A tgev challenge method based on a pig intestinal tract organoid reverse model and application thereof

Abstract

The application discloses a TGEV infection method based on a pig intestinal tract organoid reverse model and application. The TGEV infection method based on the pig intestinal tract organoid reverse model comprises the following steps: (1) after the Matrigel containing the pig intestinal tract organoid obtained through culture is peeled off from a cell culture plate and broken, the Matrigel is incubated in a 5mM EDTA PBS solution on a shaking table under the condition of 4 DEG C; (2) the supernatant is discarded through centrifugation, the precipitate is reserved, and the precipitate is resuspended in a pig colon organoid culture medium to obtain a pig intestinal tract organoid resuspension; (3) the pig intestinal tract organoid resuspension is transferred to an ultralow adsorption cell culture plate and is cultured in suspension under the condition of 37 DEG C and 5% CO2; (4) the operation of step (2) and step (3) is repeated to obtain a pig intestinal tract organoid reverse model; and (5) TGEV virus liquid is directly added to the pig intestinal tract organoid reverse model to perform soaking type infection, the infection dose is MOI=1, and the infection time is 24h.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method and application of TGEV challenge based on a porcine intestinal organoid inversion model. Background Technology

[0002] Transmissible gastroenteritis virus (TGEV) is a coronavirus that seriously threatens the swine industry. It primarily infects the small intestinal epithelial cells of piglets, causing diarrhea, dehydration, and death. In-depth research into the pathogenic mechanisms of TGEV and the development of effective prevention and control measures are crucial. Traditional two-dimensional cell line models (such as IPEC-J2) cannot realistically simulate the three-dimensional structure and complex functions of the in vivo intestine, while animal experiments suffer from drawbacks such as high cost, long duration, and ethical restrictions.

[0003] Organoid technology has provided a revolutionary in vitro tool for virological research. However, traditional three-dimensional organoids, upon maturation, form closed sac-like structures, with the apical membrane containing viral receptors encased in an internal chamber, while the basolateral membrane is exposed to the external culture medium. This "apical-inward" polarity prevents viruses from reaching the target site through simple immersion, necessitating complex and low-throughput microinjection techniques, which greatly limits their application in viral challenge and high-throughput screening.

[0004] While existing technologies have reported polarity reversal (i.e., inducing apical-out structures) in human or mouse intestinal organoids, porcine intestinal organoids differ significantly from humans or mice in growth factor requirements, matrix gel dependence, and cell biological characteristics. Directly applying existing reversal protocols leads to low survival rates, poor reversal efficiency, or model instability in porcine organoids. In other words, current technologies lack a standardized reversal and culture system specifically for the "pig colon." Therefore, there is an urgent need in this field for a stable and reliable polarity reversal model specifically for pig colon organoids, as well as a standardized TGEV challenge and evaluation system based on this model. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of the prior art and provide a method and application of TGEV challenge based on a porcine intestinal organoid inversion model.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows: The TGEV challenge method based on the porcine intestinal organoid inversion model includes the following steps: (1) After the matrix gel containing the cultured porcine intestinal organoids was peeled off from the cell culture plate and broken up, it was incubated in a shaker in PBS solution with 5 mM EDTA at 4°C. (2) After incubation in step (1), centrifuge and discard the supernatant, retain the precipitate, and then resuspend the precipitate in porcine colon organoid culture medium to obtain porcine intestinal organoid resuspension; (3) Transfer the resuspension of porcine intestinal organoids to an ultra-low adsorption cell culture plate and culture it in suspension at 37°C and 5% CO2. (4) Repeat steps (2) and (3) on the product after suspension culture in step (3) to obtain a pig intestinal organoid inversion model; (5) TGEV virus solution was directly added to the pig intestinal organoid inversion model for immersion infection. The infection dose was MOI=1 and the infection time was 24h.

[0007] Preferably, the porcine intestinal organoid is a porcine colon organoid.

[0008] Preferably, the disruption in step (1) is achieved by pipetting a pre-cooled 5 mM EDTA PBS solution onto the matrix gel.

[0009] Preferably, the conditions for incubation in the shaker in step (1) are: incubation time of 1 hour and shaker speed of 60 r / min.

[0010] Preferably, the centrifugation conditions in step (2) are 4°C, 300×g, and centrifugation for 3 minutes.

[0011] Preferably, the porcine colon organoid culture medium in step (2) is prepared by adding 1×N2, 1×B27, epidermal growth factor, glutamine, hydroxyethylpiperazine ethionine, nicotinamide, Y-27632, SB202190, CHIR99021, and A83-01 to a culture medium in which PWL038 / cell supernatant and Advanced DMEM / F12 are mixed at a volume ratio of 1:1. The concentrations of 1×N2, 1×B27, EGF, glutamine, HEPES, nicotinamide, Y-27632, SB202190, CHIR99021, and A83-01 in the complete colon organoid culture medium are 1×N2, 1×B27, 50 ng / mL, 2 mM, 10 mM, 10 mM, 10 μM, 10 μM, 2.5 μM, and 0.5 μM, respectively.

[0012] Preferably, the operation of repeating steps (2) and (3) on the product after suspension culture in step (4) refers to suspending the product in step (3) for 1-7 days, changing the porcine colon organoid culture medium every 3 days, then centrifuging the product after suspension culture according to the centrifugation conditions described in step (2), discarding the supernatant and retaining the precipitate, resuspending the precipitate with porcine colon organoid culture medium, and continuing suspension culture in an ultra-low adsorption cell culture plate at 37°C and 5% CO2 to obtain a porcine intestinal organoid inversion model.

[0013] The present invention also provides a pig intestinal organoid inversion model, which is a pig intestinal organoid inversion model prepared by the aforementioned steps (1) to (4).

[0014] The porcine intestinal organoid inversion model described in this invention can be used to screen drugs for the prevention and / or treatment of TGEV infection.

[0015] The present invention will be further described below: This invention first provides a method for constructing a specifically optimized polarity-reversed porcine colonic organoid model. By using a specific concentration of EDTA / PBS solution to gently dissociate the matrix gel at low temperature, combined with ultra-low adsorption suspension culture, a apical-facing organoid model was successfully constructed. This model solves the problem of traditional porcine intestinal organoids being difficult to directly challenge with viruses due to the encapsulated apical structure. Building upon this, this invention further provides a method for TGEV immersion challenge using this inversion model and establishes a multidimensional evaluation system encompassing intestinal physical barriers, chemical barriers, and absorption function genes. This invention provides a highly biomimetic, stable, and reliable in vitro platform for studying the pathogenic mechanism of porcine transmissible gastroenteritis virus (TGEV), screening antiviral drugs, and evaluating vaccine potency.

[0016] Traditional organoids contain their viral receptors internally, making direct viral attack difficult. This invention, however, achieves "polarity reversal" through a specific dissociation and suspension culture method, flipping the brush border (apex) of the intestinal epithelium to the outside. This allows TGEV virus to efficiently infect cells directly from the culture medium without microinjection. In other words, this invention overcomes the structural limitation of traditional intestinal organoids, which are "apex-inward," and constructs an in vitro model that more closely reflects the physiological process of viral infection. Furthermore, compared to the single IPEC-J2 cell line, this in vitro model is derived from the colonic crypts of piglets, preserving various cell types such as absorptive cells and goblet cells, as well as complex three-dimensional structures, thus more realistically reproducing the physiological characteristics of the piglet intestine.

[0017] The purpose of the challenge step in this invention is to verify the biological sensitivity of the model and simulate pathological damage. Its core application lies in providing a high-throughput platform for antiviral drug screening and pathogenic mechanism research. Experimental data show that after challenge, the model not only exhibits a significant dose-dependent increase in viral load but also accurately reproduces the intestinal barrier disruption (significant downregulation of tight junction proteins such as ZO-1 and Claudin-1) and impaired mucus secretion function (decreased MUC2 expression) caused by TGEV infection. Therefore, this invention can be widely used to evaluate the efficacy of antiviral drugs, screen nutritional supplements that can repair the intestinal barrier, and conduct in-depth research on the molecular mechanisms by which viruses interfere with intestinal absorption (such as changes in FABP2 and SI genes).

[0018] Compared to existing technologies, although organoid polarity reversal in other tissues (such as human or mouse intestines) also follows the general biological principle of "removing Matrigel to induce apical-out self-assembly," the significant difference of this invention lies in the specific optimization of key process parameters for the specific species of piglet colon (establishing a dissociation scheme of 5 mM EDTA combined with a specific time window), solving the problem of easy disintegration or low recombination efficiency of porcine cells during the reversal process. More importantly, existing technologies mostly remain at the model construction level, while this invention standardizes and integrates the reversal model with TGEV challenge and a multi-dimensional quantitative evaluation system (covering specific gene indicators such as ZO-1 and MUC2), thereby overcoming the defect that traditional porcine intestinal organoids must rely on microinjection for infection. It provides a set of in vitro screening platforms specifically for porcine coronaviruses that can realistically simulate intestinal mucus barrier damage, which is a specific application area not yet covered by current general organoid reversal technology. Attached Figure Description

[0019] Figure 1 The process of polarity reversal in piglet colon organoids is illustrated.

[0020] Figure 2 Three-dimensional images of F-actin fluorescent staining of piglet colonic polarity-reversed organoids are shown. In the images, A represents a matrix gel-embedded organoid (unreversed); B represents a reversed organoid cultured for 72 hours.

[0021] Figure 3 The images show F-actin fluorescence staining at different time points in the culture of piglet colon polarity-reversed organoids. In the images, A represents matrix gel-embedded organoids (unreversed); B represents reversed organoids cultured for 24 hours; C represents reversed organoids cultured for 48 hours; and D represents reversed organoids cultured for 72 hours.

[0022] Figure 4 The results of RT-PCR identification of polarity-reversed organoids in the colon of piglets infected with different doses of TGEV are shown.

[0023] Figure 5 The results of RT-PCR for polarity-reversed organoids in the colon of piglets infected with different doses of TGEV are shown. ZO-1, Claudin-1, and Occludin represent the intestinal barrier, SI and FABP2 represent intestinal absorption, and MUC2 represents intestinal goblet cells. Detailed Implementation

[0024] To make the objectives, technical solutions, and beneficial effects of the embodiments of the present invention clearer, further descriptions will be provided below in conjunction with specific implementations of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Other implementation methods obtained by those skilled in the art based on the embodiments disclosed in the present invention without creative effort should all fall within the scope of protection of the present invention.

[0025] Example 1 See the process of piglet colon polarity reversal organoids. Figure 1 The TGEV challenge method based on the porcine intestinal organoid inversion model includes the following: 1.1 Isolation and culture of crypt stem cells from piglet colon organoids (1) Take a colon of about 3-5 cm from a 21-day-old Duroc Large White Piglet, cut it open longitudinally, rinse the contents with tap water, place it in PBS pre-cooled at 4 °C, and transfer it to a clean bench.

[0026] (2) Place the intestinal segment in a culture dish containing 4 °C Duchenne phosphate buffer (DPBS) and gently scrape off the villi with a glass slide.

[0027] (3) Transfer the intestinal segment to a new culture dish containing 4 °C DPBS and use a glass slide to scrape out the mucosal layer containing crypts.

[0028] (4) Cut the mucosal layer into 1-2 mm fragments and place them in a 50 mL centrifuge tube containing 4 °C DPBS.

[0029] (5) Wash the fragments 15-20 times with 4 °C DPBS until the supernatant is clear.

[0030] (6) Add 2 volumes of crypt dissociation solution (using Wash media to add Gibco collagenase type 1 at a concentration of 2 mg / mL) to the fragments after removing the supernatant, so that the dissociation solution is in full contact with the fragments, and incubate at 37°C for 40 min.

[0031] (7) Shake the centrifuge tube containing the dissociation solution and fragments vigorously, then add 8 mL of Gibco DMEM / F12 medium, filter using a 70 μm sieve, and collect the supernatant containing crypts into a new centrifuge tube.

[0032] (8) Centrifuge at 4 °C and 200 g for 5 min.

[0033] (9) Remove the supernatant, add 5 mL of DMEM / F12 culture medium, and gently mix with a pipette.

[0034] (10) Repeat step (8).

[0035] (11) Repeat step (9).

[0036] (12) Repeat step (8).

[0037] (13) Remove the supernatant and add colon organoid complete culture medium and Gibco matrix gel at a volume ratio of 1:1 according to the density of the crypts under microscopic examination. Mix gently (this operation should be performed in an ice box) to obtain the crypt suspension.

[0038] (14) Spot the above crypt suspension into a well plate that has been preheated to 37 °C (wall treatment), 25-50 μL / dome.

[0039] (15) Place the above well plate in an incubator at 37 °C and 5% CO2 for 10-15 min to fix the dots.

[0040] (16) Add complete culture medium for colon organoids (volume exceeding the dot) along the well wall and incubate in an incubator at 37 °C and 5% CO2.

[0041] 1.2 Passage of piglet colon organoids (1) Preparation before operation: Pre-cool the centrifuge to 4°C, determine the passage ratio according to organoid density and growth status, and preheat the culture plate in a 37°C incubator.

[0042] (2) After culturing the plate for 7 days in step (16) of section 1.4, discard the culture medium, wash once with pre-cooled PBS containing 0.5 mM EDTA, and discard the liquid. Add 200 μL of 0.25% trypsin without phenol red (diluted from 2.5% trypsin) to each well to cover the bottom of the well, and gently blow the matrix gel with a pipette to disperse it, being careful to control the force to avoid damaging the organoid structure.

[0043] (3) Place the treated culture plate in a 37°C incubator for 5 min to digest, and determine whether the number of blows needs to be adjusted based on observation under a microscope.

[0044] (4) Add about 500 μL of Wash media to stop digestion, and blow the colonic organoids 30 times.

[0045] (5) Transfer all liquid (organoid suspension) to a 15 mL centrifuge tube and centrifuge at 200 × g, 4°C for 5 min.

[0046] (6) After discarding the supernatant, wash once with cold wash media and repeat centrifugation (a 1.5 mL centrifuge tube can be used if the cell volume is small).

[0047] (7) After centrifugation, observe the precipitate, add 50-100 μL of complete culture medium (CM) and gently resuspend by pipetting, and assess the cell density under a microscope.

[0048] (8) Perform the operation on ice, add an equal volume of pre-cooled matrix gel to the resuspended solution, mix gently to avoid generating air bubbles.

[0049] (9) Take out the preheated culture plate and spot 25 μL of the sample in the center of each well. If air bubbles appear, remove them in time.

[0050] (10) Place the culture plate after spotting in a 37°C incubator for 8-10 min to allow the droplets to solidify.

[0051] (11) After the gel solidifies, add complete culture medium for colon organoids that covers the dots along the well wall, and put it back into the incubator for further culture.

[0052] Complete colon organoid culture medium: This medium was prepared by adding 1×N2, 1×B27, epidermal growth factor (EGF), glutamine, hydroxyethylpiperazine ethanethioic acid (HEPES), nicotinamide, Y-27632, SB202190, CHIR99021, and A83-01 to a mixture of PWL038 / cell supernatant and Advanced DMEM / F12 (Gibco, Thermo Fisher Scientific) at a volume ratio of 1:1. The concentrations of 1×N2, 1×B27, EGF, glutamine, HEPES, nicotinamide, Y-27632, SB202190, CHIR99021, and A83-01 in the complete colon organoid culture medium were 1×N2, 1×B27, 50 ng / mL, 2 mM, 10 mM, 10 mM, 10 μM, 10 μM, 2.5 μM, and 0.5 μM, respectively.

[0053] 1.3 Cryopreservation of piglet colon organoids (1) The operation is the same as steps (1)-(7) in Section 1.2 “Passage of pig colon organoids”, that is, complete organoid digestion, termination reaction, blowing, transfer, centrifugation and preliminary washing.

[0054] (2) Observe the centrifuged organoid precipitate under a microscope, assess its density and morphology, and perform cryopreservation after confirming that it is in good condition.

[0055] (3) Prepare organoid cryopreservation solution by mixing colon organoid complete culture medium: Gibco fetal bovine serum: dimethyl sulfoxide (DMSO) = 7:2:1 by volume.

[0056] (4) Add the organoid suspension to an equal volume of organoid cryopreservation solution and gently blow to mix, avoiding the formation of air bubbles.

[0057] (5) Transfer the mixed organoid suspension to a cryovial, place it in a cell cryopreservation box, and freeze it at -80°C.

[0058] (6) After 24 hours, the cryopreservation tubes were transferred from -80°C to liquid nitrogen tanks for long-term storage.

[0059] 1.4 Resuscitation of piglet colonic organoids (1) Preparation before operation: Pre-cool the centrifuge to 4°C, determine the passage ratio according to organoid density and growth status, and preheat the culture plate in a 37°C incubator.

[0060] (2) Remove the cryovial from the liquid nitrogen and immediately place it in a water bath at 37°C to thaw quickly.

[0061] (3) Transfer the thawed cell suspension to a 15 mL centrifuge tube and add 5 mL Wash media and mix well.

[0062] (4) Centrifuge at 200 × g, 4°C for 5 min and discard the supernatant.

[0063] (5) Add 5 mL of fresh Wash media and wash again. Repeat centrifugation at 200 × g and 4°C for 5 min, and discard the supernatant.

[0064] (6) Observe the organoid precipitate after centrifugation, add 50-100 μL of CM culture medium, gently pipette to resuspend, and observe the cell density under a microscope.

[0065] (7) Perform the operation on ice. Add an equal volume of pre-cooled matrix gel to the cell suspension and mix gently to avoid generating air bubbles.

[0066] (8) Take out the preheated culture plate and spot 25 μL of the sample in the center of each well. Be careful to avoid generating air bubbles. If there are air bubbles, you can remove them with the pipette tip.

[0067] (9) Place the culture plate after spotting in a 37°C incubator for 8-10 min to allow the matrix gel to solidify.

[0068] (10) After the gel solidifies, add CM medium (broth medium) without dots along the well wall, and then put it back into the incubator to continue culturing.

[0069] 1.5 Cultivation of TGEV The TGEV strain TS (GenBank accession number DQ201447.1) was kindly provided by the College of Veterinary Medicine of Sichuan Agricultural University and amplified in IPEC-J2 cells.

[0070] TGEV was cultured using IPEC-J2 cells. When the cells reached approximately 90% confluence, TGEV was inoculated and cultured in a 37°C, 5% CO2 incubator. Cytopathic effects were observed daily. When more than 80% of the cells showed cytopathic effects, the cells were repeatedly frozen and thawed three times. The culture medium was collected and centrifuged at 5000 rpm for 20 min at 4°C. The supernatant was collected, and the TGEV viral titer was determined using the TCID50 method.

[0071] 1.6 Piglet Colonic Organoid Polarity Reversal (1) Porcine colon organoids were obtained by organoid differentiation culture from crypt stem cells isolated from the colonic segment of piglets. On the 7th day of culture, the original culture medium was discarded, and the cells were washed once with pre-cooled PBS containing 0.5 mM EDTA and the liquid was discarded.

[0072] (2) Use a pipette to peel the matrix gel containing piglet colon organoids from the bottom of the 24-well plate.

[0073] (3) Add 500 µL of pre-chilled 5 mM EDTA / PBS to each well of the 24-well plate, and use a P1000 pipette to blow and aspirate several times to break up the matrix gel (it is recommended to cut the tip of the pipette by 1-2 mm with scissors to avoid damaging the intact organoid sac-like structure). Transfer the solution from four wells of the 24-well plate to 15 ml conical tubes containing 10-12 ml of pre-chilled 5 mM EDTA / PBS.

[0074] (4) Incubate at 4℃ on a shaker at 60r / min for 1h.

[0075] (5) Centrifuge at 300×g for 3 min at 4℃, discard the supernatant, and resuspend the harvested porcine colon organoids in 1200 µL of porcine colon organoid culture medium per well of a 24-well plate to achieve a passage ratio of one to three. Transfer 400 µL of organoid suspension to each well of a 24-well ultra-low adsorption cell culture plate for suspension culture, shake well in a cross shape, and place in an incubator for culture at 37°C and 5% CO2.

[0076] (6) Culture for 1-7 days, change the medium every 3 days. Transfer the organoids in each well of the 24-well plate of ultra-low adsorption cell culture to a 15mL conical tube using a pipette. Centrifuge at 300×g for 3 min at 4℃. Resuspend the precipitate in fresh porcine colon organoid culture medium and then transfer it to the 24-well plate of ultra-low adsorption cell culture for suspension culture again.

[0077] 1.7 TGEV-induced colonic organoids in piglets After 24 hours of in vitro culture of piglet colonic organoids and the establishment of a polarity reversal model, they were challenged with porcine transmissible gastroenteritis virus (TGEV). The experiment was designed with one control group and three treatment groups, with 6 replicates per group (n=6). The specific grouping and treatment are as follows: (1) Negative control group: No TGEV added; (2) Low-dose infection group: 25 μL of TGEV virus solution was added to each well, and the infection dose was MOI=1; (3) Medium-dose infection group: 50 μL of TGEV virus solution was added to each well, and the infection dose was MOI=1; (4) High-dose infection group: 100 μL of TGEV virus solution was added to each well, and the infection dose was MOI=1.

[0078] After viral inoculation, the 24-well plates were incubated at 37°C with 5% CO2. Samples were collected 24 hours post-infection for subsequent RT-PCR detection. The results are as follows: Figure 4 , Figure 5 As shown, compared with the CON group, the TGEV gene expression levels of TGEV-25, TGEV-50, and TGEV-100 were significantly increased, and the TGEV gene expression level also increased significantly with increasing infection dose.

[0079] 1.8 Extraction of TGEV-induced colonic organoid mRNA from piglets (1) Cell sample collection: Transfer organoids from each well of the 24-well plate of ultra-low adsorption cell culture to a 1.5 mL sterile enzyme-free centrifuge tube using a pipette, centrifuge at 200×g for 5 min at 4℃, add 1 mL of Trizol to each tube using a pipette, and store at -20°C.

[0080] (2) mRNA extraction: a. Add 200 μL of chloroform to each tube containing 1 mL of Trizol colon organoid sample, shake vigorously to mix, let stand at room temperature for 5 min (operate in a fume hood), and centrifuge at 4°C, 12000 × g for 10 min.

[0081] b. Carefully aspirate 200-400 μL of aqueous phase (supernatant) into a new sterile enzyme-free centrifuge tube, add an equal volume of isopropanol, mix by inverting the tube, let stand for 10 min, and centrifuge at 4°C and 12000 × g for 10 min.

[0082] (3) RNA precipitation and purification: c. Discard the supernatant, pour it out slowly and horizontally, and blot the remaining liquid at the tube opening with absorbent paper. Add 1 mL of 75% ethanol to each tube, invert the tube to wash the precipitate, and centrifuge at 4°C and 7500 × g for 5 min.

[0083] d. Discard the alcohol, pour out the contents from top to bottom and absorb the remaining liquid, then immediately separate the tube. Use a pipette to remove any remaining liquid at the bottom, and open the cap to dry at room temperature for 2-3 minutes.

[0084] e. Add 10 μL of DEPC water to dissolve the RNA, incubate at 4°C for 5-10 min, and then resuspend the RNA by pipetting.

[0085] (4) RNA concentration determination: Before testing, zero the instrument with enzyme-free water. After shaking and mixing each sample, take 1 μL to detect the RNA concentration and calculate the required sample volume for subsequent experiments.

[0086] 1.9 Reverse transcription into cDNA Using a reverse transcription kit (Novizan), the RNA extracted and purified in Section 1.8, "Extraction of mRNA from colonic organoids of TGEV-infected piglets," was reverse transcribed into cDNA according to the manufacturer's instructions. The following reaction mixture was added to an enzyme-free eight-tube bundle: - 5 μL 5× HiScript II qRT SuperMix II - RNA sample (500-1000 ng / μL) - Add enzyme-free ddH2O or diethyl pyrocarbonate (DEPC) water to a total reaction volume of 20 μL. After thorough mixing, perform a brief centrifugation to concentrate the liquid at the bottom of the tube.

[0087] Place the reaction tube in the PCR instrument and set the reverse transcription program as follows: Reaction at 50°C for 15 min (reverse transcription); The reaction was terminated at 85°C for 5 seconds.

[0088] 1.10 Real-time quantitative RT-PCR detection Prepare RT-PCR premix according to Table 1 below. Using PPIA as an internal control, calculate the differential levels of each target gene as shown in Table 2 using the 2-ΔΔCt method, and perform quantitative analysis of cDNA.

[0089] Table 1 Real-time quantitative PCR reaction system

[0090] Table 2 Primer sequences used for each target gene.

[0091] 1.11 A method for assessing organ polarity of the piglet colon based on phalloid staining (1) After the piglet colon organoids were reversed for 24 hours, the suspended organoids in each well of the ultra-low adhesion plate were transferred to a 1.5 mL conical microcentrifuge tube, centrifuged at 300×g for 1 min, and the supernatant was removed.

[0092] (2) Gently wash the cells with PBS 1-2 times.

[0093] (3) Fix the cells with 4% paraformaldehyde fixative at room temperature for 10-20 min, centrifuge at 300×g for 1 min, and remove the supernatant.

[0094] (4) Wash with PBS containing 0.1% Triton X-100 2-4 times, 5 min each time.

[0095] (5) Dilute Actin-TrackerDeep Red-633 with PBS containing 1% BSA and 0.1% Triton X-100 at a ratio of 1:40. The diluted solution is the staining working solution.

[0096] (6) Add 100uL of staining working solution, incubate at room temperature in the dark for 30-60 min, centrifuge at 300×g for 1 min, and remove the supernatant.

[0097] (7) Wash with PBS containing 0.1% Triton X-100 2-4 times, 5 min each time.

[0098] (8) Resuspend the organoid in 50-100 µL of anti-fluorescence quenching agent (containing DAPI), slowly resuspend and mix well, and then drop it into the center of the bottom of the confocal dish using a pipette.

[0099] (9) After being stored in the dark at 4°C, it is used for fluorescence microscopy. The maximum excitation wavelength of DAPI in blue light is 340 nm, and the maximum emission wavelength is 488 nm; after DAPI binds to double-stranded DNA, the maximum excitation wavelength is 364 nm, and the maximum emission wavelength is 454 nm. The maximum excitation wavelength of phalloidin in red light is 630 nm, and the maximum emission wavelength is 650 nm.

[0100] (10) The subsequent procedures for 48h and 72h of piglet colon polarity reversal organoid suspension culture were the same as above. The results of phalloid staining were as follows. Figure 2 and Figure 3 As shown. In the unreversed piglet colon organoids, the red fluorescent signal (representing F-actin) is significantly concentrated on the inner side of the lumen (apical surface) (e.g. Figure 2 A and Figure 3 A), while the red fluorescent signal of the pig colon organoid after inversion treatment is concentrated on the outer side of the organoid (e.g., Figure 2 B and Figure 3(B, C, D) indicates that the cell polarity has been flipped by 180°, and the polarity reversal model of Apical-out (apical face outward) has been successfully constructed.

Claims

1. A method for challenging TGEVs based on a porcine intestinal organoid inversion model, characterized in that, The method includes the following steps: (1) After the matrix gel containing the cultured porcine intestinal organoids was peeled off from the cell culture plate and broken up, it was incubated in a shaker in PBS solution with 5 mM EDTA at 4°C. (2) After incubation in step (1), centrifuge and discard the supernatant, retain the precipitate, and then resuspend the precipitate in porcine colon organoid culture medium to obtain porcine intestinal organoid resuspension; (3) Transfer the resuspension of porcine intestinal organoids to an ultra-low adsorption cell culture plate and culture it in suspension at 37°C and 5% CO2. (4) Repeat steps (2) and (3) on the product after suspension culture in step (3) to obtain a pig intestinal organoid inversion model; (5) TGEV virus solution was directly added to the pig intestinal organoid inversion model for immersion infection. The infection dose was MOI=1 and the infection time was 24h.

2. The TGEV challenge method based on the porcine intestinal organoid inversion model as described in claim 1, characterized in that, The porcine intestinal organoids are porcine colon organoids.

3. The TGEV challenge method based on the porcine intestinal organoid inversion model as described in claim 1, characterized in that, The disruption in step (1) is achieved by pipetting a pre-cooled 5 mM EDTA PBS solution onto the matrix gel.

4. The TGEV challenge method based on the porcine intestinal organoid inversion model as described in claim 1, characterized in that, The conditions for incubation in the shaker in step (1) are: incubation time of 1 hour and shaker speed of 60 r / min.

5. The TGEV challenge method based on the porcine intestinal organoid inversion model as described in claim 1, characterized in that, The centrifugation conditions described in step (2) are 4℃, 300×g, and centrifugation for 3 minutes.

6. The TGEV challenge method based on the porcine intestinal organoid inversion model as described in claim 1, characterized in that, The porcine colon organoid culture medium described in step (2) is prepared by adding 1×N2, 1×B27, epidermal growth factor, glutamine, hydroxyethylpiperazine ethanethioic acid, nicotinamide, Y-27632, SB202190, CHIR99021, and A83-01 to a mixture of PWL038 / cell supernatant and Advanced DMEM / F12 at a volume ratio of 1:

1. The concentrations of 1×N2, 1×B27, EGF, glutamine, HEPES, nicotinamide, Y-27632, SB202190, CHIR99021, and A83-01 in the complete colon organoid culture medium are 1×N2, 1×B27, 50 ng / mL, 2 mM, 10 mM, 10 mM, 10 μM, 10 μM, 2.5 μM, and 0.5 μM, respectively.

7. The TGEV challenge method based on the porcine intestinal organoid inversion model as described in claim 1, characterized in that, The operation of repeating steps (2) and (3) after suspension culture of the product in step (3) in step (4) refers to suspending culture in step (3) for 1-7 days, changing the porcine colon organoid culture medium every 3 days, then centrifuging the product after suspension culture according to the centrifugation conditions described in step (2), discarding the supernatant and retaining the precipitate, resuspending the precipitate with porcine colon organoid culture medium, and continuing suspension culture in an ultra-low adsorption cell culture plate at 37°C and 5% CO2 to obtain a porcine intestinal organoid inversion model.

8. A porcine intestinal organoid inversion model, characterized in that, The porcine intestinal organoid inversion model is the porcine intestinal organoid inversion model prepared by steps (1) to (4) of claim 1.

9. The application of the porcine intestinal organoid inversion model as described in claim 8 in screening drugs for the prevention and / or treatment of TGEV infection.

Images