Pig jejunum crypt separation and organoid model construction method

Abstract

The invention discloses a pig jejunum crypt separation and organoid model construction method, and belongs to the technical field of animal tissue separation and culture. The method specifically comprises the following steps: intestinal tract collection, cleaning solution preparation, intestinal tract treatment, tissue trimming, tissue cleaning, digestive juice preparation, tissue digestion, cell mass separation, crypt separation, crypt counting, crypt collection, crypt inoculation and organoid culture. The porcine jejunum organ obtained by the method has a typical three-dimensional cystic structure, has complete epithelial polar and functional cell types, including microvillus, goblet cells, panning cells and endocrine cells, and shows good barrier function and drug metabolic activity. A reliable in-vitro model platform is provided for pig intestinal development, nutrient absorption, pathogen infection mechanism research and precision medical treatment, meanwhile, a technical foundation is laid for large-scale application of organoids, and the method has important scientific significance and clinical value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal tissue separation and culture, and more particularly to a method for separating porcine jejunal crypts and constructing organoid models. BACKGROUND

[0002] Crypt is a term used in zoology to describe the concave structure of organs or tissues in animals, commonly found in anatomical structures such as the digestive system and respiratory system. Its morphology and function are closely related to the organ in which it is located, and it may perform functions such as material exchange, secretion storage, or immune protection. Animal crypts, as key structures for intestinal epithelial cell renewal and differentiation, contain rich biological information. Traditional cell research methods often struggle to accurately simulate the complex physiological environment in vivo, while organoid models, as a new three-dimensional cell culture technology, can highly reproduce the microstructure and partial physiological functions of organs, providing an ideal platform for in-depth study of organ development, disease mechanisms, and drug screening.

[0003] Intestinal tissue is mainly composed of various epithelial cells, and its organoids can be generated by differentiation of pluripotent stem cells or by tissue-derived stem cells, exhibiting physiological characteristics such as secretion, absorption, barrier, and innate immune signaling. However, current methods for effectively isolating porcine jejunal crypts and successfully converting them into organoid models still have many limitations, such as damage to crypt structure during isolation, high cost of isolation and culture, and unstable success rate of organoid construction, which severely hinders the in-depth study of related research and the transformation process of clinical application.

[0004] Therefore, how to develop an efficient, stable, and suitable crypt isolation and organoid model construction method for pigs of various ages is a problem that needs to be solved by those skilled in the art. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a method for isolating porcine jejunal crypts and constructing organoid models to address the shortcomings of the prior art.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A method for isolating porcine jejunal crypts and constructing organoid models, specifically comprising the following steps: (1) Intestinal collection: pre-cool the DPBS buffer containing penicillin-streptomycin- gentamicin, and aseptically collect the middle section of the jejunum; (2) Preparation of washing solution: sterilize the DPBS solution under high pressure and pre-cool it, and prepare the DPBS solution containing 2% penicillin-streptomycin- gentamicin; (3) Intestinal tract processing: trim the intestinal tract tissue into intestinal segments, add the cleaning solution to cover the intestinal tract tissue, cut the intestinal tract tissue longitudinally, and scrape off the intestinal villi completely; (4) Tissue trimming: add the cleaning solution into the centrifugal tube, trim the intestinal tract tissue after scraping off the villi into a long strip, and then add it into the centrifugal tube; (5) Tissue cleaning: suck the jejunal tissue at the bottom of the centrifugal tube, quickly blow and beat, and then after the tissue naturally settles at the bottom of the centrifugal tube, suck the supernatant and discard it, and repeat the above steps; (6) Digestion solution preparation: put EDTA-2Na powder into sterilized DPBS solution, adjust the pH to 8.0, and then add TCEP after pre-cooling; (7) Tissue digestion: transfer the cleaned jejunal tissue into the centrifugal tube, add the digestion solution, and digest; (8) Cell cluster separation: pour out the digestion solution, add the cleaning solution, vortex, stand, suck the cleaning solution, and mark it as fraction 1, repeat the above steps 6 times, and collect 6 fractions; (9) Crypt separation: filter the collected fractions 3-5; (10) Crypt counting: dilute fractions 3-5 using the cleaning solution; (11) Crypt collection: centrifuge the crypt solution, and retain the crypt precipitate; (12) Crypt inoculation: add the culture medium to the rinsed crypt cluster, blow and scatter the crypt cluster, then add the matrix glue, uniformly blow and beat, mix, and then drop into the preheated 24-well plate; (13) Organoid culture: incubate the organoid complete medium and the inoculated 24-well plate at the same time, inject the organoid complete medium into the well, and then culture the 24-well plate.

[0007] Further, in the above step (3), the length of the intestinal segment is 5-7 cm.

[0008] Further, in the above step (4), the length of the long strip is 2-3 mm.

[0009] Further, in the above step (5), the number of times of quick blowing and beating is 15-20 times; and the number of times of repeating the above steps is 15-20 times.

[0010] Further, in the above step (6), the dosage ratio of EDTA-2Na powder, DPBS solution, and TCEP is 100 mg: 100 mL: 30 mg; and the reagent for adjusting the pH is NaOH solution.

[0011] Further, in the above step (7), the volume ratio of the jejunal tissue and the digestion solution is 1:3; and the digestion device is a horizontal shaker with a speed of 70 r / min for 20 min.

[0012] Further, in the above step (8), the cleaning solution contains 0.1% BSA; the number of vortex oscillations is 2.

[0013] Further, in the above step (10), dilution is performed to contain 20-30 crypts per 20 μL of cleaning solution.

[0014] Further, in the above step (11), the centrifugation condition is 4℃, 200g, 5min.

[0015] Further, in the above step (13), the incubation temperature is 37℃, and the incubation time is 25min; the culture condition is 37℃, 5% CO2.

[0016] According to the above technical solution, compared with the prior art, the beneficial effects of the present application are as follows: 1. The present application continuously attempts to isolate the jejunal crypts of Luchuan pigs of different ages, and optimizes the in vitro induction conditions for the differentiation of jejunal crypts into organoids by using L-wrn cells, thereby establishing a method suitable for the isolation of pig jejunal crypts and 3D organoid culture.

[0017] 2. In the process of intestinal crypt isolation, penicillin, streptomycin and gentamicin are added to avoid contamination caused by intestinal microorganisms during the later culture process. By optimizing the speed and time of the digital display horizontal shaker treatment and the number and time of vortex, the morphology of the obtained crypts is more complete, and the sphere formation rate and survival rate are higher. The method for isolating intestinal crypts of the present application is suitable for pigs of various ages and different breeds, reducing the insufficient number of crypts caused by the reduction of test samples. At the same time, the present application optimizes the culture medium components of intestinal organoids by using L-wrn cell lines, greatly reducing the cost of culture medium, and laying a good foundation for subsequent research on intestinal 3D organoids induced by crypts to study intestinal virus and bacterial infection.

[0018] 3. Luchuan pigs, as a high-quality local pig breed in my country, are characterized by their small size, tolerance to roughage, strong disease resistance, and excellent meat quality, making them an ideal model for studying pig intestinal development and immune function. This invention systematically optimizes the isolation and 3D organoid culture of jejunal crypts in Luchuan pigs, significantly improving the integrity and activity of jejunal crypts and effectively increasing crypt pellet formation efficiency and culture stability in pigs of different ages. Simultaneously, to clarify the culture conditions for porcine jejunal organoids and further reduce culture costs, this invention uses L-wrn cell conditioned medium as the source of Wnt3a, R-spondin1, and Noggin, significantly reducing the amount of exogenous recombinant proteins used, thereby greatly reducing the cost of medium preparation. The porcine jejunal organoids obtained through this method have a typical three-dimensional sac-like structure and possess complete epithelial polarity and functional cell types, including microvilli, goblet cells, Paneth cells, and endocrine cells, exhibiting good barrier function and drug metabolism activity. This invention provides a reliable in vitro model platform for the study of pig intestinal development, nutrient absorption, pathogen infection mechanisms, and precision medicine. It also lays the technical foundation for the large-scale application of organoids and has important scientific significance and clinical value. Attached Figure Description

[0019] Figure 1 A flowchart illustrating the process for generating porcine jejunal organoid culture medium using the L-wrn cell line; Figure 2 The images are of Luchuan pigs, intestinal tissue, and jejunal tissue. A represents Luchuan pigs; BC represents intestinal tissue from necropsy; and D represents jejunal tissue. Figure 3 The process involves the handling and cleaning of jejunal tissue, where AC represents the removal of villi from jejunal tissue; and D represents jejunal tissue scraped to a semi-transparent state and then trimmed. Figure 4 For cell cluster separation and fraction collection, where A represents the mechanical separation of cell clusters after tissue digestion using a vortex mixer; and B represents the collected fraction. Figure 5 These are freshly isolated porcine intestinal crypts. AB represents fresh intestinal crypts observed under a 20X inverted microscope; CD represents fresh intestinal crypts observed under a 40X inverted microscope. Red arrows in the figure indicate crypt fragments, while black arrows indicate intact crypt tissue suitable for 3D culture. Figure 6 The images show jejunal organoids (40X) formed by 3D culture of crypts, where AB represents jejunal organoids cultured to day 1 and CD represents jejunal organoids cultured to day 4. Figure 7H&E staining identification of jejunal tissue and jejunal organoids, where AB are the H&E staining results of the original jejunal tissue; CD are the H&E staining results of the jejunal organoids; in the figure, black arrows represent inflammatory cells, red arrows represent goblet cells, and blue arrows represent intestinal epithelial cells; Figure 8 Immunofluorescence identification of jejunal organoids, where ChgA is chromogranin A, Lyz is lysozyme, Villin is villin-1, and Muc2 is type II intestinal mucin. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1: Isolation and organoid culture of porcine jejunal crypts 1. Experimental materials Fresh jejunal tissue from healthy pigs, taken from 9-day-old Luchuan pigs free of specific pathogens. Figure 2 Animal A was purchased from Beiguang Village, Taigu County, Jinzhong City, Shanxi Province. After fasting for 24 hours, it was eviscerated at the Experimental Teaching Center of Animal Medicine, Shanxi Agricultural University.

[0022] 2. Separation of small recesses (1) Intestinal collection: DPBS buffer containing penicillin-streptomycin-gentamicin was added to a 50mL centrifuge tube and pre-cooled in an ice box. The mid-section of the jejunum was aseptically collected and quickly transferred to a 50mL centrifuge tube. Figure 2 (BD).

[0023] (2) Preparation of cleaning solution: Autoclave 2-3L of sterile DPBS solution and pre-cool it on an ice box. Prepare DPBS solution containing 2% penicillin-streptomycin-gentamicin in the laminar flow hood according to the sample volume.

[0024] (3) Intestinal processing: Take intestinal tissue from a 50mL centrifuge tube, trim the tissue into 5-7cm segments with surgical scissors and place them in a 15cm culture dish. Add the prepared washing solution to cover the intestinal tissue, cut the intestinal tissue longitudinally, and you can see that the intestinal villi are evenly distributed in the intestinal lining in a tongue-like shape. Use a scalpel to scrape off the intestinal villi. Figure 3 AC (Chinese)

[0025] (4) Tissue trimming: Add 30mL of cleaning solution to a 50mL centrifuge tube. Use pointed forceps to pick up the intestinal tissue after the villi have been scraped off and place it above the 50mL centrifuge tube. Use surgical scissors to trim the jejunal tissue into strips of 2-3mm and drop them evenly into the centrifuge tube.

[0026] (5) Tissue cleaning: Use a 1.5 mL Pasteur dropper to quickly aspirate the jejunal tissue that has settled to the bottom of the centrifuge tube, and quickly blow it 15-20 times. After the tissue settles naturally to the bottom of the centrifuge tube, use a Pasteur dropper to aspirate 20 mL of supernatant and discard it. Repeat the above steps 15-20 times.

[0027] (6) Preparation of Digestive Solution: Most digestive solutions for separating intestinal crypts still primarily consist of collagenase and dispersant enzymes. While enzyme preparations are superior to EDTA methods for crypt separation, their high cost makes them unsuitable for large-scale animal crypt separation. Single EDTA is insufficient to break disulfide bonds between cellular proteins during crypt separation, preventing crypts from detaching completely from the intestinal tissue. Therefore, this invention modifies and adjusts the solution to prepare a digestive solution capable of separating crypts from various animals. The digestive solution is prepared as follows: Accurately weigh 100 mg of EDTA-2Na powder and place it in 100 mL of sterile DPBS solution. Adjust the pH to 8.0 using NaOH solution; at this point, all EDTA-2Na should be observed to have dissolved. Cool the solution on ice, and accurately weigh 30 mg of TCEP and add it to the solution. The solution is now ready.

[0028] (7) Tissue digestion: Add the cleaned jejunal tissue to a 50mL centrifuge tube, and then add an appropriate amount of digestion solution at a tissue:digestion solution volume ratio of 1:3. Figure 3 (D) Place 50 mL centrifuge tubes on ice and digest on a horizontal shaker at 70 r / min for 20 min.

[0029] (8) Cell cluster separation: Remove the digested jejunal tissue from the shaker, carefully pour out the digestion solution in a laminar flow hood, add 20 mL of washing solution containing 0.1% BSA to the 50 mL centrifuge tube, place the centrifuge tube on a vortex mixer, and repeat twice. Figure 4 (A) After shaking, let the liquid stand until the intestinal tissue settles naturally to the bottom of the centrifuge tube. At this point, the washing solution will appear cloudy. Figure 4 (B) Transfer 10 mL of the washing solution to a new 50 mL centrifuge tube, place it on ice, and label it fraction 1. Repeat the above steps 6 times to collect a total of 6 fractions, and place each fraction on ice.

[0030] (9) Crypt separation: Collect 3-5 fractions in a clean bench and filter them using a 100μm filter screen.

[0031] (10) Crypt counting: Take 20 μL of each of the filtered fractions 3-5 and examine and count them under an optical microscope. Figure 5 The counting principle is based on the number of complete crypts. Combining the counting results, the fraction is diluted 3-5 times with cleaning solution to achieve an optimal density of 20-30 crypts per 20 μL of cleaning solution. Simultaneously, during this step, the matrix gel, which was stored at -80°C, is incubated directly on ice.

[0032] (11) Crypt collection: Transfer the collected crypt solution of appropriate density to 15 mL centrifuge tubes, 10 mL to each centrifuge tube, and centrifuge at 4℃ and 200g for 5 min, retaining the crypt precipitate.

[0033] 3. Preparation of porcine jejunal organoid culture medium To determine the optimal conditions for culturing porcine jejunal organoids, this invention references various mammalian organoid culture systems and optimizes them based on the characteristics of porcine tissue. Simultaneously, the use of Wnt3a, R-spondin1, and Noggin proteins secreted by the L-wrn transgenic cell line as exogenous signaling supplements not only saves costs but also significantly improves the stability and reproducibility of the culture system. Specific preparation steps are as follows... Figure 1 As shown.

[0034] 4. 3D Culture of Porcine Jejunal Organoids (1) Crypt inoculation: Preheat the 24-well plate in a 37°C culture medium, pre-cool 2-3 mL of DMEM / F12 culture medium, take 200 μL and add it to the crypts after rinsing. Use surgical scissors to trim and enlarge the tip of the 200 μL pipette, gently blow away the crypt clumps, and then transfer them to 1.5 mL EP tubes. Pre-cool on ice, then take 200 μL of matrix gel and gently add it along the wall of the EP tube. Stir first and then blow evenly 10 times. Finally, add 40 μL vertically to each well of the preheated 24-well plate.

[0035] (2) Organoid culture: The complete organoid culture medium and the inoculated 24-well plate were placed in a 37°C incubator for incubation. After 25 minutes, the plate was removed. At this time, it can be observed that the inoculated matrix gel still retains a certain fluidity. The complete organoid culture medium was slowly injected into the well at 750 μL per well along the well wall. Then the 24-well plate was placed in a 37°C, 5% CO2 environment for culture.

[0036] Example 2: Identification of porcine intestinal organoids 1. Organoid fixation (1) Organoid observation: The intestinal crypts of inoculated pigs formed 3D spherical structures as quickly as 8 hours after inoculation, such as... Figure 6As shown, standard vacuolar organoids are formed after 12 hours, and no new organoids will be generated after 24 hours. At this time, most organoids are predominantly round, with a small number being polygonal vacuolars.

[0037] (2) Organoid collection: Count the organoids in the corresponding 24-well plates under a microscope. Remove the culture medium with a pipette, add 1-2 mL of pre-chilled PBS solution to each well, and gently pipette the matrix gel. Collect approximately 1500-2000 organoids into 15 mL centrifuge tubes and incubate at 4°C for 10 min. Then centrifuge at 4°C and 200g for 5 min and discard the supernatant. Resuspend in 1 mL of pre-chilled PBS and transfer to a 1.5 mL centrifuge tube. Centrifuge at 4°C and 200g for 5 min and discard the liquid.

[0038] (3) Organoid fixation: Add 4% paraformaldehyde to the centrifuge tube, gently blow and mix well, place in the refrigerator, fix for 1 hour, centrifuge at 4°C and 200g for 5 minutes and discard the supernatant, then add pre-cooled PBS to resuspend and transfer to a new 1.5mL centrifuge tube, centrifuge at 4°C and 200g for 5 minutes and retain the precipitate.

[0039] (4) Organoid embedding: Accurately weigh 0.2g of agarose and put it into a beaker. Add 10mL of PBS and melt it completely in a microwave oven on high. After the agarose solution cools to 50-60℃, take 30μL to resuspend the organoid precipitate. Avoid generating bubbles during this process and quickly place it on ice to solidify.

[0040] (5) Paraffin sectioning: After trimming the embedded organoid paraffin blocks, place them on a tissue microtome. First, trim the paraffin blocks using a 6μm cutting interval. After the tissue is exposed, adjust the interval to 4μm for paraffin sectioning. Place the prepared paraffin sections in warm water at 42℃. After they are fully expanded, use a glass slide to retrieve the sections, and then place them on a tissue section drying apparatus at 60℃ for 1-2 hours.

[0041] 2. Organoid H&E staining (1) Dewaxing to water: Xylene I 15min → Xylene II 15min → Anhydrous ethanol I 5min → Anhydrous ethanol II 5min → 95% ethanol I 3min → 95% ethanol II 3min → 90% ethanol 3min → 80% ethanol 2min → 70% ethanol 2min → 50% ethanol 2min → Wash with distilled water for 2min.

[0042] (2) H&E staining: Hematoxylin staining solution 3 min → rinse with tap water for 7 min → differentiate with 1% hydrochloric acid alcohol for 2 s → rinse with tap water for 10 min → 70% ethanol for 2 min → 80% ethanol for 2 min → 90% ethanol for 2 min → eosin staining solution 1 min → 95% alcohol I for 4 min → 95% ethanol II for 4 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → xylene I for 10 min.

[0043] 3. Immunofluorescence identification of organoids (1) Antigen retrieval: The processed organoid paraffin sections were soaked in 3% H2O2 for 10 minutes to remove endogenous catalase. After discarding the H2O2, the sections were rinsed twice with water and then citrate buffer was added. The sections were then placed in a microwave oven and heated at level 3 for 3 minutes. If boiling was observed beforehand, the heat was turned off. This step was repeated twice.

[0044] (2) Serum blocking: After cooling to room temperature, rinse the organoid sections with tap water for 5 minutes. Repeat this step twice. Then, place the sections in sterile PBS to wash and rinse twice. After drying the PBS solution around the tissue, add serum diluted 10 times and incubate in a constant temperature incubator at 37°C for 30 minutes.

[0045] (3) Primary antibody incubation: Take out the slide from the incubator, wipe the serum around the organoid tissue on the slide with absorbent paper, add the diluted primary antibody (1:1900) and incubate overnight at 4°C.

[0046] (4) Secondary antibody incubation: After incubating with primary antibody overnight, wash with PBS for 3 min. Repeat this step 3 times. After absorbing excess liquid on the slide, add diluted fluorescent secondary antibody. Incubate in a humidified chamber at 37°C in the dark for 1 h. Wash with PBS for 3 min. Repeat this step 3 times.

[0047] (5) Fluorescent double staining: Repeat the above steps with primary antibodies of the same species, but attention should be paid to the selection of fluorescence for secondary antibodies.

[0048] (6) Counterstaining and mounting: After the secondary antibody incubation, add DAPI and incubate in the dark for 5 min. Then stain the specimen for the nucleus, wash with PBS for 3 min, repeat this step three times, and use anti-quenching mounting medium for mounting.

[0049] 4. Results like Figure 7 As shown, the H&E staining results indicate that the porcine jejunal organoids isolated in this invention have typical organoid morphology, and their tissue structure is vacuolated. The black arrows represent inflammatory cells, the blue arrows represent intestinal epithelial cells, and the red arrows represent goblet cells.

[0050] likeFigure 8 As shown, this immunofluorescence identification employed double staining to identify the cell types contained in the jejunal organoids. ChgA, chromogranin A, showed a positive result indicating that the jejunal organoids induced by this invention contain intestinal endocrine cells capable of performing corresponding secretory and digestive functions. Lyz, lysozyme, showed a positive result indicating that the jejunal organoids induced by this invention contain Paneth cells. Villin, villin-1, whose expression is limited to brush border epithelial cells, is a special structure formed by numerous microvilli, and its positive result indicates that the jejunal organoids induced by this invention possess the structural functions unique to intestinal microvilli. Muc2, type II intestinal mucin, showed a positive result indicating that the jejunal organoids induced by this invention contain goblet cells. These results demonstrate that this invention successfully constructed porcine jejunal organoids with typical histological characteristics and functional cell composition.

[0051] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for isolating porcine jejunal crypts and constructing organoid models, characterized in that, Specifically, the following steps are included: (1) Intestinal collection: The middle section of the jejunum was aseptically collected after pre-cooling DPBS buffer containing penicillin-streptomycin-gentamicin. (2) Preparation of cleaning solution: Autoclave sterile DPBS solution and pre-cool it in advance, and prepare DPBS solution containing 2% penicillin-streptomycin-gentamicin; (3) Intestinal treatment: Trim the intestinal tissue into segments, add a cleaning solution to cover the intestinal tissue, cut the intestinal tissue longitudinally, and scrape off the intestinal villi cleanly; (4) Tissue trimming: Add the cleaning solution to the centrifuge tube, trim the intestinal tissue after scraping off the villi into long strips and add it to the centrifuge tube; (5) Tissue cleaning: Aspirate the jejunal tissue that has settled to the bottom of the centrifuge tube, blow it quickly, and after the tissue settles naturally to the bottom of the centrifuge tube, aspirate and discard the supernatant, and repeat the above steps. (6) Preparation of digestion solution: Place EDTA-2Na powder in sterile DPBS solution, adjust the pH to 8.0, pre-cool, and then add TCEP; (7) Tissue digestion: Transfer the cleaned jejunal tissue to a centrifuge tube, add digestive juice, and digest; (8) Cell cluster separation: Pour out the digestion solution, add the washing solution, vortex and shake, let stand, aspirate the washing solution and label it as fraction 1. Repeat the above steps 6 times to collect a total of 6 fractions. (9) Crypt separation: Filter the collected fraction 3-5; (10) Crypt counting: Dilute fraction 3-5 with cleaning solution; (11) Crypt collection: Centrifuge the crypt solution and retain the crypt precipitate; (12) Crypt inoculation: Add the culture medium to the rinsed crypt clumps, blow away the crypt clumps, then add the matrix gel, blow evenly, mix well, and drop into the preheated 24-well plate. (13) Organoid culture: The complete organoid culture medium and the inoculated 24-well plate were incubated at the same time. The complete organoid culture medium was injected into the wells, and then the 24-well plate was cultured.

2. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (3), the length of the intestinal segment is 5-7 cm.

3. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (4), the length of the strip is 2-3 mm.

4. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (5), the number of times the rapid blowing is performed is 15-20 times; the number of times the above steps are repeated is 15-20 times.

5. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (6), the ratio of EDTA-2Na powder, DPBS solution and TCEP is 100mg:100mL:30mg; the pH adjustment reagent is NaOH solution.

6. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (7), the volume ratio of the jejunal tissue to the digestive fluid is 1:3; the digestion equipment is a horizontal shaker with a rotation speed of 70 r / min and a time of 20 min.

7. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (8), the cleaning solution contains 0.1% BSA; the vortex oscillation is performed twice.

8. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (10), the dilution is made to contain 20-30 crypts per 20 μL of cleaning solution.

9. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (11), the centrifugation conditions are 4°C, 200g, and 5min.

10. The method for isolating porcine jejunal crypts and constructing organoid models according to claim 1, characterized in that, In step (13), the incubation temperature is 37°C and the time is 25 min; the culture conditions are 37°C and 5% CO2.

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