Construction method of horse placenta organoid

Abstract

The embodiment of the invention discloses a construction method of a horse placenta organoid. The method comprises the following steps: constructing a chorionic organ; an endometrial organ is constructed; and co-inoculating the chorion-like organ and the endometrial-like organ to a low-adhesion 96-well plate with a U-shaped bottom for co-culture to form a horse placenta-like organ capable of simulating embryo implantation and placenta formation. The method breaks through the limitation that only a single type of organoid can be constructed in the prior art. A three-dimensional model capable of completely simulating a maternal-fetal interface is established by originally co-culturing a horse endometrial organ and a fluorescently-labeled trophoblast organ in a low-adhesion U-shaped plate. The technical breakthrough makes it possible to research the cell interaction in the embryo implantation process, and fills the technical blank existing in the field for a long time.

Description

Technical Field

[0001] This invention relates to the field of biological technology, specifically to a method for constructing equine placental organoids. Background Technology

[0002] Currently, studies have successfully constructed equine endometrial organoids (Verstraete et al., 2025) and equine chorionic villi organoids (Thompson et al., 2023), and their structure, function, and hormone secretion capabilities have been identified. However, the placenta is a complex organ formed by the maternal endometrium and fetal chorion. Existing technologies can only simulate the maternal or fetal portion individually and cannot reproduce the cell interactions, invasive behavior, and tissue structural changes of the placenta during implantation. Therefore, using only a single type of organoid has significant limitations in studying the molecular mechanisms related to pregnancy, implantation, or the placenta. Summary of the Invention

[0003] To address the problem that existing technologies cannot simulate the complete formation process of the equine placenta, this invention aims to establish an in vitro co-culture model of equine placental organoids that can simulate embryo implantation and placental formation, providing a reliable experimental platform for studying equine placental development, implantation mechanisms, and pregnancy-related diseases.

[0004] To fill the gap in existing equine placental organoid models, this invention provides a method for constructing equine placental organoids. The core concept of this method is: first, to construct chorionic organoids representing the fetal portion and endometrial organoids representing the maternal portion, respectively; then, through an optimized co-culture system, to simulate the embryo implantation process in vitro, forming a composite placental organoid that combines structural and functional characteristics.

[0005] Therefore, embodiments of the present invention provide a method for constructing equine placental organoids.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for constructing equine placental organoids, the method comprising:

[0008] Constructing chorionic organoids;

[0009] Constructing endometrial organoids;

[0010] Choriocarpic organoids and endometrial organoids were co-inoculated in low-adhesion U-shaped 96-well plates for co-culture to form equine placental organoids that can simulate embryo implantation and placental formation.

[0011] Furthermore, the chorionic organoids include equine chorionic band organoids or equine chorionic allantoic organoids;

[0012] The method for constructing the equine chorionic band organoid or equine chorionic band organoid includes:

[0013] Horse embryos at 30 days of gestation were harvested, and chorionic zona pellucida and allantoic chorionic villi were isolated under sterile conditions. These tissues were then separated using pre-cooled, calcium-free saline solution. 2+ Mg 2+ Wash thoroughly with PBS solution and chop into 1-2 mm pieces. 3 Small pieces;

[0014] The fragments of chorionic villi were transferred to DMEM / F12 digestion solution containing 0.4 mg / mL collagenase V and digested at 37°C for 5 minutes. Alternatively, the fragments of allantochorionic villi were transferred to DMEM / F12 digestion solution containing 1 mg / mL collagenase I and digested at 37°C for 1 hour, with intermittent gentle pipetting during digestion. After digestion, serum-containing culture medium was added to terminate digestion. The cell suspension was filtered through a 70 μm cell sieve, centrifuged, and the cell pellet was collected to obtain chorionic villi cells or allantochorionic villi cells.

[0015] The cell pellet was resuspended in trophoblast organoid medium and centrifuged. Then it was mixed with Matrigel to form a Matrigel dome in a preheated 24-well culture plate. After the colloid polymerized at 37°C, it was cultured in trophoblast organoid medium and then desorbed by Matrigel to obtain the equine choriocartilaginous organoid or equine choriocartilaginous organoid.

[0016] Furthermore, the composition of the trophoblast organoid culture medium is as follows: based on DMEM / F12, and supplemented with the following components: 1% N2 additive, 2% B27 additive, 1.25 mM N-acetyl-L-cysteine, 100 ng / mL recombinant human FGF2, 50 ng / mL recombinant human EGF, 50 ng / mL recombinant human HGF, 2 μM CHIR99021, 0.5 μM TGFβ / Alk inhibitor A83-01, 10 μM ROCK inhibitor Y-27632, 1% ITS additive, 2 mM L-glutamine, 1% penicillin and streptomycin;

[0017] The culture conditions are as follows: cultured in an incubator at 37°C and 5% CO2, with half of the culture medium replaced every 2-3 days, and organoids passaged every 7-14 days at a ratio of 1:2 to 1:4.

[0018] Furthermore, the method for constructing the endometrial organoid includes:

[0019] Endometrial tissue was collected from mares, washed with PBS, and then minced into 1-2 mm pieces. 3 Small pieces;

[0020] The fragments of endometrial tissue were transferred to DMEM / F12 digestion solution containing 1 mg / mL collagenase I and digested on a shaker at 37°C for 1.5 hours.

[0021] After digestion, serum-containing culture medium was added to stop the digestion. The cell suspension was filtered through a 100μm cell sieve, then filtered through a 40μm cell sieve and washed on the reverse side to collect the cells. After centrifugation, the cell pellet was obtained.

[0022] Cells were resuspended in pre-cooled Matrigel to form a Matrigel dome in a 24-well culture plate. Endometrial organoid complete culture medium was added to the wells to completely cover the dome for culture. The endometrial organoids were then desorbed from the Matrigel to obtain the organoids.

[0023] Furthermore, the composition of the complete endometrial organoid culture medium is as follows: based on DMEM / F12 medium, supplemented with the following components: 1% N2 additive, 2% B27 additive, 1.25 mM N-acetyl-L-cysteine, 1 mM nicotinamide, 50 ng / mL recombinant human EGF, 50 ng / mL recombinant human FGF10, 100 ng / mL recombinant human Noggin, 0.5 μM TGFβ / Alk inhibitor A83-01, 10 μM SB202190, 2 mM L-glutamine, 1% penicillin and streptomycin;

[0024] The culture conditions are as follows: cultured in an incubator at 37°C and 5% CO2, with half of the culture medium replaced every 2-3 days.

[0025] Furthermore, the cell ratio of the chorionic organoids to the endometrial organoids is 1:1;

[0026] The culture medium used for co-culture was DMEM / F12 as the basal medium, supplemented with the following components: 1% N2 additive, 2% B27 additive, 1.25mM N-acetyl-L-cysteine, 10μM ROCK inhibitor Y-27632, 100ng / mL recombinant human FGF2, 50ng / mL recombinant human EGF, 1% ITS additive, 100ng / mL BMP4, 2mM L-glutamine, 5% Matrigel, 1% penicillin and streptomycin;

[0027] The co-culture conditions were: 37℃, 5% CO2, 5% O2, with half of the culture medium replaced with fresh medium every 2 days, and co-cultured for 5 days.

[0028] The co-culture medium used in this invention retains and unifies the essential additives (N2, B27, N-acetyl-L-cysteine, glutamine, and antibiotics) and growth factors (EGF, FGF2) required by both organisms, and uses a ROCK inhibitor (Y-27632) to reduce apoptosis. 1% ITS provides additional growth support for cell contact and early interface formation; CHIR99021 and Noggin are removed to avoid overactivation or inhibition of the Wnt and BMP pathways, preventing excessive proliferation or differentiation imbalance of one organoid; 100 ng / mL BMP4 is added, a factor that plays an important role in early embryonic development and helps guide the correct interaction between the trophoblast and epithelial cells; 5% Matrigel is added to provide trace but crucial extracellular matrix in the medium, mimicking in vivo and in vitro matrix components, providing the necessary physical and biochemical support for the adhesion, migration, and three-dimensional reconstruction of both organoids.

[0029] Further, the method for desorbing the matrix gel includes: transferring the organoid and matrix gel mixture into a centrifuge tube, regurgitating it 5-10 times to break down the matrix gel, centrifuging, removing the supernatant, adding organoid recovery solution, incubating on ice for 30 minutes, and regurgitating it every 5 minutes until the matrix gel is decomposed from the organoid, centrifuging, removing the supernatant, and washing with ice-cold DMEM / F12.

[0030] Furthermore, the method further includes: fluorescently labeling the chorionic organoids prior to co-culturing.

[0031] Furthermore, the specific process of fluorescent labeling includes:

[0032] The chorionic organoids, EGFP lentivirus particles and 10 μM polyglobulin were mixed, centrifuged at 300g for 60 min, and after transduction, the viral supernatant was discarded. After washing with PBS, the cells were resuspended in matrix gel and cultured in trophoblast organoid culture medium to finally obtain fluorescently labeled trophoblast organoids that stably express EGFP.

[0033] The culture conditions are as follows: cultured in an incubator at 37°C and 5% CO2, with half of the culture medium replaced every 2-3 days, and organoids passaged every 7-14 days at a ratio of 1:2 to 1:4.

[0034] The embodiments of the present invention have the following advantages:

[0035] This invention innovatively co-cultures equine trophoblast organoids and endometrial organoids in low-adhesion U-shaped plates, utilizing an optimized culture medium to promote their contact and interaction through both physical and biochemical means. The resulting composite organoids not only structurally mimic the placenta but also functionally simulate the "invasion" and "implantation" of the chorionic zona and allantochorionic trophoblast onto the endometrium, providing a novel in vitro model for studying equine placental biology and implantation mechanisms. Furthermore, laser confocal microscopy was used to verify the structural integrity and functional characteristics of the composite placental organoids.

[0036] This invention overcomes the limitation of existing technologies that can only construct single-type organoids. By innovatively co-culturing equine endometrial organoids with fluorescently labeled trophoblast organoids in low-adhesion U-shaped plates, a three-dimensional model capable of fully simulating the maternal-fetal interface was established. This technological breakthrough makes it possible to study cell interactions during embryo implantation, filling a long-standing technological gap in this field. Attached Figure Description

[0037] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0038] Figure 1 The left side shows a schematic diagram of a low-adhesion U-shaped 96-well plate. On the right side, pink represents equine endometrial organoids and blue represents equine chorionic band organoids or equine allantochorionic band organoids. The two gradually form equine placental structures at the contact surface, and together they constitute a composite equine placental organoid.

[0039] Figure 2 The two images on the left are cultured equine endometrial organoids (EEO), the two images in the middle are cultured equine chorionic band organoids (ECG0), and the two images on the right are cultured equine allantochorionic organoids (EACO). All images were taken with an inverted phase-contrast microscope.

[0040] Figure 3 Immunofluorescence identification images of equine endometrial organoids (EEOs). (A) The top three images show the fluorescence staining of epithelial cell adhesion molecule (EpCAM). (B) The middle three images show the fluorescence staining of keratin 18 (CK-18). (C) The bottom three images show the fluorescence staining of phalloidin. All images were taken using laser confocal microscopy, and the scale bar for all images is 50 µm.

[0041] Figure 4: Fluorescently labeled (EGFP) images of equine chorionic band organoids (ECGO) and equine chorionic band organoids (EACO).

[0042] Figure 5 Image 1: Constructed equine composite placental organoids. (A) Image of composite placental organoids after co-culture of equine endometrial organoids and equine chorionic zona organoids; (B) Image of composite placental organoids after co-culture of equine endometrial organoids and equine allantochorionic zona organoids. All images were taken using a laser confocal microscopy system.

[0043] Figure 6 3D images of equine complex placental organoids. (A, B) 3D images of complex placental organoids co-cultured with equine endometrial organoids and equine chorionic villi organoids; A and B are taken from different angles. (C, D) 3D images of complex placental organoids co-cultured with equine endometrial organoids and equine allantochorionic villi organoids; C and D are taken from different angles. All images were generated by layer-by-layer scanning using a laser confocal microscopy imaging system. Detailed Implementation

[0044] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0045] Example 1: Construction of equine chorionic band organoids and equine allantoic chorionic band organoids

[0046] 1. Experimental methods and procedures

[0047] Tissue source and processing: Horse embryos at 30 days of gestation were harvested, and the chorionic band and allantoic chorionic membrane tissues were aseptically separated. The tissues were then rinsed with pre-cooled PBS (calcium-free). 2+ and Mg 2+ Thoroughly clean and cut into pieces to approximately 1-2 mm using sterile surgical scissors. 3 Small pieces.

[0048] Cell isolation: Cells were isolated using enzymatic digestion. Chorionic villus fragments were transferred to DMEM / F12 digestion solution containing collagenase V (0.4 mg / mL) and digested at 37°C for 5 minutes. Allantoic villus fragments were transferred to DMEM / F12 digestion solution containing collagenase I (1 mg / mL) and digested at 37°C for 1 hour, with intermittent gentle pipetting. After digestion, serum-containing culture medium was added to terminate the digestion. The cell suspension was filtered through a 70 μm cell sieve, centrifuged (300 g, 7 minutes), and the cell pellet was collected.

[0049] Culture and expansion: The cell pellet was resuspended in feeder organoid culture medium and centrifuged at 1500 rpm for 3 minutes. It was then mixed with Matrigel (mogengel) to form 25 μl domes in preheated 24-well plates. After the colloid polymerized at 37°C, 500 μL of feeder organoid culture medium was added to each well.

[0050] Composition of trophoblast organoid culture medium: Based on DMEM / F12 medium, with the following components added:

[0051] 1% N2 additive (Gibco);

[0052] 2% B27 additive (Gibco);

[0053] 1.25 mM N-acetyl-L-cysteine ​​(sigma Aldrich);

[0054] 100 ng / mL recombinant human FGF2 (MCE);

[0055] 50 ng / ml recombinant human EGF (MCE);

[0056] 50 ng / mL recombinant human HGF (MCE);

[0057] 2μM CHIR99021 (Stemgent);

[0058] 0.5 μM TGFβ / Alk inhibitor A83-01 (MCE);

[0059] 10 μM ROCK inhibitor Y-27632 (Enzo Life Sciences);

[0060] 1% ITS Additive (Gibco);

[0061] 2 mM L-glutamine (sigma Aldrich);

[0062] 1% penicillin and streptomycin (Gibco).

[0063] Culture conditions: Incubate at 37℃ with 5% CO2, replacing half of the culture medium every 2-3 days. Passage organoids every 7-14 days at a ratio of 1:2 to 1:4.

[0064] 2. Experimental Results

[0065] The test results are as follows Figure 2 As shown, the equine chorionic band organoids and equine chorionic band organoids established in this invention for constructing composite placental organoids are in good condition under a microscope, with a size between 40-100µm, and can be stably passaged.

[0066] Example 2: Construction and Identification of Equine Uterine Endometrial Organoids

[0067] 1. Experimental methods and procedures

[0068] Tissue source and processing: Mare uteri were obtained from the slaughterhouse and transported to the laboratory at 4°C. Endometrial tissue was harvested in a sterile environment, washed with PBS, and then minced into pieces approximately 1-2 mm in size. 3 Small pieces.

[0069] Cell isolation: Cells were isolated using enzymatic digestion. Endometrial tissue fragments were transferred to DMEM / F12 digestion solution containing collagenase I (1 mg / mL) and digested on a shaker at 37°C for 1.5 hours. After digestion, serum-containing culture medium was added to terminate the digestion. The cell suspension was filtered through a 100 μm cell sieve, followed by filtration through a 40 μm cell sieve and washing the reverse side to collect cells. The cells were then centrifuged (1500 rpm, 3 minutes) to obtain the cell pellet.

[0070] Culture and Expansion: Cells were resuspended in pre-chilled Matrigel (mogengel) to form 25 μl of Matrigel domes in 24-well plates. Each dome was covered with 500 μL of complete endometrial organoid culture medium.

[0071] Composition of complete endometrial organoid culture medium: Based on DMEM / F12 medium, with the following components added:

[0072] 1% N2 additive (Thermo Fisher, Gibco);

[0073] 2% B27 additive (Thermo Fisher, Gibco);

[0074] 1.25 mM N-acetyl-L-cysteine ​​(sigma Aldrich);

[0075] 1 mM nicotinamide (sigma Aldrich);

[0076] 50 ng / mL recombinant human EGF (MCE);

[0077] 50 ng / mL recombinant human FGF10 (Thermo Fisher);

[0078] 100 ng / mL recombinant human Noggin (Thermo Fisher);

[0079] 0.5 μM TGFβ / Alk inhibitor A83-01 (MCE);

[0080] 10μM SB202190 (sigma Aldrich);

[0081] 2 mM L-glutamine (sigma Aldrich);

[0082] 1% penicillin and streptomycin (Gibco).

[0083] Culture conditions: Incubate at 37℃ in a 5% CO2 incubator, replacing half of the culture medium with fresh medium every two days. Organoids are passaged according to growth density.

[0084] Identification: Organoids were identified by immunofluorescence staining, and their uterine epithelial origin was confirmed using anti-cytokeratin (CK-18) and epithelial cell adhesion molecule (EpCAM). Organoid cell morphology was observed using phalloidin staining.

[0085] 2. Experimental Results

[0086] The test results are as follows Figure 3 As shown, the equine endometrial organoids used in this invention for constructing composite placental organoids were in good condition under a microscope, ranging in size from 100-300 µm, and were stably passaged. Immunofluorescence identification using the endometrial marker keratin 18 (CK-18) and epithelial cell adhesion molecule (EpCAM) confirmed their origin from endometrial epithelium. Staining the cytoskeleton with phalloidin revealed an intact organoid structure and normal cell morphology. These results indicate that equine endometrial organoids can be used to construct composite placental organoids.

[0087] Example 3 Construction of composite placental organoids

[0088] 1. Experimental methods and procedures

[0089] Fluorescent labeling of trophoblast organoids: To track the attachment and invasion behavior of chorionic organoids in the co-culture system, stable fluorescent labeling of trophoblast cells is necessary before co-culture. First, equine chorionic band organoids or equine allantoic chorionic organoids are released from the matrix gel and dissociated into single-cell suspensions using a combination of mechanical pipetting and enzymatic digestion. Specifically, the organoid suspension dissociated at 4°C is repeatedly pipetted and centrifuged at 1500 rpm for 3 minutes. The supernatant is discarded, digestion solution is added, and the suspension is treated at 37°C for 15 minutes. Digestion is then terminated with DMEM / F-12 containing 10% FBS, and single cells are obtained through fine pipetting. Lentiviral transduction is then performed: Single cells are mixed with EGFP lentivirus particles and 10 μM polyglobulin, and the rotational seeding method (centrifugation at 300g for 60 min) significantly improves infection efficiency. After transduction, the viral supernatant was discarded, and the cells were washed with PBS, resuspended in matrix gel, and cultured in organoid culture medium. Finally, fluorescently labeled feeder organoids that stably expressed EGFP were obtained, laying the foundation for subsequent real-time observation of cell interactions during co-culture.

[0090] Establishment of the co-culture system: The constructed and identified equine endometrial organoids, equine chorionic band organoids, and equine allantochorionic organoids were released from the cultured matrix gel and prepared as individual organoid suspensions. Specifically, the organoid and matrix gel mixture was gently scraped off with a pipette tip and transferred to a 15 ml centrifuge tube. After disintegrating the matrix gel by pipetting 5-10 times, the tube was centrifuged at 1500 rpm for 3 minutes. After removing the supernatant, organoid recovery solution was added and the tube was incubated on ice for 30 minutes, with pipetting every 5 minutes until the matrix gel was disintegrated from the organoids. Then, the tube was centrifuged at 1500 rpm for 3 minutes, the supernatant was removed, and the tube was washed twice with ice-cold DMEM / F12. Finally, 500 ml of co-culture medium was added and the tube was transferred to a 24-well plate. Equine endometrial organoids and equine chorionic villi organoids (cell ratio 1:1), or equine endometrial organoids and equine allantochorionic villi organoids (cell ratio 1:1), were co-seeded into 96-well plates with a low-adhesion U-shaped bottom using a pipette under a stereomicroscope, and 100 μl of co-culture medium was added for 5 days of incubation. Figure 1 As shown.

[0091] Culture conditions: Incubate in an incubator at 37℃, 5% CO2, and 5% O2, replacing half of the culture medium with fresh medium every 2 days.

[0092] Model validation: Images were taken using a live-cell imaging system 5 days later. Specifically, after 5 days of culture, the resulting composite placental organoids were fixed with 4% paraformaldehyde and stained with DAPI. Finally, the formation of a composite structure similar to the maternal-fetal interface of the placenta in a mare was verified using a laser confocal microscopy system.

[0093] Co-culture medium: This medium is based on DMEM / F12 and has been optimized for co-culture characteristics.

[0094] 1% N2 additive (Thermo Fisher, Gibco); 2% B27 additive (Thermo Fisher, Gibco), 1.25 mM N-acetyl-L-cysteine ​​(sigma Aldrich), 10 μM ROCK inhibitor Y-27632 (EnzoLife Sciences), 100 ng / mL recombinant human FGF2 (MCE), 50 ng / mL recombinant human EGF (MCE), 1% ITS additive (Gibco), 100 ng / mL BMP4 (Gibco), 2 mM L-glutamine (sigma Aldrich), 5% Matrigel (Corning), and 1% penicillin and streptomycin (Gibco).

[0095] 2. Experimental Results

[0096] The test results are as follows Figure 4 , 5 and Figure 6 As shown. Figure 4 This indicates that after screening with EGFP virus infection, equine chorionic villus cells and equine allantoic chorionic villus cells can construct organoids with green fluorescent markers, which facilitates the differentiation of organoid origins in subsequent composite placental organoids. Figure 5 and Figure 6 The final result of this invention is that, after co-culturing equine endometrial organoids with EGFP-containing equine chorionic villi organoids, the chorionic villi organoids invaded the endometrial organoids, significantly altering the structure of the invaded portion and forming an in vitro-like invasive placental structure. After co-culturing equine endometrial organoids with EGFP-containing equine allantochorionic villi organoids, the allantochorionic villi organoids attached to the surface of the equine endometrial organoids, forming an in vitro-like non-invasive placental structure. These results indicate that the composite placental organoids constructed in this invention morphologically and functionally mimic embryo implantation and endometrial cup formation during early pregnancy in mares.

[0097] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method of constructing a horse placenta organoid, characterized by, The method comprises: constructing chorionic organoids; constructing endometrial organoids; co-inoculating the chorionic organoids and the endometrial organoids in a low-adhesion U-bottom 96-well plate for co-culture to form a horse placenta organoid capable of simulating embryo implantation and placenta formation.

2. The method of constructing a horse placental organoid according to claim 1, wherein, The chorionic organoids comprise horse chorionic band organoids or horse allantoic chorionic organoids; The method for constructing the horse chorionic band organoids or horse allantoic chorionic organoids comprises: The chorionic membrane band and the allantoic chorionic membrane tissue were separated from the equine embryo of 30 days of pregnancy in a sterile environment, washed thoroughly with pre-cooled PBS solution without Ca 2+ , Mg 2+ , and cut into small pieces of 1-2 mm 3 . transferring chorionic band tissue fragments into a DMEM / F12 digestion solution containing 0.4 mg / mL collagenase V and digesting at 37°C for 5 minutes, or transferring allantoic chorionic tissue fragments into a DMEM / F12 digestion solution containing 1 mg / mL collagenase I and digesting at 37°C for 1 hour with intermittent gentle blowing during the digestion; after the digestion is completed, serum-containing medium is added to terminate the digestion; the cell suspension is filtered through a 70-μm cell strainer, and the cell precipitate is collected after centrifugation to obtain chorionic band cells or allantoic chorionic cells; resuspending the cell precipitate in a trophoblast organoid culture medium and centrifuging, then mixing with Matrigel matrix glue, forming a matrix glue dome in a preheated 24-well culture plate, and after the glue is polymerized at 37°C, culturing with the trophoblast organoid culture medium, and then detaching from the matrix glue to obtain the horse chorionic band organoid or horse allantoic chorionic organoid.

3. The method for constructing a horse placenta organoid according to claim 2, wherein the composition of the trophoblast organoid culture medium is: a DMEM / F12-based medium, and the following components are added: 1% N2 supplement, 2% B27 supplement, 1.25 mM N-acetyl-L-cysteine, 100 ng / mL recombinant human FGF2, 50 ng / mL recombinant human EGF, 50 ng / mL recombinant human HGF, 2 μM CHIR99021, 0.5 μM TGFβ / Alk inhibitor A83-01, 10 μM ROCK inhibitor Y-27632, 1% ITS supplement, 2 mM L-glutamine, 1% penicillin and streptomycin; the culture condition is: culturing in a 37°C, 5% CO2 incubator, and replacing half of the culture medium every 2-3 days, and subculturing the organoids at a ratio of 1:2-1:4 every 7-14 days.

4. The method of constructing a horse placental organoid according to claim 1, wherein, The method for constructing the endometrial organoids comprises: Endometrial tissue was obtained from mares. The tissue was washed in PBS and cut into 1-2 mm 3 pieces. transferring endometrial tissue fragments into a DMEM / F12 digestion solution containing 1 mg / mL collagenase I and digesting at 37°C on a shaker for 1.5 hours; after the digestion is completed, serum-containing medium is added to terminate the digestion; the cell suspension is filtered through a 100-μm cell strainer, then filtered through a 40-μm cell strainer and washed on the reverse side to collect the cells, and the cell precipitate is obtained after centrifugation; resuspending the cells in pre-cooled Matrigel matrix glue, forming a matrix glue dome in a 24-well culture plate, adding endometrial organoid complete culture medium into the well to completely cover the dome for culture, and then detaching from the matrix glue to obtain the endometrial organoid.

5. The method of claim 4, wherein the complete endometrium organoid culture medium comprises DMEM / F12 base medium supplemented with 1% N2 supplement, 2% B27 supplement, 1.25 mM N-acetyl-L-cysteine, 1 mM nicotinamide, 50 ng / mL recombinant human EGF, 50 ng / mL recombinant human FGF10, 100 ng / mL recombinant human Noggin, 0.5 mM TGFp / Alk inhibitor A83-01, 10 mM SB202190, 2 mM L-glutamine, 1% penicillin and streptomycin. The culture condition is that the organoids are cultured in a 37°C, 5% CO2 incubator, and the medium is replaced every 2-3 days.

6. The method of claim 1, wherein the ratio of the chorionic organoids to the endometrium organoids is 1:

1. The co-culture medium comprises DMEM / F12 base medium supplemented with 1% N2 supplement, 2% B27 supplement, 1.25 mM N-acetyl-L-cysteine, 10 mM ROCK inhibitor Y-27632, 100 ng / mL recombinant human FGF2, 50 ng / mL recombinant human EGF, 1% ITS supplement, 100 ng / mL BMP4, 2 mM L-glutamine, 5% Matrigel, 1% penicillin and streptomycin. The co-culture condition is that the organoids are cultured in a 37°C, 5% CO2, 5% O2 incubator, and the medium is replaced every 2 days for 5 days. The method of detaching the Matrigel comprises transferring the organoid and Matrigel mixture into a centrifuge tube, vortexing 5-10 times to destroy the Matrigel, centrifuging, removing the supernatant, adding organoid recovery solution, incubating on ice for 30 minutes, and vortexing every 5 minutes until the Matrigel is detached from the organoids, centrifuging, removing the supernatant, and washing with ice-cold DMEM / F12. The method further comprises fluorescently labeling the chorionic organoids before co-culturing.

7. The method of constructing a horse placental organoid according to claim 2 or 4, wherein, The specific process of the fluorescent labeling comprises:

8. The method of constructing a horse placental organoid according to claim 1, wherein, mixing the chorionic organoids, EGFP lentivirus particles, and 10 mM polybrene, centrifuging at 300g for 60 minutes, discarding the virus supernatant after transduction, washing with PBS, resuspending the cells in Matrigel, and culturing in the trophoblast organoid culture medium to obtain fluorescently labeled trophoblast organoids that stably express EGFP; 9. The method of constructing a horse placental organoid according to claim 8, wherein, The culture condition is that the organoids are cultured in a 37°C, 5% CO2 incubator, and the medium is replaced every 2-3 days, and the organoids are passaged at a ratio of 1:2-1:4 every 7-14 days. ​ ​

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