Culture medium for constructing gastric cancer ovary metastasis microtumor model and application thereof

Abstract

The application discloses a culture medium for constructing a gastric cancer ovary metastasis microtumor model and application. The application relates to the technical field of biotechnology, and provides a culture medium for constructing a gastric cancer ovary metastasis microtumor model, which is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein EGF, bFGF, HGF, FGF-10, Wnt-3a, Noggin, R-spondin 1, Follistatin, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additives, cholera toxin, B27, ITS-X, Y-27632, gastrin, prostaglandin E2, beta-estradiol, progesterone, Galunisertib and a basic culture medium. The culture success rate of the gastric cancer ovary metastasis microtumor model is effectively improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a culture medium for constructing a microtumor model of gastric cancer metastasis to the ovary and its application. Background Technology

[0002] Gastric cancer (GC) is a malignant tumor with a leading incidence and mortality rate worldwide. Early diagnosis of GC is low, and most patients are diagnosed at an advanced stage. Ovarian metastasis is one of the most common forms of distant metastasis in GC, occurring in approximately 5%-15% of female patients with advanced GC, clinically most commonly as Krukenberg tumor. Krukenberg tumors are typically characterized by high malignancy, bilateral multiple lesions, a tendency to be accompanied by diffuse peritoneal seeding, and massive malignant ascites. They also respond poorly to traditional systemic chemotherapy, and the median survival is usually less than one year.

[0003] In recent years, 3D in vitro culture technologies, represented by microtumors (PTCs) and organoids, have shown great potential in personalized precision medicine for tumors. By collecting diseased tissue from patients and culturing it in vitro in three dimensions, models reflecting the heterogeneity and pathological characteristics of in situ tumors can be constructed, which can be used for preclinical drug sensitivity testing, drug screening, and basic scientific research. However, existing culture methods are mainly aimed at primary solid tumors of gastric cancer or general gynecological tumors, and have significant limitations in constructing gastric cancer ovarian metastasis models: the high heterogeneity of the microenvironment. Although Kukenberg tumors originate in the gastrointestinal tract, they undergo significant "niche adaptation" during ovarian growth. The ovarian microenvironment is rich in estrogen, progesterone, and high levels of transforming growth factor-β (TGF-β), which induce extremely severe stromal fibrosis (Desmoplasia). It is generally believed in the field that gastric cancer is a hormone-independent tumor; therefore, existing culture media do not contain estrogen / progesterone. This leads to rapid phenotypic drift (such as loss of signet ring cell characteristics) or decreased viability of metastatic cells during in vitro culture due to the lack of key microenvironmental signals. Currently, neither the general culture medium for gastric cancer nor the culture medium for gynecological tumor microtumor models takes into account the specific microenvironmental requirements of gastric cancer ovarian metastases. The general gastric cancer microtumor culture medium does not contain the characteristic hormone signals of the ovarian microenvironment, while the general gynecological tumor microtumor culture medium lacks specific adaptation to gastric tumor cells. When directly used for modeling gastric cancer ovarian metastases (such as Kukenberg tumor), it is difficult to obtain a stable and efficient microtumor model.

[0004] Therefore, given the unique biological characteristics of gastric cancer ovarian metastases, it is crucial to develop a specialized culture medium and construction method that can simulate the ovarian microenvironment and meet the growth requirements of metastatic cells. This would significantly improve the success rate of constructing a gastric cancer ovarian metastatic microtumor model and has significant clinical value and scientific research significance for conducting drug sensitivity screening and personalized treatment research on advanced gastric cancer metastases. Summary of the Invention

[0005] To effectively solve the above-mentioned technical problems, this invention provides a culture technique and supporting reagents for constructing a gastric cancer-ovarian metastasis microtumor model. The core of this technique is: (1) Gastric cancer ovarian metastasis tissue samples were treated with mild cell dissociation reagent, and the culture medium group ratio was adjusted according to the special microenvironment characteristics based on their characteristics (such as the characteristics of Kukenberg tumor) to maximize the success rate of culture.

[0006] (2) A special serum-free culture medium was prepared to better match the clinical microenvironment characteristics of "ovarian metastasis". The suspension culture system was used to allow various types of cells isolated from gastric cancer ovarian metastasis tissue to self-assemble into a cell cluster structure with multiple cell components, which is called "microtumor model of gastric cancer ovarian metastasis".

[0007] In a first aspect, the present invention claims protection for a culture medium used to construct a microtumor model of gastric cancer metastasis to the ovary.

[0008] The culture medium claimed in this invention for constructing a gastric cancer ovarian metastasis microtumor model consists of antibacterial and antifungal agents (triple antibodies), HEPES, GlutaMax, recombinant human protein EGF, recombinant human protein bFGF, recombinant human protein HGF, recombinant human protein FGF-10, recombinant human protein Wnt-3a, recombinant human protein Noggin, recombinant human protein R-spondin 1, recombinant human protein Follistatin, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 supplement, cholera toxin, B27, ITS-X, Y-27632, gastrin, prostaglandin E2 (PGE2), β-estradiol, progesterone, galunisertib, and basal medium.

[0009] The antibacterial and antifungal agents mentioned above are penicillin, streptomycin, and amphotericin B.

[0010] The final concentrations of each component in the culture medium are as follows: The final concentration of HEPES is 8-12 mM (e.g., 10 mM). The final concentration of GlutaMax is 0.8-1.2% (e.g., 1%) by volume. The final concentration of the recombinant human protein EGF is 10-100 ng / mL (e.g., 50 ng / mL). The final concentration of the recombinant human protein bFGF is 10-50 ng / mL (e.g., 20 ng / mL). The final concentration of the recombinant human protein HGF is 5-25 ng / mL (e.g., 20 ng / mL). The final concentration of the recombinant human protein FGF-10 is 5-25 ng / mL (e.g., 20 ng / mL). The final concentration of the recombinant human protein Wnt-3a is 200-300 ng / mL (e.g., 250 ng / mL). The final concentration of the recombinant human protein Noggin is 100-200 ng / mL (e.g., 100 ng / mL). The final concentration of the recombinant human protein R-spondin 1 is 250-500 ng / mL (e.g., 400 ng / mL). The final concentration of the recombinant human protein Follistatin is 50-200 ng / mL (e.g., 150 ng / mL). The final concentration of CHIR99021 is 1.5-6 μM (e.g., 3 μM). The final concentration of SB202190 is 5-10 μM (e.g., 10 μM). The final concentration of A83-01 is 0.25-1.25 μM (e.g., 1 μM). The final concentration of Primocin is 1% by volume. The final concentration of the N-acetyl-L-cysteine ​​is 1-2.5 mM (e.g., 1.5 mM). The final concentration of the nicotinamide is 5-10 mM (e.g., 10 mM). The final concentration of the N2 additive is 1% by volume. The final concentration of the cholera toxin is 0.1-1 nM (e.g., 1 nM). The final concentration of B27 is 1.5-2.5% (e.g., 2%) by volume. The final concentration of ITS-X is 0.8-1.2% (e.g., 1%) by volume. The final concentration of Y-27632 is 5-20 μM (e.g., 10 μM). The final concentration of gastrin is 5-20 nM (e.g., 10 nM). The final concentration of prostaglandin E2 is 0.05-1 μM (e.g., 0.5 μM). The final concentration of β-estradiol is 1-20 nM (e.g., 5 nM). The final concentration of progesterone is 1-200 nM (e.g., 50 nM). The final concentration of Galunisertib is 0.1-0.5 μM (e.g., 0.1 μM).

[0011] In some embodiments of the present invention, the basal culture medium is Advanced DMEM / F12 medium.

[0012] Furthermore, in the culture medium, the final concentration of penicillin in the antibacterial and antifungal agent tri-antibody can be 100-200 U / mL (e.g., 100 U / mL), the final concentration of streptomycin can be 100-200 μg / mL (e.g., 100 μg / mL), and the final concentration of amphotericin B can be 200-250 ng / mL (e.g., 250 ng / mL).

[0013] Further, the antibacterial and antifungal agent comprises the following three antibodies per milliliter: 10,000 units of penicillin (base), 10,000 μg of streptomycin (base), and 25 μg of amphotericin B. In some embodiments of the present invention, the antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100×" (e.g., Gibco#15240062, or other products with the same composition). The "Antibiotic-Antimycotic, 100×" (10,000 units of penicillin (base), 10,000 μg of streptomycin (base), and 25 μg of amphotericin B) utilizes penicillin G (sodium salt), streptomycin sulfate, and amphotericin B in 0.85% saline solution as antibacterial agents. GlutaMax is an advanced cell culture additive that can directly replace L-glutamine in cell culture media. GlutaMax is "GlutaMAX Supplement" (e.g., Gibco#35050061, or other products with the same composition). The "GlutaMAX Supplement" is composed of L-alanyl-L-glutamine, a substitute for L-glutamine, at a concentration of 200 nM, in a 0.85% NaCl solution. CHIR99021 is a highly specific glycogen synthase kinase-3 (GSK-3) inhibitor, inhibiting both GSK-3α and GSK-3β, with IC50 values ​​of 10 nM and 6.7 nM, respectively. SB202190 is "4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazolium" (e.g., Sigma#S7067, or other products with the same composition). A83-01 is "3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide" (e.g., Tocris#2939, or other products with the same composition). The Primocin is a primary cell antimicrobial agent (such as Invivogene #ant-pm-1, or other products with the same composition), an antibiotic used to protect primary cells from microbial contamination, and has killing effects against Gram-positive bacteria, Gram-negative bacteria, mycoplasma, and fungi. The N2 additive is "N-2 Supplement (100×)" (such as Gibco #17502001, or other products with the same composition).The “N-2 Supplement (100×)” contains 1 mM human transferrin (Holo) at a final concentration, 500 mg / L of recombinant insulin full chain, 0.63 mg / L of progesterone, 10 mM of putrescine, and 0.52 mg / L of selenite. The B27 is “B-27 Supplement (50×), minus vitamin A” (e.g., Gibco#12587010, or other products with the same composition). The "B-27 Supplement (50×), minus vitamin A" contains biotin, DL-α-tocopherol acetate, DL-α-tocopherol, BSA (fatty acid-free Fraction V), catalase, human recombinant insulin, human transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, and putrescine. The components are: 2HCl, sodium selenite, and triiodo-I-thyronine (T3). The solvent for ITS-X is Earle's Balanced Salt Solution (EBSS), with the following solutes and concentrations: insulin 1 g / L; transferrin 0.55 g / L; sodium selenite 0.00067 g / L; ethanolamine 0.2 g / L. Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, with Ki values ​​of 220 nM and 300 nM, respectively)" (e.g., MCE#129830-38-2, or other products with the same composition).Galunisertib, also known as LY2157299, is a potent inhibitor of TGFβ receptor 1.

[0014] Furthermore, the culture medium can exist in two forms: Firstly, the culture medium is a solution composed of the antibacterial and antifungal agent triple antibody, HEPES, GlutaMax, 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, human recombinant protein R-spondin 1, human recombinant protein Follistatin, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additive, cholera toxin, B27, ITS-X, Y-27632, gastrin, prostaglandin E2, β-estradiol, progesterone, Galunisertib, and Advanced DMEM / F12 culture medium.

[0015] After the culture medium is prepared, it needs to be filtered and sterilized using a 0.22μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

[0016] Secondly, each component in the culture medium exists independently and is prepared according to the formula when used.

[0017] Furthermore, recombinant human EGF, recombinant human bFGF, recombinant human HGF, recombinant human FGF-10, recombinant human Wnt-3a, recombinant human Noggin, recombinant human R-spondin 1, and recombinant human Follistatin can exist in stock solution (mother solution) form, specifically at a concentration of 1000 times the stock solution (mother solution). SB202190, N-acetyl-L-cysteine, Nicotinamide, Y-27632, Gastrin, Progesterone, Galunisertib, and Prostaglandin E2 (PGE2) can exist in stock solution (mother solution) form, specifically at a concentration of 1000 times the stock solution (mother solution). CHIR99021 and Cholera Toxin can exist in stock solution (mother solution) form, specifically at a concentration of 10000 times the stock solution (mother solution). A83-01, β-estradiol can exist in the form of a stock solution (mother liquor), specifically 100,000 times the stock solution (mother liquor).

[0018] The 1000× human recombinant protein EGF stock solution consists of human recombinant protein EGF, BSA and PBS, wherein the concentration of human recombinant protein EGF is 20 μg / mL, the final concentration of BSA is 0.01 g / mL, and the remainder is PBS.

[0019] The 1000× human recombinant protein bFGF stock solution consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of human recombinant protein bFGF is 20 μg / mL, the final concentration of BSA is 0.01 g / mL, and the remainder is PBS.

[0020] The 1000× human recombinant protein HGF stock solution consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of human recombinant protein HGF is 20 μg / mL, the final concentration of BSA is 0.01 g / mL, and the remainder is PBS.

[0021] The 1000× human recombinant protein FGF-10 stock solution consists of human recombinant protein FGF-10, BSA and PBS, wherein the final concentration of human recombinant protein FGF-10 is 20 μg / mL, the final concentration of BSA is 0.01 g / mL, and the remainder is PBS.

[0022] The 1000× human recombinant protein Wnt-3a stock solution consists of human recombinant protein Wnt-3a, BSA and PBS, wherein the final concentration of human recombinant protein Wnt-3a is 200 μg / mL, the final concentration of BSA is 0.01 g / mL, and the remainder is PBS.

[0023] The 1000× human recombinant protein Noggin stock solution consists 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 remainder is PBS.

[0024] The 1000× human recombinant protein R-spondin 1 stock solution consists of human recombinant protein R-spondin 1, BSA and PBS, wherein the final concentration of human recombinant protein R-spondin 1 is 250 μg / mL, the final concentration of BSA is 0.01 g / mL, and the remainder is PBS.

[0025] The 1000× human recombinant protein Follistatin stock solution consists of human recombinant protein Follistatin, BSA, and PBS, wherein the concentration of human recombinant protein Follistatin is 100 μg / mL, the final concentration of BSA is 0.01 g / mL, and the remainder is PBS.

[0026] Of the eight 1000x stock solutions mentioned above, the BSA can be present in the form of a 100x stock solution (stock solution) (prepared and used immediately). Specifically, it is composed of BSA and PBS, wherein the final concentration of BSA (Sigma#A1933) is 0.1g / mL, and the remainder is PBS.

[0027] In addition, the 1000×SB202190 stock solution is composed of SB202190 and DMSO, wherein the final concentration of SB202190 is 10mM and the balance is DMSO.

[0028] The 1000×N-acetyl-L-cysteine ​​stock solution is composed of N-acetyl-L-cysteine ​​and ultrapure water, wherein the concentration of N-acetyl-L-cysteine ​​is 0.5M, and the remainder is ultrapure water.

[0029] The 1000×Nicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of Nicotinamide is 5M and the remainder is ultrapure water.

[0030] 1000×Y-27632 is composed of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10 mM, and the remainder is ultrapure water.

[0031] The 1000× gastrin stock solution consists of gastrin and ultrapure water, with a final gastrin concentration of 10 μM and the remainder being ultrapure water.

[0032] The 1000×Progesterone stock solution consists of Progesterone and anhydrous ethanol, with a final concentration of Progesterone of 100 μM and the remainder being anhydrous ethanol.

[0033] The 1000×Galunisertib stock solution consists of galunisertib and ultrapure water, with the concentration of galunisertib at 1 mM and the remainder being ultrapure water.

[0034] The 10000×CHIR99021 stock solution is composed of CHIR99021 and DMSO, wherein the final concentration of CHIR99021 is 30mM and the balance is DMSO.

[0035] The 10000×Cholera Toxin stock solution consists of Cholera Toxin and a Cholera Toxin solution, wherein the final concentration of Cholera Toxin is 10 μM, and the remainder is the Cholera Toxin solution. The composition of the Cholera Toxin solution is as follows: each 10 mL of the Cholera Toxin solution contains Tris (1 M) pH 7.0 0.05 M, NaCl 0.2 M, sodium azide 3 mM, EDTA (0.5 M) pH 8.0 1 mM, and the remainder is ultrapure water.

[0036] The 1000×PGE2 reservoir consists of PGE2 and DMSO, with a final concentration of 1 mM for PGE2 and the remainder being DMSO.

[0037] The 100000×A83-01 stock solution is composed of A83-01 and DMSO, wherein the concentration of A83-01 is 25mM and the balance is DMSO.

[0038] The 100000×β-Estradiol stock solution is composed of β-Estradiol and anhydrous ethanol, wherein the final concentration of β-Estradiol is 1 mM, and the remainder is anhydrous ethanol.

[0039] Secondly, the present invention claims protection for a set of reagents for constructing a microtumor model of gastric cancer ovarian metastasis.

[0040] The kit of reagents claimed in this invention for constructing a microtumor model of gastric cancer with ovarian metastasis consists of the culture medium described in the first aspect above and all or part of the following: sample dissociation solution, sample preservation solution, sample washing solution, and digestion termination solution.

[0041] The sample dissociation solution is used for sample dissociation. The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV, and PBS; wherein the final concentration of collagenase I is 150-250 U / mL (e.g., 200 U / mL); the final concentration of collagenase II is 150-250 U / mL (e.g., 200 U / mL); the final concentration of collagenase IV is 150-250 U / mL (e.g., 200 U / mL); the remainder is PBS.

[0042] Furthermore, the unit U of collagenase (collagenase I, collagenase II, or collagenase IV) is defined by the enzymatic activity of the protease: at 37°C and pH 7.5, treatment of collagenase (collagenase I, collagenase II, or collagenase IV) with 1 U of protease for 5 hours can release 1 μmol of L-leucine.

[0043] The sample preservation solution can be used for temporary preservation of samples after in vitro removal, maintaining the viability of cells in the sample for a short period of time after sample removal. Once prepared, the sample preservation solution can be stored at 4°C for one month.

[0044] Further, the sample preservation solution comprises fetal bovine serum, antibacterial and antifungal agents (triple antibodies), HEPES, and HBSS; wherein the antibacterial and antifungal agents (triple antibodies) are penicillin, streptomycin, and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% (e.g., 2%) by volume; the final concentration of penicillin in the antibacterial and antifungal agents (triple antibodies) is 100-200 U / mL (e.g., 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (e.g., 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (e.g., 250 ng / mL); the final concentration of HEPES is 8-12 mM (e.g., 10 mM); the remainder is HBSS.

[0045] Further, the sample washing solution consists of antibacterial and antifungal triple antibodies and PBS; the antibacterial and antifungal triple antibodies are penicillin, streptomycin, and amphotericin B; in the sample washing solution, the final concentration of penicillin in the antibacterial and antifungal triple antibodies is 100-200 U / mL (e.g., 100 U / mL); the final concentration of streptomycin is 100-200 μg / mL (e.g., 100 μg / mL); the final concentration of amphotericin B is 200-250 ng / mL (e.g., 250 ng / mL); the remainder is PBS.

[0046] The digestion termination solution can be used to terminate the sample dissociation process. Once prepared, the digestion termination solution can be stored at 4°C for one month.

[0047] Further, the digestion termination solution consists of fetal bovine serum, antifungal antibodies, and DMEM culture medium; wherein the antifungal antibodies are penicillin, streptomycin, and amphotericin B; in the digestion termination solution, the final concentration of the fetal bovine serum is 8-12% (e.g., 10%) by volume; the final concentration of penicillin in the antifungal antibodies is 100-200 U / mL (e.g., 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (e.g., 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (e.g., 250 ng / mL); the remainder is DMEM culture medium.

[0048] Thirdly, the present invention claims protection for the use of the culture medium described in the first aspect above or the kit of reagents described in the second aspect above in constructing a microtumor model of gastric cancer metastasis to the ovary.

[0049] In some embodiments of the present invention, the gastric cancer ovarian metastasis microtumor model is a Kukenberg tumor microtumor model.

[0050] Fourthly, the present invention claims a method for culturing a microtumor model of gastric cancer metastasis to the ovary.

[0051] The method for culturing a microtumor model of gastric cancer ovarian metastasis, as claimed in this invention, may include the following steps: (a1) Dissociate the gastric cancer ovarian metastasis lesion tissue using the sample dissociation solution described in the second aspect above; (a2) Using the culture medium described in the first aspect above, the cells dissociated in step (a1) are used to form cell clusters, thus obtaining a microtumor model of gastric cancer metastasis to the ovary.

[0052] Further, in step (a1), the gastric cancer ovarian metastasis lesion tissue can be dissociated using the sample dissociation solution according to the following steps: using 1 mL of the sample dissociation solution to dissociate no more than 0.5 mg of tissue, the minced gastric cancer ovarian metastasis lesion tissue is dissociated using the sample dissociation solution at 37°C for 45 minutes to 2 hours (e.g., 1 hour).

[0053] Further, in step (a2), the cells dissociated in step (a1) can be cultured in suspension using the culture medium according to the following steps: using a cell culture container with a low adsorption surface, the cells dissociated in step (a1) are cultured in suspension using the culture medium at 37°C and 5% CO2.

[0054] The initial inoculation density can be 10. 5 pcs / cm 2 The bottom area of ​​the container, taking a six-hole plate as an example, is calculated as 10 per hole. 6 Density plating of individual cells.

[0055] Furthermore, the incubation time in step (a2) is 2-3 days.

[0056] Further, prior to step (a1), the following pretreatment steps may be included for the gastric cancer ovarian metastasis lesion tissue: washing the surface of the gastric cancer ovarian metastasis lesion tissue sample with 70-75% ethanol (by volume) for 10-30 seconds; first washing the gastric cancer ovarian metastasis lesion tissue sample with the sample washing solution described in the second aspect above 5-10 times (e.g., 5 times), then washing the gastric cancer ovarian metastasis lesion tissue sample with sterile PBS solution 5-10 times (e.g., 5 times); then removing impurities, connective tissue, adipose tissue, necrotic tissue, and other components that affect the culture of the microtumor model from the gastric cancer ovarian metastasis lesion tissue sample.

[0057] The pretreatment step for dissociation of the gastric cancer ovarian metastasis lesion tissue needs to be performed on ice, and the entire procedure needs to be completed within 10 minutes.

[0058] Further, in step (a1), after dissociating the solid tumor tissue of gastric cancer metastasis to the ovary with the sample dissociation solution, the following steps are also included: terminating the dissociation reaction with 8-15 times (e.g., 10 times) the volume of the digestion termination solution described in the second aspect above, and collecting the cell suspension; filtering the cell suspension with a 40μm or 100μm sterile cell filter to remove tissue fragments and adherent cells; centrifuging at 300-800g (e.g., 800g) at room temperature for 10-15 minutes (e.g., 10 minutes), and discarding the supernatant; then resuspending the cells with 3-5mL (e.g., 5mL) sterile PBS; then centrifuging again at 800-1000g (e.g., 800g) at room temperature for 10-15 minutes (e.g., 10 minutes), and discarding the supernatant; and then resuspending the cell pellet with the culture medium described in the first aspect above.

[0059] Furthermore, the gastric cancer ovarian metastasis lesion tissue sample undergoing the predissociation treatment is ex vivo within 12 hours and is stored in the sample preservation solution described in the second aspect above before undergoing the predissociation treatment.

[0060] In some embodiments of the present invention, the gastric cancer ovarian metastasis microtumor model is a Kukenberg tumor microtumor model.

[0061] Fifthly, the present invention claims protection for a microtumor model of gastric cancer ovarian metastasis constructed using the method described in the fourth aspect above.

[0062] Sixthly, the present invention claims the use of the microtumor model described in the fifth aspect above in screening therapeutic drugs for gastric cancer ovarian metastases.

[0063] This invention provides a method and matching reagents for extracting and culturing a microtumor model of gastric cancer-ovarian metastasis from fresh gastric cancer ovarian metastasis lesion tissue. This method has the following advantages: 1. Gastric cancer ovarian metastases are predominantly bilateral, with a large volume range, complex microenvironment, high degree of fibrosis, and low tumor content, resulting in extremely low success rates with conventional culture methods. The culture medium and method disclosed in this invention can effectively improve the culture success rate of gastric cancer ovarian metastases.

[0064] 2. This culture medium and method take into account both gastric origin characteristics and metastatic adaptability, and are suitable for gastric cancer ovarian metastatic lesions, such as Kürkenberg tumor. Attached Figure Description

[0065] Figure 1Bright-field images of microtumor microcarcinomas cultured from primary gastric cancer lesions and ovarian metastases from the same patient. The left side shows microtumor microcarcinoma microcarcinoma from the primary gastric cancer lesion; the right side shows microtumor microcarcinoma microcarcinoma from the ovarian metastases. The scale bar is 100 μm, and the objective lens magnification is 10×. Detailed Implementation

[0066] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] Example 1: Preparation of reagents for culturing a gastric cancer-ovarian metastasis microtumor model 1. Sample preservation solution The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0069] Table 1. Sample preservation solution (100mL)

[0070] After the sample preservation solution is prepared, it is aliquoted into 15mL centrifuge tubes, 5mL per tube. After aliquoting, it can be stored at 4℃ for one month. Among the three antibodies mentioned above, the final concentrations are: penicillin 100U / mL, streptomycin 100μg / mL, and amphotericin B 250ng / mL.

[0071] 2. Sample cleaning solution The specific formula for the sample cleaning solution (100 mL) is shown in Table 2. The sample cleaning solution should be prepared and used immediately.

[0072] Table 2. Sample cleaning solution (100mL)

[0073] 3. Sample dissociation solution The specific formula for the sample dissociation solution (10 mL) is shown in Table 3. The sample dissociation solution should be prepared and used immediately.

[0074] Table 3. Sample dissociation solution (10 mL)

[0075] Table 3 shows the preparation of collagenase stock solutions as shown in Tables 4 to 6.

[0076] Table 4. 10× Collagenase I Stock Solution (100mL)

[0077] After preparing the 10× collagenase I stock solution, aliquot it into 1.5mL sterile centrifuge tubes, 1mL per tube. This stock solution can be stored at -20℃ for extended periods.

[0078] Table 5. 10× Collagenase II Stock Solution (100mL)

[0079] After preparing the 10× collagenase II stock solution, aliquot it into 1.5mL sterile centrifuge tubes, 1mL per tube. This stock solution can be stored at -20℃ for extended periods.

[0080] Table 6. 10× Collagenase IV Stock Solution (100mL)

[0081] After preparing the 10× collagenase IV stock solution, aliquot it into 1.5mL sterile centrifuge tubes, 1mL per tube. This stock solution can be stored at -20℃ for extended periods.

[0082] In Tables 4, 5 and 6, the unit U of collagenase (collagenase I, collagenase II or collagenase IV) is defined by the enzyme activity of the protease: at 37°C and pH 7.5, treatment of collagenase (collagenase I, collagenase II or collagenase IV) with 1 U of protease for 5 hours can release 1 μmol of L-leucine.

[0083] 4. Digestion Termination Solution The specific formula for the digestion termination solution (100 mL) is shown in Table 7.

[0084] Table 7. Digestion Termination Solution (100mL)

[0085] After preparation, the digestion termination solution can be stored at 4°C for one month.

[0086] 5. Culture medium for constructing a gastric cancer-ovarian metastasis microtumor model The specific formulation of the culture medium (100 mL) used to construct the gastric cancer ovarian metastasis microtumor model is shown in Table 8.

[0087]

[0088] After preparing the microtumor culture medium for gastric cancer and ovarian metastases, it should be filtered through a 0.22μm syringe filter for sterilization. The prepared medium should be stored at 4°C protected from light and used within two weeks. To maintain the activity of each component, it is recommended to aliquot and store it.

[0089] Table 8 shows the preparation of human recombinant protein stock solutions as shown in Tables 10-17 (BSA stock solution preparation is shown in Table 9; 100×BSA solution should be prepared fresh for immediate use), CHIR99021 stock solution preparation is shown in Table 18, SB202190 stock solution preparation is shown in Table 19, A83-01 stock solution preparation is shown in Table 20, N-acetyl-L-cysteine ​​stock solution preparation is shown in Table 21, and Y-27632 stock solution preparation is shown in Table 22; Cholera toxin (Cholera The preparation of Toxin reservoir is shown in Table 23, the preparation of Nicotinamide reservoir is shown in Table 25, the preparation of Gastrin reservoir is shown in Table 26, the preparation of PGE2 reservoir is shown in Table 27, the preparation of β-estradiol reservoir is shown in Table 28, the preparation of Progesterone reservoir is shown in Table 29, and the preparation of Galunisertib reservoir is shown in Table 30.

[0090] Table 9. 100×BSA solution (1 mL)

[0091] Table 10. 1000× Human Recombinant Protein EGF Stock Solution (5 mL)

[0092] After preparing the 1000× human recombinant protein EGF stock solution, aliquot it into 1.5mL sterile centrifuge tubes. This stock solution can be stored at -80℃ for a long time.

[0093] Table 11. 1000× Human Recombinant Protein bFGF Stock Solution (2.5 mL)

[0094] After preparing the 1000× human recombinant protein bFGF stock solution, aliquot it into 1.5mL sterile centrifuge tubes. This stock solution can be stored at -80℃ for a long time.

[0095] Table 12. Stock solution of 1000× human recombinant protein HGF (5 mL)

[0096] After preparing the 1000× human recombinant protein HGF stock solution, aliquot it into 1.5mL sterile centrifuge tubes. This stock solution can be stored at -80℃ for a long time.

[0097] Table 13. 1000× Human Recombinant Protein FGF-10 Stock Solution (5 mL)

[0098] The 1000× human recombinant protein FGF-10 stock solution can be aliquoted into 1.5mL centrifuge tubes. After aliquoting, it can be stored at -80℃ for extended periods.

[0099] Table 14. Stock solution of 1000× human recombinant protein Wnt-3a (2.5 mL)

[0100] The 1000× human recombinant protein Wnt-3a stock solution can be aliquoted into 1.5 mL centrifuge tubes. After aliquoting, it can be stored at -80℃ for extended periods.

[0101] Table 15. 1000× Human Recombinant Protein Noggin Stock Solution (5 mL)

[0102] After preparing the 1000× human recombinant protein Noggin stock solution, aliquot it into 1.5mL sterile centrifuge tubes. This stock solution can be stored at -80℃ for a long time.

[0103] Table 16. Stock solution of 1000× human recombinant protein R-spondin 1 (4 mL)

[0104] After preparing the 1000× human recombinant protein R-spondin 1 stock solution, aliquot it into 1.5mL sterile centrifuge tubes. This stock solution can be stored at -80℃ for a long time.

[0105] Table 17. Stock solution of 1000× human recombinant protein Follistatin (5 mL)

[0106] After preparing the 1000× human recombinant protein Follistatin stock solution, aliquot it into 1.5mL sterile centrifuge tubes. This stock solution can be stored at -80℃ for a long time.

[0107] Table 18. 10000×CHIR99021 stock solution (1.16 mL)

[0108] After preparing the 10000×CHIR99021 stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored at -20℃ for a long time.

[0109] Table 19, 1000×SB202190 stock solution (1.51 mL)

[0110] After preparing the 1000×SB202190 stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored for a long time at -20℃.

[0111] Table 20, 100000×A83-01 stock solution (1.05 mL)

[0112] After preparing the 100000×A83-01 stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored for a long time at -20℃.

[0113] Table 21. 1000×N-acetyl-L-cysteine ​​stock solution (5 mL)

[0114] After preparing the 1000×N-acetyl-L-cysteine ​​stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored for a long time at -20℃.

[0115] Table 22, 1000×Y-27632 stock solution (3.125 mL)

[0116] After preparing the 1000×Y-27632 stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored for a long time at -80℃.

[0117] Table 23, 10000×Cholera Toxin stock solution (10mL)

[0118] After preparing the 1000×Cholera Toxin stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored for a long time at -20℃.

[0119] The specific formulation of the Cholera Toxin solution in Table 23 is shown in Table 24.

[0120] Table 24. Cholera Toxin solution (10 mL)

[0121] After preparing the Cholera Toxin solution, dispense it into 0.5 mL sterile centrifuge tubes. This solution can be stored at -20°C for an extended period.

[0122] Table 25. 1000×Nicotinamide stock solution (4 mL)

[0123] After preparing the 1000×Nicotinamide stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored at -20℃ for a long period of time.

[0124] Table 26. 1000×Gastrin Stock Solution (48 mL)

[0125] After preparing the 1000× gastrin stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored for a long time at -20℃.

[0126] Table 27, 1000×PGE2 reservoir solution (14.19 mL)

[0127] The 10000×PGE2 stock solution can be dispensed into 1.5mL centrifuge tubes. After dispensing, it can be stored at -80℃ for a long time.

[0128] Table 28. 100000×β-Estradiol stock solution (18.36 mL)

[0129] The 100,000×β-Estradiol stock solution can be aliquoted into 0.5 mL centrifuge tubes. After aliquoting, it can be stored at -20°C for extended periods.

[0130] Table 29. 1000×Progesterone stock solution (15.9mL)

[0131] The 1000×Progesterone stock solution can be aliquoted into 0.5mL centrifuge tubes. After aliquoting, it can be stored at -20℃ for extended periods.

[0132] Table 30, 1000×Galunisertib stock solution (13.5 mL)

[0133] After preparing the 1000×Galunisertib stock solution, dispense it into 0.5mL sterile centrifuge tubes. This stock solution can be stored at -80℃ for a long period of time.

[0134] Example 2: Obtaining surgical specimens of primary gastric cancer and ovarian transplant samples 1. We collaborate with top-tier hospitals to obtain samples through researcher-initiated clinical research, and the collaboration has undergone formal medical ethics review.

[0135] 2. The attending physician selected patients for enrollment according to the clinical indications specified in the medical guidelines, and selected appropriate samples for in vitro culture based on intraoperative clinical indications. The sample selection criteria were: primary gastric cancer with ovarian metastases, clinically diagnosed as Kukenberg tumor. For patients who underwent simultaneous resection of the primary tumor and ovarian metastases during surgery, the primary lesion tissue sample exceeded 20 mg, and the ovarian metastasis tissue sample exceeded 5 mg.

[0136] 3. All enrolled cases are uniformly coded using the format of sample collection date + the last four digits of the patient's hospital number. For example, a sample submitted on January 1, 2020, with patient hospital number T001537474, would have a sample experiment number of 202001017474. The attending physician provides basic clinical information such as the patient's gender, age, medical history, family history, smoking history, pathological stage and type, and clinical diagnosis.

[0137] 4. After the tumor tissue is removed during surgery, a sample collection specialist collects fresh specimens in a sterile environment in the operating room. Samples should be collected from areas rich in fresh blood vessels, avoiding areas with poor cell viability such as necrotic tissue, adipose tissue, and fibrotic tissue. The collected samples are placed in a pre-cooled sample preservation solution (see Example 1) at 4°C. The sample preservation tube containing the sample is temporarily stored on ice and transported to the laboratory within 12 hours for further processing, with the temperature controlled between 2-8°C during transport.

[0138] Example 3: Pretreatment of tissue samples before dissociation The following operations must be performed on ice, and the entire operation must be completed within 10 minutes.

[0139] All surgical instruments used in the following procedures must be sterilized by high-temperature steam (120°C, 20 minutes) and dried before use.

[0140] 1. After weighing the sample, clean the sample surface with medical alcohol (75% by volume) for 10 to 30 seconds.

[0141] 2. Wash the sample 5 times with sample cleaning solution, and then wash the sample 5 times with sterile PBS solution.

[0142] 3. Using ophthalmic scissors, ophthalmic forceps, scalpels, and other instruments, carefully remove the adipose tissue, connective tissue, and necrotic tissue from the sample.

[0143] Example 4: Dissociation of Tissue Samples All surgical instruments used in the following examples must be sterilized by high-temperature steam (120°C, 20 minutes) and dried before use.

[0144] 1. Use ophthalmic scissors to cut the tissue into 0.5mm pieces. 3 Small pieces on the left and right.

[0145] 2. Treat the tissue with the sample dissociation solution (see Example 1). For tissue samples no larger than 0.5 mg, use 1 mL of sample dissociation solution. For tissue samples larger than 0.5 mg, add 0.1 mL of sample dissociation solution for every 0.1 mg increase in tissue weight. The sample dissociation solution treatment conditions are 37°C and the dissociation time is 1 hour. During the dissociation process, observe the sample dissociation under a microscope every 15 minutes until most cells are observed to have detached from the tissue.

[0146] 3. Terminate the dissociation reaction with 10 times the volume of digestion termination solution (see Example 1). After filtering the cell suspension through a 100μm sterile cell filter to remove tissue fragments and adherent cells, centrifuge at 800g at room temperature for 10 minutes and discard the supernatant.

[0147] 4. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0148] 5. Resuspend the cell pellet in the gastric cancer-ovarian metastasis microtumor model culture medium (see Example 1), count the cells, and determine the cell viability by trypan blue staining. Cells with a viability greater than 70% can be seeded and cultured.

[0149] Example 5: Culture of a microtumor model 1. A low-attachment-surface was used for suspension culture of a gastric cancer-ovarian metastasis microtumor model. The culture medium used was the same as that used in Table 8 of Example 1 for culturing the gastric cancer-ovarian metastasis microtumor model (wherein, the final concentration of HEPES was 10 mM; the final concentration of GlutaMax was 1% v / mL; 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 Wnt-3a was 250 ng / mL; the final concentration of human recombinant protein Noggin was 100 ng / mL; and the final concentration of human recombinant protein R-spondin was 100 ng / mL). The final concentration of 1 is 400 ng / mL; the final concentration of the recombinant human protein Follistatin is 150 ng / mL; the final concentration of CHIR99021 is 3 μM; the final concentration of SB202190 is 10 μM; the final concentration of A83-01 is 1 μM; the final concentration of N-acetyl-L-cysteine ​​is 1.5 mM; the final concentration of Nicotinamide is 10 mM; the final concentration of Cholera Toxin is 1 nM; the final concentration of Y-27632 is 10 μM; the final concentration of Gastrino is 10 nM; the final concentration of PGE2 is 0.5 μM; the final concentration of β-estradiol is 5 nM; the final concentration of progesterone is 50 nM; and the final concentration of Galunisertib is 0.1 μM. Taking a six-well plate as an example, 10 [units of solution] per well are used. 6 Cells were seeded at a density of 1000 cells / well, with 2-3 mL of culture medium per well. After seeding, the cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0150] 2. Observe the cell status daily until the cells form clumps with a diameter of about 100μm. After that, change the culture medium every 2-3 days to maintain the growth of microtumors.

[0151] like Figure 1 As shown, within the first 48 hours of culture, various cell types derived from cancer tissue spontaneously aggregated and self-assembled into cell clumps of 100 μm in size, which we call a microtumor model. The total number of microtumor cell clumps can reach 10. 5 -10 6 This method has been tested on a large number of samples, and the success rate of culturing microtumor models from different gastric cancer surgical samples (primary gastric cancer lesions and gastric cancer ovarian metastases) can reach 70%.

[0152] Example 6: Comparison of the ability of different culture media to form microtumor structures in primary gastric cancer lesions and ovarian metastases of gastric cancer. In this embodiment, the procedures for culturing the microtumor models for all samples were completely identical (as described above), with only the culture medium formulations differing. In addition to the culture medium shown in Table 8 of this invention (with the same formulation as in Example 5), the following three control culture media were used for comparative testing: (1) Control culture medium A: Table 9 in the specification of Chinese Patent 202111136701.4 (Invention title: A method for culturing gastric cancer microtumor cell model) (i.e. Table 31 below).

[0153]

[0154] The final concentration of the recombinant human protein EGF was 50 ng / mL; the final concentration of the recombinant human protein bFGF was 20 ng / mL; the final concentration of the recombinant human protein HGF was 20 ng / mL; the final concentration of the recombinant human protein FGF-10 was 20 ng / mL; the final concentration of the recombinant human protein Wnt-3a was 200 ng / mL; the final concentration of the recombinant human protein Noggin was 100 g / mL; and the final concentration of the recombinant human protein R-spondin was... The final concentration of 1 is 400 ng / mL; the final concentration of the human recombinant protein IL-2 is 20 ng / mL; the final concentration of the human recombinant protein IL-15 is 20 ng / mL; the final concentration of CHIR99021 is 3 μM; the final concentration of SB202190 is 10 μM; the final concentration of A83-01 is 1 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 1 nM; the final concentration of Y-27632 is 10 μM; and the final concentration of Gastrino is 10 nM.

[0155] (2) Control culture medium B: 202410996481.X (Invention title: A culture medium and culture method for culturing a microtumor model of gastric cancer liver metastasis) is shown in Table 8 of the instruction manual (i.e. Table 32 below).

[0156]

[0157] The final concentrations of the recombinant human protein EGF were 50 ng / mL; the final concentrations of the recombinant human protein bFGF were 20 ng / mL; the final concentrations of the recombinant human protein HGF were 20 ng / mL; the final concentrations of the recombinant human protein Noggin were 100 ng / mL; the final concentrations of the recombinant human protein R-spondin 1 were 400 ng / mL; the final concentrations of the recombinant human protein FGF-10 were 20 ng / mL; the final concentrations of the recombinant human protein Wnt-3a were 200 ng / mL; the final concentrations of the SB202190 were 10 μM; the final concentrations of the CHIR99021 were 3 μM; the final concentrations of the A83-01 were 1 μM; the final concentrations of the N-acety-L-cysteine ​​were 1 mM; the final concentrations of the Nicotinamide were 10 mM; and the final concentrations of the Cholera... The final concentration of Toxin was 0.5 nM; the final concentration of Y-27632 was 10 μM; and the final concentration of Gastrin was 10 nM.

[0158] (3) Control culture medium C: 202211316153.8 (Invention title: A method for culturing a gynecological tumor microtumor model and the culture medium used therein) is shown in Table 22 of the instruction manual (i.e. Table 33 below).

[0159]

[0160] The primary gastric cancer lesions and ovarian metastatic lesions of the same patient were cultured using the culture medium shown in Table 8 of this invention, as well as the three control culture media shown in Tables 31, 32, and 33 (see above for the specific procedure). The number and size of microtumors were observed, and the results are shown in Tables 34 and 35.

[0161] As can be seen from Table 34, when culturing primary gastric cancer samples, the control culture medium A in Table 31 has a higher success rate in microtumor culture, a greater number of cell clusters obtained, and a more stable size.

[0162]

[0163] As shown in Table 35, culturing ovarian metastatic lesions is more difficult than culturing primary gastric cancer lesions, especially control medium B. Although it is also a medium for metastatic lesions, its success rate is low, making it unsuitable for culturing microtumors of gastric cancer-ovarian metastases. Furthermore, gynecological tumor media are also unsuitable for culturing gastric cancer-ovarian metastases. The media in Table 8 of this invention exhibit higher success rates, better cell cluster numbers and sizes, and stronger overall culture stability in the culture of microtumors from gastric cancer-ovarian metastases.

[0164] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A culture medium for constructing a gastric cancer-ovarian metastasis microtumor model, characterized in that: The culture medium consists of antibacterial and antifungal agents (triple antibodies), HEPES, GlutaMax, recombinant human protein EGF, recombinant human protein bFGF, recombinant human protein HGF, recombinant human protein FGF-10, recombinant human protein Wnt-3a, recombinant human protein Noggin, recombinant human protein R-spondin 1, recombinant human protein Follistatin, CHIR99021, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additive, cholera toxin, B27, ITS-X, Y-27632, gastrin, prostaglandin E2, β-estradiol, progesterone, Galunisertib, and basal medium; wherein the antibacterial and antifungal agents (triple antibodies) are penicillin, streptomycin, and amphotericin B; the final concentrations of each component in the culture medium are as follows: The final concentration of HEPES is 8-12 mM; The final concentration of GlutaMax is 0.8-1.2% by volume. The final concentration of the recombinant human 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 recombinant human protein HGF is 5-25 ng / mL; The final concentration of the recombinant human protein FGF-10 is 5-25 ng / mL; The final concentration of the recombinant human protein Wnt-3a is 200-300 ng / mL; The final concentration of the recombinant human protein Noggin is 100-200 ng / mL; The final concentration of the human recombinant protein R-spondin 1 is 250-500 ng / mL; The final concentration of the human recombinant protein Follistatin is 50-200 ng / mL; The final concentration of CHIR99021 is 1.5-6 μM; The final concentration of SB202190 is 5-10 μM; The final concentration of A83-01 is 0.25-1.25 μM; The final concentration of Primocin is 1% by volume. The final concentration of the N-acetyl-L-cysteine ​​is 1-2.5 mM; The final concentration of the nicotinamide is 5-10 mM; The final concentration of the N2 additive is 1% by volume. The final concentration of the cholera toxin is 0.1-1 nM; The final concentration of B27 is 1.5-2.5% by volume. The final concentration of ITS-X is 0.8-1.2% by volume. The final concentration of Y-27632 is 5-20 μM; The final concentration of gastrin is 5-20 nM; The final concentration of prostaglandin E2 is 0.05-1 μM; The final concentration of β-estradiol is 1-20 nM; The final concentration of progesterone is 1-200 nM; The final concentration of Galunisertib is 0.1-0.5 μM.

2. The culture medium according to claim 1, characterized in that: The basal culture medium is Advanced DMEM / F12 medium.

3. The culture medium according to claim 1 or 2, characterized in that: In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agent tri-antibody is 100-200 U / mL, the final concentration of streptomycin is 100-200 μg / mL, and the final concentration of amphotericin B is 200-250 ng / mL.

4. A complete set of reagents for constructing a microtumor model of gastric cancer metastasis to the ovary, characterized in that: The complete set of reagents consists of the culture medium described in any one of claims 1-3 and all or part of the following: sample dissociation solution, sample preservation solution, sample washing solution, and digestion termination solution.

5. The complete set of reagents according to claim 4, characterized in that: The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV, and PBS; wherein the final concentrations of collagenase I, collagenase II, and collagenase IV are all 150-250 U / mL; the remainder is PBS; and / or The sample preservation solution comprises fetal bovine serum, antibacterial and antifungal agents (triple antibodies), HEPES, and HBSS; wherein the antibacterial and antifungal agents (triple antibodies) are penicillin, streptomycin, and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% by volume; the final concentration of the HEPES is 8-12 mM; the remainder is the HBSS; and / or The sample washing solution consists of a triple antibody (antibacterial and antifungal agent) and PBS; wherein the triple antibody is penicillin, streptomycin, and amphotericin B; and / or The digestion termination solution consists of fetal bovine serum, antibacterial and antifungal agents (antibiotics, antibiotics, and amphotericin B), and DMEM culture medium. The antibacterial and antifungal agents are penicillin, streptomycin, and amphotericin B. The final concentration of the fetal bovine serum in the digestion termination solution is 8-12% by volume, with the remainder being the DMEM culture medium.

6. The use of the culture medium according to any one of claims 1-3 or the reagent kit according to claim 4 or 5 in constructing a gastric cancer ovarian metastasis microtumor model.

7. The application according to claim 6, characterized in that: The gastric cancer-ovarian metastasis microtumor model is the Kukenberg tumor microtumor model.

8. A method for constructing a microtumor model of gastric cancer metastasis to the ovary, characterized in that: The method includes the following steps: (a1) Dissociating gastric cancer ovarian metastatic lesion tissue using the sample dissociation solution described in claim 4 or 5; (a2) The single cells dissociated in step (a1) are suspended in the culture medium described in any one of claims 1-3 to form cell clusters, thereby obtaining a gastric cancer ovarian metastasis microtumor model.

9. The method according to claim 8, characterized in that: The gastric cancer-ovarian metastasis microtumor model is the Kukenberg tumor microtumor model.

10. A gastric cancer ovarian metastasis microtumor model constructed using the method described in claim 8 or 9.

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