CN111808815A

CN111808815A - Method for culturing primary cells of gastric cancer solid tumor

Info

Publication number: CN111808815A
Application number: CN201910289073.XA
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 method for culturing primary cells of gastric cancer solid tumors. The invention provides a method for culturing primary cells of gastric cancer solid tumors and a matched reagent, and the core of the technology is as follows: (1) the stomach cancer solid tumor tissue is treated by a mild cell dissociation reagent, so that the activity of cancer cells in the tissue is ensured to the greatest extent; (2) preparing a special serum-free culture medium, and culturing tumor cells derived from the gastric cancer solid tumor in vitro by using a suspension culture system, 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 has wide application prospect in the fields of research and clinical diagnosis and treatment of gastric cancer.

Description

Method for culturing primary cells of gastric cancer solid tumor

Technical Field

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

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, and the core of the technology is as follows: (1) the stomach cancer solid tumor tissue is treated by a mild cell dissociation reagent, so that the activity of cancer cells in the tissue is ensured to the greatest extent; (2) preparing a special serum-free culture medium, and culturing tumor cells derived from the gastric cancer solid tumor in vitro by using a suspension culture system, so that the interference of normal cells is eliminated to the maximum extent while the normal amplification of the cancer cells is ensured.

In a first 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: carrying out suspension culture on the primary gastric cancer solid tumor cells by using a primary gastric cancer solid tumor cell culture medium;

the culture medium of the gastric cancer solid tumor primary cells is prepared from antibacterial antifungal agents of three antibiotics (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, Non-essential amino acids (Non-essential amino acids), human recombinant protein EGF, human recombinant protein bFGF and human recombinant protein HGF. Human recombinant protein FGF-10, 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-quinolinyl) -1H-pyrazole-1-carbothioamide), Primocin^TMN-acetyl-L-cysteine (N-acetyl-L-cysteine), nicotine (Nicotinamide), N-2Supplement, cortisol, B27, ITS-X (Insulin, Transferrin, Selenium, EthanolamineSolution), Gastin (Gastin 1), Y-27632 and Advanced DMEM/F12. Wherein the final concentration of penicillin in the three antibiotics of the antibacterial and antifungal agents in the primary cell culture medium of the gastric cancer solid tumor is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the three antibiotics of the antibacterial and antifungal agents in the primary cell culture medium of the gastric cancer solid tumor is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent in the primary cell culture medium of the gastric cancer solid tumor is 250-250ng/mL (such as 250 ng/mL); the final concentration of the HEPES in the gastric cancer solid tumor primary cell culture medium is 8-12mM (e.g., 10 mM); the final concentration of the GlutaMax in the gastric cancer solid tumor primary cell culture medium is 0.8-1.2% (e.g. 1%,% represents volume percentage); glycine in the non-essential amino acid is present in the culture medium of the gastric cancer solid tumor primary cells at a concentration of 80-120 μ M (e.g., 10 μ M); the concentration of the L-alanine in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M (such as 10 mu M); the concentration of L-asparagine in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 μ M (such as 10 μ M); the concentration of the L-aspartic acid in the non-essential amino acid in the gastric cancer solid tumor primary cell culture medium is 80-120 mu M (such as 10 mu M); the concentration of the L-glutamic acid in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M (such as 10 mu M); the concentration of L-proline in the non-essential amino acid in the gastric cancer solid tumor primary cell culture medium is 80-120 mu M (such as 10 mu M); the concentration of the L-serine in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M (such as 10 mu M); the human recombinant protein EGF is used for treating the gastric cancer solid tumorThe final concentration in the primary cell culture medium is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF in the culture medium of the gastric cancer solid tumor primary cells is 10-50 ng/mL; the final concentration of the human recombinant protein HGF in the culture medium of the gastric cancer solid tumor primary cells is 5-25 ng/mL; the final concentration of the human recombinant protein FGF-10 in the culture medium of the gastric cancer solid tumor primary cells is 5-25 ng/mL; the final concentration of the human recombinant protein Wnt-3a in the culture medium of the gastric cancer solid tumor primary cell is 200-300 ng/mL; the final concentration of the human recombinant protein Noggin in the primary cell culture medium of the gastric cancer solid tumor is 100-200 ng/mL; the final concentration of the SB202190 in the gastric cancer solid tumor primary cell culture medium is 5-10 μ M; the final concentration of the A83-01 in the culture medium of the gastric cancer solid tumor primary cells is 0.25-1.25 mu M; the final concentration of the Primocin in the gastric cancer solid tumor primary cell culture medium is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine (N-acetyl-L-cysteine) in the culture medium of the gastric cancer solid tumor primary cells is 0.5-2 mM; the final concentration of nicotine (Nicotinamide) in the gastric cancer solid tumor primary cell culture medium is 5-10 mM; the final concentration of the N-2Supplement in the culture medium of the gastric cancer solid tumor primary cells is 1 percent (volume percentage); the final concentration of the cortisol in the primary cell culture medium for the gastric cancer solid tumors is 20-50 ng/mL; the final concentration of the B27 in the primary cell culture medium of the gastric cancer solid tumor is 1.5-2.5% (such as 2%,% represents volume percentage content); the final concentration of ITS-X in the culture medium of the gastric cancer solid tumor primary cell is 0.8-1.2% (such as 1%,% represents volume percentage content); the final concentration of the Gastrin (Gastrin 1) in the gastric cancer solid tumor primary cell culture medium is 8-12nM (e.g., 10 nM); the final concentration of the Y-27632 in the culture medium of the gastric cancer solid tumor primary cells is 5-20 mu M; the balance is Advanced DMEM/F12 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 three-resistant (penicillin-streptomycin-amphotericin B) of the antibacterial antifungal agent is' Antibiotic-Antimycin, 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 non-essential amino acids are composed as follows: the non-essential amino acids comprise 750 μ g glycine, 890 μ g L-alanine, 1320 μ g L-asparagine, 1330 μ g L-aspartic acid, 1470 μ g L-glutamic acid, 1150 μ g L-proline, 1050 μ g L-serine per ml, and the solvent is water (the concentrations of the various amino acids mentioned above per ml of the non-essential amino acids are 10 mM). The Primocin is an antibacterial agent (such as Invivogen # ant-pm-1 or other products with the same composition) for primary cells, is an antibiotic for protecting the primary cells from being polluted by microorganisms, and has 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), and Catalase (Catalase)) Human recombinant insulin (Human recombinant insulin), Human Transferrin (Human Transferrin), Superoxide Dismutase (Superoxide Dismutase), Corticosterone (Corticosterone), D-Galactose (D-Galactose), Ethanolamine hydrochloride (Ethanolamine HCl), reduced glutathione (glutathione), L-Carnitine HCl, Linoleic Acid (Linoleic Acid), Linolenic Acid (Linolenic Acid), Progesterone (Progesterone), Putrescine (Putrescine2HCl), Sodium Selenite (Sodium Selenite), triiodothyronine (T3 (trio-I-thyronine)). The ITS-X solvent is EBSS solution (Earle's balanced salt solution), and the solutes and the concentrations are as follows: 1g/L of insulin; 0.55g/L of transferrin; 0.00067g/L sodium selenite; ethanolamine 0.2 g/L. 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 nonessential amino acids are under the brand code Gibco # 11140050; 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 of the human recombinant protein FGF-10 is Peprotech AF-100-26-100; the brand 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 Primocin^TMThe brand of (1) is Invivogen # ant-pm-1; the brand and cargo number of the N-acetyl-L-cysteine is Sigma # A9165; the brand of the Nicotinamide is Sigma # N0636; the brand goods number of the N-2Supplement is Gibco # 17502001; the brand of cortisol is Sigma # H0888; the brand name of B27 is Gibco # 12587010; the ITS-X brand has a goods number of Gibco # 51500056; the brand name of the gastrin is NJpeptide # Pep 12307; the brand goods number of the Y-27632 is MCE # 129830-38-2; the brand of the Advanced DMEM/F12 medium is Gibco # 12634010.

The gastric cancer solid tumor primary cells are obtained by dissociating gastric cancer solid tumor tissues by using sample dissociation liquid.

The sample dissociation liquid consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I in the dissociation solution of the sample is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase II in the dissociation solution of the sample is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase IV in the dissociation solution of the sample is 50-150U/mL (such as 100U/mL); the balance being PBS.

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

In a specific embodiment of the present invention, the brand name of collagenase I is Gibco # 17100-017; the brand of collagenase II is Gibco # 17101-015; the brand goods number of the collagenase IV is Gibco # 17104-; the PBS was branded under Gibco # 21-040-CVR.

Further, the tissue of the gastric cancer solid tumor can be dissociated by the sample dissociation solution according to the method comprising the following steps: cutting the solid tumor tissue of gastric cancer (e.g. into 0.8-1.2 mm) according to the dosage of 0.1-0.3mL (e.g. 0.1mL) of the sample dissociation solution per mg of tissue³Small pieces of (a) were treated with the sample dissociation solution preheated at 37 ℃ in advance, and sample dissociation was performed 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.

Further, in the method, the gastric cancer solid tumor primary cell culture medium can be used for suspension culture of the gastric cancer solid tumor primary cell according to a method comprising the following steps: using a cell culture container made of Polystyrene (abbreviated as PS), Polycarbonate (abbreviated as PC), polymethyl methacrylate (abbreviated as PMMA), COC resin, cyclic Olefin Polymer (abbreviated as COP) or low-adsorption surface (abbreviated as LAS), or performing CYTOP modification on the cell culture container, culturing the primary gastric cancer solid tumor cells in a suspension manner by using the primary gastric cancer solid tumor cell culture medium, wherein the primary gastric cancer solid tumor cells are cultured in a suspension manner 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.

The culture vessel includes a standard-sized cell culture dish, a standard-sized cell culture well plate, a self-designed and processed cell culture consumable (a microplate chip as shown in FIG. 7 in example 19), and the like.

Further, the method for CYTOP modification is as follows: firstly, pure oxygen etching is carried out on the cell culture container, the etching condition is 20W, and the etching time is 3 minutes. Then, the surface of the cell culture container (such as a culture dish or a culture plate) is covered with an appropriate amount (taking a 96-well plate as an example, 20 μ L of each well, the appropriate amount refers to completely covering the bottom of the culture dish) of 1% CYTOP solution, and the 1% CYTOP solution can be used after being completely dried.

Still further, the composition of the 1% CYTOP solution is as follows: each 100mL of the 1% CYTOP solution contained 1mL of CYTOP, with the balance being fluoro oil.

Wherein the CYTOP is perfluoro (1-butenylvinylether) polymer. The fluoro oil may be a fluoro oil of brand 3M # FC40, or other product of the same composition.

In a particular embodiment of the invention, the CYTOP brand code is specifically Asashi glass # CTL-809M; the brand goods number of the fluorine oil is specifically 3M # FC 40.

Wherein the initial seeding density may be10⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density of individual cells was plated.

Further, the method may further comprise a step of performing a pretreatment for dissociation of the gastric cancer solid tumor tissue as follows: cleaning the surface of the gastric cancer solid tumor tissue sample by using ethanol with the volume percentage of 70-75% (such as 75%) for 10-30 seconds; washing the gastric cancer solid tumor tissue sample 10-20 times (such as 10 times) with a sample washing solution, and washing the gastric cancer solid tumor tissue sample 5-10 times (such as 5 times) with a sterile PBS solution; then removing impurities, connective tissues, adipose tissues, necrotic tissues and other components which influence the culture of the primary cells in the gastric cancer solid tumor tissue sample.

Wherein the sample cleaning solution consists of an antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) and PBS; wherein the final concentration of penicillin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the sample washing solution is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the sample washing solution is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the sample washing solution is 250-500ng/mL (such as 250 ng/mL); the balance being PBS.

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 antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is under the brand code Gibco # 15240062; the PBS was branded under Gibco # 21-040-CVR.

The step of pretreatment for dissociation of the gastric cancer solid tumor tissue needs to be operated on ice, and the whole operation step needs to be completed within 10 minutes.

Furthermore, the time for separating the gastric cancer solid tumor tissue sample subjected to the dissociation pretreatment needs to be within 2 hours, and the gastric cancer solid tumor tissue sample is preserved in a sample preservation solution before the dissociation pretreatment.

Wherein the sample preservation solution consists of fetal calf serum, three antibiotics (penicillin-streptomycin-amphotericin B), HEPES and HBSS (Hank's balanced salt solution); wherein the final concentration of the fetal calf serum in the sample preservation solution is 1-5% (such as 2%,% represents volume percentage content); the final concentration of penicillin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the sample preservation 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 sample preservation solution is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the sample preservation solution is 250-500ng/mL (such as 250 ng/mL); the final concentration of the HEPES in the sample preservation solution is 8-12mM (e.g., 10 mM); the balance being HBSS.

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 a solventIs composed of

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 brand of HEPES is Gibco # 15630080; the HBSS is sold under the brand name Gibco # 14170161.

Further, in the method, after the dissociation treatment of the gastric cancer solid tumor tissue by the sample dissociation solution, the method may further include the following steps: terminating the dissociation reaction with 8-15 (e.g., 10) times the volume of the digestion stop solution, and collecting the cell suspension; filtering the cell suspension with a 100 μm or 40 μm sterile cell strainer to remove tissue debris and adherent cells; 800-1000g (e.g., 800g) of the suspension is centrifuged at room temperature for 10-15 minutes (e.g., 10 minutes), and the supernatant is discarded; then resuspend the cells in 3-5mL (e.g., 5mL) sterile PBS; centrifuging at room temperature for 10-15 min (such as 10 min) again at 800-; then, the gastric cancer solid tumor primary cell culture medium is used for resuspending cell pellets, and the cell state is observed under a microscope for cell counting.

Wherein the digestion stop solution consists of fetal calf serum, antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) and DMEM culture 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: every milli of Chinese characterLiters contain 10000 units of penicillin (base), 10000. mu.g streptomycin (base) and 25. mu.g 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.

Further, in the method, the following steps may be further included: 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 cell digestive juice adopted when the passage is carried out consists of the following components: each 10mL of the cell digest contained 4-6mL (e.g., 5mL) of Accutase, a final concentration of 5mM EDTA (i.e., 10. mu.L of 0.5M EDTA), 1.5-2.5mL (e.g., 2mL) of TrypLE Express, and the balance PBS.

Further, the Accutase is StemPro^TMAccutase^TMCell DissociationReagent "(e.g., Gibco # A11105-01, or other product of the same composition). The Accutase is a single-component enzyme, and is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is' TrypLE^TMExpressEnzyme (1X), no phenol red "(e.g., Gibco #12604013, or other products of the same composition). The TrypLE^TMExpress Enzyme (1X), no phenol red "contains 200mg/L KCl and 200mg/L KH₂PO₄8000mg/L NaCl, 2160mg/L Na₂HPO₄·7H₂O, 457.6mg/L EDTA; also contains recombinant proteinAn enzyme.

In a specific embodiment of the invention, the brand name of the Accutase is Gibco # A11105-01; the brand name of the 0.5M EDTA is Invitrogen # AM 9261; the brand goods number of the TrypLE Express is Gibco # 12604013; the PBS was branded under Gibco # 21-040-CVR.

Further, the digestion temperature used for the passage was 37 ℃.

Further, the digestion stop solution used in the passage is the digestion stop solution described above.

More specifically, the step of performing said passaging is carried out: collecting cell mass to be passaged, 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 cell, stopping digestion reaction with the digestion stopping solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting cell suspension; resuspending the cell pellet with the gastric carcinoma solid tumor primary cell culture medium after centrifugation, counting, and then suspension culturing the cells using a culture vessel with a low adsorption surface (initial seeding density can be 10)⁵Per 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 freezing solution adopted during 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 frozen, 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 cell, terminating the digestion reaction with the digestion terminating solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting 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); suspending the cell pellet with the gastric cancer solid tumor primary cell culture medium 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 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 specifically comprises the culture medium for the primary cells of the gastric cancer solid tumor and at least one of the following reagents: the sample dissociation solution, the sample preservation solution, the cell digestion solution, the sample washing solution, the digestion stop solution, the cell cryopreservation solution and the 1% CYTOP solution described above.

The sample preservation solution can be used for temporarily preserving a sample after the sample is separated, and can maintain the activity of cells in the sample in a short time after the sample is separated. The sample preservation solution can be preserved for 1 month at 4 ℃ after being prepared.

The sample washing solution can be used for washing and disinfecting a sample. The sample cleaning solution needs to be ready for use.

The sample dissociation liquid can be used for dissociation of a sample, and can dissociate gastric cancer solid tumor primary cells in the sample from tissues. The sample dissociation solution is prepared, wherein collagenase I, collagenase II and collagenase IV can be stored in a stock solution (mother solution) at-20 ℃ for a long time, and specifically 10 or 20 times of the stock solution (mother solution). The 10 × collagenase I stock consists of the collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; a 10 × collagenase II stock solution consists of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; the balance being PBS; 20 × collagenase IV stock consists of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance being PBS. The enzyme activities of collagenase I, collagenase II and collagenase IV are defined above.

The cell digestive juice can be used for digestion and passage of cell masses and can digest gastric cancer tumor masses into single cells. The cell digestive juice is required to be prepared immediately.

The digestion stop solution can be used for stopping the dissociation of the sample or the digestion process of the cells. The prepared digestion stop solution can be stored for one month at 4 ℃.

The gastric cancer solid tumor primary cell culture medium can be used for culturing gastric cancer solid tumor primary cells. The primary cell culture medium for the gastric cancer solid tumor needs to be sterilized by filtration through a 0.22 mu M needle filter (Millipore SLGP033RS) after being prepared, and can be stored for two weeks at 4 ℃. 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 Wnt-3a and the human recombinant protein Noggin can be stored in a stock solution (mother solution) form at-80 ℃ for a long time, and particularly can be stored in a stock solution (mother solution) of 1000 times. SB202190, N-acetyl-L-cysteine, Nicotinamide, cortisol, gastrin and Y-27632 can be stored in stock solution (mother solution) form at-20 deg.C for a long period, specifically 1000 times of stock solution (mother solution). A83-01 can be stored in stock solution (mother liquor) at-20 deg.C for a long period, specifically 100000 times of 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 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. The 1000 Xcortisol stock solution consists of cortisol, absolute ethyl alcohol and ultrapure water, wherein the final concentration of the cortisol is 25 mu g/mL, the final concentration of the absolute ethyl alcohol is 5% (volume percentage content), and the balance is the ultrapure water. 1000 times gastrin is composed of gastrin and ultrapure water, wherein the concentration of gastrin is 10 μ M, and the balance is 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.

The cell freezing medium needs to be prepared at present. Wherein the 1% methylcellulose solution can be stored for a long period of time at 4 ℃.

In a third aspect, the invention claims the use of the kit as described hereinbefore for culturing primary cells of solid tumors of gastric cancer.

Furthermore, the gastric cancer can be primary gastric cancer, the clinical stage is stage II, stage III or stage IV (according to the stage TNM), or various pathologically typed gastric cancer or gastric cancer metastasis focuses, and the weight of the operation sample is 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 primary tumor cells from fresh gastric cancer solid tumor tissues and a matched reagent, 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).

FIG. 6 shows the results of in vitro drug sensitivity tests using primary gastric cancer cells cultured according to the present invention.

FIG. 7 is a diagram of a microplate chip design of the invention.

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 formulation of human recombinant protein stocks is shown in tables 11 to 16, the formulation of SB202190 stock is shown in Table 17, the formulation of A83-01 stock is shown in Table 18, the formulation of N-acetyl-L-cysteine stock is shown in Table 19, the formulation of Nicotinamide stock is shown in Table 20, the formulation of cortisol stock is shown in Table 21, the formulation of gastrin stock is shown in Table 22, and the formulation of Y-27632 stock is shown in Table 23. 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 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 161000 × stock solution of human recombinant protein Noggin (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 171000 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 18100000 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 191000 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 201000 XNicotinamide 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 211000 cortisol stock solution (100mL)

1000 Xcortisol stock solution is prepared and then subpackaged with 1.5mL sterile centrifuge tubes, and the stock solution can be stored at-20 ℃ for a long time.

TABLE 221000 × Gastrin stock solution (48mL)

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

TABLE 231000 XYY-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 ℃. 7. Cell cryopreservation liquid

The specific formulation of the cell culture medium is shown in Table 24.

TABLE 24 cell cryopreservation solution

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

In table 24, the preparation of the 1% methylcellulose solution is shown in table 25.

TABLE 251% methylcellulose solution (10mL)

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

8. 1% CYTOP solution

TABLE 261% CYTOP solution (100mL)

After the 1% CYTOP solution is prepared, the product can be stored for a long time at normal temperature.

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. The suspension culture of the gastric cancer primary cells was carried out by using a low-adsorption surface (low-adsorption surface), and the culture medium used was the culture medium of the gastric cancer solid tumor primary cells in example 1 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 250 ng/mL; the final concentration of human recombinant protein Noggin was 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 was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10. mu.M), taking a six-hole plate as an example, according to 10 holes⁶Individual 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. Culturing the gastric cancer primary cells by using a low-adsorption surface (low-adsorption-surface), wherein the culture medium is the gastric cancer solid tumor primary cell culture medium in example 1, and the culture medium is a six-well plate, and each well is 10⁶Individual 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, resuscitating each cell tube into a 3.5cm dish at 37 deg.C and 5% CO₂The 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, the gastric cancer solid tumor primary cell mass obtained by culturing the gastric cancer solid tumor primary cell culture medium in example 1 (wherein the final concentration of human recombinant protein EGF is 20ng/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 Wnt-3a is 200ng/mL, the final concentration of human recombinant protein Noggin is 100ng/mL, the final concentration of SB202190 is 5 muM, the final concentration of A83-01 is 1 muM, the final concentration of N-acetyl-L-steine is 1mM, the final concentration of Nicotinamide is 10mM, the final concentration of cortisol is 25ng/mL, and the final concentration of Y-27632 is 10 muM) was collected by centrifugation at 800g, and fixed by 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);

a gastric cancer cell mass obtained by culturing 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 25ng/mL, the final concentration of human recombinant protein HGF is 25ng/mL, the final concentration of human recombinant protein FGF-10 is 25ng/mL, the final concentration of human recombinant protein Wnt-3a is 300ng/mL, the final concentration of human recombinant protein Noggin is 200ng/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 cortisol is 25ng/mL, and the final concentration of Y-27632 is 10 muM) is subjected to immunofluorescence staining, and anti-CK 8+ CK18 is used for characterizing epithelial cells from which the source.

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 27).

TABLE 27 immunofluorescence staining identification of primary cultures of gastric cancer samples

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 tissues, 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 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 1 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 8mM, the final concentration of cortisol is 25ng/mL, the final concentration of Y-27632 is 8 muM) is used for culturing for a period of time to form cell clusters with the diameter of more than 100μm and is marked as P0 generation cells, then, the results are sequentially marked as P1, P2, … and Pn according to the number of passages. 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 28. 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 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 1 muM, the final concentration of N-acetyl-L-cysteine is 1mM, the final concentration of Nicotinamide is 8mM, the final concentration of cortisol is 25ng/mL, and the final concentration of Y-27632 is 8 muM).

TABLE 28 Primary cell culture Medium formulation (100mL) for testing

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 29:

TABLE 29 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 30. Wherein, the scheme E is the formula adopted in the invention, and is specifically shown in the table 1.

Table 30 sample preservation solution for test (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 31:

TABLE 31 cultivation of different specimen-preserving solutions

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 32. Wherein, the scheme D is the formula adopted in the invention, and is specifically shown in the table 3.

TABLE 32 sample dissociation solution 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 33:

TABLE 33 incubation of different sample dissociation solutions

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 fluids tested are shown in table 34. Wherein, the scheme D is the formula adopted in the invention, and is shown in the table 7.

TABLE 34 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 35:

TABLE 35 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.

Example 16 culture of gastric cancer Primary tumor cells Using cell culture consumables of different materials

The procedure for primary culture was completely identical in all samples in this example (see above), and only the cell culture consumables (unmodified) were different (Table 36).

TABLE 36 influence of unmodified culture consumables of different materials on gastric cancer primary tumor cell culture

Note: polystyrene (abbreviated PS), Polycarbonate (abbreviated PC), polymethyl methacrylate (abbreviated PMMA), COC resin, cyclic olefin Polymer (abbreviated COP), low-adsorption surface (LAS).

As can be seen from table 36: the culture consumables made of different materials have certain influence on the culture success rate of the gastric cancer solid tumor primary cells, wherein the culture success rate of the low-absorption surface (LAS) is the highest.

Example 17 culture of gastric cancer Primary tumor cells with CYTOP-modified cell culture consumables

In this example, the procedures of all primary cultures were identical (see above), the CYTOP modification method was identical, and only the materials of the cell culture consumables were different (table 37).

The CYTOP modification method comprises the following steps: firstly, pure oxygen etching is carried out on the cell culture container, the etching condition is 20W, and the etching time is 3 minutes. Then, the surface of the culture dish or the culture plate is covered with an appropriate amount of 1% CYTOP solution (taking a 96-well plate as an example, 20 mu L of each well, and the appropriate amount refers to the condition of completely covering the bottom of the culture dish), and the CYTOP solution can be used after being completely dried.

TABLE 37 influence of CYTOP-modified different materials CYTOP-modified culture consumables on gastric cancer primary tumor cell culture

As can be seen from table 37: it can be seen that CYTOP modification can effectively improve the success rate of culture of various materials.

Example 18 drug susceptibility testing with gastric cancer Primary tumor cells

The chemotherapeutic drugs Irinotecan, 5-fluorourcil and Oxaliplatin used in this example are all Selleck products.

The Celltiter-Glo cell viability assay kit mentioned in this example is a Promega product.

The primary gastric cancer cells cultured by the invention are used for in vitro drug sensitivity test: using a standard size 96-well low-adsorption cell culture plate according to the formula of 10⁵Primary cell inoculation was performed at a density per well, with 5 drug concentration gradients per drug, n-3. After the drug is added, the temperature of the cells is 37 ℃ and the CO content is 5 percent₂Incubate under conditions for 7 days. After the action of the drug is finished, the cell viability of each well is detected by using a Celltiter-Glo cell viability detection kit. The results of the experiment are shown in FIG. 6. The results show that: the primary gastric cancer cell obtained by the method can be used for in vitro drug sensitivity detection.

Example 19 microplate chip processing

In this example, a microplate chip for culturing the gastric cancer solid tumor primary cells of the present invention is obtained by processing a PMMA material (or PS, PC, COC, COP, LAS, or the like) by injection molding. The chip can be used for primary gastric cancer cell culture and in-vitro drug sensitivity detection experiments. FIG. 7 shows the design of the microplate chip.

In the practical application process, the PMMA material (or PS, PC, COC, COP, LAS and other materials) is used to prepare the structure of the microplate chip shown in the design drawing of FIG. 7, and then the surface of the microplate chip is subjected to CYTOP modification by the CYTOP modification method (see example 17), so that the microplate chip for culturing the gastric cancer primary cells is obtained.

Claims

1. A method for culturing primary cells of gastric cancer solid tumors comprises the following steps: carrying out suspension culture on the primary gastric cancer solid tumor cells by using a primary gastric cancer solid tumor cell culture medium;

the culture medium of the primary solid tumor cells of the gastric cancer consists of three antibacterial antifungal agents, HEPES, GlutaMax, nonessential amino acids, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotine, N-2Supplement, cortisol, B27, ITS-X, gastrin, Y-27632 and Advanced DMEM/F12 culture medium; wherein the final concentration of penicillin in the three antibiotics of the antibacterial and antifungal agents in the primary cell culture medium of the gastric cancer solid tumor is 100-200U/mL; the final concentration of streptomycin in the three antibiotics of the antibacterial and antifungal agents in the primary cell culture medium of the gastric cancer solid tumor is 100-200 mug/mL; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent in the primary cell culture medium of the gastric cancer solid tumor is 250 ng/mL; the final concentration of the HEPES in the gastric cancer solid tumor primary cell culture medium is 8-12 mM; the final concentration of the GlutaMax in the gastric cancer solid tumor primary cell culture medium is 0.8-1.2% (volume percentage content); the concentration of glycine in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M; the concentration of the L-alanine in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M; the concentration of the L-asparagine in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M; the concentration of the L-aspartic acid in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M; the concentration of the L-glutamic acid in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M; the concentration of L-proline in the non-essential amino acid in the gastric cancer solid tumor primary cell culture medium is 80-120 mu M; the concentration of the L-serine in the non-essential amino acid in the culture medium of the gastric cancer solid tumor primary cells is 80-120 mu M; the final concentration of the human recombinant protein EGF in the primary cell culture medium of the gastric cancer solid tumor is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF in the culture medium of the gastric cancer solid tumor primary cells is 10-50 ng/mL; the final concentration of the human recombinant protein HGF in the culture medium of the gastric cancer solid tumor primary cells is 5-25 ng/mL; the final concentration of the human recombinant protein FGF-10 in the culture medium of the gastric cancer solid tumor primary cells is 5-25 ng/mL; the final concentration of the human recombinant protein Wnt-3a in the culture medium of the gastric cancer solid tumor primary cell is 200-300 ng/mL; the final concentration of the human recombinant protein Noggin in the primary cell culture medium of the gastric cancer solid tumor is 100-200 ng/mL; the final concentration of the SB202190 in the gastric cancer solid tumor primary cell culture medium is 5-10 μ M; the final concentration of the A83-01 in the culture medium of the gastric cancer solid tumor primary cells is 0.25-1.25 mu M; the final concentration of the Primocin in the gastric cancer solid tumor primary cell culture medium is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine in the gastric cancer solid tumor primary cell culture medium is 0.5-2 mM; the final concentration of nicotine in the gastric cancer solid tumor primary cell culture medium is 5-10 mM; the final concentration of the N-2Supplement in the culture medium of the gastric cancer solid tumor primary cells is 1 percent (volume percentage); the final concentration of the cortisol in the primary cell culture medium for the gastric cancer solid tumors is 20-50 ng/mL; the final concentration of the B27 in the primary cell culture medium of the gastric cancer solid tumor is 1.5-2.5% (volume percentage); the final concentration of ITS-X in the culture medium of the gastric cancer solid tumor primary cells is 0.8-1.2% (volume percentage content); the final concentration of the gastrin in the gastric cancer solid tumor primary cell culture medium is 8-12 nM; the final concentration of the Y-27632 in the culture medium of the gastric cancer solid tumor primary cells is 5-20 mu M; the balance is Advanced DMEM/F12 medium.

2. The method of claim 1, wherein: the primary gastric cancer solid tumor cell is obtained by dissociating gastric cancer solid tumor tissue by using sample dissociation liquid;

the sample dissociation liquid consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase II in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase IV in the dissociation liquid of the sample is 50-150U/mL; the balance being PBS;

further, the tissue of the gastric cancer solid tumor is dissociated by the sample dissociation solution according to the method comprising the following steps: treating the sheared gastric cancer solid tumor tissue by using the sample dissociation liquid preheated at 37 ℃ in advance according to the dosage of 0.1-0.3mL of the sample dissociation liquid per mg of tissue, and dissociating the sample at 37 ℃ for 15 minutes to 3 hours.

3. The method according to claim 1 or 2, characterized in that: the method comprises the following steps of performing suspension culture on the gastric cancer solid tumor primary cells by using the gastric cancer solid tumor primary cell culture medium: using a cell culture container M to culture the gastric cancer solid tumor primary cells in a suspension manner by using the gastric cancer solid tumor primary cell culture medium at 37 ℃ and 5% CO₂Culturing under the condition, and replacing the culture medium every 2-4 days;

the cell culture vessel M is any one of: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a cell culture container made of COC resin, a cell culture container made of cyclic olefin polymer, or a cell culture container with a low adsorption surface; (II) subjecting the cell culture vessel of (I) to CYTOP modification;

further, the cell culture container is a cell culture dish, a cell culture pore plate or a micropore plate chip for cell culture;

further, in the (II), the cell culture vessel in the (I) is subjected to CYTOP modification according to a method comprising the following steps: carrying out pure oxygen etching on the cell culture container in the step (I), wherein the etching condition is that the power is 20W, and the etching time is 3 minutes; then covering the surface of the cell culture container with 1% CYTOP solution, and airing the 1% CYTOP solution to finish the CYTOP modification;

still further, the composition of the 1% CYTOP solution is as follows: each 100mL of the 1% CYTOP solution contained 1mL of LCYTOP, the balance being fluoro oil.

4. A method according to any one of claims 1-3, characterized in that: the method further comprises the following step of performing dissociation pretreatment on the gastric cancer solid tumor tissue: cleaning the surface of the gastric cancer solid tumor tissue sample by using ethanol with the volume percentage of 70-75%; sequentially cleaning the gastric cancer solid tumor tissue samples by using a sample cleaning solution and a sterile PBS solution;

specifically, the sample cleaning solution consists of an antibacterial antifungal agent triantion and PBS; wherein the final concentration of penicillin in the three antibiotics of the antibacterial and antifungal agents in the sample cleaning solution is 100-200U/mL; the final concentration of streptomycin in the three antibiotics of the antibacterial and antifungal agents in the sample cleaning solution is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent in the sample cleaning solution is 250-500 ng/mL; the balance being PBS.

5. The method of claim 4, wherein: the in vitro time of the gastric cancer solid tumor tissue sample subjected to the dissociation pretreatment is less than 2 hours, and the gastric cancer solid tumor tissue sample is preserved in a sample preservation solution before the dissociation pretreatment;

specifically, the sample preservation solution consists of fetal calf serum, three antibiotics of antibacterial and antifungal agents, HEPES and HBSS; wherein the final concentration of the fetal calf serum in the sample preservation solution is 1-5% (volume percentage content); the final concentration of penicillin in the three antibiotics of the antibacterial antifungal agent in the sample preservation solution is 100-200U/mL; the final concentration of streptomycin in the three antibiotics of the antibacterial and antifungal agents in the sample preservation solution is 100-200 mug/mL; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent in the sample preservation solution is 250-500 ng/mL; the final concentration of the HEPES in the sample preservation solution is 8-12 mM; the balance being HBSS.

6. The method according to any one of claims 1-5, wherein: in the method, after the dissociation treatment of the gastric cancer solid tumor tissue by using the sample dissociation solution, the method further comprises the following steps: terminating the dissociation reaction by using a digestion termination solution, and collecting cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; resuspending the cells with sterile PBS after centrifugation; re-centrifuging, and then re-suspending the cell sediment by using the gastric cancer solid tumor primary cell culture medium;

specifically, the digestion stop solution consists of fetal calf serum, an antibacterial antifungal agent three-antibody and a DMEM culture medium; wherein the final concentration of the fetal calf serum in the digestion stop solution is 8-12% (volume percentage); 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 balance is DMEM medium.

7. The method according to any one of claims 1-6, wherein: in the process of dissociating the gastric cancer solid tumor tissue by the sample dissociation solution, the method further comprises the following steps: 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 cell digest used for the passage was composed as follows: every 10mL of the cell digestive juice contains 4-6mL of Accutase, EDTA with the final concentration of 5mM, 1.5-2.5mL of TrypLE Express and the balance of PBS; and/or

The use of a digestion stopping solution for said passaging, which is the digestion stopping solution according to claim 6;

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.

8. A kit for culturing primary cells of gastric cancer solid tumors, which comprises the culture medium of the primary cells of gastric cancer solid tumors of any one of claims 1 to 7 and at least one of the following reagents: the sample dissociation solution, the sample preservation solution, the cell digestion solution, the sample washing solution, the digestion stop solution, the cell cryopreservation solution and the 1% CYTOP solution according to any one of claims 1 to 7.

9. Use of a kit of parts according to claim 8 for culturing primary cells of solid tumors of gastric cancer.

10. The method or kit or use 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.