Method for preparing 3d model of maternal-fetal interface by constructing endometrial organoids in vitro

Abstract

The application discloses a preparation method of a 3D model of a maternal-fetal interface constructed by an endometrial organoid in vitro, and the method comprises the following steps: establishing a primary endometrial epithelial organoid and an endometrial stromal cell line in vitro; improving an extracellular matrix for assembling endometrial gland-like organoids and stromal cells; the constructed endometrial organoid model comprises gland cells and stromal cells; the endometrial organoid model is cultured by using a gas-liquid interface, so that the endometrial organoid model has a cavity epithelium and a gland structure; the endometrial organoid model simulates a menstrual cycle of the endometrium and performs function identification. The endometrial organoid model has the cavity epithelium and the gland epithelium for the first time, the tissue structure, the cell composition, the hormone-induced change, the gene expression characteristics of the organoid are similar to those in the body, and the organoid can reproduce the receptivity genes and the dynamicity of cilia which are necessary for embryo implantation in the body; the organoid can be used as a model for studying embryo implantation regulation, endometrial diseases and regeneration mechanisms.

Description

Technical Field

[0001] This invention belongs to the fields of reproductive medicine and biotechnology, specifically relating to a method for preparing a 3D model of the maternal-fetal interface for in vitro construction of endometrial organoids. Background Technology

[0002] Compared to other mammals, human fertility is low. Even for healthy women, the chance of conceiving naturally each menstrual cycle is only 30%. According to human fertility statistics, one in six couples is diagnosed with infertility, and among these couples, approximately 25% are diagnosed with unexplained infertility. Although assisted reproductive technologies are used clinically to improve fertility rates, the success rate is only between 40-55%, with implantation failure being the limiting factor. Because our understanding of embryo implantation is still unclear, we need to better understand the interaction between the mother and fetus, and the importance of endometrial receptivity for embryo implantation, in order to further improve pregnancy outcomes and increase pregnancy rates for infertile couples.

[0003] The endometrium, serving as the "soil" for embryo implantation, is crucial for successful pregnancy. It is a multi-layered structure composed of an outer layer of luminal epithelium and an inner layer of glandular epithelium. The former is the implantation site where the embryo interacts, while the latter provides a nutrient environment for the embryo. During a woman's menstrual cycle, the endometrium undergoes a highly dynamic process under the influence of estrogen and progesterone, experiencing shedding, regeneration, and differentiation each cycle. Abnormal endometrial remodeling and regeneration can lead to infertility, recurrent miscarriage, endometrial tumors, inflammation, thin endometrium, and endometriosis. Although the pathology of these diseases is well understood, the molecular and cellular mechanisms involved remain unresolved. Therefore, analyzing the cellular and molecular mechanisms involved in the physiological and pathological conditions of the endometrium is essential for a better understanding of this dynamic organ and its associated diseases, and can contribute to the development of novel therapies. Currently, most research models use immortalized cell lines. After long-term passage, immortalized cell lines may differ phenotyped and genotyped from their primary sources, failing to encompass the nature and heterogeneity of these diseases, thus hindering scientific and clinical progress. In 2017, the establishment of human and mouse endometrial glandular epithelial organoid models provided new insights for in vitro research. In vivo, cells exist in a complex microenvironment with intricate signaling and cell-cell interactions, crucial for establishing, maintaining, and regulating cellular phenotypes and functions. Organoids, obtained directly from endometrial biopsies, are considered to be more phenotypically and physiologically similar to tissue cells, and the 3D culture modality better reflects their physiological functions. Due to the limitations of translating research results from animal models into humans, our understanding of the pathogenesis of human endometrial diseases remains limited. The development of organoids advances precision medicine in the 21st century, enabling personalized treatment using organoid models with a defined genetic background. Endometrial organoids will become promising tools for a wide range of biomedical applications, from disease modeling to personalized medicine, accelerating our understanding of the molecular and cellular mechanisms involved in endometrial development and disease. However, most in vitro endometrial models described to date are limited to single endometrial cells or lack luminal epithelial structures, failing to truly simulate the three-dimensional structure and function of the in vivo endometrium.

[0004] Therefore, in order to solve the above problems, this paper proposes a method for preparing a 3D model of the maternal-fetal interface by constructing endometrial organoids in vitro. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention presents a method for preparing a 3D model of the maternal-fetal interface using endometrial organoids in vitro. This invention establishes a microenvironment for communication and dialogue between endometrial glandular epithelial organoids and endometrial stromal cells, overcoming the limitation of single epithelial organoids failing to realistically simulate the characteristics of the menstrual cycle, and verifying the consistency of hormonal response with in vivo data. Furthermore, this invention combines the characteristics of Matrigel and collagen to obtain an extracellular matrix superior to that obtained from culturing endometrial glandular organoids and endometrial stromal cells separately. Using a unique gas-liquid interface culture method, an endometrial organoid model simultaneously possessing luminal and glandular epithelium was established.

[0006] To achieve the above-mentioned technical effects, the present invention is implemented through the following technical solution: a method for preparing a 3D model of an in vitro maternal-fetal interface for constructing endometrial organoids, characterized by comprising the following steps:

[0007] S1. Establish primary endometrial epithelial organoids and endometrial stromal cell lines in vitro;

[0008] S2. Improve the extracellular matrix used to assemble endometrial gland-like organoids and stromal cells;

[0009] S3. The constructed endometrial organoid model includes glands and stromal cells;

[0010] S4. Use the gas-liquid interface to culture the endometrial organoid model to give it luminal epithelial and glandular structures;

[0011] S5. Endometrial organoid models simulate the menstrual cycle and functional identification of the endometrium.

[0012] Furthermore, the culture medium for the endometrial epithelial organoids in S1 is called expansion medium (ExM), which includes Advanced DMEM / F12, N2 supplement, B27 supplement minus vitamin A, penicillin / streptomycin, 0.5-2mM N-Acetyl-L-cysteine, 0.5-3mM M-glutamine, 30-70ng / ml EGF, 50-200ng / ml Noggin, 50-100ng / ml Rspondin-1, 50-200ng / ml FGF-10, 20-80ng / ml HGF, 200-1000nM A83-01, 5-20nM nicotinamide, and 5-15nM Y27632.

[0013] Furthermore, the culture medium for endometrial stromal cells in S1 consists of 5-20% fetal bovine serum and 0.5-5 μg / ml L-ascorbic acid.

[0014] Furthermore, in S2, endometrial gland-like epithelial organoids and stromal cells are mixed in a certain proportion; the extracellular matrix of the assembled endometrial gland-like epithelial organoids and stromal cells is improved, and the extracellular matrix is ​​a mixture of Matrigel and type I collagen; after assembly, the two are placed in the upper chamber of a Transwell chamber or a culture dish and incubated at 37°C for more than 1 hour, and culture medium is added after complete solidification.

[0015] Furthermore, in S3, an endometrial organoid luminal epithelial structure is constructed. After the assembly of the two in S2 is solidified, the endometrial organoids in ExM culture are collected in a low-adsorption centrifuge tube, and the extracellular matrix is ​​removed by repeated gentle pipetting and centrifugation. The obtained cell pellet is resuspended in ExM medium, and then added to the upper chamber of the Transwell chamber, and ExM medium with the same composition is added to the lower chamber.

[0016] Furthermore, in S4, the endometrial organoid assembly is cultured using an air-liquid interface. The endometrial organoid model constructed in S3 is placed in a Transwell chamber of a 24-well plate, with both the upper and lower chambers containing ExM medium for 0-4 days. Subsequently, a special air-liquid interface culture method is used, in which the liquid in the upper chamber of the Transwell is removed, keeping the upper part of the endometrial organoid model in contact with air and the lower part in contact with the culture medium to obtain nutrients. The air-liquid interface culture method is used for 4-20 days.

[0017] Furthermore, the structure and function of the endometrial organoid model in S5 were verified by in vitro treatment with 5-15 nM β-estradiol (E2) for 3-8 days, followed by gas-liquid interface culture and differentiation for 4-20 days, simulating the proliferative phase of the menstrual cycle; after treatment with 5-15 nM β-estradiol (E2) for 3-8 days, 1 μM progesterone (P4) and 1 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP) were added for 3-8 days, followed by gas-liquid interface culture and differentiation for 4-20 days, simulating the secretory phase of the menstrual cycle; through single-cell transcriptome analysis and immunofluorescence staining, comparison with endometrial tissues of different menstrual phases confirmed the similarity of the organoid's tissue structure, cell composition, hormone-induced changes, and gene expression characteristics to those in vivo, and demonstrated its ability to reproduce the receptivity genes and cilia dynamics necessary for embryo implantation in vivo.

[0018] The beneficial effects of this invention are:

[0019] This invention utilizes endometrial epithelial and stromal cells to construct an organoid model with similar cell composition in vivo, explores the effects of stromal cells on the growth, proliferation, and gene phenotype of gland-like organoids, verifies the consistency of hormone response with in vivo, and improves the shortcomings of the original single gland-like organoids in simulating menstrual cycle changes.

[0020] This invention also combines the characteristics of Matrigel and collagen to obtain an extracellular matrix more suitable for in vitro organoid culture; combined with the gas-liquid interface culture method, it has for the first time established an endometrial organoid model with both luminal epithelium and glandular epithelium. Through single-cell transcriptome analysis and immunofluorescence staining, compared with endometrial tissues of different menstrual stages, it was confirmed that the tissue structure, cellular composition, hormone-induced changes, and gene expression characteristics of the organoid are similar to those in vivo, and it can reproduce the receptivity genes and cilia dynamics necessary for embryo implantation in vivo; it can be used as a model for studying embryo implantation regulation, endometrial diseases, and regeneration mechanisms. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 In vitro construction of endometrial epithelial organoids and endometrial stromal cell lines;

[0023] Figure 2 Improve the extracellular matrix of the assembled endometrial organoid model;

[0024] Figure 3 In vitro endometrial organoid models simulate the proliferative phase;

[0025] Figure 4 In vitro endometrial organoid models simulate the secretory phase;

[0026] Figure 5 Single-cell atlas analysis reveals the similarities and differences between in vivo and in vitro endometrial tissues. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1

[0029] Preparation of culture medium for endometrial glandular epithelial organoids and endometrial stromal cells:

[0030] (1) Preparation of endometrial epithelial organoid culture medium (abbreviated as ExM), the components of which include Advanced DMEM / F12, N2 supplement, B27 supplement minus vitamin A, penicillin / streptomycin, 0.5-2mM N-Acetyl-L-cysteine, 0.5-3mM L-glutamine, 30-70ng / ml EGF, 50-200ng / ml Noggin, 50-100ng / ml Rspondin-1, 50-200ng / ml FGF-10, 20-80ng / ml HGF, 200-1000nM A83-01, 5-20nM nicotinamide and 5-15nM Y27632.

[0031] (2) The endometrial stromal cell culture medium consists of DMEM, 5-20% fetal bovine serum and 0.5-5 μg / ml L-ascorbic acid.

[0032] Example 2

[0033] Establishment and functional identification of endometrial gland-like epithelial organoids in vitro:

[0034] (1) Tissue was collected in PBS phosphate buffer and then subjected to cell separation within 2-4 hours. Cell culture was performed in a humidified incubator at 37°C with 5% CO2. Centrifugation and incubation were performed at room temperature unless otherwise specified. The obtained endometrial biopsy tissue was finely shredded with surgical scissors for 5-10 minutes and collected in 50 ml centrifuge tubes. 5-20 ml of separation culture medium (RPMI 1640 medium containing 0.5-5 U / ml neutral protease / dispersin II and 0.2-2 mg / ml collagenase V, and 5-20% FBS) was added. The tubes were incubated at 37°C with shaking for 30-60 minutes (observed under a microscope for a large number of glands). The supernatant was filtered through one or more 70 μm cell sieves, and the filtered cell sieves were washed several times with 1640 medium.

[0035] (2) Invert the cell sieve, and backwash the glands remaining on the top of the cell sieve into a culture dish using basal culture medium. Centrifuge to remove the supernatant, and resuspend the precipitate in pre-chilled 50-100% Matrigel. Transfer 20-30 μl of the resuspension to each well of a 48-well plate, incubate at 37°C for at least 30 minutes, and add 200-300 μl of ExM culture medium to the wells for further culture.

[0036] (3) Subculture of endometrial gland-like epithelial organoids: Gently scrape Matrigel containing endometrial gland-like epithelial organoids into a low-adsorption centrifuge tube using a pipette tip. Centrifuge to remove the supernatant, add 100-500 μL of Advanced DMEM / F12 to resuspend the pellet, and gently pipette 100-500 times. Centrifuge to remove the supernatant, ensuring the centrifuge tube is not inverted. Repeat the above steps, resuspending the cell pellet in pre-chilled 50-100% Matrigel. Transfer 20-30 μL of the resuspended solution to each well of a 48-well plate and incubate at 37°C for at least 30 minutes. Add 200-300 μL of ExM culture medium to the wells for subsequent subculture. Routinely, change the medium every 2 days and subculture every 6-10 days.

[0037] (4) Cryopreservation of endometrial gland-like epithelial organoids: Gently scrape Matrigel encapsulating endometrial gland-like epithelial organoids into a low-adsorption centrifuge tube using a pipette tip. Centrifuge to remove the supernatant, add 100-500 μL of Advanced DMEM / F12 to resuspend the precipitate, and gently pipette 100-500 times. Centrifuge to remove the supernatant, ensuring the centrifuge tube is not inverted. Repeat the above steps using pre-chilled Recovery media. TM Resuspend the cell pellet in the cell culture cryopreservation medium, and add the cell suspension to cryovials, 500 μl / tube, each tube containing 20-100 gland-like organoids. Then place the cryovials containing the cell suspension in a programmed cooling box and freeze overnight at -80°C, before transferring them to liquid nitrogen for long-term storage.

[0038] (5) Primary endometrial glandular epithelial organoids were obtained according to step (2). After two generations of subculture according to step (3), three groups were set up for the experiment. The first group collected glandular organoids cultured in ExM. The second group collected glandular organoids treated with 5-15 nM β-estradiol (E2) under ExM culture conditions. The third group collected glandular organoids treated with 5-15 nM β-estradiol (E2), 0.5-2 μM progesterone (P4), and 0.5-2 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP) under ExM culture conditions. Epithelial markers and hormone response markers were detected and confirmed by immunofluorescence imaging. Figure 1 The results show that the endometrial epithelial organoids we established specifically express the epithelial markers CK7 and E-cadherin, and can be passaged in vitro for a long time. Figure 1 EI indicates that in vitro-developed endometrial epithelial organoids can respond to the effects of estrogen and progesterone. Under the influence of estrogen, they can be induced to differentiate into ciliated cells, and the proliferation of epithelial organoids is promoted under the influence of hormones.

[0039] Example 3

[0040] Establishment and functional identification of in vitro endometrial stromal cell lines:

[0041] (1) After filtration of the suspension according to step (1) of Example 2, the cells in the filtrate are collected and cultured in endometrial stromal cell culture medium for several days. After the cells adhere and grow, the primary endometrial stromal cell line is obtained.

[0042] (2) Passaging of endometrial stromal cells: Endometrial stromal cells were digested into single cells using 50% TrypLE, centrifuged to remove the supernatant, and then resuspended in endometrial stromal cell culture medium. The cells were seeded into 6 cm culture dishes at a density of 1×10^4-2.5×10^4 cells / cm2. During subsequent passage culture, the medium was changed every 2 days, and the cells were passaged every 6-10 days.

[0043] (3) Cryopreservation of endometrial stromal cells: Endometrial stromal cells were digested into single cells using 50% TrypLE solution. After centrifugation and removal of supernatant, the cell pellet was resuspended in pre-cooled cryopreservation solution. The cell suspension was added to cryovials at a rate of 500 μl / tube, with each tube containing 5 × 10^5 - 2 × 10^6 cells. The cryovials containing the cell suspension were then placed in a programmed cooling box and stored overnight at -80°C before being transferred to liquid nitrogen for long-term storage.

[0044] (4) After two generations of culture, decidualization was performed using 0.5-2 μM P4 and 0.5-2 μM cAMP. The endometrial stromal cells were qualitatively identified by immunofluorescence staining, and the expression levels of decidualization markers were confirmed by real-time quantitative PCR. Figure 1 JL indicates that the primary endometrial stromal cells obtained in 2D culture mode express Vimentin as well as stromal cell-specific markers COL6A3, FN1 and LUM. Figure 1 j,m indicates that after progesterone decidualization treatment, stromal cells changed from a spindle shape to a polygon shape, and quantitative real-time PCR confirmed that the expression levels of PRL and IGFBP1 were significantly increased. Figure 1 n, p indicates that the proliferative capacity of stromal cells weakened after decidualization treatment, and the expression of PGR and Connexin43 decreased while the expression of FOXO1 increased.

[0045] Example 4

[0046] In vitro assembly of endometrial organoids and stromal cells to construct the luminal epithelial structure of an endometrial organoid model:

[0047] (1) Establishment of endometrial gland-like organoids and endometrial stromal cells: As described in Examples 1 and 2, endometrial gland-like epithelial organoids and endometrial stromal cells cultured in ExM were passaged as described in Examples 1 and 2, and stromal cells and gland cells were collected respectively.

[0048] (2) Following the steps of Example 2 (3) cell passage, after the second centrifugation to remove the supernatant, 100-500 μL of Advanced DMEM / F12 was added to resuspend the precipitate. The precipitate was gently pipetted 100-500 times and stored on ice. The old culture medium of the endometrial stromal cells was removed, and the endometrial stromal cells were digested into single cells using 50% TrypLE. After centrifugation to remove the supernatant, the cells were mixed into the pre-chilled endometrial gland-like organoids at a ratio of 3×10^5-8×10^5 cells. After centrifugation to remove the supernatant, the cells were resuspended in the pre-chilled gland-like organoid and matrix mixture at a ratio of 1:1:(0.5-2)(v / v) (Matrigel:Type I collagen:Advanced DMEM / F12). 20-50 μL of the mixture was placed in the upper chamber of a 24-well plate and placed at 37°C for at least 30 minutes until solidification before proceeding to the next step. like Figure 2 This indicates that the novel extracellular matrix (MAC) developed in vitro, combining the characteristics of Matrigel and type I collagen, improves the extracellular matrix. Young's modulus measurements revealed that the MAC's stiffness is similar to that of tissues. Immunofluorescence staining determined the morphology and proliferative capacity of epithelial organoids, showing that the improved MAC is more conducive to the formation of columnar epithelium and the growth of stromal cells.

[0049] (3) Take another 1-2 wells of gland-like organoids cultured in ExM medium into a low-adsorption centrifuge tube, and obtain the cell pellet of gland-like organoids by passage centrifugation according to step (3) of Example 2. Resuspend the cell pellet with 50-200 μL of ExM medium and add it to the upper chamber of the Transwell of the solidified cell mixing gel in step (2). Add 200-500 μL of ExM medium to the corresponding lower chamber.

[0050] Example 5

[0051] Endometrial organoid assemblages were cultured using an air-liquid interface:

[0052] (1) If the endometrial organoid model constructed in Example 3 is cultured for 0-3 days as described above, 5-15 nM β-estradiol (E2) is added to the ExM medium and the endometrial organoid model is treated for 3-8 days. Then the liquid in the upper chamber of the Transwell is removed, and the lower chamber contains 200-500 μL of ExM medium. The medium is changed every 2 days and cultured at the gas-liquid interface for 4-20 days. Figure 3 The results showed that the proliferative phase hormone treatment flowchart for in vitro simulated menstrual cycles was compared with that of conventional culture at the same time as a control. In in vitro simulations of the proliferative phase of menstrual cycles, endometrial organoids, compared with organoids cultured in a liquid environment, showed that an air-liquid balance was more conducive to the formation of columnar epithelium. Figure 3 d and e indicate that after 15 days of differentiation, the development of ciliated cells is more similar to that in vivo. Figure 3 f indicates that endometrial cells are regulated by estrogen, with estrogen receptor (ESR) and progesterone receptor (PGR) showing high expression in the in vitro organoid model, consistent with in vivo characteristics.

[0053] (2) If the secretory phase of the menstrual cycle is simulated in vitro, the endometrial organoid model constructed in Example 3 is cultured for 0-3 days as above. Then, 5-15 nM β-estradiol (E2) is added to ExM medium to treat the endometrial organoid model for 3-8 days. Then, 0.5-2 μM progesterone (P4) and 0.5-2 μM 8-bromoadenosine 3′,5′-cyclic monophosphate (cAMP) are added to treat the endometrial organoid model for another 3-8 days. Then, the liquid in the upper chamber of the Transwell is removed, and the lower chamber is kept with 200-500 μL of medium. The medium is changed every 2 days, and the gas-liquid interface is cultured for 4-20 days. Figure 4 The results showed that the hormone treatment flowchart for the secretory phase of the in vitro simulated menstrual cycle was compared with that of conventional culture at the same time point as a control. In the in vitro endometrial organoid model simulating the secretory phase, the air-liquid level culture method, compared to the conventional liquid culture method, also promoted the formation of columnar cavitary epithelium. Figure 4 d and e indicate that progesterone antagonizes the effects of estrogen, reducing the proportion of ciliated cells to facilitate embryo adhesion and implantation. For example... Figure 4 f indicates that, under the influence of progesterone, the expression of PGR and ESR in epithelial cells decreases, but PGR is continuously expressed in stromal cells. The dynamic changes of estrogen and progesterone receptors with the menstrual cycle are similar to those in vivo. During the secretory phase, endometrial epithelial organoids also specifically express PAEP at high levels.

[0054] Example 6

[0055] Identification of endometrial organoid assembly models:

[0056] (1) We used different extracellular matrix to encapsulate endometrial organoid models and treated them with estrogen for 3-8 days. Complete endometrial organoid models were carefully obtained from the upper chamber of the Transwell. Sample stiffness was measured using a nanoindenter. To ensure the accuracy of statistical analysis, three biological replicate experiments were performed.

[0057] (2) In vitro endometrial organoid models simulate the proliferative and secretory phases of the menstrual cycle. Gently aspirate the lower chamber medium from the Transwell. Fix with 4% paraformaldehyde for 1-3 hours, dehydrate with 20% sucrose for 30-60 minutes, then embed with OCT. After freezing the OCT, cut 5-15 μm frozen sections using a cryostat and collect them on adhesive slides. Wash the slides with PBS to remove OCT. Add 100-200 μl of 3% BSA containing 0.3-0.5% Triton X-100 to each slide for transmembrane blocking at room temperature for 3-4 hours or overnight at 4°C. Incubate the primary antibody overnight at 4°C. The next day, after washing with PBS, add the corresponding species' secondary antibody (1:500) and DAPI (1:1000) for labeling at room temperature for 2 hours. Wash three times with PBS for 10 minutes each time. Add 20 μl of 20% glycerol, seal with a coverslip, and then analyze and image using a Leica SP8 confocal microscope and Leica X software.

[0058] (3) An in vitro endometrial organoid model simulates the proliferative and secretory phases of the menstrual cycle. After a period of estrogen and progesterone administration, the model is carefully removed from the Transwell chamber and placed in 0.1-1 mg / ml type I collagenase. The mixture is incubated at 37°C for 30-60 minutes, gently agitated every ten minutes to dissociate the gel mixture. After centrifugation and removal of the supernatant, Advanced DMEM / F12 medium is added and the precipitate is gently pipetted. The supernatant is removed again, and 50% TrypLE is added. The precipitate is digested at 37°C for 30-60 minutes. The cell clusters are gently pipetted to allow rapid precipitation until all cell clusters are separated into single cells. The precipitate is harvested by centrifugation and resuspended in 0.1% bovine serum albumin to prevent single-cell adhesion. Cell viability is assessed by trypan blue staining and cell counting. Figure 5 Based on single-cell transcriptome sequencing results, single-cell data from an in vitro constructed endometrial organoid model were integrated and compared with data from in vivo epithelial cells and stromal cells. In vitro and in vivo cell data were further subdivided into ciliated cells, non-ciliated cells, and stromal cells. This confirmed that the cellular composition, gene markers of subpopulations, and functions of the organoids were similar to those in vivo.

[0059] The conclusion is that co-culturing endometrial glandular organoids with stromal cells improves the composition of endometrial cells. By improving the extracellular matrix and combining the characteristics of matrix gel and collagen, it not only promotes the growth of epithelial organoids but also enhances their rigidity, providing better adhesion sites for stromal cells to proliferate and differentiate. Differentiation is achieved through a unique gas-liquid interface culture method, constructing tissue structures in vitro that are more similar to those in vivo, exhibiting both luminal epithelial-like and glandular epithelial-like structures. Immunofluorescence staining and single-cell transcriptome sequencing analysis reveal that the in vitro constructed endometrial organoid model can form tissue structures that conform to in vivo characteristics and can simulate the dynamic changes in genes during the menstrual cycle in vitro. This model can serve as a novel approach for studying embryo implantation regulation, the mechanisms of endometrial disease, the mechanisms of endometrial cyclic regeneration, and drug screening.

Claims

1. A method for preparing a 3D model of a maternal-fetal interface constructed in vitro from an endometrial organoid, characterized in that, The method comprises the following steps: S1, in vitro, establishing a primary endometrial glandular epithelial organoid and an endometrial stromal cell line; S2, mixing the endometrial glandular epithelial organoid with the stromal cells, and using MAC to improve the extracellular matrix for assembling the endometrial glandular epithelial organoid and the stromal cells; the MAC is a mixture of Matrigel: collagen type I: Advanced DMEM / F12 in a ratio of 1:1:(0.5-2) (v / v); S3, after the assembly of S2 is solidified, another 1-2 holes of the endometrial glandular epithelial organoid cultured in the ExM medium are collected in a low adsorption centrifuge tube, and the extracellular matrix is removed by repeated gentle blowing and centrifugation to obtain a precipitate of the glandular epithelial cells; The cell precipitate is resuspended with 50-200 μL of ExM medium, and is added to the upper chamber of the Transwell, and 200-500 μL of ExM medium is added to the lower chamber; the upper chamber and the lower chamber are both cultured with the ExM medium for 0-4 days; the composition of the ExM medium comprises Advanced DMEM / F12, N2 supplement, B27 supplement minus vitamin A, penicillin / streptomycin, 0.5-2 mM N-Acetyl-L-cysteine, 0.5-3 mM L-glutamine, 30-70 ng / ml EGF, 50-200 ng / ml Noggin, 50-100 ng / ml Rspondin-1, 50-200 ng / ml FGF-10, 20-80 ng / ml HGF, 200-1000 nM A83-01, 5-20 nM nicotinamide, and 5-15 nM Y27632; Then, the liquid in the upper chamber of the Transwell is removed, and the endometrial organoid model is cultured in a gas-liquid interface mode for 4-20 days, in which the upper part of the endometrial organoid model is in contact with air, and the lower part is in contact with the culture medium; S4, simulating the menstrual cycle of the endometrium and functional identification of the endometrial organoid model.

2. The method for preparing a 3D model of an endometrial organoid for in vitro construction of a maternal-fetal interface according to claim 1, characterized in that: The composition of the culture medium of the endometrial stromal cells in S1 is 5-20% fetal bovine serum and 0.5-5 μg / ml L-ascorbic acid.

3. The method for preparing a 3D model of an endometrial organoid for in vitro construction of a maternal-fetal interface according to claim 1, characterized in that: The S4 endometrial organoid model structure and function verification, in vitro administration of 5-15nM β-estradiol (E2) treatment 3-8 days, gas-liquid interface culture differentiation 4-20 days, simulate the proliferation phase of menstrual cycle; after administration of 5-15nM β-estradiol (E2) treatment 3-8 days, then add 1 μM progesterone (P4) and 1 μM 8-bromo adenosine 3', 5'-cyclic monophosphate (cAMP) treatment 3-8 days, gas-liquid interface culture differentiation 4-20 days, simulate the secretory phase of menstrual cycle; through single cell transcriptome analysis and immunofluorescence staining, compared with endometrial tissue in different menstrual phase, confirm that the organoid tissue structure, cell composition, hormone-induced changes, gene expression characteristics and in vivo similarity, and can reproduce in vivo embryo implantation necessary receptivity genes and cilia dynamic.

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