A culture medium for sheep endometrial organoids and its culture method

Abstract

The present invention discloses a culture medium for sheep endometrial organoids and a culture method thereof. The formulation of the culture medium includes a basal medium, cell growth factors, Wnt / β-catenin signaling pathway activators, BMP inhibitors, antioxidants and protectants, nutrients and cell culture additives, and auxiliary components. Culturing sheep endometrial organoids using the culture medium developed by the present invention can be used to optimize sheep breeding techniques, help study the improvement of the breeding efficiency and offspring quality of sheep breeds, and has potential economic value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a culture medium for sheep endometrial organoids and a culture method thereof. Background Art

[0002] In recent years, the research focus has gradually shifted to in vitro organ modeling. Organoids, based on stem cell cultures derived from human or animal tissues, are cultured in artificial basement membrane matrix using three-dimensional cell culture technology, and can effectively simulate the physiological reactivity, stable phenotype, and genetic characteristics of the originating tissue.

[0003] Two-dimensional cell culture cannot effectively simulate the complex in vivo environment, with differences in cell type, polarity, and spatial structure, thus limiting the in-depth study of complex diseases. In addition, animal models are restricted by ethical limitations, high costs, and long preparation cycles. In contrast, organoids have significant advantages in studying normal organ development, disease simulation, and drug effect prediction, providing a new research approach for personalized regenerative medicine.

[0004] Endometrial organoids (EO) are self-organizing 3D aggregates of endometrial cells, representing the structure and function of the endometrium. Endometrial organoids mimic normal epithelial function and are affected by reproductive hormones. In 2017, a breakthrough was made in the generation of endometrial organoids. Endometrial cells have molecular and functional characteristics, with clonality and long-term expandability. Boretto et al. seeded endometrial cells in 70% basement membrane matrix and cultured them with various cytokine components and Wnt concentrations. The cells expanded and rapidly formed organoid structures that could be expanded long-term. Under the action of ovarian hormones, the organoids expressed endometrium-related molecular markers and hormone-responsive genes. The organoids were genetically stable after long-term passage.

[0005] EO has opened up a new way for endometrial research and provided an excellent opportunity to study the endometrium in an unprecedented way, which will accelerate the understanding of the molecular and cellular mechanisms involved in endometrial development and diseases. Although corresponding endometrial organoids have been successfully constructed and applied in humans and mice, there is no report on the construction and application of sheep endometrial organoids. Summary of the Invention

[0006] Aiming at the technical problems existing in the current construction and culture technology of sheep endometrial organoids, the present invention provides a culture medium for sheep endometrial organoids and a culture method thereof.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] In a first aspect, the present invention claims a culture medium for ovine endometrial organoids, and the formulation of the culture medium comprises a basal medium, cell growth factors, a Wnt / β-catenin signaling pathway activator, a BMP inhibitor, an antioxidant and protective agent, a nutrient and cell culture additive, and an auxiliary component;

[0009] The basal medium is DMEM;

[0010] The cell growth factors include: FGF10 at 50 - 100 ng / ml, EGF at 30 - 50 ng / ml, and HGF at 30 - 50 ng / ml;

[0011] The Wnt / β-catenin signaling pathway activator includes: Rspondin-1 at 100 - 500 ng / ml and Wnt3a at 100 - 500 ng / ml;

[0012] The BMP inhibitor is Noggin at 50 - 200 ng / ml;

[0013] The antioxidant and protective agent includes: nicotinamide at 10 - 200 nM and cysteine at 1 - 1.5 mM;

[0014] The nutrient and cell culture additive includes: 1×transferrin, 1×N2, 1×B27, HEPES at 1 mM, and GlutaMAX at 200 nM;

[0015] The auxiliary component is A83-01 at 300 - 600 nM.

[0016] Preferably, the formulation of the culture medium comprises the following components: DMEM, FGF10 at 50 - 100 ng / ml, EGF at 30 - 50 ng / ml, HGF at 30 - 50 ng / ml, Rspondin-1 at 100 - 500 ng / ml, Wnt3a at 100 - 500 ng / ml, Noggin at 50 - 200 ng / ml, nicotinamide at 10 - 200 nM, cysteine at 1 - 1.5 mM, 1×transferrin, 1×N2, 1×B27, HEPES at 1 mM, GlutaMAX at 200 nM, and A83-01 at 300 - 600 nM.

[0017] In a second aspect, the present invention claims the application of the above-mentioned culture medium for ovine endometrial organoids in culturing ovine endometrial organoids.

[0018] In a third aspect, the present invention claims a method for culturing ovine endometrial organoids, and the culturing method comprises the following steps:

[0019] (1)Prepare a cell suspension using the epithelial cells of the ovine endometrial tissue suspended in DMEM. Mix the cell suspension with Matrigel to form a mixture.

[0020] (2)Drop the mixture onto a preheated cell culture plate to form gel droplets. Place it in an incubator until the gel droplets solidify. Then add the above-mentioned ovine endometrial organoid medium to the cell culture plate and culture to obtain ovine endometrial organoids.

[0021] Furthermore, the method for preparing the epithelial cells of the ovine endometrial tissue in the above method is as follows: Dissect the ovine endometrial tissue, soak it in DPBS containing 2% (v / v) double antibiotics for washing, and then cut it into tissue fragments in the shape of minced meat; Add tissue digestion solution to the tissue fragments, incubate with shaking at 37 °C until 90% of the tissue is dissolved, and then add DMEM containing 10% (v / v) serum to terminate digestion; Filter through a 100 μm nylon mesh cell filter to remove undigested tissue fragments; Pass the filtrate through a 40 μm cell filter to separate and remove stromal cells, and collect the epithelial cells of the ovine endometrial tissue.

[0022] Furthermore, in step (1) of the above method, the cell concentration of the cell suspension is 1.0 - 3.0 × 10 6 / ml; the volume ratio of the cell suspension to Matrigel is 1:4.

[0023] Furthermore, in step (1) of the above method, culture at 37 °C and 5% CO2 for 10 min until the gel droplets reach a semi-solid state, and then invert the cell culture plate for 10 min to make the gel droplets completely solidify.

[0024] Furthermore, in step (2) of the above method, add the above-mentioned ovine endometrial organoid medium to the cell culture plate and continue to culture at 37 °C and 5% CO2 to obtain ovine endometrial organoids.

[0025] The detailed process of the technical solution of the present invention specifically includes the following steps:

[0026] I: Method for constructing ovine endometrial organoids

[0027] A. Collect healthy ovine uterine tissue, wash the outer surface of the uterus 2 - 3 times with physiological saline containing 0.2% (0.2 g / 100 ml) gentamicin sulfate, cut off the two uterine horns and place them on a sterile surgical cloth in a safety cabinet. Longitudinally open the uterine horns with sterile scissors and then wash the endometrium with DPBS containing 2% (v / v) double antibiotics.

[0028] B. Dissect the surface of the endometrium using sterile scissors or a sterile scalpel and forceps, peel the endometrial tissue from the underlying myometrium, soak the dissected small tissue pieces in DPBS containing 2% (v / v) double antibiotics for washing, and then collect them into a 4 ml centrifuge tube, add a small amount of DMEM, and use sterile scissors to cut the tissue pieces into a minced meat-like state.

[0029] C. Transfer the processed tissue fragments to a 15 ml centrifuge tube, add an appropriate amount of tissue digestion solution (2 mg / ml collagenase I), incubate in a 37 °C water bath for 1 hour, intermittently oscillate until 90% of the tissue is dissolved, and then add DMEM containing 10% (v / v) serum to terminate digestion.

[0030] D. Filter the obtained mixture through a 100 μm nylon mesh cell filter to remove undigested tissue fragments.

[0031] E. Filter the filtrate through a 40 μm cell filter to separate stromal cells. Since epithelial cells are too large to pass through a 40 μm cell filter, they remain on the filter.

[0032] F. Invert the 40 μm cell filter, and use 10 ml DMEM to wash the epithelial cells into a clean 50 ml tube and centrifuge. Add an appropriate amount of DMEM to resuspend the cells and mix evenly according to the volume ratio of 20% cell suspension / 80% Matrigel.

[0033] G. Pipette 40 μl of the mixed solution and drop it onto a 24-well plate preheated half an hour in advance to form a gel droplet. Place the culture plate in a 37 °C, 5% CO2 incubator for 10 min to allow the gel droplet to reach a semi-solidified state, and then invert the culture plate for another 10 min to make the gel droplet completely solidify.

[0034] H. Take out each well and add 500 μl / well of ovine endometrial organoid medium. Place it in a 37 °C, 5% CO2 incubator for culture, observe the organoids, and change the medium once every 2 - 3 days.

[0035] II: Subculture and Amplification of Ovine Endometrial Organoids

[0036] A. Take out the well-grown, high-density organoids that need to be subcultured from the incubator, suck out the medium completely, wash them once with DPBS, and pay attention to adding DPBS while adhering to the wall to avoid washing up the gel droplets.

[0037] B. After sucking out the DPBS completely, add 1 ml of DPBS to the gel droplet and pipette the gel droplet about ten times to disperse the Matrigel.

[0038] C. Collect the contents from more than four wells into a 15-ml centrifuge tube and add pre-cooled DPBS to 10 - 12 ml. Place it in a -20°C refrigerator for 5 - 8 min.

[0039] D. Take it out and place it in a centrifuge pre-cooled to 4°C at 300 rpm / min for 5 min to remove the supernatant.

[0040] E. Add 1 ml of pre-cooled DPBS to the tube, and use a blue pipette tip with a yellow pipette tip to pipette the liquid 200 - 300 times. Aspirate a small amount of liquid for microscopic examination. Generally, at this time, the organoids have been dispersed into cell clusters of about 10 - 30 cells.

[0041] F. Supplement pre-cooled DPBS to 6 ml, centrifuge at 300 rpm / min for 5 min, discard the supernatant, and resuspend the cells. Mix the cell suspension and Matrigel according to the volume ratio of 20% cell suspension / 80% Matrigel.

[0042] G. Aspirate 40 μl of the mixture and drop it onto a 24-well plate pre-warmed half an hour ago to form a gel drop. Incubate it upright in a 37°C, 5% CO₂ incubator for 10 minutes, then invert it for 10 minutes to solidify the gel drop and prevent cell adhesion.

[0043] H. After the gel drop solidifies, slowly add 500 μl of ovine endometrial organoid medium along the wall of the cell culture well to submerge the entire gel drop. Place it in a 37°C, 5% CO₂ incubator for culture, and change the medium every 2 - 3 days.

[0044] III: Cryopreservation of Ovine Endometrial Organoids

[0045] A. Select organoids after 2 - 3 passages for cryopreservation.

[0046] B. Wash the organoids twice with pre-cooled DPBS. After sucking out all the DPBS, add 1 ml of DPBS to the gel drop and pipette the gel drop about ten times to disperse the Matrigel.

[0047] C. Collect the contents from more than four wells into a 15-ml centrifuge tube and add pre-cooled DPBS to 10 - 12 ml. Place it in a -20°C refrigerator for 5 - 8 min.

[0048] D. Take it out and place it in a centrifuge pre-cooled to 4°C at 300 rpm / min for 5 min to remove the supernatant.

[0049] E. Add 1 ml of pre-cooled DPBS, pipette the liquid 50 - 80 times with a pipette tip, then supplement pre-cooled DPBS to 6 ml, centrifuge at 300 rpm / min for 5 min, and discard the supernatant.

[0050] F. Resuspend with serum-free cryopreservation medium, add 1 ml of the mixed suspension to each cryotube, label it, place it in a gradient freezing box, leave it overnight at -80 °C, and then store it in liquid nitrogen for long-term preservation.

[0051] IV: Thaw and recover the cryopreserved ovine endometrial organoids

[0052] A. Take out the endometrial organoids stored in the liquid nitrogen tank, and quickly and gently thaw them in a 37 °C water bath until there is a small ice cube left in the tube.

[0053] B. Transfer the thawed organoid suspension to a 15 ml centrifuge tube containing 4 ml of DMEM, mix well, place it in a pre-cooled centrifuge at 4 °C at 300 rpm for 5 min, and remove the supernatant.

[0054] C. Mix the cell suspension and Matrigel according to the volume ratio of 20% cell suspension / 80% Matrigel.

[0055] D. Pipette 40 μl of the mixed solution and drop it onto a 24-well plate preheated half an hour in advance to form a gel droplet. Incubate it upright in a 37 °C incubator for 10 minutes and then invert it for 10 minutes to solidify the gel droplet and prevent cell adhesion.

[0056] E. After the gel droplet solidifies, slowly add 500 μl of ovine endometrial organoid medium along the wall of the cell culture well to submerge the entire gel droplet. Incubate it in a 37 °C, 5% CO2 incubator, and change the medium every 2 - 3 days.

[0057] V. Organoid medium prepared for ovine endometrial organoids

[0058] A. The medium composition is: basal medium, cell growth factors, Wnt / β-catenin signaling pathway activator, BMP inhibitor, antioxidant and protectant, nutrient and cell culture additive, other auxiliary components.

[0059] B. The specific components are: DMEM, FGF10, EGF, HGF, Rspondin-1, Wnt3a, Noggin, Niacinamide, cysteine, Transferrin, N2, B27, HEPES, GlutaMAX, A83-01.

[0060] C. The concentration of cell growth factor FGF10 is 50 - 100 (such as 50, 60, 70, 80, 90, 100) ng / ml, the concentration of EGF is 30 - 50 (such as 30, 40, 50) ng / ml, and the concentration of HGF is 30 - 50 (such as 30, 40, 50) ng / ml.

[0061] D. The concentration of the Wnt / β-catenin signaling pathway activator Rspondin-1 is 100 - 500 (e.g., 100, 200, 300, 400, 500) ng / ml, and the concentration of Wnt3a is 100 - 500 (e.g., 100, 200, 300, 400, 500) ng / ml.

[0062] E. The concentration of the BMP inhibitor Noggin is 50 - 200 (e.g., 50, 100, 150, 200) ng / ml.

[0063] F. The concentration of the antioxidant and protector nicotinamide is 10 - 200 (e.g., 10, 50, 100, 150, 200) nM, and the concentration of cysteine is 1 - 1.5 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5) mM.

[0064] G. Add 1× transferrin, 1× N2, 1× B27, the concentration of HEPES is 1 mM, and the concentration of GlutaMAX is 200 nM to the nutrient and cell culture additive.

[0065] H. The concentration of the other auxiliary component A83 - 01 is 300 - 600 (e.g., 300, 400, 500, 600) nM.

[0066] Advantages of the present invention

[0067] (1) Simulating the real physiological environment: This kind of organoid can simulate the physiological structure and function of the sheep endometrium, providing an ideal experimental model for studying the reproductive physiology, endocrine regulation and related diseases of sheep, and making up for the limitations of traditional animal experiments.

[0068] (2) Facilitating disease research and treatment: Through this kind of organoid, the pathological state in the sheep body can be more accurately simulated, promoting the research on endometrium-related diseases (such as endometritis, endometriosis, etc.), and providing an important platform for developing new treatment methods.

[0069] (3) Replacing animal experiments: In aspects such as drug screening, chemical substance toxicity testing and gene function research, using this kind of organoid can effectively reduce the need for experimental animals, meet the requirements of modern medical ethics, and reduce the use of animal experiments.

[0070] (4) Promoting the development of the sheep breeding industry: This kind of organoid can be used to optimize the breeding technology of sheep, help research improve the breeding efficiency and offspring quality of sheep breeds, and has potential economic value.

[0071] (5) Improving the successful culture efficiency of sheep endometrial organoids: The culture medium provided by the present invention is more targeted at the culture of sheep endometrial organoids and can efficiently form cystic organoids with a diameter of more than 200 μm. Description of the Drawings

[0072] Figure 1 Light microscopy image of the sheep endometrial organoids cultured in Example 1; the scale bar is 100 μm.

[0073] Figure 2 Light microscopy image of the subcultured, thawed and revived sheep endometrial organoids; the scale bar is 100 μm.

[0074] Figure 3 Light microscopy image and PAS staining of the sheep endometrial organoids before and after physiological hormone treatment; the scale bar is 100 μm.

[0075] Figure 4 Detection of changes in related proteins of the sheep endometrial organoids before and after physiological hormone treatment.

[0076] Figure 5 Immunofluorescence image of the sheep endometrial organoids; the scale bar is 100 μm.

[0077] Figure 6 Ultrastructure observation image of the sheep endometrial organoids.

[0078] Figure 7 Comparison chart of the effects of culturing sheep endometrial organoids with different culture medium formulations; the scale bar is 500 μm. Detailed Description of the Invention

[0079] The technical solutions of the present invention will be further described in detail below in conjunction with the examples and related drawings. It should be noted, however, that the embodiments of the present invention are not limited to this, and relevant personnel can implement them in other different forms without departing from the purpose and spirit of the present invention.

[0080] Source of raw materials:

[0081] DMEM: Dulbecco's Modified Eagle Medium, modified Eagle medium.

[0082] FGF10: Fibroblast Growth Factor 10, fibroblast growth factor 10.

[0083] EGF: Epidermal Growth Factor, epidermal growth factor.

[0084] HGF: Hepatocyte Growth Factor

[0085] Rspondin-1: R-spondin 1, also known as RSPO1.

[0086] Wnt3a: Wnt3a protein.

[0087] Noggin: Nog protein.

[0088] Nicotinamide: Nicotinamide, vitamin B3, and also the precursor of NAD + and NADP + precursor.

[0089] Cysteine: Cysteine.

[0090] Transferrin: Transferrin.

[0091] N2: N2 medium supplement.

[0092] B27: B27 medium supplement.

[0093] HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

[0094] GlutaMAX: GlutaMAX, an alternative to L-glutamine.

[0095] A83-01: 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolyl)-1H-pyrazole-1-carbothioamide, CAS NO. 909910-43-6, a commercially available product.

[0096] Matrigel: Matrigel.

[0097] Among them, DMEM, transferrin, N2, B27, HEPES, and GlutaMAX were purchased from Gibco, FGF10, EGF, HGF, Rspondin-1, Wnt3a, and Noggin were purchased from Beijing Sino Biological Inc., nicotinamide, cysteine, and A83-01 were purchased from MCE, and Matrigel was purchased from Boman Bio.

[0098] Example 1 Preparation of Ovine Endometrial Organoid Medium

[0099] Formulation 1: DMEM, FGF10 50 ng / ml, EGF 50 ng / ml, HGF 50 ng / ml, Rspondin-1 100 ng / ml, Wnt3a 100 ng / ml, Noggin 50 ng / ml, nicotinamide 10 nM, cysteine 1 mM, 1×transferrin, 1×N2, 1×B27, HEPES 1 mM, GlutaMAX 200 nM, A83-01 500 nM.

[0100] Formulation 2: DMEM, FGF10 50 ng / ml, EGF 50 ng / ml, HGF 50 ng / ml, Rspondin-1 200 ng / ml, Wnt3a 100 ng / ml, Noggin 100 ng / ml, nicotinamide 10 nM, cysteine 1 mM, 1×transferrin, 1×N2, 1×B27, HEPES 1 mM, GlutaMAX 200 nM, A83-01 500 nM.

[0101] Formulation 3: DMEM, FGF10 100 ng / ml, EGF 40 ng / ml, HGF 50 ng / ml, Rspondin-1 500 ng / ml, Wnt3a 100 ng / ml, Noggin 100 ng / ml, nicotinamide 10 nM, cysteine 1 mM, 1×transferrin, 1×N2, 1×B27, HEPES 1 mM, GlutaMAX 200 nM, A83-01 500 nM.

[0102] Example 2 Construction of Ovine Endometrial Organoids

[0103] 1. Isolation of Ovine Endometrial Epithelial and Stromal Cells

[0104] 1.1 Select healthy ovine uterine tissue and place it in 37 °C physiological saline containing 0.2% (0.2 g / 100 ml) gentamicin sulfate (obtained by directly adding a commercially available gentamicin sulfate injection with an active ingredient content of 5 ml: 0.2 g to physiological saline at a final concentration of 5% by volume. The production company of the gentamicin sulfate injection: Hefei Dubang Biopharmaceutical Co., Ltd., product number: Qadmszy3) and transport it to the laboratory using an incubator device within 3 hours after slaughter.

[0105] 1.2 After washing the outer surface of the uterus 2 - 3 times with the above - mentioned normal saline containing gentamicin sulfate at 37 °C, excise the two uterine horns and place them on a sterile surgical cloth in a safety cabinet. After longitudinally opening the uterine horns with sterile scissors, wash the endometrium with DPBS containing 2% double - antibody (v / v, double - antibody is the commercially available penicillin - streptomycin mixture, production company: Gibco, catalog number: 15070063).

[0106] 1.3 Using sterile ophthalmic forceps for assistance, separate the endometrium and myometrium with sterile scissors. Immerse the separated endometrial tissue in DPBS containing 2% double - antibody and wash it 2 - 3 times, then transfer it to a 4 - ml centrifuge tube, add a small amount of DMEM, and cut it into minced meat - like with sterile scissors.

[0107] 1.4 Transfer the processed minced - meat - like tissue fragments to a 15 - ml centrifuge tube, add tissue digestion solution (2 mg / ml collagenase I) to the minced - meat - like tissue, adding 4 ml of tissue digestion solution for every 1 ml of minced - meat - like tissue. Place it in a 37 °C water bath and incubate with constant shaking for 1 h until 90% of the tissue is dissolved. Add DMEM containing 10% serum (v / v, serum production company: Gibco, catalog number: A3161002C) to terminate the digestion.

[0108] 1.5 First, filter out the undigested tissue fragments with a 100 - μm nylon mesh cell filter, and then use a 40 - μm nylon mesh cell filter to separate endometrial epithelial cells and stromal cells. Due to the different cell sizes, stromal cells will be in the filtrate, while epithelial cells are too large to pass through the 40 - μm cell filter and remain on the filter mesh. Flip the filter upside - down and rinse the epithelial cells into a clean 50 - ml tube with 10 ml of DMEM to obtain endometrial epithelial cells.

[0109] 2. Culture of ovine endometrial organoids

[0110] 2.1 Centrifuge the rinsed liquid, and the precipitate at the bottom of the tube is endometrial epithelial cells. Resuspend it with DMEM to obtain a cell suspension, and perform cell counting. Adjust the cell concentration to be 1.0 - 3.0 × 10 6 / ml.

[0111] 2.2 Mix the cell suspension (EXM) and Matrigel in a volume ratio of 2:8. Take 40 μl of the mixed solution and drop it onto a pre - warmed 24 - well plate half an hour in advance to form a gel droplet. Place the culture plate upright in a 37 °C, 5% CO2 incubator for 10 minutes to make the gel droplet reach a semi - solid state, and then invert the culture plate for 10 minutes to solidify the gel droplet and prevent cell adhesion.

[0112] 2.3 After the glue droplets solidify, slowly add 500 μl / well of the ovine endometrial organoid medium prepared according to Formulation 1 of Example 1 along the wall of the cell culture well to submerge the entire glue droplet. Incubate in an incubator at 37 °C and 5% CO2, and change the medium every 2 - 3 days.

[0113] 4. Experimental results

[0114] The endometrial organoids cultured in this experiment by the above methods and the medium used, as Figure 1 shown, it can be seen that the formed and growing changes of the cultured ovine endometrial organoids on the first day, the third day, the fifth day, and the seventh day have a high formation rate, and the overall diameter is 100 - 300 μm on the seventh day.

[0115] Example 3 Passage of ovine endometrial organoids

[0116] 1. Select ovine endometrial organoids with an overall diameter growing to more than 100 μm, that is, ovine endometrial organoids cultured for 7 - 9 days for passage.

[0117] 2. Remove the glue and release the ovine endometrial organoids: Take the organoids out of the incubator, suck out the medium completely, then add DPBS along the wall to wash once, avoiding flushing the glue droplets. After sucking out the DPBS completely, add 1 ml of DPBS to the glue droplet and pipette the glue droplet about ten times to disperse the Matrigel. Collect the contents of more than four wells into a 15 ml centrifuge tube and add pre-cooled DPBS to 10 - 12 ml. Place it in a -20 °C refrigerator for 5 - 8 min. Take it out and place it in a pre-cooled centrifuge at 4 °C at 300 rpm / min for 5 min, and remove the supernatant.

[0118] 3. Passage: Add 1 ml of pre-cooled DPBS to the tube, and use a blue pipette tip with a yellow pipette tip to pipette the liquid 200 - 300 times. Pipette a small amount of liquid for microscopic examination. Generally, at this time, the organoids have been dispersed into cell clusters of about 10 - 30 cells. Add pre-cooled DPBS to 6 ml, centrifuge at 300 rpm / min for 5 min, discard the supernatant, and resuspend the cells. Mix the cell suspension and Matrigel according to the volume ratio of 20% cell suspension / 80% Matrigel. The passage ratio is 1:2 - 1:3. Take 40 μl of the mixed solution and drop it onto a 24-well plate preheated half an hour in advance to form glue droplets, place it upright in an incubator at 37 °C and 5% CO2 for 10 minutes, then invert it for 10 minutes to solidify the glue droplets and avoid cell adhesion to the wall. After the glue droplets solidify, slowly add 500 μl of the ovine endometrial organoid medium prepared according to Formulation 1 of Example 1 along the wall of the cell culture well to submerge the entire glue droplet. Incubate in an incubator at 37 °C and 5% CO2, and change the medium every 2 - 3 days.

[0119] 4. Cryopreservation: Select the organoids after 2 - 3 passages for cryopreservation. Wash the organoids twice with pre - cooled DPBS. After sucking out the DPBS completely, add 1 ml of DPBS to the gel drop and pipette the gel drop about ten times to disperse the Matrigel. Collect the contents in more than four wells into a 15 - ml centrifuge tube and add pre - cooled DPBS to 10 - 12 ml. Place it in a - 20 °C refrigerator for 5 - 8 min. Take it out and place it in a 4 °C pre - cooled centrifuge at 300 rpm / min for 5 min to remove the supernatant. Add 1 ml of pre - cooled DPBS to the tube, gently pipette the liquid 50 - 80 times with a blue pipette tip to disrupt the organoid structure, supplement pre - cooled DPBS to 6 ml, centrifuge at 300 rpm / min for 5 min, and discard the supernatant. Resuspend with serum - free cryopreservation solution, add 1 ml of the mixed suspension to each cryotube, mark it, then put it into a gradient freezing box, keep it at - 80 °C overnight, and then store it in liquid nitrogen for long - term preservation.

[0120] 5. Thawing and recovery: Take out the endometrial organoids stored in the liquid nitrogen tank, and quickly and gently shake them in a 37 °C water bath until there is a small ice lump left in the tube. Transfer the thawed organoid suspension to a 15 - ml centrifuge tube containing 4 ml of DMEM, mix well, place it in a 4 °C pre - cooled centrifuge at 300 rpm / min for 5 min to remove the supernatant. Mix the cell suspension and Matrigel according to the volume ratio of 20% cell suspension / 80% Matrigel. Pipette 40 μl of the mixed solution and drop it onto a 24 - well plate pre - heated half an hour ago to form a gel drop. Incubate it upright in a 37 °C incubator for 10 minutes, then invert it for 10 minutes to solidify the gel drop and prevent cell adhesion. After the gel drop solidifies, slowly add 500 μl of the sheep endometrial organoid medium prepared according to Formula 1 of Example 1 along the cell culture well wall to submerge the entire gel drop. Place it in a 37 °C, 5% CO2 incubator for culture, and change the medium every 2 - 3 days.

[0121] 6. Experimental results

[0122] The culture results of different passages and thawing and recovery of sheep endometrial organoids are as Figure 2 shown. The results show that sheep endometrial organoids can still grow normally and maintain good morphology, growth rate and biological characteristics after passage, thawing and recovery.

[0123] Example 4 Verification of the physiological hormone response of sheep endometrial organoids

[0124] 1. Select the ovine endometrial organoids at passage 3 and culture them normally until the sixth day, then divide them into three groups: the control group, the estrogen treatment group, and the progesterone treatment group. In the control group, the culture medium was changed normally. For the estrogen treatment group and the progesterone treatment group, the culture medium of ovine endometrial organoids containing 10 nM estrogen was used to treat for two days. On the eighth day, the control group still changed the culture medium normally, the estrogen treatment group changed to the culture medium of ovine endometrial organoids containing 10 nM estrogen, and the progesterone treatment group changed to the culture medium of ovine endometrial organoids containing 1 μM progesterone. During each medium change, the morphology and size changes were observed, and samples were collected on the tenth day. The culture medium of the ovine endometrial organoids was the ovine endometrial organoid medium prepared according to Formula 1 in Example 1.

[0125] 2. On the tenth day, the organoids were collected, embedded in agar, sectioned, and stained with PAS to detect the changes in the secretory function of the organoids (such as Figure 3 ).

[0126] 3. On the tenth day, after collecting the organoids and removing the glue, the proteins were extracted by RIPA lysis, and the changes in the expression levels of hormone-responsive related proteins were detected by Western Blot (such as Figure 4 ).

[0127] 4. Experimental results

[0128] The results of the reaction changes of ovine endometrial organoids after treatment with physiological hormones are as shown in Figure 3 and 4 . Figure 3 The results showed that treatment with estrogen would accelerate the growth rate of the organoids, making the diameter of the organoids increase; while treatment with progesterone would enhance the secretory function of the organoids, thus secreting more mucus. Figure 4 The results showed that estrogen treatment would increase the protein expression levels of estrogen receptors and progesterone receptors in the organoids, while progesterone treatment would significantly increase the expression level of mucin in the organoids. These results all indicated that the ovine endometrial organoids constructed in this experiment could simulate the corresponding responses and changes of the normal body endometrium to physiological hormone treatment.

[0129] Example 5 Immunofluorescence verification of ovine endometrial organoids

[0130] 1. Select the ovine endometrial organoids at passage 3 and culture them normally until the seventh day. Samples were collected, the glue was removed to release the organoids, and they were embedded in agar and sectioned.

[0131] 2. After the sections were dewaxed, permeabilized, antigen repaired, blocked, incubated with primary antibody, incubated with secondary antibody, nuclear stained, and sealed, the results were observed using a confocal microscope.

[0132] 3. Experimental results

[0133] Immunofluorescence staining was performed on ovine endometrial organoids for verification, and the results are as Figure 5 shown. The ovine endometrial organoids obtained by culturing in this experiment could express endometrial glandular epithelial markers EPCAM, CK19, and E-cadherin.

[0134] Example 6 Electron Microscopic Observation of the Ultrastructure of Ovine Endometrial Organoids

[0135] 1. Select the ovine endometrial organoids passaged to the 3rd generation and culture them normally for 7 days. Add DPBS along the wall and wash once, avoiding flushing the gel droplets. After sucking out the DPBS completely, add 1 ml of DPBS to the gel droplets and pipette the gel droplets about 10 times to disperse the Matrigel. Collect the contents of more than four wells into a 15 ml centrifuge tube and add pre-cooled DPBS to 10 - 12 ml. Place it in a -20 °C refrigerator for 5 - 8 min.

[0136] 2. Take it out and place it in a centrifuge pre-cooled at 4 °C at 300 rpm / min for 5 min to remove the supernatant. The obtained precipitate is the ovine endometrial organoids in a complete form. Add pre-cooled electron microscopy fixative and fix it in a 4 °C refrigerator for 24 h, then send it to the electron microscopy room to prepare samples for electron microscopy observation.

[0137] 3. Observe the ultrastructure of the ovine endometrial organoid samples with a transmission electron microscope.

[0138] 4. Experimental Results

[0139] The results of electron microscopic observation of the ultrastructure of ovine endometrial organoids are as Figure 6 shown. The tubular structure of the organoids is pseudostratified columnar epithelium, and there are obvious and numerous microvilli structures (black arrows) on the inner side; there are tight junctions (white arrows) between the cells of the endometrial structure; the cytoplasm is rich in mitochondria with clear structures visible (orange arrows), and there are a large number of secretory vesicles (green arrows).

[0140] Comparative Example 1

[0141] Referring to the endometrial organoid culture medium formula disclosed in the patent CN113957036A, prepare the endometrial organoid culture medium according to the final concentration composition shown as follows: 50 ng / ml of EGF, 100 ng / ml of Noggin, 500 ng / ml of R-spondin 1, 100 ng / ml of Wnt3a, 50 ng / ml of FGF9, 30 ng / ml of KIAA1199, 50 ng / ml of sox2, 15 nM of isoflavone, 200 nM of CHIR99021, 15 μM of A83-01, 10 μM of RKI-1477, 100 μg / ml of primary cell antibiotics. After culturing the endometrial organoids for 7 days, observe the growth morphology under a microscope, and the results are as Figure 7as shown in A of

[0142] Prepare the endometrial organoid culture medium according to Formulation 1 in Example 1. After culturing the endometrial organoids for seven days, observe the growth morphology under a microscope. The results are as Figure 7 shown in A of

[0143] Statistically analyze the growth diameter and the formation rate of cystic organoids (the formation of organoids is a cystic structure lined by columnar epithelium, and secretions can be seen in the lumen) of the organoids cultured from two endometrial organoid culture media with different ratios. The results are as Figure 7 shown in B and C of Figure 7 B), and more cystic Figure 7 C) endometrial organoids, indicating that the endometrial organoid culture medium formula proposed in the present invention is more suitable for the culture of sheep endometrial organoids.

[0144] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. Each specific technical feature can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

[0145] In addition, for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. A culture medium for sheep endometrial organoids, characterized in that, The formulation of this culture medium consists of the following components: DMEM, FGF10 at 50 - 100 ng / ml, EGF at 30 - 50 ng / ml, HGF at 30 - 50 ng / ml, Rspondin-1 at 100 - 500 ng / ml, Wnt3a at 100 - 500 ng / ml, Noggin at 50 - 200 ng / ml, nicotinamide at 10 - 200 nM, cysteine at 1 - 1.5 mM, 1×transferrin, 1×N2, 1×B27, HEPES at 1 mM, GlutaMAX at 200 nM, and A83-01 at 300 - 600 nM.

2. Use of the ovine endometrial organoid culture medium according to claim 1 in culturing ovine endometrial organoids.

3. A method for culturing sheep endometrial organoids, characterized in that, This culturing method comprises the following steps: (1) Suspending epithelial cells of ovine endometrial tissue with DMEM to prepare a cell suspension, and mixing the cell suspension with Matrigel to form a mixture; (2) Dropping the mixture onto a preheated cell culture plate to form gel drops, placing them in an incubator until the gel drops solidify, and then adding the ovine endometrial organoid culture medium according to claim 1 to the cell culture plate for culturing to obtain ovine endometrial organoids.

4. The culturing method of the ovine endometrial organoids according to claim 3, wherein The preparation method of the epithelial cells of the ovine endometrial tissue is as follows: After stripping the ovine endometrial tissue and soaking it in DPBS containing 2% double antibiotics for washing, cutting it into tissue fragments in the shape of minced meat; adding tissue enzymatic digestion solution to the tissue fragments and incubating them at 37 °C with shaking until 90% of the tissue is dissolved, and then adding DMEM containing 10% serum to terminate digestion; Filtering with a 100 μm nylon mesh cell filter to remove undigested tissue fragments; Passing the filtrate through a 40 μm cell filter to separate and remove stromal cells, and collecting the epithelial cells of the ovine endometrial tissue.

5. The culturing method of the ovine endometrial organoids according to claim 3, wherein The cell concentration of the cell suspension described in step (1) is 1.0 - 3.0 × 10 6 / ml; the volume ratio of the cell suspension to the Matrigel is 1:

4.

6. The culturing method of the ovine endometrial organoids according to claim 3, wherein, In step (2), culturing at 37 °C and 5% CO2 for 10 min until the gel drops reach a semi-solidified state, and then inverting the cell culture plate for 10 min to make the gel drops completely solidify.

7. The culturing method of the ovine endometrial organoids according to claim 3, characterized in that, In step (2), adding the ovine endometrial organoid culture medium to the cell culture plate and continuing to culture under the conditions of 37 °C and 5% CO2 to obtain ovine endometrial organoids.

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