A culture system, culture method and application of gastrointestinal tumor organoids

Abstract

The present invention relates to the technical field of organoid culture, and for the first time relates to a culture system and a culture method for gastrointestinal tumor organoids. The culture system is composed of adding antioxidant active traditional Chinese medicine extracts such as naringin or polydatin to the existing culture system. Using this culture system to culture gastrointestinal tumor organoids can improve the primary survival rate and the proliferation rate after passage of the gastrointestinal tumor organoids. In addition, the present invention also provides an application of the culture system for gastrointestinal tumor organoids, using the culture system and the gastrointestinal cancer organoids cultured thereby to perform drug screening on small molecule targeted anti-tumor drugs, providing a better in vitro research model for subsequent anti-tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and more specifically, it relates to a culture system, a culture method and an application of gastrointestinal tumor organoids. Background Art

[0002] Gastric cancer and colorectal cancer are common malignant tumors in clinical practice in China, with high incidence and mortality rates ranking among the top. However, at present, there are significant differences in anti-tumor efficacy among individuals. Therefore, a good preclinical model is needed to evaluate the drug efficacy. Currently, the main preclinical models are cell line models and patient-derived tumor xenograft animal models (PDX). However, cell line models derived from homogeneous tumor cells cannot simulate the microenvironment of human-derived tumors, and animal models have characteristics such as high experimental complexity and long cycle. In recent years, the emerging organoid preparation and culture technology has the ability to self-organize into "miniature organs" similar to the original tissues. Therefore, organoid models derived from tumor patients are highly similar to in vivo organs and tissues, can better retain the gene mutation spectrum of parental tumors, and can make up for the deficiencies of traditional culture technologies in retaining primary tumor information and simulating treatment response prediction.

[0003] However, at present, the success rate of the gastrointestinal tumor organoid culture system is not high enough. Relevant research shows that the success rate of gastric cancer organoid culture is 31 - 70.9%, and the success rate of colorectal cancer organoid culture is 68 - 85.7%. At the same time, the existing gastrointestinal tumor organoid culture system has problems such as low survival rate of primary cells and decreased proliferation rate after passage. Summary of the Invention

[0004] In order to solve the problems of low survival rate of primary organoids and decreased proliferation rate after passage under the existing gastrointestinal tumor organoid culture system, the purpose of the present invention is to provide a culture system for gastrointestinal tumor organoids.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] A culture system for gastrointestinal tumor organoids, comprising a basal medium and an antioxidant active traditional Chinese medicine extract; the antioxidant active traditional Chinese medicine extract is one or both of polydatin and naringin.

[0007] Existing studies have shown that naringin has the ability to scavenge free radicals and enhance the activities of antioxidant substances in the body, such as superoxide dismutase (SOD) and glutathione peroxidase (GPX), thereby improving the antioxidant function and promoting cell and tissue growth. Polydatin can exert antioxidant effects, protect cells and tissues, and enhance the antioxidant capacity of the body through various ways, such as scavenging free radicals in the body, inhibiting lipid peroxidation, increasing the activity of antioxidant enzymes, and regulating the level of antioxidant substances.

[0008] The inventors' research shows that adding naringin or polydatin to the existing gastrointestinal tumor organoid culture medium can increase the primary culture survival rate of gastrointestinal tumor organoids and the proliferation ability after passage, providing a better in vitro research model for subsequent anti-tumor treatment. However, adding quercetin, which also has antioxidant activity, to the existing gastrointestinal tumor organoid culture medium has the opposite effect at certain concentrations.

[0009] Preferably, the basal medium is based on Advanced DMEM / F12 medium, which contains Glutamax at a final concentration of 1%, N-acetyl-l-cysteine at 1.25 mmol / L, Wnt-3a at 20 - 100 ng / mL, R-Spondin 1 at 100 ng / mL, Noggin at 100 ng / mL, HEPES at 10 mmol / L, 1× N2 and B27, nicotinamide at 10 mmol / L, gastrin at 5 - 10 nmol / L, EGF at 50 ng / mL, FGF10 at 100 ng / mL, A8301 at 5 μmol / L, CHIR99021 at 3 μmol / L, and Y27632 at 10 μmol / L.

[0010] Specifically, the basal medium is based on Advanced DMEM / F12 medium, which contains Glutamax at a final concentration of 1%, N-acetyl-l-cysteine at a final concentration of 1.25 mmol / L, and Wnt-3a at a final concentration of 20 - 100 ng / mL, and the other components are set similarly.

[0011] Preferably, when the basal medium is used to culture gastric cancer organoids, the final concentration of Wnt-3a is 100 ng / mL, and the final concentration of gastrin is 10 nmol / L.

[0012] Preferably, when the basal medium is used to culture colorectal cancer organoids, the final concentration of Wnt-3a is 20 ng / mL, and the final concentration of gastrin is 5 nmol / L.

[0013] Preferably, the traditional Chinese medicine extract with antioxidant activity is added to the basal medium at a final concentration of 500 ng / mL.

[0014] Preferably, the traditional Chinese medicine extract with antioxidant activity is naringin.

[0015] Another object of the present invention is to provide a method for culturing gastrointestinal tumor organoids, which comprises the following steps: obtaining tumor cells from fresh gastric cancer tissue or intestinal cancer tissue; resuspending the tumor cells in a pre-cooled mixture composed of tumor organoid medium and Matrigel matrix glue at a volume ratio of 1:2, adjusting the cell concentration to 2500 - 4000 cells / 50 μL, and inoculating them into a cell culture plate; placing the cell culture plate in a cell incubator at 37°C and 5% carbon dioxide for 25 min for solidification, and then using the culture system provided above in the present invention to perform in vitro culture on gastrointestinal cancer organoids, replacing the corresponding culture system every 2 - 3 days, and observing the morphology of the organoids under a light microscope after 10 days of culture.

[0016] Another object of the present invention is to provide an application of a culture system for gastrointestinal tumor organoids, using the aforementioned culture system and the gastrointestinal cancer organoids cultured thereby to perform drug screening on small molecule targeted anti-tumor drugs.

[0017] The present invention has the following beneficial effects: By adding the traditional Chinese medicine extracts naringin or polydatin with antioxidant activity to the gastrointestinal tumor organoid culture system, the in vitro culture conditions of gastrointestinal tumor organoids are optimized, enabling the gastrointestinal tumor organoids to have higher survival rates, stronger proliferation abilities, and providing a good in vitro research model for subsequent anti-tumor treatments. Description of the Drawings

[0018] Figure 1 are the bright field cell photos of the control group, polydatin group, naringin group, and quercetin group in gastric cancer organoids;

[0019] Figure 2 are the relative cell survival rate graphs of the control group, polydatin group, naringin group, and quercetin group in gastric cancer organoids;

[0020] Figure 3 are the bright field cell photos of the control group, polydatin group, naringin group, and quercetin group in intestinal cancer organoids;

[0021] Figure 4 are the relative cell survival rate graphs of the control group, polydatin group, naringin group, and quercetin group in intestinal organoids;

[0022] Figure 5 are the bright field cell photos of the control group, polydatin group, naringin group, and quercetin group in P4 generation gastric cancer organoids;

[0023] Figure 6 It is a graph of the relative cell viability of the control group, polydatin group, naringin group, and quercetin group in P4-generation gastric cancer organoids;

[0024] Figure 7 It is a bright-field cell photograph of the control group, polydatin group, naringin group, and quercetin group in P4-generation colorectal cancer organoids;

[0025] Figure 8 It is a graph of the relative cell viability of the control group, polydatin group, naringin group, and quercetin group in P4-generation intestinal organoids;

[0026] Figure 9 It is a dose-effect curve graph of gastric cancer organoids from different patient sources against different drugs;

[0027] Figure 10 It is a dose-effect curve graph of colorectal cancer organoids from different patient sources against different drugs. Detailed implementation mode

[0028] The present invention will be further described below in conjunction with the embodiments, but the scope of protection required by the present invention is not limited to the scope described in the embodiments. Embodiment 1

[0029] A culture system for gastric tumor organoids, comprising a basal medium and an antioxidant-active traditional Chinese medicine extract; wherein, the antioxidant-active traditional Chinese medicine extract is polydatin.

[0030] Among them, the basal medium is based on Advanced DMEM / F12 medium, which contains Glutamax with a final concentration of 1%, N-acetyl-l-cysteine at 1.25 mmol / L, Wnt-3a at 20 - 100 ng / mL, R-Spondin 1 at 100 ng / mL, Noggin at 100 ng / mL, HEPES at 10 mmol / L, N2 and B27 at 1×, Nicotinamide at 10 mmol / L, Gastrin at 5 - 10 nmol / L, EGF at 50 ng / mL, FGF10 at 100 ng / mL, A8301 at 5 μmol / L, CHIR99021 at 3 μmol / L, and Y27632 at 10 μmol / L. Polydatin is added to the basal medium at final concentrations of 100, 500, 1000, and 1500 ng / mL respectively.

[0031] When the above-mentioned culture system is used to culture gastric cancer organoids, the final concentration of Wnt-3a in the basal medium is preferably 100 ng / mL, and the final concentration of Gastrin is preferably 10 nmol / L. The basal medium at this time is denoted as the optimized gastric cancer organoid medium I.

[0032] When the above-mentioned culture system is used to culture colorectal cancer organoids, the final concentration of Wnt-3a in the basal medium is preferably 20 ng / mL, and the final concentration of Gastrin is preferably 5 nmol / L. The basal medium at this time is denoted as the optimized colorectal cancer organoid medium I. Example 2

[0033] The difference from Example 1 is that the antioxidant active traditional Chinese medicine extract is naringin; the other components and their respective contents are the same as those in Example 1. Example 3

[0034] The difference from Example 1 is that the antioxidant active traditional Chinese medicine extract is quercetin; the other components and their respective contents are the same as those in Example 1.

[0035] Comparative Example 1

[0036] The difference from Example 1 is that the antioxidant active traditional Chinese medicine extract polydatin is not added; the other components and their respective contents are the same as those in Example 1. The unoptimized gastric cancer organoid medium (the final concentration of Wnt-3a in the basal medium is 100 ng / mL, and the final concentration of Gastrin is 10 nmol / L) and the unoptimized colorectal cancer organoid medium (the final concentration of Wnt-3a in the basal medium is 20 ng / mL, and the final concentration of Gastrin is 5 nmol / L) are obtained.

[0037] Referring to relevant literature, polydatin, naringin, and quercetin with different concentrations (100, 500, 1000, 1500 ng / mL) are respectively added to the optimized gastric cancer organoid medium I and the optimized colorectal cancer organoid medium I, and the resuscitated gastric cancer and colorectal cancer organoids are cultured. The CellTiter-Glo 3D luminescence cell viability detection kit is used to detect the cell viability, and the concentration of the traditional Chinese medicine extract added to the optimized medium is determined. The results are shown in Table 1 and Table 2.

[0038] Table 1 Effects of the concentration of antioxidant active traditional Chinese medicine extract on the cell viability of gastric tumor organoids

[0039] Group Basic composition Polygonin Naringin Quercetin Cell viability Control group Gastric cancer organoid medium I 0 0 0 / Experimental group 1 Gastric cancer organoid medium I 100 ng / mL 0 0 / Experimental group 2 Gastric cancer organoid medium I 500 ng / mL 0 0 + Experimental group 3 Gastric cancer organoid medium I 1000 ng / mL 0 0 / Experimental group 4 Gastric cancer organoid medium I 1500 ng / mL 0 0 - Experimental group 5 Gastric cancer organoid medium I 0 100 ng / mL 0 / Experimental group 6 Gastric cancer organoid medium I 0 500 ng / mL 0 + Experimental group 7 Gastric cancer organoid medium I 0 1000 ng / mL 0 / Experimental group 8 Gastric cancer organoid medium I 0 1500 ng / mL 0 - Experimental group 9 Gastric cancer organoid medium I 0 0 100 ng / mL / Experimental group 10 Gastric cancer organoid medium I 0 0 500 ng / mL / Experimental group 11 Gastric cancer organoid medium I 0 0 1000 ng / mL / Experimental group 12 Gastric cancer organoid medium I 0 0 1500 ng / mL -

[0040] Table 2 Effects of the concentration of antioxidant active traditional Chinese medicine extract on the cell viability of intestinal tumor organoids

[0041] Group Basic composition Polygonin Naringin Quercetin Cell viability Control group Colorectal cancer organoid medium I 0 0 0 / Experimental group 1 Colorectal cancer organoid medium I 100 ng / mL 0 0 / Experimental group 2 Colorectal cancer organoid medium I 500 ng / mL 0 0 ++ Experimental group 3 Colorectal cancer organoid medium I 1000 ng / mL 0 0 / Experimental group 4 Colorectal cancer organoid medium I 1500 ng / mL 0 0 - Experimental group 5 Colorectal cancer organoid medium I 0 100 ng / mL 0 / Experimental group 6 Colorectal cancer organoid medium I 0 500 ng / mL 0 ++ Experimental group 7 Colorectal cancer organoid medium I 0 1000 ng / mL 0 / Experimental group 8 Colorectal cancer organoid medium I 0 1500 ng / mL 0 - Experimental group 9 Colorectal cancer organoid medium I 0 0 100 ng / mL / Experimental group 10 Colorectal cancer organoid medium I 0 0 500 ng / mL / Experimental group 11 Colorectal cancer organoid medium I 0 0 1000 ng / mL - Experimental group 12 Colorectal cancer organoid medium I 0 0 1500 ng / mL -

[0042] Note: In Table 1 and Table 2, "-" indicates a decrease of 0 - 50%, " / " indicates no significant change, "+" indicates an increase of 0 - 50%, and "++" indicates an increase of 50 - 100%.

[0043] As can be seen from Table 1 and Table 2, the optimal final concentration of the antioxidant active traditional Chinese medicine extract in the optimized gastric cancer organoid medium I and the optimized intestinal cancer organoid medium I above is 500 ng / mL for both.

[0044] The optimized gastric cancer organoid medium I containing 500 ng / mL of the antioxidant active traditional Chinese medicine extract is denoted as gastric cancer organoid medium II; the optimized intestinal cancer organoid medium I containing 500 ng / mL of the antioxidant active traditional Chinese medicine extract is denoted as intestinal cancer organoid medium II.

[0045] Application Example 1: Optimization of the culture system of primary gastrointestinal tumor organoids

[0046] A method for culturing gastrointestinal tumor organoids, which comprises the following steps:

[0047] (1) Fresh gastric cancer tissues and intestinal cancer tissues are collected respectively. The specimen acquisition methods include surgery and biopsy, and they are transported on ice using tissue transport fluid. The tissue transport fluid is Advanced DMEM / F12 basal medium, and 1% (V / V%) Glutamax, 10 mmol / L HEPES, and 2% (V / V%) penicillin / streptomycin solution are added.

[0048] (2) Use sterilized scissors to cut off excess tissues such as muscle and fat, and wash the tumor tissues with pre-cooled physiological saline 1 - 2 times. Cut the tumor tissue strips into pieces within 1 mm, and wash them several times with pre-cooled physiological saline in a centrifuge tube.

[0049] (3) After washing several times and standing still, discard the supernatant, and place the tissue precipitate in tissue digestive fluid for digestion. The tissue digestive fluid consists of basal medium Advanced DMEM / F12, 2.5 mg / mL of type IV collagenase, 0.1 mg / mL of deoxyribonuclease 1, and 2% (V / V%) penicillin / streptomycin solution. Place it in a 37°C incubator and shake for digestion for 50 minutes.

[0050] (4) Add an equal volume of pre-cooled digestion termination fluid to terminate digestion. The digestion termination fluid consists of complete cell medium (basal medium Advanced DMEM / F12 + 10% fetal bovine serum), 10 μmol / L of cell detachment and apoptosis protector Y27632, and 1% (V / V%) penicillin / streptomycin solution.

[0051] (5) The cell suspension was filtered through a 100-μm filter and centrifuged at 300 g for 10 minutes at 4°C. The supernatant was discarded, and the precipitate was washed twice with pre-cooled physiological saline. The finally collected precipitate was the tumor cells.

[0052] (6) The tumor cells were resuspended in a pre-cooled mixture composed of tumor organoid medium and Matrigel matrix gel at a volume ratio of 1:2. The cell concentration was adjusted to 2500 - 4000 cells / 50 μL and seeded in a cell culture plate. According to the settings before and after optimization, 4 groups of organoids were set up (control group, polydatin group, naringin group, and quercetin group), with 3 wells in each group. In the control group, the obtained gastric cancer cells were cultured using the unoptimized gastric cancer organoid medium in Comparative Example 1, and the obtained intestinal cancer cells were cultured using the unoptimized intestinal cancer organoid medium in Comparative Example 1; in the polydatin group, the obtained gastric cancer cells were cultured using the gastric cancer organoid medium II containing polydatin, and the obtained intestinal cancer cells were cultured using the intestinal cancer organoid medium II containing polydatin; in the naringin group, the obtained gastric cancer cells were cultured using the gastric cancer organoid medium II containing naringin, and the obtained intestinal cancer cells were cultured using the intestinal cancer organoid medium II containing naringin; in the quercetin group, the obtained gastric cancer cells were cultured using the gastric cancer organoid medium II containing quercetin, and the obtained intestinal cancer cells were cultured using the intestinal cancer organoid medium II containing quercetin.

[0053] (7) The cell culture plate was placed in a cell culture incubator at 37°C and 5% carbon dioxide for 25 minutes for solidification.

[0054] (8) Each was cultured using the corresponding culture system, and the corresponding culture system was replaced every 2 - 3 days. After 10 days of culture, the morphology of the organoids in different groups was observed under a light microscope.

[0055] The results showed that, see Figures 1 - 4 , compared with the control group, more surviving gastric and intestinal cancer organoids appeared in the experimental groups supplemented with polydatin and naringin. The CellTiter-Glo 3D luminescence cell viability detection kit was used to detect the cell viability of different groups under an enzyme-labeled instrument, and the GraphPad Prism 8.3.0 software was used to calculate and analyze the cell survival rate of each group. In gastric cancer, the primary organoids in the polydatin and naringin groups showed higher cell survival rates, and the differences were statistically significant (P = 0.0037, P = 0.0093; Figures 1 - 2 ), and there was no significant difference in the cell viability of the primary organoids in the quercetin group and the control group (P = 0.4669, Figures 1 - 2 ). In intestinal cancer, the primary organoids in the polydatin and naringin groups showed higher cell survival rates, and the differences were statistically significant (P = 0.0018, P = 0.0042; Figures 3 - 4), the viability of primary organoid cells in the quercetin group was not significantly different from that in the control group (P = 0.059, Figures 3 - 4 ).

[0056] Application Example 2: Optimization of the culture system for passaged gastrointestinal tumor organoids

[0057] A method for culturing gastrointestinal tumor organoids, comprising the following steps:

[0058] (1) Take out 2 frozen gastric cancer and 2 frozen colorectal cancer organoids from liquid nitrogen, thaw them in a 37 °C water bath. When the ice crystals are about to melt, take out the water bath, add 1 mL of complete cell medium (Advanced DMEM / F12 supplemented with 10% (V / V%) fetal bovine serum and 1% (V / V%) penicillin / streptomycin solution), mix well and aspirate into a 15 mL centrifuge tube, and centrifuge at 300 g for 10 minutes at 4 °C. Discard the supernatant and wash twice with normal saline.

[0059] (2) Resuspend the tumor cells in a pre-cooled mixture composed of tumor organoid medium and Matrigel matrix glue at a volume ratio of 1:2, adjust the cell concentration to 2500 - 4000 cells / 50 μL, and inoculate into a cell culture plate. Set up 4 groups of organoids according to before and after optimization (control group, polydatin group, naringin group, and quercetin group).

[0060] (3) Place it in a cell culture incubator at 37 °C and 5% carbon dioxide for 25 min to solidify. Cultivate using the corresponding culture system respectively, and replace the corresponding culture system every 2 - 3 days.

[0061] (4) Determine the passage culture cycle according to the growth rate, passage 2 generations, observe and record the size of organoids on the 1st day and the 7th day under a light microscope for different groups of gastric cancer and colorectal cancer organoids, and calculate their size using ImageJ.

[0062] The results showed that, see Figures 5 - 8 , in gastric cancer, compared with the control group, more and larger organoids appeared in the P4 - generation organoids of the polydatin, naringin, and quercetin groups, and the differences were statistically significant (P = 0.0004, P < 0.0001, P = 0.0044, Figures 5 - 6 ); in colorectal cancer, compared with the control group, more and larger organoids appeared in the P4 - generation organoids of the polydatin and naringin groups, while in the quercetin group, the P4 - generation organoids were significantly smaller ( Figures 7 - 8 ), and the differences were all statistically significant (P = 0.0157, P = 0.0004, P < 0.0001; Figures 7 - 8 ).

[0063] The comparison of the primary survival rate of organoids and the proliferation ability after passage before and after optimizing the gastric cancer organoid culture system is shown in Table 3. The comparison of the primary survival rate of organoids and the proliferation ability after passage before and after optimizing the colorectal cancer organoid culture system is shown in Table 4.

[0064] Table 3 Ability table of gastric cancer organoid culture system before and after optimization

[0065] Group Basic composition Polygonin Naringin Quercetin Primary survival ability Passage proliferation ability Control group Unoptimized gastric cancer organoid medium 0 0 0 / / Polygonin group Gastric cancer organoid medium I 500 ng / mL 0 0 + ++ Naringin group Gastric cancer organoid medium I 0 500 ng / mL 0 + +++ Quercetin group Gastric cancer organoid medium I 0 0 500 ng / mL / ++

[0066] Table 4 Ability table of colorectal cancer organoid culture system before and after optimization

[0067] Group Basic composition Polygonin Naringin Quercetin Primary survival ability Passage proliferation ability Control group Unoptimized colorectal cancer organoid medium 0 0 0 / / Polygonin group Colorectal cancer organoid medium I 500 ng / mL 0 0 ++ + Naringin group Colorectal cancer organoid medium I 0 500 ng / mL 0 ++ ++ Quercetin group Colorectal cancer organoid medium I 0 0 500 ng / mL / --

[0068] Note: In Table 3 and Table 4, "--" indicates a decrease of 50 - 100%, "-" indicates a decrease of 0 - 50%, " / " indicates no significant change, "+" indicates an increase of 0 - 50%, "++" indicates an increase of 50 - 100%, and "+++" indicates an increase of 100 - 150%. The basal medium in Table 1 is the unoptimized gastric cancer organoid medium in Comparative Example 1, and the basal medium in Table 2 is the unoptimized colorectal cancer organoid medium in Comparative Example 1.

[0069] The results in Table 3 and Table 4 show that compared with before optimization and adding different traditional Chinese medicine extracts, adding 500 ng / mL naringin can better optimize the gastric cancer and colorectal cancer organoid media, significantly enhancing the primary culture survival rate of gastrointestinal tumor organoids and the proliferation ability after passage.

[0070] Application Example 3: Drug screening test for small molecule targeted anti-tumor drugs

[0071] Select the optimized gastric cancer organoid medium II and optimized colorectal cancer organoid medium II with 500 ng / mL naringin added in Example 2, and conduct drug sensitivity tests for small molecule targeted anti-tumor drugs respectively. The steps are as follows:

[0072] (1) Collect fresh specimens from different gastric cancer patients and different colorectal cancer patients, and culture and expand them to the 3rd generation using the optimized gastrointestinal tumor organoid medium.

[0073] (2) After culturing for 3 days, aspirate the organoid medium (i.e., the optimized gastrointestinal tumor organoid medium), and prepare gastric cancer organoid media and colorectal cancer organoid media for 5 small molecule targeted anti-tumor drugs such as fruquintinib, regorafenib, apatinib, sunitinib, and sorafenib according to the drug concentration gradients of 0, 0.008, 0.016, 0.080, 0.400, 2, and 10 μmol / L. Conduct drug sensitivity tests, with 3 replicates for each concentration of each drug, and set positive and negative control groups respectively.

[0074] (3) After 3 days, aspirate the culture medium (i.e., gastric cancer organoid culture medium or colorectal cancer organoid culture medium containing different drug concentrations), add 50 µL of Advanced DMEM / F-12 culture medium, and then add 50 µL of CellTiter-Glo® 3D cell viability assay reagent. Transfer to a multimode microplate reader and set the corresponding parameters. The positive control group is defined as 0% cell viability, and the negative control (solvent control) is defined as 100% cell viability. The calculation formula is: Cell viability = (well value - average positive control value) / (average negative control value - average positive control value) * 100%. Plot the dose-effect curve.

[0075] The results showed that for colorectal and gastric cancer organoids cultured in the optimized culture medium, organoids from different patients showed different responses to drugs. In gastric cancer, organoids from patient G001 showed higher drug sensitivity to fruquintinib, regorafenib, apatinib, and sunitinib, and stronger drug resistance to sorafenib compared with organoids from patient G002 ( Figure 9 ); in colorectal cancer, organoids from patient C001 showed higher drug sensitivity to regorafenib and sorafenib, and stronger drug resistance to fruquintinib, apatinib, and sunitinib compared with organoids from patient C002 ( Figure 10 ).

[0076] In summary, by adding naringin or polydatin, traditional Chinese medicine extracts with antioxidant activity, to the gastrointestinal tumor organoid culture system, the present invention optimized the in vitro culture conditions of gastrointestinal tumor organoids, enabling gastrointestinal tumor organoids to have higher survival rates and stronger proliferation abilities, and providing a good in vitro research model for subsequent anti-tumor treatment.

[0077] The above are only the preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A gastrointestinal tumor organoid culture system, characterized in that: The method comprises a basic culture medium and a Chinese herbal medicine extract with antioxidant activity; the Chinese herbal medicine extract with antioxidant activity is added to the basic culture medium at a final concentration of 500 ng / mL; the Chinese herbal medicine extract with antioxidant activity is polydatin or naringin; The basic culture medium is based on Advanced DMEM / F12 culture medium, which contains Glutamax, N-acetyl-l-cysteine, Wnt-3a, R-Spondin 1, Noggin, HEPES, N2 and B27, Nicotinamide, Gastrin, EGF, FGF10, A8301, CHIR99021 and Y27632.

2. The culture system according to claim 1, characterized in that The basic culture medium is based on Advanced DMEM / F12 culture medium, which contains Glutamax with a final concentration of 1%, 1.25mmol / L N-acetyl-l-cysteine, 20-100ng / mL Wnt-3a, 100ng / mL R-Spondin 1, 100ng / mL Noggin, 10mmol / L HEPES, 1× N2 and B27, 10mmol / L Nicotinamide, 5-10nmol / L Gastrin, 50ng / mL EGF, 100ng / mL FGF10, 5µmol / L A8301, 3µmol / L CHIR99021 and 10µmol / L Y27632.

3. The culture system according to claim 2, characterized in that: When used for culturing gastric cancer organoids, the final concentration of Wnt-3a in the basal culture medium is 100 ng / mL, and the final concentration of Gastrin is 10 nmol / L.

4. The culture system according to claim 2, characterized in that: When used for culturing intestinal cancer organoids, the final concentration of Wnt-3a in the basal culture medium is 20 ng / mL, and the final concentration of Gastrin is 5 nmol / L.

5. The culture system according to claim 1, characterized in that: The Chinese medicinal extract with antioxidant activity is naringin.

6. A method for culturing gastrointestinal tumor organoids, characterized in that: Gastrointestinal cancer organoids are cultured in vitro using the culture system described in any one of claims 1 to 5.

7. An application of a gastrointestinal tumor organoid culture system, characterized in that: Small molecule targeted anti-tumor drugs are screened using the culture system and gastrointestinal cancer organoids cultured therein as described in any one of claims 1 to 5.

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