Model construction method based on animal endometrial epithelium organ and application thereof

Abstract

The invention discloses a model building method based on animal endometrial epithelium organoid and application of the model building method, and relates to the technical field of cell engineering and organoid. According to the method, healthy cow endometrial tissue serves as a raw material, a model is built through the steps of pretreatment, enzymolysis, cell cleaning and screening, seed gum culture, passage, cryopreservation resuscitation and the like, and the model is built through Hamp; e, verifying morphological integrity through dyeing, and detecting E-cadherin expression through IHC dyeing to confirm cell specificity. The animal organ model restores the in-vivo three-dimensional microenvironment and species specificity, overcomes the limitation of traditional two-dimensional culture and mouse models, can accurately simulate the pathological process of endometritis, is applied to research on the pathogenesis of endometritis of dairy cows, can also efficiently screen anti-inflammatory drugs and evaluate the drug effect, and has a wide application prospect. A reliable in-vitro tool is provided for disease prevention and treatment, and healthy development of the dairy cow breeding industry is assisted.

Description

Technical Field

[0001] This invention relates to the fields of cell engineering and organoid technology, specifically to a method for constructing organoid models based on animal endometrial epithelium and its application. Background Technology

[0002] Organoids are miniature organ models formed by the self-organization of cells through in vitro culture technology. Their core principle is to utilize the self-renewal and directed differentiation capabilities of stem cells or pluripotent progenitor cells. In a culture environment containing specific growth factors and matrix materials, they gradually develop into three-dimensional structures with organ characteristics, which can simulate the structure and function of real organs. They are formed by the differentiation of stem cells or specific tissue cells under specific conditions and have similar cell types, spatial arrangements and some physiological functions to natural organs. They are mainly used in disease research, drug testing and regenerative medicine.

[0003] Currently, research on the pathogenesis of endometritis in dairy cows and the development of anti-inflammatory drugs still mainly relies on traditional two-dimensional cell culture and mouse models. The former cannot simulate the three-dimensional structure of the endometrium and the microenvironment of intercellular interactions in vivo, while the latter has significant species differences from dairy cows, resulting in distorted simulation of pathological reactions and insufficient specificity and reliability of experimental results. This makes it difficult to accurately reveal the pathogenesis of the disease and efficiently screen anti-inflammatory drugs suitable for dairy cows, thus restricting breakthroughs in the prevention and treatment of endometritis in dairy cows and continuing to cause huge economic losses to the dairy farming industry, such as decreased reproductive performance and reduced milk production. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for constructing a model of organoids based on animal endometrial epithelium and its application, thus solving the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for constructing an organoid model based on animal endometrial epithelium, wherein the organoid model construction includes the following steps: S1. Select healthy dairy cows to remove the uterus, clean the surface of the uterus with pre-cooled tissue cleaning solution, remove fat, connective tissue and residual blood, and cut to obtain tissue fragments with a diameter of 0.5-1mm. S2. Place the tissue fragment suspension in a centrifuge tube and centrifuge. After centrifugation, discard the supernatant and add 10 mL of enzymatic hydrolysis solution I to resuspend the tissue fragments. Transfer the resuspended tissue fragments to a 6 cm culture dish and enzymatically hydrolyze the tissue fragments at 37 °C. Observe the tissue fragments under a microscope every 3-10 min with a Pasteur pipette until no obvious tissue fragments are found and a large number of dissociated glandular structures appear. The tissue suspension is then obtained. S3. Transfer the tissue suspension to a 15mL centrifuge tube, add the tissue washing solution to 10mL to terminate the enzymatic hydrolysis, centrifuge and discard the supernatant, then repeat the washing 2-3 times with the tissue washing solution. After each washing, centrifuge and discard the supernatant. After washing and centrifuging, add 500-1000μL of erythrocyte lysis buffer to the cell pellet, mix by pipetting and incubate on ice, add 5 times the volume of pre-cooled organoid washing solution to terminate the lysis, and centrifuge to collect the cell pellet. S4. Resuspend the cell pellet in the organoid washing solution, collect the completely digested cell clusters by natural sedimentation, and filter them through a 100μm cell sieve to remove extracellular matrix impurities to obtain filtrate. Transfer the filtrate to a centrifuge tube, centrifuge, discard the supernatant, add 1mL of organoid culture medium to resuspend the cells to obtain a cell suspension. S5. Using a 24-well low-adhesion culture plate as a carrier, prepare a 50 μL droplet for each well of the culture plate. Mix the cell suspension with an equal volume of matrix gel and add it to the well of the culture plate. Pre-cure at 37°C for 5 min, then incubate by inversion for 20-30 min to allow the matrix gel to completely solidify. After complete solidification, add >500 μL of organoid culture medium containing 1X additive I along the well wall of the culture plate. After culturing for 48 h, replace with organoid culture medium without additive I. Then replace the organoid culture medium every 2-3 days. S6. Primary culture of organoids for 7-15 days, until 500 organoids are obtained, each 100-200 μm in size, with clear boundaries, and in good growth condition. Then, passage is performed. The culture medium in the wells of the culture plate is aspirated, and the organoid washing solution is added to wash the gel droplets. After aspirating the washing solution, 500 μL of organoid recovery solution is added to each well, and the matrix gel is dispersed. The mixture is incubated at room temperature for 5 minutes to obtain a mixture. The mixture is collected into a 15 mL centrifuge tube, and residual cells in the wells are washed with the organoid washing solution and transferred to a centrifuge tube. After 3-4 passages to remove contaminating cells, the organoids are obtained.

[0006] Preferably, the dissociation of the organoid includes the following steps: Add 1 mL of organoid culture medium containing 1X additive I to the cells of the organoid, resuspend them, and observe them under a microscope after pipetting 5-50 times. If the cells are single cells or small cell clusters, dissociation is complete. If there are glandular organoids with solid-like growth in the cells, add 500 μL of enzymatic digestion solution II to resuspend the cells and transfer them to the 24-well low-adhesion culture plate. Incubate at 37°C for 2–15 min, pipetting 5–10 times every 1–3 min until the cells form small cell clusters. Add 9.5 mL of pre-cooled organoid washing solution to terminate the digestion. Centrifuge and discard the supernatant. Resuspend the cells in 1 mL of organoid culture medium containing 1X additive I to complete dissociation.

[0007] Preferably, the organoid model construction further includes a post-processing step, specifically including the following steps: After passage and culture for 3-5 days, the organoids are collected and dissociated to obtain small cell clusters. After centrifugation and discarding the supernatant, the cells are added to cryovials. 500-1000 μL of organoid cryopreservation solution is added to the cryovials. The cryovials are placed in a programmed cooling box and cooled at -80°C for 24 hours before being transferred to a liquid nitrogen tank for storage. When the organoids are resuscitated, the cryopreservation tubes are removed from the liquid nitrogen tank, thawed in a water bath at 37°C and mixed by blowing and agitating 3-5 times. The suspension of the organoids is transferred to a centrifuge tube containing 5 mL of the organoid washing solution and centrifuged for 5 min at 4°C and 200-300 g. The supernatant is discarded. Steps S4-S6 are repeated to complete the resuscitation of the organoids.

[0008] Preferably, the identification of the organoid construction model includes the following steps: H&E staining: After fixing, embedding, and sectioning the organoid samples, the slides were baked in a 65°C oven for 1 hour. They were then dewaxed in three xylene tanks, each for 15 minutes. After hydration, they were treated with a gradient of 100%, 95%, and 75% ethanol for 3-5 minutes at each concentration. After hydration, they were stained with hematoxylin stain for 5 minutes and then rinsed with tap water for 5 minutes. After staining, the sample is differentiated by 1% hydrochloric acid alcohol for 3-5 seconds, then blued in ammonia water for 1-3 seconds, stained with eosin solution for 1 minute, rinsed with tap water, and then dehydrated by 75%, 95%, and 100% ethanol in gradients, with each concentration of ethanol dehydration treatment lasting 3 minutes. Finally, after clearing with xylene for 3 minutes, neutral resin was added to seal the slide, and the morphology of the organoid was observed under a microscope to confirm whether the organoid construction was successful or failed. IHC staining: After dewaxing the organoid sections, antigen retrieval was performed at 100℃ for 20 min using ER2 antigen retrieval solution. After retrieval, the sections were treated with 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase, and then blocked with blocking solution for 10 min. Then add E-cadherin antibody diluted 1:5000 and incubate at room temperature for 60 min; then add goat anti-rabbit HRP secondary antibody and incubate at room temperature for 30 min. Finally, develop the stain with DAB working solution for 5 minutes, counterstain with hematoxylin for 5 minutes, rinse with tap water, and repeat the dehydration, clearing and mounting steps of H&E staining. Observe the expression of E-cadherin under a microscope to confirm whether the organoid construction was successful or failed.

[0009] Preferably, the uterus is removed within 30 minutes after slaughter of the dairy cow, washed with physiological saline, and then placed in a tissue preservation solution for preservation and transportation. The tissue preservation solution is kept at 4°C for storage and transportation. The uterine body tissue cutting operation is performed under sterile conditions, using sterile scissors to cut 100-200 times, controlling the diameter of the tissue fragments to be 0.5-1mm, with no excessively large tissue fragments remaining.

[0010] Preferably, in step S3, during cell washing and red blood cell lysis, the centrifugation parameters are controlled as follows: temperature 4°C, centrifugal force 200-300g, and centrifugation time 5min. After adding the red blood cell lysis buffer, blow the mixture 5-10 times to ensure that the cell pellet is completely mixed. Let it stand on ice for 3 minutes to avoid excessive lysis and damage to the target cells.

[0011] Preferably, the sedimentation time for the natural sedimentation method in step S4 is 10-15 min. The natural sedimentation method is used to collect dense, fully digested cell clusters. The cell sieve is operated under aseptic conditions. After filtration, the cell sieve screen is rinsed twice with the organoid washing solution to ensure that no target cells remain on the cell sieve.

[0012] Preferably, in step S5, the culture plate containing the cell seed gel is preheated at 37°C for 30 minutes before culturing. When mixing the cell suspension with the matrix gel, blow the mixture 3-5 times, and the mixing time should not exceed 2 minutes to avoid premature solidification of the matrix gel. The gel droplets are added to the culture plate, and the culture plate is incubated upright for 5 minutes to prevent movement during pre-curing. The cell density of the gel droplets in each well of the culture plate is controlled to be 10,000-50,000 cells / 50 μL.

[0013] Preferably, the contaminating cells in step S6 include fibroblasts and uterine myocytes. During passage, the contaminating cells are removed by allowing the cells to stand for 2-4 hours after inoculation. The specific procedure is as follows: After passage and inoculation, the culture plate is left to stand for 2-4 hours until the fibroblasts and myocytes adhere to the well walls of the culture plate. The supernatant and non-adherent organoid cell clusters in the wells are gently aspirated, and organoid culture medium without additive I is added for further culture. After 3-4 passages of this operation, the contaminating cells are removed, and the organoids of high purity endometrial glands are obtained.

[0014] An animal endometrial epithelial organoid model was constructed using a method based on animal endometrial epithelial organoids. This animal endometrial epithelial organoid model is used in the study of the pathogenesis of bovine endometritis, the screening of anti-inflammatory drugs for bovine endometritis, and the evaluation of their efficacy.

[0015] This invention provides a method for constructing an organoid model based on animal endometrial epithelium and its application. It has the following beneficial effects: (1) The constructed bovine endometrial epithelial organoid adopts a three-dimensional culture mode, which can realistically reproduce the glandular structure and intercellular interaction microenvironment of the endometrium in animals, achieving the effect of accurately simulating the physiological and pathological state of bovine endometrium. It solves the problem that traditional two-dimensional cell culture cannot replicate the three-dimensional microenvironment and the pathological reaction simulation is not realistic. It provides a practical research carrier for revealing the pathogenesis of bovine endometritis in depth and accurately, and further clarifies the core law of disease occurrence and development, providing an accurate research basis for subsequent diagnosis and treatment of the disease.

[0016] (2) This type of organ is derived from the healthy endometrial tissue of dairy cows, which fully preserves the cellular properties and tissue characteristics unique to the dairy cow species. It avoids the bias of cross-species model experiments and overcomes the defects of large differences between mouse models and dairy cow species and insufficient specificity of experimental results. It makes the anti-inflammatory drug screening experiment more in line with the actual physiology of dairy cows, significantly improves the reliability and suitability of drug screening, and provides a precise in vitro evaluation system for developing truly effective anti-inflammatory drugs suitable for dairy cows.

[0017] (3) The established organoid culture system has the characteristics of long-term stable culture and normal growth after cryopreservation and thawing. It can continuously provide standardized and consistent in vitro models for related research, thereby reducing the cost of experimental reproducibility and improving research efficiency. It promotes the efficient and coordinated advancement of disease mechanism research, drug screening and prevention and control program optimization, and ultimately effectively reduces the losses in the dairy farming industry caused by endometritis, such as decreased reproductive performance and reduced milk production, and provides strong technical support for the healthy and sustainable development of the industry. Attached Figure Description

[0018] Figure 1 These are micrographs of cells before and after enzymatic digestion in an embodiment of the present invention; Wherein: A represents tissue containing blood vessels (red arrow) and red blood cells before enzymatic digestion; B represents tissue containing some irregular glands (red arrow) before enzymatic digestion; C represents tissue with dissociated blood vessel-like structures (blue arrow) after enzymatic digestion; and D represents glandular organoids (blue arrow) after enzymatic digestion. Figure 2 These are micrographs of cell counting and seed gelation in an embodiment of the present invention; Where: A represents cell count, with a viable cell rate of approximately 69%; B represents cells collected after enzymatic hydrolysis following seeding, which contain a small number of organoids (red arrows). Figure 3 Microscopic images showing the results of organoid passage, cryopreservation, and resuscitation in embodiments of the present invention; Among them: A is day 4 of P0 generation, with a small number of organoids formed, some of which are thin-walled single-layered cysts (blue arrows), and some are solid gland-like structures (red arrows); B is day 4 of P1 generation, with an increase in the number of organoids, an increase in cystic organoids (blue arrows), and a small number of mixed cell clusters (red triangles); C is day 7 after resuscitation (P2 generation). Figure 4 Microscopic images of organoid identification results according to an embodiment of the present invention; Where: A represents the H&E results, with the organoids showing intact morphology (red arrow); B represents the IHC results, with the organoids expressing E-cadherin protein (blue arrow). Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 Experimental materials Experimental samples: Healthy dairy cows slaughtered by Ningxia State Farms Beef and Mutton Food Co., Ltd. were selected. The uterus was quickly removed within 30 minutes after slaughter. After rinsing the surface with physiological saline to remove residual impurities, the samples were immediately placed in tissue preservation solution and transported to the laboratory at 4°C. Main reagents: Tissue washing solution (Jiyanbiotech, B002), enzyme digestion solution I (Jiyanbiotech, D001), enzyme digestion solution II (Jiyanbiotech, D002), organoid washing solution (Jiyanbiotech, B004), erythrocyte lysis buffer (Jiyanbiotech, B005), organoid culture medium (Jiyanbiotech, S001), matrix gel (Jiyanbiotech, M001), organoid recovery solution (Jiyanbiotech, B008), E-cadherin antibody (Wuhan Sanying Biotechnology Co., Ltd., 20874-1-AP), ER2 antigen retrieval solution (Leica, AR9640-CN), goat anti-rabbit HRP antibody (Beijing Biosen Biotechnology Co., Ltd., bs-0294R-HRP), DAB colorimetric reagent kit (Wuhan Saiwei Biotechnology Co., Ltd., G1212), trypan blue staining solution, neutral resin, hematoxylin staining solution, eosin staining solution, 3% hydrogen peroxide solution, and blocking solution; Experimental equipment: 24-well low-adhesion culture plate, 6cm culture dish, 1.5mL centrifuge tube, 15mL centrifuge tube, sterile scissors, Pasteur pipette, 1mL pipette tip, cell sieve (100μm), programmed cooling box, liquid nitrogen container, 37℃ constant temperature water bath, 4℃ centrifuge, 37℃ constant temperature incubator and optical microscope.

[0021] Experimental steps Primary culture Tissue pretreatment: The uterine body transported to the laboratory was removed and rinsed three times with pre-cooled tissue cleaning solution. It was then transferred to a 6cm culture dish on a sterile operating table. Surrounding fat, connective tissue and residual blood were removed with sterile forceps. The treated uterine body tissue was then transferred to a 1.5mL centrifuge tube and quickly cut 100-200 times with sterile scissors to make the tissue fragments 0.5-1mm in diameter. Tissue enzymatic hydrolysis: Transfer the suspension of the shredded tissue fragments to a 15mL centrifuge tube. Set the centrifuge parameters to 4℃, 250g, and 5min. After discarding the supernatant, add 10mL of enzymatic hydrolysis solution I to resuspend the tissue fragments. Transfer the suspension to a new 6cm culture dish and place it in a 37℃ incubator for enzymatic hydrolysis. Remove the culture dish every 3-10min, gently blow the tissue suspension with a Pasteur pipette, and observe under an optical microscope until no obvious intact tissue fragments are visible in the field of view and a large number of dissociated glandular structures appear. Then, terminate the enzymatic hydrolysis. Cell washing and erythrocyte lysis: Transfer the enzymatically digested tissue suspension to a 15 mL centrifuge tube, add pre-chilled tissue washing solution to 10 mL, gently invert to mix and terminate the enzymatic activity, centrifuge at 4°C and 250 g for 5 min, discard the supernatant, add 5 mL of pre-chilled tissue washing solution, resuspend the cell pellet and centrifuge again. Repeat this washing step 2-3 times, discarding the supernatant after each centrifugation. Add 500-1000 μL of erythrocyte lysis solution to the final cell pellet, and pipette 5-10 times to thoroughly mix the cell pellet. Place on ice for 3 min, then add 5 times the volume of pre-chilled organoid washing solution, gently invert the centrifuge tube to terminate the lysis, centrifuge at 4°C and 250 g for 5 min, discard the supernatant and collect the cell pellet. Cell screening and counting: Resuspend the cell pellet in 1 mL of pre-cooled organoid washing buffer, transfer the cell suspension to a new 15 mL centrifuge tube, allow it to settle naturally at room temperature for 10-15 min, collect the enzymatically digested cell clusters in the supernatant, filter the collected cell suspension through a 100 μm cell sieve, rinse the sieve twice with a small amount of organoid washing buffer to remove extracellular matrix impurities, collect the filtrate into a 15 mL centrifuge tube, centrifuge at 250 g for 5 min at 4 °C, discard the supernatant, add 1 mL of organoid culture medium to resuspend the cells, obtain the cell suspension, take 10 μL of the cell suspension and mix it with 10 μL of trypan blue staining solution, add it to a cell counting plate, and detect and count the number of viable cells under an optical microscope; Cell seeding: Preheat the 24-well low-adhesion culture plate to 37°C for 30 minutes. Take an appropriate amount of cell suspension and mix it with the matrix gel in an equal volume ratio. Gently pipette 3-5 times with a 1mL pipette tip to mix thoroughly, avoiding air bubbles. The entire process should not take more than 2 minutes. Quickly add the mixture to the center of the preheated 24-well plate at a rate of 50μL per well, controlling the cell density to 10,000-50,000 cells / 50μL drop. If the cells are intact glandular structures or large cell clusters, adjust the density to 300-500 cells / 50μL. Place the 24-well plate upright in the 37°C incubator and incubate for 5 minutes for pre-curing. Then gently invert the plate and continue incubating for 20-30 minutes to allow the matrix gel to completely solidify. Maintenance culture: After the matrix gel has completely solidified, slowly add more than 500 μL of organoid culture medium containing 1X Additive I along the well wall of the 24-well plate, avoiding dispersing the gel droplets. After 48 hours of culture, replace the medium with organoid culture medium without Additive I, and then replace with fresh medium every 2-3 days. During the culture process, to prevent the medium from evaporating, add sterile water between the culture plates. Depending on the growth of the organoids, decide whether to passage within 7-15 days of culture.

[0022] Organoid passage When the primary culture (P0 generation) reaches 7-15 days, and approximately 500 organoids are observed to have formed under an optical microscope, with a size of about 100-200 μm, clear boundaries, and good growth status, then the passage operation is carried out. Organoid collection: Aspirate the culture medium from the wells of a 24-well plate, add pre-chilled organoid washing buffer to gently wash the surface of the gel droplets, aspirate the washing buffer, add organoid recovery buffer to each well at a ratio of 500 μL organoid recovery buffer to 50 μL of gel droplet per well, gently disperse the matrix gel with a 1 mL pipette tip, incubate at room temperature for 5 min, collect the mixture in the wells into a 15 mL centrifuge tube, wash the residual cells in the wells with a small amount of organoid washing buffer, transfer the washing buffer to the same centrifuge tube, add 2 times the volume of pre-chilled organoid washing buffer, place on ice for 10 min, centrifuge at 4 °C and 250 g for 5 min, and discard the supernatant.

[0023] Organoid dissociation: Add 1 mL of organoid culture medium containing 1X additive I to the cell pellet, and gently pipette 5-50 times with a 1 mL pipette tip. Observe the cell state under an optical microscope. If the cells are mostly single cells and small cell clusters, proceed directly to the next step. If there are glandular organoids with solid-like growth, add 500 μL of enzymatic digestion solution II to resuspend the cell pellet, transfer it to a 24-well plate, and incubate at 37°C for 2-15 min, pipetting 5-10 times every 1-3 min until the cells are in a small cell cluster state. Immediately add 9.5 mL of pre-cooled organoid washing solution to stop the digestion, centrifuge at 4°C and 250 g for 5 min, discard the supernatant, and resuspend the cells in 1 mL of organoid culture medium containing 1X additive I. Subsequent culture: Following the cell counting, seeding, and maintenance culture steps described above for primary culture, complete the organoid passage. After passage and seeding, let the 24-well plate stand for 2-4 hours until fibroblasts, myocytes, and other contaminating cells adhere to the well walls. Then, gently aspirate the supernatant and any unattached organoid cell clusters from the wells. Add fresh organoid culture medium without additive I and continue culturing. After 3-4 passages, contaminating cells in the culture system will be largely removed, resulting in a high-purity endometrial gland organoid composition.

[0024] Organoid cryopreservation When the organoids are cultured for 3-5 days after passage, and approximately 500 organoids of 100-200 μm in size are observed under an optical microscope and are in good growth condition, cryopreservation is performed. Following the collection and dissociation steps in organoid passage, small cell clusters were obtained. The cell suspension was transferred to a 15mL centrifuge tube, centrifuged at 4℃ and 250g for 5min, and the supernatant was discarded. Depending on the cell count, add organoid cryopreservation solution to the centrifuge tubes, adding 500-1000 μL of solution to each tube, ensuring each tube contains 0.5-5 × 10⁻⁶ cells.5 For each cell / cluster, place the cryovial in a programmed cooling box and place it in a -80°C freezer for gradient cooling at a rate of -1°C / min. After 24 hours, transfer the cryovial to a liquid nitrogen tank and store it at -196°C for long-term storage.

[0025] Organoid resuscitation Remove the target cryovial from the liquid nitrogen tank and quickly transfer it to the sterile cell handling room. Immediately place it in a 37°C constant temperature water bath to thaw. Gently shake the cryovial to accelerate the thawing. Control the thawing time to 1-2 minutes until the cryopreservation solution is completely thawed. Gently pipette the cell suspension 3-5 times with a 1mL pipette tip to mix it. Transfer the thawed cell suspension to a 15 mL centrifuge tube containing 5 mL of organoid washing solution. Centrifuge at 250 g for 5 min at 4 °C, discarding the supernatant. Optional steps include cell screening and counting as described in primary culture to check cell viability, requiring a viability ≥ 60%. Then, follow the cell seeding and maintenance culture steps as described in primary culture to complete the organoid resuscitation culture.

[0026] Organoid identification H&E staining: Select organoid samples in good growth condition, fix and embed them to prepare paraffin sections, bake the sections in a 65℃ oven for 1 hour, and then dewax them in three xylene solutions for 15 minutes each. Then, hydrate them with 100%, 95%, and 75% graded ethanol for 3-5 minutes each. After hydration, stain with hematoxylin for 5 minutes, rinse with tap water for 5 minutes, differentiate with 1% hydrochloric acid alcohol for 3-5 seconds, then immerse in ammonia water for 1-3 seconds to regain blue color, stain with eosin for 1 minute, rinse with tap water, dehydrate with 75%, 95%, and 100% graded ethanol for 3 minutes each, and finally clear with xylene for 3 minutes. Mount the sections with neutral resin and observe the organoid morphology under an optical microscope. IHC staining: After dewaxing organoid paraffin sections with xylene and hydration with graded ethanol, antigen retrieval was performed in a boiling water bath at 100℃ for 20 min using ER2 antigen retrieval solution. After cooling to room temperature, the sections were treated with 3% hydrogen peroxide solution in the dark for 10 min to block endogenous peroxidase activity. Blocking solution was added and the sections were blocked at room temperature for 10 min. E-cadherin antibody diluted 1:5000 was added and the sections were incubated at room temperature for 60 min. The sections were washed three times with PBS buffer for 5 min each time. Goat anti-rabbit HRP antibody was added and the sections were incubated at room temperature for 30 min. The sections were washed three times with PBS buffer for 5 min each time. The working solution from the DAB staining kit was added and the sections were developed for 5 min. The staining depth was controlled under an optical microscope. After the staining was completed, the sections were rinsed with distilled water to stop the staining. The sections were counterstained with hematoxylin stain for 5 min and rinsed with tap water. The dehydration, clearing, and mounting steps of H&E staining were repeated. E-cadherin expression was observed under an optical microscope.

[0027] Experimental results Reference Figure 1 - Figure 4 After being cultured using the above method, a small number of organoids were observed to form under an optical microscope on the 4th day of primary culture (P0 generation). Some of these organoids were thin-walled, single-layered cystic structures, while others were solid gland-like structures. After passage 3-4, contaminating cells were basically eliminated, the purity of the organoids was significantly improved, and the morphology was stable, maintaining a typical gland-like structure. On the 7th day after cryopreservation and thawing, the organoids were still able to grow normally without obvious structural disintegration. H&E staining results showed that the organoids had intact morphology and orderly cell arrangement. IHC staining results showed that the cell membrane region of the organoids showed obvious positive signals, confirming that they could express E-cadherin normally, indicating that the bovine endometrial epithelial organoids were successfully constructed.

[0028] Example 2 Application of bovine endometrial epithelial organoid models Application in the study of the pathogenesis of bovine endometritis The P3 generation bovine endometrial epithelial organoids constructed in Example 1 were inoculated into 24-well low-adhesion culture plates. After the organoids stabilized, they were divided into experimental and control groups. The experimental group was supplemented with bacterial culture of common pathogens of bovine endometritis (such as Escherichia coli) to a final concentration of 1×10⁻⁶. 6 CFU / mL, the control group was given an equal volume of sterile culture medium; Both groups were cultured under the same conditions for 24 hours. After the culture was completed, the morphological and structural changes of the organoids in the two groups were observed by H&E staining, and the expression levels of inflammatory factors (such as IL-6 and TNF-α) were detected by IHC staining. The damaging effects of pathogenic bacteria on the endometrial epithelial organoids of dairy cows and the activation mechanism of inflammatory response were compared and analyzed. Compared with the control group without pathogens, the experimental group with common pathogens of bovine endometritis (such as Escherichia coli) showed significant structural damage to organoids and a significant increase in the expression of inflammatory factors. This confirms that the organoid model can accurately simulate the pathological damage process of bovine endometritis and can directly reflect the damage mechanism of pathogens to the endometrial epithelium and the activation pattern of inflammatory response, providing reliable in vitro experimental evidence for revealing the pathogenesis of bovine endometritis.

[0029] Application in the screening and efficacy evaluation of anti-inflammatory drugs for bovine endometritis The P3 generation bovine endometrial epithelial organoids constructed in Example 1 were selected and inoculated into 24-well low-adhesion culture plates. After the organoids grew stably, Escherichia coli culture (final concentration 1×10⁻⁶) was added. 6 (CFU / mL), cultured at 37℃ for 24 hours to construct an in vitro inflammation model; The constructed inflammation model was divided into a model group, a positive drug control group, and a test drug group. The model group was added with an equal volume of sterile culture medium, the positive drug control group was added with known effective anti-inflammatory drugs (such as cephalosporins), and the test drug group was added with different concentration gradients of the anti-inflammatory drugs to be screened. All groups were cultured under the same culture conditions for 48 hours. After the culture, the organoid structure repair was observed by H&E staining, and the expression level of inflammatory factors was detected by IHC staining or ELISA. With the model group as the control, the anti-inflammatory effects and optimal concentrations of different test drugs were evaluated, providing a reliable in vitro model for the screening and efficacy evaluation of anti-inflammatory drugs for bovine endometritis. The organoids in the model group maintained a state of inflammatory damage, while the positive drug control group and the medium and high concentration test drug groups were able to repair organoid damage and reduce the expression of inflammatory factors to varying degrees. Moreover, the repair effect of the test drug showed a concentration-dependent effect, and the anti-inflammatory repair effect of the high concentration test drug was better than that of the positive drug. This indicates that the organoid model can effectively distinguish the anti-inflammatory activity of different drugs and different concentrations of the same drug, and can achieve efficient screening and quantitative evaluation of anti-inflammatory drugs. It provides a precise and efficient in vitro evaluation system for the development of anti-inflammatory drugs for bovine endometritis.

[0030] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention.

Claims

1. A method for constructing a model based on animal endometrial epithelial organoids, characterized in that: The organoid model construction includes the following steps: S1. Select healthy dairy cows to remove the uterus, clean the surface of the uterus with pre-cooled tissue cleaning solution, remove fat, connective tissue and residual blood, and cut to obtain tissue fragments with a diameter of 0.5-1mm. S2. Place the tissue fragment suspension in a centrifuge tube and centrifuge. After centrifugation, discard the supernatant and add 10 mL of enzymatic hydrolysis solution I to resuspend the tissue fragments. Transfer the resuspended tissue fragments to a 6 cm culture dish and enzymatically hydrolyze the tissue fragments at 37 °C. Observe the tissue fragments under a microscope every 3-10 min with a Pasteur pipette until no obvious tissue fragments are found and a large number of dissociated glandular structures appear. The tissue suspension is then obtained. S3. Transfer the tissue suspension to a 15mL centrifuge tube, add the tissue washing solution to 10mL to terminate the enzymatic hydrolysis, centrifuge and discard the supernatant, then repeat the washing 2-3 times with the tissue washing solution. After each washing, centrifuge and discard the supernatant. After washing and centrifuging, add 500-1000μL of erythrocyte lysis buffer to the cell pellet, mix by pipetting and incubate on ice, add 5 times the volume of pre-cooled organoid washing solution to terminate the lysis, and centrifuge to collect the cell pellet. S4. Resuspend the cell pellet in the organoid washing solution, collect the completely digested cell clusters by natural sedimentation, and filter them through a 100μm cell sieve to remove extracellular matrix impurities to obtain filtrate. Transfer the filtrate to a centrifuge tube, centrifuge, discard the supernatant, add 1mL of organoid culture medium to resuspend the cells to obtain a cell suspension. S5. Using a 24-well low-adhesion culture plate as a carrier, prepare a 50 μL droplet for each well of the culture plate. Mix the cell suspension with an equal volume of matrix gel and add it to the well of the culture plate. Pre-cure at 37°C for 5 min, then incubate by inversion for 20-30 min to allow the matrix gel to completely solidify. After complete solidification, add >500 μL of organoid culture medium containing 1X additive I along the well wall of the culture plate. After culturing for 48 h, replace with organoid culture medium without additive I. Then replace the organoid culture medium every 2-3 days. S6. Primary culture of organoids for 7-15 days, until 500 organoids are obtained, each 100-200 μm in size, with clear boundaries, and in good growth condition. Then, passage is performed. The culture medium in the wells of the culture plate is aspirated, and the organoid washing solution is added to wash the gel droplets. After aspirating the washing solution, 500 μL of organoid recovery solution is added to each well, and the matrix gel is dispersed. The mixture is incubated at room temperature for 5 minutes to obtain a mixture. The mixture is collected into a 15 mL centrifuge tube, and residual cells in the wells are washed with the organoid washing solution and transferred to a centrifuge tube. After 3-4 passages to remove contaminating cells, the organoids are obtained.

2. The method for constructing a model of organoids based on animal endometrial epithelium according to claim 1, characterized in that: The dissociation of the organoids includes the following steps: Add 1 mL of organoid culture medium containing 1X additive I to the cells of the organoid, resuspend them, and observe them under a microscope after pipetting 5-50 times. If the cells are single cells or small cell clusters, dissociation is complete. If there are glandular organoids with solid-like growth in the cells, add 500 μL of enzymatic digestion solution II to resuspend the cells and transfer them to the 24-well low-adhesion culture plate. Incubate at 37°C for 2–15 min, pipetting 5–10 times every 1–3 min until the cells form small cell clusters. Add 9.5 mL of pre-cooled organoid washing solution to terminate the digestion. Centrifuge and discard the supernatant. Resuspend the cells in 1 mL of organoid culture medium containing 1X additive I to complete dissociation.

3. The method for constructing a model of organoids based on animal endometrial epithelium according to claim 1, characterized in that: The organoid model construction also includes a post-processing step, specifically the following steps: After passage and culture for 3-5 days, the organoids are collected and dissociated to obtain small cell clusters. After centrifugation and discarding the supernatant, the cells are added to cryovials. 500-1000 μL of organoid cryopreservation solution is added to the cryovials. The cryovials are placed in a programmed cooling box and cooled at -80°C for 24 hours before being transferred to a liquid nitrogen tank for storage. When the organoids are resuscitated, the cryopreservation tubes are removed from the liquid nitrogen tank, thawed in a water bath at 37°C and mixed by blowing and agitating 3-5 times. The suspension of the organoids is transferred to a centrifuge tube containing 5 mL of the organoid washing solution and centrifuged for 5 min at 4°C and 200-300 g. The supernatant is discarded. Steps S4-S6 are repeated to complete the resuscitation of the organoids.

4. The method for constructing a model of organoids based on animal endometrial epithelium according to claim 1, characterized in that: The identification of the organoid model includes the following steps: H&E staining: After fixing, embedding, and sectioning the organoid samples, the slides were baked in a 65°C oven for 1 hour. They were then dewaxed in three xylene tanks, each for 15 minutes. After hydration, they were treated with a gradient of 100%, 95%, and 75% ethanol for 3-5 minutes at each concentration. After hydration, they were stained with hematoxylin stain for 5 minutes and then rinsed with tap water for 5 minutes. After staining, the sample is differentiated by 1% hydrochloric acid alcohol for 3-5 seconds, then blued in ammonia water for 1-3 seconds, stained with eosin solution for 1 minute, rinsed with tap water, and then dehydrated by 75%, 95%, and 100% ethanol in gradients, with each concentration of ethanol dehydration treatment lasting 3 minutes. Finally, after clearing with xylene for 3 minutes, neutral resin was added to seal the slide, and the morphology of the organoid was observed under a microscope to confirm whether the organoid construction was successful or failed. IHC staining: After dewaxing the organoid sections, antigen retrieval was performed at 100℃ for 20 min using ER2 antigen retrieval solution. After retrieval, the sections were treated with 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase, and then blocked with blocking solution for 10 min. Then add E-cadherin antibody diluted 1:5000 and incubate at room temperature for 60 min; then add goat anti-rabbit HRP secondary antibody and incubate at room temperature for 30 min. Finally, develop the stain with DAB working solution for 5 minutes, counterstain with hematoxylin for 5 minutes, rinse with tap water, and repeat the dehydration, clearing and mounting steps of H&E staining. Observe the expression of E-cadherin under a microscope to confirm whether the organoid construction was successful or failed.

5. The method for constructing a model of organoids based on animal endometrial epithelium according to claim 1, characterized in that: The uterus was removed from the dairy cow within 30 minutes after slaughter, washed with physiological saline, and then placed in a tissue preservation solution for preservation and transportation. The tissue preservation solution was kept at 4°C for storage and transportation. The uterine body tissue cutting operation is performed under sterile conditions, using sterile scissors to cut 100-200 times, controlling the diameter of the tissue fragments to be 0.5-1mm, with no excessively large tissue fragments remaining.

6. The method for constructing a model of organoids based on animal endometrial epithelium according to claim 1, characterized in that: In step S3, during cell washing and erythrocyte lysis, the centrifugation parameters are controlled as follows: temperature 4℃, centrifugal force 200-300g, and centrifugation time 5min. After adding the red blood cell lysis buffer, blow the mixture 5-10 times to ensure that the cell pellet is completely mixed. Let it stand on ice for 3 minutes to avoid excessive lysis and damage to the target cells.

7. The method for constructing an organoid model based on animal endometrial epithelium according to claim 1, characterized in that: In step S4, the natural sedimentation method for cell screening takes 10-15 minutes. The natural sedimentation method is used to collect dense clusters of completely digested cells. The cell sieve is operated under aseptic conditions. After filtration, the cell sieve screen is rinsed twice with the organoid washing solution to ensure that no target cells remain on the cell sieve.

8. The method for constructing a model of organoids based on animal endometrial epithelium according to claim 1, characterized in that: In step S5, the culture plate containing the cell seed gel is preheated at 37°C for 30 minutes before culturing. When mixing the cell suspension with the matrix gel, blow the mixture 3-5 times, and the mixing time should not exceed 2 minutes to avoid premature solidification of the matrix gel. The gel droplets are added to the culture plate, and the culture plate is incubated upright for 5 minutes to prevent movement during pre-curing. The cell density of the gel droplets in each well of the culture plate is controlled to be 10,000-50,000 cells / 50 μL.

9. The method for constructing a model of organoids based on animal endometrial epithelium according to claim 1, characterized in that: The contaminating cells mentioned in step S6 include fibroblasts and uterine myocytes. During passage, these contaminating cells are removed by allowing the cells to stand for 2-4 hours after inoculation. The specific procedure is as follows: After passage and inoculation, the culture plate is left to stand for 2-4 hours until the fibroblasts and myocytes adhere to the well walls of the culture plate. The supernatant and non-adherent organoid cell clusters in the wells are gently aspirated, and organoid culture medium without additive I is added for further culture. After 3-4 passages of this operation, the contaminating cells are removed, and the organoids of high purity endometrial glands are obtained.

10. An animal endometrial epithelial organoid model constructed by the model construction method based on animal endometrial epithelial organoids as described in any one of claims 1-9, wherein the animal endometrial epithelial organoid model is used in the study of the pathogenesis of bovine endometritis, the screening of anti-inflammatory drugs for bovine endometritis, and the evaluation of their efficacy.

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