Preparation method and application of ox tongue mucous membrane organoid

Abstract

The invention discloses a preparation method and application of a bovine tongue mucous membrane organoid, and relates to the technical field of organoid separation and culture. The preparation method of the ox tongue mucosa organoid comprises the following steps: treating fetal ox tongue tip tissues with digestive enzymes Dispase I, Collagenase II and Collagenase IV, separating ox tongue mucosa, cutting into pieces, treating again with pancreatin, and sieving to remove tissues and impurities; and continuously culturing by adopting a three-dimensional culture technology-matrigel embedding method and an organoid culture medium to obtain the final ox tongue mucous membrane organoid. The ox tongue mucous membrane organoid prepared by the preparation method disclosed by the invention has normal cell proliferation, differentiation and metabolism functions, and can be stably passaged under an in-vitro culture condition; the method can be widely applied to various related scientific researches such as FMDV infection mechanism research, drug screening and vaccine evaluation.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid isolation and culture, and in particular to a method for preparing bovine tongue mucosal organoids and applications thereof. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease that severely harms even-toed ungulates. FMDV spreads rapidly and infects a wide range of animals, causing significant economic losses to the global livestock industry.

[0003] Currently, research on FMDV relies primarily on traditional cell culture and animal models. However, traditional cell lines have limitations in simulating the complex physiological environment and intercellular interactions in the body, and cannot fully reflect the true infection process of the virus in the host. While animal models can better simulate the overall situation of viral infection, they are subject to issues such as high cost, long experimental cycles, complex procedures, and animal ethics.

[0004] Organoids are three-dimensional cell structures cultured in vitro that closely mimic the physiological and pathological characteristics of organs in vivo. In recent years, organoid technology has made significant progress in multiple fields, providing powerful new tools for disease research and drug development. However, there have been no reports using organoid models in FMDV research. In particular, there is a lack of knowledge on the construction of organoids targeting the bovine tongue mucosa, a key target organ for FMDV infection. Furthermore, the difficulty of cultivating bovine tongue mucosal organoids that exhibit normal cell proliferation, differentiation, and metabolic functions and can be stably passaged under in vitro culture conditions has been further increased. Summary of the Invention

[0005] The present invention provides a method for preparing bovine tongue mucosal organoids and their applications. By employing a unique preparation process, the bovine tongue mucosa is digested, enzymatically treated, and cultured in a specialized culture medium. Ultimately, the resulting culture produces bovine tongue mucosal organoids that exhibit normal cell proliferation, differentiation, and metabolic functions and can be stably passaged under in vitro culture conditions. These bovine tongue mucosal organoids can be widely used in various scientific studies related to FMDV infection. The present invention is specifically implemented through the following technologies.

[0006] A method for preparing bovine tongue mucosal organoids comprises the following steps:

[0007] Fetal bovine tongue tip tissue was collected, washed with a buffer solution containing antibiotics, and then immersed in a basal medium without antibiotics and incubated at 4°C for 6-8 h.

[0008] The first digestion process was performed using digestive enzyme solution to separate the mucosa and muscle layers of the bovine tongue;

[0009] The bovine tongue mucosa was cleaned, cut into pieces, and digested for the second time using a pancreatic enzyme solution. After the second digestion, the filtrate was collected by sieving and the precipitate was collected by centrifugation.

[0010] embedding the precipitate in matrigel, and adding the organoid culture medium for culturing after the matrigel solidifies;

[0011] The digestive enzymatic hydrolysis solution includes Dispase I, Collagenase II and Collagenase IV.

[0012] Furthermore, the digestion enzymatic solution includes Dispase I 1-2 mg / ml, Collagenase II 1-2 mg / ml and Collagenase IV 1-2 mg / ml.

[0013] Furthermore, the digestion enzymatic solution includes Dispase I 2 mg / ml, Collagenase II 2 mg / ml and Collagenase IV 1 mg / ml.

[0014] Furthermore, the conditions of the first digestion treatment are 35-37° C., 180-220 rpm, and digestion for 0.5-1 h.

[0015] Furthermore, the conditions of the first digestion treatment are 37° C., 200 rpm, and digestion for 1 hour.

[0016] Furthermore, the mass fraction of pancreatic enzyme in the pancreatic enzyme solution is 0.05-0.25%.

[0017] Furthermore, the mass fraction of pancreatic enzyme in the pancreatic enzyme solution is 0.125%.

[0018] Furthermore, the components of the organoid culture medium include: Advanced DMEM / F12+++ medium, GlutaMAX-I 1X, penicillin 100 U / ml, streptomycin 0.1 mg / ml, HEPES 20mM, B27 Supplement 1×, RSPO1 25 ng / ml, N-acetyl-L-cysteine ​​2 mM, Nicotinamide 15 mM, recombinant human FGF2 20 ng / ml, recombinant human EGF 50ng / ml, ROCK inhibitor Y-27632 10 μmol.

[0019] Furthermore, the components of the organoid culture medium include: Advanced DMEM / F12+++ medium, GlutaMAX-I 1X, penicillin 100 U / ml, streptomycin 0.1 mg / ml, HEPES 20mM, B27 Supplement 1×, RSPO1 25 ng / ml, N-acetyl-L-cysteine ​​2 mM, Nicotinamide 15 mM, recombinant human FGF2 20 ng / ml, CHIR99021 0.3 μM, and ROCK inhibitor Y-27632 10 μmol.

[0020] Furthermore, the culture conditions in the organoid culture medium are 37° C. and 5% CO 2 .

[0021] The present invention also provides a bovine tongue mucosal organoid prepared by any of the above-mentioned preparation methods.

[0022] The bovine tongue mucosal organoids provided by the preparation method of the present invention are composed of a variety of bovine tongue mucosa-related cell types. These cells maintain normal cellular functions and interactions in the organoids, and have a layered structure and physiological functions similar to those of natural bovine tongue mucosa. For example, the basal cell layer, spinous cell layer, and surface keratinized cell layer can simulate the normal tissue structure of the bovine tongue mucosa. The bovine tongue mucosal organoids provided by the present invention can express markers specific to the bovine tongue mucosa, have normal cell proliferation, differentiation, and metabolic functions, and can grow stably for a long time under in vitro culture conditions.

[0023] After 7-10 days of culture in organoid culture medium, bovine tongue mucosal organoids begin to form and gradually grow and expand. Once the organoids reach an appropriate size and number, they can be subcultured or used for subsequent experiments.

[0024] The present invention also provides an application of bovine tongue mucosal organoids prepared by any of the preparation methods described above, which can be used for in vitro screening and / or evaluation of anti-FMDV drugs, or for research on FMDV infection mechanisms not for the purpose of disease diagnosis and treatment, or for the preparation and / or evaluation of FMDV vaccines.

[0025] The application of the bovine tongue mucosal organoids provided by the present invention is to inoculate the cultured bovine tongue mucosal organoids with FMDV. The inoculation method can be to drip the virus solution directly onto the surface of the organoid, or other commonly used virus inoculation methods. For example, the inoculated virus titer can be specifically selected to be 10 -3 -10 -6The infection time can be selected to be 2-5 hours. During the infection process, the virus infection status can be preliminarily judged by observing the morphological changes of the organoids.

[0026] When using bovine tongue mucosal organoids to study viral infection mechanisms, researchers use bovine tongue mucosal organoids and commercially available FMDV to study the infection mechanism of FMDV in the laboratory, including but not limited to viral adsorption, entry, replication, transcription, and translation. For example, real-time quantitative PCR is used to detect viral nucleic acid replication in organoids and analyze changes in viral gene expression. Immunofluorescence staining and Western blot are used to detect the expression and distribution of viral proteins and to investigate the impact of viral infection on intracellular signaling pathways in organoids.

[0027] When using bovine tongue mucosal organoids for anti-FMDV drug screening and / or testing, anti-FMDV drugs can be screened and evaluated using the organoids as a model. Specifically, the drug to be screened can be added to an FMDV-infected organoid culture system to observe its inhibitory effect on viral infection. For example, the drug's antiviral activity and cytotoxicity can be assessed by measuring viral nucleic acid and protein expression levels, cytopathic effect, and cell survival rate. The bovine tongue mucosal organoids can also be used to study the mechanism of action of anti-FMDV drugs, providing experimental evidence for the development of new anti-FMDV drugs.

[0028] When bovine tongue mucosal organoids are used in the preparation and / or evaluation of FMDV vaccines, the immunogenicity and protective efficacy of the FMDV vaccine can be assessed on the bovine tongue mucosal organoids. Specifically, vaccine antigens can be co-incubated with bovine tongue mucosal organoids to detect immune response indicators produced by the organoids, such as cytokine secretion. Furthermore, FMDV can be used to challenge the vaccine-treated organoids to observe the protective effect of the vaccine on the organoids, providing important reference for vaccine development and optimization.

[0029] The present invention also provides a modeling method for bovine tongue mucosal organoids using virus transfection, wherein the bovine tongue mucosal organoids are prepared using the preparation method of any one of claims 1 to 7; the virus is any virus that infects the bovine tongue mucosa; the bovine tongue mucosal organoids are placed on a culture plate, and a preparation containing the virus is dripped onto the bovine tongue mucosal organoids, incubated, and cultured after replacing the organoid culture medium to complete the modeling of the virus-infected transfected bovine tongue mucosal organoids.

[0030] Furthermore, in the above modeling method, the infection condition is incubation with the virus at 37°C for 2-5 hours; and the continued culture condition after replacing the organoid culture medium is cultured in an environment of 37°C and 5% CO2.

[0031] In the above-mentioned modeling method of the present invention, the selected virus is applicable not only to FMDV virus, but also to other viruses that can infect the bovine tongue mucosa.

[0032] Compared with the existing technology, the benefits of the present invention are: the present invention provides a set of preparation methods for bovine tongue mucosal organoids. By selecting special digestion treatment and culture medium containing specific growth factors and nutrients for cultivation, the obtained bovine tongue mucosal organoids have normal cell proliferation, differentiation and metabolic functions, and can be stably propagated under in vitro culture conditions; they can be widely used in FMDV infection research, related anti-FMDV drug and FMDV vaccine screening / evaluation experiments, etc., providing a new and effective experimental model for the research of FMDV-related prevention and control technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 HE-stained images of bovine tongue mucosal tissue (left) and bovine tongue mucosal organoids (right). As can be seen from the images, the layered structure of bovine tongue mucosal tissue and bovine tongue mucosal organoids is consistent.

[0034] Figure 2 Bovine tongue mucosal organoids were cultured for passage 0 ( Figure 2 a) 5th generation ( Figure 2 b) and 10th generation ( Figure 2 c) The results of passage.

[0035] Figure 3 Photos of bovine tongue mucosal organoids before (left) and after (right) FMDV infection. As can be seen, viral infection causes cell swelling, lysis, and shedding.

[0036] Figure 4 Results of qPCR detection of viral nucleic acid in bovine tongue mucosal organoids infected with FMDV. The figure shows the relative levels of viral nucleic acid in bovine tongue mucosal organoids at different time points after FMDV infection, as measured by real-time quantitative PCR, indicating viral replication in the organoids.

[0037] Figure 5 Immunofluorescence detection of FMDV infection in bovine tongue mucosal organoids.

[0038] Figure 6 To optimize the culture medium for HE detection of bovine tongue mucosal organoids; Figure 6 a is before optimization; Figure 6 b is after optimization. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] In some embodiments of the present invention, a method for preparing bovine tongue mucosal organoids is provided, comprising the following steps:

[0041] Fetal bovine tongue tip tissue was collected, washed with a buffer solution containing antibiotics, and then immersed in a basal medium without antibiotics and incubated at 4°C for 6-8 h.

[0042] The first digestion process was performed using digestive enzyme solution to separate the mucosa and muscle layers of the bovine tongue;

[0043] The bovine tongue mucosa was cleaned, cut into pieces, and digested for the second time using a pancreatic enzyme solution. After the second digestion, the filtrate was collected by sieving and the precipitate was collected by centrifugation.

[0044] embedding the precipitate in matrigel, and adding the organoid culture medium for culturing after the matrigel solidifies;

[0045] The digestive enzymatic hydrolysis solution contains digestive enzymes Dispase I, Collagenase II and Collagenase IV.

[0046] Optionally, in the above digestion enzymatic solution, the specific concentrations of various enzymes are Dispase I 1-2 mg / ml, Collagenase II 1-2 mg / ml, and Collagenase IV 1-2 mg / ml.

[0047] Specifically, in the above digestion enzymatic solution, the specific concentrations of various enzymes are Dispase I 2 mg / ml, Collagenase II 2 mg / ml and Collagenase IV 1 mg / ml.

[0048] Optionally, the conditions for the first digestion treatment are 35-37° C., 180-220 rpm, digestion for 0.5-1 h. For example, the conditions for the first digestion treatment are specifically: 37° C., 200 rpm, digestion for 1 h.

[0049] Optionally, in the above preparation method, the mass fraction of pancreatic enzyme in the pancreatic enzyme solution is 0.05-0.25%. For example, the mass fraction of pancreatic enzyme in the pancreatic enzyme solution is specifically 0.125%.

[0050] Optionally, in the above preparation method, the components of the organoid culture medium include: Advanced DMEM / F12+++ medium, GlutaMAX-I 1X, penicillin 100 U / ml, streptomycin 0.1 mg / ml, HEPES 20mM, B27 Supplement1×, RSPO1 25 ng / ml, N-acetyl-L-cysteine ​​2 mM, Nicotinamide 15 mM, recombinant human FGF2 20ng / ml, recombinant human EGF 50ng / ml, ROCK inhibitor Y-27632 10 μmol.

[0051] For example, the components of organoid culture medium specifically include: Advanced DMEM / F12+++ medium, GlutaMAX-I 1X, penicillin 100 U / ml, streptomycin 0.1 mg / ml, HEPES 20mM, B27 Supplement 1×, RSPO1 25ng / ml, N-acetyl-L-cysteine ​​2mM, Nicotinamide 15mM, recombinant human FGF2 20ng / ml, CHIR99021 0.3μM, ROCK inhibitor Y-27632 10μmol.

[0052] Optionally, in the above preparation method, the culture conditions in the organoid culture medium can adopt commonly used organoid culture methods, for example, specifically: culture at 37°C and 5% CO2.

[0053] When the bovine tongue mucosal organoids prepared by the above preparation method are actually used, they can be used for in vitro screening and / or evaluation of anti-FMDV drugs, or for studying the FMDV infection mechanism, or for the preparation and / or evaluation of FMDV vaccines.

[0054] For example, cultured bovine tongue mucosal organoids can be inoculated with FMDV to construct a model of FMDV infection of bovine tongue mucosa.

[0055] Specifically, the inoculation method can be to directly drip the virus solution onto the surface of the organoid, or other commonly used virus inoculation methods. For example, the inoculated virus titer can be specifically selected to be 10 -3 -10 -6 The infection condition can be incubated with the virus at 37°C for 2-5 hours. During the infection process, the morphological changes of the organoids can be observed to preliminarily determine the infection status of the virus.

[0056] For example, when bovine tongue mucosal organoids are used to study viral infection mechanisms, using bovine tongue mucosal organoids and commercially available FMDV, the infection mechanism of FMDV can be studied in the laboratory. It should be noted that this infection mechanism includes, but is not limited to, FMDV adsorption, invasion, replication, transcription, and translation.

[0057] Specifically, real-time quantitative PCR technology was used to detect the replication level of viral nucleic acid in organoids, and the expression changes of viral genes were analyzed; immunofluorescence staining, Western blot and other methods were used to detect the expression and distribution of viral proteins, and the impact of viral infection on the intracellular signaling pathways of organoids was studied.

[0058] For example, when using bovine tongue mucosal organoids for anti-FMDV drug screening and / or testing, the bovine tongue mucosal organoids can be used as a model to screen and evaluate anti-FMDV drugs. Specifically, the drug to be screened can be added to the FMDV-infected organoid culture system to observe the drug's inhibitory effect on viral infection.

[0059] Specifically, the antiviral activity and cytotoxicity of drugs can be evaluated by detecting indicators such as the expression levels of viral nucleic acids and proteins, cytopathic effects, and cell survival rates; the bovine tongue mucosal organoids can also be used to study the mechanism of action of anti-FMDV drugs, providing an experimental basis for the development of new anti-FMDV drugs.

[0060] For another example, when bovine tongue mucosal organoids are used for the preparation and / or evaluation of FMDV vaccines, the immunogenicity and protective effect of the FMDV vaccine are evaluated on bovine tongue mucosal organoids.

[0061] Specifically, vaccine antigens can be co-incubated with bovine tongue mucosal organoids to detect immune response-related indicators produced by the organoids, such as cytokine secretion; FMDV can also be used to attack vaccine-treated organoids to observe the protective effect of the vaccine on the organoids, providing important reference for vaccine development and optimization.

[0062] Example 1: Preparation of Bovine Tongue Mucosal Organoids

[0063] 1. Source of Materials

[0064] The bovine tongue mucosa biomaterial used in this example was obtained from a healthy cow fetus, and the tongue tip was harvested under sterile conditions to ensure that the obtained tissue had good cell activity and differentiation ability. The main reagents used in this example are as follows.

[0065] PBS buffer: purchased from Thermo Fisher.

[0066] MEM basal medium: purchased from Thermo Fisher.

[0067] Digestive enzyme solution: Contains digestive enzymes Dispase I (tissue dispase), Collagenase II (collagenase II) and Collagenase IV (collagenase IV), which are purchased from Thermo Fisher.

[0068] Pancreatic enzyme solution: purchased from Thermo Fisher.

[0069] Organoid culture medium: Purchased from Thermo Fisher Scientific. Organoid culture medium contains: Advanced DMEM / F12+++ medium, GlutaMAX-I 1×, penicillin 100 U / ml, streptomycin 0.1 mg / ml, HEPES 20 mM, B27 Supplement 1×, RSPO1 (R-spondin 1) 25 ng / ml, N-acetyl-L-cysteine ​​2 mM, Nicotinamide 15 mM, Recombinant human FGF2 20 ng / ml, Recombinant human EGF 50 ng / ml, ROCK inhibitor Y-27632 10 μM.

[0070] Digestion solution for passaging: purchased from Thermo Fisher, containing 0.25% trypsin and 0.02% EDTA.

[0071] 2. Organoid Preparation Method

[0072] (1) Immediately place the removed tongue tip tissue sample into a sterile centrifuge tube containing pre-cooled PBS buffer containing antibiotics (penicillin 100 U / ml, streptomycin 0.1 mg / ml) and quickly bring it back to the laboratory for subsequent processing.

[0073] Transfer the tissue sample to a sterile culture dish and wash it 3-5 times with PBS buffer containing antibiotics to remove blood, mucus and impurities on the surface.

[0074] (2) Soak the tissue sample in antibiotic-free MEM basal medium and incubate at 4°C for 8 h.

[0075] (3) After the tissue sample incubation is completed, use digestion enzyme solution (containing Dispase I 2mg / ml, Collagenase II 1mg / ml, Collagenase IV 1mg / ml) to digest at 37°C with shaking (220 rpm) for 1 h; the mucosal layer and muscle layer of the bovine tongue are completely separated.

[0076] (4) After cleaning the mucosal layer with PBS buffer, cut it into small pieces of 1 mm × 1 mm and incubate it in 0.125% pancreatic enzyme at 37°C for 30 min, gently shaking it every 5 min.

[0077] (5) After digestion, add MEM medium containing 10% fetal bovine serum to terminate digestion, and gently blow the tissue blocks with a pipette to form a single-cell suspension.

[0078] (6) Filter the single-cell suspension through a 70 μm cell sieve to remove undigested tissue fragments; transfer the filtrate to a centrifuge tube, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0079] (7) Resuspend the cell pellet after centrifugation in organoid culture medium and adjust the cell concentration to 2×10 5 Using the three-dimensional culture technology-Matrigel embedding method, 10 μL of cell suspension was thoroughly mixed with 90 μL of Matrigel to evenly embed the cells in the Matrigel.

[0080] (8) The mixed solution was evenly added to each well of a 24-well culture plate and incubated in a 37°C incubator for 30 min to allow the matrix gel to solidify.

[0081] (9) After the matrix gel solidifies, add 500 μL of organoid culture medium to each well and place the culture plate in an incubator at 37°C and 5% CO2.

[0082] After 2 days of culture, organoids began to form. During the culture process, the culture medium was replaced every 2 days and the growth of the organoids was observed.

[0083] The culture results are as follows Figure 1 As shown in the figure, it can be seen that the layered structure of bovine tongue mucosal tissue is consistent with that of bovine tongue mucosal organoids.

[0084] 3. Subculture of Organoids

[0085] When organoids grow to a diameter of approximately 300-500 μm, they can be subcultured.

[0086] (1) When passaged, use a pipette to remove the organoids from the culture wells and place them in a digestion solution containing 0.25% trypsin and 0.02% EDTA. Digest them in a 37°C constant temperature shaker for 5 minutes to dissociate the organoids into single cell suspensions or small cell clusters.

[0087] (2) Following steps (7) to (9) of the above-mentioned organoid preparation method, the cell suspension or cell cluster is re-seeded into a new culture plate for culture.

[0088] The results of subculture are as follows Figure 2 As shown, it can be seen that the morphology of bovine tongue mucosal organoids at generation P0 is consistent with that of generations P5 and P10 after subculture, and they still maintain the ability to proliferate.

[0089] Example 2: Application of Bovine Tongue Mucosal Organoids in FMDV Infection Research

[0090] 1. Virus preparation

[0091] The FMDV strain (Akesu / 58 / 2002, GenBank accession number AF511039) was amplified and cultured in BHK-21 cells. The virus solution was harvested, the virus titer was determined, and the virus solution was stored at -80°C for future use.

[0092] 2. Infection Experiment

[0093] Select the bovine tongue mucosal organoids obtained in Example 1, which are in good growth state and have a diameter of about 300-500 μm, remove the culture medium, and add the bovine tongue mucosal organoids to each well of the well plate. Add 10 -4 (You can usually choose 10 -6 -10 -3 ) 500 μL of FMDV virus solution was added to evenly cover the organoids with the virus, incubated at 37°C for 5 h, and then replaced with fresh organoid culture medium.

[0094] The culture plate was placed in an incubator at 37°C and 5% CO2, and organoid samples were collected at 0, 24, and 48 h after infection for subsequent experimental analysis.

[0095] The results are as follows Figure 3 As shown, viral infection can be seen to cause cell swelling, lysis, and shedding in bovine tongue mucosal organoids.

[0096] Example 3: Study on FMDV infection mechanism

[0097] 1. Preparation of FMDV virus infection samples

[0098] Select the bovine tongue mucosal organoids obtained in Example 1, which are in good growth state and have a diameter of about 300-500 μm, remove the culture medium, and add the bovine tongue mucosal organoids to each well of the well plate. Add 10 -4 (You can usually choose 10 -6 -10 -3 ) 500 μL of FMDV virus solution was added to evenly cover the organoids with the virus, incubated at 37°C for 5 h, and then replaced with fresh organoid culture medium.

[0099] The culture plate was placed in an incubator at 37°C and 5% CO2, and organoid samples were collected at 0, 24, and 48 h after infection for subsequent experimental analysis.

[0100] 2. Viral nucleic acid detection

[0101] (1) The total RNA from bovine tongue mucosal organoids at different time points after infection was extracted using the Trizol method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0102] (2) Using cDNA as a template, FMDV-specific primers were used for real-time quantitative PCR amplification.

[0103] The total reaction volume was 20 μL, including 10 μL SYBR Green PCR Master Mix, 0.5 μL each of upstream and downstream primers, 2 μL cDNA template, and 7 μL ddH2O.

[0104] The reaction conditions were as follows: pre-denaturation at 95°C for 30 s, followed by denaturation at 95°C for 5 s and annealing at 60°C for 30 s, for a total of 40 cycles.

[0105] By comparing the Ct values ​​at different time points, the relative content of viral nucleic acid was calculated and the replication dynamics of the virus in organoids were analyzed.

[0106] The results are as follows Figure 4 As shown in the figure, the relative content of viral nucleic acid in bovine tongue mucosal organoids at different time points after FMDV infection was detected using real-time quantitative PCR technology, indicating that FMDV virus replicates in bovine tongue mucosal organoids.

[0107] 3. Viral protein detection

[0108] Bovine tongue mucosal organoids at different time points after infection were fixed with 4% paraformaldehyde and then subjected to immunofluorescence staining.

[0109] Organoids were first treated with 0.1% Triton X-100 for 10 min to increase the permeability of the cell membrane.

[0110] The membranes were then blocked with 5% BSA for 30 min, and then FMDV-specific primary antibodies (purchased from ThermoFisherScientific) were added and incubated at 4°C overnight.

[0111] The next day, the cells were washed three times with PBS for 5 min each time, and then fluorescently labeled secondary antibodies (purchased from ThermoFisherScientific) were added and incubated at room temperature for 1 h.

[0112] Finally, the nuclei were stained with DAPI for 5 min, washed three times with PBS, and then observed and photographed under a fluorescence microscope to detect the expression and distribution of viral proteins in organoid cells.

[0113] The results are as follows Figure 5 As shown in the figure, as time goes by, the virus shows a trend of penetration from the outside to the inside, gradually invading the internal structure of the bovine tongue mucosal organoid.

[0114] Example 4

[0115] The passaging method for bovine tongue mucosal organoids in this example is essentially the same as that in Example 1. However, to increase the number of passages and meet library construction requirements, recombinant human EGF was removed from the organoid culture medium in Example 1, and 0.3 μM CHIR99021 (GSK-3α / β inhibitor) was added.

[0116] The experimental results of the above examples show that after systematic culture medium optimization, bovine tongue mucosal organoids exhibit significant passage stability and can be continuously and stably passaged for more than 10 generations, fully meeting the cell bank construction standards.

[0117] HE staining was used to compare the morphology of organoids before and after optimization ( Figure 6 ) found that the optimized bovine tongue mucosal organoids showed significant morphological differences from those before optimization. The pre-optimized bovine tongue mucosal organoids were excessively keratinized in the center, forming a hollow structure. The optimized bovine tongue mucosal organoids had a solid center. This indicates that the optimized organoids are better able to maintain cell stemness, a property that provides a good foundation for their continued proliferation. This gives the optimized bovine tongue mucosal organoids greater application value and research potential in subsequent functional studies, drug screening, and other diverse experiments.

[0118] The above specific embodiments confirm that the preparation method of the present invention can be used to successfully prepare bovine tongue mucosal organoids, and bovine tongue mucosal organoids can be used for FMDV infection research, drug screening, and vaccine evaluation experiments, providing a new and effective experimental model for research in FMDV-related fields.

[0119] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A method for preparing bovine tongue mucosal organoids, characterized in that: The following steps are involved: Fetal bovine tongue tip tissue was collected, washed with a buffer solution containing antibiotics, and then immersed in a basal medium without antibiotics and incubated at 4°C for 6-8 h. The first digestion process was performed using digestive enzyme solution to separate the mucosa and muscle layers of the bovine tongue; The bovine tongue mucosa was cleaned, cut into pieces, and digested for the second time using a pancreatic enzyme solution. After the second digestion, the filtrate was collected by sieving and the precipitate was collected by centrifugation. embedding the precipitate in matrigel, and adding the organoid culture medium for culturing after the matrigel solidifies; The digestive enzymatic hydrolysis solution contains digestive enzymes Dispase I, Collagenase II and Collagenase IV.

2. The method for preparing bovine tongue mucosal organoids according to claim 1, characterized in that: The digestion enzymatic solution includes Dispase I 1-2 mg / ml, Collagenase II 1-2 mg / ml and Collagenase IV 1-2 mg / ml; Furthermore, the digestion enzymatic solution includes Dispase I 2 mg / ml, Collagenase II 2 mg / ml and Collagenase IV 1 mg / ml.

3. The method for preparing bovine tongue mucosal organoids according to claim 1, characterized in that: The conditions for the first digestion treatment are 35-37°C, 180-220 rpm, digestion for 0.5-1 h; Furthermore, the conditions for the first digestion treatment are 37° C., 200 rpm, and digestion for 1 hour.

4. The method for preparing bovine tongue mucosal organoids according to claim 1, characterized in that: The mass fraction of pancreatic enzyme in the pancreatic enzyme solution is 0.05-0.25%; Furthermore, the mass fraction of pancreatic enzyme in the pancreatic enzyme solution is 0.125%.

5. The method for preparing bovine tongue mucosal organoids according to claim 1, characterized in that: The components of the organoid culture medium include: Advanced DMEM / F12+++ medium, GlutaMAX-I 1X, penicillin 100 U / ml, streptomycin 0.1 mg / ml, HEPES 20 mM, B27 Supplement 1×, RSPO1 25 ng / ml, N-acetyl-L-cysteine ​​2 mM, Nicotinamide 15 mM, recombinant human FGF2 20 ng / ml, recombinant human EGF 50 ng / ml, ROCK inhibitor Y-27632 10 μmol; Alternatively, the components of the organoid culture medium include: Advanced DMEM / F12+++ medium, GlutaMAX-I1X, penicillin 100 U / ml, streptomycin 0.1 mg / ml, HEPES 20 mM, B27 Supplement 1×, RSPO1 25 ng / ml, N-acetyl-L-cysteine ​​2 mM, Nicotinamide 15 mM, recombinant human FGF2 20 ng / ml, CHIR990210.3 μM, and ROCK inhibitor Y-27632 10 μmol.

6. The method for preparing bovine tongue mucosal organoids according to claim 1, characterized in that: The culture conditions in the organoid culture medium were 37° C. and 5% CO 2 .

7. A bovine tongue mucosal organoid prepared by the preparation method according to any one of claims 1 to 6.

8. A bovine tongue mucosal organoid prepared by the preparation method according to any one of claims 1 to 6, or a use of the bovine tongue mucosal organoid prepared according to claim 12, characterized in that: Used for in vitro screening and / or evaluation of anti-FMDV drugs, or for research on FMDV infection mechanisms not for the purpose of disease diagnosis and treatment, or for the preparation and / or evaluation of FMDV vaccines.

9. A method for modeling bovine tongue mucosal organoids using viral transfection, characterized in that: The bovine tongue mucosal organoid is prepared by the preparation method of any one of claims 1 to 7; the virus is any virus that infects the bovine tongue mucosa; the bovine tongue mucosal organoid is placed on a culture plate, and the preparation containing the virus is dripped onto the bovine tongue mucosal organoid, incubated, and the culture medium is replaced and then cultured to complete the modeling of the virus-infected transfected bovine tongue mucosal organoid.

10. The method for modeling bovine tongue mucosal organoids using viral transfection according to claim 9, characterized in that: The infection conditions are incubation with the virus at 37°C for 2-5 hours; the continued culture conditions after replacing the organoid culture medium are cultured at 37°C and 5% CO2 environment.

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