A culture medium for constructing a colorectal cancer peritoneal metastasis microtumor cell model and its application

Abstract

The present invention discloses a culture medium for constructing a colorectal cancer peritoneal metastasis microtumor cell model and its application. The present invention relates to the field of biotechnology and provides a culture medium for culturing a colorectal cancer peritoneal metastasis microtumor model, comprising antibacterial and antifungal agents, triple anti-antibiotics, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein Noggin, human recombinant protein R-spondin 1, human recombinant protein GDNF, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additive, cholera toxin, B27, ITS-X, Y-27632, Galunisertib, and a basal culture medium. The culture medium and culture method disclosed in the present invention can effectively improve the culture success rate of colorectal cancer peritoneal metastasis and lesions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium for constructing a colorectal cancer peritoneal metastasis microtumor cell model and application thereof. Background Art

[0002] Colorectal cancer is a malignant tumor that poses a serious threat to human health. Peritoneal metastasis is a major form of metastasis of colorectal cancer, and approximately 10-20% of colorectal cancer patients will eventually develop peritoneal metastasis. After radical surgery, 4-19% of patients will still develop peritoneal metastasis during the follow-up period, and 40%-50% of deaths in patients with postoperative recurrence are directly related to the progression of peritoneal metastasis. Patients with peritoneal metastasis have a poor prognosis, with a median overall survival time of only 6-9 months and a 5-year survival rate of less than 20%. Therefore, peritoneal metastasis is also one of the main causes of death in patients with colorectal cancer.

[0003] Chemotherapy based on classic cytotoxic drugs is one of the basic treatments for colorectal cancer. However, chemotherapy drugs have limited efficacy against peritoneal metastases, often only slowing their progression. For patients with extensive peritoneal metastases, there are no effective drug treatment options. This places new demands on the research and personalized treatment of peritoneal metastases in colorectal cancer.

[0004] Colorectal cancer is a complex systemic disease. The biological processes underlying its development, progression, recurrence, and metastasis, as well as its heterogeneity, drug resistance, and tumor immune responses, remain largely unknown. The causes and progression of peritoneal metastasis in colorectal cancer vary significantly from individual to individual, and the tumor microenvironment of peritoneal metastases differs significantly from that of the primary colorectal cancer lesion. Therefore, using primary cell cultures of peritoneal metastases as models for personalized, precision research is a growing trend in colorectal cancer research, including in diagnosis and treatment. Therefore, developing tumor models that accurately reflect the characteristics of patient peritoneal metastases is crucial. Existing primary tumor cell culture techniques, such as 2D, 3D, and reprogrammed cultures, all face challenges, to varying degrees, including extremely long culture cycles, low success rates, difficulty removing contaminating cells, and an inability to recreate the tumor microenvironment. Peritoneal metastases of colorectal cancer exhibit distinct microenvironmental characteristics from the primary lesion, necessitating distinct culture conditions. Summary of the Invention

[0005] In order to effectively solve the above technical problems, the present invention provides a new colorectal cancer peritoneal metastasis lesion micro-tumor model culture technology and supporting reagents. The core of this technology is: (1) using a mild cell dissociation reagent to treat the solid tumor tissue of colorectal cancer peritoneal metastasis, adjusting the culture medium component ratio according to the microenvironment characteristics of the colorectal cancer peritoneal metastasis lesion, and ensuring the vitality of various types of cells in the tissue to the greatest extent; (2) preparing a special serum-free culture medium, and using a suspension culture system to make various types of cells isolated from the colorectal cancer peritoneal metastasis lesion tissue self-assemble to form a cell cluster structure with multiple cell components, which is called a "colorectal cancer peritoneal metastasis micro-tumor model".

[0006] In a first aspect, the present invention claims a culture medium for culturing a microtumor model of peritoneal metastasis of colorectal cancer.

[0007] The culture medium for culturing a microtumor model of peritoneal metastasis of colorectal cancer claimed in the present invention is composed of three antibacterial and antifungal agents, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein Noggin, human recombinant protein R-spondin 1, human recombinant protein GDNF, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 supplement, cholera toxin, B27, ITS-X, Y-27632, Galunisertib and basal culture medium; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B.

[0008] In the culture medium, the final concentration of HEPES is 8-12 mM (such as 10 mM); the final concentration of GlutaMax is 0.8-1.2% (such as 1%) by volume; the final concentration of the human recombinant protein EGF is 10-100 ng / mL (such as 50 ng / mL); the final concentration of the human recombinant protein bFGF is 10-50 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein HGF is 5-25 ng / mL (such as 20 ng / mL); the final concentration of the human recombinant protein Noggin is 100-200 ng / mL (such as 100 ng / mL); the final concentration of the human recombinant protein R-spondin is 100-200 ng / mL (such as 100 ng / mL); The final concentration of 1 is 250-500 ng / mL (such as 400 ng / mL); the final concentration of the human recombinant protein GDNF is 50-100 ng / mL (such as 100 ng / mL); the final concentration of SB202190 is 5-10 μM (such as 10 μM); the concentration of A83-01 is 0.25-1.25 μM (such as 1 μM); the final concentration of Primocin is 1% by volume; the concentration of N-acetyl-L-cysteine ​​is 0.5-2 mM (such as 1 mM); the final concentration of nicotinamide is The final concentration of the N2 additive is 1% by volume; the final concentration of the cholera toxin is 0.1-1nM (such as 0.5nM); the final concentration of the B27 is 1.5-2.5% (such as 2%) by volume; the final concentration of the ITS-X is 0.8-1.2% (such as 1%) by volume; the final concentration of the Y-27632 is 5-20μM (such as 10μM); and the final concentration of the Galunisertib is 0.1-0.5μM (such as 0.1μM).

[0009] Furthermore, GlutaMax is an advanced cell culture supplement 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 ingredient of "GlutaMAX™ Supplement" is L-alanyl-L-glutamine, a substitute for L-glutamine, at a concentration of 200 nM, and the solvent is 0.85% NaCl solution. SB202190 is "4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole" (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). Primocin is an antimicrobial agent for primary cells (such as Invivogene #ant-pm-1, or other products with the same composition). It is an antibiotic used to protect primary cells from microbial contamination and is effective against Gram-positive and Gram-negative bacteria, mycoplasmas, and fungi. The N2 supplement is "N-2 Supplement (100X)" (such as Gibco #17502001, or other products with the same composition). The N-2 Supplement (100X) contains a final concentration of 1 mM human transferrin (Holo), 500 mg / L recombinant insulin full chain, 0.63 mg / L progesterone, 10 mM putrescine, and 0.52 mg / L selenite. The B27 is "B-27™ Supplement (50X), minus vitamin A" (such as Gibco #12587010, or other products with the same composition).The "B-27™ Supplement (50X), 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 hydrochloride, glutathione (reduced), L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine 2HCl), sodium selenite, and triiodothyronine (T3). The solvent for ITS-X is EBSS (Earle's balanced salt solution), and the solutes and concentrations are as follows: insulin 1g / L; transferrin 0.55g / L; sodium selenite 0.00067g / L; and ethanolamine 0.2g / L. Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor with Ki of 220nM and 300nM, respectively)" (e.g., MCE#129830-38-2, or other products with the same composition). Galunisertib, also known as LY2157299, is a potent inhibitor of TGFβ receptor 1.

[0010] Furthermore, the basal culture medium may be Advanced DMEM / F12 culture medium.

[0011] In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents is 100-200 U / mL (such as 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (such as 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (such as 250 ng / mL).

[0012] Furthermore, the antibacterial and antifungal triple antibiotic is "Antibiotic-Antimycotic, 100X" (such as Gibco #15240062, or other products with the same composition). Each milliliter of "Antibiotic-Antimycotic, 100X" contains 10,000 units of penicillin (base), 10,000 μg of streptomycin (base), and 25 μg of amphotericin B, using penicillin G (sodium salt), streptomycin sulfate, and amphotericin B in the form of a 0.85% saline solution as antifungal agents.

[0013] Furthermore, the culture medium may exist in two forms:

[0014] First, the culture medium is a solution prepared by mixing the antibacterial and antifungal agents triple antibody, HEPES, GlutaMax, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF, the human recombinant protein Noggin, the human recombinant protein R-spondin 1, the human recombinant protein GDNF, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 supplement, cholera toxin, B27, ITS-X, Y-27632, Galunisertib, and the basal culture medium.

[0015] After the culture medium is prepared, it needs to be sterilized by filtration using a 0.22 μM 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, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein Noggin, human recombinant protein R-spondin 1, and human recombinant protein GDNF can be present in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). SB202190, N-acetyl-L-cysteine, Nicotinamide, Y-27632, and Galunisertib can be present in the form of a stock solution (mother solution), specifically a 1000-fold stock solution (mother solution). Cholera Toxin can be present in the form of a stock solution (mother solution), specifically a 10,000-fold stock solution (mother solution). A83-01 can be present in the form of a stock solution (mother solution), specifically a 100,000-fold stock solution (mother solution).

[0018] The 1000× 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 μg / mL, the final concentration of the 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 the human recombinant protein bFGF is 20 μg / mL, the final concentration of the 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 the human recombinant protein HGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0021] The 1000× 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 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0022] 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 the human recombinant protein R-spondin 1 is 100 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0023] The 1000× human recombinant protein GDNF stock solution consists of human recombinant protein GDNF, BSA and PBS, wherein the final concentration of the human recombinant protein GDNF is 200 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the remainder is PBS.

[0024] Among the six 1000x stock solutions, the BSA can be present in the form of a 100x stock solution (mother solution) (prepared and used immediately), specifically composed of BSA and PBS, wherein the final concentration of BSA (Sigma #A1933) is 0.1 g / mL, and the remainder is PBS.

[0025] In addition, 1000×SB202190 is composed of SB202190 and DMSO, wherein the final concentration of SB202190 is 10 mM and the remainder is DMSO.

[0026] The 1000×N-acetyl-L-cysteine ​​stock solution consists of N-acetyl-L-cysteine ​​and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine ​​is 0.5 M and the remainder is ultrapure water.

[0027] The 1000× Nicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of Nicotinamide is 5M and the remainder is ultrapure water.

[0028] The 1000× Galunisertib stock solution consists of Galunisertib and ultrapure water, wherein the concentration of Galunisertib is 1 mM and the remainder is ultrapure water.

[0029] 1000×Y-27632 consists of Y-27632 and ultrapure water, wherein the concentration of Y-27632 is 10 mM and the remainder is ultrapure water.

[0030] 10000× Cholera Toxin stock solution consists of Cholera Toxin and Cholera Toxin dissolving solution, where the concentration of Cholera Toxin is 10 μM, and the remainder is the Cholera Toxin dissolving solution. The Cholera Toxin dissolving solution is composed of the following: 0.05 M Tris (1 M) pH 7.0, 0.2 M NaCl, 3 mM sodium azide, 1 mM EDTA (0.5 M) pH 8.0, and the remainder is ultrapure water per 10 mL of the Cholera Toxin dissolving solution.

[0031] The 100000×A83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25 mM and the remainder is DMSO.

[0032] In a second aspect, the present invention claims a kit of reagents for culturing a microtumor model of peritoneal metastasis of colorectal cancer.

[0033] The reagent set for culturing a microtumor model of peritoneal metastasis of colorectal cancer claimed in the present invention 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.

[0034] Furthermore, 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 (such as 200 U / mL); the final concentration of collagenase II is 150-250 U / mL (such as 200 U / mL); the final concentration of collagenase IV is 150-250 U / mL (such as 200 U / mL); and the remainder is PBS.

[0035] The unit U of collagenase (the collagenase I, the collagenase II, or the collagenase IV) is defined by the enzymatic activity of the protease: 1 μmol of L-leucine can be released when the collagenase (the collagenase I, the collagenase II, or the collagenase IV) is treated with 1 U of protease at 37°C and pH 7.5 for 5 hours.

[0036] Furthermore, the sample preservation solution is composed of fetal bovine serum, three antibacterial and antifungal agents, HEPES and HBSS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% (such as 2%) by volume; the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200 U / mL (such as 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (such as 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (such as 250 ng / mL); the final concentration of the HEPES is 8-12 mM (such as 10 mM); and the remainder is HBSS.

[0037] Furthermore, the sample cleaning solution is composed of three antibacterial and antifungal agents and PBS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibacterial and antifungal agents is 100-200 U / mL (such as 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (such as 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (such as 250 ng / mL); the remainder is PBS.

[0038] Furthermore, the digestion stop solution is composed of fetal bovine serum, three antifungal antibodies and DMEM culture medium; wherein the three antibacterial and antifungal antibodies are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% (such as 10%) by volume; the final concentration of penicillin in the three antibacterial and antifungal antibodies is 100-200 U / mL (such as 100 U / mL), the final concentration of streptomycin is 100-200 μg / mL (such as 100 μg / mL), and the final concentration of amphotericin B is 200-250 ng / mL (such as 250 ng / mL); the remainder is DMEM culture medium.

[0039] In a third aspect, the present invention claims the use of the culture medium described in the first aspect or the set of reagents described in the second aspect in constructing a microtumor model of peritoneal metastasis of colorectal cancer.

[0040] In a fourth aspect, the present invention claims a method for constructing a microtumor model of peritoneal metastasis of colorectal cancer.

[0041] The method for constructing a micro-tumor model of peritoneal metastasis of colorectal cancer claimed in the present invention may include the following steps:

[0042] (a1) dissociating solid tumor tissue of colorectal cancer peritoneal metastasis using the sample dissociation solution described in the second aspect above;

[0043] (a2) Using the culture medium described in the first aspect above, the single cells dissociated in step (a1) are suspended and cultured to form cell clusters, thereby obtaining a microtumor model of peritoneal metastasis of colorectal cancer.

[0044] In step (a1), the solid tumor tissue of peritoneal metastasis of colorectal cancer can be dissociated using the sample dissociation solution according to a method comprising the following steps: using no more than 0.5 mg of tissue per 1 mL of the sample dissociation solution, the minced solid tumor tissue of peritoneal metastasis of colorectal cancer is dissociated using the sample dissociation solution at 37° C. for a dissociation time of 15 minutes to 2 hours (e.g., 1 hour).

[0045] In step (a2), the cells dissociated from (a1) can be suspended and cultured using the culture medium according to a method comprising the following steps: using a cell culture container with a low adsorption surface, suspending and culture the cells dissociated from (a1) using the culture medium, and culturing the cells at 37° C. and 5% CO 2 .

[0046] Among them, the initial seeding density can be 10 5 pieces / cm 2 The bottom area of ​​the container, taking a six-well plate as an example, is 10 per well. 6 Cells were plated at a density of 100 cells.

[0047] Furthermore, the culturing time in step (a2) is 2-3 days.

[0048] Furthermore, before step (a1), the following steps may be included for pre-dissociation treatment of the solid tumor tissue of colorectal cancer peritoneal metastasis: washing the surface of the solid tumor tissue sample of colorectal cancer peritoneal metastasis with 70-75% (such as 75%) ethanol by volume for 10-30 seconds; washing the solid tumor tissue sample of colorectal cancer peritoneal metastasis 5-10 times (such as 5 times) with the sample washing solution described in the second aspect above, and washing the solid tumor tissue sample of colorectal cancer peritoneal metastasis 5-10 times (such as 5 times) with a sterile PBS solution; and then removing impurities, connective tissue, adipose tissue, necrotic tissue and other components that affect micro-tumor culture in the solid tumor tissue sample of colorectal cancer peritoneal metastasis.

[0049] The step of pre-dissociation treatment of the solid tumor tissue of colorectal cancer peritoneal metastasis needs to be performed on ice, and the entire operation needs to be completed within 10 minutes.

[0050] Furthermore, in step (a1), after the solid tumor tissue of colorectal cancer peritoneal metastasis is dissociated with the sample dissociation solution, the following steps are also included: terminating the dissociation reaction with 8-15 times (such as 10 times) the volume of the digestion stop solution described in the second aspect above, and collecting the cell suspension; filtering the cell suspension with a 100μm or 40μm sterile cell strainer to remove tissue fragments and adherent cells; centrifuging at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; then resuspending the cells with 3-5mL (such as 5mL) of sterile PBS; centrifuging again at 800-1000g (such as 800g) at room temperature for 10-15 minutes (such as 10 minutes), and discarding the supernatant; and then resuspending the cell pellet with the culture medium described in the first aspect above.

[0051] The solid tumor tissue sample of colorectal cancer peritoneal metastasis that undergoes the pre-dissociation treatment is isolated from the body within 12 hours and has been stored in the sample preservation solution described in the second aspect before the pre-dissociation treatment.

[0052] In a fifth aspect, the present invention claims protection for a micro-tumor model of peritoneal metastasis of colorectal cancer constructed using the method described in the fourth aspect above.

[0053] The present invention has the beneficial effects of: small-sized peritoneal metastases from colorectal cancer, high fibrosis, and low tumor content, whereas conventional primary cell culture methods have extremely low success rates. The culture medium and culture method disclosed in the present invention can effectively improve the success rate of culturing peritoneal metastases from colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Bright field images of microtumors obtained by culture from samples of primary colorectal cancer and peritoneal metastasis from the same patient. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0056] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0057] Example 1: Preparation of reagents for culturing a colorectal cancer peritoneal metastasis microtumor model

[0058] 1. Sample preservation solution

[0059] The specific formula of sample preservation solution (100 mL) is shown in Table 1.

[0060]

[0061] After the sample preservation solution is prepared, aliquot it into 15 mL centrifuge tubes, 5 mL per tube. The aliquots can be stored at 4°C for 1 month.

[0062] 2. Sample cleaning solution

[0063] The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

[0064]

[0065] The sample cleaning solution must be prepared and used immediately.

[0066] 3. Sample dissociation solution

[0067] The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

[0068]

[0069] Note: The sample dissociation buffer should be prepared and used immediately.

[0070] In Table 3, the preparation of collagenase stock solution is shown in Tables 4 to 6.

[0071]

[0072] After preparing the 10× collagenase I stock solution, aliquot it into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored long-term at -20°C.

[0073]

[0074] After preparing the 10× collagenase II stock solution, aliquot it into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored long-term at -20°C.

[0075]

[0076] After preparing the 10× collagenase IV stock solution, aliquot it into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored long-term at -20°C.

[0077] In Tables 4, 5, and 6, the unit U of collagenase (the collagenase I, the collagenase II, or the collagenase IV) is defined by the enzymatic activity of the protease: 1 μmol of L-leucine can be released when the collagenase (the collagenase I, the collagenase II, or the collagenase IV) is treated with 1 U of protease at 37° C., pH 7.5, for 5 hours.

[0078] 4. Digestion stop solution

[0079] The specific formula of the digestion stop solution (100 mL) is shown in Table 7.

[0080]

[0081] After preparation, the digestion stop solution can be stored at 4°C for one month.

[0082] 5. Culture medium for culturing colorectal cancer peritoneal metastasis microtumor model

[0083] The specific formula of the culture medium (100 mL) used to culture the colorectal cancer peritoneal metastasis microtumor model is shown in Table 8.

[0084]

[0085] After the colorectal cancer peritoneal metastasis microtumor model culture medium is prepared, it is sterilized by filtration using a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

[0086] In Table 8, the preparation of human recombinant protein stock solutions is shown in Tables 10 to 15 (the preparation of BSA stock solution is shown in Table 9), the preparation of SB202190 stock solution is shown in Table 16, the preparation of A83-01 stock solution is shown in Table 17, the preparation of N-acetyl-L-cysteine ​​stock solution is shown in Table 18, the preparation of Nicotinamide stock solution is shown in Table 19, the preparation of Cholera Toxin stock solution is shown in Table 20, the preparation of Y-27632 stock solution is shown in Table 22, and the preparation of Galunisertib stock solution is shown in Table 23.

[0087]

[0088] 100× BSA solution is prepared and used immediately.

[0089]

[0090] After the 1000× human recombinant protein EGF stock solution is prepared, it is packaged in 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0091]

[0092] After the 1000× human recombinant protein bEGF stock solution is prepared, it is divided into 1.5 mL sterile centrifuge tubes and can be stored at -80°C for a long time.

[0093]

[0094] After the 1000× human recombinant protein HGF stock solution is prepared, it is divided into 1.5 mL sterile centrifuge tubes and can be stored at -80°C for a long time.

[0095]

[0096] After the 1000× human recombinant protein Noggin stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0097]

[0098] After the 1000× human recombinant protein R-spondin 1 stock solution is prepared, it is divided into 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0099]

[0100] After the 1000× human recombinant protein GDNF stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0101]

[0102] After the 1000×SB202190 stock solution is prepared, it is divided into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0103]

[0104] After the 1000×A83-01 stock solution is prepared, it is divided into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0105]

[0106] After the 1000× N-acetyl-L-cysteine ​​stock solution is prepared, it is divided into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0107]

[0108] After the 1000× Nicotinamide stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0109]

[0110] After the 1000× Cholera Toxin stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.

[0111] The specific formula of the Cholera Toxin dissolving solution in Table 20 is shown in Table 21.

[0112]

[0113] After the Cholera Toxin solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The solution can be stored at -20°C for a long time.

[0114]

[0115] After the 1000×Y-27632 stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0116]

[0117] After the 1000× Galunisertib stock solution is prepared, it is dispensed into 0.5 mL sterile centrifuge tubes. The stock solution can be stored at -80°C for a long time.

[0118] Example 2: Obtaining surgical specimens of primary colorectal cancer and peritoneal metastasis

[0119] 1. Cooperate with tertiary hospitals to obtain samples through researcher-initiated clinical research, and the cooperation has passed formal medical ethics review.

[0120] 2. The attending physician selects patients for enrollment according to the clinical indications specified in the medical guidelines and selects appropriate specimens for in vitro culture based on the intraoperative clinical indications. The specimen selection criteria are as follows: patients with primary colorectal cancer accompanied by peritoneal metastasis, who undergo simultaneous resection of the primary tumor and peritoneal metastasis during surgery, with a primary lesion tissue sample exceeding 20 mg and a peritoneal metastasis tissue sample exceeding 5 mg.

[0121] 3. All included cases will be uniformly coded using the sample collection date plus the last four digits of the patient's hospitalization number. For example, if a sample was provided on January 1, 2020, and the patient's hospitalization number is T001537474, the sample experiment number will be 202001017474. The attending physician will provide basic clinical information such as the patient's gender, age, medical history, family history, smoking history, pathological stage and classification, and clinical diagnosis. Information related to patient privacy, such as the patient's name and ID number, will be omitted and replaced with a unified experiment number. The experiment number is named based on the eight-digit sample collection date plus the last four digits of the patient's hospitalization number.

[0122] 4. After the tumor tissue is removed during surgery, a sample collection specialist collects fresh specimens in the sterile environment of the operating room. Samples should be collected from fresh, vascularized areas, avoiding areas with poor cell activity, such as necrotic tissue, adipose tissue, and fibrotic tissue. The collected sample is placed in a sample preservation solution (see Example 1) that has been pre-chilled to 4°C. The sample tube containing the sample is temporarily stored on ice and transported to the laboratory for further processing within 12 hours. The temperature during transportation is controlled at 2-8°C.

[0123] Example 3: Tissue sample pre-dissociation treatment

[0124] The following steps need to be performed on ice and the entire process should be completed within 10 minutes.

[0125] The surgical instruments used in the following operations must be sterilized with high-temperature steam (120°C, 20 minutes) and dried before use.

[0126] 1. After weighing the sample, clean the sample surface with medical alcohol (75% by volume) for 10 to 30 seconds.

[0127] 2. Wash the sample 5 times with sample cleaning solution and 5 times with sterile PBS solution.

[0128] 3. Use ophthalmic scissors, ophthalmic tweezers, scalpel and other instruments to carefully remove the fat tissue, connective tissue and necrotic tissue from the sample.

[0129] Example 4: Tissue Sample Dissociation

[0130] The surgical instruments used in the following examples must be sterilized with high-temperature steam (120°C, 20 minutes) and dried before use.

[0131] 1. Use ophthalmic scissors to cut the tissue into 0.5mm pieces. 3 Small pieces around.

[0132] 2. Treat the tissue with sample dissociation solution (see Example 1). For tissue samples up to 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. Treat the tissue with sample dissociation solution at 37°C for 1 hour. Observe the sample under a microscope every 15 minutes until most cells are observed to have detached from the tissue.

[0133] 3. Terminate the dissociation reaction with 10 times the volume of digestion stop solution (see Example 1). Filter the cell suspension through a 100 μm sterile cell strainer to remove tissue debris and adherent cells. Centrifuge at 800 g for 10 minutes at room temperature and discard the supernatant.

[0134] 4. Resuspend the cells in 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0135] 5. Resuspend the cell pellet in the colorectal cancer peritoneal metastasis microtumor model culture medium (see Example 1), count the cells, and determine the cell viability by trypan blue staining. If the isolated cells have a viability greater than 70%, they can be used for cell inoculation and culture.

[0136] Example 5: Microtumor Model Cultivation

[0137] 1. A low-attachment surface was used to perform suspension culture of a colorectal cancer peritoneal metastasis microtumor model. The culture medium used was the culture medium for culturing a colorectal cancer peritoneal metastasis microtumor model in Table 8 of Example 1 (wherein the final concentration of the human recombinant protein EGF was 50 ng / mL; the final concentration of the human recombinant protein bFGF was 20 ng / mL; the final concentration of the human recombinant protein HGF was 20 ng / mL; the final concentration of the human recombinant protein Noggin was 100 ng / mL; the final concentration of the human recombinant protein R-spondin 1 was 400 ng / mL; the final concentration of the human recombinant protein GDNF was 100 ng / mL; the final concentration of the SB202190 was 10 μM; the final concentration of the A83-01 was 1 μM; the final concentration of the N-acetyl-L-cysteine ​​was 1 mM; the final concentration of the nicotinamide was 10 mM; and the final concentration of the Cholera The final concentration of Toxin is 0.5nM; the final concentration of Y-27632 is 10μM, and the final concentration of Galunisertib is 0.1μM). Taking a six-well plate as an example, 10 cells per well were added. 6 The cells were plated at a density of 100 cells / well, with 2-3 mL of culture medium used per well. The inoculated cells were cultured in a cell culture incubator at 37°C and 5% CO2.

[0138] 2. Observe the cell status every day until the cells form clumps of about 100 μm in diameter. Then replace the culture medium every 2-3 days to maintain the microtumor growth state.

[0139] like Figure 1 As shown in Figure 2, within the first 48 hours of culture, various cell types derived from cancer tissues spontaneously aggregated and self-assembled into cell clusters of 100 μm in size, which we call microtumor models. The total number of microtumor cell clusters 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 using different colorectal cancer surgical samples (colorectal cancer primary lesions and peritoneal metastasis samples) can reach 70%.

[0140] Example 6. Comparison of the ability of different culture media to form microtumor structures in primary colorectal cancer lesions and peritoneal metastasis samples

[0141] The micro-tumor model cultivation procedures for all samples in this example were identical (see above), with only the culture medium formulations varying. In addition to the culture medium shown in Table 8 of the present invention (with the same formulation as in Example 5), the following two control culture media were also used for comparative testing:

[0142] (1) Control culture medium A: as shown in Table 9 of the specification of Chinese Patent 202111135386.3 (CN113817682B, Invention Name: A Method for Cultivating a Colorectal Cancer Microtumor Cell Model) (i.e., as shown in Table 24 below).

[0143]

[0144] In control culture medium A, the final concentration of the human recombinant protein EGF is 50 ng / mL; the final concentration of the human recombinant protein bFGF is 20 ng / mL; the final concentration of the human recombinant protein HGF is 20 ng / mL; the final concentration of the human recombinant protein Noggin is 100 ng / mL; the final concentration of the human recombinant protein R-spondin 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 SB202190 is 10 μM; the final concentration of cortisol is 25 ng / mL; the final concentration of Forskolin is 5 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 is 10 μM. The final concentration of Toxin was 0.5 nM; the final concentration of Y-27632 was 10 μM.

[0145] That is, the only difference between the control culture medium A and the culture medium shown in Table 8 of the present invention (the specific formula is the same as that of Example 5) is that the culture medium formula of the present invention does not contain the human recombinant protein IL-2, human recombinant protein IL-15, cortisol, and Forskolin involved in the control culture medium A, and the culture medium formula of the present invention newly contains human recombinant protein GDNF and Galunisertib.

[0146] (2) Control culture medium B: as shown in Table 8 of the specification of Chinese Patent 202410996217.6 (CN118638735A; Invention Name: A Culture Medium and Culture Method for Cultivating a Colorectal Cancer Liver Metastasis Microtumor Model) (i.e., as shown in Table 25 below).

[0147]

[0148] In control culture medium B, the final concentration of the human recombinant protein EGF is 50 ng / mL; the final concentration of the human recombinant protein bFGF is 20 ng / mL; the final concentration of the human recombinant protein HGF is 20 ng / mL; the final concentration of the human recombinant protein Noggin is 100 ng / mL; the final concentration of the human recombinant protein R-spondin 1 is 400 ng / mL; 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 0.5 nM; and the final concentration of Y-27632 is 10 μM.

[0149] That is, the only difference between the control culture medium B and the culture medium shown in Table 8 of the present invention (the specific formula is the same as that of Example 5) is that the culture medium of the present invention is newly added with human recombinant protein GDNF and Galunisertib.

[0150] The culture medium shown in Table 8 of the present invention and the two control culture media shown in Tables 24 and 25 were used to culture the primary colon cancer lesions and peritoneal metastases of the same patient (see the above-mentioned procedures for the specific process). The number and size of microtumors were observed. The results are shown in Tables 26 to 31:

[0151]

[0152]

[0153]

[0154] It can be seen from Tables 26 to 28 that when culturing primary colorectal cancer samples, the control culture medium A in Table 24 has a higher micro-tumor culture success rate, and the number and size of the cultured cell clusters are also more advantageous.

[0155]

[0156]

[0157]

[0158] As can be seen from Tables 29 to 31, compared with primary colorectal cancer lesions, the culture of peritoneal metastases is more difficult, and the number and size of microtumors obtained by the three culture media are smaller than those of primary lesions. However, the culture medium in Table 8 of the present invention has a higher culture success rate in the culture of microtumors of peritoneal metastases of colorectal cancer lesions. It can be seen that compared with the control culture medium formula, the addition of the two new components of human recombinant protein GDNF and Galunisertib in the present invention significantly improved the success rate of microtumor culture of peritoneal metastases of colorectal cancer lesions, and the size of the microtumors obtained by the present invention is also relatively more advantageous.

[0159] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A culture medium for culturing a microtumor model of peritoneal metastasis of colorectal cancer, characterized in that: The culture medium is composed of three antibacterial and antifungal antibodies, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein Noggin, human recombinant protein R-spondin 1, human recombinant protein GDNF, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, N2 additive, cholera toxin, B27, ITS-X, Y-27632, Galunisertib and basal culture medium; wherein the three antibacterial and antifungal antibodies are penicillin, streptomycin and amphotericin B; In the culture medium, 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 human recombinant protein EGF is 10-100 ng / mL; the final concentration of human recombinant protein bFGF is 10-50 ng / mL; the final concentration of human recombinant protein HGF is 5-25 ng / mL; the final concentration of human recombinant protein Noggin is 100-200 ng / mL; the final concentration of human recombinant protein R-spondin is 100-200 ng / mL. The final concentration of 1 is 250-500 ng / mL; the final concentration of the human recombinant protein GDNF is 50-100 ng / mL; the final concentration of the SB202190 is 5-10 μM; the concentration of the A83-01 is 0.25-1.25 μM; the final concentration of the primocin is 1% by volume; the concentration of the N-acetyl-L-cysteine ​​is 0.5-2 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 the B27 is 1.5-2.5% by volume; the final concentration of the ITS-X is 0.8-1.2% by volume; the final concentration of the Y-27632 is 5-20 μM; and the final concentration of the galunisertib is 0.1-0.5 μM. The basic culture medium is Advanced DMEM / F12 culture medium.

2. The culture medium according to claim 1, wherein: In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agents 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.

3. A kit for culturing a microtumor model of peritoneal metastasis of colorectal cancer, comprising the culture medium of claim 1 or 2 and all or part of the following: Sample dissociation solution, sample preservation solution, sample washing solution and digestion termination solution.

4. The reagent set according to claim 3, characterized in that: The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I is 150-250 U / mL; the final concentration of the collagenase II is 150-250 U / mL; the final concentration of the collagenase IV is 150-250 U / mL; and the remainder is PBS.

5. The reagent set according to claim 3, characterized in that: The sample preservation solution is composed of fetal bovine serum, three antibiotics (antibacterial and antifungal agents), HEPES and HBSS; wherein the three antibiotics (antibacterial and antifungal agents) 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 penicillin in the three antibiotics (antibacterial and antifungal agents) 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; the final concentration of the HEPES is 8-12 mM; and the remainder is the HBSS.

6. The reagent kit according to any one of claims 3 to 5, characterized in that: The sample cleaning solution is composed of three antibacterial and antifungal agents and PBS; wherein the three antibacterial and antifungal agents are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the three antibacterial and antifungal agents 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; the remainder is the PBS.

7. The reagent kit according to any one of claims 3 to 5, characterized in that: The digestion stop solution is composed of fetal bovine serum, three antifungal antibodies and DMEM culture medium; wherein the three antibacterial and antifungal antibodies are penicillin, streptomycin and amphotericin B; in the digestion stop solution, the final concentration of the fetal bovine serum is 8-12% by volume; the final concentration of penicillin in the three antibacterial and antifungal antibodies 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; the remainder is the DMEM culture medium.

8. Use of the culture medium according to claim 1 or 2 or the reagent set according to any one of claims 3 to 7 in constructing a microtumor model of peritoneal metastasis of colorectal cancer.

9. A method for constructing a microtumor model of peritoneal metastasis of colorectal cancer, comprising the following steps: (a1) dissociating solid tumor tissue of colorectal cancer peritoneal metastasis using the sample dissociation solution described in claim 4; (a2) Using the culture medium of claim 1 or 2 to suspend and culture the single cells dissociated in step (a1) to form cell clusters, thereby obtaining a microtumor model of peritoneal metastasis of colorectal cancer.

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