Culture medium for constructing colorectal cancer peritoneal metastasis micro-tumor cell model and application thereof

Abstract

The invention discloses a culture medium for constructing a colorectal cancer peritoneal metastasis micro-tumor cell model and application of the culture medium. The invention relates to the technical field of biology, and provides a culture medium for culturing a micro-tumor model for colorectal cancer peritoneal metastasis. The invention discloses a cholera toxin immunopotentiator, which is composed of an antibacterial and antifungal agent three antibody, HEPES, GlutaMax, a human recombinant protein EGF, a human recombinant protein bFGF, a human recombinant protein HGF, a human recombinant protein Noggin, a human recombinant protein R-spondin 1, a human recombinant protein GDNF, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, nicotinamide, an N2 additive, cholera toxin, B27, ITS-X, Y-27632, Galunissertib and a basal culture medium. The culture medium and the culture method disclosed by the invention can effectively improve the culture success rate of colorectal cancer peritoneal metastasis and focus combination.

Description

Technical Field

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

[0002] Colorectal cancer is a malignant tumor that seriously threatens human health. Peritoneal metastasis is a major form of metastasis of colorectal cancer. 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 the deaths of postoperative recurrence patients are directly related to the progression of peritoneal metastasis. The prognosis of patients with peritoneal metastasis is poor, and the median overall survival time is only 6-9 months, and the 5-year survival rate is less than 20%. Therefore, peritoneal metastasis is also one of the main causes of death of colorectal cancer patients.

[0003] Chemotherapy based on classical cytotoxic drugs is one of the basic treatment methods for colorectal cancer, but the efficacy of chemotherapy drugs on peritoneal metastasis lesions is limited. In many cases, it can only delay the progression speed of peritoneal metastasis lesions. For patients with extensive peritoneal metastasis, there is no effective drug treatment plan. This poses new requirements for the research and individualized treatment of peritoneal metastasis of colorectal cancer.

[0004] Colorectal cancer is a complex systemic disease. Currently, little is known about the biological processes such as the occurrence, development, recurrence and metastasis of this disease, as well as the heterogeneity, drug resistance, and tumor immune response mechanisms. The causes and development processes of peritoneal metastasis of colorectal cancer have strong individual differences, and there are significant differences in the tumor microenvironment between peritoneal metastasis lesions and the primary colorectal cancer lesions. Therefore, using primary cell cultures of peritoneal metastasis lesions of colorectal cancer as a model for individualized precision research is a trend in the field of colorectal cancer research and even in the field of colorectal cancer diagnosis and treatment. Therefore, it is crucial to develop a tumor model that can accurately reflect the characteristics of patients' peritoneal metastasis lesions. Existing primary tumor cell culture techniques mainly include several types such as 2D culture, 3D culture, and reprogramming culture. These methods all face problems to varying degrees, such as extremely long culture cycles, low culture success rates, difficulty in removing contaminating cells, and inability to reproduce the tumor microenvironment. The peritoneal metastasis lesions of colorectal cancer have completely different microenvironmental characteristics from the primary lesions, so different requirements are also placed on the culture conditions. Summary of the Invention

[0005] In order to effectively solve the above technical problems, the present invention provides a new culture technique and supporting reagents for a micro-tumor model of colorectal cancer peritoneal metastasis. The core of this technique is as follows: (1) treating the solid tumor tissue of colorectal cancer peritoneal metastasis with a mild cell dissociation reagent, and adjusting the composition ratio of the culture medium according to the characteristics of the microenvironment of colorectal cancer peritoneal metastasis lesions, which maximally ensures the viability of various types of cells in the tissue; (2) preparing a special serum-free culture medium, and using a suspension culture system to enable various types of cells isolated from the colorectal cancer peritoneal metastasis lesion tissue to self-assemble into cell cluster structures with multiple cell components, which is called the "micro-tumor model of colorectal cancer peritoneal metastasis".

[0006] In a first aspect, the present invention claims to protect a culture medium for culturing a micro-tumor model of colorectal cancer peritoneal metastasis.

[0007] The culture medium for culturing a micro-tumor model of colorectal cancer peritoneal metastasis claimed by the present invention is composed of an antibacterial and antifungal agent triple antibiotic, 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 a basal medium; wherein, the antibacterial and antifungal agent triple antibiotic is penicillin, streptomycin and amphotericin B.

[0008] In the said 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 human recombinant protein EGF is 10 - 100 ng / mL (such as 50 ng / mL); the final concentration of human recombinant protein bFGF is 10 - 50 ng / mL (such as 20 ng / mL); the final concentration of human recombinant protein HGF is 5 - 25 ng / mL (such as 20 ng / mL); the final concentration of human recombinant protein Noggin is 100 - 200 ng / mL (such as 100 ng / mL); the final concentration of human recombinant protein R-spondin 1 is 250 - 500 ng / mL (such as 400 ng / mL); the final concentration of 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 5 - 10 mM (such as 10 mM); the final concentration of N2 additive is 1% by volume; the final concentration of cholera toxin is 0.1 - 1 nM (such as 0.5 nM); the final concentration of B27 is 1.5 - 2.5% (such as 2%) by volume; the final concentration of ITS-X is 0.8 - 1.2% (such as 1%) by volume; the final concentration of Y-27632 is 5 - 20 μM (such as 10 μM); the final concentration of Galunisertib is 0.1 - 0.5 μM (such as 0.1 μM).

[0009] Furthermore, the GlutaMax is an advanced cell culture additive that can directly replace L-glutamine in the cell culture medium. The GlutaMax is "GlutaMAX™ Supplement" (such as Gibco #35050061, or other products with the same composition). The composition of the "GlutaMAX™ Supplement" is L-alanyl-L-glutamine, which is a substitute for L-glutamine, with a concentration of 200 nM and a solvent of 0.85% NaCl solution. The SB202190 is "4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole" (such as Sigma #S7067, or other products with the same composition). The A83-01 is "3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide" (such as Tocris #2939, or other products with the same composition). The Primocin is an antibacterial agent for primary cells (such as Invivogene #ant-pm-1, or other products with the same composition), an antibiotic used to protect primary cells from microbial contamination, which has a killing effect on Gram-positive bacteria, Gram-negative bacteria, mycoplasma, and fungi. The N2 additive is "N-2 Supplement(100X)" (such as Gibco #17502001, or other products with the same composition). The "N-2 Supplement(100X)" contains human holo-transferrin with a final concentration of 1 mM, recombinant insulin full chain with a concentration of 500 mg / L, progesterone with a concentration of 0.63 mg / L, putrescine with a concentration of 10 mM, and selenite with a concentration of 0.52 mg / L. 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 HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, and T3 (triodo-I-thyronine). The solvent of the ITS-X is EBSS solution (Earle's balanced salt solution), and the solutes and their concentrations are as follows: insulin 1 g / L; transferrin 0.55 g / L; sodium selenite 0.00067 g / L; ethanolamine 0.2 g / L. The 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)" (such as MCE#129830-38-2, or other products with the same composition). The Galunisertib, also known as LY2157299, is an effective inhibitor of TGFβ receptor 1.

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

[0011] In the medium, the final concentration of penicillin in the antibacterial and antifungal agent 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] Further, the triple antibiotic-antimycotic agent 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 0.85% saline solution as the antifungal agent.

[0013] Further, the culture medium can exist in two forms: One is that the culture medium is a solution prepared by mixing the triple antibiotic-antimycotic agent, the HEPES, the 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, the SB202190, the A83-01, the Primocin, the N-acetyl-L-cysteine, the nicotinamide, the N2 supplement, the cholera toxin, the B27, the ITS-X, the Y-27632, the Galunisertib, and the basal medium.

[0014] After the culture medium is prepared, it needs to be filtered and sterilized with a 0.22 μM syringe filter (Millipore SLGP033RS) and can be stored at 4°C for two weeks.

[0015] The other is that each component in the culture medium exists separately and is prepared according to the formula when in use.

[0016] Furthermore, 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, and the human recombinant protein GDNF can exist in the form of a stock solution (mother liquor), specifically a 1000-fold stock solution (mother liquor). SB202190, N-acetyl-L-cysteine, nicotinamide, Y-27632, and Galunisertib can exist in the form of a stock solution (mother liquor), specifically a 1000-fold stock solution (mother liquor). Cholera Toxin can exist in the form of a stock solution (mother liquor), specifically a 10,000-fold stock solution (mother liquor). A83-01 can exist in the form of a stock solution (mother liquor), specifically a 100,000-fold stock solution (mother liquor).

[0017] The 1000× human recombinant protein EGF stock solution is composed of human recombinant protein EGF, BSA, and PBS. Among them, the final concentration of the human recombinant protein EGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the balance is PBS.

[0018] The 1000× human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA, and PBS. Among them, 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 balance is PBS.

[0019] The 1000× human recombinant protein HGF stock solution is composed of human recombinant protein HGF, BSA, and PBS. Among them, the final concentration of the human recombinant protein HGF is 20 μg / mL, the final concentration of the BSA is 0.01 g / mL, and the balance is PBS.

[0020] The 1000× human recombinant protein Noggin stock solution is composed of human recombinant protein Noggin, BSA, and PBS. Among them, 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 balance is PBS.

[0021] The 1000× human recombinant protein R-spondin 1 stock solution is composed of human recombinant protein R-spondin 1, BSA, and PBS. Among them, 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 balance is PBS.

[0022] The 1000× human recombinant protein GDNF stock solution is composed of human recombinant protein GDNF, BSA, and PBS. Among them, 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 balance is PBS.

[0023] Among the above six 1000-fold stock solutions, the BSA can exist in the form of a 100-fold stock solution (mother liquor) (prepared and used immediately). Specifically, it is composed of BSA and PBS. Among them, the final concentration of BSA (Sigma#A1933) is 0.1 g / mL, and the balance is PBS.

[0024] In addition, 1000× SB202190 is composed of SB202190 and DMSO. Among them, the final concentration of SB202190 is 10 mM, and the balance is DMSO.

[0025] The 1000×N-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of N-acetyl-L-cysteine is 0.5 M, and the balance is ultrapure water.

[0026] The 1000×Nicotinamide stock solution consists of Nicotinamide and ultrapure water, wherein the concentration of Nicotinamide is 5 M, and the balance is ultrapure water.

[0027] The 1000×Galunisertib stock solution consists of Galunisertib and ultrapure water, wherein the concentration of Galunisertib is 1 mM, and the balance is ultrapure water.

[0028] The 1000×Y-27632 consists of Y-27632 and ultrapure water, wherein the concentration of Y-27632 is 10 mM, and the balance is ultrapure water.

[0029] The 10000×Cholera Toxin stock solution consists of Cholera Toxin and Cholera Toxin dissolution solution, wherein the concentration of Cholera Toxin is 10 μM, and the balance is the Cholera Toxin dissolution solution. The Cholera Toxin dissolution solution is composed as follows: every 10 mL of the Cholera Toxin dissolution solution contains Tris (1 M) pH7.0 0.05 M, NaCl 0.2 M, sodium azide 3 mM, EDTA (0.5 M) pH8.0 1 mM, and the balance is ultrapure water.

[0030] The 100000×A83-01 stock solution consists of A83-01 and DMSO, wherein the concentration of A83-01 is 25 mM, and the balance is DMSO.

[0031] In a second aspect, the present invention claims to protect a set of reagents for culturing a micro-tumor model of colorectal cancer peritoneal metastasis.

[0032] The set of reagents for culturing a micro-tumor model of colorectal cancer peritoneal metastasis claimed by the present invention consists of all or part of the culture medium described in the first aspect above and the following: sample dissociation solution, sample preservation solution, sample cleaning solution, and digestion termination solution.

[0033] Furthermore, the sample dissociation solution is composed 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); the balance is PBS.

[0034] Wherein, the unit U of collagenase (collagenase I or collagenase II or collagenase IV) is defined by the enzyme activity of the protease: under the conditions of 37°C and pH 7.5, treating collagenase (collagenase I or collagenase II or collagenase IV) with 1 U of protease for 5 hours can release 1 μmol of L-leucine.

[0035] Furthermore, the sample preservation solution is composed of fetal bovine serum, the triple antibiotic-antifungal agent, HEPES, and HBSS; wherein, the triple antibiotic-antifungal agent is penicillin, streptomycin, and amphotericin B; in the sample preservation solution, the final concentration of fetal bovine serum is 1 - 5% (such as 2%) by volume; the final concentration of penicillin in the triple antibiotic-antifungal agent 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), the final concentration of amphotericin B is 200 - 250 ng / mL (such as 250 ng / mL); the final concentration of HEPES is 8 - 12 mM (such as 10 mM); the balance is HBSS.

[0036] Furthermore, the sample washing solution is composed of the triple antibiotic-antifungal agent and PBS; wherein, the triple antibiotic-antifungal agent is penicillin, streptomycin, and amphotericin B; in the sample washing solution, the final concentration of penicillin in the triple antibiotic-antifungal agent 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), the final concentration of amphotericin B is 200 - 250 ng / mL (such as 250 ng / mL); the balance is PBS.

[0037] Further, the digestion termination solution is composed of fetal bovine serum, triple antibiotics as an antifungal agent, and DMEM medium; wherein, the triple antibiotics as an antibacterial and antifungal agent are penicillin, streptomycin, and amphotericin B; in the digestion termination solution, the final concentration of the fetal bovine serum is 8-12% (such as 10%) by volume percentage; the final concentration of penicillin in the triple antibiotics as an antibacterial and antifungal agent 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 balance is DMEM medium.

[0038] In a third aspect, the present invention claims the use of the medium described in the first aspect above or the kit of reagents described in the second aspect above in constructing a microtumor model of colorectal cancer peritoneal metastasis.

[0039] In a fourth aspect, the present invention claims a method for constructing a microtumor model of colorectal cancer peritoneal metastasis.

[0040] The method for constructing a microtumor model of colorectal cancer peritoneal metastasis claimed by the present invention may include the following steps: (a1) Dissociate the solid tumor tissue of colorectal cancer peritoneal metastasis with the sample dissociation solution described in the second aspect above; (a2) Suspension-culture the single cells dissociated in step (a1) with the medium described in the first aspect above to form cell clusters, thereby obtaining the microtumor model of colorectal cancer peritoneal metastasis.

[0041] In step (a1), the method of dissociating the solid tumor tissue of colorectal cancer peritoneal metastasis with the sample dissociation solution may include the following steps: According to the dosage of 1 mL of the sample dissociation solution not exceeding 0.5 mg of tissue, use the sample dissociation solution to dissociate the solid tumor tissue of colorectal cancer peritoneal metastasis after being minced at 37 °C for 15 minutes to 2 hours (such as 1 hour).

[0042] In step (a2), the method of suspension-culturing the cells dissociated in (a1) with the medium may include the following steps: Use a cell culture container with a low-attachment surface, and suspension-culture the cells dissociated in (a1) with the medium at 37 °C and 5% CO2.

[0043] Among them, the initial seeding density may be 10 5 cells / cm 2 of the bottom area of the container. Taking a six-well plate as an example, plate at a density of 10 6 cells per well.

[0044] Further, the culturing time in step (a2) is 2 - 3 days.

[0045] Further, before step (a1), the following steps for pre - treatment before dissociation of the solid tumor tissue of colorectal cancer peritoneal metastasis may also be included: washing the surface of the solid tumor tissue sample of colorectal cancer peritoneal metastasis with ethanol with a volume percentage of 70 - 75% (such as 75%) for 10 - 30 seconds; washing the solid tumor tissue sample of colorectal cancer peritoneal metastasis with the sample washing solution described in the second aspect above 5 - 10 times (such as 5 times), and washing the solid tumor tissue sample of colorectal cancer peritoneal metastasis with a sterile PBS solution 5 - 10 times (such as 5 times); then removing components that affect micro - tumor culture, such as impurities, connective tissue, adipose tissue, necrotic tissue, etc. from the solid tumor tissue sample of colorectal cancer peritoneal metastasis.

[0046] The steps for pre - treatment before dissociation of the solid tumor tissue of colorectal cancer peritoneal metastasis need to be operated on ice, and the whole operation steps need to be completed within 10 minutes.

[0047] Further, in step (a1), after dissociating the solid tumor tissue of colorectal cancer peritoneal metastasis with the sample dissociation solution, the following steps are also included: terminating the dissociation reaction with a digestion termination solution with a volume 8 - 15 times (such as 10 times) that of the second aspect above, and collecting the cell suspension; filtering the cell suspension with a 100μm or 40μm sterile cell filter to remove tissue debris 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; then resuspending the cell pellet with the medium described in the first aspect above.

[0048] The ex - vivo time of the solid tumor tissue sample of colorectal cancer peritoneal metastasis for which the pre - treatment before dissociation is carried out is within 12 hours, and it has been stored in the sample preservation solution described in the second aspect above before the pre - treatment before dissociation.

[0049] In the fifth aspect, the present invention claims to protect a micro - tumor model of colorectal cancer peritoneal metastasis constructed by the method described in the fourth aspect above.

[0050] Advantages of the present invention: The volume of the colorectal cancer peritoneal metastasis lesion is small, the fibrosis degree is high, and the tumor content is low. The success rate of conventional primary cell culture methods is extremely low. The culture medium and culture method disclosed in the present invention can effectively improve the culture success rate of colorectal cancer peritoneal metastasis lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Bright-field images of microtumors obtained by culturing samples from the primary focus and peritoneal metastases of colorectal cancer in the same patient. Detailed implementation manners

[0052] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0053] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0054] Example 1: Preparation of reagents for culturing a peritoneal metastasis microtumor model of colorectal cancer 1. Sample preservation solution The specific formula of the sample preservation solution (100 mL) is shown in Table 1.

[0055]

[0056] After the sample preservation solution is prepared, it is dispensed into 15 mL centrifuge tubes, 5 mL per tube. After dispensing, it can be stored at 4 °C for 1 month.

[0057] 2. Sample cleaning solution The specific formula of the sample cleaning solution (100 mL) is shown in Table 2.

[0058]

[0059] The sample cleaning solution needs to be prepared and used immediately.

[0060] 3. Sample dissociation solution The specific formula of the sample dissociation solution (10 mL) is shown in Table 3.

[0061]

[0062] Note: The sample dissociation solution needs to be prepared and used immediately.

[0063] In Table 3, the preparation of the collagenase stock solution is shown in Tables 4 to 6.

[0064]

[0065] After the 10× collagenase I stock solution is prepared, it is dispensed into 1.5 mL sterile centrifuge tubes, 1 mL per tube. This stock solution can be stored at -20 °C for a long time.

[0066]

[0067] 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.

[0068]

[0069] 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.

[0070] In Tables 4, 5, and 6, the unit U of collagenase (the collagenase I or the collagenase II or the collagenase IV) is defined by the enzyme activity of the protease: Under the conditions of 37°C and pH 7.5, treating the collagenase (the collagenase I or the collagenase II or the collagenase IV) with 1 U of protease for 5 hours can release 1 μmol of L-leucine.

[0071] 4. Digestion termination solution The specific formula of the digestion termination solution (100 mL) is shown in Table 7.

[0072]

[0073] After preparing the digestion termination solution, it can be stored at 4°C for one month.

[0074] 5. Medium for culturing the colorectal cancer peritoneal metastasis microtumor model The specific formula of the medium for culturing the colorectal cancer peritoneal metastasis microtumor model (100 mL) is shown in Table 8.

[0075]

[0076] After preparing the medium for the colorectal cancer peritoneal metastasis microtumor model, filter it through a 0.22 μM needle filter (Millipore SLGP033RS) to sterilize, and it can be stored at 4°C for two weeks.

[0077] In Table 8, the preparation of the human recombinant protein stock solution is shown in Tables 10 - 15 (the preparation of the BSA stock solution is shown in Table 9), the preparation of the SB202190 stock solution is shown in Table 16, the preparation of the A83-01 stock solution is shown in Table 17, the preparation of the N-acetyl-L-cysteine stock solution is shown in Table 18, the preparation of the Nicotinamide stock solution is shown in Table 19, the preparation of the Cholera Toxin stock solution is shown in Table 20, and the preparation of the Y-27632 stock solution is shown in Table 22, and the preparation of the Galunisertib stock solution is shown in Table 23.

[0078]

[0079] The 100×BSA solution should be prepared and used immediately.

[0080]

[0081] After the 1000× human recombinant protein EGF stock solution is prepared, it is aliquoted into 1.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0082]

[0083] After the 1000× human recombinant protein bEGF stock solution is prepared, it is aliquoted into 1.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0084]

[0085] After the 1000× human recombinant protein HGF stock solution is prepared, it is aliquoted into 1.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0086]

[0087] After the 1000× human recombinant protein Noggin stock solution is prepared, it is aliquoted into 1.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0088]

[0089] After the 1000× human recombinant protein R-spondin 1 stock solution is prepared, it is aliquoted into 1.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0090]

[0091] After the 1000× human recombinant protein GDNF stock solution is prepared, it is aliquoted into 1.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0092]

[0093] After the 1000× SB202190 stock solution is prepared, it is aliquoted into 0.5 mL sterile centrifuge tubes. This stock solution can be stored at -20°C for a long time.

[0094]

[0095] After the 1000× A83-01 stock solution is prepared, it is aliquoted into 0.5 mL sterile centrifuge tubes. This stock solution can be stored at -20°C for a long time.

[0096]

[0097] After the preparation of 1000×N-acetyl-L-cysteine stock solution, it was aliquoted into 0.5 mL sterile centrifuge tubes. This stock solution can be stored at -20°C for a long time.

[0098]

[0099] After the preparation of 1000×Nicotinamide stock solution, it was aliquoted into 0.5 mL sterile centrifuge tubes. This stock solution can be stored at -20°C for a long time.

[0100]

[0101] After the preparation of 1000×Cholera Toxin stock solution, it was aliquoted into 0.5 mL sterile centrifuge tubes. This stock solution can be stored at -20°C for a long time.

[0102] The specific formula of the Cholera Toxin dissolution solution in Table 20 is shown in Table 21.

[0103]

[0104] After the preparation of Cholera Toxin dissolution solution, it was aliquoted into 0.5 mL sterile centrifuge tubes. This solution can be stored at -20°C for a long time.

[0105]

[0106] After the preparation of 1000×Y-27632 stock solution, it was aliquoted into 0.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0107]

[0108] After the preparation of 1000×Galunisertib stock solution, it was aliquoted into 0.5 mL sterile centrifuge tubes. This stock solution can be stored at -80°C for a long time.

[0109] Example 2. Obtaining surgical specimens of primary colorectal cancer and peritoneal metastasis samples 1. Collaborated with a tertiary hospital to obtain samples through a researcher-initiated clinical study, and the cooperation was carried out through formal medical ethics review.

[0110] 2. The attending physician selected the enrolled patients according to the clinical indications specified in the medical guidelines and selected appropriate samples for in vitro culture according to the intraoperative clinical indications. The selection criteria for the samples were: patients with primary colorectal cancer accompanied by peritoneal metastasis lesions, and those who had both the primary tumor and peritoneal metastasis lesions resected during the operation, with the primary lesion tissue sample exceeding 20 mg and the peritoneal metastasis lesion tissue sample exceeding 5 mg.

[0111] 3. All enrolled cases are uniformly coded in the way of the sample collection date + the last four digits of the patient's hospital admission number. For example, for the sample provided on January 1, 2020, and the patient's hospital admission number is T001537474, the sample experiment number is 202001017474. The attending physician provides the patient's basic clinical information such as gender, age, medical history, family history, smoking history, pathological stage classification, and clinical diagnosis. Information related to patient privacy such as the patient's name and ID number is hidden and replaced with a unified experiment number. The naming principle of the experiment number is the eight-digit date of the collected sample + the last four digits of the patient's hospital admission number.

[0112] 4. After the tumor tissue is removed during the operation, the sample collection specialist collects fresh specimens in the sterile environment of the operating room. When collecting samples, it is necessary to select fresh parts with rich blood vessels and avoid parts with poor cell activity such as necrotic tissue, adipose tissue, and fibrotic tissue. The collected samples are placed in the sample preservation solution (see Example 1) pre-cooled to 4°C in advance. The sample preservation tube containing the sample is temporarily stored on ice and transported to the laboratory for the next operation within 12 hours. The temperature is controlled at 2 - 8°C during transportation.

[0113] Example 3. Pretreatment before dissociation of tissue samples The following operations need to be carried out on ice, and the whole operation procedure needs to be completed within 10 minutes.

[0114] All surgical instruments used in the following operations need to be sterilized by high-temperature steam (120°C, 20 minutes) in advance and dried before use.

[0115] 1. After weighing the sample, clean the surface of the sample with medical alcohol (volume percentage content 75%) for 10 to 30 seconds.

[0116] 2. Clean the sample 5 times with the sample cleaning solution and 5 times with sterile PBS solution.

[0117] 3. Use instruments such as ophthalmic scissors, ophthalmic forceps, and scalpel to carefully peel off the adipose tissue, connective tissue, and necrotic tissue in the sample.

[0118] Example 4. Dissociation of tissue samples All surgical instruments used in the following examples need to be sterilized by high-temperature steam (120°C, 20 minutes) in advance and dried before use.

[0119] 1. Use ophthalmic scissors to cut the tissue into small pieces about 0.5mm 3 in size.

[0120] 2. Treat the tissue with the sample dissociation solution (see Example 1). For tissues with a size not exceeding 0.5 mg, use 1 mL of the sample dissociation solution. For tissues with a size exceeding 0.5 mg, an additional 0.1 mL of the sample dissociation solution is required for every 0.1 mg increase in tissue weight. The treatment conditions of the sample dissociation solution are 37°C and a dissociation time of 1 hour. During the dissociation process, observe the dissociation of the sample under a microscope every 15 minutes until most cells are detached from the tissue.

[0121] 3. Terminate the dissociation reaction with 10 volumes of the digestion termination solution (see Example 1). After filtering 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.

[0122] 4. Resuspend the cells with 5 mL of sterile PBS, centrifuge at 800 g for 10 minutes at room temperature, and discard the supernatant.

[0123] 5. Resuspend the cell pellet with the medium for the colorectal cancer peritoneal metastasis microtumor model (see Example 1), perform cell counting, and determine the cell viability by trypan blue staining. The isolated cells with a viability greater than 70% can be used for cell inoculation and culture.

[0124] Example 5. Microtumor model culture 1. Use a low-attachment surface for suspension culture of the colorectal cancer peritoneal metastasis microtumor model. The medium used is the medium for culturing the colorectal cancer peritoneal metastasis microtumor model in Table 8 of Example 1 (where the final concentration of human recombinant protein EGF is 50 ng / mL; the final concentration of human recombinant protein bFGF is 20 ng / mL; the final concentration of human recombinant protein HGF is 20 ng / mL; the final concentration of human recombinant protein Noggin is 100 ng / mL; the final concentration of human recombinant protein R-spondin 1 is 400 ng / mL; the final concentration of human recombinant protein GDNF is 100 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; the final concentration of Y-27632 is 10 μM, and the final concentration of Galunisertib is 0.1 μM). Taking a six-well plate as an example, plate at a density of 10 6 cells per well, and the medium volume per well is 2 - 3 mL. Incubate the seeded cells in a cell culture incubator at 37°C and 5% CO2.

[0125] 2. Observe the cell status every day until the cells form cell aggregates with a diameter of about 100 μm, and then change the culture medium every 2-3 days to maintain the growth state of the microtumors.

[0126] As Figure 1 shown, within the first 48 hours of culture, various different types of cells derived from cancer tissues spontaneously aggregate and self-assemble to form cell aggregate structures of 100 μm in size, which we call the microtumor model. The total number of microtumor cell aggregates can reach 10 5 -10 6 . This method has been tested with a large number of samples, and the success rate of culturing the microtumor model for different colorectal cancer surgical samples (primary colorectal cancer and peritoneal metastasis samples) can reach 70%.

[0127] Example 6. Comparison of the ability of different culture media to form microtumor structures in primary colorectal cancer samples and colorectal cancer peritoneal metastasis samples The operation method and process for culturing the microtumor model of all samples in this example are exactly the same (refer to the description above), and only the culture medium formula is different. In addition to the culture medium shown in Table 8 of the present invention (the specific formula is the same as that in Example 5), the following two control culture media are also used for the comparative test: (1) Control culture medium A: as shown in Table 9 of the specification of Chinese Patent 202111135386.3 (CN113817682B, invention name: a method for culturing a colorectal cancer microtumor cell model) (i.e., as shown in Table 24 below).

[0128]

[0129] In control culture medium A, the final concentration of human recombinant protein EGF is 50 ng / mL; the final concentration of human recombinant protein bFGF is 20 ng / mL; the final concentration of human recombinant protein HGF is 20 ng / mL; the final concentration of human recombinant protein Noggin is 100 ng / mL; the final concentration of human recombinant protein R-spondin 1 is 400 ng / mL; the final concentration of human recombinant protein IL-2 is 20 ng / mL; the final concentration of 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 Toxin is 0.5 nM; the final concentration of Y-27632 is 10 μM.

[0130] That is, compared with the medium shown in Table 8 of the present invention (the specific formula is the same as that in Example 5), the control medium A only differs in that: the human recombinant protein IL-2, human recombinant protein IL-15, cortisol, and Forskolin involved in the control medium A are not contained in the medium formula of the present invention, and the human recombinant protein GDNF and Galunisertib are newly added to the medium formula of the present invention.

[0131] (2) Control medium B: as shown in Table 8 of the specification of Chinese Patent 202410996217.6 (CN118638735A; Invention Title: A Medium and Cultivation Method for Culturing a Colorectal Cancer Liver Metastasis Microtumor Model) (i.e., as shown in Table 25 hereinafter).

[0132]

[0133] In the control 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; the final concentration of Y-27632 is 10 μM.

[0134] That is, compared with the medium shown in Table 8 of the present invention (the specific formula is the same as that in Example 5), the control medium B only differs in that: the human recombinant protein GDNF and Galunisertib are newly added to the medium of the present invention.

[0135] Using the medium shown in Table 8 of the present invention and the two control media shown in Tables 24 and 25, the primary colorectal cancer lesions and peritoneal metastasis lesions of the same patient are cultured respectively (the specific process is as described above), and the number and size of the microtumors are observed. The results are shown in Tables 26 to 31:

[0136]

[0137]

[0138] As can be seen from Tables 26 to 28, when culturing colorectal cancer primary site samples, the control medium A in Table 24 has a higher success rate of microtumor culture, and the number and size of the cell clusters obtained by culture are also more advantageous.

[0139]

[0140]

[0141]

[0142] As can be seen from Tables 29 to 31, compared with colorectal cancer primary sites, the culture of peritoneal metastasis lesions is more difficult, and the number and size of microtumors obtained with the three media are less than those of the primary sites. However, the medium in Table 8 of the present invention has a higher success rate of microtumor culture in the culture of colorectal cancer peritoneal metastasis lesions. It can be seen that compared with the control medium formula, after adding two components, human recombinant protein GDNF and Galunisertib, to the present invention, the success rate of microtumor culture in colorectal cancer peritoneal metastasis lesions is significantly improved, and the size of the microtumors obtained by the present invention is also relatively more advantageous.

[0143] 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 unnecessary experiments, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any changes, uses or improvements of the present invention, including changes made with conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A culture medium for culturing a microtumor model of colorectal cancer peritoneal metastasis, characterized in that: The culture medium is composed of the antibacterial and antifungal agent triple antibiotic, 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 a basal medium; wherein, the antibacterial and antifungal agent triple antibiotic is 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% (v / v); 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 1 is 250-500 ng / mL; the final concentration of human recombinant protein GDNF is 50-100 ng / mL; the final concentration of SB202190 is 5-10 μM; the concentration of A83-01 is 0.25-1.25 μM; the final concentration of Primocin is 1% (v / v); the concentration of N-acetyl-L-cysteine is 0.5-2 mM; the final concentration of nicotinamide is 5-10 mM; the final concentration of N2 supplement is 1% (v / v); the final concentration of cholera toxin is 0.1-1 nM; the final concentration of B27 is 1.5-2.5% (v / v); the final concentration of ITS-X is 0.8-1.2% (v / v); the final concentration of Y-27632 is 5-20 μM; the final concentration of Galunisertib is 0.1-0.5 μM.

2. The culture medium according to claim 1, characterized in that: The basal 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 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 kit of reagents for culturing a micro-tumor model of peritoneal metastasis of colorectal cancer, comprising all or part of the culture medium described in any one of claims 1-3 and the following: Sample dissociation solution, sample preservation solution, sample washing solution and digestion termination solution.

5. The kit of reagents according to claim 4, wherein: The sample dissociation solution is composed of collagenase I, collagenase II, collagenase IV and PBS; wherein, the final concentration of collagenase I is 150-250 U / mL; the final concentration of collagenase II is 150-250 U / mL; the final concentration of collagenase IV is 150-250 U / mL; the balance is the PBS.

6. The kit of reagents according to claim 4, wherein: The sample preservation solution is composed of fetal bovine serum, triple antibiotic-antimycotic, HEPES and HBSS; wherein, the triple antibiotic-antimycotic is penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% (v / v); the final concentration of penicillin in the triple antibiotic-antimycotic 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 HEPES is 8-12 mM; the balance is the HBSS.

7. The kit of reagents according to any one of claims 4-6, characterized in that: The sample cleaning solution is composed of triple antibiotic-antimycotic and PBS; wherein, the triple antibiotic-antimycotic is penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the triple antibiotic-antimycotic 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 balance is the PBS; and / or The digestion termination solution is composed of fetal bovine serum, triple antibiotic-antimycotic and DMEM medium; wherein, the triple antibiotic-antimycotic is penicillin, streptomycin and amphotericin B; in the digestion termination solution, the final concentration of the fetal bovine serum is 8-12% (v / v); the final concentration of penicillin in the triple antibiotic-antimycotic 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 balance is the DMEM medium.

8. Use of the medium according to any one of claims 1-3 or the kit of reagents according to any one of claims 4-7 in constructing a micro-tumor model of peritoneal metastasis of colorectal cancer.

9. A method for constructing a micro-tumor model of peritoneal metastasis of colorectal cancer, comprising the following steps: (a1) Dissociating the solid tumor tissue of peritoneal metastasis of colorectal cancer with the sample dissociating solution according to claim 4 or 5; (a2) Suspending and culturing the single cells dissociated in step (a1) with the medium according to any one of claims 1-3 to form cell clusters, thereby obtaining the micro-tumor model of peritoneal metastasis of colorectal cancer.

10. A micro-tumor model of peritoneal metastasis of colorectal cancer constructed by using the method according to claim 9.

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