Pig intestine organoid and lymphocyte co-culture system as well as construction method and application thereof

Abstract

The invention discloses a pig intestine organoid and lymphocyte co-culture system as well as a construction method and application thereof, and belongs to the technical field of virus culture. The co-culture system is obtained by co-culturing porcine ileum organs and porcine small intestine epithelium lymphocytes. By using the co-culture system, the PEDV still can infect the organoid under the condition of not adding pancreatin, and by using the co-culture system to separate and culture the PEDV wild strain, the pathological change of the organoid can be observed during blind passage of the fourth generation, and at the moment, the pathological change does not appear on Vero cells. Reverse transcription-polymerase chain reaction (RT-PCR) identification finds that a stripe can be amplified after a PEDV wild strain is continuously passed for five generations on the co-culture system, and no stripe can be amplified after the PEDV is continuously and blindly passed for five generations on Vero cells no matter whether pancreatin is added or not. The proposal of the invention is of great significance to understanding the pathogenesis of PEDV and developing new drugs and therapies for preventing PEDV infection.

Description

Technical Field

[0001] The present invention relates to an intestinal organoid for in vitro culture of PEDV, in particular to a co-culture system of porcine intestinal organoid and lymphocyte for in vitro culture of PEDV, and also to a construction method of the co-culture system and an application of the co-culture system in in vitro culture of PEDV. The present invention belongs to the technical field of virus culture. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a major pathogenic virus in the pig intestine. It is highly contagious and is the main cause of morbidity and mortality in piglets, causing serious economic losses to the pig industry. However, the virus mainly infects and proliferates in vitro in non-intestinal Vero cells. However, Vero cells are derived from African green monkey kidneys, and the relevant biological information that may be produced after PEDV infection may not be consistent with that in the pig intestine. The role of Vero cells in the innate immune response should be carefully considered, and it cannot represent the true mechanism of action of PEDV in vivo. PEDV wild virus directly isolated from the sample needs to be blindly propagated on Vero cells many times before the cell-adapted strain can be obtained, and the cell-adapted strain often mutates and changes the characteristics of the original strain. More importantly, in the current in vitro cultured cell lines, PEDV needs to be cut by trypsin pretreatment to expose its antigen binding sites before further infection can occur, which is obviously not completely consistent with the natural infection of PEDV, thus limiting the study of the pathogenic mechanism of PEDV and the regulation of the host's innate immune response.

[0003] Researchers have established several porcine small intestinal epithelial cell lines, such as porcine small intestinal epithelial cells IPEC-J2 and ileal epithelial cells IPI-21, which are derived from porcine jejunum and ileum, respectively. However, the virus infection rates in IPEC-J2 and IP-21 cells are very low, and these cell models cannot completely replace the rich intestinal epithelial cell types and complex structures. In addition, the in vitro isolation and culture of epidemic strains is the basis for studying PEDV, which usually requires the additional addition of pancreatic enzymes. PEDV strains are generally dependent on pancreatic enzymes. Wild PEDV viruses directly isolated from samples require multiple blind passages on cells to obtain cell-tamed strains, and cell-tamed strains often mutate and change the characteristics of the original strains, which restricts the study of in vitro viral characteristics and vaccine production. Therefore, it is particularly necessary to establish a cell model that can simulate the in vivo small intestinal epithelium. The emergence of intestinal organoids provides a possibility for solving the above problems.

[0004] Intestinal organoids are in vitro culture models composed of multiple intestinal cell types, including fully mature goblet cells, enterocytes, Paneth cells, and endocrine cells. They have a petal-like structure with a hollow cavity, contain villi and crypt-like domains, and are capable of long-term self-renewal. They can be stably cryopreserved and revived, providing a unique platform for studying host-pathogen interactions in in vitro systems. A recent study showed that porcine intestinal organoids (PIOs) are closer to epithelial tissue than porcine intestinal epithelial cells (IPECs) and are physiologically closer to in vivo conditions than existing cell lines. However, because the microvilli of intestinal epithelial cells in intestinal organoids are completely built into the sphere under current 3D culture conditions, most pathogenic microorganisms that use the apical membrane of the intestinal villi to invade the body are difficult to infect the organoids, making it difficult to conduct research on related pathogen-host interactions.

[0005] To solve the above problems, researchers have established several solutions, such as forming 2D monolayer intestinal organoids by treating them with trypsin and then adding pathogenic microorganisms; injecting pathogenic microorganisms directly into the intestinal organoid cavity, etc. However, although intestinal organoids cultured in vitro have the complex cell structure phenotype of the gastrointestinal tract, they lack the participation of immune cells compared to the real gastrointestinal tract.

[0006] In order to achieve the in vitro culture of primary PEDV, the present invention established for the first time a co-culture system of porcine ileal organoids and porcine intestinal intraepithelial lymphocytes, which provides a reliable platform for exploring the biological characteristics and pathogenic mechanisms of PEDV-infected intestinal cells. It is of great significance for understanding the pathogenesis of PEDV in weaned piglets and developing drugs or methods to prevent PEDV infection. Summary of the invention

[0007] The object of the present invention is to provide a co-culture system of porcine intestinal organoids and lymphocytes that can be used for in vitro culture of PEDV, as well as a construction method and application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical means: A method for constructing a co-culture system of porcine intestinal organoids and lymphocytes of the present invention comprises the steps of co-culturing porcine ileal organoids and porcine small intestinal intraepithelial lymphocytes, wherein the co-culture of porcine ileal organoids and porcine small intestinal intraepithelial lymphocytes is carried out according to the following steps: (1) Resuscitation of porcine small intestinal intraepithelial lymphocytes: Take out the porcine small intestinal intraepithelial lymphocyte cryopreservation tube from liquid nitrogen, place it in a 37°C water bath until the cell suspension is completely thawed, transfer the cell suspension to a centrifuge tube, add RPMI1640, mix well, centrifuge, and discard the supernatant; (2) Porcine ileal organoids are obtained by organoid differentiation and culture of crypt stem cells isolated from the intestinal segments of piglets' ileum. After culturing the porcine ileal organoids for 1-2 days, the original culture medium is discarded, and pre-cooled PBS is added to blow the intestinal organoids into the centrifuge tube. The bottom of the well is rinsed with pre-cooled PBS, and the wells are added to the same centrifuge tube. Centrifuge and discard the supernatant. (3) 100 porcine ileal organoids were combined with 10 5 The small intestinal intraepithelial lymphocytes were mixed, added to DMEM / F12 culture medium, and incubated in an incubator at 37°C with 5% CO2 for 30 min; (4) After the incubation is completed, the cell suspension is centrifuged at 1500 rpm / min for 5 min, the cell pellet is resuspended in matrix gel and transferred to a 48-well cell culture plate. The 48-well plate is placed in an incubator at 37°C and 5% CO2 for incubation for 15 min, and then 200 μL of IntestiCult™ human intestinal organoid growth medium containing 1% v / v penicillin-streptomycin mixture and 100 U / mL IL-2 is added to each culture well containing cells. The cell culture plate is placed in an incubator at 37°C and 5% CO2 for incubation to obtain the porcine intestinal organoid and lymphocyte co-culture system.

[0009] Preferably, the porcine ileum organoid is prepared by the following steps: (1) Thaw the Matrigel on ice, pre-cool PBS and add 2% v / v penicillin-streptomycin mixture to the pre-cooled PBS for the following experiments; (2) A 5-10 cm ileum segment was cut from a 7-day-old pig and placed in a cell culture dish containing the above-mentioned pre-cooled PBS; (3) Flush the contents of the ileum until the intestinal contents are completely removed, and then discard the flushing fluid; (4) Add the above pre-cooled PBS to another new culture dish, place the washed intestinal segment into the culture dish, cut it into small segments of 1-3 mm, transfer the tissue suspension containing the intestinal segment to a 50 mL centrifuge tube, and allow the intestinal fragments to settle naturally; (5) Remove the upper layer of liquid in the centrifuge tube and add the above pre-cooled PBS; repeat this washing step until the supernatant becomes clear; (6) After removing the supernatant, resuspend the intestinal segment in 10 mL of mild cell dissociation buffer and incubate on a shaker at 100 rpm for 20 min at room temperature; (7) After incubation, allow the intestinal segments to settle naturally, discard the supernatant, and then add 10 mL of the above pre-cooled PBS containing 1% v / v fetal bovine serum (FBS) and move the intestinal fragments up and down; (8) After most of the intestinal fragments have settled, collect the supernatant, filter it through a 70 μm filter, and inject the filtrate into a 15 mL centrifuge tube. Repeat this step 4 to 5 times. (9) Centrifuge the filtrate at 1200 rpm for 5 min at 4°C, discard the supernatant, and collect the crypt fragment precipitate; suspend the crypt fragment precipitate in DMEM / F12 medium and count the cells; (10) Centrifuge the suspension at 1200 rpm for 5 minutes at 4°C, discard the supernatant, add IntestiCult™ human intestinal organoid growth medium and Matrigel to the crypt stem cells at a ratio of 25 μL IntestiCult™ human intestinal organoid growth medium and 25 μL Matrigel per 50 crypt stem cells, and transfer 50 μL of the crypt stem cell suspension to a cell culture plate; (11) Place the cell culture plate in a 37°C CO2 incubator and incubate for 15 min to allow the matrix gel to solidify. Then, add 200 μL of IntestiCult™ human intestinal organoid growth medium containing 1% v / v penicillin-streptomycin mixture to each culture well containing crypt stem cells. (12) Place the cell culture plate in an incubator at 37°C containing 5% carbon dioxide; replace the IntestiCult™ human intestinal organoid growth medium every 3-4 days. After 7-8 days, the crypt stem cells proliferate and differentiate into complete 3D porcine ileal organoids with villus-like epithelial cells and crypt region domains with bud-like structures.

[0010] Wherein, preferably, the porcine intestinal intraepithelial lymphocytes are prepared by the following steps: (1) Take 10-15 cm of the ileum of a 21-day-old piglet and place it in PBS containing 2% v / v penicillin-streptomycin mixture; (2) Immerse the small intestine in a culture dish on ice containing 2% v / v penicillin-streptomycin mixture in PBS, use forceps to remove fat, lymphatic tissue and Peyer's patches (PP), and rinse the intestinal segment with a 1 mL pipette tip; (3) Replace the culture dish with a new one and add PBS containing 2% v / v penicillin-streptomycin mixture, cut the intestinal segment into 3±0.5 cm×0.5±0.5 cm in size, and transfer it to a 50 mL centrifuge tube; (4) Add ice-cold PBS containing 2% v / v penicillin-streptomycin mixture, shake for 5-15 seconds, and discard the supernatant after the intestinal fragments settle naturally. Repeat 3-5 times until the supernatant is clear. (5) Transfer the intestinal segment to Hanks-dithiothreitol (DTT) solution, incubate at 37°C constant temperature shaker at 150 rpm / min for 20 min, and discard the solution. The Hanks-dithiothreitol (DTT) solution contains 30 mL Hanks, 5 mmol DTT and 2% v / v FBS; (6) Add 30 mL of Hanks-EDTA digestion solution, incubate at 37°C on a shaker for 25 min, filter through a 100 μm cell strainer in a 50 mL centrifuge tube, collect the supernatant, and repeat this step three times with new digestion solution. The Hanks-EDTA digestion solution contains 30 mL of Hanks, 5 mmol EDTA, and 2% v / v FBS. (7) Add washing solution to the supernatant obtained in step (6), incubate at 37°C on a shaker for 20 min, and collect the cells through a 100 μm cell strainer into the same 50 mL centrifuge tube. The washing solution contains 20 mL Hanks, 2 mmol / L-glutamine, and 2% v / v FBS. (8) Centrifuge the collected supernatant at 600 g / min for 10 min at 4°C to obtain a cell pellet; (9) Rinse a 15 mL centrifuge tube with 1 mL FBS in advance, then slowly add 66% v / v-44% v / v-25% v / v Percoll solution from bottom to top, slowly add the cell pellet obtained in step (8) to the upper layer of Percoll solution, centrifuge at 600 g / min at 4°C for 30 min, and collect the lymphocytes between the 44% v / v-66% v / v Percoll solution layers; (10) Wash the cells twice with RPMI-1640 and resuspend them in 1 mL of RPMI-1640 complete medium. Count the cells using a counting plate and adjust the concentration to 2×10 6 cells / well, and then spread them into a 96-well cell culture plate and cultured in a 37°C incubator to obtain the porcine intestinal intraepithelial lymphocytes; wherein the RPMI-1640 complete culture medium is an RPMI-1640 culture medium containing 10% v / v FBS and 1% v / v penicillin-streptomycin mixture.

[0011] The co-culture system of porcine intestinal organoids and lymphocytes constructed according to any of the methods described above is also within the protection scope of the present invention.

[0012] Furthermore, the present invention also proposes the use of the porcine intestinal organoid and lymphocyte co-culture system in culturing porcine epidemic diarrhea virus.

[0013] Among them, preferably, the porcine epidemic diarrhea virus is a porcine epidemic diarrhea virus cell-acclimated strain or a porcine epidemic diarrhea virus wild strain.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The in vitro culture of PEDV strictly requires pancreatic enzyme treatment so that the virus can effectively infect cells, promote viral proliferation and accelerate the formation of cytopathic diseases. Finally, pancreatic enzyme releases virus particles from the cell surface. However, in real pig intestines, intestinal infection can be achieved without the addition of pancreatic enzymes. In the co-culture system of pig intestinal organoids and lymphocytes of the present invention, PEDV can still infect organoids without the addition of pancreatic enzymes. 2. Currently, the commonly used cell line for the isolation and culture of wild PEDV strains is Vero cells. However, PEDV needs to be domesticated and adapted on Vero cells before it can be used later. When the co-culture system of the present invention is used to isolate and culture wild PEDV strains, organoid lesions can be observed in the fourth blind passage. At this time, no lesions appear on Vero cells. RT-PCR identification shows that wild PEDV strains can amplify bands in organoids for five consecutive passages. However, regardless of whether pancreatin is added, PEDV cannot amplify bands in five consecutive blind passages on Vero cells. 3. The pig intestinal organoid and lymphocyte co-culture system proposed in the present invention provides a reliable platform for exploring the biological characteristics and pathogenic mechanism of PEDV-infected intestinal cells, which is of great significance for understanding the pathogenesis of PEDV in weaned piglets and developing new drugs and new therapies to prevent PEDV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 For the isolation and culture of porcine ileal organoids; Among them, A. Organoids cultured for 0 days; B. Organoids cultured for 3 days; C. Organoids cultured for 5 days; D. Organoids cultured for 8 days; Figure 2 This is a pig ileum organoid after 7 days of recovery; Figure 3 For the isolation of intestinal intraepithelial lymphocytes; Figure 4 CD3 for flow cytometry + The proportion of lymphocytes; Figure 5 The effects of intestinal intraepithelial lymphocytes and IL-2 on the growth of porcine ileal organoids; Among them, A. Morphology of intestinal intraepithelial lymphocytes in IntestiCult™ human intestinal organoids growth medium; B. Morphology of porcine ileal organoids in IntestiCult™ human intestinal organoids growth medium; C. Morphology of porcine ileal organoids in IntestiCult™ human intestinal organoids growth medium with the addition of IL-2; D. Morphology of porcine ileal organoids in IntestiCult™ human intestinal organoids growth medium with the addition of IL-2 and intestinal intraepithelial lymphocytes; Figure 6 To identify the co-culture system of porcine ileal organoids and intestinal intraepithelial lymphocytes; Figure 7 The morphological changes of porcine ileal organoids and Vero cells after infection with PEDV; Among them, A. Co-culture system (Mock); B. Co-culture system + PEDV + pancreatin; C. Co-culture system + PEDV; D. Porcine ileal organoids + PEDV + pancreatin; E. Porcine ileal organoids + PEDV; F. Vero cells + PEDV; Figure 8 Indirect immunofluorescence was used to prove the infection of the co-culture system by PEDV; Among them, A. Co-culture system (Mock); B. Co-culture system + PEDV + pancreatin; C. Porcine ileal organoids + PEDV + pancreatin; D. Co-culture system + PEDV; E. Porcine ileal organoids + PEDV; F. Vero cells + PEDV; Fig. 9 Indirect immunofluorescence was used to prove the infection of the co-culture system by PEDV; Among them, A. Co-culture system (Mock); B. Porcine ileal organoids + PEDV; C. Co-culture system + PEDV; Fig.10 The replication of strong and weak strains of PEDV in porcine ileal organoids; Among them, A. standard curve; B. PEDV in cells; C. PEDV in supernatant; Fig.11 The co-localization detection results of different cell subpopulations in PEDV-infected porcine ileal organoids; Fig.12 The morphological changes of organoids after infection with PEDV; Among them, A. Porcine ileum organoids (Mock); B. Co-culture system (Mock); C. Porcine ileum organoids + PEDV; D. Co-culture system + PEDV; Fig.13 Indirect immunofluorescence was used to prove the infection of the co-culture system by the wild-type PEDV strain B18 during the fourth blind passage; Fig.14 The results of RT-PCR test of the 1st to 5th generation of PEDV wild-type virus in the co-culture system and Vero cells infected with the virus were obtained by blind transmission in vitro; Wherein, M is DNA marker 2000; 1-5: RT-PCR test results of the co-culture system infected with PEDV blind passage 1-5 generations of strains; 6-10: RT-PCR test results of Vero cells infected with PEDV blind passage 1-5 generations of strains with the addition of trypsin; 11-15: RT-PCR test of Vero cells infected with PEDV blind passage 1-5 generations without the addition of trypsin; 16: negative control. DETAILED DESCRIPTION

[0016] The present invention is further described below by way of examples, the purpose of which is only to provide a better understanding of the research content of the present invention rather than to limit the protection scope of the present invention.

[0017] Example 1 Construction of a co-culture system of porcine intestinal organoids and lymphocytes

[0018] 1 Experimental methods

[0019] 1.1 Isolation and culture of 3D piglet ileal organoids According to the culture method of Li Liang et al. [Li L, Fu F, Guo S, et al. Porcine intestinal enteroids: a new model for studying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response [J]. Journal of virology, 2019, 93 (5): e01682-18.], the porcine ileum organoids derived from intestinal crypt stem cells were isolated and cultured from the intestinal tissue of 7-day-old Duroc piglets with appropriate modifications. The specific operation is as follows: (1) Thaw the Matrigel on ice, pre-cool PBS and add 2% v / v penicillin-streptomycin mixture to the pre-cooled PBS for the following experiments; (2) A 5-10 cm ileum segment was cut from a 7-day-old pig and placed in a cell culture dish containing the above-mentioned pre-cooled PBS; (3) Flush the contents of the ileum until the intestinal contents are completely removed, and then discard the flushing fluid; (4) Add the above pre-cooled PBS to another new culture dish, place the washed intestinal segment into the culture dish, cut it into small segments of 1-3 mm, transfer the tissue suspension containing the intestinal segment to a 50 mL centrifuge tube, and allow the intestinal fragments to settle naturally; (5) Remove the upper layer of liquid in the centrifuge tube and add the above pre-cooled PBS; repeat this washing step until the supernatant becomes clear; (6) After removing the supernatant, resuspend the intestinal segment in 10 mL of Gentle Cell Dissociation Reagent (Cat. No. 07174, Stem Cell Technologies) and incubate on a shaker at 100 rpm for 20 min at room temperature. (7) After incubation, allow the intestinal segments to settle naturally, discard the supernatant, and then add 10 mL of the above pre-cooled PBS containing 1% v / v fetal bovine serum (FBS) and move the intestinal fragments up and down; (8) After most of the intestinal fragments have settled, collect the supernatant, filter it through a 70 μm filter, and inject the filtrate into a 15 mL centrifuge tube. Repeat this step 4 to 5 times. (9) Centrifuge the filtrate at 1200 rpm for 5 min at 4°C, discard the supernatant, and collect the crypt fragment precipitate; suspend the crypt fragment precipitate in DMEM / F12 medium and count the cells; (10) Centrifuge the suspension at 1200 rpm for 5 minutes at 4°C, discard the supernatant, add 25 μL IntestiCult™ Human Intestinal Organoid Growth Medium (Human, product number 06010, brand Stem Cell Technologies) and 25 μL Matrigel to every 50 crypt stem cells, add IntestiCult™ Human Intestinal Organoid Growth Medium and Matrigel to the crypt stem cells, and transfer 50 μL of the crypt stem cell suspension to a cell culture plate; (11) Place the cell culture plate in a 37°C CO2 incubator and incubate for 15 min to allow the matrix gel to solidify. Then, add 200 μL of IntestiCult™ human intestinal organoid growth medium containing 1% v / v penicillin-streptomycin mixture to each culture well containing crypt stem cells. (12) Place the cell culture plate in an incubator at 37°C containing 5% carbon dioxide and culture. Replace the medium with new IntestiCult™ Human Intestinal Organoid Growth Medium every 3 to 4 days.

[0020] 1.2 Passaging of 3D piglet ileal organoids After intestinal stem cells have been cultured for about 7 days and differentiated into branched intestinal organoids, they can be passaged. The specific passage steps are as follows: (1) Gently remove the culture medium surrounding the dome in the well to prepare for the next step; (2) Add 1 mL of pre-cooled PBS to each well and incubate it in the well for 1 min to create a lower temperature environment (less than 5°C) for the cells, which is conducive to the dissolution of the matrix gel; (3) Use a 1 mL pipette to gently pipette the mixture of PBS and intestinal organoid cells about 10 to 20 times to ensure that the cell mixture is evenly suspended; (4) Transfer the suspension to a 15 mL centrifuge tube, rinse the well with an appropriate amount of PBS, and then add the rinse solution to the same centrifuge tube; (5) Centrifuge at 1500 rpm for 5 min at 4°C and then gently discard the supernatant. (6) Add an appropriate amount of 0.25% EDTA-trypsin, place the centrifuge tube in a 37°C water bath, and shake it every 2 minutes for 5 minutes to promote cell digestion; (7) Add an appropriate amount of DMEM / F12 culture medium containing 10% v / v serum (purchased from Cell Technology Co., Ltd.) and use a pipette to disperse the cells; (8) Centrifuge the tube again at 1500 rpm for 5 min at room temperature and discard the supernatant. (9) Add IntestiCult™ Human Intestinal Organoid Growth Medium and Matrigel to the crypt stem cells at a ratio of 25 μL IntestiCult™ Human Intestinal Organoid Growth Medium and 25 μL Matrigel per 50 crypts. Transfer 50 μL of the crypt stem cell suspension to a 48-well cell culture plate and form a complete arc in the well; (10) Place the 48-well cell culture plate in a 37°C CO2 incubator and incubate for 15 min to allow the matrix gel to solidify. Then, add 200 μL of IntestiCult™ Human Intestinal Organoid Growth Medium containing 1% v / v penicillin-streptomycin mixture to each culture well containing crypt stem cells; (11) Add sterile PBS to the edge wells; (12) Place the cell culture plate in an incubator at 37°C containing 5% carbon dioxide and culture. Replace the medium with new IntestiCult™ Human Intestinal Organoid Growth Medium every 3 to 4 days.

[0021] 1.3 Cryopreservation of 3D piglet ileal organoids After the intestinal organoids have grown for about 7 days, use 0.25% EDTA-trypsin to digest the ileal organoids into single cells according to the above-mentioned passaging steps, digest at 37°C for 5 min, then add DMEM medium containing 10% v / v FBS to terminate the digestion, discard the supernatant after centrifugation, add an appropriate amount of cell freezing solution (90% v / v FBS + 10% v / v DMSO) to the cell pellet, transfer the mixed liquid to a cryopreservation tube, place the cell freezing box at -80°C for freezing, and transfer to a liquid nitrogen tank after 24 hours.

[0022] 1.4 Recovery of 3D piglet ileal organoids The steps for resuscitation of intestinal organoids after freezing are similar to those for general cell recovery. The specific steps are as follows: After taking out the cryotube, transfer it to a 37°C water bath as soon as possible and shake it continuously until the cell suspension is completely thawed. Transfer the suspension to a 15mL centrifuge tube, slowly add 9 mL of DMEM / F12, mix gently, and centrifuge at 1200 rpm for 5 min. Then culture according to the above intestinal organoid subculture steps.

[0023] 1.5 Isolation and culture of porcine intestinal intraepithelial lymphocytes (1) Take 10-15 cm of the ileum of a 21-day-old piglet and place it in PBS containing 2% v / v penicillin-streptomycin mixture; (2) Immerse the small intestine in a culture dish on ice containing 2% v / v penicillin-streptomycin mixture in PBS, use forceps to remove fat, lymphatic tissue and Peyer's patches (PP), and rinse the intestinal segment with a 1 mL pipette tip; (3) Replace the culture dish with a new one and add PBS containing 2% v / v penicillin-streptomycin mixture, cut the intestinal segment into 3±0.5 cm×0.5±0.5 cm in size, and transfer it to a 50 mL centrifuge tube; (4) Add ice-cold PBS containing 2% v / v penicillin-streptomycin mixture, shake for 5-15 seconds, and discard the supernatant after the intestinal fragments settle naturally. Repeat 3-5 times until the supernatant is clear. (5) Transfer the intestinal segment to Hanks-dithiothreitol (DTT) solution, incubate at 37°C constant temperature shaker at 150 rpm / min for 20 min, and discard the solution. The Hanks-dithiothreitol (DTT) solution contains 30 mL Hanks, 5 mmol DTT and 2% v / v FBS; (6) Add 30 mL of Hanks-EDTA digestion solution, incubate at 37°C on a shaker for 25 min, filter through a 100 μm cell strainer in a 50 mL centrifuge tube, collect the supernatant, and repeat this step three times with new digestion solution. The Hanks-EDTA digestion solution contains 30 mL of Hanks, 5 mmol EDTA, and 2% v / v FBS. (7) Add washing solution to the supernatant obtained in step (6), incubate at 37°C on a shaker for 20 min, and collect the cells through a 100 μm cell strainer into the same 50 mL centrifuge tube. The washing solution contains 20 mL Hanks, 2 mmol / L-glutamine, and 2% v / v FBS. (8) Centrifuge the collected supernatant at 600 g / min for 10 min at 4°C to obtain a cell pellet; (9) Rinse a 15 mL centrifuge tube with 1 mL FBS in advance, then slowly add 66% v / v-44% v / v-25% v / v Percoll solution from bottom to top, slowly add the cell pellet obtained in step (8) to the upper layer of Percoll solution, centrifuge at 600 g / min at 4°C for 30 min, and collect the lymphocytes between the 44% v / v-66% v / v Percoll solution layers; (10) Wash the cells twice with RPMI-1640 and resuspend them in 1 mL of RPMI-1640 complete medium. Count the cells using a counting plate and adjust the concentration to 2×10 6 cells / well, and then spread them into a 96-well cell culture plate and cultured in a 37°C incubator to obtain the porcine intestinal intraepithelial lymphocytes; wherein the RPMI-1640 complete culture medium is an RPMI-1640 culture medium containing 10% v / v FBS and 1% v / v penicillin-streptomycin mixture.

[0024] 1.6 Flow cytometry Take 10 lymphocytes from the isolated 5, placed in a 2 mL EP tube. Add 50 μL of 1:100 diluted anti-CD3 antibody solution and let it stand at room temperature for 30 min to ensure sufficient reaction. After the reaction, centrifuge the sample and remove the supernatant, then wash the sample twice with PBS, add 50 μL FITC-labeled goat anti-rabbit IgG, let it stand at room temperature in the dark for 30 min, and then wash the sample twice with PBS. After completing the above operations, analyze the sample on a flow cytometer. At the same time, set up two groups of controls, the first group was incubated with only the secondary antibody, and the second group was incubated with PBS.

[0025] 1.7 Co-culture of porcine ileal organoids and small intestinal intraepithelial lymphocytes (1) Resuscitation of porcine small intestinal intraepithelial lymphocytes: Take out the porcine small intestinal intraepithelial lymphocyte cryopreservation tube from liquid nitrogen, place it in a 37°C water bath until the cell suspension is completely thawed, transfer the cell suspension to a centrifuge tube, add RPMI1640, mix well, centrifuge, and discard the supernatant; (2) After culturing the porcine ileal organoids for 1-2 days, discard the original culture medium, add pre-cooled PBS to blow the intestinal organoids into the centrifuge tube, rinse the bottom of the well with pre-cooled PBS, add it to the same centrifuge tube, centrifuge, and discard the supernatant; (3) 100 porcine ileal organoids were combined with 10 5 The small intestinal intraepithelial lymphocytes were mixed, added to DMEM / F12 culture medium, and incubated in an incubator at 37°C with 5% CO2 for 30 min; (4) After the incubation is completed, the cell suspension is centrifuged at 1500 rpm / min for 5 min, the cell pellet is resuspended in matrix gel and transferred to a 48-well cell culture plate. The 48-well plate is placed in an incubator at 37°C and 5% CO2 for incubation for 15 min, and then 200 μL of IntestiCult™ human intestinal organoid growth medium containing 1% v / v penicillin-streptomycin mixture and 100 U / mL IL-2 is added to each culture well containing cells. The cell culture plate is placed in an incubator at 37°C and 5% CO2 for incubation to obtain the porcine intestinal organoid and lymphocyte co-culture system.

[0026] 1.8 Indirect immunofluorescence (1) Aspirate the original culture medium of the organoids, add pre-cooled PBS, and incubate at room temperature for 1 min; (2) Use a 1 mL pipette to aspirate the mixture of PBS and intestinal organoid cells, transfer it to a centrifuge tube, and let it stand for 10 min; (3) Centrifuge at 1500 rpm for 5 min at 4°C. After centrifugation, gently pour out and discard the supernatant. (4) Add 1 mL of 4% paraformaldehyde (PFA) to the organoid pellet, mix it evenly with a pipette, and then place it on ice or at 4°C for 1 h to 2 h (no more than 10 h). (5) After centrifugation at 1500 rpm for 5 min, use a pipette to discard the supernatant and keep only the organoid pellet; (6) After discarding the supernatant, add 1 mL of PBS, let the tube stand for 5 min and centrifuge at 1200 rpm for 3 min to precipitate again. Repeat this step twice. (7) Discard PBS, add 300-500 μL of 0.25% TritonX-100, and permeabilize at room temperature for 20-30 min; (8) Add 1 mL of PBS to wash twice, let the tube stand for 5 min, and centrifuge at 1200 rpm for 3 min before collecting the precipitate; (9) Add 200 μL of 5% BSA (bovine serum albumin, dissolved in PBS) and block at room temperature for 60 min. Aspirate and discard the supernatant, leaving only the precipitate. (10) Primary antibodies: Rabbit anti-CD3 polyclonal antibody (Beijing Bioson Biotechnology Co., Ltd., bs-10498R), rabbit anti-Ki-67 polyclonal antibody (Boster, PB9026), rabbit anti-mucin2 polyclonal antibody (Abcam, Ab272692), rabbit anti-villin polyclonal antibody (Boster, PB9457), rabbit anti-Lgr5 polyclonal antibody (Novus, NBP1-28904) and mouse anti-PEDV S protein polyclonal antibody were used as primary antibodies for detecting lymphocytes, proliferating cells, goblet cells, intestinal villus epithelial cells, intestinal stem cells and PEDV. The primary antibody solution (solvent is 5% BSA) was added to the organoid precipitate to completely cover it, and incubated at 4°C overnight. After incubation, it was washed three times with 1 mL of PBST. Each time, the tube was allowed to stand for 5 min and centrifuged at 400 rpm for 3 min before collecting the precipitate. (11) After washing, discard the supernatant and add 50 µL of FITC-labeled goat anti-mouse IgG and TRITC-labeled goat anti-rabbit IgG secondary antibodies (solvent is 5% BSA) to the organoid precipitate suspension. Incubate at room temperature for 1 h in the dark. After incubation, wash repeatedly with 1 mL of PBST for 3 times. Let the tube stand for 5 min each time and centrifuge at 400 rpm for 3 min before collecting the precipitate. (12) After washing, discard the supernatant and incubate with 1:1000 diluted DAPI solution (solvent is PBS) at room temperature in the dark for 10-15 min, or incubate with CD3 antibody labeled with rabbit IgG antibody labeling kit at room temperature in the dark for 1 h. After incubation, wash three times with 1 mL of PBST; (13) Use an immunohistochemistry pen to delineate the area (so that the 3D structure of the organoid is not destroyed by the glass slide), discard the supernatant, leave 20 uL, and transfer the organoid sediment to the delineated area of ​​the slide. Use absorbent paper or a 200 µL pipette to gently absorb excess water (be careful not to absorb the organoid), and drop anti-fluorescence quenching sealing medium on the top. Put vacuum grease into a 10mL syringe and apply vacuum grease on the four corners of the slide as a gasket. Cover with a coverslip. Use tweezers to gently press the corners of the coverslip until the coverslip contacts the sealing agent; (14) After stabilization, dry in a dark place overnight or fix with nail polish and store at room temperature for short term or at -20°C for long term.

[0027] 1.9 PEDV-infected intestinal organoids and intestinal intraepithelial lymphocyte co-culture system After 1-2 days of co-culture, collect the organoids in cold DMEM / F12 into a centrifuge tube, centrifuge at 1300 rpm / min for 5 minutes, remove the supernatant, and add PEDV adsorption solution (DMEM + PEDV virus solution). After 2 hours of virus adsorption at 37℃ 5% CO2, wash twice with excess DMEM / F12 to remove unbound virus. Resuspend the cell pellet in 30 µL of Matrigel and place it in a 48-well cell culture plate. Incubate the well plate in an incubator (37℃ and 5% CO2) for 15 minutes, and add 200 µL of intestinal organoid culture medium containing 100U / mL IL-2 and 1% v / v penicillin-streptomycin mixture.

[0028] 1.10 RNA extraction Extract PEDV and cell RNA according to the instructions of the Fijet RNA extraction kit: (1) Collect the infected cells into EP tubes and retain the supernatant after centrifugation.

[0029] (2) Add 500 µL of RA2 solution to the EP tube, mix thoroughly by inverting for 5-10 times (until the lysate is clear), and let stand for 1 min.

[0030] (3) Aspirate or pour all the supernatant into the inner tube (which has been inserted into the outer tube) and centrifuge at 12,000 rpm / min for 1 min.

[0031] (4) Remove the inner cannula, discard the liquid in the outer cannula, and put it back into the inner cannula. Add 500 µl of washing solution and centrifuge at 12000 rpm / min for 1 min.

[0032] (5) Repeat step 4 and wash again.

[0033] (6) Remove the inner cannula, discard the liquid in the outer cannula, and put it back into the inner cannula without adding washing solution. Centrifuge at 12,000 rpm / min for 1 min.

[0034] (7) Move the inner tube into a new 1.5 ml EP tube and add 25-50 µl of elution buffer to the center of the membrane. After standing at room temperature for 1 min, centrifuge at 12,000 rpm / min for 1 min to obtain total RNA.

[0035] 1.11 Reverse transcription of RNA According to the instructions of the RNA reverse transcription kit, the extracted RNA was reverse transcribed to obtain the required cDNA. The specific steps are as follows: take 22 µL of RNA in a sterilized centrifuge tube, add 5 µL of Oligo DT and mix well, then place in a water bath at 75°C for 10 min, and then immediately place in an ice bath for 5 min. Then add 8 µL of RT Buffer, 5 µL of dNTPs, 5 µL of dNTPs mixture, 2 µL of RTAce and 2 µL of RRI to the centrifuge tube, mix well, place in a 42°C water bath for 1 h, then transfer to a 72°C water bath for 10 min, and then place in an ice bath for 5 min to obtain cDNA.

[0036] 1.12 Primer design and synthesis The required PEDV gene design primers were retrieved from NCBI, and multiple pairs of specific primers were designed using Oligo 6.0 software and synthesized by Jilin Kumei Biotechnology Co., Ltd. Table 1 shows the primer sequences used in this study.

[0037]

[0038] 1.13 Real-time quantitative PCR detection Prepare the premix according to Table 2 below. GAPDH was used as the internal reference. -∆∆Ct Methods The difference level of each target gene was calculated and quantitative analysis was performed.

[0039]

[0040] 1.14 Data Analysis Our laboratory has previously constructed a PEDV virus standard plasmid bacteria, activated it and extracted the plasmid, then diluted the plasmid in multiples for fluorescence quantitative detection. Based on the cycle number (CT) of plasmids at different dilution concentrations, we finally obtained the standard curve of porcine PEDV virus copy number. The calculation formula is: Lg (virus copy number) / μL=-0.2072 (Ct value)+11.634. Substitute the Ct value into the formula to obtain the specific value of the virus copy number.

[0041] GraphPad Prism 8.0 software was used for statistical analysis. The statistical data showed a normal distribution. All data are the mean ± standard deviation of 3 independent experiments (n = 3). Statistical analysis was performed using the t-test method. When the P value was < 0.05, the difference was significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001.

[0042] 2 Results 2.1 Establishment of 3D piglet ileum organoid culture system 2.1.1 Isolation and culture of 3D piglet ileal organoids In this study, crypt stem cells were isolated from the ileum of 7-day-old piglets, placed in matrix gel, and cultured in organoid culture medium for organoid differentiation. After 3 days of culture, it was found that the small intestinal crypt stem cells proliferated and differentiated into cystic structures with cavities. The cystic cavity continued to expand and budding occurred on the 5th day of culture. After about 8 days, budding and organoid volume increased significantly. At this time, the crypt stem cells have proliferated and differentiated into complete 3D intestinal organoids with villus-like epithelial cells and bud-like crypt area domains, such as Figure 1 The results showed that intestinal crypt stem cells from ileum segments can grow and differentiate into porcine ileum organoids.

[0043] 2.1.2 Subculture and cryopreservation of 3D piglet ileal organoids The ileal organoids that were clearly differentiated after 5-7 days of culture were digested and recovered, and after being frozen, they were subjected to conventional resuscitation culture and continuous subculture. The results showed that porcine ileal organoids could be successfully revived after freezing, and their structure was the same as that of the ileal organoids differentiated from crypt stem cells isolated from primary piglet intestines. A single revived ileal organoid cell could grow into a branched porcine ileal organoid within 7 days, still having a central cavity and villus-like structure epithelial cells and bud-like structure crypt region domains, such as Figure 2 Thus, this study established a 3D intestinal organoid culture system derived from porcine ileum intestinal crypt stem cells, which can be cryopreserved, revived and serially passaged.

[0044] 2.2 Isolation and culture of porcine intestinal intraepithelial lymphocytes In this study, ileum segments were isolated from 21-day-old weaned piglets, and primary intestinal intraepithelial lymphocytes were obtained by density gradient centrifugation. The cells were cultured in 96-well plates, and round lymphocytes were observed, such as Figure 3 The separated intraepithelial lymphocytes were detected by flow cytometry, as shown in Figure 4 As shown, the purity of CD3+ IEL cells was 26.7%. The results showed that porcine intestinal intraepithelial lymphocytes were successfully isolated and can be used for the establishment of subsequent co-culture systems.

[0045] 2.3 Establishment of a co-culture system of porcine ileal organoids and small intestinal intraepithelial lymphocytes 2.3.1 Screening of culture conditions for co-culture system To investigate whether T cell growth factor IL-2 would affect the growth morphology of porcine ileal organoids, we added lymphocytes or IL-2 after culturing porcine ileal organoids for 2 days and observed the growth status of cells under different conditions under a microscope on the 4th, 6th, and 8th days of culture. Figure 5 Figure A shows the cell morphology of lymphocytes under normal culture conditions; Figure 5B shows the morphological changes of porcine ileal organoids under normal culture conditions. In the co-culture system of porcine ileal organoids and intestinal intraepithelial lymphocytes, IL-2 needs to be added to stimulate the activity of lymphocytes. Therefore, we first added IL-2 to the culture medium of porcine ileal organoids. The results are shown in Figure 5B. Figure 5 As shown in Figure C, we can find that IL-2 can promote the growth and maturation of porcine ileal organoids. IL-2 was further added to the co-culture system of porcine ileal organoids and lymphocytes. Figure 5 As shown in D, the effect of adding intestinal intraepithelial lymphocytes and IL-2 on the overall growth morphology of porcine ileal organoids is the same as that of adding IL-2 alone. The above results indicate that T cell growth factor IL-2 promotes the growth and maturation of porcine ileal organoids in the co-culture system.

[0046] 2.3.2 Establishment and identification of the co-culture system of porcine ileal organoids and small intestinal intraepithelial lymphocytes In order to further confirm the successful establishment of the co-culture system of porcine ileal organoids and intestinal intraepithelial lymphocytes, this study first fixed the porcine ileal organoids after 3 days of co-culture, then performed immunofluorescence staining for the intestinal intraepithelial lymphocyte marker CD3, and observed the staining results of porcine ileal organoids under a fluorescence microscope. The results showed that CD3 could be located in porcine ileal organoids, such as Figure 6 As shown. It was demonstrated that intestinal intraepithelial lymphocytes can grow together in porcine ileal organoids, and the co-culture system was successfully established.

[0047] 2.4 Co-culture system of PEDV-infected porcine ileal organoids and small intestinal intraepithelial lymphocytes 2.4.1 PEDV can still infect the co-culture system without adding trypsin The isolation and culture of wild-type PEDV strains strictly require in vitro pancreatic enzyme treatment so that the virus can effectively infect cells, promote viral proliferation and accelerate the formation of cytopathic effects. Ultimately, pancreatic enzymes release viral particles from the cell surface. In order to explore whether intestinal intraepithelial lymphocytes assist PEDV in infecting organoids, and whether intestinal organoids show similar damage to the small intestinal epithelium in vivo, this study used PEDV virus liquid adsorbed for 2 hours with or without the addition of pancreatic enzymes, and then infected the co-culture system and Vero cells. Cytopathic effects of PEDV-infected porcine ileal organoids and Vero cells were observed and identified by indirect immunofluorescence. The results showed that PEDV could not infect Vero without the addition of pancreatic enzymes, which ruled out the possibility that this strain was a non-pancreatic enzyme-dependent strain. Regardless of whether pancreatic enzymes were added, PEDV could not directly infect organoids, but PEDV could infect porcine ileal organoids in the co-culture system, and cavity shrinkage and obvious cell fragments appeared. It can be seen that PEDV infection of the co-culture system caused lesions in porcine ileal organoids, such as Figure 7 To further verify the infection of PEDV in the porcine ileal organoids in the co-culture system without the addition of pancreatic enzymes, mouse anti-PEDV-S protein antibodies were used to perform immunofluorescence detection on porcine ileal organoids infected for 18 h. The results showed that the presence of PEDV could be located in the porcine ileal organoids when the co-culture system was infected without the addition of pancreatic enzymes, as shown in Figure 2. Figure 8 It was shown that PEDV can still infect porcine ileal organoids without the addition of pancreatic enzymes, indicating that this infection may be mediated by lymphocytes.

[0048] 2.4.2 Immunofluorescence identification of lymphocytes involved in PEDV infection of porcine ileal organoids To further verify that PEDV infection of porcine ileal organoids is mediated by intestinal intraepithelial lymphocytes, this study used PEDV to infect porcine ileal organoids with or without intestinal intraepithelial lymphocytes, and used mouse anti-PEDV-S protein antibodies and intestinal intraepithelial lymphocyte marker CD3 to perform immunofluorescence detection on porcine ileal organoids infected for 18 h. The results showed that PEDV was located in porcine ileal organoids co-cultured with small intestinal intraepithelial lymphocytes, such as Fig. 9 As shown. It proves that intestinal intraepithelial lymphocytes are involved in PEDV infection of porcine ileal organoids.

[0049] 2.4.3 Determination of proliferation curves of strong and weak PEDV strains in porcine ileal organoids In order to determine the replication of porcine ileal organoids in the co-culture system after infection with strong / weak PEDV strains, this study collected infected porcine ileal organoids and culture supernatants at different time points after infection and used RT-qPCR to substitute the PEDV standard curve ( Fig.10 The results showed that after PEDV infection, the PEDV genome copy number in the cells of the pig ileal organoids increased significantly. Both the strong PEDV strain SX and the weak PEDV strain CV777 reached a peak of about 3.1×10 at 24 h after infection. 6 copies / mL( Fig.10 B); The viral load in the culture supernatant continued to increase with the duration of culture. The strong PEDV strain SX reached its peak at 36 h after infection, and the weak strain CV777 reached its peak at 24 h after infection ( Fig.10 C). The above results indicate that PEDV successfully infected porcine ileal organoids in the co-culture system and established effective viral replication and infection.

[0050] 2.4.4 Identification of cell subsets in porcine ileal organoids in the PEDV-infected co-culture system In order to further study the infection characteristics of PEDV in porcine ileal organoids in the co-culture system, this study used antibodies against four cell protein surface markers, Ki67, Muc2, villin 1 and Lgr5, and anti-PEDV-S protein antibodies, and performed double immunofluorescence labeling co-localization detection on proliferating cells, goblet cells, villus epithelial cells, intestinal stem cells and PEDV-infected cells. The experimental results are shown in Figure 2. Fig.11 As shown. The results showed that PEDV protein was significantly co-localized with Ki67-positive proliferating cells, Muc2-positive goblet cells, villi epithelial cells, and Lgr5-positive intestinal stem cells, indicating that PEDV has tropism for proliferating cells, goblet cells, villi epithelial cells, and intestinal stem cells. The results showed that PEDV can infect multiple cell subsets of porcine ileal organoids in the co-culture system.

[0051] 2.5 Co-culture system can be used as a PEDV wild-type virus infection model 2.5.1 Morphological changes of porcine ileal organoids infected with wild-type PEDV in co-culture system In recent years, the isolation and culture of PEDV has been a major problem restricting the research on the virus. At present, the commonly used cell line for the isolation and culture of wild strains of PEDV is Vero cells. However, PEDV needs to be domesticated and adapted on Vero cells before subsequent use. In particular, the prevalent strains of PEDV in recent years are still difficult to adapt to cells after adding trypsin to the commonly used cell line Vero, and cannot represent the true reaction of PEDV in the intestine in vivo. In this study, the co-culture system and Vero cells were infected with the wild strain of PEDV B18 without adding trypsin, and the virus liquid was recovered and blindly propagated. The cytopathic effect of porcine ileal organoids infected with PEDV was observed every 12 hours. The results showed that obvious lesions of porcine ileal organoids could be observed when PEDV was blindly propagated to the fourth generation, but no lesions had yet appeared on Vero cells, such as Fig.12 To further verify the infection of the fourth-generation PEDV wild-type strain B18 on the porcine ileal organoids in the co-culture system, mouse anti-PEDV-S protein and rabbit anti-CD3 antibodies were used to perform immunofluorescence detection on the porcine ileal organoids in the co-culture system infected for 36 h. The results showed that PEDV could be located in the porcine ileal organoids at the fourth generation of blind transmission, such as Fig.13 It was confirmed that the co-culture system is more suitable for the isolation and culture of PEDV wild strains than traditional Vero cells, and the co-culture system of porcine ileal organoids and porcine small intestinal intraepithelial lymphocytes can be better used as a PEDV wild strain infection model.

[0052] 2.5.2 RT-PCR identification of PEDV wild-type virus in pig ileal organoids and Vero infected with PEDV in co-culture system In order to investigate whether wild-type PEDV strains can be continuously passaged in porcine ileal organoids and Vero cells in the co-culture system, the collected virus fluid was blindly passaged and tested by RT-PCR in the co-culture system and Vero cells at a ratio of 1:10. Fig.14 As shown in the figure, the results show that without the addition of trypsin, PEDV can be detected in the co-culture system for five blind passages, and the target band can be seen at 146 bp. Regardless of whether trypsin is added, PEDV was not detected in Vero cells for five blind passages. This further confirms that the co-culture system is more suitable as an infection model for PEDV wild strains than traditional Vero cells.

Claims

1. A method for constructing a co-culture system of porcine intestinal organoids and lymphocytes, characterized in that: The method comprises the step of co-culturing porcine ileal organoids with porcine small intestinal intraepithelial lymphocytes, wherein the co-culturing of porcine ileal organoids with porcine small intestinal intraepithelial lymphocytes is carried out according to the following steps: (1) Resuscitation of porcine small intestinal intraepithelial lymphocytes: Take out the porcine small intestinal intraepithelial lymphocyte cryopreservation tube from liquid nitrogen, place it in a 37°C water bath until the cell suspension is completely thawed, transfer the cell suspension to a centrifuge tube, add RPMI 1640, mix well, centrifuge, and discard the supernatant; (2) Porcine ileal organoids are obtained by organoid differentiation and culture of crypt stem cells isolated from the intestinal segments of piglets' ileum. After culturing the porcine ileal organoids for 1-2 days, the original culture medium is discarded, and pre-cooled PBS is added to blow the intestinal organoids into the centrifuge tube. The bottom of the well is rinsed with pre-cooled PBS, and the wells are added to the same centrifuge tube. Centrifuge and discard the supernatant. (3) 100 porcine ileal organoids were combined with 10 5 The small intestinal intraepithelial lymphocytes were mixed, added to DMEM / F12 culture medium, and incubated in an incubator at 37°C with 5% CO2 for 30 min; (4) After the incubation is completed, the cell suspension is centrifuged at 1500 rpm / min for 5 min, the cell pellet is resuspended in matrix gel and transferred to a 48-well cell culture plate. The 48-well plate is placed in an incubator at 37°C and 5% CO2 for incubation for 15 min, and then 200 μL of IntestiCult™ human intestinal organoid growth medium containing 1% v / v penicillin-streptomycin mixture and 100 U / mL IL-2 is added to each culture well containing cells. The cell culture plate is placed in an incubator at 37°C and 5% CO2 for incubation to obtain the porcine intestinal organoid and lymphocyte co-culture system.

2. The method according to claim 1, characterized in that The porcine ileum organoids are prepared by the following steps: (1) Thaw the Matrigel on ice, pre-cool PBS and add 2% v / v penicillin-streptomycin mixture to the pre-cooled PBS for the following experiments; (2) A 5-10 cm ileum segment was cut from a 7-day-old pig and placed in a cell culture dish containing the above-mentioned pre-cooled PBS; (3) Flush the contents of the ileum until the intestinal contents are completely removed, and then discard the flushing fluid; (4) Add the above pre-cooled PBS to another new culture dish, place the washed intestinal segment into the culture dish, cut it into small segments of 1-3 mm, transfer the tissue suspension containing the intestinal segment to a 50 mL centrifuge tube, and allow the intestinal fragments to settle naturally; (5) Remove the upper layer of liquid in the centrifuge tube and add the above pre-cooled PBS; repeat this washing step until the supernatant becomes clear; (6) After removing the supernatant, resuspend the intestinal segment in 10 mL of mild cell dissociation buffer and incubate on a shaker at 100 rpm for 20 min at room temperature; (7) After incubation, allow the intestinal segments to settle naturally, discard the supernatant, and then add 10 mL of the above pre-cooled PBS containing 1% v / v fetal bovine serum (FBS) and move the intestinal fragments up and down; (8) After most of the intestinal fragments have settled, collect the supernatant, filter it through a 70 μm filter, and inject the filtrate into a 15 mL centrifuge tube. Repeat this step 4 to 5 times. (9) Centrifuge the filtrate at 1200 rpm for 5 min at 4°C, discard the supernatant, and collect the crypt fragment precipitate; suspend the crypt fragment precipitate in DMEM / F12 medium and count the cells; (10) Centrifuge the suspension at 1200 rpm for 5 minutes at 4°C, discard the supernatant, add IntestiCult™ human intestinal organoid growth medium and Matrigel to the crypt stem cells at a ratio of 25 μL IntestiCult™ human intestinal organoid growth medium and 25 μL Matrigel per 50 crypt stem cells, and transfer 50 μL of the crypt stem cell suspension to a cell culture plate; (11) Place the cell culture plate in a 37°C CO2 incubator and incubate for 15 min to allow the matrix gel to solidify. Then, add 200 μL of IntestiCult™ human intestinal organoid growth medium containing 1% v / v penicillin-streptomycin mixture to each culture well containing crypt stem cells. (12) Place the cell culture plate in an incubator at 37°C containing 5% carbon dioxide; replace the IntestiCult™ human intestinal organoid growth medium every 3-4 days. After 7-8 days, the crypt stem cells proliferate and differentiate into complete 3D porcine ileal organoids with villus-like epithelial cells and crypt region domains with bud-like structures.

3. The method according to claim 1, characterized in that The porcine intestinal intraepithelial lymphocytes are prepared by the following steps: (1) Take 10-15 cm of the ileum of a 21-day-old piglet and place it in PBS containing 2% v / v penicillin-streptomycin mixture; (2) Immerse the small intestine in a culture dish on ice containing 2% v / v penicillin-streptomycin mixture in PBS, use forceps to remove fat, lymphatic tissue and Peyer's patches (PP), and rinse the intestinal segment with a 1 mL pipette tip; (3) Replace the culture dish with a new one and add PBS containing 2% v / v penicillin-streptomycin mixture, cut the intestinal segment into 3±0.5 cm×0.5±0.5 cm in size, and transfer it to a 50 mL centrifuge tube; (4) Add ice-cold PBS containing 2% v / v penicillin-streptomycin mixture, shake for 5-15 seconds, and discard the supernatant after the intestinal fragments settle naturally. Repeat 3-5 times until the supernatant is clear. (5) Transfer the intestinal segment to Hanks-dithiothreitol (DTT) solution, incubate at 37°C constant temperature shaker at 150 rpm / min for 20 min, and discard the solution. The Hanks-dithiothreitol (DTT) solution contains 30 mL Hanks, 5 mmol DTT and 2% v / v FBS; (6) Add 30 mL of Hanks-EDTA digestion solution, incubate at 37°C on a shaker for 25 min, filter through a 100 μm cell strainer in a 50 mL centrifuge tube, collect the supernatant, and repeat this step three times with new digestion solution. The Hanks-EDTA digestion solution contains 30 mL of Hanks, 5 mmol EDTA, and 2% v / v FBS. (7) Add washing solution to the supernatant obtained in step (6), incubate at 37°C on a shaker for 20 min, and collect the cells through a 100 μm cell strainer into the same 50 mL centrifuge tube. The washing solution contains 20 mL Hanks, 2 mmol / L-glutamine, and 2% v / v FBS. (8) Centrifuge the collected supernatant at 600 g / min for 10 min in a 4°C centrifuge to obtain a cell pellet; (9) Rinse a 15 mL centrifuge tube with 1 mL FBS in advance, then slowly add 66% v / v-44% v / v-25% v / v Percoll solution from bottom to top, slowly add the cell pellet obtained in step (8) to the upper layer of Percoll solution, centrifuge at 600 g / min at 4°C for 30 min, and collect the lymphocytes between the 44% v / v-66% v / v Percoll solution layers; (10) Wash the cells twice with RPMI-1640 and resuspend them in 1 mL of RPMI-1640 complete medium. Count the cells using a counting plate and adjust the concentration to 2×10 6 cells / well, and then spread them into a 96-well cell culture plate and cultured in a 37°C incubator to obtain the porcine intestinal intraepithelial lymphocytes; wherein the RPMI-1640 complete culture medium is an RPMI-1640 culture medium containing 10% v / v FBS and 1% v / v penicillin-streptomycin mixture.

4. A co-culture system of porcine intestinal organoids and lymphocytes constructed according to the method described in any one of claims 1 to 3.

5. Use of the porcine intestinal organoid and lymphocyte co-culture system according to claim 4 in culturing porcine epidemic diarrhea virus.

6. The use according to claim 5, characterized in that The porcine epidemic diarrhea virus is a porcine epidemic diarrhea virus cell domesticated strain or a porcine epidemic diarrhea virus wild strain.

Images