CN108728399A - External organoid 3D based on mouse difference section small intestine is cultivated, passed on, freezing, recovering and identification method - Google Patents

Abstract

The invention discloses a method for in vitro organoid 3D culture, subculture, cryopreservation, recovery and identification based on different sections of the small intestine of mice. The method includes: (1) isolating the crypts of the mouse duodenum, jejunum and ileum; (2) 3D culturing the crypts of the mouse duodenum, jejunum and ileum to form Organoids; (3) passage of mouse small intestine organoids; (4) cryopreservation of mouse small intestine organoids; (5) recovery of mouse small intestine organoids; (6) preparation of frozen sections of mouse small intestine organoids; Immunofluorescent labeling and identification of frozen sections of mouse intestinal organoids. Compared with the prior art, the present invention is based on small intestinal crypts containing stem cells, optimizes the crypt extraction method, in vitro culture system and culture medium, and includes complete methods of culture, subculture, cryopreservation, recovery and identification. The small intestine organoids obtained by this method can be repeatedly passaged, and the passaged organoids have stable properties.

Description

3D culture, passaging, cryopreservation and replication of in vitro organoids based on different segments of mouse small intestine Su and identification method

technical field

The invention relates to a method for in vitro organoid 3D culture, passage, cryopreservation, recovery and identification based on different sections of the small intestine of mice.

Background technique

The small intestine is the largest nutrient digestion and absorption organ in the human body. Due to its important role in metabolic disorders such as obesity and diabetes, there is growing interest in intestinal nutrient absorption, transport, perception, and hormone release. Currently, the in vitro models used for the study of nutrient sensing, transport, absorption and local hormone regulation in the small intestine are mainly divided into two categories: one is based on intestinal tissue, including ring method, everted intestinal sac, and Uss perfusion. Chamber and isolated intestinal perfusion, the above models can be used for the interaction of microorganisms, epithelial cells, immune cells, nerve cells and other cells in tissues under normal physiological conditions; one type is based on tumor cell lines, including various A transformed intestinal epithelial cell line, such as Caco-2, HT29, NCI-H716, STC-1, etc., the above Caco-2 cell model combined with transmembrane analysis technology (Transwell) can produce polarized epithelial layers for in vitro epithelial Cell Response Studies.

However, the above-mentioned in vitro research models of intestinal function have certain defects: the models based on intestinal tissue, because the above-mentioned models need to obtain isolated tissues, and it is impossible to obtain sufficient quality and quantity of tissues when the number of experimental subjects is limited, and the in vitro The organization is time-effective, so there is only a limited time for functional testing, and the repeatability and stability of the experiment are poor. At the same time, in order to obtain sufficient data, the number of animals used is high. The cell-based model has a single cell composition and cannot truly reproduce the interaction between intestinal cells and between cells and the cell matrix.

Contents of the invention

In view of this, the purpose of the present invention is to provide a method for 3D culture, passage, cryopreservation, recovery and identification of in vitro organoids based on different sections of the small intestine of mice. This method is different from the prior art in that it optimizes the crypt extraction method, in vitro culture system and medium, and includes complete methods of culture, subculture, cryopreservation, recovery and identification. Furthermore, the small intestinal organoids obtained by this culture method can be repeatedly passaged, and the passaged organoids have stable properties.

To achieve the above object, the present invention provides the following technical solutions:

A method for in vitro organoid 3D culture, passage, cryopreservation, and recovery based on different segments of the small intestine of mice, comprising the following steps:

Step 1. Collect the small intestine of an 8-10-week-old male C57BL/6 mouse, put it into a pre-cooled calcium- and magnesium-free phosphate buffer solution, and place it on ice; remove the residual mesentery of the small intestine with ophthalmic forceps, Cut the small intestine longitudinally along the longitudinal axis from the duodenum, wash with pre-cooled calcium and magnesium ion-free phosphate buffer solution to remove the chyme in the intestine, and obtain the cleaned small intestine; divide the cleaned small intestine into three equal parts The 4 cm small intestine at the front end close to the stomach was cut as the duodenum segment, the 4 cm middle part of the middle small intestine was the jejunum segment, and the 4 cm rear end close to the ileocecum was the ileum segment; the three small intestine parts were After immersing in absolute ethanol for 5 minutes, use a sterile cover glass to gently scrape the three sections of the small intestine 2-3 times along the villi of the small intestine; wash the three sections of the small intestine with pre-cooled calcium- and magnesium-ion-free phosphate buffer solution; Ophthalmic scissors: Cut the three sections of small intestine into 3-5 mm small sections along the longitudinal axis, wash the duodenum section in centrifuge tube 1 containing 8 ml of pre-cooled phosphate buffer solution without calcium and magnesium ions, and clean the jejunum Place the segment in centrifuge tube 2 containing 8 ml of pre-cooled calcium and magnesium ion-free phosphate buffer solution for washing, and place the ileum segment in centrifuge tube 3 containing 8 ml of pre-cooled calcium and magnesium ion-free phosphate buffer solution To clean, gently invert the centrifuge tubes 1, 2 and 3 back and forth 10-15 times; after cleaning, place the centrifuge tubes 1, 2 and 3 on ice to allow the small intestine fragments to settle naturally in the centrifuge tubes 1 and 2 and 3 bottoms, and then remove the supernatant; add 8 ml of pre-cooled EDTA solution with a concentration of 2 mM and a pH of 8.0 to centrifuge tubes 1 and 2 respectively, bury them horizontally in an ice box, and shake The bed was shaken at a speed of 40-50rpm for 30 minutes for digestion; after adding 8 ml of pre-cooled EDTA solution with a concentration of 5mM and a pH of 8.0 to the centrifuge tube 3, bury it horizontally in an ice box and shake it with a rotary The bed was shaken at a speed of 40-50rpm for 45 minutes for digestion; the centrifuge tubes 1, 2 and 3 were placed on ice to settle naturally, and the supernatant was removed; the pre-cooled calcium-free , Magnesium ion phosphate buffer solution, wash centrifuge tubes 1, 2 and 3 upside down for 10-15 times respectively;

Step 2: Place the centrifuge tubes 1, 2 and 3 cleaned in step 1 on ice to settle naturally. After removing the supernatant, add 7.5 ml of pre-cooled calcium and magnesium ion-free phosphate buffer solution, and shake gently for 50 The second time, the crypts in the small intestine fragments were removed, and then centrifuge tubes 1, 2 and 3 were placed on ice to settle naturally, and the supernatants of centrifuge tubes 1, 2 and 3 were combined and collected into 50 ml centrifuge tube 4; using 100 Filter the supernatant in the centrifuge tube 4 with a cell sieve with a micron pore size, and transfer the filtrate to the 50 ml centrifuge tube 5; repeat several times until there are less than 10 crypts in the collected liquid in the centrifuge tube 4 per 100 microliters , stop collecting;

Step 3, centrifuge the centrifuge tube 5 containing crypts obtained in step 2 at a speed of 150g and a temperature of 4°C for 10 minutes; remove the supernatant in the centrifuge tube 5, and add 3 ml of precooled calcium- and magnesium-ion-free phosphoric acid After the salt buffer solution, get the crypt suspension, pipette 20 microliters of the crypt suspension, drop it on the glass slide, count the number of crypts under an inverted microscope; transfer the crypt suspension to a 15ml centrifuge tube 6 Centrifuge at 150g and 4°C for 10 minutes, remove the supernatant in centrifuge tube 6 to obtain crypt pellets, and place the crypt pellets on ice;

Step 4: Add a corresponding volume of pre-thawed composite gel solution on ice to the crypt pellet obtained in Step 3 at a dose of 50 microliters per well, and gently blow on ice with a pre-cooled pipette tip to form a uniform gel. Small intestinal crypt complex gel suspension 1; inoculate the small intestinal crypt complex gel suspension 1 in a 24-well culture plate 1 preheated at 37°C in a volume of 50 microliters per well; place the 24-well culture plate 1 at temperature In a carbon dioxide incubator at 37°C for 30 minutes; after the composite gel is completely polymerized, add 500 microliters of complete medium to each well of 24-well culture plate 1, and replace the complete medium every 3 days to obtain the primary small intestine organoids;

Step five, 6 days after inoculation in step four, remove the medium in the 24-well culture plate in step four, then add 500 microliters of pre-cooled calcium and magnesium ion-free phosphate buffered saline buffer solution to each well and place it on ice, Use a pipette gun to repeatedly blow the composite gel in each well until it breaks to obtain a composite gel suspension 2; absorb the entire composite gel suspension 2 with a syringe and push it out, repeat once to make the crypt similar to the structure of the small intestine organoid Release to obtain dissociated small intestinal organoid suspension 1; transfer the dissociated small intestinal organoid suspension 1 to a 15ml centrifuge tube 7, centrifuge at 200g and 4°C for 10 minutes, and remove the supernatant in centrifuge tube 7 ; Add 150-250 microliters of pre-thawed composite gel solution on ice to the centrifuge tube 7 containing the dissociated small intestinal organoid suspension 1, and blow it 3-4 times with a pipette gun to make a pre-thawed composite gel solution on ice. Gel suspension 3; inoculate the composite gel suspension 3 in a preheated 24-well culture plate 2 at 37°C at a volume of 50 microliters per well; place the 24-well culture plate 2 in a carbon dioxide incubator at a temperature of 37°C for 30 Minutes; after the composite gel is completely polymerized, add 500 microliters of complete medium to each well of 24-well culture plate 2, and replace the complete medium every 3 days to obtain passaged small intestinal organoids;

Step 6. After 3 days of passage of small intestine organoids in step 5, remove the medium from the passage small intestine organoids in 24-well culture plate 2, add 500 microliters of pre-cooled calcium and magnesium ion-free phosphate buffer solution, and place on ice above, use a pipette gun to repeatedly blow the composite gel in each well until it breaks to obtain a composite gel suspension 3; Centrifuge for 10 minutes, remove the supernatant in centrifuge tube 8; add 1 ml of cryopreservation solution to centrifuge tube 8 to obtain small intestinal organoid resuspension; use 2-3 wells of small intestinal organoid resuspension as a group, transfer Put it into a 2ml cryopreservation tube, put it in a cryopreservation box containing isopropanol, store it at -80°C for 24 hours, and then put it in liquid nitrogen for long-term storage to obtain cryopreserved small intestinal organoids;

Step 7. Take out the cryopreservation tube containing the small intestinal organoid resuspension from the liquid nitrogen, and immediately put it in a 37°C water bath for instant dissolution. After the small intestinal organoid resuspension is completely melted, transfer the small intestinal organoid resuspension Add pre-cooled basal medium to 5 ml into 15 ml centrifuge tube 9, centrifuge at 200 g for 10 minutes; remove the supernatant of centrifuge tube 9, add 100 microliters of composite gel solution to obtain small intestinal organoids Resuscitation resuspension; inoculate small intestinal organoid resuspension in a volume of 50 microliters per well in preheated 24-well culture plate 3 at 37°C; place 24-well culture plate 3 in a carbon dioxide incubator at 37°C 30 minutes; after the composite gel was completely polymerized, 500 microliters of complete medium was added to each well of 24-well culture plate 3, and the complete medium was replaced every 3 days to obtain a revived small intestinal organoid.

Further, the composite gel liquid described in steps 4, 5 and 7 is extracted from mouse sarcoma, and is composed of laminin, collagen IV, nestin, heparan sulfate proteoglycan, transforming growth factor-β, epidermal growth factor, Composition of insulin growth factor, fibroblast growth factor and tissue plasminogen activator.

The complete medium described in steps four, five and seven consists of R-spondin conditioned medium, Noggin conditioned medium, DMEM/F12 medium, 4-hydroxyethylpiperazineethanesulfonic acid, L-alanyl-L- Composition of glutamine dipeptide, penicillin, streptomycin, epidermal growth factor, neuronal cell culture supplements N2 and B27, Rho-associated forming protein serine/threonine kinase coiled-coil inhibitor Y27632.

The basal medium described in step seven consists of DMEM/F12 medium, 4-hydroxyethylpiperazineethanesulfonic acid, L-alanyl-L-glutamine dipeptide, penicillin and streptomycin.

The cryopreservation solution described in step 6 consists of DMEM/F12 medium, 4-hydroxyethylpiperazineethanesulfonic acid, L-alanyl-L-glutamine dipeptide, penicillin, streptomycin, fetal bovine serum and Composed of dimethyl sulfoxide.

The present invention also provides an in vitro organoid identification method based on different sections of the small intestine of mice, which includes the following steps:

Step 1. Select any small intestinal organoid among primary, subcultured and resuscitated small intestinal organoids as the identification object, remove the medium after 6 days of culture, add 500 microliters of pre-cooled calcium and magnesium ion-free phosphate buffer solution, and place on ice above, use a pipette gun to repeatedly blow the composite gel in each well until it breaks to obtain a composite gel suspension 4; transfer the composite gel suspension 4 to a 15 ml centrifuge tube 10, and centrifuge at a speed of 200g for 10 minutes ; After removing the supernatant of the centrifuge tube 9, add paraformaldehyde with a solubility of 4% pre-cooled on ice in advance, and fix it at a temperature of 20-25° C. for 15 minutes; Centrifuge at a speed of 200g for 10 minutes to remove the supernatant; add calcium and magnesium ion-free phosphate buffer solution to the centrifuge tube 10, remove the supernatant after centrifuging at a speed of 200g for 5 minutes, and repeat 3 times; Add 2 ml of methylene blue solution with a solubility of 0.01% to the precipitate in tube 10, and incubate at a temperature of 20-25°C for 20 minutes; after the incubation is completed, centrifuge the centrifuge tube 10 at a speed of 200g for 10 minutes, and remove the supernatant solution; in the centrifuge tube 10, add calcium-free and magnesium ion-free phosphate buffer solution, remove the supernatant after centrifuging at a speed of 200g for 5 minutes, and repeat 3 times; 20% sucrose solution, placed overnight at 4°C for 12 hours to obtain a centrifuge tube 10 containing the small intestine organoid sucrose solution;

Step 2. Transfer the small intestinal organoid sucrose solution in centrifuge tube 10 to 1.5 ml centrifuge tube 11, centrifuge at 4000 rpm for 30-60 seconds, add 50-100 microliters of cryosection embedding agent, and gently suspend , stand still at a temperature of 20-25°C for 20-30 minutes; after the small intestinal organoids have settled in the 1.5 ml centrifuge tube 11, place the centrifuge tube 11 on dry ice for 1-3 minutes; remove the block of small intestinal organoids from the centrifuge tube Take it out in 11, place the side containing the small intestinal organoid downward, place it in the embedding box containing the frozen section embedding agent for secondary embedding, and freeze it with dry ice for 1-3 minutes to obtain the small intestinal organoid embedding; the quick freezing is completed Finally, slice the small intestinal organoid embedding material with a thickness of 8-10 microns using a frozen microtome to obtain the small intestinal organoid embedding slice 1; place the small intestinal organoid embedding slice 1 on a drying machine, and heat After drying for 30 minutes, the dried small intestinal organoid-embedded slice 2 was prepared, and the small intestinal organoid-embedded slice 2 was put into a slice box, and stored in a -80°C refrigerator for later use;

Step 3. Take the small intestinal organoid-embedded slice 2 obtained in step 2 out of the -80°C refrigerator, place it on a drying machine, and bake it at 42°C for 20 minutes; then, place the small intestinal organoid-embedded slice 2 on In the incubation box, wash with phosphate buffer solution for 10 minutes; then, remove the phosphate buffer solution, add antigen blocking solution, and incubate at a temperature of 20-25°C for 30-45 minutes to obtain small intestinal organoid embedded slice 3; Add the primary antibody to the organoid-embedded slice 3, and place it overnight at 4°C for 12 hours to obtain the small intestine organoid-embedded slice 4;

Step 4. Wash the small intestinal organoid-embedded slice 4 obtained in step 3 repeatedly with phosphate buffer solution for 3 times, each time for 5 minutes, to obtain the small intestinal organoid-embedded slice 5; add the small intestinal organoid embedded slice 5 to The secondary antibody corresponding to the primary antibody in step 3 was incubated at 20-25°C for 30 minutes to obtain the small intestinal organoid-embedded slice 6; the small intestinal organoid-embedded slice 6 was repeatedly washed 4 times with phosphate buffered saline, 5 minutes each time; then, add 2-(4-amidinophenyl)-6-indoleamidine dihydrochloride solution and incubate at 20-25°C for 5 minutes; wash with ultrapure water for 1-2 times, 5 minutes each time, to obtain the small intestinal organoid-embedded slice 7; finally, seal the small intestinal organoid-embedded slice 7 with a mounting agent to obtain the small intestinal organoid-embedded slice;

Step 5: Using a laser confocal microscope to observe the mounted small intestinal organoids obtained in Step 4 to obtain immunofluorescence images and identify them.

The present invention has the following advantages and beneficial effects:

Compared with prior art methods, the method provided by the invention has the following significant advantages and progress:

1. The technical method of the present invention optimizes the selection positions of different segments of the mouse small intestine, optimizes the extraction process, and can ensure the smooth completion of small intestinal organoid culture in vitro.

2. The technical method of the present invention has carried out a breakthrough optimization of key biochemical reagents such as the culture system, composite gel solution, complete medium and basal medium used in the in vitro culture system of mouse small intestinal organoids, thereby ensuring that the small intestinal organoids Organs can achieve normal culture, repeated passage, cryopreservation and repeated resuscitation in vitro, and have passage stability.

3. The technical method of the present invention, in addition to providing a complete in vitro culture method, also provides a complete in vitro passage, cryopreservation and recovery method, which ensures the recycling of small intestinal organoids in vitro.

4. The technical method of the present invention optimizes the in vitro identification method of small intestinal organoids, and can clarify the reliability of small intestinal organoids.

Description of drawings

Figure 1 is a schematic diagram of collection of small intestine segments.

Figure 2 is a diagram of the in vitro culture growth of small intestinal organoids.

Fig. 3 is a graph showing the growth of small intestinal organoids cultured in vitro for 12 passages.

Figure 4 is an immunofluorescence identification diagram of small intestinal organoids.

specific embodiment

The present invention discloses a method for 3D culture, subculture, cryopreservation, recovery and identification of in vitro organoids based on different sections of the small intestine of mice. Those skilled in the art can learn from the content of this article and appropriately improve the parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply this invention.

In order to illustrate the present invention better, the following examples illustrate.

Example 1:

Step 1. Euthanize a 9-week-old male C57BL/6 mouse with carbon dioxide and dislocate the cervical vertebrae; wet the abdomen of the mouse with 70% ethanol solution, and cut the abdominal cavity of the mouse to the extracranial genitalia by dissecting , cut the bilateral abdominal muscle tissue and extend the incision to the ribs, cut the duodenum from the pyloric sphincter, pull the small intestine out of the abdominal cavity and cut off the small intestine at the ileum and cecum; put the collected small intestine in pre-cooled Place it on ice in a phosphate buffer solution without calcium and magnesium ions; remove the residual mesentery of the small intestine with ophthalmic forceps, cut the small intestine longitudinally from the duodenum along the longitudinal axis, and use pre-cooled calcium and magnesium ion-free phosphate Wash with salt buffer solution to remove the chyme in the intestinal tract to obtain the cleaned small intestine; divide the cleaned small intestine into three equal parts, cut the 4 cm small intestine at the front end close to the stomach as the duodenum section, and the middle section of the small intestine in the middle The first 4 cm is the jejunum segment, and the 4 cm rear end close to the ileocecum is the ileum segment; Figure 1 is a schematic diagram of the collection of small intestine segments. Among them, 1 is the cutting position of the pyloric sphincter, 2 is the cutting position of the ileum and cecum, 3 is the duodenum segment, 4 is the jejunum segment, and 5 is the ileum segment.

After immersing the three sections of small intestine in absolute ethanol for 5 minutes, gently scrape the three sections of small intestine along the villi surface of the small intestine with a sterilized cover glass for 3 times; wash the three sections with pre-cooled calcium- and magnesium-ion-free phosphate buffer solution Small intestine: Use ophthalmic scissors to cut the three sections of small intestine into small sections of 4 mm along the longitudinal axis, put the duodenum section in centrifuge tube 1 containing 8 ml of pre-cooled calcium- and magnesium-ion-free phosphate buffer solution, and wash it. Clean the jejunum segment in centrifuge tube 2 containing 8 ml of pre-cooled calcium and magnesium ion-free phosphate buffer solution, and place the ileum segment in centrifuge tube 3 containing 8 ml of pre-cooled calcium and magnesium ion-free phosphate buffer solution During cleaning, gently invert centrifuge tubes 1, 2 and 3 back and forth 15 times; after cleaning, place centrifuge tubes 1, 2 and 3 on ice to allow small intestine fragments to settle naturally 3 bottom, and then remove the supernatant; add 8 ml of pre-cooled EDTA solution with a concentration of 2 mM and a pH of 8.0 to centrifuge tubes 1 and 2 respectively, bury them in an ice box horizontally and use a rotary shaker Shake at a speed of 45 rpm for 30 minutes for digestion; add 8 ml of pre-cooled EDTA solution with a concentration of 5 mM and a pH of 8.0 to the centrifuge tube 3, and bury it horizontally in an ice box with a rotary shaker at 45 rpm. Shake at a high speed for 45 minutes for digestion; place centrifuge tubes 1, 2 and 3 on ice to settle naturally, remove the supernatant; add pre-cooled calcium- and magnesium-free phosphate to centrifuge tubes 1, 2 and 3 respectively After buffering the solution, centrifuge tubes 1, 2 and 3 were washed by inverting up and down 15 times respectively.

Step 2: Place the centrifuge tubes 1, 2 and 3 cleaned in step 1 on ice to settle naturally. After removing the supernatant, add 7.5 ml of pre-cooled calcium and magnesium ion-free phosphate buffer solution, and shake gently for 50 The second time, the crypts in the small intestine fragments were removed, and then centrifuge tubes 1, 2 and 3 were placed on ice to settle naturally, and the supernatants of centrifuge tubes 1, 2 and 3 were combined and collected into 50 ml centrifuge tube 4; using 100 Filter the supernatant in the centrifuge tube 4 with a cell sieve with a micron pore size, and transfer the filtrate to the 50 ml centrifuge tube 5; repeat several times until there are less than 10 crypts in the collected liquid in the centrifuge tube 4 per 100 microliters , stop collecting.

Step 3, centrifuge the centrifuge tube 5 containing crypts obtained in step 2 at a speed of 150g and a temperature of 4°C for 10 minutes; remove the supernatant in the centrifuge tube 5, and add 3 ml of precooled calcium- and magnesium-ion-free phosphoric acid After the salt buffer solution, the crypt suspension was obtained; the crypt suspension was transferred to a 15 ml centrifuge tube 6 and centrifuged at 150g and 4°C for 10 minutes, and the supernatant in the centrifuge tube 6 was removed to obtain the crypt precipitate , and place the crypt pellet on ice.

Step 4: Add a corresponding volume of pre-thawed composite gel solution on ice to the crypt pellet obtained in Step 3 at a dose of 50 microliters per well, and gently blow on ice with a pre-cooled pipette tip to form a uniform gel. Small intestinal crypt complex gel suspension 1; inoculate the small intestinal crypt complex gel suspension 1 in a 24-well culture plate 1 preheated at 37°C in a volume of 50 microliters per well; place the 24-well culture plate 1 at temperature In a carbon dioxide incubator at 37°C for 30 minutes; after the composite gel is completely polymerized, add 500 microliters of complete medium to each well of 24-well culture plate 1, and replace the complete medium every 3 days to obtain the primary small intestine organoids;

Figure 2 is the tissue morphology of the small intestinal organoids on the first day and the fourth day of in vitro culture. Figure 2 A is a representative image of the primary isolated duodenal crypt morphology, A1 is a representative image of the duodenal crypt formed after 4 days of culture; B is a representative image of the primary isolated jejunal crypt morphology Image, B1 is a representative image of organoids formed from jejunal crypts after 4 days of culture; C is a representative image of the morphology of primary isolated ileal crypts, and C1 is a representative image of organoids formed from ileal crypts after 4 days of culture.

It can be seen from Figure 2 that small intestinal organoids have stable tissue growth in vitro.

Step 5: 6 days after inoculation in step 4, add 500 microliters of pre-cooled calcium- and magnesium-ion-free phosphate buffered saline buffer solution to each well of the 24-well culture plate 1 in step 4 and place it on ice. The composite gel in each hole was blown repeatedly with a gun until it was broken to obtain a composite gel suspension 2; the entire composite gel suspension 2 was sucked up with a syringe and pushed out, and repeated once to release the crypt-like structure from the small intestine organoid to obtain Dissociate the small intestinal organoid suspension 1; transfer the dissociated small intestinal organoid suspension 1 to a 15 ml centrifuge tube 7, centrifuge at a speed of 200g and a temperature of 4°C for 10 minutes, remove the supernatant in the centrifuge tube 7; Add 175 microliters of pre-thawed composite gel solution on ice to the centrifuge tube 7 of the dissociated small intestinal organoid suspension 1, and pipette 3-4 times with a pipette gun to prepare the pre-thawed composite gel suspension 3 on ice; Inoculate the composite gel suspension 3 into a preheated 24-well culture plate 2 at 37°C according to a volume of 50 microliters per well; place the 24-well culture plate 2 in a carbon dioxide incubator at a temperature of 37°C for 30 minutes; After the gel was completely polymerized, 500 microliters of complete medium was added to each well of the 24-well culture plate 2, and the complete medium was replaced every 3 days to obtain a passage small intestinal organoid.

Step 6. Repeat step 5 for 11 times to obtain small intestinal organoids with 13 passages. Figure 3 is the histological diagram of the small intestinal organoids with 12 passages on the first day and the fourth day of in vitro culture.

Figure 3D is a representative image of similar structures of crypts after dissociation of duodenal organoids after 12 passages, D1 is a representative image of organoids after 4 days of subculture; E is the dissociation of jejunum organoids after 12 passages Representative images of similar structures of crypts, D1 is a representative image of organoids after 4 days of subculture; F is a representative image of similar structures of crypts after dissociation of ileal organoids after passage 12, D1 is a representative image of organoids after 4 days of subculture image.

It can be seen from Figure 3 that the small intestinal organoids can still have stable tissue growth after 12 passages in vitro.

Step 7. After 3 days of passage of small intestinal organoids in step 5, remove the medium from the passaged small intestinal organoids in 24-well culture plate 2, add 500 microliters of pre-cooled calcium and magnesium ion-free phosphate buffer solution, and place on ice above, use a pipette gun to repeatedly blow the composite gel in each well until it breaks to obtain a composite gel suspension 3; Centrifuge for 10 minutes, remove the supernatant in centrifuge tube 8; add 1 ml of cryopreservation solution to centrifuge tube 8 to obtain small intestinal organoid resuspension; use 2-well small intestinal organoid resuspension as a group, transfer to a Put them in a 2ml cryopreservation tube, put them in a cryopreservation box containing isopropanol, store them at -80°C for 24 hours, and then put them in liquid nitrogen for long-term storage to obtain cryopreserved small intestinal organoids.

Step 8. Take out the cryopreservation tube containing the small intestinal organoid resuspension in step 7 from the liquid nitrogen, and immediately put it in a 37°C water bath for instant dissolution. After the small intestinal organoid resuspension is completely melted, transfer the small intestinal organoid Resuspend the liquid into a 15 ml centrifuge tube 9, add pre-cooled basal medium to 5 ml, and centrifuge at a speed of 200 g for 10 minutes; after removing the supernatant of the centrifuge tube 9, add 100 microliters of composite gel solution to obtain Resuspension of small intestinal organoid resuscitation; inoculate the resuspension of small intestinal organoid resuscitation into a preheated 24-well culture plate 3 at 37°C in a volume of 50 μl per well; place the 24-well culture plate 3 in carbon dioxide at a temperature of 37°C 30 minutes in the incubator; after the composite gel was completely polymerized, 500 microliters of complete medium was added to each well of 24-well culture plate 3, and the complete medium was replaced every 3 days to obtain resuscitated small intestinal organoids.

Step 9: Using the first-generation small intestinal organoids from Step 4 as the identification object to label the intestinal epithelial cell marker protein E-cadherin, the absorption cell marker protein villin (Villin), and the Paneth cell marker protein lysozyme ( Lysozyme), goblet cell marker protein mucin (Mucin2), endocrine cell marker protein chromogranin A (Chromogranin A) and L-type cell marker protein glucagon-like peptide (GLP-1) protein as marker proteins, cultured for 6 One day later, remove the medium, add 500 microliters of pre-cooled calcium- and magnesium-ion-free phosphate buffer solution, place it on ice, and repeatedly blow the composite gel in each well with a pipette until it breaks to obtain a composite gel suspension 4 ; Transfer the composite gel suspension 4 to a 15 ml centrifuge tube 10, and centrifuge at a speed of 200g for 10 minutes; after removing the supernatant of the centrifuge tube 9, add polyol with a solubility of 4% pre-cooled on ice in advance Polyoxymethylene, fixed at a temperature of 20°C for 15 minutes; after the fixation, centrifuge the centrifuge tube 10 at a speed of 200g for 10 minutes to remove the supernatant; add calcium and magnesium ion-free phosphate buffer solution, centrifuge at 200g for 5 minutes, remove the supernatant, and repeat 3 times; then add 1 ml of methylene blue solution with a solubility of 0.01% to the precipitate in the centrifuge tube 10, and incubate at a temperature of 20°C 20 minutes; after the incubation is completed, centrifuge the centrifuge tube 10 at a speed of 200g for 10 minutes to remove the supernatant; add calcium and magnesium ion-free phosphate buffer solution to the centrifuge tube 10, and centrifuge at a speed of 200g for 5 minutes Finally, remove the supernatant, and repeat 3 times; then add 1 ml of sucrose solution with a solubility of 20% to the precipitate in the centrifuge tube 10, and place it overnight at a temperature of 4° C. for 12 hours to obtain a sucrose solution containing small intestinal organoids. Centrifuge tube 10.

Step 10. Transfer the small intestinal organoid sucrose solution in the centrifuge tube 10 to a 1.5 ml centrifuge tube 11, centrifuge at 4000 rpm for 45 seconds, add 50-100 microliters of cryosection embedding agent, suspend gently, and place in Stand still for 25 minutes at a temperature of 20°C; after the small intestinal organoids settle in the 1.5 ml centrifuge tube 11, freeze them with dry ice for 2 minutes; take out the block of small intestinal organoids from the centrifuge tube 11, with the side containing the small intestinal organoids facing down, Embedded for the second time in an embedding box containing frozen section embedding agent, and quick-frozen with dry ice for 30 seconds to obtain the small intestinal organoid embedding; The thickness of the small intestine organoid embedding slice 1 was obtained; the small intestinal organoid embedding slice 1 was placed on a drying machine, and after drying at 42°C for 30 minutes, the dried small intestinal organoid embedding slice was obtained 2. Put the small intestinal organoid-embedded slice 2 into a slice box and store it in a -80°C refrigerator for later use.

Step 11. Take the small intestinal organoid-embedded slice 2 obtained in step 10 out of the -80°C refrigerator, place it on a drying machine, and bake it at 42°C for 20 minutes; then, place the small intestinal organoid-embedded slice 2 In the incubation box, wash with phosphate buffer solution for 10 minutes; then, remove the phosphate buffer solution, add antigen blocking solution, and incubate at 20°C for 40 minutes to obtain small intestinal organoid embedded slice 3; The primary antibody was added to the embedded slice 3, and left overnight at 4°C for 12 hours to obtain the small intestinal organoid embedded slice 4.

Step 12. Wash the small intestinal organoid-embedded slice 4 obtained in step 11 repeatedly with phosphate buffered saline solution for 3 times, each time for 5 minutes, to obtain the small intestinal organoid-embedded slice 5; Add the secondary antibody corresponding to the primary antibody in step 11, and incubate at 20°C for 30 minutes to obtain the small intestinal organoid-embedded slice 6; wash the small intestinal organoid-embedded slice 6 with phosphate buffer solution repeatedly 4 times , 5 minutes each time; then, add 2-(4-amidinophenyl)-6-indoleamidine dihydrochloride solution and incubate at 20°C for 5 minutes; wash with ultrapure water twice, each After 5 minutes, the small intestinal organoid-embedded section 7 was obtained; finally, the small intestinal organoid-embedded section 7 was mounted with a mounting agent to obtain the small intestinal organoid-embedded section.

In step 13, the small intestinal organoid obtained in step 12 is observed with a laser confocal microscope, and immunofluorescence images are obtained and identified.

Figure 4 is a diagram of the identification results of small intestinal organoids.

G, H, I, P, Q, and R in Figure 4 are intestinal epithelial cell marker protein cadherin (E-cadherin), absorption cell marker protein villin (Villin), Paneth cell marker protein lysozyme (Lysozyme), respectively. ), goblet cell marker protein Mucin (Mucin2), endocrine cell marker protein Chromogranin A (Chromogranin A) and L-type cell marker protein glucagon-like peptide (GLP-1) marked different types of intestinal cells in Representative images of immunofluorescence expression in duodenal organoids; G0, H0, I0, P0, Q0, R0 are the immunofluorescence expression of different types of intestinal cells marked by the above marker proteins in mouse duodenal tissue Fluorescent representative images;

J, K, L, S, T, and U in Figure 4 are the intestinal epithelial cell marker protein E-cadherin (E-cadherin), the absorption cell marker protein villin (Villin), the Paneth cell marker protein lysozyme (Lysozyme ), goblet cell marker protein Mucin (Mucin2), endocrine cell marker protein Chromogranin A (Chromogranin A) and L-type cell marker protein glucagon-like peptide (GLP-1) marked different types of intestinal cells in Representative immunofluorescence images expressed in jejunal organoids; J0, K0, L0, S0, T0, and U0 are representative immunofluorescence images expressed in mouse jejunum tissues by different types of intestinal cells labeled with the above marker proteins.

M, N, O, V, W, and X in Figure 4 are intestinal epithelial cell marker protein cadherin (E-cadherin), absorption cell marker protein villin (Villin), Paneth cell marker protein lysozyme (Lysozyme), respectively. ), goblet cell marker protein Mucin (Mucin2), endocrine cell marker protein Chromogranin A (Chromogranin A) and L-type cell marker protein glucagon-like peptide (GLP-1) marked different types of intestinal cells in Representative immunofluorescence images expressed in ileal organoids; M0, N0, O0, V0, W0, and X0 are representative immunofluorescence images of expression of different types of intestinal cells labeled with the above marker proteins in mouse jejunum tissue.

It can be seen from Figure 4 that, the same as the small intestinal villi in mice, the small intestinal organoids in vitro are also composed of a single layer of epithelial cells; and the cell composition and structure are the same as those in the small intestinal villi in vivo, and differentiate a variety of different types of intestinal epithelial cells, including Intestinal absorptive cells, Paneth cells, goblet cells, endocrine cells and L-type cells.

Therefore, it can be seen from the above steps and results that the methods for in vitro organoid 3D culture, passage, cryopreservation, recovery and identification based on different sections of the small intestine of mice provided by the present invention are stable, reliable and effective.

The above is only a specific embodiment of the present invention, and it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (6)

1. the external organoid 3D based on mouse difference section small intestine is cultivated, passes on, is frozen, method for resuscitation, it is characterised in that packet Include following steps：

Step 1: collect one 8-10 week greatly male C57BL/6 mouse small intestine, be put into precooling without calcium and magnesium ion phosphoric acid In salt buffer solution, it is placed on ice；Small intestine is removed with ophthalmic tweezers and remains mesenterium, by small intestine from duodenum section along longitudinal axis longitudinal direction It splits, cleans the chyme in removal enteron aisle without calcium and magnesium ion phosphate buffer solution with precooling, what is cleaned up is small Intestines；The small intestine that finishes of cleaning is divided into trisection, respectively clip front end close to 4 centimetres of small intestines of stomach as duodenum section, 4 centimetres of the middle part of interlude small intestine is jejunal segment, and rear end is ileal segment close to 4 centimetres of ileocecum portion；By three sections of small intestines Three sections of small intestine sites are gently scraped along intestinal villi face 2-3 times in position with being impregnated in absolute ethyl alcohol after five minutes with disinfection coverslip；With Precooling cleans three sections of small intestines without calcium and magnesium ion phosphate buffer solution；Three sections of small intestines are cut into 3-5 millis along the longitudinal axis with eye scissors Duodenum section is put in clear in the centrifuge tube 1 without calcium and magnesium ion phosphate buffer solution being pre-chilled containing 8 milliliters by the segment of rice It washes, jejunal segment is put in the centrifuge tube 2 without calcium and magnesium ion phosphate buffer solution being pre-chilled containing 8 milliliters and is cleaned, by ileal segment It is put in the centrifuge tube 3 without calcium and magnesium ion phosphate buffer solution containing 8 milliliters of precoolings and cleans, respectively by centrifuge tube when cleaning 1, it 2 and 3 gently overturns 10-15 times back and forth；After cleaning, centrifuge tube 1,2 and 3 is respectively placed in makes small ramenta intestinonun natural on ice Centrifuge tube 1,2 and 3 bottoms are fallen to, supernatant is then removed；Respectively in centrifuge tube 1 and 2 be added 8 milliliters precooling it is a concentration of After the edta solution that 2mM, pH are 8.0, laterally it is embedded in ice chest and is shaken with the speed of 40-50rpm with Clothoid type shaking table It swings 30 minutes and is digested；A concentration of 5mM of 8 milliliters of precoolings is added in centrifuge tube 3, the ethylenediamine tetra-acetic acid that pH is 8.0 is molten After liquid, laterally it is embedded in ice chest and is digested within 45 minutes with the velocity fluctuation of 40-50rpm with Clothoid type shaking table；By centrifuge tube 1,2 It is respectively placed in natural subsidence on ice with 3, removes supernatant；Be separately added into centrifuge tube 1,2 and 3 precooling without calcium and magnesium ion After phosphate buffer solution, centrifuge tube 1,2 and 3 is turned upside down 10-15 times respectively and is cleaned；

The centrifuge tube 1,2 and 3 that completion is cleaned in step 1 is placed in natural subsidence on ice by step 2, after removing supernatant, respectively Be added 7.5 milliliters precooling without calcium and magnesium ion phosphate buffer solution, gentle agitation 50 times deviates from the crypts in small ramenta intestinonun, After centrifuge tube 1,2 and 3 is then placed in natural subsidence on ice, centrifuge tube 1,2 and 3 supernatants are merged and collected to 50 milliliters of centrifugations In pipe 4；The supernatant in centrifuge tube 4 is filtered using the cell sieve of 100 micron pore sizes, filtrate is transferred to 50 milliliters of centrifugations In pipe 5；Repeatedly, when being less than 10 crypts in the collection liquid in every 100 microlitres of centrifuge tubes 4, stop collecting；

Step 3: by the centrifuge tube 5 containing crypts obtained in step 2 to be centrifuged 10 minutes at a temperature of 150g rotating speeds and 4 DEG C； Remove centrifuge tube 5 in supernatant, be added 3 milliliters precooling without calcium and magnesium ion phosphate buffer solution after, obtain crypts suspension, Liquid-transfering gun draws 20 microlitres of crypts suspensions, and drop counts crypts quantity on glass slide under inverted microscope；Crypts suspension is shifted To, to be centrifuged 10 minutes at a temperature of 150g rotating speeds and 4 DEG C, the supernatant in removal centrifuge tube 6 obtains crypts in 15 milliliters of centrifuge tubes 6 Precipitation, and crypts precipitation is placed on ice；

Step 4: the pre- on ice of respective volume is added according to the dosage in 50 microlitres of every hole in the crypts precipitation obtained into step 3 The plural gel liquid of thawing, with precooling liquid transfer gun head, gently piping and druming forms uniform crypts of small intestine plural gel suspension 1 on ice；It will Crypts of small intestine plural gel suspension 1 is inoculated according to 50 microlitres of every hole volume in 24 well culture plates 1 of 37 DEG C of preheatings；24 holes are trained Plate 1 is supported to be placed in the carbon dioxide incubator that temperature is 37 DEG C 30 minutes；Gel to be composite completely polymerization after, then to 24 holes cultivate 500 microlitres of complete mediums are added in every hole of plate 1, replaces a complete medium within every 3 days, obtains primary small intestine organoid；

Step 5: 6 days after step 4 inoculation, culture medium in 24 well culture plates, is then added in every hole in removal step four 500 microlitres precooling be placed on ice without calcium and magnesium ion phosphate buffer solution, blow and beat the compound of each hole repeatedly with liquid-transfering gun Gel obtains plural gel suspension 2 to being crushed；It is released after drawing whole plural gel suspensions 2 with syringe, is repeated once, makes Crypts similar structures are discharged from small intestine organoid, obtain dissociation small intestine organoid suspension 1；It will dissociation small intestine organoid suspension 1 It is transferred in 15 milliliters of centrifuge tubes 7, to be centrifuged 10 minutes at a temperature of 200g rotating speeds and 4 DEG C, removes supernatant in centrifuge tube 7；To The 150-250 microlitres of plural gel liquid melted in advance on ice is added in centrifuge tube 7 containing dissociation small intestine organoid suspension 1, with shifting Liquid rifle is blown and beaten 3-4 times and the plural gel suspension 3 melted in advance on ice is made；By plural gel suspension 3 according to 50 microlitres of every hole volume It is inoculated in 37 DEG C of 24 well culture plates 2 of preheating；24 well culture plates 2 are placed in the carbon dioxide incubator that temperature is 37 DEG C 30 points Clock；After gel to be composite polymerization completely, then 500 microlitres of complete mediums are added into every hole of 24 well culture plates 2, every 3 days more A complete medium is changed, passage small intestine organoid is obtained；

Step 6: after step 5 small intestine organoid passes on 3 days, the passage small intestine organoid in 24 well culture plates 2 is removed into training After supporting base, being placed on ice without calcium and magnesium ion phosphate buffer solution for 500 microlitres of precoolings of addition is blown and beaten repeatedly with liquid-transfering gun The plural gel in each hole obtains plural gel suspension 3 to being crushed；Plural gel suspension 3 is transferred to 15 milliliters of centrifuge tubes 8 In, to be centrifuged 10 minutes at a temperature of 200g rotating speeds and 4 DEG C, remove supernatant in centrifuge tube 8；1 milliliter of jelly is added in centrifuge tube 8 After liquid storage, small intestine organoid re-suspension liquid is obtained；With the holes 2-3 small intestine organoid re-suspension liquid for one group, it is transferred to 2 milliliters of cryopreservation tubes In, be put in the freezing storing box containing isopropanol, -80 DEG C place 24 hours after be put into liquid nitrogen and preserve for a long time, obtain freezing small intestine class Organ；

Step 7: the cryopreservation tube for having small intestine organoid re-suspension liquid is taken out from liquid nitrogen, it is immediately placed in 37 DEG C of water-bath middling speeds It is molten, it after small intestine organoid re-suspension liquid is melted completely, shifts in small intestine organoid re-suspension liquid to 15 milliliters of centrifuge tubes 9, is added pre- Cold basal medium centrifuges 10 minutes to 5 milliliters under the conditions of rotating speed 200g；After the supernatant for removing centrifuge tube 9, it is added 100 Microlitre plural gel liquid, obtains small intestine organoid recovery re-suspension liquid；By small intestine organoid recovery re-suspension liquid according to 50 microlitres of every hole Volume is inoculated in 37 DEG C of 24 well culture plates 3 of preheating；24 well culture plates 3 are placed in the carbon dioxide incubator that temperature is 37 DEG C 30 minutes；After gel to be composite polymerization completely, then 500 microlitres of complete mediums are added into every hole of 24 well culture plates 3, every 3 It replaces a complete medium, obtains recovery small intestine organoid.

2. according to the method described in claim 1, it is characterized in that Step 4: plural gel liquid described in five and seven extracts from childhood Rat meat tumor, by Fibronectin, collagen IV, nestin, heparan sulfate proteoglycan, transforming growth factor-β, epidermis Growth factor, insulin-like growth factor, fibroblast growth factor and tissue plasminogen activator's composition.

3. according to the method described in claim 1, it is characterized in that Step 4: complete medium is by R- described in five and seven Spondin conditioned mediums, Noggin conditioned mediums, DMEM/F12 culture mediums, 4- hydroxyethyl piperazineethanesulfonic acids, the third ammonia of L- Acyl-L-Glutamine dipeptides, penicillin, streptomysin, epidermal growth factor, neuronal cell culture replenishers N2 and B27, Rho Correlation forms Protein Serine/threonine kinase coiled coil inhibitor Y27632 compositions.

4. according to the method described in claim 1, it is characterized in that basal medium described in step 7 is cultivated by DMEM/F12 Base, 4- hydroxyethyl piperazineethanesulfonic acids, Ala-Gln dipeptides, penicillin and streptomysin composition.

5. according to the method described in claim 1, it is characterized in that frozen stock solution described in step 6 is by DMEM/F12 culture mediums, 4- Hydroxyethyl piperazineethanesulfonic acid, Ala-Gln dipeptides, penicillin, streptomysin, fetal calf serum and dimethyl sulfoxide (DMSO) group At.

6. a kind of external organoid identification method based on mouse difference section small intestine, it is characterised in that including following step Suddenly：

Step 1: selecting any small intestine organoid in primary, passage and recovery small intestine organoid as identification object, culture 6 Culture medium is removed after it, liquid-transfering gun is used in being placed on ice without calcium and magnesium ion phosphate buffer solution for 500 microlitres of precoolings of addition The plural gel in each hole is blown and beaten repeatedly to being crushed, and obtains plural gel suspension 4；Plural gel suspension 4 is transferred to 15 milliliters In centrifuge tube 10, centrifuged 10 minutes under the conditions of rotating speed 200g；After the supernatant for removing centrifuge tube 9, addition is pre-chilled on ice in advance Solubility be 4% paraformaldehyde, fix 15 minutes under the conditions of 20-25 DEG C of temperature；After fixation, by centrifuge tube 10 It is centrifuged 10 minutes under the conditions of rotating speed 200g, removes supernatant；It is added in centrifuge tube 10 molten without calcium and magnesium ion phosphate-buffered Liquid centrifuges under the conditions of rotating speed 200g and removes supernatant after five minutes, is repeated 3 times；Then to being added in the precipitation in centrifuge tube 10 The methylene blue solution that 2 milliliters of solubility are 0.01% is incubated 20 minutes under the conditions of 20-25 DEG C of temperature；It, will be from after the completion of incubation Heart pipe 10 centrifuges 10 minutes under the conditions of rotating speed 200g, removes supernatant；It is added without calcium and magnesium ion phosphate in centrifuge tube 10 Buffer solution centrifuges under the conditions of rotating speed 200g and removes supernatant after five minutes, is repeated 3 times；Then to the precipitation in centrifuge tube 10 The middle sucrose solution that 1 milliliter of solubility is added and is 20% is placed 12 hours under the conditions of 4 DEG C of temperature, is obtained containing small intestine class overnight The centrifuge tube 10 of organ sucrose solution；

Step 2: the small intestine organoid sucrose solution in centrifuge tube 10 is transferred in 1.5 milliliters of centrifuge tubes 11, in rotating speed 50-100 microlitres of frozen section embedding medium is added after being centrifuged 30-60 seconds under the conditions of 4000rpm, after gently suspending, in temperature 20-25 It is 20-30 minutes static under the conditions of DEG C；After small intestine organoid falls to 1.5 milliliters of centrifuge tubes 11, centrifuge tube 11 is placed in dry ice It is quick-frozen 1-3 minutes upper；Blocking small intestine organoid is taken out from centrifuge tube 11, the side of the organoid containing small intestine is downward, is placed in Secondary embedding in the embedded box of the embedding medium containing frozen section obtained small intestine organoid embedded object with dry ice quick-frozen 1-3 minutes；Speed After the completion of jelly, freezing microtome is used to be sliced small intestine organoid embedded object with 8-10 microns of thickness, obtains small intestine class device Official's embedded section 1；Small intestine organoid embedded section 1 is placed in and is dried on piece machine, after being dried 30 minutes at a temperature of 42 DEG C, drying is made Small intestine organoid embedded section 2 is put into slice box, is preserved in -80 DEG C of refrigerators standby by small intestine organoid embedded section 2 afterwards With；

Step 3: the small intestine organoid embedded section 2 obtained in step 2 is taken out from -80 DEG C of refrigerators, it is placed in and dries on piece machine, It is dried 20 minutes at a temperature of 42 DEG C；Then, small intestine organoid embedded section 2 is put in and is incubated in box, it is clear with phosphate buffer solution It washes 10 minutes；Then, phosphate buffer solution is removed, antigen blockade liquid is added, 30-45 points are incubated under the conditions of 20-25 DEG C of temperature Clock obtains small intestine organoid embedded section 3；Primary antibody is added in small intestine organoid embedded section 3, under the conditions of 4 DEG C of temperature overnight It places 12 hours, obtains small intestine organoid embedded section 4；

Step 4: the small intestine organoid embedded section 4 obtained in step 3 is used phosphate buffer solution repeated washing 3 times, often Secondary 5 minutes, obtain small intestine organoid embedded section 5；It is added in small intestine organoid embedded section 5 corresponding with primary antibody in step 3 Secondary antibody, be incubated 30 minutes under the conditions of 20-25 DEG C of temperature, obtain small intestine organoid embedded section 6；By small intestine organoid embedded section 6 use phosphate buffer solution repeated washing 4 times, every time 5 minutes；Then, two hydrochloric acid of 2- (4- carbamimido-phenyls) -6- indoles amidine is added Salting liquid is incubated 5 minutes under the conditions of 20-25 DEG C of temperature；With ultra-pure water to clean 1-2 times, 5 minutes every time, small intestine organoid is obtained Embedded section 7；Mounting finally is carried out to small intestine organoid embedded section 7 with mountant, obtains small intestine organoid mounting；

Step 5 observes the small intestine organoid mounting obtained in step 4 using laser confocal microscope, and acquisition is exempted from Epidemic disease fluorescent image.