Intestinal organ culture system and application thereof in combined stem cell regeneration therapy

Abstract

The invention discloses an intestinal organoid culture system and application thereof in a combined stem cell regeneration therapy, and the intestinal mucosal injury is repaired by utilizing the regeneration capacity of intestinal organoids cultured in vitro and crypt stem cells, so that intestinal diseases are relieved and treated. The intestinal organoids and the combined stem cells have obvious treatment and repair effects on ulcerative colitis of mice, and the combined stem cells of the small intestine organoids and the crypts have better effects on repair of intestinal inflammation damage than pure organoids. Therefore, the intestinal tracts and the combined stem cells have good effects and application prospects in preparation of medicines for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the culture and application of intestinal stem cells, and in particular to an intestinal organoid culture system and its application in combined stem cell regenerative therapy. Background Art

[0002] In recent years, the development of intestinal organoid technology has provided new tools for studying the function and fate of intestinal stem cells, evaluating the repair effect of therapeutic measures on intestinal epithelial cells, and exploring new therapeutic strategies for intestinal diseases. Intestinal organoids are micro-organs constructed by intestinal stem cells or crypts in a specific three-dimensional environment (such as culture matrix gel). Their appearance is similar to intestinal tissue and contains all mature cell types in the intestine, such as enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. They are highly similar to intestinal tissue in vivo in structure and function. Compared with traditional two-dimensional cell culture, intestinal organoids can better simulate the structure and function of intestinal tissue, including the spatial distribution of cells, interactions between cells, and interactions with the surrounding environment. In addition, intestinal organoids can be cultured and passaged in vitro for a long time, with high genetic stability and strong plasticity, and can be used to study the mechanisms of genetic diseases and cell differentiation as well as drug screening and evaluation. Therefore, intestinal organoids have great potential in intestinal disease research, drug development, and precision medicine, and are expected to be more widely used in basic medicine and clinical medicine in the future.

[0003] Intestinal stem cell therapy is a new treatment for intestinal diseases with the potential to regulate immune responses and promote tissue regeneration. Previous studies have shown that intestinal stem cells can selectively migrate to damaged tissues and inflammatory sites, promote regeneration and angiogenesis of damaged intestinal epithelium, improve and regulate intestinal inflammation, and rebuild intestinal barriers and immune tolerance. Combination stem cell therapy refers to the combined use of different types of stem cells to achieve better therapeutic effects. For example, the combined use of intestinal stem cells and mesenchymal stem cells can promote the repair and regeneration of intestinal epithelial cells. Different stem cells have different stem differentiation potentials and tissue repair capabilities. Combination stem cell therapy can integrate different stem cell treatment advantages, so it has broad application prospects in the treatment of intestinal diseases, such as inflammatory bowel disease, intestinal tumors, intestinal fibrosis stenosis, and short bowel syndrome. However, there is currently a lack of an efficient intestinal organoid regeneration culture system, which cannot meet the needs of combined stem cell therapy for intestinal stem cells. Therefore, it is of great scientific significance and application value to develop an efficient intestinal organoid regeneration culture system and apply it to combined stem cell therapy.

[0004] Combination stem cell therapy, as the future direction for the treatment of intestinal diseases, shows great potential, but it still faces many challenges and requires further improvement and innovation. In terms of stem cell culture and differentiation, the efficiency of stem cells differentiating into specific cell types (such as intestinal epithelial cells) needs to be improved to ensure a sufficient number of cells for treatment. Existing culture methods rely on complex combinations of culture media and growth factors, and require long-term culture, resulting in high costs, cumbersome operations, and difficulty in large-scale production for clinical treatment. This limits the scope of application and accessibility of combination stem cell therapy. In addition, there is heterogeneity between stem cell populations, and different stem cells may exhibit different fates during differentiation, which may affect the effectiveness of treatment. Heterogeneity may cause transplanted cells to be unable to effectively repair damaged intestinal tissue, or cause tissue dysfunction.

[0005] Traditional stem cell therapy usually injects stem cells into the patient's body, but due to the lack of precise targeting methods, it is difficult for cells to accurately locate the damaged area and reach the target tissue or organ, resulting in low transplant efficiency and poor treatment effect, which limits its application in repairing specific tissue damage. For example, in diseases such as Crohn's disease or ulcerative colitis, the damaged areas may be distributed in different parts of different intestinal segments, and cells need to be accurately transplanted to these areas to effectively repair the damaged tissue.

[0006] In existing stem cell therapies, immunosuppressants are commonly used auxiliary drugs, but they have large side effects, which can lead to decreased immunity, increased risk of infection, and may affect the functions of other organs. At the same time, long-term use of immunosuppressants may cause side effects such as opportunistic infections and tumorigenesis, which limits their safety. In addition, growth factors play an important role in promoting stem cell proliferation and differentiation, but their efficacy is affected by cell type and microenvironment, and it is difficult to guarantee that they are effective for all patients, and it is difficult to be widely popularized. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to construct an intestinal organoid. Another purpose of the present invention is to provide a combined stem cell comprising an intestinal organoid and a crypt. Another purpose of the present invention is to provide the use of the intestinal organoid and the combined stem cell in the preparation of a drug for ulcerative colitis.

[0008] Technical solution: Application of small intestinal crypts and / or intestinal organoids in the preparation of drugs for the treatment of ulcerative colitis.

[0009] In the application, the drug is an enteral drug.

[0010] The method for preparing a suspension of small intestinal crypts and intestinal organoids comprises the following steps:

[0011] (1) Remove the outer membrane, blood vessels and fat from the small intestine of the rodent near the stomach, and flush out the feces and intestinal contents;

[0012] (2) Place the treated intestine in a buffer solution at 2-8°C and repeatedly blow and rinse to remove impurities;

[0013] (3) The cleaned intestine is cut longitudinally and cut into small segments of 1-5 mm;

[0014] (4) Add a mild cell dissociation reagent to the small intestinal segment, shake well, and separate the small intestinal crypts;

[0015] (5) Resuspend the tissue fragments in a 2-8°C buffer containing BSA and pipette up and down; let stand until most of the intestinal tissue fragments settle to the bottom, pour out the supernatant, and repeat the steps until the supernatant is clear;

[0016] (6) adding 2-8°C buffer containing BSA to resuspend the intestinal segments, filtering the cell suspension using a 60-80 μm filter, and collecting the filtrate;

[0017] (7) Discard the filter, label the filtrate as “Fraction 1”, and store it on ice;

[0018] (8) Repeat steps (6)-(7) three times to obtain fractions 2-4 respectively; collect fractions 3 and 4;

[0019] (9) After centrifuging fraction 3 and / or fraction 4, add the fraction to a 2-8° C. buffer containing BSA and resuspend the fraction to obtain a crypt suspension; or mix fraction 3 and / or fraction 4 with matrix gel to prepare a mixed solution;

[0020] (10) Add the mixed solution dropwise to the well plate to form a 3D culture system matrix gel ball. After the ball solidifies, add the organoid growth medium;

[0021] (11) The well plate is placed in an incubator for organoid culture to obtain an intestinal organoid suspension.

[0022] In the method, the rodent is selected from mice. Preferably, the rodent is a mouse.

[0023] In the method, the 2-8°C buffer is a 2-8°C PBS buffer.

[0024] In the method, the BSA content in the 2-8° C. buffer containing BSA is 0.05-0.15% by volume.

[0025] An intestinal organoid regeneration system drug is an intestinal administration preparation prepared by adding pharmaceutically acceptable excipients to intestinal organoids and / or small intestinal crypts.

[0026] A combination of stem cells that combines small intestinal crypts and intestinal organoids.

[0027] The application of the combined stem cells in the preparation of medicine for treating ulcerative colitis.

[0028] Application of small intestinal crypts and intestinal organoids in the preparation of drugs for the treatment of ulcerative colitis.

[0029] A method for establishing an intestinal organoid regeneration system comprises the following steps:

[0030] (1) Remove the outer membrane, blood vessels and fat from the small intestine of the rodent near the stomach, and flush out the feces and intestinal contents;

[0031] (2) Place the treated intestine in a buffer solution at 2-8°C and repeatedly blow and rinse to remove impurities;

[0032] (3) The cleaned intestine is cut longitudinally and cut into small segments of 1-5 mm;

[0033] (4) Add a mild cell dissociation reagent to the small intestinal segment, shake well, and separate the intestinal crypts;

[0034] (5) Resuspend the tissue fragments in a 2-8°C buffer containing BSA and pipette up and down; let stand until most of the intestinal tissue fragments settle to the bottom, pour out the supernatant, and repeat the steps until the supernatant is clear;

[0035] (6) adding 2-8°C buffer containing BSA to resuspend the intestinal segments, filtering the cell suspension using a 60-80 μm filter, and collecting the filtrate;

[0036] (7) Discard the filter, label the filtrate as “Fraction 1”, and store it on ice;

[0037] (8) Repeat steps (6)-(7) three times to obtain fractions 2-4 respectively; collect fractions 3 and 4;

[0038] (9) After centrifuging fraction 3 and / or fraction 4, add the fraction to a 2-8° C. buffer containing BSA and resuspend the fraction to obtain a crypt suspension; or mix fraction 3 and / or fraction 4 with matrix gel to prepare a mixed solution;

[0039] (10) Add the mixed solution dropwise to the well plate to form a 3D culture system matrix gel ball. After the ball solidifies, add the organoid growth medium;

[0040] (11) The well plate is placed in an incubator for organoid culture to obtain an intestinal organoid suspension.

[0041] Further preferably, the present invention develops a mouse intestinal organoid regeneration culture system including the preparation of organoid crypt suspension and organoid enema administration. The establishment of the intestinal organoid regeneration culture system includes the following steps:

[0042] 1. Culture Methods of Intestinal Organoids

[0043] S1: The mice were killed by cervical dislocation.

[0044] S2: Open the abdomen and chest cavity, and use surgical scissors to remove about 15 cm of small intestine at the end close to the stomach.

[0045] S3: Use pointed forceps to remove the membrane, blood vessels and fat on the outside of the intestine and flush out the feces and intestinal contents.

[0046] S4: Place the treated intestine in ice-cold (2-8°C) PBS and repeatedly blow and rinse to remove impurities.

[0047] S5: The cleaned intestine was cut longitudinally and cut into 2 mm segments.

[0048] S6: At room temperature, add Gentle Cell Dissociation Reagent (StemCell) to the small intestinal segment and shake gently to separate the intestinal crypts.

[0049] S7: Resuspend the tissue fragments in 10 mL of cold (2-8°C) PBS containing 0.1% BSA and pipette up and down three times. Let stand until most of the intestinal tissue fragments settle to the bottom (about 30 seconds), pour out the supernatant, and repeat step S7 15-20 times until the supernatant is clear.

[0050] S8: Add 10 mL of cold (2-8°C) PBS containing 0.1% BSA to resuspend the intestinal segment, filter the cell suspension using a 70 μm filter, and collect the filtrate in a clean 50 mL conical tube.

[0051] S9: Discard the filter, label the filtrate as "Fraction 1", and store on ice.

[0052] S10: Repeat S8-S9 three times to obtain fractions 2-4, respectively.

[0053] S11: Observe and evaluate each fraction under a microscope. Usually, fractions 3 and 4 contain more crypts suitable for culture.

[0054] S12: Fraction 3 and fraction 4 were combined, and Matrigel (Corning) and the fraction were mixed in a ratio (1:1.2) according to the amount of 500 crypts / well to prepare a mixed solution.

[0055] S13: Add the mixed solution to the center of the 24-well plate (60 μL / well) to form a 3D culture system Matrigel ball. After the ball solidifies, add the full IntestiCult organoid growth medium (800 μL / well, Stem Cell).

[0056] S14: Place the 24-well plate in an incubator for organoid culture, change the culture medium every 3 days, and subculture every 6 days.

[0057] 2. Isolation of Small Intestinal Crypts

[0058] Consistent with the above-mentioned steps for isolating intestinal organoids (Method 1: Culture method of intestinal organoids, S1-S10), fractions 3 and 4 were collected and combined. After centrifugation, cold (2-8°C) PBS containing 0.1% BSA was added to resuspend the crypts in the fractions. The morphology of the crypts was observed under a microscope and counted (note that the crypts obtained in this step need to be used immediately).

[0059] 3. Preparation of Organoid and Crypt Suspensions

[0060] S1: 1.5 hours before the planned infusion, add 5.0 mL of 0.1% BSA / PBS solution (0.1% (wt / vol)) into a 15 mL centrifuge tube.

[0061] S2: Collect the organoid suspension cultured in method 1 and the crypt suspension obtained in method 2, mix them in a 1:1 volume ratio (prepare 500 organoids and 500 crypts for each recipient mouse), and add them to the centrifuge tube prepared in step S1.

[0062] S3: The suspension was centrifuged at 500 g for 3 minutes, and the supernatant was discarded. The suspension was washed with 2.0 mL of 0.1% BSA / PBS solution, and mixed by pipetting, and centrifuged again and the supernatant was discarded.

[0063] S4: Resuspend the pellet in 200 μL of Matrigel / PBS solution (5% (vol / vol)), transfer to a wetted 1.5 mL EP tube, and store on ice until use (usually within 1 hour).

[0064] 4. Infusion method of intestinal combined stem cell therapy

[0065] S1: An appropriate amount of Avertin solution was injected intraperitoneally to anesthetize the mice (the mice were fasted for 12 hours before administration).

[0066] S2: Connect the rectal catheter to a 1 mL syringe and slowly draw up 200 μL of crypt / organoid suspension.

[0067] S3: Dip the catheter into the lubricant PEG400 and insert it smoothly into the mouse anus to a depth of about 3-5 cm.

[0068] S4: After slowly injecting the organoid / crypt suspension, invert the mouse with the anus facing upward for about 1 minute and apply medical gel to the skin at the anus to seal the anus.

[0069] S5: Appropriate dose of Avertin: Generally, the dose range of Avertin is 200-250 mg / kg. The actual dose will be adjusted according to the mouse species, age, gender, weight and experimental requirements.

[0070] 5. Evaluation of the repair effect of intestinal organoid regeneration system on intestinal damage:

[0071] S1: At the end of the experiment, recipient mice were sacrificed and the colon was removed intact.

[0072] S2: Rinse the colon contents using cold PBS.

[0073] S3: The colon is incised longitudinally opposite the mesentery attachment.

[0074] S4: Flush the colon thoroughly with PBS.

[0075] S5: Place the opened colon in a 10 cm dish with the epithelium facing up.

[0076] S6: Observe the colon under a fluorescent stereomicroscope and identify the site containing the graft.

[0077] S7: Place the colon section containing the graft on the culture dish, avoiding wrinkles or twists.

[0078] S8: Wrap the colon tissue in another culture dish.

[0079] S9: Fix the tissue in 4% paraformaldehyde overnight to preserve endogenous fluorescence.

[0080] S10: The fixed tissue was immersed in 20% sucrose / PBS solution overnight for cryopreservation and subsequent pathological analysis.

[0081] S11: Based on the measured distance, the number of slices required to be cut is calculated to reach the site containing the transplant.

[0082] S12: Use a freezing microtome to slice at a thickness of 30 μm until the site containing the graft is reached.

[0083] S13: Adjust the slice thickness to 8 μm and continue slicing.

[0084] S14: Every 10th section was examined for the presence of grafts using a fluorescence microscope.

[0085] S15: The sections containing the grafts were subjected to pathological analysis.

[0086] The present invention uses EGFP fluorescent mice (B6-H11-CAG-EGFP) to extract ileal crypts and culture small intestinal organoids, and uses C57 mice to establish a dextran sulfate sodium (2.5% DSS)-induced ulcerative colitis model, and administers organoids or organoids plus crypt suspensions rectal administration by enema, and observes the tissue repair effect and clinical indication changes of pathologically damaged parts of mice by fluorescence tracing and pathological analysis. By comparing the index data of colorectal length, body weight, disease activity index, pathological sections and fluorescent sections of mice in different groups, the repair effect of small intestinal organoids and crypts on ulcerative colitis damage is evaluated and compared.

[0087] The key technical points of the present invention are: 1. Extraction of intestinal stem cells (crypts) and culture of organoids; 2. Preparation of organoid (crypt) suspension; 3. Establishment of rectal administration method; 4. Establishment of intestinal organoid regeneration system. 5. Evaluation of the repair effect of intestinal organoid regeneration system on ulcerative colitis damage.

[0088] The present invention utilizes the regenerative capacity of intestinal organoids and crypt stem cells cultured in vitro to repair intestinal mucosal damage, thereby alleviating and treating intestinal diseases. The therapy achieves the purpose of alleviating and treating damaging intestinal inflammation by infusing intestinal organoids or combined stem cells (organoids + crypt stem cells) separated and cultured in vitro into the damaged intestinal part of mice with induced enteritis, allowing them to integrate with the damaged intestinal epithelium, repair and restore intestinal function.

[0089] Beneficial effects: (1) The present invention shows that, compared with the control group, the DSS-induced mouse enteritis group showed pathological manifestations such as weight loss, indicating that the DSS-induced enteritis model was successful. Compared with the DSS enteritis untreated group, after stem cell transplantation by rectal administration, the weight loss of mice in the organoid group (Crypt) and the combined stem cell group (Crypt+Organoid) was significantly slowed down, the weight recovery in the recovery period was faster, the degree of increase in the disease activity index was reduced, the degree of shortening of the colorectal length was reduced, and the pathological sections showed that the degree of inflammation was reduced. Compared with the organoid administration group, the combined stem cell administration group had more obvious damage repair for enteritis. (2) The results of GFP fluorescence color development and H&E stained tissue sections further confirmed that the transplanted crypt fragments successfully entered the mouse intestine and reached the damaged parts of the ileum and colon, effectively participating in and promoting the repair of the damaged wound surface. The above invention shows that both intestinal organoid transplantation and combined stem cell transplantation have a more significant therapeutic and repair effect on ulcerative colitis in mice, and the combined stem cell therapy of ileal organoids and colon crypts is superior to simple organoid transplantation in repairing intestinal inflammatory damage. This indicates that the combined stem cell transplantation technology of the present invention has good effects and application prospects in the repair and regeneration process of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Diagram of the strategy for DSS-induced ulcerative colitis and organoid combined stem cell transplantation;

[0091] Figure 2 Record the growth status of organoids from Day 0 to Day 7;

[0092] Figure 3 The changes in body weight, disease activity index, and colorectal length pathological indicators of mice after DSS-induced colitis and organoid combined stem cell transplantation;

[0093] Figure 4 H&E and AB-PAS staining of the colon of mice in each group;

[0094] Figure 5 Fluorescent mouse crypts and isolated and cultured organoids under fluorescent and normal fields of view;

[0095] Figure 6 Fluorescence staining of ileum and colon tissue sections after transplantation of fluorescent murine organoids and crypts into enteritis mice. DETAILED DESCRIPTION

[0096] Example 1 Crypt extraction and organoid culture of the present invention

[0097] After 2 EGFP mice and 2 C57 mice were killed by cervical dislocation (all animals were SPF grade), a small intestine of about 15 cm in length was taken from the end close to the stomach. Use pointed forceps to remove the membrane, blood vessels and fat on the outside of the intestine. After repeated blowing and rinsing with cold PBS, cut it into small segments of about 2 mm, and use a mild cell dissociation reagent GCDR to separate the mouse intestinal crypts at room temperature. After blowing with a PBS solution containing 0.1% BSA, let it stand and settle, filter with a 70μm filter, and collect the filtrate in a clean 50mL conical tube. Discard the filter and mark the filtrate as "Fraction 1". Place the fraction on ice. Repeat this step three times to obtain fractions 2-4.

[0098] Fractions with a high proportion of villi, single cells, and debris are not suitable for organoid culture. Usually, fractions 3 and 4 are usually enriched with more crypts suitable for subsequent culture. At the same time, we combined fractions 3 and 4, mixed them with Matrigel in proportion (usually 1:1, but considering that some 0.1% BSA solution cannot be completely removed during the extraction process, a 1:1.2 ratio with a higher Matrigel content is used, so that the solidified structure of the Matrigel can be more stable and not easy to split and fragment), and made a 3D three-dimensional culture system ball in the center of the 24-well plate. This saves culture medium while ensuring the number of organoids.

[0099] Day 0: Extract and separate crypts, which are arranged in long strips. Figure 2 , Day0;

[0100] Day 1: The crypts form a spherical shape. Figure 2 , Day1;

[0101] Day 2: The sphere begins to grow and small buds begin to grow, and intestinal organoids are formed. Figure 2 , Day2;

[0102] Day 3: During the growth and differentiation process, the organoids sprout more and longer, and begin to stack together. Figure 2 , Day3;

[0103] Day 5: Organoid buds become longer and more numerous, the organoids further grow larger, and metabolites are excreted from the cell body. Figure 2 , Day5;

[0104] Day 7: Organoids basically stop growing, metabolite excretion increases, cell bodies turn gray-black, and need to be passaged. Figure 2 , Day7;

[0105] Different from simple organoid transplantation, we explored mixing the directly extracted crypts with cultured mature organoids, processing them into cell suspensions for subsequent stem cell combination transplantation injections.

[0106] Example 2 Establishment of ulcerative colitis disease model and stem cell rectal transplantation

[0107] 32 SPF grade C57 mice, 6-8 weeks old, weighing between 18-22g. Feeding conditions: indoor temperature 25-27℃, humidity 50%-70%, light and dark conditions 12h cycle each. In the SPF environment, clean the cages once a day, replace new bedding, add feed, and use boiled cold water to drink, ensuring that water and feed are freely available. The experiment was conducted after one week of adaptive training of mice. The 32 mice were divided into four groups, with 8 mice in each group. Except for the control group (CTL), the mice in other groups were given drinking water containing 2.5% DSS for 7 days. Organoids or combined stem cells were given to the treated mice starting from the fifth day of DSS treatment. Starting from the eighth day, the mice were given normal drinking water until the tenth day when they were killed and dissected. The colorectum was measured for length, and the colorectal tissue was collected for pathological examination. During this period, the mice were weighed every day, the viscosity of the feces was observed, and the feces of the mice were taken for occult blood. For strategies for DSS-induced ulcerative colitis and organoid combined stem cell transplantation, see Figure 1 The grouping of mice is shown in Table 1.

[0108] Table 1: Experimental grouping

[0109]

[0110] The weight changes of the DSS model group and the CTL group were presented in the form of a line graph. Figure 3 As shown, the weight of mice in the DSS group decreased significantly, and after being killed, it was found that the length of their colorectum was smaller than that of the CTL group, indicating that the DSS modeling was successful. After rectal stem cell transplantation on Day 5, the weight of mice in the organoid group and the combined stem cell group showed a slower weight loss, faster weight recovery during the remission period, a lower increase in the disease activity index, a lower shortening of the colorectal length, and pathological sections showed a reduction in the degree of inflammation. Moreover, compared with the organoid administration group, the combined stem cell administration group can more significantly reduce DSS-induced enteritis damage, and the damage repair effect on enteritis is more obvious. This suggests that combined stem cell transplantation is a potential therapy for intestinal inflammatory diseases.

[0111] Example 3 HE and AB-PAS staining

[0112] HE staining: Dewax the slices in xylene I and xylene II for 10 minutes, then use anhydrous ethanol, 95% ethanol, 80% ethanol, 75% ethanol for 5 minutes each, and wash with distilled water for 5 minutes. Add hematoxylin stain on the slices, and wash off the excess stain with tap water after 5 minutes. Use 1% hydrochloric acid ethanol to differentiate and wash off the excess stain in the cells. Add eosin stain and wash off the excess stain after 1 minute. Wash the slices with anhydrous ethanol 3 times, 5 minutes each time, and then soak in xylene 2 times, 5 minutes each time. Put the slices in a fume hood to dry first, then add neutral gum to seal, and do not produce bubbles. Take pictures under a microscope.

[0113] AB-PAS staining: paraffin sections are dewaxed to water: the sections are immersed in xylene for 10 minutes, xylene for 10 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 75% ethanol for 5 minutes, and then washed with tap water; Alcian blue staining: the tissue pen draws a circle around the tissue, and the reagent 1 in the Alcian blue-glycogen staining kit is evenly added to the tissue, and incubated at room temperature for 20 minutes. Wash with water for about 30 seconds; periodic acid oxidation: the reagent 2 in the Alcian blue-glycogen staining kit is evenly added to the tissue, and incubated at room temperature for 20 minutes. Wash with water for 2-3 minutes; Schiff's solution staining: prepare the reagent 3 Schiff's reagent working solution, evenly add it to the tissue, and incubate at room temperature for 5 minutes. Slowly rinse the slides with tap water for 5 minutes; hematoxylin counterstaining: put the slices into the hematoxylin solution, stain for 1 minute, wash with tap water to turn blue; dehydrate and transparently seal the slides: immerse the slices in anhydrous ethanol for 2 minutes-anhydrous ethanol for 2 minutes-anhydrous ethanol for 2 minutes-xylene for 5 minutes-xylene for 5 minutes. Take out the slices, drop an appropriate amount of neutral gum on the slices, and cover them with a glass slide; take pictures under a microscope.

[0114] The results of HE and AB-PAS staining of the colon pathology of mice after DSS-induced colitis and organoid combined with stem cell transplantation are shown in Figure 4 The results showed that compared with the DSS-untreated group, the pathological inflammation of the colon in the organoid group and the combined stem cell group was reduced, and the degree of intestinal mucosal barrier damage was reduced. Compared with the organoid group, the combined stem cell group showed better therapeutic effects.

[0115] Example 4 In vivo fluorescence colorimetry of crypts and organoids

[0116] After the crypts of EGPF fluorescent mice were extracted and isolated, they were directly observed in the living cell state. The cells were placed under a fluorescence microscope, a specific wavelength was used to excite EGFP (usually 488nm blue light), and then the emitted green fluorescence (usually 509nm) was observed through a filter. After a few days of culture, the fluorescence was still relatively strong, indicating that the fluorescence of the mice used as organoid donors was sustained and stable, and traceability could be observed. The results are as follows Figure 5 shown.

[0117] Example 5 Fluorescence staining of tissue sections

[0118] 2.5% DSS was used to induce enteritis in mice. After five days, the drinking water was changed to normal. The small intestinal crypts and organoids of fluorescent mice were transplanted. After the tenth day, the mice were killed, and the colorectal and ileal tissues of the mice were obtained. After frozen sections, fluorescence observation was performed. The fluorescence color development process of tissue sections includes the following steps: first, the tissue is fixed, dehydrated, sectioned, and antigen repaired, and then a blocking agent is used to prevent nonspecific binding. Next, DAPI nuclear staining is performed, and then the slides are sealed with an antifade agent to prevent photobleaching of the fluorescent signal. Finally, the images are observed and collected under a fluorescence microscope. Photobleaching must be avoided throughout the process, and the conditions of each step must be optimized to obtain the best fluorescent signal and image quality. See the results. Figure 6 After transplanting mouse crypts and organoids, some crypts and intestinal epithelium of the damaged intestine showed green fluorescent signals. This suggests that after intestinal organoids and crypts are transplanted into the mouse colon, they can spontaneously embed into the damaged parts of the colon and small intestine to repair the damage.

Claims

1. Application of small intestinal crypts and / or intestinal organoids in the preparation of drugs for the treatment of ulcerative colitis.

2. The use according to claim 1, characterized in that: The drug is an enteral drug.

3. A method for preparing a suspension of small intestinal crypts and intestinal organoids, characterized in that: The following steps are involved: (1) Remove the outer membrane, blood vessels and fat from the small intestine of the rodent near the stomach, and flush out the feces and intestinal contents; (2) Place the treated intestine in a buffer solution at 2-8°C and repeatedly blow and rinse to remove impurities; (3) The cleaned intestine is cut longitudinally and cut into small segments of 1-5 mm; (4) Add a mild cell dissociation reagent to the small intestinal segment, shake well, and separate the small intestinal crypts; (5) Resuspend the tissue fragments in a 2-8°C buffer containing BSA and pipette up and down; let stand until most of the intestinal tissue fragments settle to the bottom, pour out the supernatant, and repeat the steps until the supernatant is clear; (6) adding 2-8°C buffer containing BSA to resuspend the intestinal segments, filtering the cell suspension using a 60-80 μm filter, and collecting the filtrate; (7) Discard the filter, label the filtrate as "Fraction 1", and store it on ice; (8) Repeat steps (6)-(7) three times to obtain fractions 2-4, respectively; Fractions 3 and 4 were collected; (9) After centrifuging fraction 3 and / or fraction 4, add the fraction to a 2-8° C. buffer containing BSA and resuspend the fraction to obtain a crypt suspension; or mix fraction 3 and / or fraction 4 with matrix gel to prepare a mixed solution; (10) Add the mixed solution dropwise to the well plate to form a 3D culture system matrix gel ball. After the ball solidifies, add the organoid growth medium; (11) The well plate is placed in an incubator for organoid culture to obtain an intestinal organoid suspension.

4. The method according to claim 3, characterized in that The rodent is selected from mice.

5. The method according to claim 3, characterized in that: The 2-8°C buffer is a 2-8°C PBS buffer.

6. The method according to claim 3, characterized in that: The BSA content in the 2-8° C. buffer containing BSA is 0.05-0.15% by volume.

7. A drug for intestinal organoid regeneration system, characterized in that: It is an enteral administration preparation prepared by adding pharmaceutically acceptable excipients to small intestinal crypts and / or intestinal organoids.

8. A combined stem cell, characterized in that: It is a combination of small intestinal crypts and intestinal organoids.

9. Use of the combined stem cells according to claim 8 in the preparation of a drug for treating ulcerative colitis.

10. Application of small intestinal crypts and intestinal organoids in the preparation of drugs for the treatment of ulcerative colitis.

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