CN111808816A - A medium for culturing gastric cancer solid tumor primary cells - Google Patents

Abstract

The invention discloses a culture medium for culturing gastric cancer solid tumor primary cells. The present invention provides a method for culturing gastric cancer solid tumor primary cells and supporting reagents. The present invention prepares a special serum-free culture medium, and uses a suspension culture system to culture tumor cells derived from gastric cancer solid tumors in vitro, so as to ensure the normal expansion of cancer cells. At the same time, the interference of normal cells is excluded to the greatest extent. The gastric cancer primary cell culture obtained by the method of the present invention can be used for various cell-level in vitro experiments, next-generation sequencing, construction of animal models, construction of cell lines and the like. It can be predicted that this culture method and the special culture medium provided by the present invention have broad application prospects in the fields of gastric cancer research and clinical diagnosis and treatment.

Description

A medium for culturing gastric cancer solid tumor primary cells

technical field

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

Background technique

Gastric cancer is one of the most common malignant tumors that seriously threaten human health. my country is a country with a high incidence of gastric cancer, and the incidence and deaths of gastric cancer account for 42.6% and 45% of the global incidence and deaths, respectively. The incidence of gastric cancer in my country is 11.8%, ranking fourth among all malignant tumors. The mortality rate of gastric cancer was 22.0%, ranking fifth among all malignant tumors. With the development of economy, improvement of living standards and changes in lifestyle, the incidence of gastric cancer will continue to rise. In addition, gastric cancer has a high risk of recurrence and metastasis. More than 50% of gastric cancer patients will experience different degrees of recurrence and metastasis several months to several years after radical treatment.

Although scientific research and medical institutions all over the world have invested heavily in the research on the etiology and development of gastric cancer, humans still know little about this disease. Gastric cancer is a complex disease. Its occurrence and development is a dynamic process, involving the interaction of many signal molecules, forming a complex molecular regulation network, and is also affected by external environmental factors. The etiology and occurrence and development of gastric cancer have strong individual differences and cannot be generalized. Therefore, it is a trend in the field of gastric cancer research and even the field of gastric cancer diagnosis and treatment to use the primary cell culture of gastric cancer solid tumor as a model for individualized and precise research.

The existing primary tumor cell culture technologies mainly include 2D culture, 3D culture, and reprogramming culture. These methods all face the problems of extremely long culture cycle, low culture success rate, and difficulty in removing miscellaneous cells to varying degrees.

SUMMARY OF THE INVENTION

In order to effectively solve the above technical problems, the present invention provides a new gastric cancer solid tumor primary cell culture technology and supporting reagents. The present invention prepares a special serum-free culture medium, and utilizes a suspension culture system to culture gastric cancer solid tumor-derived tumor cells. In vitro culture to ensure the normal expansion of cancer cells while maximizing the exclusion of normal cell interference.

In a first aspect, the present invention claims a medium for culturing gastric cancer solid tumor primary cells.

The medium for culturing gastric cancer solid tumor primary cells provided by the present invention is composed of three antibodies against antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF , Human recombinant protein HGF, Human recombinant protein FGF-10, Human recombinant protein R-spondin, Human recombinant protein Wnt-3a, Human recombinant protein Noggin, SB202190(4-(4-Fluorophenyl)-2-(4-hydroxyl Phenyl)-5-(4-pyridinyl)-1H-imidazole), A83-01(3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole- 1-carbothioamide), Primocin, N-acetyl-L-cysteine (N-acetyl-L-cysteine), Nicotinamide (Nicotinamide), N-2Supplement, Cholera Toxin (Cholera Toxin), B27, Y-27632, Gastrin (Gastrin) and AdvancedDMEM/F12 medium composition.

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

抗真菌剂。所述GlutaMAX为“GlutaMAX^TMSupplement”(如Gibco#35050061，或与其组成相同的其他产品)。所述“GlutaMAX^TMSupplement”的成分为L-alanyl-L-glutamine，是L-glutamine的替代物，浓度为200nM，溶剂为0.85％NaCl溶液。所述Primocin^TM为原代细胞用抗菌剂(如Invivogene#ant-pm-1，或与其组成相同的其他产品)，用于保护原代细胞免受微生物污染的抗生素，对革兰氏阳性菌、革兰氏阴性菌、支原体和真菌均有杀伤作用。所述N-2Supplement为“N-2Supplement(100X)”(如Gibco#17502001，或与其组成相同的其他产品)。所述“N-2Supplement(100X)”中含有终浓度为1mM的人全铁转铁蛋白(Human Transferrin(Holo))、500mg/L的重组胰岛素全链(Insulin Recombinant Full Chain)、0.63mg/L的孕酮(Progesterone)、10mM的腐胺(Putrescine)、0.52mg/L的亚硒酸盐(Selenite)。所述B27为“B-27^TM Supplement(50X),minus vitamin A”(如Gibco#12587010，或与其组成相同的其他产品)。所述“B-27^TMSupplement(50X),minus vitamin A”中含有生物素(Biotin)、DL-α-生育酚乙酸酯(DLAlpha Tocopherol Acetate)、DL-α-生育酚(DL Alpha-Tocopherol)、BSA(fatty acidfree Fraction V)、过氧化氢酶(Catalase)、人重组胰岛素(Human RecombinantInsulin)、人转铁蛋白(Human Transferrin)、超氧化物歧化酶(Superoxide Dismutase)、皮质酮(Corticosterone)、D-半乳糖(D-Galactose)、乙醇胺盐酸(Ethanolamine HCl)、还原型谷胱甘肽(Glutathione(reduced))、左旋肉碱盐酸(L-Carnitine HCl)、亚油酸(Linoleic Acid)、亚麻酸(Linolenic Acid)、孕酮(Progesterone)、腐胺(Putrescine2HCl)、亚硒酸钠(Sodium Selenite)、三碘甲状腺原氨酸(T3(triodo-I-thyronine))。所述GlutaMAX是一种高级细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。所述GlutaMAX为“GlutaMAX^TM Supplement”(如Gibco#35050061，或与其组成相同的其他产品)。所述Y-27632为“Y-27632dihydrochloride(一种ATP竞争性的ROCK-I和ROCK-II抑制剂，Ki分别为220nM和300nM)”(如MCE#129830-38-2，或与其组成相同的其他产品)。Further, the composition of the antibacterial and antifungal tertiary antibody (penicillin-streptomycin-amphotericin B) is as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B. The antibacterial and antifungal tertiary antibody (Penicillin-Streptomycin-Amphotericin B) is "Antibiotic-Antimycotic, 100X" (eg Gibco #15240062, or other products of the same composition). The "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, using penicillin G (sodium salt) in 0.85% saline, streptomycin sulfate and amphotericin B as

Antifungal agent. The GlutaMAX is "GlutaMAX ^™ Supplement" (eg Gibco #35050061, or other products of the same composition). Said "GlutaMAX ^™ Supplement" contains L-alanyl-L-glutamine, a substitute for L-glutamine, at a concentration of 200 nM in a 0.85% NaCl solution. The Primocin ^TM is an antibacterial agent for primary cells (such as Invivogene#ant-pm-1, or other products with the same composition), an antibiotic used to protect primary cells from microbial contamination, against Gram-positive bacteria, Gram-negative bacteria, mycoplasma and fungi have a killing effect. The N-2Supplement is "N-2Supplement (100X)" (eg Gibco #17502001, or other products with the same composition). The "N-2 Supplement (100X)" contains a final concentration of 1mM Human Transferrin (Holo), 500mg/L Insulin Recombinant Full Chain (Insulin Recombinant Full Chain), 0.63mg/L Progesterone (Progesterone), 10 mM putrescine (Putrescine), 0.52 mg/L selenite (Selenite). The B27 is "B-27 ^TM Supplement (50X), minus vitamin A" (eg Gibco #12587010, or other products with the same composition). The "B-27 ^TM Supplement (50X), minus vitamin A" contains biotin (Biotin), DL-α-tocopherol acetate (DLAlpha Tocopherol Acetate), DL-α-tocopherol (DL Alpha-Tocopherol) ), BSA (fatty acidfree Fraction V), Catalase, Human Recombinant Insulin, Human Transferrin, Superoxide Dismutase, Corticosterone , D-Galactose, Ethanolamine HCl, Glutathione (reduced), L-Carnitine HCl, Linoleic Acid, Linolenic Acid (Linolenic Acid), Progesterone (Progesterone), Putrescine (Putrescine2HCl), Sodium Selenite (Sodium Selenite), Triiodothyronine (T3 (triodo-I-thyronine)). The GlutaMAX is an advanced cell culture supplement that directly replaces L-glutamine in cell culture media. The GlutaMAX is "GlutaMAX ^™ Supplement" (eg Gibco #35050061, or other products of the same composition). The Y-27632 is "Y-27632dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor with Ki of 220nM and 300nM, respectively)" (such as MCE#129830-38-2, or its composition other products).

In a specific embodiment of the present invention, the brand article number of the antibacterial and antifungal tertiary antibody (penicillin-streptomycin-amphotericin B) is Gibco#15240062; the brand article number of the HEPES is Gibco#15630080; the The brand product number of GlutaMAX is Gibco#35050061; the brand product number of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand product number of the human recombinant protein bFGF is Peprotech AF-100-18B-50; The brand product number of the recombinant protein HGF is Peprotech AF-100-39-100; the brand product number of the human recombinant protein FGF-10 is PeprotechAF-100-26-100; the brand product number of the human recombinant protein R-spondin is Shanghai Jin An #CD83; the brand article number of the human recombinant protein Wnt-3a is R&D5036-WN-500; the brand article number of the human recombinant protein Noggin is Shanghai Jinan #C018; the brand article number of the SB202190 is Sigma#S7067; The brand product number of the A83-01 is Tocris#2939; the brand product number of the Primocin is Invivogene#ant-pm-1; the brand product number of the N-acetyl-L-cysteine is Sigma#A9165; the brand product number of the Nicotinamide It is Sigma#N0636; the N-2Supplement brand product number is Gibco#17502001; the Cholera Toxin brand product number is Listlab#100B; the B27 brand product number is Gibco#12587010; the Y-27632 brand product number is MCE#129830-38-2; the brand product number of the Gastrin is NJPeptide#Pep12307; the brand product number of the Advanced DMEM/F12 medium is Gibco#12634010.

Further, the existence forms of the medium for culturing gastric cancer solid tumor primary cells can be two:

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

After the medium is prepared, it needs to be filtered and sterilized with a 0.22 μM syringe filter (Millipore SLGP033RS), and can be stored at 4° C. for two weeks.

Second, each component in the medium for culturing gastric cancer solid tumor primary cells exists alone, and is prepared according to the formula when used.

Further, the human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein R-spondin, human recombinant protein Wnt-3a and human recombinant protein Noggin can be stored in liquid ( Mother liquor) form (-80 ℃ can be stored for a long time), specifically, it can be a 1000 times stock solution (mother liquor). SB202190, N-acetyl-L-cysteine, Nicotinamide, Y-27632 and gastrin can exist in stock solution (mother solution) (long-term storage at -20°C), specifically 1000 times stock solution (mother solution). Cholera Toxin can exist in the form of a stock solution (mother solution) (it can be stored for a long time at -20°C), specifically, a 10,000-fold stock solution (mother solution). A83-01 can exist in the form of a stock solution (mother solution) (it can be stored for a long time at -20°C), specifically a 100,000-fold stock solution (mother solution).

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

1000× human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 μg/mL, the final concentration of BSA is 0.01 g/mL, and the balance is PBS.

The 1000× human recombinant protein HGF stock solution is composed of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 μg/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is PBS.

The 1000× human recombinant protein FGF-10 stock solution is composed of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of the human recombinant protein FGF-10 is 20 μg/mL, and the final concentration of the BSA is 0.01 g/mL mL, and the remainder is PBS.

1000× human recombinant protein R-spondin stock solution is composed of human recombinant protein R-spondin, BSA and PBS, wherein the final concentration of the human recombinant protein R-spondin is 250 μg/mL, and the final concentration of the BSA is 0.01 g/mL mL, and the remainder is PBS.

1000× human recombinant protein Wnt-3a stock solution is composed of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of the human recombinant protein Wnt-3a is 200 μg/mL, and the final concentration of the BSA is 0.01 g/mL mL, and the remainder is PBS.

1000× human recombinant protein Noggin stock solution is composed of human recombinant protein Noggin, BSA and PBS, wherein the final concentration of human recombinant protein Noggin is 100 μg/mL, the final concentration of BSA is 0.01 g/mL, and the balance is PBS.

In the above-mentioned five kinds of 1000-fold storage solutions, the BSA can exist in the form of 100-fold storage solution (mother liquor) (for current use), which is specifically composed of BSA and PBS, and the final concentration of BSA (Sigma#A1933) is 0.1g /mL, and the remainder is PBS.

In addition, 1000×SB202190 stock solution consists of SB202190 and DMSO, wherein the final concentration of SB202190 is 10 mM, and the balance is DMSO.

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

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

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

10000×Cholera Toxin stock solution consists of Cholera Toxin and Cholera Toxin dissolving solution, wherein the final concentration of Cholera Toxin is 10 μM, and the balance is Cholera Toxin dissolving solution. The Cholera Toxin dissolving solution is composed as follows: every 10 mL of the Cholera Toxin dissolving solution contains Tris (1M) pH 7.0 0.05M, NaCl 0.2M, sodium azide 3mM, EDTA (0.5M) pH 8.0 1mM, the balance is equal. for ultrapure water.

1000×Y-27632 consists of Y-27632 and ultrapure water, where the final concentration of Y-27632 is 10mM, and the remainder is ultrapure water.

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

In a second aspect, the present invention claims a kit of reagents for culturing gastric cancer solid tumor primary cells.

The kit of reagents for culturing gastric cancer solid tumor primary cells provided by the present invention contains the medium described above and at least one of the following reagents: the digestion termination solution and the cell cryopreservation solution described below.

In the third aspect, the present invention claims the application of the culture medium or the complete set of reagents in culturing gastric cancer solid tumor primary cells.

In a fourth aspect, the present invention claims a method for culturing gastric cancer solid tumor primary cells.

The method for culturing gastric cancer solid tumor primary cells provided by the present invention may specifically include the following steps: using the medium to suspend and culture gastric cancer solid tumor primary cells.

Further, the method includes the steps of: using a culture vessel with a low-attachment-surface, using the culture medium to suspend the primary cells of the gastric cancer solid tumor, 37° C., 5% CO ₂ conditions The cells are cultured at low temperature, and the medium is changed every 2-4 days (eg, 3 days) until the cells form clumps with a diameter of 50-80 μm (eg, 80 μm).

Wherein, the initial seeding density can be 10 ⁵ cells/cm ² of the bottom area of the container. Taking a six-well plate as an example, the plate is plated at a density of 10 ⁶ cells per well.

Further, the method may further comprise the step of: when the primary cells of solid gastric cancer form a mass with a diameter of 50-80 μm (eg, 80 μm), passage the primary cells of solid gastric cancer.

Wherein, the digestion stop solution used in the passage (it can be stored at 4° C. for one month after preparation) is cultured by fetal bovine serum, antibacterial and antifungal three antibodies (penicillin-streptomycin-amphotericin B) and DMEM base composition; wherein, the final concentration of the fetal bovine serum in the digestion stop solution is 8-12% (eg 10%, % means volume percentage); the antibacterial and antifungal agent tertiary antibody (penicillin-chain The final concentration of penicillin in the digestion stop solution is 100-200 U/mL (such as 100 U/mL); The final concentration of streptomycin B) in the digestion stop solution is 100-200 μg/mL (such as 100 μg/mL); The final concentration of amphotericin B in the digestion stop solution in B) is 250-500 ng/mL (eg, 250 ng/mL); the remainder is DMEM medium.

抗真菌剂。Further, the composition of the antibacterial and antifungal tertiary antibody (penicillin-streptomycin-amphotericin B) is as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B. The antibacterial and antifungal tertiary antibody (Penicillin-Streptomycin-Amphotericin B) is "Antibiotic-Antimycotic, 100X" (eg Gibco #15240062, or other products of the same composition). The "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, using penicillin G (sodium salt) in 0.85% saline, streptomycin sulfate and amphotericin B as

Antifungal agent.

In a specific embodiment of the present invention, the brand product number of the fetal bovine serum is Gibco#16000-044; the brand product number of the antibacterial and antifungal tertiary antibody (Penicillin-Streptomycin-Amphotericin B) is Gibco# 15240062; the brand product number of the DMEM medium is Gibco#11965-092.

More specifically, the step of carrying out the passage: collecting the cell pellets to be passaged, washing the cell pellets with sterile PBS solution after centrifugation, centrifuging again, and then resuspending the cell pellets with cell digestion solution, at 37° C. Digestion is carried out until the cell clumps are digested into single cells, the digestion reaction is terminated with the digestion stop solution (the amount of which can be 5-10 times, such as 10 times the volume), and the cell suspension is collected; after centrifugation, use the Resuspend the cell pellet in the medium, count, and then use a culture vessel with a low adsorption surface to suspend the cultured cells (the initial seeding density can be 10 ⁵ cells/cm ² container bottom area, taking a six-well plate as an example, 10 ⁶ cells per well density of cells plated) at 37° C., 5% CO ₂ . All centrifugation in the above-mentioned passaging steps can specifically be centrifugation at 800-1000g (eg, 800g) at room temperature for 10-20 minutes (eg, 10 minutes).

Further, the method may further include the step of cryopreserving and/or resuscitating the gastric cancer solid tumor primary cells after 2-3 passages and expansions.

Wherein, the cell cryopreservation solution used in the cryopreservation (to be prepared and used immediately) is composed of Advanced DMEM/F12 medium, DMSO and 1% methylcellulose solution; wherein, the Advanced DMEM/F12 medium , The volume ratio of the DMSO and the 1% methylcellulose solution is 20:2:(0.8-1.2), such as 20:2:1; the 1% methylcellulose solution is a concentration of 1 g/ 100ml of methylcellulose in water.

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

Further, the specific steps of performing the cryopreservation: collecting the cell aggregates to be frozen, washing the cell aggregates with sterile PBS solution after centrifugation, centrifuging again, and then resuspending the cell aggregates with the cell digestion solution , digest at 37°C until the cell clumps are digested into single cells, use the digestion stop solution (the amount of which can be 5-10 times, such as 10 times the volume) to stop the digestion reaction, and collect the cell suspension; After centrifugation, use the cell cryopreservation solution to resuspend the cell pellet at a density of 0.5-2×10 ⁶ /mL (eg, 10 ⁶ /mL), freeze it in a gradient cooling box overnight, and then transfer it to liquid nitrogen for long-term storage. All centrifugation in the above freezing steps can be specifically centrifuged at 800-1000g (eg, 800g) at room temperature for 10-20 minutes (eg, 10 minutes).

Further, the specific steps of carrying out the recovery: take out the cryopreservation tube containing the cells to be recovered from the liquid nitrogen, rapidly thaw the cells in sterile water at 37-39°C (eg, 37°C); centrifuge (eg, 800-1000g). , such as 800g room temperature centrifugation for ^5-10 minutes, such as 10 minutes), resuspend the cell pellet with the medium, and then use a culture vessel with a low adsorption surface to suspend the culture cells (the initial seeding density can be 105/ ^cm2 vessel bottom area), cells (10 ⁶ cells per tube) were recovered to a 3.5 cm culture dish), and the culture conditions were 37° C., 5% CO ₂ .

In the first aspect, the second aspect, the third aspect and the fourth aspect, the gastric cancer can specifically be primary gastric cancer, the clinical stage is stage II, stage III or stage IV (according to TNM stage), or various pathological stages Typed gastric cancer or gastric cancer metastases, with surgical specimens weighing more than 20 mg.

In the present invention, all of the above PBS can be 1×PBS, pH 7.3-7.5. Its specific composition is as follows: the solvent is water, the solute and the concentration are: KH ₂ PO ₄ 144 mg/L, NaCl 9000 mg/L, and Na ₂ HPO ₄ ·7H ₂ O 795 mg/L.

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

1. The amount of tissue samples is small, only about 20mg of gastric cancer surgical samples;

1. The amount of tissue samples is small, only about 20mg of gastric cancer surgical samples;

2. It can be used for the culture of primary tumor cells of primary tumor of gastric cancer, as well as the culture of primary tumor cells of metastatic lesions of gastric cancer;

3. The culture period is short, and it only takes 3-10 days to obtain gastric cancer primary tumor cells of the order of 10 ⁷ ;

4. The culture stability is high, and the success rate of in vitro culture of qualified gastric cancer surgical specimens by this method is as high as 70%;

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

The gastric cancer primary cell culture obtained by the method of the present invention can be used for various cell-level in vitro experiments, next-generation sequencing, construction of animal models, construction of cell lines and the like. It is foreseeable that this culture method has broad application prospects in the field of gastric cancer research and clinical diagnosis and treatment.

Description of drawings

Figure 1 shows single cells obtained from gastric cancer tissue after treatment. Scale bar is 100 μm, 100x magnification.

Figure 2 shows the cell mass obtained after primary culture of gastric cancer tissue. Scale bar is 100 μm, 100x magnification.

Figure 3 is a HE staining image of gastric cancer cell aggregate sections obtained after primary culture of gastric cancer tissue. Scale bar is 100 μm, 200 times magnification.

Figure 4 is an immunofluorescence staining diagram of cancer cell mass obtained after primary culture of gastric cancer tissue. Scale bar is 50 μm, 200 times magnification

Figure 5 shows that copy number variation (CNV) analysis based on sequencing results shows that the copy number variation of each generation of gastric cancer primary cell cultures (P1, P2, P3, P4, P5) and primary gastric cancer tumor tissue (Tumor) is highly consistent .

Detailed ways

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Preparation of reagents for culturing gastric cancer primary cells

1. Sample Preservation Solution (100mL)

The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

Table 1 Sample Preservation Solution (100mL)

After the preparation of the sample preservation solution is completed, use 15mL centrifuge tubes for aliquots, each tube of 5mL. After aliquoting, it can be stored at 4°C for 1 month.

2. Sample cleaning solution (100mL)

The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

Table 2 Sample cleaning solution (100mL)

The sample cleaning solution needs to be prepared and used immediately.

3. Sample dissociation solution (10mL)

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

Table 3 Sample dissociation solution (10mL)

Note: The sample dissociation solution is ready for use.

In Table 3, the preparations of collagenase stock solutions are shown in Tables 4-6.

Table 4 10× Collagenase I stock solution (100mL)

After the 10× collagenase I stock solution is prepared, it is divided into 1.5 mL sterile centrifuge tubes, each tube being 1 mL. This stock solution can be stored long-term at -20°C.

Table 5 10× Collagenase II stock solution (100mL)

After the 10× collagenase II stock solution was prepared, it was divided into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored long-term at -20°C.

Table 6 20× Collagenase IV stock solution (100mL)

After the 20× collagenase IV stock solution is prepared, aliquot into 1.5 mL sterile centrifuge tubes, each tube being 1 mL. This stock solution can be stored long-term at -20°C.

In Table 4, Table 5 and Table 6, the enzymatic activity of protease is used to define the unit U of collagenase (the collagenase I or the collagenase IV): at 37°C, pH 7.5, treated with 1 U of protease Collagenase (the collagenase I or the collagenase IV) can release 1 μmol of L-leucine for 5 hours.

4. Cell Digestion Solution (10mL)

The specific formula of cell digestion solution (10 mL) is shown in Table 7.

Table 7 Cell Digestion Solution (10mL)

The cell digestion solution is ready to use.

5. Digestion stop solution (100mL)

The specific formula of digestion stop solution (100 mL) is shown in Table 8.

Table 8 Digestion Stop Solution (100mL)

After the digestion stop solution is prepared, it can be stored at 4°C for one month.

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

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

Table 9 Gastric cancer solid tumor primary cell culture medium (100mL)

After the preparation of the gastric cancer solid tumor primary cell culture medium was completed, it was sterilized by filtration with a 0.22 μM syringe filter (MilliporeSLGP033RS), and it could be stored at 4°C for two weeks.

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

Table 10 100×BSA solution (1mL)

100×BSA solution is prepared and used now.

Table 11 1000× human recombinant protein EGF stock solution (5mL)

After the 1000× human recombinant protein EGF stock solution is prepared, it is divided into 1.5mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 12 1000× human recombinant protein bFGF stock solution (2.5mL)

After the 1000× human recombinant protein bEGF stock solution is prepared, it is divided into 1.5mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 13 1000× human recombinant protein HGF stock solution (5mL)

After the 1000× human recombinant protein HGF stock solution is prepared, it is divided into 1.5mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 14 1000× human recombinant protein FGF-10 stock solution (5mL)

After the 1000× human recombinant protein FGF-10 stock solution is prepared, it is divided into 1.5mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 15 1000× human recombinant protein R-spondin stock solution (4mL)

After the 1000× human recombinant protein R-spondin stock solution is prepared, it is divided into 1.5mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 16 1000× human recombinant protein Wnt-3a stock solution (2.5mL)

After the 1000× human recombinant protein Wnt-3a stock solution is prepared, it is divided into 1.5mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 17 1000× Human Recombinant Protein Noggin Stock Solution (5mL)

After the 1000× human recombinant protein Noggin stock solution is prepared, it is divided into 1.5mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

Table 18 1000×SB202190 stock solution (1.51mL)

After the 1000×SB202190 stock solution is prepared, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

Table 19 100000×A83-01 stock solution (1.05mL)

After the 1000×A83-01 stock solution is prepared, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

Table 20 1000×N-acetyl-L-cysteine stock solution (5mL)

After the 1000×N-acetyl-L-cysteine stock solution is prepared, aliquot into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

Table 21 1000×Nicotinamide stock solution (4mL)

After the 1000×Nicotinamide stock solution is prepared, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

Table 22 10000×Cholera Toxin stock solution (1.17mL)

After the 1000×Cholera Toxin stock solution is prepared, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

In Table 22, the formula of Cholera Toxin solution is shown in Table 23.

Table 23 Cholera Toxin solution (10mL)

After the Cholera Toxin solution is prepared, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

Table 24 1000×Y-27632 stock solution (3.125mL)

After the 1000×Y-27632 stock solution is prepared, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

Table 25 1000×Gastrin stock solution (48mL)

After the 1000×Gastrin stock solution is prepared, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

7. Cell cryopreservation

The specific formula of the cell cryopreservation solution is shown in Table 26.

Table 26 Cell cryopreservation solution

Cell cryopreservation solution is ready to use.

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

Table 27 1% methylcellulose solution (10mL)

The 1% methylcellulose solution can be stored at 4°C for a long time after preparation.

Example 2. Acquisition of postoperative specimens for gastric cancer

1. Cooperate with the top three hospitals, and the cooperation has passed the formal medical ethics review.

2. The attending physician selects the patients according to the clinical indications stipulated in the medical guidelines, and selects appropriate samples for in vitro culture according to the intraoperative clinical indications. The selection criteria for the samples are: primary gastric cancer, and the pathological stage is stage II. , Stage III or IV, gastric cancer or gastric cancer metastases of various pathological types, and the surgical specimen weight of gastric cancer exceeds 20 mg.

3. The attending physician provides basic clinical information such as gender, age, medical history, family history, smoking history, pathological staging, and clinical diagnosis of the patient. The patient's name, ID number and other information related to the patient's privacy are hidden and replaced with a unified experimental number. The naming principle of the experimental number is the eight-digit date of the sample collection + the last four digits of the patient's hospitalization number. For example, for the sample provided on January 1, 2018, the patient's hospital number is T001512765, and the sample experiment number is 201801012765.

4. During the operation, the surgeon collects fresh specimens in the sterile environment of the operating room and places them in the sample preservation solution (see Example 1) prepared in advance. The samples were temporarily stored on ice after ex vivo, and transported to the laboratory within two hours for the next step.

Example 3. Treatment before dissociation of gastric cancer tissue samples

The following procedures need to be performed on ice, and the entire procedure needs to be completed within 10 minutes.

The surgical equipment used in the following operations must be sterilized by high temperature and high pressure in advance, and can be used after drying.

1. Weigh the sample.

2. Wash the surface of the sample with 75% (volume percent) ethanol for 10 to 30 seconds.

3. Wash the sample 10 times with sample wash solution and 5 times with sterile PBS solution.

4. Use ophthalmic scissors, ophthalmic forceps, scalpel and other equipment to carefully peel off the adipose tissue, connective tissue and necrotic tissue in the sample.

Example 4. Dissociation of gastric cancer tissue samples

The surgical equipment used in the following embodiments all need to be sterilized by high temperature and high pressure in advance, and can be used after drying.

1. Use ophthalmic scissors to cut the tissue into small pieces of about 1mm ³ .

2. According to the dosage of 0.1 mL of sample dissociation solution (see Example 1) per mg of tissue, treat the chopped tissue samples with the sample dissociation solution preheated at 37 °C, and dissociate the samples at 37 °C. 15 minutes to 3 hours away from time. Dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

3. Terminate the dissociation reaction with 10 times the volume of the digestion stop solution (see Example 1), and collect the cell suspension.

4. Filter the cell suspension with a 40 μm sterile cell strainer to remove tissue debris and adherent cells.

5. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

6. Resuspend the cells with 5 mL of sterile PBS, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

7. Resuspend the cell pellet with the gastric cancer solid tumor primary cell culture medium (see Example 1), observe the cell state under a microscope, and count the cells.

As shown in Figure 1, in the dissociated single cell suspension, in addition to tumor cells, a large number of other cells of various types, such as erythrocytes, lymphocytes, fibroblasts, etc., are mixed. One of the advantages of this method is that in the subsequent culture process, only cancer cells can be expanded in large quantities, while the proportion of other cells gradually decreases or even disappears, and finally gastric cancer primary tumor cells with higher purity are obtained.

Example 5. Culture of gastric cancer primary cells

1. Use a low-attachment-surface for the suspension culture of gastric cancer primary cells, and the medium used is the gastric cancer solid tumor primary cell culture medium in Example 1 (wherein, the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein HGF is 20ng/mL; the final concentration of human recombinant protein FGF-10 is 20ng/mL; the final concentration of human recombinant protein R-spondin The final concentration of human recombinant protein Wnt-3a is 250 ng/mL; the final concentration of human recombinant protein Noggin is 100 ng/mL; the final concentration of SB202190 is 10 μM; the final concentration of A83-01 is 0.5 μM; N The final concentration of -acetyl-L-cysteine is 1 mM; the final concentration of Nicotinamide is 10 mM; the final concentration of Cholera Toxin is 0.1 nM; the final concentration of Y-27632 is 10 μM; the final concentration of Gastrin is 10 nM). For example, plate at a density of 10 ⁶ cells per well and culture in a cell incubator at 37°C, 5% CO ₂ .

2. Observe the cell status every day, and replace the medium every 3 days until the cells form clumps with a diameter of about 80 μm.

As shown in Figure 2, after 3-10 days of culture, the cancer cells expanded massively to form cell clumps with a diameter of 80 μm, the total number of tumor cells could exceed 10 ⁷ , and the numbers of other types of cells were significantly reduced or even disappeared. The method has been tested by a large number of samples, and the success rate of in vitro culture of gastric cancer primary tumor cells can reach 80%.

Example 6. Passaging of gastric cancer primary cells

1. Collect the cell clumps in the culture dish, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

2. Wash the cell pellet with sterile PBS solution, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

3. Resuspend the cell pellet with cell digestion solution (see Example 1) and digest at 37°C. The digestion of the cell clumps was observed under the microscope every 5 minutes until the cell clumps were all digested into single cells.

4. The dissociation reaction was terminated with 10 times the volume of the digestion stop solution (see Example 1), and the cell suspension was collected.

5. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

6. The cell pellet was resuspended in the gastric cancer solid tumor primary cell culture medium, and the cells were counted.

7. Use a low-attachment-surface for culturing gastric cancer primary cells, and the medium used is the gastric cancer solid tumor primary cell culture medium in Example 1. Taking a six-well plate as an example, press 10 per well. Plated at a density of ⁶ cells and cultured in a cell incubator at 37°C, 5% CO ₂ .

Example 7. Cryopreservation of gastric cancer primary cells

The primary gastric cancer cells in suspension culture can be cryopreserved after 2-3 passages and expansions:

1. Collect the cell clumps in the culture dish, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

2. Wash the cell pellet with sterile PBS solution, centrifuge at 800g for 10 minutes at room temperature, and discard the supernatant.

3. Resuspend the cell pellet with cell digestion solution (see Example 1) and digest at 37°C. The digestion of the cell clumps was observed under the microscope every 15 minutes until the cell clumps were all digested into single cells.

4. Terminate the dissociation reaction with 10 times the volume of the digestion stop solution (see Example 1), collect the cell suspension, and count the cells.

5. Centrifuge at 800g for 10 minutes at room temperature and discard the supernatant.

6. Use cell cryopreservation solution (see Example 1) to resuspend the cell pellet at a density of 10 ⁶ /mL. Each tube of 2mL cryopreservation tube has 1mL cell suspension. After overnight cryopreservation in a gradient cooling box, transfer to liquid nitrogen for medium and long-term storage. save.

Example 8. Recovery of gastric cancer primary cells

Gastric cancer primary cells preserved in liquid nitrogen can be recovered:

1. Prepare sterile water at 37°C five minutes in advance.

2. Remove the cryovial from the liquid nitrogen, and quickly thaw the cells in sterile water at 37°C.

3. Centrifuge at 800g at room temperature for 10 minutes, and discard the supernatant.

4. Resuspend the cell pellet with gastric cancer solid tumor primary cell culture medium (see Example 1), and use a low-adsorption surface for gastric cancer primary cell culture. The cells in each tube are recovered to a 3.5cm culture dish, 37°C, 5% CO. ₂ conditions in a cell incubator.

Example 9. HE staining identification of gastric cancer primary cells

Description of the reagent consumables used in the following examples:

HE staining kit (Beijing Soleibo Biotechnology Co., Ltd., #G1120);

Cationic anti-off slides (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.);

Xylene, methanol, acetone (Beijing Chemical Reagent Company, analytical grade);

Neutral resin glue (Beijing Yili Fine Chemicals Co., Ltd.).

1. The gastric cancer solid tumor primary cell culture medium in Example 1 (wherein, the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein HGF The final concentration of human recombinant protein FGF-10 is 20ng/mL; the final concentration of human recombinant protein R-spondin is 500ng/mL; the final concentration of human recombinant protein Wnt-3a is 250ng/mL; The final concentration of human recombinant protein Noggin is 100ng/mL; the final concentration of SB202190 is 10 μM; the final concentration of A83-01 is 0.5 μM; the final concentration of N-acetyl-L-cysteine is 1 mM; the final concentration of Nicotinamide is 10 mM; Cholera The final concentration of Toxin was 0.1 nM; the final concentration of Y-27632 was 10 μM; the final concentration of Gastrin was 10 nM), and the clumps of gastric cancer solid tumor primary cells were cultured and fixed with 4% paraformaldehyde. Cell pellets were paraffin-embedded and sectioned at a thickness of 5 μm.

2. The paraffin sections were soaked in xylene solution and incubated at room temperature for 5 minutes for deparaffinization. After repeating 3 times, the sections were rinsed twice with deionized water.

3. Immerse the sections in absolute ethanol and incubate for 10 minutes at room temperature, repeat twice.

4. Immerse ginger slices in 95% ethanol and incubate for 10 minutes at room temperature. After repeating twice, rinse the slices twice with deionized water.

5. When the water on the slide is slightly dry, add 100 μL of hematoxylin staining solution for 1 min.

6. Aspirate the hematoxylin staining solution and wash the slides 3 times with tap water.

7. Add 100 μL of differentiation solution dropwise to differentiate for 1 mins.

8. Aspirate the differentiation solution, wash the slide twice with tap water and once with distilled water.

9. Absorb the water on the surface of the slide, add 200 μL of eosin staining solution dropwise for 40s.

10. Aspirate the eosin staining solution, rinse with 75%, 80%, 90%, and 100% ethanol for 20s, 20s, 40s, and 40s.

11. After the ethanol is dry, add 50 μL of xylene dropwise to permeabilize the cells.

12. After the xylene is completely dried, add a drop of neutral resin glue dropwise, cover the slide with a coverslip, observe and take pictures under a microscope.

Figure 3 shows the HE staining effect of gastric cancer primary tumor cells cultured in vitro. It can be seen that these cells generally have the characteristics of cancer cells such as high nucleocytoplasmic ratio, deep nuclear staining, intranuclear chromatin condensation, multinucleation, and uneven cell size. , dozens to hundreds of tumor cells aggregate to form tumor cell clumps with a certain three-dimensional structure.

Example 10. Identification of primary gastric cancer cells by immunofluorescence staining

Description of reagents used in the following examples:

Paraformaldehyde (Beijing Chemical Reagent Company, analytical grade), dissolve paraformaldehyde powder with ultrapure water to make 4% (4g/100mL) paraformaldehyde solution;

Methanol, dimethyl sulfoxide (Beijing Chemical Reagent Company, analytical grade);

Hydrogen peroxide (Beijing Chemical Reagent Company, 35%);

Methanol, dimethyl sulfoxide, and 35% hydrogen peroxide were mixed in a ratio of 4:4:1 (volume ratio) to prepare Dan's rinse solution;

Bovine serum albumin (Sigma, #A1933), dissolve bovine serum albumin in PBS solution to make 3% (3g/100mL) BSA solution;

Immunofluorescence primary antibody (Abeam, #ab17139);

Immunofluorescence secondary antibody (CST, #4408);

Hoechst dye solution (Beijing Soleibo Biotechnology Co., Ltd., #C0021);

The gastric cancer solid tumor primary cell culture medium in Example 1 (wherein, the final concentration of human recombinant protein EGF is 50 ng/mL; the final concentration of human recombinant protein bFGF is 20 ng/mL; the final concentration of human recombinant protein HGF is 20 ng/mL; The final concentration is 20ng/mL; the final concentration of human recombinant protein FGF-10 is 20ng/mL; the final concentration of human recombinant protein R-spondin is 500ng/mL; the final concentration of human recombinant protein Wnt-3a is 250ng/mL; The final concentration of recombinant protein Noggin is 100ng/mL; the final concentration of SB202190 is 10 μM; the final concentration of A83-01 is 0.5 μM; the final concentration of N-acetyl-L-cysteine is 1 mM; the final concentration of Nicotinamide is 10 mM; Cholera Toxin The final concentration of gastrin was 0.1 nM; the final concentration of Y-27632 was 10 μM; the final concentration of Gastrin was 10 nM) for immunofluorescence staining of the cultured gastric cancer cell clumps, and the primary antibody was CK8+CK18 to characterize epithelial-derived cells.

1. Collect the cell clumps in the culture dish, wash with PBS, resuspend the cell pellet with 4% paraformaldehyde, and fix it overnight at 4°C.

2. Centrifuge at 800 g to discard the supernatant, resuspend the cell pellet with pre-cooled methanol solution, and place on ice for 1 hour.

3. Centrifuge at 800g to discard the supernatant, resuspend the cell pellet in Dan's rinsing solution, and place at room temperature for 2 hours.

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

5. Centrifuge at 800 g to discard the supernatant, suspend the cell pellet with 3% BSA solution, and block at room temperature for 2 hours.

6. At a ratio of 1:500, dilute the primary antibody with 3% BSA solution, and resuspend the cell pellet with antibody diluent (3% BSA solution), and keep the primary antibody at 4°C overnight.

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

8. Dilute the secondary antibody with 3% BSA solution at a ratio of 1:2000, and resuspend the cell pellet with antibody diluent (3% BSA solution) for 2 hours at room temperature.

9. Centrifuge at 800 g to discard the supernatant, and wash the cell pellet with PBS solution 5 times for 20 minutes each time.

10. Add 100× Hoechst staining solution at a volume ratio of 1/100, and stain at room temperature for 20 minutes.

11. Wash the cell pellet twice with PBS solution for 10 minutes each time. The staining of cell clumps was observed using a confocal laser microscope.

Figure 4 shows the effect of immunofluorescence staining of gastric cancer primary tumor cell clumps cultured in vitro. It can be seen that the cells constituting the cell clumps are all positive for CK8/CK18 and are of epithelial origin, confirming that the cultured cells obtained by this method are CK8/CK18 positive. high-purity tumor cells. The primary cultures of 20 gastric cancer samples were identified by immunofluorescence staining, and the statistical results showed that in the gastric cancer primary cells obtained by this method, the proportion of tumor cells reached 70%-93% (Table 28).

Table 28 Identification of primary cultures of gastric cancer samples by immunofluorescence staining

Example 11. Gastric cancer primary cell culture and primary tumor tissue

The DNA extraction procedure mentioned in the following examples was carried out using Tiangen blood/tissue/cell genome extraction kit (DP304).

The library construction procedures mentioned in the following examples were carried out using the NEB DNA Sequencing Library Construction Kit (E7645).

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

1. Obtain gastric cancer solid tumor samples, and before in vitro culture operation, first take 10 mg of gastric cancer solid tumor samples for DNA extraction, library construction and whole-genome high-throughput sequencing (WGS), with a sequencing depth of 300×, and the remaining solid tumor samples are used for gastric cancer progenitors. Cells were cultured in vitro.

2. After gastric cancer tissue treatment, use the gastric cancer solid tumor primary cell culture medium in Example 1 (wherein, the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein The final concentration of HGF is 20ng/mL; the final concentration of human recombinant protein FGF-10 is 20ng/mL; the final concentration of human recombinant protein R-spondin is 500ng/mL; the final concentration of human recombinant protein Wnt-3a is 250ng/mL ; the final concentration of human recombinant protein Noggin is 100ng/mL; the final concentration of SB202190 is 10 μM; the final concentration of A83-01 is 0.5 μM; the final concentration of N-acetyl-L-cysteine is 1 mM; the final concentration of Nicotinamide is 10 mM; The final concentration of Cholera Toxin is 0.1 nM; the final concentration of Y-27632 is 10 μM; the final concentration of Gastrin is 10 nM) After a period of culture, the formation of cell clumps with a diameter of more than 100 μm is recorded as P0 generation cells, and then according to the number of passages It is denoted as P1, P2, ..., Pn in turn. From the P1, P2, P3, P4, P5 generation of gastric cancer primary tumor cell culture, 10 ⁶ cells were taken each, and DNA extraction, library construction and whole-genome high-throughput sequencing (WGS) were performed with a sequencing depth of 300×.

3. Copy number variation analysis (CNV) was performed on the sequencing results of each group to compare the copy number variation between the primary gastric cancer tumor tissue and each generation of gastric cancer primary cell cultures. As shown in Figure 5, each generation of gastric cancer primary cells The copy number variation of the culture (P1, P2, P3, P4, P5) and the primary gastric cancer tumor tissue (Tumor) is highly consistent, so the gastric cancer primary cells obtained by this method can represent the real situation of the patient's primary tumor.

Example 12. Comparison of success rate of culture of different primary cell culture media

The operation method and process of primary culture of all samples in this example are completely the same (refer to the above), and only the medium formula is different. The various primary cell culture media tested are shown in Table 29. Wherein scheme D is the formula adopted in the present invention, specifically shown in Table 9 (wherein, the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein HGF is 20ng/mL; the final concentration of human recombinant protein FGF-10 is 20ng/mL; the final concentration of human recombinant protein R-spondin is 500ng/mL; the final concentration of human recombinant protein Wnt-3a is 250ng/mL; the final concentration of human recombinant protein Noggin The final concentration of SB202190 is 10 μM; the final concentration of A83-01 is 0.5 μM; the final concentration of N-acetyl-L-cysteine is 1 mM; the final concentration of Nicotinamide is 10 mM; the final concentration of Cholera Toxin 0.1 nM; the final concentration of Y-27632 was 10 μM; the final concentration of Gastrin was 10 nM).

Table 29 Test primary cell culture medium formula (100mL)

After the primary cell culture medium was prepared, it was sterilized by filtration with a 0.22 μM syringe filter (Millipore SLGP033RS) and stored at 4°C for two weeks.

Each of the four primary cell culture medium regimens treated 20 samples, and the samples were processed and cultured according to the methods described in Examples 3, 4, and 5. After 10 days of culture, the success rate of gastric cancer solid tumor primary cell culture was calculated as shown in Table 30. Show:

Table 30 Cultivation of different mediums

It can be seen that the primary cell culture medium has a great influence on the culture success rate of gastric cancer primary cells, and the gastric cancer solid tumor primary cell culture medium (Table 9) used in the present invention can stimulate the cancer in the gastric cancer solid tumor tissue samples to the greatest extent. Cell proliferation and improve the success rate of primary cell culture of gastric cancer solid tumor.

Embodiment 13. Comparison of the success rate of culture of different sample preservation solutions

In this example, the operation method and process of primary culture of all samples are completely the same (refer to the above), and only the formula of the sample preservation solution is different. The various sample preservation solutions tested are listed in Table 31. Wherein scheme E is the formula adopted in the present invention, and is specifically shown in Table 1.

Table 31 Test sample preservation solution formula (100mL)

After the preparation of various sample preservation solutions in the above table is completed, use 15mL centrifuge tubes for aliquoting, each tube is 5mL. After aliquoting, it can be stored at 4°C for 1 month.

Each of the five sample preservation solutions treated 20 samples. The samples were temporarily stored in the sample preservation solution at 4°C after ex vivo. After 2 hours in vitro, the samples were processed and cultured according to the methods described in Examples 3, 4 and 5. , after 10 days of culture, the success rate of gastric cancer solid tumor primary cell culture is calculated as shown in Table 32:

Table 32 Culture conditions of different sample preservation solutions

It can be seen that the formula of the sample preservation solution has a great influence on the success rate of the primary cell culture of gastric cancer solid tumor. activity and improve the success rate of cultivation.

Example 14. Comparison of success rate of dissociation solution culture of different samples

In this example, the operation method and process of primary culture of all samples are completely the same (refer to the above-mentioned), and only the sample dissociation solution formula is different. The various sample dissociation buffers tested are shown in Table 33. Wherein scheme D is the formula adopted in the present invention, and is specifically shown in Table 3.

Table 33 Test sample dissociation solution formula (10mL)

The sample dissociation solution is ready to use.

Twenty samples of gastric cancer solid tumor tissue blocks weighing more than 100 mg were selected and divided into four equal parts. The above four sample dissociation solutions were used for sample processing and culture operations according to the methods described in Examples 3, 4, and 5. After 10 days of culture, the success rate of gastric cancer solid tumor primary cell culture is calculated as shown in Table 34:

Table 34 Dissociation fluid culture of different samples

It can be seen that the formula of the sample dissociation solution has a great influence on the success rate of the primary cell culture of gastric cancer solid tumor. , to improve the success rate of gastric cancer solid tumor primary cell culture.

Example 15. Comparison of success rate of passage of different cell digests

In this example, all the samples in the primary cell passaging method and process are completely the same (refer to the above), and only the cell digestion solution formula is different. The various sample dissociation buffers tested are shown in Table 35. Wherein scheme D is the formula adopted in the present invention, and is specifically shown in Table 7.

Table 35 Test Cell Digestion Solution (10mL)

The cell digestion solution is ready to use.

Twenty cases of successfully cultured gastric cancer samples were selected, and the primary cells of gastric cancer solid tumor obtained by culture were successively passaged according to the method described in Example 6 using the above-mentioned four cell digestion solutions respectively. Passaging was performed every time the cancer cells expanded to form cell clusters with a diameter of 100 μm (no more than 10 times), and the maximum number of passages was recorded. The statistical results are shown in Table 36:

Table 36 Culture conditions of different cell digests

It can be seen that the formula of the cell digestion solution has a great influence on the success rate of the passage of primary cells of gastric cancer solid tumors. Serial passages can be performed to maintain the viability of gastric cancer solid tumor primary cells.

Claims (10)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

and/or

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

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

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

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