A83-01(3- (6-Methyl-2-pyridyl) -N-phenyl-4- (4-quinolyl) -1H-pyrazozole-1-carbothioamide), Primocin, N-acetyl-L-cysteine (N-acetyl-L-cysteine), Nicotine (Nicotinamide), N-2Supplement, Cholera Toxin (Cholera Toxin), B27, Y-27632, Gastrin (Gastrin) and advanced DMEM/F12 medium.

Wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-200 mu g/mL (such as 100 mu g/mL); the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the final concentration of GlutaMax is 0.8-1.2% (e.g., 1%,% represents volume percent); the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein FGF-10 is 5-25 ng/mL; the final concentration of the human recombinant protein R-spondin is 250-500 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL; the final concentration of the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the SB202190 is 5-10 μ M; the final concentration of the A83-01 is 0.25-1.25 mu M; the final concentration of the Primocin is, for example, 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine (N-acetyl-L-cysteine) is 0.5-2 mM; the final concentration of nicotine (Nicotinamide) is 5-10 mM; the final concentration of the N-2Supplement is 1 percent (volume percentage); the final concentration of the Cholera Toxin (Cholera Toxin) is 0.1-1 nM; the final concentration of B27 is 1.5-2.5% (e.g., 2%,% indicates volume percent); the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the Gastrin (Gastrin) is 5-20 nM; the balance is Advanced DMEM/F12 medium. The final concentrations of the above substances are all the final concentrations in the culture medium for culturing the gastric cancer solid tumor primary cells.

Further, the composition of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is as follows: each ml contains 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B. The antibacterial antifungal agent is three-resistant (Penicillium notatum)Biotin-streptomycin-amphotericin B) is "Antibiotic-Antibiotic, 100X" (e.g., Gibco #15240062, or other products of the same composition). The "Antibiotic-Antibiotic, 100X" contained 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B per ml, using penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline as the active ingredients

An antifungal agent. The GlutaMAX is GlutaMAX^TMSupplement "(e.g., Gibco #35050061, or other products of the same composition). The "GlutaMAX^TMThe Supplement "was composed of L-allyl-L-glutamine as a substitute for L-glutamine at a concentration of 200nM in a 0.85% NaCl solution. The Primocin^TMAntibiotics used for protecting primary cells from microbial contamination, which are antibacterial agents for primary cells (such as Invivogen # ant-pm-1, or other products having the same composition), have killing effects on gram-positive bacteria, gram-negative bacteria, mycoplasma, and fungi. The N-2Supplement is "N-2 Supplement (100X)" (e.g., Gibco #17502001, or other products of the same composition). The "N-2 Supplement (100X)" contained Human total Transferrin (Human Transferrin (Holo)) at a final concentration of 1mM, 500mg/L Recombinant Insulin Full Chain (Insulin Recombinant Full Chain), 0.63mg/L Progesterone (Progesterone), 10mM Putrescine (Putrescine), and 0.52mg/L Selenite (Selenite). The B27 is' B-27^TMSupplement (50X), minus vitamin A "(e.g., Gibco #12587010, or other products of the same composition). Said "B-27^TMExample 50X, minus vitamin A "contains Biotin (Biotin), DL-Alpha-tocopheryl Acetate (DLalpha Tocopherol Acetate), DL-Alpha-Tocopherol (DL Alpha-Tocopherol), BSA (fat acid fragment V), Catalase (Catalase), Human recombinant insulin (Human recombinant insulin), Human Transferrin (Human Transferrin), Superoxide Dismutase (Superoxide Dismutase), Corticosterone (Corticterone), D-Galactose (D-Galactose), Ethanolamine hydrochloric acid (Ethanolamine HCl), glutathione reduced (Glutathione), carnitine L-saltAcid (L-Carnitine HCl), Linoleic Acid (Linoleic Acid), Linolenic Acid (Linolenic Acid), Progesterone (Progesterone), Putrescine (Putrescine2HCl), Sodium Selenite (Sodium Selenite), triiodothyronine (T3 (triodo-I-thyronine)). The GlutaMAX is a high-grade cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMAX is GlutaMAX^TMSupplement "(e.g., Gibco #35050061, or other products of the same composition). Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor with Ki of 220nM and 300nM, respectively)" (e.g. MCE #129830-38-2, or other products of the same composition).

In a specific embodiment of the invention, the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is under the brand code Gibco # 15240062; the brand of HEPES is Gibco # 15630080; the brand name of GlutaMAX is Gibco # 35050061; the brand of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the brand of the human recombinant protein HGF is Peprotech AF-100-39-100; the brand code of the human recombinant protein FGF-10 is PeprotechAF-100-26-100; the brand goods number of the human recombinant protein R-spondin is Shanghai nearshore # CD 83; the brand and commodity number of the human recombinant protein Wnt-3a is R & D5036-WN-500; the brand goods number of the human recombinant protein Noggin is Shanghai near shore # C018; the brand of the SB202190 is Sigma # S7067; the brand name of A83-01 is Tocris # 2939; the brand name of the Primocin is Invivogen # ant-pm-1; the brand and cargo number of the N-acetyl-L-cysteine is Sigma # A9165; the brand of Nicotinamide is Sigma # N0636; the brand goods number of the N-2Supplement is Gibco # 17502001; the brand name of Cholera Toxin is Listlab # 100B; the brand name of B27 is Gibco # 12587010; the brand goods number of the Y-27632 is MCE # 129830-38-2; the brand goods number of Gastrin is NJPeptide # Pep 12307; the brand of the Advanced DMEM/F12 medium is Gibco # 12634010.

Further, the culture medium for culturing the gastric cancer solid tumor primary cells may exist in two forms:

the culture medium for culturing the primary cells of the gastric cancer solid tumor is a solution prepared by mixing the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B), the HEPES, the GlutaMax, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein R-spondin, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the SB202190, the A83-01, the Primocin, the N-acetyl-L-cysteine, the nicotine, the N-2Supplement, the cholera toxin, the B27, the Y-27632, the gastrin and the Advanced DMEM/F12 culture medium.

The media was prepared and sterilized by filtration through a 0.22 μ M needle filter (Millipore SLGP033RS) and stored at 4 ℃ for two weeks.

Secondly, each component of the culture medium for culturing the gastric cancer solid tumor primary cells exists independently and is prepared according to a formula when in use.

Furthermore, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein R-spondin, the human recombinant protein Wnt-3a and the human recombinant protein Noggin can exist in a stock solution (mother solution) form, can be stored for a long time at the temperature of minus 80 ℃, and can be specifically 1000 times of the stock solution (mother solution). SB202190, N-acetyl-L-cysteine, Nicotinamide, Y-27632 and gastrin can exist in stock solution (mother liquor) form (-20 deg.C for long-term storage), specifically 1000 times stock solution (mother liquor). Cholera Toxin can exist in a stock solution (mother liquor) form (the stock solution can be stored for a long time at the temperature of 20 ℃), and specifically can be 10000 times of the stock solution (the mother liquor). A83-01 can exist in stock solution (mother liquor) form (long-term storage at-20 deg.C), specifically 100000 times stock solution (mother liquor).

The 1000 Xhuman recombinant protein EGF stock solution consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The stock solution of 1000 Xhuman recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein HGF stock solution consists of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant proteins HGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein FGF-10 stock solution consists of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein R-spondin stock solution is composed of human recombinant protein R-spondin, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin is 250 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 Xhuman recombinant protein Wnt-3a stock solution consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

The 1000 multiplied human recombinant protein Noggin stock solution consists of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

In the five 1000-fold stock solutions, the BSA can be present (ready for formulation) in the form of 100-fold stock solution (mother liquor), and specifically consists of BSA and PBS, wherein the final concentration of BSA (Sigma # A1933) is 0.1g/mL, and the balance is PBS.

Additionally, the 1000 × SB202190 stock consisted of SB202190 and DMSO, with the final concentration of SB202190 being 10mM, the balance being DMSO.

The 100000 XA 83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25mM, and the balance is DMSO.

The 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is the ultrapure water.

The 1000 XNicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of the Nicotinamide is 5M, and the balance is the ultrapure water.

10000 XCholera Toxin stock solution consists of Cholera Toxin and Cholera Toxin solution, wherein the final concentration of Cholera Toxin is 10 μ M, and the rest is Cholera Toxin solution. The Cholera Toxin dissolving solution comprises the following components: each 10mL of the Cholera Toxin solution contained Tris (1M) pH 7.00.05M, NaCl0.2M, sodium azide (3 mM), EDTA (0.5M) pH 8.01mM, and the balance ultrapure water.

1000 XY-27632 consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.

1000 × gastrin is composed of gastrin and sterile water, wherein the final concentration of gastrin is 10 μ M, and the balance is sterile water.

In a second aspect, the invention claims a kit for culturing primary cells of gastric carcinoma solid tumors.

The kit for culturing the primary cells of the gastric cancer solid tumor provided by the invention comprises the culture medium and at least one of the following reagents: digestion stop solutions and the cell cryopreservation solution described below.

In a third aspect, the invention claims the use of the culture medium or the kit for culturing primary cells of gastric carcinoma solid tumors.

In a fourth aspect, the invention claims a method of culturing primary cells of gastric carcinoma solid tumors.

The method for culturing the primary cells of the gastric cancer solid tumor provided by the invention specifically comprises the following steps: and (3) performing suspension culture on the gastric cancer solid tumor primary cells by using the culture medium.

Further, the method comprises the steps of: using a culture container with a low-adsorption surface (low-adsorption surface), performing suspension culture on the gastric cancer solid tumor primary cells by using the culture medium at 37 ℃ and 5% CO₂Culturing is carried out under conditions in which the medium is changed every 2 to 4 days (e.g., 3 days) until the cells form a mass of 50 to 80 μm (e.g., 80 μm) in diameter.

Wherein the initial seeding density may be 10⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density of individual cells was plated.

Further, the method may further comprise the steps of: and (3) when the primary gastric cancer solid tumor cells form a mass with the diameter of 50-80 mu m (such as 80 mu m), carrying out passage on the primary gastric cancer solid tumor cells.

Wherein, the digestion stop solution adopted during the passage (can be stored for one month at 4 ℃ after being prepared) consists of fetal calf serum, three antibiotics (penicillin-streptomycin-amphotericin B) of an antibacterial antifungal agent and a DMEM medium; wherein the final concentration of the fetal calf serum in the digestion stop solution is 8-12% (such as 10%,% represents volume percentage content); the final concentration of penicillin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the digestion stop solution is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) in the digestion stop solution is 100-200 [ mu ] g/mL (such as 100 [ mu ] g/mL); the final concentration of amphotericin B in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the digestion stop solution is 250-500ng/mL (such as 250 ng/mL); the balance is DMEM medium.

Further, the composition of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is as follows: each ml contains 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B. The antimicrobial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is "antibacterial-antibacterial, 100X" (e.g., Gibco #15240062, or other products of the same composition). The "Antibiotic-Antibiotic, 100X" contained 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B per ml, using penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline as the active ingredients

An antifungal agent.

In a specific embodiment of the invention, the brand of fetal bovine serum is Gibco # 16000-; the brand code of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is Gibco # 15240062; the DMEM medium is sold under the brand name Gibco # 11965-092.

More specifically, the step of performing said passaging is carried out: collecting cell masses to be passaged, washing the cell masses by using a sterile PBS solution after centrifugation, then centrifuging, then re-suspending the cell masses by using a cell digestion solution, digesting at 37 ℃ until the cell masses are digested into single cells, stopping digestion reaction by using the digestion stop solution (the dosage can be 5-10 times, for example, 10 times of the volume), and collecting cell suspension; after centrifugation, the cell pellet is resuspended with the medium, counted, and cells are then suspension cultured using a culture vessel with a low adsorption surface (initial seeding density can be 10⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density plating of individual cells), culture conditions were 37 ℃ and 5% CO₂. All the centrifugation in the above-mentioned passaging step may be specifically 800-1000g (e.g., 800g) at room temperature for 10-20 minutes (e.g., 10 minutes).

Further, the method can also comprise the step of performing cryopreservation and/or resuscitation on the gastric cancer solid tumor primary cells after the expansion of 2-3 passages.

Wherein, the cell frozen stock solution (which is used as the frozen stock solution) adopted during the freezing is composed of Advanced DMEM/F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2), such as 20:2: 1; the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

In a specific embodiment of the invention, the Advanced DMEM/F12 medium is under the brand code Gibco # 12634010; the brand code of the DMSO is Sigma # D2438; the brand of methylcellulose is Sigma # M7027.

Further, the specific steps of the cryopreservation are as follows: collecting cell mass to be cryopreserved, centrifuging, washing the cell mass with sterile PBS solution, centrifuging, suspending the cell mass with the cell digestive juice, digesting at 37 deg.C until the cell mass is digested into single cells, and adding the digestion stop solution (in an amount capable of being used as the digestion stop solution)5-10 times, e.g., 10 times, volume) to terminate the digestion reaction and collect the cell suspension; centrifuging, and freezing the cells at 0.5-2 × 10⁶/mL (e.g., 10)⁶mL), and transferring the cell sediment to liquid nitrogen for long-term storage after the cell sediment is frozen and stored overnight by a gradient cooling box. All the centrifugation in the above freezing step may be specifically 800-1000g (e.g., 800g) at room temperature for 10-20 minutes (e.g., 10 minutes).

Further, the specific steps of performing the resuscitation are: taking out the freezing tube containing the cells to be rescued from the liquid nitrogen, and rapidly thawing the cells in sterile water at 37-39 deg.C (such as 37 deg.C); after centrifugation (e.g., 800-⁵Per cm²Bottom area of container), cells per tube (10)⁶Respectively) reviving to 3.5cm culture dish), culturing at 37 deg.C and 5% CO₂。

In the first, second, third and fourth aspects, the gastric cancer may be in particular primary gastric cancer, clinically staged as stage II, III or IV (staged as TNM), or various pathologically typed gastric cancers or metastatic lesions of gastric cancer, a sample with a surgical specimen weight of more than 20 mg.

In the present invention, all of the above PBS's may be 1 XPBS, pH 7.3-7.5. The concrete composition is as follows: the solvent is water, and the solute and the concentration are as follows: KH (Perkin Elmer)₂PO₄144mg/L，NaCl 9000mg/L，Na₂HPO₄·7H₂O 795mg/L。

The invention provides a method for extracting and culturing gastric cancer solid tumor primary cells from fresh gastric cancer solid tumor tissues and a special culture medium, and the method has the following advantages:

1. the dosage of the tissue sample is less, and only about 20mg of gastric cancer operation sample is needed;

2. can be used for culturing primary tumor cells of gastric cancer primary tumors and can also be used for culturing primary tumor cells of gastric cancer metastasis focuses;

3. the culture period is short, and only 3-10 days are needed to obtain 10⁷Magnitude order gastric cancer primary tumor cells;

4. the culture stability is high, and the success rate of in vitro culture of the qualified gastric cancer operation specimen by using the method is up to 70 percent;

5. the cell purity is high, the proportion of cancer cells in the gastric cancer primary cell culture obtained by the method can reach 70-95%, and the interference of mixed cells is less.

The gastric cancer primary cell culture obtained by the method can be used for in-vitro experiments, next generation sequencing, animal model construction, cell line construction and the like of various cell levels. It is expected that the culture method has wide application prospect in the fields of research and clinical diagnosis and treatment of gastric cancer.

Drawings

FIG. 1 shows single cells obtained after treatment of gastric cancer tissues. The scale is 100 μm, 100 times magnification.

FIG. 2 shows the cell masses obtained after primary culture of gastric cancer tissues. The scale is 100 μm, 100 times magnification.

FIG. 3 is a staining chart of HE (high affinity binding) section of gastric cancer cell mass obtained after primary culture of gastric cancer tissue. The scale is 100 μm, 200 times magnification.

FIG. 4 is an immunofluorescence staining pattern of cancer cell masses obtained after primary culture of gastric cancer tissues. The scale is 50 μm, 200 times magnification

FIG. 5 shows that copy number variation analysis (CNV) performed according to the sequencing results shows that the copy number variation of each generation of gastric cancer primary cell cultures (P1, P2, P3, P4, P5) is highly consistent with that of primary gastric cancer Tumor tissue (Tumor).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of reagents for culturing gastric cancer Primary cells

1. Sample preservation solution (100mL)

The specific formulation of the specimen preservation solution (100mL) is shown in table 1.

TABLE 1 sample preservation solution (100mL)

After the preparation of the sample preservation solution is completed, the sample preservation solution is subpackaged by 15mL centrifuge tubes, and each tube is 5 mL. Can be stored at 4 deg.C for 1 month after subpackaging.

2. Sample cleaning solution (100mL)

The specific formulation of the sample rinse (100mL) is shown in table 2.

TABLE 2 sample rinse (100mL)

The sample cleaning solution needs to be prepared for use.

3. Sample dissociation liquid (10mL)

The specific formulation of the sample dissociation solution (10mL) is shown in table 3.

TABLE 3 sample dissociation solution (10mL)

Note: the sample dissociation liquid is prepared for use.

In Table 3, the formulation of collagenase stocks is shown in tables 4-6.

TABLE 410 collagenase I stock solution (100mL)

After preparing the 10 Xcollagenase I stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 510 collagenase II stock solution (100mL)

After preparing the 10 Xcollagenase II stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 620 collagenase IV stock solution (100mL)

After preparing 20 Xcollagenase IV stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

In tables 4, 5 and 6, the unit U of collagenase (said collagenase I or said collagenase IV) is defined by the enzymatic activity of the protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

4. Cell digestive juice (10mL)

The specific formulation of the cell digest (10mL) is shown in Table 7.

TABLE 7 cell digest (10mL)

The cell digestive juice is prepared for use.

5. Digestive stop solution (100mL)

The specific formulation of the digestion-stopping solution (100mL) is shown in Table 8.

TABLE 8 digestive stop solution (100mL)

The digestion stop solution can be stored for one month at 4 ℃ after being prepared.

6. Stomach cancer solid tumor primary cell culture medium (100mL)

The specific formulation of the gastric cancer solid tumor primary cell culture medium (100mL) is shown in table 9.

TABLE 9 gastric carcinoma solid tumor Primary cell culture Medium (100mL)

After the preparation of the gastric cancer solid tumor primary cell culture medium, the medium was sterilized by filtration using a 0.22 μ M needle filter (millipore slgp033RS) and stored at 4 ℃ for two weeks.

In Table 9, the preparation of human recombinant protein stocks is shown in tables 11 to 17, the preparation of SB202190 stock is shown in Table 18, the preparation of A83-01 stock is shown in Table 19, the preparation of N-acetyl-L-cysteine stock is shown in Table 20, the preparation of Nicotinamide stock is shown in Table 21, the preparation of Cholera Toxin stock is shown in Table 22, the preparation of Y-27632 stock is shown in Table 24, and the preparation of Gastrin stock is shown in Table 25. The 100 × BSA solutions required to formulate these stock solutions are shown in table 10.

TABLE 10100 XBSA solution (1mL)

The 100 × BSA solution is ready for use.

TABLE 111000 × stock solution of human recombinant protein EGF (5mL)

After 1000 Xhuman recombinant protein EGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 121000 × stock solution of human recombinant protein bFGF (2.5mL)

After 1000 Xhuman recombinant protein bEGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 131000 Xhuman recombinant protein HGF stock solution (5mL)

1000 Xthe human recombinant protein HGF stock solution is prepared and subpackaged by a sterile centrifuge tube of 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 141000 × stock solution of human recombinant protein FGF-10 (5mL)

After 1000 Xhuman recombinant protein FGF-10 stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 151000 Xhuman recombinant protein R-spondin stock solutions (4mL)

After 1000 Xhuman recombinant protein R-spondin stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 161000 Xhuman recombinant protein Wnt-3a stock solution (2.5mL)

1000 Xthe human recombinant protein Wnt-3a stock solution is prepared and then subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 171000 × human recombinant protein Noggin stock solution (5mL)

1000 times of human recombinant protein Noggin stock solution is prepared and then subpackaged by a 1.5mL sterile centrifuge tube, and the stock solution can be stored for a long time at the temperature of minus 80 ℃.

TABLE 181000 XSB 202190 stock solution (1.51mL)

After preparing the stock solution of 1000 XSB 202190, the stock solution can be stored for a long time at-20 ℃ by subpackaging with a 0.5mL sterile centrifuge tube.

TABLE 19100000 XA 83-01 stock solution (1.05mL)

After preparing a stock solution of 1000 XA 83-01, the stock solution can be stored for a long time at-20 ℃ by dispensing with a 0.5mL sterile centrifuge tube.

TABLE 201000 XN-acetyl-L-cysteine stock solutions (5mL)

After preparing a stock solution of 1000 XN-acetyl-L-cysteine, subpackaging the stock solution by using a sterile centrifuge tube with the volume of 0.5mL, and storing the stock solution at the temperature of 20 ℃ below zero for a long time.

TABLE 211000 Nicotinamide stock solutions (4mL)

1000 XNicotinamide stock solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be stored for a long time at the temperature of minus 20 ℃.

TABLE 2210000 XCholera Toxin stock solution (1.17mL)

After preparing a stock solution of 1000 XCholera Toxin, subpackaging the stock solution by using a sterile centrifuge tube of 0.5mL, and storing the stock solution at the temperature of 20 ℃ below zero for a long time.

In table 22, the formulation of the Cholera Toxin dissolution solution is shown in table 23.

TABLE 23 Cholera Toxin solution (10mL)

The Cholera Toxin dissolving solution is prepared and then subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be stored for a long time at the temperature of minus 20 ℃.

TABLE 241000 XY-27632 stock solution (3.125mL)

After preparing the stock solution of 1000 XY-27632, the stock solution is subpackaged by a sterile centrifuge tube of 0.5mL and can be stored for a long time at the temperature of minus 20 ℃.

TABLE 251000 × Gastrin stock solution (48mL)

After preparing 1000 XGastrin stock solution, subpackaging with a sterile centrifuge tube of 0.5mL, and storing the stock solution at-20 ℃ for a long time.

7. Cell cryopreservation liquid

The specific formulation of the cell culture medium is shown in Table 26.

TABLE 26 cell cryopreservation solution

The cell frozen stock solution is prepared for use at present.

In table 26, the preparation of the 1% methylcellulose solution is shown in table 27.

TABLE 271% methylcellulose solution (10mL)

The 1% methyl cellulose solution can be stored for a long time at 4 ℃ after being prepared.

Example 2 acquisition of postoperative specimens for gastric cancer

1. In cooperation with the Hospital, the cooperative development passed a formal medical ethical examination.

2. The attending physician selects patients to be grouped according to clinical indications specified by medical guidelines and selects appropriate samples for in vitro culture according to the clinical indications in surgery, the selection criteria of the samples are as follows: primary gastric cancer, pathological stages of II, III or IV, various pathologically typed gastric cancer or gastric cancer metastasis focuses, and samples with the weight of gastric cancer operation specimens more than 20 mg.

3. The primary physician provides basic clinical information such as sex, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc. of the patient. The name, the identification card number and other information of the patient related to the privacy of the patient are hidden and replaced by a uniform experiment number, and the naming principle of the experiment number is eight-digit numerical date of the collected sample plus four digits after the patient is hospitalized. For example, if the sample is provided on 1/2018, the hospitalization number of the patient is T001512765, and the sample experiment number is 201801012765.

4. During the operation, fresh specimens are collected by the surgeon in a sterile environment in the operating room and placed in a previously prepared specimen preservation solution (see example 1). The samples were kept temporarily on ice after being isolated and transported to the laboratory within two hours for further processing.

Example 3 pretreatment for dissociation of gastric cancer tissue sample

The following operations required working on ice and the entire procedure required completion within 10 minutes.

The surgical instruments used in the following operations all need to be sterilized in advance at high temperature and high pressure and can be used after being dried.

1. The samples were weighed.

2. The sample surface was rinsed with 75% (volume percent) ethanol for 10 to 30 seconds.

3. The samples were washed 10 times with sample wash and 5 times with sterile PBS solution.

4. The fat tissue, connective tissue and necrotic tissue in the sample are carefully stripped off with the aid of an ophthalmic scissors, an ophthalmic forceps, a scalpel and the like.

Example 4 gastric cancer tissue sample dissociation

The surgical instruments used in the following examples were sterilized at high temperature and high pressure in advance and dried before use.

1. Cutting the tissue into pieces of 1mm by using an ophthalmic scissors³The left and right small blocks.

2. The minced tissue samples were treated with a sample dissociation solution preheated at 37 ℃ in advance at a dose of 0.1mL of the sample dissociation solution (see example 1) per mg of tissue, and dissociation was carried out at 37 ℃ for 15 minutes to 3 hours. The dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

3. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 1) and the cell suspension was collected.

4. The cell suspension was filtered through a 40 μm sterile cell strainer to remove tissue debris and adherent cells.

5. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

6. The cells were resuspended in 5mL sterile PBS, centrifuged at 800g for 10 minutes at room temperature, and the supernatant discarded.

7. The cell pellet was resuspended in gastric carcinoma solid tumor primary cell culture medium (see example 1), and the cell status was observed under a microscope for cell counting.

As shown in FIG. 1, the dissociated single cell suspension contains a large amount of various types of cells, such as erythrocytes, lymphocytes, and fibroblasts, in addition to tumor cells. One of the advantages of the method is that in the subsequent culture process, only cancer cells can be greatly amplified, and the proportion of other cells is gradually reduced or even disappears, so that the gastric cancer primary tumor cells with higher purity are finally obtained.

Example 5 culture of gastric cancer Primary cells

1. Gastric cancer primary cell suspension culture was performed using a low-adsorption surface (low-adsorption-surface), which was the gastric cancer solid tumor primary cell culture medium of example 1 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; human recombinant protein EGF wasThe final concentration of bFGF is 20 ng/mL; the final concentration of human recombinant protein HGF is 20 ng/mL; the final concentration of the human recombinant protein FGF-10 is 20 ng/mL; the final concentration of the human recombinant protein R-spondin is 500 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 250 ng/mL; the final concentration of the human recombinant protein Noggin is 100 ng/mL; the final concentration of SB202190 was 10. mu.M; the final concentration of A83-01 was 0.5. mu.M; the final concentration of N-acetyl-L-cysteine is 1 mM; the final concentration of Nicotinamide is 10 mM; cholera Toxin final concentration of 0.1 nM; the final concentration of Y-27632 is 10. mu.M; final concentration of gastin 10nM), 10 per well, for example, in a six well plate⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

2. The cell status was observed every day, and the medium was changed every 3 days until the cells formed clumps of about 80 μm in diameter.

As shown in FIG. 2, after 3-10 days of culture, cancer cells are greatly expanded to form cell masses with the diameter of 80 μm, and the total number of tumor cells can exceed 10⁷The number of other types of cells is significantly reduced or even eliminated. Through a large number of sample tests, the success rate of in vitro culture of the gastric cancer primary tumor cells can reach 80%.

Example 6 passaging of gastric carcinoma Primary cells

1. The cell pellet was collected from the dish, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

2. The cell pellet was washed with sterile PBS solution, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 5 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 1) and the cell suspension was collected.

6. And (4) resuspending the cell sediment by using a gastric cancer solid tumor primary cell culture medium, and counting the cells.

7. Use of lowCulturing the gastric cancer primary cells on an adsorption surface (low-attachment-surface), wherein the used culture medium is the gastric cancer solid tumor primary cell culture medium in example 1, and a six-well plate is taken as an example, and 10 cells are arranged in each well⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 7 cryopreservation of gastric cancer Primary cells

After the suspension culture of the gastric cancer primary cells is subjected to passage amplification for 2-3 times, the gastric cancer primary cells can be frozen:

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 15 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and the cell suspension was collected and counted.

6. Cell cryopreservation (see example 1) at 10⁶Resuspending the cell sediment at a density of/mL, freezing 1mL of cell suspension in each tube of a 2mL freezing tube, freezing overnight by using a gradient cooling box, and transferring the cell sediment into liquid nitrogen for long-term storage.

Example 8 recovery of gastric cancer Primary cells

The stomach cancer primary cells preserved in liquid nitrogen can be recovered:

1. sterile water at 37 ℃ was prepared five minutes in advance.

2. The vial was removed from the liquid nitrogen and the cells were rapidly thawed in sterile water at 37 ℃.

3. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

4. Resuspending the cell pellet with gastric carcinoma solid tumor primary cell culture medium (see example 1), culturing gastric carcinoma primary cells using a low adsorption surface, and resuscitating the cells to 3.5 per tubeIn a cm dish, 5% CO at 37 ℃₂The culture was carried out in a cell culture incubator under the conditions.

Example 9 HE staining identification of gastric cancer Primary cells

The reagent consumables used in the following examples are illustrated:

HE staining kit (beijing solibao biotechnology limited, # G1120);

cation anticreep slide (Beijing China fir Jinqiao Biotech limited);

xylene, methanol, acetone (Beijing chemical reagent company, analytical pure);

neutral resin adhesive (fine chemicals, GmbH, Beijing).

1. 800g of solid tumor cells cultured in the culture medium for primary gastric cancer solid tumors (wherein the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein R-spondin is 500ng/mL, the final concentration of human recombinant protein Wnt-3a is 250ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 10 μ M, the final concentration of A83-01 is 0.5 μ M, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of Cholera Toxin is 0.632 nM, the final concentration of Y-27nM primary tumor cell is 10 μ M, and the final concentration of Gastrin is 10nM) in example 1 were collected by centrifugation, fix with 4% paraformaldehyde. The pellet of cells was embedded in paraffin and sliced to a thickness of 5 μm.

2. Paraffin sections were incubated in xylene solution for 5 minutes at room temperature for deparaffinization, repeated 3 times, and the sections were rinsed 2 times with deionized water.

3. Sections were incubated in absolute ethanol for 10 min at room temperature, repeated twice.

4. Ginger slices were incubated in 95% ethanol for 10 minutes at room temperature, and after repeating twice, the slices were rinsed twice with deionized water.

5. When the water on the slide is slightly dry, 100 mu L of hematoxylin staining solution is added for staining for 1 mins.

6. The hematoxylin stain was aspirated and the slides were washed 3 times with tap water.

7. 100 mu L of differentiation solution is added dropwise for differentiation for 1 mins.

8. The differentiation medium was aspirated off, and the slides were washed sequentially 2 times with tap water and 1 time with distilled water.

9. The water on the surface of the slide is sucked off, and 200 mu L of eosin dye solution is dripped to stain the slide for 40 s.

10. Absorbing eosin dye solution, rinsing and dehydrating with 75%, 80%, 90% and 100% ethanol for 20s, 40s and 40 s.

11. After the ethanol was dried, 50. mu.L of xylene was added dropwise for cell permeation.

12. After xylene is completely dried, a drop of neutral resin adhesive is added dropwise, and the piece is mounted by a cover glass, observed under a microscope and photographed.

Fig. 3 shows the HE staining effect of the primary tumor cells of gastric cancer obtained by in vitro culture, and it can be seen that these cells generally have the characteristics of cancer cells such as high nuclear-mass ratio, deep nuclear staining, chromatin condensation in the nucleus, multinucleate, and uneven cell size, and several tens to several hundreds of tumor cells aggregate to form tumor cell masses with certain three-dimensional structures.

Example 10 immunofluorescence staining identification of gastric cancer Primary cells

The reagents used in the following examples are illustrative:

paraformaldehyde (Beijing chemical reagent company, analytical pure) was dissolved in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;

methanol, dimethyl sulfoxide (Beijing chemical reagent company, analytical pure);

hydrogen peroxide (beijing chemicals, 35%);

mixing methanol, dimethyl sulfoxide and 35% hydrogen peroxide according to a volume ratio of 4:4:1 to prepare a Dan's rinsing solution;

bovine serum albumin (Sigma, # A1933) was dissolved in PBS to prepare a 3% (3g/100mL) BSA solution;

immunofluorescence primary anti-antibody (Abcam, # ab 17139);

immunofluorescent secondary antibody (CST, # 4408);

hoechst dye liquor (Beijing Sorleibao Biotech limited, # C0021);

the gastric cancer solid tumor primary cell culture medium in example 1 (wherein the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein R-spondin is 500ng/mL, the final concentration of human recombinant protein Wnt-3a is 250ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 10 μ M, the final concentration of A83-01 is 0.5 μ M, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of Cholera Toxin is 0.632 nM, the final concentration of Y-27nM is 10 μ M, and the final concentration of Gastrin is 10nM) was cultured in the following steps to obtain the gastric cancer cell foci, primary antibody is CK8+ CK18, characterizing cells of epithelial origin.

1. The cell pellet was collected from the dish, washed once with PBS, resuspended in 4% paraformaldehyde and fixed overnight at 4 ℃.

2. The supernatant was discarded by centrifugation at 800g, and the cell pellet was resuspended in a precooled methanol solution and placed on ice for 1 hour.

3. Centrifuging at 800g, discarding supernatant, resuspending cell pellet with Dan's rinsing solution, and standing at room temperature for 2 hr.

4. The supernatant was discarded by centrifugation at 800g, and the cells were washed sequentially with 75%, 50%, 25% (volume percent) methanol diluted with PBS for 10 minutes each.

5. The supernatant was discarded by centrifugation at 800g, and the cell pellet was suspended in 3% BSA solution and blocked at room temperature for 2 hours.

6. According to the following steps: 500, primary antibody was diluted with 3% BSA solution and the cell pellet was resuspended with antibody dilution (3% BSA solution) and primary antibody was incubated overnight at 4 ℃.

7. The supernatant was discarded by centrifugation at 800g, and the cell pellet was washed 5 times with PBS solution for 20 minutes each.

8. According to the following steps: 2000, the secondary antibody was diluted with 3% BSA solution and the cell pellet was resuspended in antibody dilution (3% BSA solution) and the secondary antibody was incubated at room temperature for 2 hours.

9. The supernatant was discarded by centrifugation at 800g, and the cell pellet was washed 5 times with PBS solution for 20 minutes each.

10. Adding 100 Xhoechst dye solution according to the volume ratio of 1/100, and dyeing for 20 minutes at room temperature.

11. The cell pellet was washed 2 times with PBS solution for 10 minutes each. The staining of the cell mass was observed using a confocal laser microscope.

FIG. 4 shows the effect of immunofluorescence staining of gastric cancer primary tumor cell mass cultured in vitro, and it can be seen that the cells constituting the cell mass are all CK8/CK18 positive and are epithelial in origin, confirming that the tumor cells cultured by the method are high in purity. Immunofluorescence staining identification was performed on 20 primary cultures of gastric cancer samples, and statistical results show that the proportion of tumor cells in the gastric cancer primary cells obtained by the method reaches 70% -93% (table 28).

TABLE 28 identification of gastric cancer samples by immunofluorescence staining of primary cultures

Example 11 gastric cancer Primary cell cultures and Primary tumor tissues

The DNA extraction procedure mentioned in the examples below was performed using the tiangen blood/tissue/cell genome extraction kit (DP 304).

The pooling procedures mentioned in the examples below were performed using the NEB DNA sequencing pooling kit (E7645).

The high throughput sequencing referred to in the examples below refers to the Illumina HiSeq X-ten sequencing platform.

1. Obtaining a gastric cancer solid tumor sample, before in vitro culture operation, firstly taking 10mg of the gastric cancer solid tumor sample for DNA extraction, establishing a library and whole genome high throughput sequencing (WGS), wherein the sequencing depth is 300 times, and the rest solid tumor sample is used for in vitro culture of gastric cancer primary cells.

2. After the treatment of the gastric cancer tissue, the primary cell culture medium of the gastric cancer solid tumor in example 1 (wherein, the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein R-spondin is 500ng/mL, the final concentration of human recombinant protein Wnt-3a is 250ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 10 muM, the final concentration of A83-01 is 0.5 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of Cholera Toxin is 0.632 nM, the final concentration of Y-27muM, and the final concentration of Gastrin is 10nM) is cultured for a period of time, the cell masses with a diameter of 100 μm or more were designated as P0 generation cells, and then designated as P1, P2, … and Pn in the order of the number of generations. 10 of each of the primary tumor cell cultures of gastric cancer of generations P1, P2, P3, P4 and P5⁶And (3) carrying out DNA extraction, library construction and whole genome high throughput sequencing (WGS) with the sequencing depth of 300X.

3. Copy number variation analysis (CNV) is respectively carried out on each group of sequencing results, copy number variation between the primary gastric cancer Tumor tissue and each generation of gastric cancer primary cell culture is compared, as shown in FIG. 5, copy number variation conditions of each generation of gastric cancer primary cell culture (P1, P2, P3, P4 and P5) and the primary gastric cancer Tumor tissue (Tumor) are highly consistent, so that the gastric cancer primary cells obtained by the method can represent the real condition of the primary Tumor of a patient.

Example 12 comparison of success rates of different primary cell cultures

The procedures of all primary cultures were identical (see above) and only the media formulations were different. The various primary cell culture media tested are shown in table 29. The formula adopted in the invention is shown in a scheme D, and concretely shown in a table 9 (wherein the final concentration of human recombinant protein EGF is 50ng/mL, the final concentration of human recombinant protein bFGF is 20ng/mL, the final concentration of human recombinant protein HGF is 20ng/mL, the final concentration of human recombinant protein FGF-10 is 20ng/mL, the final concentration of human recombinant protein R-spondin is 500ng/mL, the final concentration of human recombinant protein Wnt-3a is 250ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 10 muM, the final concentration of A83-01 is 0.5 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of Cholera Toxin is 0.1nM, the final concentration of Y-27632 is 10 muM, and the final concentration of strin is 10 nM).

TABLE 29 Primary cell culture Medium formulation for testing (100mL)

After the primary cell culture medium preparation is complete, it is sterile filtered through a 0.22 μ M needle filter (Millipore SLGP033RS) and can be stored at 4 ℃ for two weeks.

20 samples were treated in each of the four primary cell culture medium protocols, and the sample treatment and culture operations were performed according to the methods described in examples 3, 4, and 5, and the success rate of culturing primary cells of gastric cancer solid tumors after 10 days of culture was counted as shown in table 30:

TABLE 30 cultivation in different media

The primary cell culture medium for gastric cancer solid tumors (table 9) can stimulate cancer cell proliferation in tissue samples of gastric cancer solid tumors to the maximum extent, and the success rate of culturing the primary cells for gastric cancer solid tumors is improved.

Example 13 comparison of success rates of culture in different specimen-preserving solutions

In this example, the procedures of all sample primary culture procedures are completely identical (see the above description), and only the formula of the sample preservation solution is different. The various sample preservation solutions tested are shown in table 31. Wherein, the scheme E is the formula adopted in the invention, and is specifically shown in the table 1.

Table 31 sample preservation solution formula for testing (100mL)

After the preparation of the various sample preserving solutions in the above table is completed, the samples are subpackaged by 15mL centrifuge tubes, 5mL for each tube. Can be stored at 4 deg.C for 1 month after subpackaging.

Each of the five sample preservation solutions treats 20 samples, the samples are temporarily stored in the sample preservation solution at 4 ℃ after being separated in vitro, after being separated in vitro for 2 hours, the sample treatment and culture operations are carried out according to the methods described in the embodiments 3, 4 and 5, and the success rate of culturing primary cells of the gastric cancer solid tumors is counted after 10 days of culture as shown in table 32:

TABLE 32 incubation of different sample stocks

The success rate of the culture of the primary cells of the gastric cancer solid tumor is greatly influenced by the formula of the sample preserving fluid, and the sample preserving fluid (shown in table 1) used by the invention can protect the activity of the cancer cells in the tissue sample of the gastric cancer solid tumor to the greatest extent and improve the success rate of the culture.

Example 14 comparison of the culture powers of different dissociation solutions

The procedures of all primary culture methods in this example are identical (see the above description), and only the sample dissociation solution formula is different. The various sample dissociation solutions tested are shown in table 33. Wherein, the scheme D is the formula adopted in the invention, and is specifically shown in the table 3.

TABLE 33 sample dissociation fluid formulation for testing (10mL)

The sample dissociation liquid is prepared for use.

Samples with the weight of over 100mg of the tissue mass of 20 cases of the gastric cancer solid tumor are selected, evenly divided into four parts, and the four sample dissociation liquids are respectively used for sample treatment and culture operation according to the methods described in the examples 3, 4 and 5. After 10 days of culture, the success rate of primary cell culture of gastric cancer solid tumors is counted as the following table 34:

TABLE 34 incubation of dissociation liquid of different samples

The success rate of the primary cell culture of the gastric cancer solid tumor can be greatly influenced by the formula of the sample dissociation solution, and the sample dissociation solution (table 3) used in the invention can separate the cancer cells in the tissue of the gastric cancer solid tumor to the maximum extent, so that the success rate of the primary cell culture of the gastric cancer solid tumor is improved.

Example 15 comparison of success rates of different cell digests

The procedure of the primary cell passage operation method is completely consistent in all samples in this example (refer to the above description), and only the cell digestion solution formula is different. The various sample dissociation solutions tested are shown in table 35. Wherein, the scheme D is the formula adopted in the invention, and is shown in the table 7.

TABLE 35 cell digest formulations for testing (10mL)

The cell digestive juice is prepared for use.

And (3) selecting 20 successfully cultured gastric cancer samples, and carrying out continuous passage operation on the primary gastric cancer solid tumor cells obtained by culture by using the four cell digestive juices according to the method in the embodiment 6. Passages were performed (no more than 10) each time the cancer cells expanded to form a cell mass of 100 μm in diameter, and the maximum number of passages was recorded. The statistical results are shown in Table 36:

TABLE 36 different cell digesta cultures

The cell digestive juice formula has great influence on the success rate of the passage of the gastric cancer solid tumor primary cells, and the cell digestive juice (table 7) used in the invention can gently dissociate the cancer cells in the cell masses, so that the sample can be continuously passed and the activity of the gastric cancer solid tumor primary cells can be maintained.

Claims

1. A culture medium for culturing primary cells of gastric cancer solid tumors, which is characterized in that: the culture medium consists of antibacterial antifungal agent three-antibody, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein R-spondin, human recombinant protein Wnt-3a, human recombinant protein Noggin, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, N-2Supplement, cholera toxin, B27, Y-27632, gastrin and Advanced DMEM/F12 culture medium;

wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250 ng/mL; the final concentration of the HEPES is 8-12 mM; the final concentration of the GlutaMax is 0.8-1.2% (volume percentage); the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein FGF-10 is 5-25 ng/mL; the final concentration of the human recombinant protein R-spondin is 250-500 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL; the final concentration of the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the SB202190 is 5-10 μ M; the final concentration of the A83-01 is 0.25-1.25 mu M; the final concentration of the Primocin is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine is 0.5-2 mM; the final concentration of nicotine is 5-10 mM; the final concentration of the N-2Supplement is 1 percent (volume percentage); the final concentration of cholera toxin is 0.1-1 nM; the final concentration of B27 is 1.5-2.5% (volume percentage); the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the gastrin is 5-20 nM; the balance is Advanced DMEM/F12 medium.

2. The culture medium according to claim 1, wherein: the culture medium is a solution formed by mixing the antibacterial antifungal agent triantion, the HEPES, the GlutaMax, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein FGF-10, the human recombinant protein R-spondin, the human recombinant protein Wnt-3a, the human recombinant protein Noggin, the SB202190, the A83-01, the Primocin, the N-acetyl-L-cysteine, the nicotine, the N-2Supplement, the cholera toxin, the B27, the Y-27632, the gastrin and the Advanced DMEM/F12 culture medium.

3. The culture medium according to claim 1, wherein: the components of the medium are present separately.

4. The culture medium of claim 3, wherein: said human recombinant protein EGF, said human recombinant protein bFGF, said human recombinant protein HGF, said human recombinant protein FGF-10, said human recombinant protein R-spondin, said human recombinant protein Wnt-3a, said human recombinant protein Noggin, said SB202190, said A83-01, said N-acetyl-L-cysteine, said nicotine, said cholera toxin, said Y-27632, and said gastrin are present in stock solution;

specifically, the mother liquor is 1000-100000 times of mother liquor;

the 1000 multiplied human recombinant protein EGF stock solution consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

the stock solution of 1000 multiplied human recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

the 1000 Xhuman recombinant protein HGF stock solution consists of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

the 1000 Xhuman recombinant protein FGF-10 stock solution consists of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

1000 Xthe stock solution of the human recombinant protein R-spondin consists of the human recombinant protein R-spondin, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin is 250 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

the 1000 Xhuman recombinant protein Wnt-3a stock solution consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

the 1000 multiplied human recombinant protein Noggin stock solution consists of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of the human recombinant protein Noggin is 100 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

the 1000 × SB202190 stock consists of SB202190 and DMSO, wherein the final concentration of SB202190 is 10mM, the balance being DMSO;

the 100000 XA 83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of the A83-01 is 25mM, and the balance is DMSO;

the 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is water;

1000 × nicotine stock solution is composed of nicotine and water, wherein the concentration of nicotine is 5M, and the balance is water;

10000 Xcholera toxin stock solution consists of cholera toxin and cholera toxin solution, wherein the final concentration of the cholera toxin is 10 μ M, and the rest is cholera toxin solution; the cholera toxin dissolving solution comprises the following components: each 10mL of the cholera toxin dissolving solution contains Tris (1M) with pH 7.00.05M, NaCl with 0.2M, sodium azide with 3mM, EDTA (0.5M) with pH 8.01mM and the balance of water;

1000 XY-27632 consists of Y-27632 and water, wherein the final concentration of Y-27632 is 10mM, and the balance is water;

1000 × gastrin is composed of gastrin and water, wherein the final concentration of gastrin is 10 μ M, and the balance is water.

5. The culture medium according to claim 4, wherein: in the 1000 × human recombinant protein EGF solution, the 1000 × human recombinant protein bFGF solution, the 1000 × human recombinant protein HGF solution, the 1000 × human recombinant protein FGF-10 solution, the 1000 × human recombinant protein R-spondin solution, the 1000 × human recombinant protein Wnt-3a solution, and 1000 × human recombinant protein Noggin, the BSA is present in a mother solution;

specifically, the BSA exists in the form of 100 times of mother liquor; 100 × BSA solution consisted of BSA and PBS; wherein the final concentration of BSA was 0.1g/mL, and the balance was PBS.

6. Kit for culturing primary cells of solid tumors of gastric cancer, comprising a culture medium according to any one of claims 1 to 5 and at least one of the following reagents: the digestion stopping solution and the cell freezing medium according to claim 8 or 9.

7. Use of the culture medium of any one of claims 1-5 or the kit of claim 6 for culturing primary cells of gastric carcinoma solid tumors.

8. A method for culturing primary cells of gastric cancer solid tumors comprises the following steps: culturing in suspension the primary gastric cancer solid tumor cells in the culture medium of any one of claims 1 to 5;

specifically, the method comprises the following steps: using a culture vessel with a low adsorption surface, using the culture medium to culture the gastric cancer solid tumor primary cells in suspension at 37 ℃ and 5% CO₂Culturing under the condition, and replacing the culture medium every 2-4 days.

9. The method of claim 8, wherein: the method further comprises the steps of: when the primary gastric cancer solid tumor cells form lumps with the diameter of 50-80 mu m, carrying out passage on the primary gastric cancer solid tumor cells;

specifically, the digestion stop solution adopted during the passage consists of fetal calf serum, three antibiotics of an antibacterial antifungal agent and a DMEM medium; wherein the final concentration of the fetal calf serum in the digestion stop solution is 8-12%; the final concentration of penicillin in the three antibiotics of the antibacterial and antifungal agents in the digestion stop solution is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent in the digestion stop solution is 100-200 mu g/mL; the final concentration of amphotericin B in the antibacterial antifungal agent triantion in the digestion stop solution is 250-500 ng/mL; the rest is DMEM culture medium;

and/or

The method also comprises the step of performing cryopreservation and/or resuscitation on the gastric cancer solid tumor primary cells after the expansion of 2-3 passages;

specifically, the cell cryopreservation solution adopted in the cryopreservation process consists of an Advanced DMEM/F12 culture medium, DMSO and a 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2); the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

10. The culture medium or use or method according to any one of claims 1 to 9, wherein: the gastric cancer is primary gastric cancer, the clinical stage is II stage, III stage or IV stage, or various pathologically typed gastric cancer or gastric cancer metastasis focus.