Sheep endometrial organ culture medium and culture method thereof

Abstract

The invention discloses a sheep endometrial organ culture medium and a culture method thereof. The formula of the culture medium comprises a basic culture medium, a cell growth factor, a Wnt / beta-catenin signal channel activating factor, a BMP inhibitor, an antioxidant and protective agent, a nutrition and cell culture medium additive and auxiliary components. The culture medium developed by the invention is used for culturing the endometrial organs of the sheep, can be used for optimizing the breeding technology of the sheep, helps to research and improve the breeding efficiency and offspring quality of sheep species, and has potential economic value.

Description

Technical Field

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

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

[0003] Two-dimensional cell culture cannot effectively simulate the complex environment in the body, and there are differences in cell types, polarity, and spatial structure, which limits the in-depth study of complex diseases. In addition, animal models are limited by moral and ethical restrictions, 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 path for personalized regenerative medicine.

[0004] Endometrial organoids (EOs) are self-organizing 3D aggregates of endometrial cells that represent the structure and function of the endometrium. Endometrial organoids mimic normal epithelial function and are influenced by reproductive hormones. In 2017, a breakthrough was made in the generation of endometrial organoids, which have molecular and functional characteristics of endometrial cells and are clonogenic and long-term scalable. Boretto et al. seeded endometrial cells in 70% Matrigel and cultured them with various cytokine components and Wnt concentrations. The cells expanded and rapidly formed organoid structures capable of long-term expansion. Under the action of ovarian hormones, the organoids expressed endometrial-related molecular markers as well as hormone-responsive genes. The organoids were genetically stable after long-term passaging.

[0005] EO has opened up new avenues 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 endometrial organoids have been successfully constructed and applied in humans and mice, there are no reports on the construction and application of sheep endometrial organoids. Summary of the invention

[0006] In view of the technical problems existing in the current sheep endometrial organoid construction and culture technology, the present invention provides a sheep endometrial organoid culture medium and a culture method thereof.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention claims protection for a sheep endometrial organoid culture medium, the formula of which comprises a basal culture medium, cell growth factors, Wnt / β-catenin signaling pathway activating factors, BMP inhibitors, antioxidants and protective agents, nutrients and cell culture medium additives and auxiliary components;

[0009] The basal culture medium is DMEM;

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

[0011] The Wnt / β-catenin signaling pathway activating factors include: Rspondin-1 100-500 ng / ml and Wnt3a 100-500 ng / ml;

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

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

[0014] The nutrient and cell culture medium supplements include: 1× transferrin, 1× N2, 1× B27, HEPES 1mM and GlutaMAX 200 nM;

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

[0016] Preferably, the formula of the culture medium contains the following components: DMEM, FGF10 50-100 ng / ml, EGF 30-50 ng / ml, HGF 30-50 ng / ml, Rspondin-1 100-500ng / ml, Wnt3a 100-500 ng / ml, Noggin50-200 ng / ml, nicotinamide 10-200nM, cysteine ​​1-1.5 mM, 1×transferrin, 1×N2, 1×B27, HEPES1mM, GlutaMAX 200 nM and A83-01 300-600 nM.

[0017] In a second aspect, the present invention seeks to protect the use of the above-mentioned sheep endometrial organoid culture medium in culturing sheep endometrial organoids.

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

[0019] (1) epithelial cells of sheep endometrial tissue are suspended in DMEM to prepare a cell suspension, and the cell suspension is mixed with matrix gel to prepare a mixed solution;

[0020] (2) Dropping the mixed solution into a preheated cell culture plate to form a gel droplet, placing the droplet in an incubator until the gel droplet solidifies, and then adding the above-mentioned sheep endometrial organoid culture medium into the cell culture plate for culturing to obtain sheep endometrial organoids.

[0021] Furthermore, the preparation method of the epithelial cells of the sheep endometrial tissue in the above method is: peeling the sheep endometrial tissue and soaking it in DPBS containing 2% (v / v) double antibody for washing, then cutting it into meat-like tissue fragments; adding tissue enzymatic solution to the tissue fragments, shaking and incubating at 37°C until the tissue is 90% dissolved, and then adding DMEM containing 10% (v / v) 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 sheep endometrial tissue.

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

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

[0024] Furthermore, in step (2) of the above method, the sheep endometrial organoid culture medium is added to the cell culture plate and culture is continued at 37° C. and 5% CO2 to obtain the sheep endometrial organoid.

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

[0026] 1. Method for constructing sheep endometrial organoids

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

[0028] B. Use sterile scissors or a sterile scalpel and forceps to dissect the endometrial surface, peel off the endometrial tissue from the underlying myometrium, soak the resulting small tissue strips in DPBS containing 2% (v / v) double antibody for washing, then collect them into a 4 ml centrifuge tube, add a small amount of DMEM and use sterile scissors to cut the tissue strips into a meat paste.

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

[0030] D. Filter the resulting mixture through a 100 μm nylon mesh cell strainer to remove undigested tissue debris.

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

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

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

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

[0035] II: Passaging and expansion of sheep endometrial organoids

[0036] A. Take out the organoids that are growing well, have a high density, and need to be passaged from the incubator, aspirate the culture medium, and wash them with DPBS. Be careful to add DPBS so that they adhere to the wall and avoid washing up the gel droplets.

[0037] B. After aspirating the DPBS, add 1 ml of DPBS to the gel drop and blow the gel drop about ten times to disperse the matrix gel.

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

[0039] D. Take out the tube and place it in a 4℃ precooled centrifuge at 300 rpm / min for 5 min, then 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 aspirate the liquid 200-300 times. Aspirate a small amount of liquid for microscopic examination. Generally, the organoid has been dispersed into cell clusters of about 10-30 cells at this time.

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

[0042] G. Pipette 40 μl of the mixed solution and drop it into a 24-well plate preheated half an hour in advance to form a gel drop. Place it upright for 10 minutes and inverted for 10 minutes in a 37°C, 5% CO2 incubator to allow the gel drop to solidify and prevent cells from adhering to the wall.

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

[0044] 3. Cryopreservation of sheep endometrial organoids

[0045] A. Select organoids that have been passaged 2-3 times for cryopreservation.

[0046] B. Wash the organoids twice with pre-cooled DPBS. After aspirating the DPBS, add 1 ml of DPBS to the gel droplet and pipette the gel droplet about ten times to disperse the matrix gel.

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

[0048] D. Take out the tube and place it in a 4 ℃ precooled centrifuge at 300 rpm / min for 5 minutes, then remove the supernatant.

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

[0050] F. Add serum-free freezing solution to resuspend, add 1 ml of mixed suspension to each cryotube, mark and place in a gradient freezing box, store at -80℃ overnight, and then store in liquid nitrogen for long-term storage.

[0051] 4. Thawing and resuscitation of cryopreserved sheep endometrial organoids

[0052] A. Take out the endometrial organoids stored in the liquid nitrogen tank and thaw them quickly in a 37°C water bath by shaking gently until only a small ice block remains in the tube.

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

[0054] C. Mix the cell suspension and matrix gel in a volume ratio of 20% cell suspension / 80% matrix gel.

[0055] D. Pipette 40 μl of the mixed solution into a 24-well plate preheated half an hour in advance to form a gel drop. Place it upright for 10 minutes and inverted for 10 minutes in a 37°C incubator to allow the gel drop to solidify and prevent cells from adhering to the wall.

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

[0057] 5. Organoid Culture Medium for Sheep Endometrial Organoids

[0058] A. The culture medium consists of: basal culture medium, cell growth factors, Wnt / β-catenin signaling pathway activators, BMP inhibitors, antioxidants and protective agents, nutrients and cell culture medium additives, and other auxiliary components.

[0059] B. Specific ingredients 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 (e.g. 50, 60, 70, 80, 90, 100) ng / ml, the concentration of EGF is 30-50 (e.g. 30, 40, 50) ng / ml, and the concentration of HGF is 30-50 (e.g. 30, 40, 50) ng / ml.

[0061] D. The concentration of 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 BMP inhibitor Noggin is 50-200 (e.g., 50, 100, 150, 200) ng / ml.

[0063] F. The concentration of antioxidant and protective agent 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. Nutrition and cell culture medium supplements: 1× transferrin, 1× N2, 1× B27, HEPES at a concentration of 1 mM, and GlutaMAX at a concentration of 200 nM.

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

[0066] Beneficial effects of the present invention

[0067] (1) Simulating the real physiological environment: This type of organ 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) Assisting disease research and treatment: This type of organ can more accurately simulate the pathological state in sheep, promote research on endometrial-related diseases (such as endometritis, endometriosis, etc.), and provide an important platform for the development of new treatments.

[0069] (3) Replacement of animal experiments: In drug screening, chemical toxicity testing, and gene function research, the use of this type of organ can effectively reduce the demand for experimental animals, comply with modern medical ethics, and reduce the use of animal experiments.

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

[0071] (5) Improving the efficiency of successful culture 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is a light microscopic image of the sheep endometrial organoid cultured in Example 1; the scale bar is 100 μm.

[0073] Figure 2 Light microscopic image of sheep endometrial organoids that have been passaged, thawed and revived; the scale bar is 100μm.

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

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

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

[0077] Figure 6 This is an ultrastructural observation diagram of sheep endometrial organoids.

[0078] Figure 7 A comparison of the effects of culturing sheep endometrial organoids using different culture medium formulas; the scale bar is 500μm. DETAILED DESCRIPTION

[0079] The technical solution of the present invention is further described in detail below in conjunction with the embodiments and related drawings. However, it is worth noting that the implementation of the present invention is not limited to this, and relevant personnel can implement it in other different forms without violating 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-factor 1, also known as RSPO1.

[0086] Wnt3a: Wnt3a protein.

[0087] Noggin: Nog protein.

[0088] Nicotinamide: Nicotinamide, vitamin B3, also 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-pyridyl)-N-phenyl-4-(4-quinolyl)-1H-pyrazole-1-carbothioamide, CAS NO. 909910-43-6, a conventional commercial product.

[0096] Matrigel: Matrigel.

[0097] 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 Technology Co., Ltd., nicotinamide, cysteine, and A83-01 were purchased from MCE, and Matrigel was purchased from BiomilliBio Corporation.

[0098] Example 1 Preparation of Sheep Endometrial Organoid Culture Medium

[0099] Formula 1: DMEM, FGF10 50 ng / ml, EGF 50 ng / ml, HGF 50 ng / ml, Rspondin-1 100ng / ml, Wnt3a 100 ng / ml, Noggin 50 ng / ml, Nicotinamide 10nM, Cysteine ​​1mM, 1× Transferrin, 1×N2, 1×B27, HEPES 1mM, GlutaMAX 200 nM, A83-01 500nM.

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

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

[0102] Example 2 Construction of sheep endometrial organoids

[0103] 1. Isolation of sheep endometrial epithelial and stromal cells

[0104] 1.1 Healthy sheep uterine tissue was selected and placed in 37 ℃ physiological saline containing 0.2% (0.2g / 100ml) gentamicin sulfate within 3 hours of slaughter (the commercially available gentamicin sulfate injection with an active ingredient content of 5ml:0.2g was directly added to the physiological saline at a final concentration of 5% by volume; the gentamicin sulfate injection was produced by Hefei Dubang Biopharmaceutical Co., Ltd., item number: Qadmszy3) and transported to the laboratory in an incubator.

[0105] 1.2 After washing the outer surface of the uterus 2-3 times with the above-mentioned 37 ℃ saline containing gentamicin sulfate, cut the uterine horns on both sides and place them on the sterile surgical cloth in the safety cabinet. Use sterile scissors to open the uterine horns longitudinally and wash the endometrium with DPBS containing 2% double antibody (v / v, double antibody is a commercially available penicillin-streptomycin mixture, produced by Gibco, product number: 15070063).

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

[0107] 1.4 Transfer the processed meat-like tissue fragments to a 15 ml centrifuge tube and add tissue enzymatic solution (2 mg / ml collagenase I) to the meat-like tissue. Add 4 ml tissue enzymatic solution for every 1 ml meat-like tissue. Place in a 37 ℃ water bath and shake at constant temperature for 1 h until the tissue is 90% dissolved. Add DMEM containing 10% serum (v / v, serum manufacturer: Gibco, catalog number: A3161002C) to terminate the digestion.

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

[0109] 2. Cultivation of Sheep Endometrial Organoids

[0110] 2.1 Centrifuge the rinsed liquid, and the precipitate at the bottom of the tube is the endometrial epithelial cells. Add DMEM to resuspend the cells to obtain a cell suspension, count the cells, and adjust the cell concentration to 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 mixture and drop it in a 24-well plate preheated half an hour in advance to form a gel drop. Place the culture plate upright in a 37°C, 5% CO2 incubator for 10 minutes to allow the gel drop to reach a semi-solidified state, and then invert the culture plate for 10 minutes to allow the gel drop to solidify and prevent cells from adhering to the wall.

[0112] 2.3 After the gel drop solidifies, 500 μl / well of the sheep endometrial organoid culture medium prepared according to the formula 1 of Example 1 is slowly added along the wall of the cell culture well to submerge the entire gel drop. The culture is placed in a 37°C, 5% CO2 incubator for culture, and the medium is changed every 2-3 days.

[0113] 4. Experimental Results

[0114] In this experiment, endometrial organoids were cultured using the above methods and culture medium, such as Figure 1 As shown, the formation and growth changes of the cultured sheep endometrial organoids on the first, third, fifth, and seventh days can be seen, with a high formation rate and an overall diameter of 100-300 μm on the seventh day.

[0115] Example 3 Passaging of Sheep Endometrial Organoids

[0116] 1. Select sheep endometrial organoids that have grown to a total diameter of more than 100 μm, that is, sheep endometrial organoids on the 7th to 9th day of culture for subculture.

[0117] 2. Remove the gel and release the sheep endometrial organoids: Take the organoids out of the incubator, aspirate the culture medium, and wash them with DPBS along the wall to avoid washing up the gel droplets. After aspirating the DPBS, add 1 ml of DPBS to the gel droplets and blow the gel droplets about ten times to disperse the matrix gel. Collect the above contents from four wells in a 15 ml centrifuge tube and add pre-cooled DPBS to 10-12 ml. Place in a -20 ℃ refrigerator for 5-8 min. Take out and place in a 4℃ pre-cooled centrifuge at 300 rpm / min for 5 min, and remove the supernatant.

[0118] 3. Passaging: Add 1 ml of pre-cooled DPBS to the tube, and use a blue gun tip with a yellow gun tip to blow and aspirate the liquid 200-300 times. Aspirate a small amount of liquid for microscopic examination. Generally, the organoids have been blown into cell clusters of about 10-30 cells at this time. Add pre-cooled DPBS to 6 ml centrifuge at 300 rpm / min for 5 minutes, discard the supernatant, and resuspend the cells. Mix the cell suspension and matrix gel at a volume ratio of 20% cell suspension / 80% matrix gel. The passaging ratio is 1:2-1:3. Take 40 μl of the mixed solution and drop it in a 24-well plate preheated half an hour in advance to form a gel drop. Place it upright in a 37°C, 5% CO2 incubator for 10 minutes, and invert it for 10 minutes to solidify the gel drop and prevent cells from adhering to the wall. After the gel drop solidifies, slowly add 500 μl of sheep endometrial organoid culture medium prepared according to formula 1 of Example 1 against the wall of the cell culture well to submerge the entire gel drop. Culture the cells in a 37°C, 5% CO2 incubator and change the medium every 2-3 days.

[0119] 4. Cryopreservation: Select organoids that have been passaged 2-3 times for cryopreservation. Wash the organoids twice with pre-cooled DPBS, aspirate the DPBS, add 1 ml DPBS to the gel drop, blow the gel drop about ten times, and blow away the matrix gel. Collect the contents of the above four wells in a 15 ml centrifuge tube and add pre-cooled DPBS to 10-12 ml. Place in a -20 ℃ refrigerator for 5-8 min. Take it out and put it in a 4 ℃ pre-cooled centrifuge at 300 rpm / min for 5min, and remove the supernatant. Add 1 ml pre-cooled DPBS to the tube, use a blue pipette tip to gently blow the liquid 50-80 times to destroy the organoid structure, add pre-cooled DPBS to 6 ml centrifuge at 300 rpm / min for 5min, and discard the supernatant. Add serum-free cryopreservation solution to resuspend, add 1 ml of the mixed suspension to each cryopreservation tube, mark it, and put it in a gradient cryopreservation box, at -80 ℃ overnight, and then store it in liquid nitrogen for long-term storage.

[0120] 5. Thawing and recovery: Take out the endometrial organoids stored in the liquid nitrogen tank, and quickly shake them gently in a 37 ℃ water bath to thaw until a small ice block remains in the tube. Transfer the thawed organoid suspension to a 15 ml centrifuge tube with 4 ml DMEM, mix well, put it in a 4 ℃ pre-cooled centrifuge at 300 rpm / min for 5 minutes, and remove the supernatant. Mix the cell suspension and matrix gel according to the volume ratio of 20% cell suspension / 80% matrix gel. Pipette 40 μl of the mixed solution droplet into a 24-well plate preheated half an hour in advance to form a gel droplet, place it upright in a 37 ℃ incubator for 10 minutes, and invert it for 10 minutes to solidify the gel droplet and prevent the cells from adhering to the wall. After the gel droplet solidifies, slowly add 500 μl of the sheep endometrial organoid culture medium prepared according to the formula 1 of Example 1 along the wall of the cell culture well to submerge the entire gel droplet. Place it in a 37 ℃, 5% CO2 incubator for culture, and change the medium every 2-3 days.

[0121] 6. Experimental Results

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

[0123] Example 4: Verification of Physiological Hormone Response of Sheep Endometrial Organoids

[0124] 1. Select the sheep endometrial organoids of the third generation, and divide them into three groups on the sixth day of normal culture, namely the control group, the estrogen treatment group, and the progesterone treatment group. The control group changes the medium normally, and the estrogen treatment group and the progesterone treatment group are replaced with sheep endometrial organoid culture medium containing 10nM estrogen for two days. On the eighth day, the control group still changes the medium normally, the estrogen treatment group changes the sheep endometrial organoid culture medium containing 10nM estrogen, and the progesterone treatment group changes the sheep endometrial organoid culture medium containing 1 μM progesterone. During each change of the medium, the morphology and size changes are observed, and samples are collected on the tenth day. The sheep endometrial organoid culture medium is a sheep endometrial organoid culture medium prepared according to Formula 1 of Example 1.

[0125] 2. On the tenth day, the organoids were collected and sliced ​​after embedding in agar, and PAS staining was used to detect changes in the secretory function of the organoids (e.g. Figure 3 ).

[0126] 3. After collecting the organoids and removing the gel on the tenth day, the proteins were extracted by RIPA lysis, and the changes in the expression of hormone response-related proteins were detected by Western Blot (e.g. Figure 4 ).

[0127] 4. Experimental results

[0128] The results of the response changes of sheep endometrial organoids after treatment with physiological hormones are as follows Figure 3 , 4 shown. Figure 3 The results showed that estrogen treatment of organoids accelerated the growth rate of organoids and increased the diameter of the organoids; while progesterone treatment enhanced the secretory function of the organoids, thereby secreting more mucus. Figure 4 The results showed that estrogen treatment increased the protein expression of estrogen receptors and progesterone receptors in organoids, while progesterone treatment significantly increased the expression of mucin in organoids. These results indicate that the sheep endometrial organoids constructed in this experiment can simulate the response and changes of normal endometrium to physiological hormone treatment.

[0129] Example 5 Immunofluorescence Validation of Sheep Endometrial Organoids

[0130] 1. Select sheep endometrial organoids that have been passaged to the third generation and culture them normally until the seventh day. Collect the samples, remove the gelatin to release the organoids, and then embed them in agar and slice them.

[0131] 2. After the sections have gone through the steps of dewaxing, permeabilization, antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, nuclear staining, and sealing, use confocal microscopy to observe the results.

[0132] 3. Experimental results

[0133] Immunofluorescence staining was performed on sheep endometrial organoids, and the results were as follows Figure 5 As shown. The sheep endometrial organoids cultured in this experiment can express endometrial glandular epithelial markers EPCAM, CK19, and E-cadherin.

[0134] Example 6 Electron microscopic observation of the ultrastructure of sheep endometrial organoids

[0135] 1. Select the sheep endometrial organoids that have been passaged to the third generation and culture them normally until the seventh day. Add DPBS along the wall to wash once to avoid washing up the gel droplets. After aspirating the DPBS, add 1 ml of DPBS to the gel droplets and blow the gel droplets about ten times to disperse the matrix gel. Collect the above contents from four wells in a 15 ml centrifuge tube and add pre-cooled DPBS to 10-12 ml. Place in a -20 ℃ refrigerator for 5-8 min.

[0136] 2. Take out and place in a 4℃ precooled centrifuge at 300 rpm / min for 5 minutes, and remove the supernatant. The resulting precipitate is a complete sheep endometrial organoid. Add precooled electron microscopy fixative and fix it in a 4℃ refrigerator for 24 hours before sending it to the electron microscopy room to prepare samples for electron microscopy observation.

[0137] 3. Ultrastructural observation of sheep endometrial organoid samples using transmission electron microscopy.

[0138] 4. Experimental Results

[0139] The results of electron microscopy observation of the ultrastructure of sheep endometrial organoids are as follows Figure 6 As shown, the tubular structure of the organoid is a pseudostratified columnar epithelium with obvious and numerous microvilli structures on the inner side (black arrows); there are tight junctions between the endometrial structural cells (white arrows); the cytoplasm is rich in mitochondrial structures that are clearly 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 CN113957036A, the endometrial organoid culture medium was prepared according to the final concentration composition as follows: 50ng / ml EGF, 100ng / ml Noggin, 500ng / m1 R-spondin 1, 100ng / m1 Wnt3a, 50ng / ml FGF9, 30ng / m1 KIAA1199, 50ng / m1 sox2, 15nM isoflavones, 200nM CHIR99021, 15μM A83-01, 10μM RKI-1477, 100ug / m1 primary cell antibiotics. After seven days of endometrial organoid culture, the growth morphology was observed under a microscope. The results are as follows: Figure 7As shown in A.

[0142] The endometrial organoid culture medium was prepared according to the formula 1 in Example 1. After the endometrial organoid culture was completed for seven days, the growth morphology was observed under a microscope. Figure 7 As shown in A.

[0143] The growth diameter and cystic organoid formation rate (organoid formation is a cystic structure lined by columnar epithelium, with secretions visible in the lumen) of organoids cultured with two different ratios of endometrial organoid culture medium were statistically analyzed. The results are as follows Figure 7 As shown in B and C in the figure. The culture medium provided by the present invention can cultivate a larger diameter ( Figure 7 B) More cystic ( Figure 7 C), 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-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. Each specific technical feature can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0145] In addition, it is apparent to those skilled in the art that several modifications and improvements may be made without departing from the concept of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.

Claims

1. A sheep endometrial organoid culture medium, characterized in that The formula of the culture medium includes basal culture medium, cell growth factors, Wnt / β-catenin signaling pathway activators, BMP inhibitors, antioxidants and protective agents, nutrients and cell culture medium supplements, and auxiliary components; The basal culture medium is DMEM; The cell growth factors include: FGF10 50-100 ng / ml, EGF 30-50 ng / ml and HGF 30-50 ng / ml; The Wnt / β-catenin signaling pathway activating factors include: Rspondin-1 100-500 ng / ml and Wnt3a100-500 ng / ml; The BMP inhibitor is Noggin 50-200 ng / ml; The antioxidant and protective agent comprises: nicotinamide 10-200nM and cysteine ​​1-1.5 mM; The nutrient and cell culture medium supplements include: 1× transferrin, 1× N2, 1× B27, HEPES 1mM and GlutaMAX 200 nM; The auxiliary component is A83-01 300-600 nM.

2. The sheep endometrial organoid culture medium according to claim 1, characterized in that The culture medium comprises the following components in its formula: DMEM, FGF10 50-100 ng / ml, EGF 30-50 ng / ml, HGF 30-50 ng / ml, Rspondin-1 100-500 ng / ml, Wnt3a 100-500 ng / ml, Noggin 50-200 ng / ml, nicotinamide 10-200 nM, cysteine ​​1-1.5 mM, 1× transferrin, 1× N2, 1× B27, HEPES 1 mM, GlutaMAX 200 nM and A83-01 300-600 nM.

3. Use of the sheep endometrial organoid culture medium according to any one of claims 1 to 2 in culturing sheep endometrial organoids.

4. A method for culturing sheep endometrial organoids, characterized in that: The culture method comprises the following steps: (1) epithelial cells of sheep endometrial tissue are suspended in DMEM to prepare a cell suspension, and the cell suspension is mixed with matrix gel to prepare a mixed solution; (2) Dropping the mixed solution into a preheated cell culture plate to form a gel droplet, placing the droplet in an incubator until the gel droplet solidifies, and then adding the sheep endometrial organoid culture medium according to claim 1 or 2 into the cell culture plate for culturing to obtain sheep endometrial organoids.

5. The method for culturing sheep endometrial organoids according to claim 4, characterized in that: The preparation method of epithelial cells of sheep endometrial tissue is as follows: peeling the sheep endometrial tissue and soaking it in DPBS containing 2% double antibody for washing, then chopping it into meat-like tissue fragments; adding tissue enzymatic solution to the tissue fragments, shaking and incubating at 37° C. until the tissue is 90% dissolved, and then adding DMEM containing 10% serum to terminate digestion; Filter through a 100 μm nylon mesh cell strainer to remove undigested tissue debris; The filtrate was passed through a 40 μm cell strainer to separate and remove the stromal cells, and the epithelial cells of the sheep endometrial tissue were collected.

6. The method for culturing sheep endometrial organoids according to claim 4, characterized in that: The cell concentration of the cell suspension in step (1) is 1.0-3.0 × 10 6 / ml; the volume ratio of the cell suspension to the matrix gel is 1:

4.

7. The method for culturing sheep endometrial organoids according to claim 4, characterized in that: In step (2), the cells were cultured at 37°C and 5% CO2 for 10 min until the gel droplet reached a semi-solidified state, and then the cell culture plate was inverted for 10 min to allow the gel droplet to completely solidify.

8. The method for culturing sheep endometrial organoids according to claim 4, characterized in that: In step (2), the sheep endometrial organoid culture medium is added to the cell culture plate and culture is continued at 37° C. and 5% CO 2 to obtain sheep endometrial organoids.

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