Kit and method for constructing small intestine organoids and application of kit and method in evaluation of medicines affecting intestinal stem cells

Abstract

The invention belongs to the technical field of organoids, and particularly relates to a method for evaluating the influence of intervention measures such as drugs on the proliferation capacity of intestinal stem cells through proliferation indexes of the organoids and application of the method. The invention discloses a kit for efficiently, simply, conveniently and effectively culturing and constructing intestinal organs and evaluating the multiplication capacity of intestinal stem cells, which can better fit in-vivo tissues and cell microenvironments, as well as a method and application of the kit. The method can be used for detecting the influence of different intervention measures on the proliferation activity of the small intestine stem cells more accurately and more stably, and is beneficial to deeply researching and developing new drugs and researching the drug sensitivity and the drug action mechanism.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoids, and specifically includes a kit and method for constructing small intestinal organoids and their application in evaluating drugs affecting intestinal stem cells, especially a method and application for evaluating the influence of interventions such as drugs on the proliferation ability of intestinal stem cells through organoid proliferation indexes. Background Art

[0002] The intestine is an important and crucial organ in the human digestive system, responsible for the digestion and absorption of most nutrients in the human body. It is also the first line of defense against external toxic and harmful substances and an important immune organ of the human body. Imbalances in intestinal structure or intestinal epithelial homeostasis can lead to diseases such as diarrhea, bleeding, and infection. Corresponding abnormal states are usually caused by the loss or abnormal function of intestinal stem cells. Small intestinal stem cells are responsible for the normal tissue renewal of the small intestinal epithelium and tissue repair under abnormal conditions, and their proliferation ability is the basic condition for the normal function of small intestinal stem cells. After acute exposure to ionizing radiation, acute intestinal ischemia-reperfusion, or in the state of intestinal aging, the combined effects of multiple mechanisms such as increased ROS levels, aggravated damage to cellular DNA and protein macromolecules, or direct cell damage lead to the loss of small intestinal stem cells or a sharp decline in their proliferation ability, further leading to disorders of small intestinal epithelial homeostasis, destruction of intestinal structure, and obstacles to intestinal tissue repair, and then leading to digestive system diseases and even multi-system diseases. Therefore, evaluating the proliferation ability of small intestinal stem cells is a direct, effective, and fundamental evaluation method, and it is also the basic and necessary content for the study of the mechanisms and treatments of digestive system diseases.

[0003] Organoid technology is an advanced technology for biomedical research. It is a three-dimensional cell model cultured in the laboratory that is similar in structure and function to human organs. It is usually constructed from stem cells or tissue-specific cells, can simulate the physiological characteristics of real organs, and can achieve long-term stable subculture. Organoid technology has unique advantages. Compared with traditional two-dimensional culture models, organoids can simulate the tissue cell microenvironment and approach the state of cells and genomic expression in tissues to the greatest extent. Compared with animal models, organoid operation is simpler and more efficient, more conducive to high-throughput experiments, and can also largely avoid animal ethics issues. In addition, organoids can be cultured from the cells of patients to help doctors formulate personalized treatment plans according to the specific conditions of patients. Organoid technology has broad prospects in the field of biomedicine and is expected to bring major breakthroughs in disease research, drug development, and regenerative medicine.

[0004] Currently, the evaluation methods for intestinal tissue repair or the proliferative ability of intestinal stem cells mainly include the staining of proliferative molecular markers in tissue sections (such as Ki67 and PCNA immunohistochemistry), as well as cell cycle detection and molecular experiments (Western blot, RT-qPCR) after cell culture, and the detection of proliferative molecular markers, etc. However, all of the above methods have obvious limitations. For example, the tissue section staining method and molecular experiment method can only detect the expression status of molecular markers under specific spatio-temporal conditions, and due to the mixing of non-proliferative non-stem cell components in the tissue sections and tissue extraction samples, false positive and false negative results are likely to occur due to differences in experimental operations. Moreover, single-type cell culture has obvious defects because the cell line lacks representativeness and cannot directly simulate the state of small intestinal stem cells in the tissue. Therefore, there is an urgent need for a new method for effectively and efficiently detecting the proliferative ability of intestinal stem cells.

[0005] To solve the deficiencies of the prior art, the present invention discloses a kit for detecting the proliferative ability of intestinal stem cells, which is efficient, simple, effective and more compatible with the in vivo tissue and cell microenvironment. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to find a method and application for detecting the proliferative ability of intestinal stem cells that are efficient, simple, effective and more compatible with the in vivo tissue and cell microenvironment.

[0007] The present invention belongs to the technical field of organoids, especially a method and application for evaluating the influence of interventions such as drugs on the proliferative ability of intestinal stem cells through organoid proliferation indexes. The present invention discloses a kit, its method and use for efficiently, simply and effectively culturing and constructing intestinal organoids and evaluating drugs and the like for the proliferative ability of intestinal stem cells, which are more compatible with the in vivo tissue and cell microenvironment. Using the method of the present invention, the influence of different interventions on the proliferative activity of small intestinal stem cells can be detected more accurately and stably, which is helpful for more in-depth research and development of new drugs, research on drug sensitivity and drug action mechanisms.

[0008] The animal tissues related to the present invention can be the small intestine, colon, etc. of mammals, and the corresponding intestinal crypts are small intestinal crypts, colon crypts, etc., especially small intestinal crypts; the animal tissues contain stem cells, namely adult small intestinal stem cells, adult colon stem cells, etc. of mammals.

[0009] The kit method for evaluating the proliferative ability of intestinal stem cells based on intestinal organoids provided by the present invention is specifically as follows:

[0010] The present invention discloses an intestinal organoid kit, and the combined reagents in the kit include intestinal crypt extraction reagents, intestinal organoid culture reagents, and proliferative ability detection reagents, which specifically include:

[0011] R1: Intestinal crypt extraction reagent, including D-PBS, digestion medium, and washing medium.

[0012] R2: Intestinal organoid culture reagent, including organoid medium and Matrigel.

[0013] R3: Proliferation ability detection reagent, including detection primary antibody, detection secondary antibody, and chromogenic reagent.

[0014] Furthermore, in the R1 intestinal crypt extraction reagent, the main component of the digestion medium is EDTA; the main components of the washing medium are fetal bovine serum and Penicillin-Streptomycin;

[0015] In the R2 intestinal organoid culture reagent, the specific components of the organoid medium include DME / F12, L-Glutamin, HEPES, B27, N2, N-Acetyl-L-cysteine, Glutamax, Primocin, Mouse-EGF, Human R-spondin1, Mouse-noggin;

[0016] The main components of the R3 proliferation ability detection reagent are the primary antibodies against Ki67 and PCNA, the secondary antibody labeled with HRP, and the DAB chromogenic reagent.

[0017] The present invention also discloses the experimental method of the above intestinal organoid kit, as well as the method for evaluating the influence of interventions such as drugs on the proliferation ability of intestinal stem cells based on intestinal organoids. Specifically as follows:

[0018] S1: Take animal tissues, digest, and centrifuge to obtain crudely extracted intestinal crypts;

[0019] S2: Through centrifugation and filtration through a cell sieve, obtain purified intestinal crypts without debris, and perform counting;

[0020] S3: Resuspend the intestinal crypts in a Matrigel solution to obtain a Matrigel-crypt mixture, pipette evenly and plate it in a well plate to form a "Matrigel-organoid dome", and continue culturing after solidification and addition of the medium;

[0021] S4: Model establishment, drug administration, observe the growth of organoids, record the change in organoid size as proliferation index I;

[0022] S5: After model establishment and drug administration, collect the "Matrigel-organoid dome", section it to obtain organoid sections, and perform PCNA and Ki67 immunohistochemical staining as proliferation indices II and III.

[0023] Further, the above experimental method and the method for evaluating the influence of interventions such as drugs on the proliferation ability of intestinal stem cells based on intestinal organoids are as follows:

[0024] S1: Take fresh mouse tissues and perform digestion and centrifugation treatment by specific methods to obtain crudely extracted active intestinal crypts.

[0025] S2: Obtain debris-free active purified intestinal crypts through specific centrifugation and cell sieve filtration, and perform counting.

[0026] S3: Resuspend the intestinal crypts in a Matrigel solution to obtain a Matrigel-crypt mixture. After pipetting evenly, plate it in a well plate to form a "Matrigel-organoid dome". After solidification and addition of the culture medium, continue culturing.

[0027] S4: After 2 days of culture, perform intervention for modeling and drug administration, continuously observe the growth of organoids, and record the size change of organoids as proliferation index I.

[0028] S5: On the third day after modeling and drug administration, collect the "Matrigel-organoid dome", prepare frozen sections, and after obtaining the frozen sections of organoids, perform PCNA and Ki67 immunohistochemical staining as proliferation indices II and III.

[0029] Further, the method for evaluating the influence of interventions such as drugs on the proliferation ability of intestinal stem cells based on intestinal organoids according to the present invention:

[0030] The animal tissues in step S1 can be the small intestine, colon, etc. of mammals, and the corresponding intestinal crypts are: small intestinal crypts, colon crypts, etc., especially small intestinal crypts; the animal tissues contain stem cells, that is, adult small intestinal stem cells, adult colon stem cells, etc. of mammals. That is to say, the intestinal organoids in S1 are intestinal organoids constructed from adult small intestinal stem cells or adult colon stem cells of mammals.

[0031] For the crypt purification method by centrifugation in S2, the centrifuge speed is first 100×g for 1 min once and 50×g for 1 min multiple times.

[0032] For the crypt purification method using a cell sieve in S2, the sizes of the cell sieves are 100 μm and 40 μm.

[0033] Before the modeling treatment in S3, a pretreatment is also included. The pretreatment is to add drugs, etc. to a specific well plate. The control groups in S3 include a normal control group and a model control group.

[0034] The volume of the "Matrigel-organoid dome" structure in S3 is 20 μl. The well plate is selected as a 48-well plate with 1 "Matrigel-organoid dome" per well, or a 12-well plate with 3 "Matrigel-organoid domes" per well.

[0035] The standard growth curve plotted from the sizes of the organoids in the normal group in S4 is used as Proliferation Index I.

[0036] In S5, "Matrigel-organoid dome" enrichment is performed on the third day after modeling or drug administration treatment, and organoid sections are obtained in the form of frozen sections. In S5, when obtaining Proliferation Indexes II and III, they are obtained through PCNA and Ki67 immunohistochemical staining experiments.

[0037] The present invention provides a method and application for evaluating the influence of interventions such as drugs on the proliferation ability of intestinal stem cells based on intestinal organoids, including: using the aforementioned kit for the method of evaluating the influence or improvement of interventions such as drugs on the proliferation ability of intestinal stem cells, and further for the use in evaluating the intestinal protection or damage effects of interventions such as drugs based on intestinal organoids.

[0038] The present invention can be used for the evaluation of drugs that have direct or indirect effects on the intestine, including the evaluation of drugs that affect intestinal stem cells, such as in aspects of stem cell proliferation, reduction, intestinal protection, intestinal damage, etc. For example, it is applied to ionizing radiation (nuclear radiation, tumor radiation, etc.) and drugs for the treatment of ionizing radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Growth history of a single small intestinal organoid under ionizing radiation (proliferation inhibition) and treatment with drugs for treatment;

[0040] Figure 2 Change in growth area-time of small intestinal organoids under normal conditions;

[0041] Figure 3 Change in growth area-time of small intestinal organoids under ionizing radiation (proliferation inhibition);

[0042] Figure 4 Change in growth area-time of small intestinal organoids under treatment with drugs for the treatment of ionizing radiation;

[0043] Figure 5 Growth changes of small intestinal organoids under different treatment conditions (Proliferation Index I);

[0044] Figure 6 Growth curves of small intestinal organoids under different treatment conditions (Proliferation Index I);

[0045] Figure 7 Immunohistochemical staining of Ki67 and PCNA in frozen sections;

[0046] Figure 8 Quantitative analysis of Ki67 and PCNA immunohistochemical staining (proliferation indices II and III). Specific implementation manners

[0047] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the specific implementation manners of the present invention will be clearly and completely described below.

[0048] Example 1 Preparation of intestinal crypt extraction reagent

[0049] The intestinal crypt extraction reagent includes a digestion medium, a washing medium, and D-PBS, where:[[]]

[0050] Digestion medium: 2 mM EDTA, diluted and prepared with D-PBS;

[0051] Washing medium: 1% double antibody (penicillin + streptomycin), 10% fetal bovine serum (FBS), diluted and prepared with DPBS.

[0052] Example 2 Preparation of intestinal organoid culture reagent

[0053] The intestinal organoid culture reagent includes Matrigel that plays a supporting role during the formation process and an organoid culture medium that plays a nutritional support role during the formation process. Among them, the organoid culture medium is prepared as follows:

[0054]

[0055]

[0056] Example 3 Preparation of proliferation ability detection reagent (including but not limited to Ki67 and PCNA)

[0057] The proliferation ability detection reagent includes conventional reagents for frozen sections and immunohistochemical reagents. Among them, the immunohistochemical reagents include:

[0058] ① Antigen retrieval solution (heat retrieval): 10 mM sodium citrate, 0.05% Tween 20, pH 6.0, diluted and prepared with ddH2O, and the pH is adjusted with HCl;

[0059] ② Membrane-breaking solution: 0.5% Triton-X100, diluted and prepared with ddH2O;

[0060] ③ Endogenous peroxidase blocking solution: 3% hydrogen peroxide, diluted and prepared with ddH2O;

[0061] ④ Nonspecific antigen blocking solution: 3% BSA, diluted and prepared with PBS;

[0062] ⑤ Detection of antigen primary antibody: Ki67 and PCNA primary antibodies (rabbit source), diluted and prepared with PBS at a dilution ratio of 1:100;

[0063] ⑥ Detection of antigen secondary antibody: Goat anti-rabbit secondary antibody, diluted and prepared with PBS at a dilution ratio of 1:100;

[0064] ⑦ Detection chromogenic reagent: 5 mg of DAB (3,3'-diaminobenzidine), 10 μL of 30% hydrogen peroxide, made up to 10 mL with PBS.

[0065] Example 4 Crude extraction of small intestinal crypts

[0066] S1: Take fresh mouse tissues, perform digestion and centrifugation by specific methods to obtain crude active small intestinal crypts. Specifically, after sacrificing the mouse, dissect the abdomen, cut a 10-cm small intestine starting from 2 cm below the stomach, and transfer it to a sterile and low-temperature condition for operation. Carefully remove the attached blood vessels, omentum, and fat of the intestine, and then rinse the intestinal lumen with D-PBS pre-cooled at 4°C to remove intestinal contents. Subsequently, turn the small intestine inside out to expose the small intestinal epithelium, gently scrape the villus part of the small intestine with a glass slide to expose the small intestinal crypts. Then place the small intestine into D-PBS (containing double antibodies) pre-cooled at 4°C and rinse until the rinse solution is clear. After rinsing, transfer the small intestine to the digestion medium, lyse it on ice for 30 min, and then perform a water bath at 37°C for 5 min. Subsequently, place the digested small intestinal tissue into the washing medium, shake and mix well, and filter it through a 100-μm filter membrane to obtain active small intestinal crypts in the liquid phase.

[0067] Example 5 Purification of small intestinal crypts

[0068] S2: Through centrifugation under specific conditions and filtration through a cell sieve, obtain debris-free active purified small intestinal crypts and perform counting. Specifically, first, centrifuge the small intestinal crypt suspension at 100×g for 1 min, discard the supernatant, resuspend it with 5 ml of washing medium, and then centrifuge at 100×g for 1 min and resuspend 3 times. Subsequently, intercept the debris-free active purified small intestinal crypts through a 40-μm cell sieve, backwash the filter membrane with 1 ml of washing medium to prepare an active purified small intestinal crypt suspension. After counting under a microscope, centrifuge at 100×g for 1 min and discard the supernatant to obtain purified small intestinal crypts.

[0069] Example 6 Matrigel-organoid domes

[0070] S3: Add the matrix gel solution to the small intestinal crypts and resuspend them to obtain a Matrigel-crypt mixture. After blowing and beating evenly, spread it on the well plate to make a "Matrigel-organoid vault". After solidification and the addition of culture medium, continue to culture. Specifically, add an appropriate volume of Matrigel (such as 20ul / single well of a 48-well plate) according to the amount of precipitation, and mix it with a pipette tip pre-cooled at -20℃ to avoid bubbles. Pipette 20μl of the matrix gel crypt suspension and add it to the center of each well of the 48-well plate preheated at 37℃ in advance, and invert it in the incubator for 10 minutes to solidify. After solidification, add 400ul R2 small intestinal organoid culture medium to each well.

[0071] Example 7 Modeling and Drug Administration

[0072] S4: After 2 days of culture, intervention was performed to establish the model and administer the drug, and the growth of the organoids was continuously observed, and the changes in organoid size were recorded as proliferation indicators I. Specifically, the medium was changed every 2 days, and the drug administration could be continued during the process according to the experimental conditions.

[0073] Example 8 Immunohistochemistry

[0074] S5: On the third day after modeling and drug treatment, the "Matrigel-organoid vault" was collected for frozen section preparation. After obtaining the organoid frozen sections, PCNA and Ki67 immunohistochemical staining were performed as proliferation indicators II and III. Specifically, the R2 small intestinal organoid culture medium was first removed, D-PBS was washed once, and 4% paraformaldehyde was added to fix at 4°C for 30 minutes. After fixation, D-PBS was washed twice, and the Matrigel-organoid vault was carefully scraped with a cell scraper, collected in the mold and frozen sections were made. After the frozen sections were warmed up at room temperature for 1 hour, they were washed 3 times with PBS and then antigen repair was performed. After repair, 0.5% Triton-X100 was used for membrane permeabilization, 3% hydrogen peroxide solution was used for endogenous peroxide blocking after PBS washing, and 3% BSA was used for nonspecific antigen blocking after PBS washing. The cells were incubated at room temperature for 1 hour, and then the blocking solution was removed. The primary antibody of Ki67 or PCNA was incubated at 4°C overnight. The cells were warmed up for 1 hour on the second day, and the secondary antibody was incubated at room temperature for 1 hour after PBS washing. After washing the secondary antibody, conventional DAB color development was performed to obtain proliferation indicators II and III.

[0075] Example 9 Evaluation of the effects of ionizing radiation and ionizing radiation treatment drugs on the proliferation activity of small intestinal stem cells

[0076] S1: Fresh mouse intestinal tissue (10 cm of small intestine cut from 2 cm below the stomach) was collected and digested and centrifuged using a specific method to obtain crudely extracted active small intestinal crypts.

[0077] S2: Through centrifugation under specific conditions and cell mesh filtration, the fragmented and active purified small intestinal crypts are obtained and counted.

[0078] S3: Resuspend the small intestinal crypts in 200 μl of Matrigel solution to obtain a "Matrigel-crypt mixture". After pipetting evenly with a pre-chilled pipette tip at -20°C, add 20 μl of the "Matrigel-crypt mixture" to each well of a 48-well plate (preheated to 37°C) to prepare a "Matrigel-organoid dome". After solidifying the dome at 37°C for 10 min, add 400 μl of R2 small intestinal organoid medium to each well and culture under the conditions of 37°C and 5% carbon dioxide.

[0079] S4: On the 3rd day of culture, irradiate with 6 Gy of γ-rays (0.80 Gy / min). Administer the positive rescue drug once 1 h before irradiation, continuously observe the growth of the organoids, record the changes in the size of the organoids, and obtain proliferation index I ( Figures 1 - 5 ).

[0080] S5: Collect the "Matrigel-organoid dome" on the third day after modeling and drug administration, prepare frozen sections. After obtaining the frozen sections of the organoids, perform PCNA and Ki67 immunohistochemical staining as proliferation indices II and III. Specifically, first remove the R2 small intestinal organoid medium, wash once with D-PBS, add 4% paraformaldehyde, and fix at 4°C for 30 min. After fixation, wash twice with D-PBS, carefully scrape the Matrigel-organoid dome with a cell scraper, collect it into a mold, and make frozen sections. After the frozen sections are rewarmed at room temperature for 1 h, wash three times with PBS and then perform antigen repair. After repair, permeabilize with 0.5% Triton-X100, wash with PBS, block endogenous peroxidase with 3% hydrogen peroxide solution, wash with PBS, block non-specific antigens with 3% BSA, incubate at room temperature for 1 h, then remove the blocking solution, incubate with Ki67 or PCNA primary antibody at 4°C overnight. On the next day, rewarm for 1 h, wash with PBS, and then incubate with the secondary antibody at room temperature for 1 h. After washing the secondary antibody, develop color with conventional DAB to obtain proliferation indices II and III ( Figures 6 - 7 ).

[0081] As shown in Example 9, Figure 1 , Figure 3 , Figure 5 and Figure 6 , 6 Gy of γ-ray ionizing radiation can significantly reduce the proliferation activity of small intestinal stem cells and affect the normal growth state of small intestinal organoids. The proliferation index I of this kit in the ionizing radiation modeling group is significantly different from that of the CON group, which is in line with the academic consensus.

[0082] As shown in Example 9, Figure 1 , Figure 2 , Figure 4 , Figure 5 and Figure 6As can be seen, the ionizing radiation treatment drug can significantly alleviate the growth damage of small intestinal organoids caused by 6 Gy γ-ray ionizing radiation and rescue the proliferative vitality of small intestinal stem cells. The proliferation index I of this kit in the positive drug group is close to that of the CON group and is significantly different from that of the ionizing radiation group, which is consistent with our previous data and the academic consensus.

[0083] As can be seen from Example 9, Figure 7 and Figure 8 ionizing radiation significantly reduces the proliferative activity of small intestinal stem cells, while the positive drug for ionizing radiation treatment shows a significant restorative effect on proliferative activity. The results of proliferation index II and proliferation index III of this kit corresponding thereto are consistent with those of proliferation index I.

[0084] In summary, the content of the present invention is not limited to the embodiments. Those skilled in the art in the same field can easily propose other embodiments within the technical guiding ideology of the present invention, but such embodiments are all included within the scope of the present invention.

[0085] It should be understood that for those of ordinary skill in the art, certain improvements or transformations can be made according to the above R1-R3, S1-S5, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. An intestinal organoid kit, comprising an intestinal crypt extraction reagent, an intestinal organoid culture reagent, and a proliferation ability detection reagent, wherein: The composition of the intestinal crypt extraction reagent includes: D-PBS, digestion medium, and washing medium; The composition of the intestinal organoid culture reagent includes: organoid medium, Matrigel; The composition of the proliferation ability detection reagent includes: detection primary antibody, detection secondary antibody, and chromogenic reagent.

2. The intestinal organoid kit according to claim 1, wherein: For the proliferation ability detection reagent, wherein: the detection primary antibody includes but is not limited to Ki67 and PCNA; the detection secondary antibody is HRP-labeled; the chromogenic reagent is DAB chromogenic reagent.

3. A method for evaluating a drug's effect on the proliferation ability of intestinal stem cells according to the intestinal organoid kit described in any one of claims 1 to 2, comprising: S1: Take animal tissue, digest it, and centrifuge to obtain crudely extracted intestinal crypts; S2: Through centrifugation and filtration through a cell sieve, obtain purified intestinal crypts free of debris, and perform counting; S3: Resuspend the intestinal crypts in a Matrigel solution to obtain a Matrigel-crypt mixture, pipette evenly and plate it in a well plate to form a "Matrigel-organoid dome", and continue culturing after solidification and addition of the medium; S4: Establish a model, administer the drug, observe the growth of the organoids, and record the change in the size of the organoids as proliferation index I; S5: After the model establishment and drug administration, collect the "Matrigel-organoid dome", section it to obtain organoid sections, and perform immunohistochemical staining with PCNA and Ki67 as representatives as proliferation indices II and III.

4. The drug evaluation method according to claim 3, characterized in that: The animal tissue in step S1 is: any one or a combination of several of the small intestine and colon of mammals.

5. The drug evaluation method according to claim 4, wherein: The animal tissue in step S1 contains stem cells, and the stem cells are adult small intestine stem cells and adult colon stem cells of mammals.

6. The drug evaluation method according to claim 3, wherein: The intestinal crypts in step S1 are: small intestine crypts and colon crypts.

7. According to the drug evaluation method described in claim 3, wherein: For the centrifugation in S2, the centrifuge speed is first 100×g for 1 min once, 50×g for 1 min once or more; For the cell sieve in S2, the size of the cell sieve is: 100μm and 40μm.

8. According to the drug evaluation method described in claim 3, wherein: Before the model establishment treatment in S3, it also includes a pretreatment, which is to add a drug, etc. to a specific well plate; The structure volume of the "Matrigel-organoid dome" in S3 is 20 - 100 μl, and the well plate is selected from: 1 "Matrigel-organoid dome" per well in a 48-well plate or 3 "Matrigel-organoid domes" per well in a 12-well plate.

9. According to the drug evaluation method described in claim 3, wherein: The standard growth curve plotted from the size of the organoids in the normal group in S4 is used as proliferation index I.

10. According to the drug evaluation method described in claim 3, wherein: In S5, on the third day after the model establishment and drug administration, the "Matrigel-organoid dome" is enriched, and the organoid sections are obtained in the form of frozen sections; When obtaining proliferation indexes II and III in S5, they are obtained through PCNA and Ki67 immunohistochemical staining experiments, but are not limited to these two indexes.

11. Use of the kit according to any one of claims 1-2 in evaluating drugs affecting the intestine.

12. Use of the kit according to claim 11 in evaluating drugs affecting the proliferation of intestinal stem cells.

13. Use of the kit according to any one of claims 1-2 in evaluating ionizing radiation and drugs for treating ionizing radiation.

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