Culture system, culture method and application of gastrointestinal tumor organoid

Abstract

The invention relates to the technical field of organoid culture, and relates to a culture system and a culture method for gastrointestinal tumor organoids for the first time, the culture system is formed by adding antioxidant active traditional Chinese medicine extracts such as naringin or polydatin on the basis of an existing culture system, when the culture system is used for culturing the gastrointestinal tumor organoid, the primary survival rate of the gastrointestinal tumor organoid and the proliferation rate after passage can be improved. Besides, the invention further provides application of the culture system of the gastrointestinal tumor organoid, the culture system and the cultured gastrointestinal tumor organoid are used for screening small-molecule targeted anti-tumor drugs, and a better in-vitro research model is provided for subsequent anti-tumor treatment.

Description

Technical Field

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

[0002] Gastric cancer and intestinal cancer are common malignant tumors in clinical practice in my country, with the highest incidence and mortality rates. However, at present, there are large differences in anti-tumor efficacy between individuals, so good preclinical models are needed to evaluate drug efficacy. The main preclinical models are cell line models and patient-derived tumor xenograft animal models (PDX), but cell line models derived from homogeneous tumor cells cannot simulate the microenvironment of human tumors, and animal models have the characteristics of high experimental complexity and long cycles. In recent years, the emerging organoid preparation and culture technology has the ability to self-organize to form "micro-organs" similar to the original tissue. Therefore, the organoid model derived from tumor patients is highly similar to the organs and tissues in the body, and can better preserve the gene mutation spectrum of the parental tumor, which can make up for the shortcomings of traditional culture technology in retaining primary tumor information and simulating treatment response prediction.

[0003] However, at present, the success rate of gastrointestinal tumor organoid culture system is still not high enough. Relevant studies have shown that the success rate of gastric cancer organoid culture is 31-70.9%, and the success rate of intestinal cancer organoid culture is 68-85.7%. At the same time, the existing gastrointestinal tumor organoid culture system has problems such as low primary cell survival rate and decreased proliferation rate after passaging. Summary of the invention

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

[0005] To achieve the above object, the present invention adopts the following technical solution: A gastrointestinal tumor organoid culture system comprises a basal culture medium and a Chinese herbal extract with antioxidant activity; the Chinese herbal extract with antioxidant activity is one or both of polydatin and naringin.

[0006] Studies have shown that naringin has the ability to scavenge free radicals and enhance the activity of antioxidant substances superoxide dismutase (SOD) and glutathione peroxidase (GPX) in the body, thereby improving antioxidant function and promoting cell and tissue growth. Polydatin can play an antioxidant role in multiple 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, thereby protecting cells and tissues and enhancing the body's antioxidant capacity.

[0007] 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 capacity after subculture, 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 a counter-effect at certain concentrations.

[0008] Preferably, the basal 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.

[0009] Specifically, the basic culture medium is based on Advanced DMEM / F12 culture medium, which contains Glutamax with a final concentration of 1%, N-acetyl-l-cysteine ​​with a final concentration of 1.25 mmol / L, Wnt-3a with a final concentration of 20 to 100 ng / mL, and other components are similarly set.

[0010] Preferably, when the basal culture 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.

[0011] Preferably, when the basal culture medium is used to culture intestinal cancer organoids, the final concentration of Wnt-3a is 20 ng / mL, and the final concentration of Gastrin is 5 nmol / L.

[0012] Preferably, the Chinese herbal medicine extract with antioxidant activity is added to the basal culture medium at a final concentration of 500 ng / mL.

[0013] Preferably, the Chinese herbal extract with antioxidant activity is naringin.

[0014] 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 mixed solution composed of tumor organoid culture medium and Matrigel matrix gel in a volume ratio of 1:2, adjusting the cell concentration to 2500-4000 cells / 50 μL, and inoculating them in a cell culture plate; placing the cell culture plate in a cell culture incubator at 37°C and 5% carbon dioxide for solidification for 25 minutes, and then culturing the gastrointestinal cancer organoids in vitro using the culture system provided by the present invention, replacing the corresponding culture system every 2-3 days, and observing the organoid morphology under a light microscope after 10 days of culture.

[0015] Another object of the present invention is to provide an application of a gastrointestinal tumor organoid culture system, and to use the aforementioned culture system and the cultured gastrointestinal cancer organoids to screen small molecule targeted anti-tumor drugs.

[0016] The present invention has the following beneficial effects: by adding naringin or polydatin, a traditional Chinese medicine extract with antioxidant activity, to the gastrointestinal tumor organoid culture system, the in vitro culture conditions of the gastrointestinal tumor organoids are optimized, so that the gastrointestinal tumor organoids have a higher survival rate and stronger proliferation ability, and provide a good in vitro research model for subsequent anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are bright field photos of cells in the control group, polydatin group, naringin group, and quercetin group in gastric cancer organoids; Figure 2 is a graph of relative cell survival rates of the control group, polydatin group, naringin group, and quercetin group in gastric cancer organoids; Figure 3 These are bright field photos of cells in the control group, polydatin group, naringin group, and quercetin group in colorectal cancer organoids; Figure 4 is a graph of relative cell survival rates of the control group, polydatin group, naringin group, and quercetin group in intestinal organoids; Figure 5 These are bright field photos of cells in the control group, polydatin group, naringin group, and quercetin group in the P4 generation gastric cancer organoids; Figure 6This is a graph of the relative cell survival rates of the control group, polydatin group, naringin group, and quercetin group in the P4 generation of gastric cancer organoids; Figure 7 These are bright field photos of cells in the control group, polydatin group, naringin group, and quercetin group in the P4 generation of colorectal cancer organoids; Figure 8 is a graph of relative cell survival rates of the control group, polydatin group, naringin group, and quercetin group in the P4 generation enteroids; Fig. 9 This is a dose-effect curve of gastric cancer organoids from different patients to different drugs; Fig.10 This is a dose-effect curve of colorectal cancer organoids from different patients to different drugs. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with embodiments, but the scope of protection claimed by the present invention is not limited to the scope described in the embodiments. Example 1

[0019] A gastric tumor organoid culture system comprises a basal culture medium and a Chinese herbal extract with antioxidant activity; wherein the Chinese herbal extract with antioxidant activity is polydatin.

[0020] The basal medium was based on Advanced DMEM / F12 medium, which contained 1% Glutamax, 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. Polydatin was added to the basal medium at final concentrations of 100, 500, 1000, and 1500ng / mL, respectively.

[0021] When the above culture system is used to culture gastric cancer organoids, the final concentration of Wnt-3a in the basal culture medium is preferably 100 ng / mL, and the final concentration of Gastrin is preferably 10 nmol / L. The basal culture medium at this time is recorded as optimized gastric cancer organoid culture medium I.

[0022] When the above culture system is used to culture intestinal cancer organoids, the final concentration of Wnt-3a in the basal culture medium is preferably 20 ng / mL, and the final concentration of Gastrin is preferably 5 nmol / L. The basal culture medium at this time is recorded as optimized intestinal cancer organoid culture medium I. Example 2

[0023] The difference from Example 1 is that the Chinese herbal extract with antioxidant activity is naringin; the other components and their respective contents are the same as those in Example 1. Example 3

[0024] The difference from Example 1 is that the Chinese herbal extract with antioxidant activity is quercetin; the other components and their respective contents are the same as those in Example 1.

[0025] Comparative Example 1 The difference from Example 1 is that polydatin, a Chinese herbal extract with antioxidant activity, is not added; the remaining components and their respective contents are the same as those in Example 1. An unoptimized gastric cancer organoid culture medium (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) and an unoptimized intestinal cancer organoid culture medium (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) are obtained.

[0026] Referring to relevant literature, different concentrations (100, 500, 1000, 1500 ng / mL) of polydatin, naringin, and quercetin were added to the optimized gastric cancer organoid culture medium I and the optimized intestinal cancer organoid culture medium I, respectively, and the resuscitated gastric cancer and intestinal cancer organoids were cultured. The CellTiter-Glo 3D luminescent cell viability assay kit was used to detect cell viability, and the concentration of the Chinese medicine extract added to the optimized culture medium was determined. The results are shown in Tables 1 and 2.

[0027] Table 1 Effect of concentration of Chinese herbal extracts with antioxidant activity on cell viability of gastric tumor organoids Group Basic composition Polydatin Naringin Quercetin Cell viability Control group Gastric Cancer Organoid Culture Medium I 0 0 0 / Experimental Group 1 Gastric Cancer Organoid Culture Medium I 100ng / mL 0 0 / Experimental Group 2 Gastric Cancer Organoid Culture Medium I 500ng / mL 0 0 + Experimental Group 3 Gastric Cancer Organoid Culture Medium I 1000ng / mL 0 0 / Experimental Group 4 Gastric Cancer Organoid Culture Medium I 1500ng / mL 0 0 - Experimental Group 5 Gastric Cancer Organoid Culture Medium I 0 100ng / mL 0 / Experimental Group 6 Gastric Cancer Organoid Culture Medium I 0 500ng / mL 0 + Experimental Group 7 Gastric Cancer Organoid Culture Medium I 0 1000ng / mL 0 / Experimental Group 8 Gastric Cancer Organoid Culture Medium I 0 1500ng / mL 0 - Experimental Group 9 Gastric Cancer Organoid Culture Medium I 0 0 100ng / mL / Experimental Group 10 Gastric Cancer Organoid Culture Medium I 0 0 500ng / mL / Experimental Group 11 Gastric Cancer Organoid Culture Medium I 0 0 1000ng / mL / Experimental Group 12 Gastric Cancer Organoid Culture Medium I 0 0 1500ng / mL - Table 2 Effect of concentration of Chinese herbal extracts with antioxidant activity on cell viability of intestinal tumor organoids Group Basic composition Polydatin Naringin Quercetin Cell viability Control group Intestinal Cancer Organoid Culture Medium I 0 0 0 / Experimental Group 1 Intestinal Cancer Organoid Culture Medium I 100ng / mL 0 0 / Experimental Group 2 Intestinal Cancer Organoid Culture Medium I 500ng / mL 0 0 ++ Experimental Group 3 Intestinal Cancer Organoid Culture Medium I 1000ng / mL 0 0 / Experimental Group 4 Intestinal Cancer Organoid Culture Medium I 1500ng / mL 0 0 - Experimental Group 5 Intestinal Cancer Organoid Culture Medium I 0 100ng / mL 0 / Experimental Group 6 Intestinal Cancer Organoid Culture Medium I 0 500ng / mL 0 ++ Experimental Group 7 Intestinal Cancer Organoid Culture Medium I 0 1000ng / mL 0 / Experimental Group 8 Intestinal Cancer Organoid Culture Medium I 0 1500ng / mL 0 - Experimental Group 9 Intestinal Cancer Organoid Culture Medium I 0 0 100ng / mL / Experimental Group 10 Intestinal Cancer Organoid Culture Medium I 0 0 500ng / mL / Experimental Group 11 Intestinal Cancer Organoid Culture Medium I 0 0 1000ng / mL - Experimental Group 12 Intestinal Cancer Organoid Culture Medium I 0 0 1500ng / mL - Note: In Table 1 and Table 2, “-” means a decrease of 0-50%, “ / ” means no significant change, “+” means an increase of 0-50%, and “++” means an increase of 50-100%.

[0028] It can be seen from Tables 1 and 2 that the optimal final concentration of the antioxidant active Chinese herbal extracts in the optimized gastric cancer organoid culture medium I and the optimized intestinal cancer organoid culture medium I are both 500 ng / mL.

[0029] The optimized gastric cancer organoid culture medium I containing 500 ng / mL of the antioxidant Chinese herbal extract was recorded as gastric cancer organoid culture medium II; the optimized intestinal cancer organoid culture medium I containing 500 ng / mL of the antioxidant Chinese herbal extract was recorded as intestinal cancer organoid culture medium II.

[0030] Application Example 1: Optimization of the Culture System for Primary Gastrointestinal Tumor Organoids A method for culturing gastrointestinal tumor organoids, comprising the following steps: (1) Fresh gastric cancer tissue and intestinal cancer tissue were collected separately. The specimens were obtained by surgery and biopsy and transported on ice using tissue transport fluid. The tissue transport fluid was Advanced DMEM / F12 basal culture medium supplemented with 1% (V / V%) Glutamax, 10mmol / L HEPES, and 2% (V / V%) penicillin / streptomycin solution.

[0031] (2) Use high-temperature sterilized scissors to cut off excess tissue such as muscle and fat, and wash the tumor tissue 1 to 2 times with pre-cooled saline. Cut the tumor tissue into strips less than 1 mm and wash it several times in a centrifuge tube with pre-cooled saline.

[0032] (3) After washing several times, let it stand, discard the supernatant, and place the tissue pellet in a tissue digestion solution for digestion. The tissue digestion solution consists of the basic culture medium Advanced DMEM / F12, 2.5 mg / mL type IV collagenase, 0.1 mg / mL deoxyribonuclease 1, and 2% (V / V%) penicillin / streptomycin solution. Place it in a 37°C incubator and shake it for 50 minutes.

[0033] (4) Add an equal volume of pre-cooled digestion stop solution to terminate the digestion. The digestion stop solution consists of complete cell culture medium (basal culture medium Advanced DMEM / F12 + 10% fetal bovine serum), 10 µmol / L cell apoptosis protectant Y27632, and 1% (V / V%) penicillin / streptomycin solution.

[0034] (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 cells were washed twice with pre-cooled saline. The precipitate collected was the tumor cells.

[0035] (6) Resuspend the tumor cells in a pre-cooled mixture of tumor organoid culture medium and Matrigel matrix gel at a volume ratio of 1:2, adjust the cell concentration to 2500-4000 cells / 50μL, and inoculate them in a cell culture plate. Set up 4 groups of organoids (control group, polydatin group, naringin group, and quercetin group) according to the optimization, with 3 wells in each group. In the control group, the gastric cancer cells obtained above were cultured using the non-optimized gastric cancer organoid culture medium in Comparative Example 1, and the intestinal cancer cells obtained above were cultured using the non-optimized intestinal cancer organoid culture medium in Comparative Example 1; in the polydatin group, the gastric cancer cells obtained above were cultured using the gastric cancer organoid culture medium II containing polydatin, and the intestinal cancer cells obtained above were cultured using the intestinal cancer organoid culture medium II containing polydatin; in the naringin group, the gastric cancer cells obtained above were cultured using the gastric cancer organoid culture medium II containing naringin, and the intestinal cancer cells obtained above were cultured using the intestinal cancer organoid culture medium II containing naringin; in the quercetin group, the gastric cancer cells obtained above were cultured using the gastric cancer organoid culture medium II containing quercetin, and the intestinal cancer cells obtained above were cultured using the intestinal cancer organoid culture medium II containing quercetin.

[0036] (7) Place the cell culture plate in a cell culture incubator at 37°C and 5% carbon dioxide for 25 minutes.

[0037] (8) Each group of organoids was cultured using the corresponding culture system, and the corresponding culture system was changed every 2 to 3 days. After 10 days of culture, the morphology of the organoids in different groups was observed under a light microscope.

[0038] The results show that Figure 1 to Figure 4 Compared with the control group, more surviving gastric cancer and intestinal cancer organoids appeared in the experimental group with the addition of polydatin and naringin. The CellTiter-Glo 3D luminescent cell viability detection kit was used to detect the cell viability of different groups under an ELISA instrument, and the cell survival rate of each group was calculated and analyzed using GraphPad Prism 8.3.0 software. 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; Figure 1-2 ), there was no significant difference in the primary organoid cell viability between the quercetin group and the control group (P=0.4669, Figure 1-2 In colorectal cancer, 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; Figure 3-4 ), there was no significant difference in the primary organoid cell viability between the quercetin group and the control group (P=0.059, Figure 3-4 ).

[0039] Application Example 2: Optimization of the Culture System for Passaging Gastrointestinal Tumor Organoids A method for culturing gastrointestinal tumor organoids, comprising the following steps: (1) Take out two gastric cancer and two intestinal cancer organoids frozen in liquid nitrogen and thaw them in a 37°C water bath. When the ice crystals are about to melt, remove them from the water bath and add 1 mL of complete cell culture medium (Advanced DMEM / F12 supplemented with 10% (V / V%) fetal bovine serum and 1% (V / V%) penicillin / streptomycin solution). Mix well and transfer to a 15 mL centrifuge tube. Centrifuge at 300 g for 10 minutes at 4°C. Discard the supernatant and wash twice with saline.

[0040] (2) Tumor cells were resuspended in a pre-cooled mixture of tumor organoid culture medium and Matrigel matrix gel in a volume ratio of 1:2, and the cell concentration was adjusted to 2500-4000 cells / 50μL, and inoculated into a cell culture plate. Four groups of organoids were set up before and after optimization (control group, polydatin group, naringin group, and quercetin group).

[0041] (3) Place in a cell culture incubator at 37°C and 5% carbon dioxide for 25 minutes to solidify. Use the corresponding culture system for culture and change the corresponding culture system every 2 to 3 days.

[0042] (4) The subculture cycle was determined based on the growth rate. After two subcultures, the organoid sizes of gastric cancer and intestinal cancer organoids of different groups were observed under a light microscope on the first and seventh days, and their sizes were calculated using ImageJ.

[0043] The results show that Figures 5 to 8 In gastric cancer, compared with the control group, more and larger organoids appeared in the P4 generation of polydatin, naringin, and quercetin groups, and the differences were statistically significant (P=0.0004, P<0.0001, P=0.0044, Figure 5-6 ); In colorectal cancer, compared with the control group, more and larger organoids appeared in the P4 generation of polydatin and naringin groups, while in the quercetin group, the P4 generation of organoids was significantly smaller ( Figure 7-Figure 8 ), the differences were statistically significant (P=0.0157, P=0.0004, P<0.0001; Figure 7-Figure 8 ).

[0044] The comparison of the primary survival rate and proliferation capacity of gastric cancer organoid culture system before and after optimization is shown in Table 3. The comparison of the primary survival rate and proliferation capacity of intestinal cancer organoid culture system before and after optimization is shown in Table 4.

[0045] Table 3 Capacity of gastric cancer organoid culture system before and after optimization Group Basic composition Polydatin Naringin Quercetin Primary survival ability Subculture proliferation capacity Control group Non-optimized gastric cancer organoid culture medium 0 0 0 / / Polydatin group Gastric Cancer Organoid Culture Medium I 500ng / mL 0 0 + ++ Naringin group Gastric Cancer Organoid Culture Medium I 0 500ng / mL 0 + +++ Quercetin group Gastric Cancer Organoid Culture Medium I 0 0 500ng / mL / ++ Table 4 Capacity of colorectal cancer organoid culture system before and after optimization Group Basic composition Polydatin Naringin Quercetin Primary survival ability Subculture proliferation capacity Control group Non-optimized culture medium for colorectal cancer organoids 0 0 0 / / Polydatin group Intestinal Cancer Organoid Culture Medium I 500ng / mL 0 0 ++ + Naringin group Intestinal Cancer Organoid Culture Medium I 0 500ng / mL 0 ++ ++ Quercetin group Intestinal Cancer Organoid Culture Medium I 0 0 500ng / mL / -- 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 culture medium in Table 1 is the non-optimized gastric cancer organoid culture medium in Comparative Example 1, and the basal culture medium in Table 2 is the non-optimized intestinal cancer organoid culture medium in Comparative Example 1.

[0046] The results in Tables 3 and 4 show that compared with before optimization and adding different Chinese medicine extracts, the addition of 500 ng / mL naringin can better optimize the culture medium of gastric cancer and intestinal cancer organoids, and significantly enhance the survival rate of primary culture of gastrointestinal tumor organoids and the proliferation ability after passaging.

[0047] Application Example 3: Small molecule targeted anti-tumor drug screening test The optimized gastric cancer organoid culture medium II and the optimized intestinal cancer organoid culture medium II after adding 500 ng / mL naringin in Example 2 were selected to perform drug sensitivity tests on small molecule targeted anti-tumor drugs, respectively, and the steps are as follows: (1) Fresh specimens from different gastric cancer patients and different intestinal cancer patients were collected, and the optimized gastrointestinal tumor organoid culture medium was used to culture and expand them to the third generation.

[0048] (2) After 3 days of culture, the organoid culture medium (i.e., the optimized gastrointestinal tumor organoid culture medium) was removed, and gastric cancer organoid culture medium and intestinal cancer organoid culture medium containing five small molecule targeted anti-tumor drugs, including furiquintinib, regorafenib, apatinib, sunitinib, and sorafenib, were prepared according to the drug concentration gradient of 0, 0.008, 0.016, 0.080, 0.400, 2, and 10 µmol / L. Drug sensitivity tests were performed, and three replicates were made for each concentration of each drug, with positive and negative control groups set up respectively.

[0049] (3) After 3 days, remove the culture medium (i.e., gastric cancer organoid culture medium or intestinal cancer organoid culture medium containing different drug concentrations), add 50µL of Advanced DMEM / F-12 culture medium, and then add 50µL of CellTiter-Glo® 3D cell viability detection reagent. Transfer to a multifunctional 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%. Draw a dose-effect curve.

[0050] The results showed that the colorectal cancer and gastric cancer organoids cultured in optimized culture medium showed different responses to drugs from different patients. In gastric cancer, the organoids from patient G001 showed higher sensitivity to fruquintinib, regorafenib, apatinib and sunitinib, and showed stronger resistance to sorafenib compared with the organoids from patient G002 ( Fig. 9 ); in colorectal cancer, organoids from patient C001 showed higher sensitivity to regorafenib and sorafenib, but stronger resistance to fruquintinib, apatinib, and sunitinib compared to organoids from patient C002 ( Fig.10 ).

[0051] In summary, the present invention optimizes the in vitro culture conditions of gastrointestinal tumor organoids by adding Chinese herbal extracts naringin or polydatin with antioxidant activity to the gastrointestinal tumor organoid culture system, so that the gastrointestinal tumor organoids have a higher survival rate and stronger proliferation ability, and provide a good in vitro research model for subsequent anti-tumor treatment.

[0052] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A gastrointestinal tumor organoid culture system, characterized in that: The invention comprises a basic culture medium and a Chinese herbal medicine extract with antioxidant activity; the Chinese herbal medicine extract with antioxidant activity is one or both of polydatin and naringin.

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 the basic culture 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.

4. The culture system according to claim 2, characterized in that: When the basal culture medium is used to culture intestinal cancer organoids, the final concentration of Wnt-3a 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 antioxidant active Chinese herbal medicine extract is added to the basic culture medium at a final concentration of 500 ng / mL.

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

7. 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 6.

8. An application of a gastrointestinal tumor organoid culture system, characterized in that: Small molecule targeted anti-tumor drugs are screened using the culture system described in any one of claims 1 to 6 and the gastrointestinal cancer organoids cultured therein.

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