Co-culture model based on endometrial stromal cell organoid and follicle organoid as well as construction method and application of co-culture model

Abstract

The invention discloses a co-culture model based on endometrial stromal cell organoid and follicle organoid as well as a construction method and application of the co-culture model, and belongs to the technical field of reproductive medicine and cell engineering. The invention discloses a construction method of a co-culture model based on an endometrial stromal cell organ and a follicle organ. The construction method comprises a culture method of the follicle organ, a culture method of the endometrial stromal cell organ and co-culture of the follicle organ and the endometrial stromal cell organ. The 3D follicle-like organ and endometrial stromal cell-like organ co-culture system provided by the invention can reproduce in-vivo physiological conditions more truly, can be used for exploring a deciduous molecular mechanism, and also has the potential of constructing a high-throughput detection platform for drugs and environmental toxicants.

Description

Technical Field

[0001] This invention relates to the fields of reproductive medicine and cell engineering technology, and more specifically to a co-culture model of endometrial stromal cell organoids and follicle organoids, its construction method and application. Background Technology

[0002] The human endometrium is a highly dynamic tissue that undergoes changes in response to hormonal fluctuations throughout the menstrual cycle. In a woman's lifetime, the endometrium undergoes approximately 400-50 cyclical changes, including menstrual shedding, repair, proliferation, and secretion phases. These cyclical events are precisely regulated by estrogen and progesterone secreted by the ovaries, and are accompanied by autocrine and paracrine signaling to establish a suitable microenvironment for pregnancy. After ovulation, endometrial stromal cells (ESCs) undergo morphological and functional differentiation, transforming into decidual cells—a process known as decidualization. Decidualization not only depends on progesterone-progesterone receptor signaling but also involves a complex network of regulation by transcription factors and cytokines such as cAMP, FOXO1, HOXA10, and HAND2. Disorders of decidualization are considered a significant cause of infertility, recurrent miscarriage, and uteroplacental diseases.

[0003] Currently, most studies on endometrial decidualization rely on two-dimensional ESC adherent culture systems, inducing decidualization through the exogenous addition of estrogen (Estradiol, E2), progesterone (P4), and cAMP, and using... IGFBP1 , PRL , FOXO1 Upregulation of marker expression was used as the main criterion. However, the two-dimensional culture system has obvious limitations: (1) its hormone stimulation is added artificially at a single time point, lacking the dynamic temporal and cyclical nature of hormone secretion during follicular development, and cannot truly reflect the endocrine regulation of the ovarian-uterine axis in vivo; (2) ESCs lack three-dimensional structure and extracellular matrix support in the two-dimensional plane, resulting in insufficient intercellular interactions and difficulty in simulating the real endometrial microenvironment; (3) in the detection of drugs or environmental factors, the predictive ability of this system for actual in vivo responses is significantly insufficient.

[0004] In recent years, with the development of organoids and 3D culture technology, researchers have gradually established a variety of endometrial organoid models and spheroid / organoid models. For example, endometrial epithelial cell (EECs) organoids can maintain long-term expansion and hormone response characteristics and are widely used for basic research and disease modeling; co-culture models using synthetic matrix or micromolds are also used to explore the interaction between epithelial and stromal cells; in the study of endometriosis, endometrial spheroid models are used to simulate lesion formation and invasion. These advances show that 3D models are significantly superior to traditional 2D culture systems in many ways. First, the 3D environment can provide spatial support, maintain cell-cell tight junctions and cell-matrix interactions, and make the cell morphology, polarity and differentiation state more similar to in vivo conditions; in contrast, cells in 2D adherent culture are usually flattened and lose their original tissue characteristics. Second, 3D models can better simulate the extracellular matrix, nutrients and oxygen gradients in the body, resulting in more consistent signal transduction and gene / protein expression profiles with in vivo tissues. Studies have shown that the gene expression patterns formed in 3D models are highly consistent with clinical samples, while 2D models often exhibit molecular characteristics that are inconsistent with in vivo conditions. Finally, 3D systems are more predictive and reliable in drug screening and environmental factor evaluation, as their response patterns are more similar to in vivo phenotypes, reducing the false positive or false negative results caused by 2D models.

[0005] However, there is currently a lack of a co-culture experimental system that can integrate the dynamic hormone secretion of ovarian follicle organoids and the decidualization process of 3D endometrial stromal cell organoid models. Traditional 3D endometrial organoid research mainly focuses on the modeling of epithelial or diseased tissues, while ovarian follicle organoid systems are mainly used for oocyte maturation and follicular endocrine research, and the two have not been effectively combined.

[0006] Therefore, it is an urgent problem for those skilled in the art to provide a co-culture model based on 3D endometrial stromal cell endometrial organoids and ovarian follicle organoids, a construction method thereof and an application thereof. SUMMARY

[0007] Therefore, it is an urgent problem for those skilled in the art to provide a co-culture model based on 3D endometrial stromal cell endometrial organoids and ovarian follicle organoids, a construction method thereof and an application thereof.

[0008] The system can: (1) significantly dynamically simulate the decidualization process of endometrial stromal cells, make up for the shortcomings of traditional two-dimensional models; (2) provide a new model for studying the molecular mechanism of endometrial decidualization; (3) provide a high-throughput and repeatable experimental system for drug screening, detection of the effect of environmental pollutants / toxins on endometrial function.

[0009] In order to achieve the above object, the technical scheme adopted by the present application is as follows: 1) 3D follicle organoid culture method and system (1) Follicle isolation

[0010] Under sterile conditions, the mouse ovary is peeled off, and the ovary is placed in a dissection dish containing L15 medium.

[0011] The complete follicle is separated by mechanical method (fine needle picking) or enzyme digestion method (Liberase 25 μg / mL + DNAse I 200 μg / mL).

[0012] The separated follicle is rinsed with PBS and ready for use. (2) Sodium alginate capsule embedding

[0013] Prepare 0.5% (w / v) sodium alginate solution (dissolved in sterile PBS, stirred overnight, and filtered to remove bacteria).

[0014] The follicle is suspended in the sodium alginate solution, and cross-linked with CaCl2 cross-linking solution (50 mM CaCl2 + 140 mM NaCl) for 2 min to form capsules with a diameter of about 300-400 μm. The capsules are transferred to a low adsorption culture plate for culture. (3) Culture system

[0015] Culture temperature: 37℃; gas environment: 5% CO2, 95% humidity. Replace half of the culture medium every 48 hours. (4) Follicle culture medium composition formula

[0016] Basic medium: alpha-MEM (containing GlutaMAX) or DMEM / F12; provides nutritional ingredients.

[0017] Fetal bovine serum (FBS): 10%; provides growth factors.

[0018] Insulin-transferrin-selenium supplement (ITS): 1x; promotes cell metabolism and antioxidant.

[0019] BSA: 3 mg / mL; stabilizes the culture environment.

[0020] Fetuin: 10 mg / mL; prevent zona pellucida hardening.

[0021] FSH: 5-100 mIU / mL; promote follicle development.

[0022] E2: 10 nM (optimize follicle growth environment).

[0023] Antibiotic: Penicillin-Streptomycin (100 IU / mL); inhibit bacteria.

[0024] When follicles are cultured to the 8th day, add hCG (1.5-10 IU / mL) to induce ovulation and luteinization (also can add hCG from the 7th day to the 9th day, which does not affect subsequent ovulation and luteinization). (5) Ovulation and luteinization simulation

[0025] When follicles grow to the antral stage, remove the sodium alginate capsule or digest it with Alginate Lyase.

[0026] Transfer to mature medium (Mature Medium): α-MEM + 10% FBS + FSH (10 mIU / mL) + EGF (10 ng / mL) + hCG (1.5-10 IU / mL).

[0027] Culture for 12-16 h can induce ovulation, and continue to culture for 48 h can induce luteinization and secretion of progesterone. (6) Culture device

[0028] Low-adsorption 96-well or 48-well culture plates, with a single or small number of follicle capsules in each well; combined with a CO2 incubator and an inverted microscope, used to maintain culture and observe follicle development.

[0029] Alternative method for follicle three-dimensional culture: Without departing from the core idea of the present application, the three-dimensional culture method of follicles is not limited to sodium alginate embedding, and the following methods or combinations thereof can also be used: such as hanging-drop culture.

[0030] Principle: Place the isolated follicle in a hanging drop (20-40 μL / drop) inverted on the cover of the culture plate, and maintain the three-dimensional structure of the follicle by surface tension.

[0031] Features: No need for external support, follicles can maintain complete spherical shape; easy to observe the growth and development of early follicles.

[0032] Application: commonly used for small volume culture and development mechanism research. 2) Acquisition and culture method of primary human endometrial stromal cells (1) Tissue source

[0033] Endometrial tissues were obtained from clinical curettage, hysterectomy or abortion specimens with ethical approval and informed consent.

[0034] After collection, tissues were placed in cold PBS containing antibiotics and transported to the laboratory at 4℃. (2) Cell isolation

[0035] Under sterile conditions, tissues were cut into approximately 1mm 3 pieces.

[0036] The pieces were digested with a digestion solution (Collagenase I 1mg / mL + DNase I 0.1mg / mL) for 30-60min at 37℃.

[0037] The digestion solution was filtered through a 70μm cell strainer and the filtrate was mainly endometrial stromal cells.

[0038] The filtrate was collected, centrifuged and resuspended in DMEM / F12 complete medium to obtain primary human endometrial stromal cells (HESCs). (3) Cell preservation and recovery

[0039] The primary cells obtained can be used directly for three-dimensional spheroid culture. They can also be preserved by cryopreservation (cryopreservation solution is 90% FBS + 10% DMSO) and stored in liquid nitrogen for long-term storage. When used, the cryopreserved cells are recovered and can also be used for spheroid culture after subculture and expansion. (4) Three-dimensional spheroid culture

[0040] Prepare 2% (w / v) agarose micro-molds (96-well, 48-well or 12-well 3D printed molds).

[0041] 3D printed molds are MicroTissues ® 3D Petri Dish ® micro-mold spheroids purchased from MicroTissues.

[0042] Inoculate the cell suspension (2-5×10 3 cells / well) into the micro-wells. After 24-48h of culture with spheroid culture medium, the cells naturally aggregate to form stable three-dimensional spheroids.

[0043] Spheroid culture medium composition formula: MammoCult TM medium (Stem Cell TMtechnologies): 450 ml; MammoCult TM Proliferation Supplement(Stem Cell TM Technologies): 50 ml; Heparin storage solution (Sigma): 1 mL, final concentration 4 µg / ml; Hydrocortisone storage solution (Sigma): 2.5 ml, final concentration 0.48 μg / mL; Antibiotic (Thermofisher): Penicillin and streptomycin, final concentration 100 IU / mL.

[0044] During the pelleting stage, no cAMP, E2, or P4 is added; the pellet is maintained solely with the basal culture medium MammoCult™ Human Medium (serum-free).

[0045] Incubation temperature: 37 ℃; gas environment: 5% CO2.

[0046] Endometrial organoid replacement options: Without departing from the core concept of this invention, the method for preparing three-dimensional spheres of endometrial stromal cells (HESC / ESC) is not limited to agarose micromolds, and the following alternatives can also be used: self-aggregation of spheres using ultra-low adhesion (ULA) round-bottomed well plates.

[0047] Key steps: Prepare a single-cell suspension of ESC cells (2-10 × 10⁶ cells recommended). 3 Cells (cells / well) were seeded in ULA round-bottom 96 / 384-well plates and cultured at 37 ℃ and 5% CO2 for 24-72 h to self-aggregate into spheres.

[0048] Features: Universal equipment, easy to operate, suitable for high throughput. 3) Preparation method of agarose micromold (1) Raw materials and equipment

[0049] Agarose powder (analytical grade or molecular biology grade); PBS buffer (calcium and magnesium free); sterile petri dishes or multi-well plates (such as 12-well, 96-well, or special sphere molds); prefabricated silicone rubber molds or commercially available micromold templates (such as microcolumn array molds); autoclave.

[0050] Preparation of agarose solution: Weigh out agarose at a mass-to-volume ratio of 1.5%-2% (w / v) and add it to PBS buffer. Heat (microwave or electric furnace) until the agarose is completely dissolved and the solution is clear and transparent. Cool the solution to approximately 60°C and immediately unmold. (2) Mold forming

[0051] Place pre-sterilized silicone rubber mold into a petri dish or well plate. Pour the molten agarose solution to cover the surface of the mold and fill the pores. Let it stand at room temperature for 10-20 min until the agarose is fully solidified. Carefully remove the silicone mold to form a regular microwell array in the agarose. (3) Sterilization and storage

[0052] The finished agarose micromold can be sterilized by 70% ethanol or UV irradiation for 30 min, and can be soaked in sterile PBS.

[0053] The sterilized mold can be stored in a humid environment at 4°C for 1-2 weeks, and placed in an incubator before use. (4) Method of use

[0054] Drop the cell suspension onto the surface of the agarose micromold, and the cells will settle into the microwells under the action of gravity.

[0055] Each well can accommodate about 2-5 x 10 3 cells, and after 24-48 h the cells will aggregate in the microwells and form three-dimensional spheroids.

[0056] The agarose itself is a non-adherent substrate, ensuring that the cells cannot adhere and spread, thereby maintaining the spheroid state. 4) Establishment of a co-culture system for 3D follicles and endometrial stromal cell organoids (1) Material preparation

[0057] Follicle organoids that have been cultured for 6 days and developed to the early antral stage (obtained by sodium alginate capsule culture); Endometrial stromal cell organoids that have been formed in agarose micromolds (cultured for 24-48 h, with stable structure); Co-culture base solution: DMEM / F12 (100 mL) + FBS 10% (10 mL) + ITS 1x (1 mL) + penicillin-streptomycin (100 IU / mL); Addition factors: cAMP (0.5 mM); E2 (10 nM), P4 (1 μM). (2) Co-culture method

[0058] When the follicles are cultured for 6 days, they are introduced into wells containing endometrial organoid-stromal cell spheroids, and co-culture is initiated.

[0059] The endometrial organoids are kept in the agarose mold, and the follicle organoids are directly suspended in the same culture medium.

[0060] Under co-culture conditions, the hormones secreted by the follicles can directly act on the endometrial organoids, simulating the physiological interaction between the ovary and the uterus. (3) Culture conditions

[0061] Temperature: 37℃; Gas environment: 5% CO2.

[0062] Culture cycle: every 48h, half of the culture solution was replaced from day 6 to day 8.

[0063] Innovative explanation: The follicle organoids cultured for 6 days can be co-cultured with endometrial organoids to ensure that the changes in hormone secretion are synchronized with the endometrial response; The EPC group establishes a hormone control by adding E2 (day 6) and P4 (day 8) exogenously; The co-culture group relies only on the dynamic secretion pattern of the follicle itself, which is closer to the physiological state in vivo; Compared with traditional two-dimensional hormone induction, this system can significantly enhance the decidualization process of endometrial stromal cells and has potential applications in environmental toxicant and drug detection. 4) Detection and verification methods (1) Molecular level detection

[0064] Real-time quantitative PCR (qPCR): Total RNA is extracted from endometrial stromal cell organoids, reverse transcribed into cDNA, and the expression level of decidualization-related genes is detected.

[0065] Representative markers: IGFBP1 、 PRL 、 LEFTY1 、 FOXO1 . (2) Protein level detection

[0066] Immunofluorescence staining: Using antibodies against IGFBP1, fluorescent staining of endometrial stromal cell organoids is performed to observe protein expression and localization. (3) Morphological detection

[0067] H&E staining: Paraffin-embedded sections of spheroids are observed for cell morphology and spheroid structure; decidualization cells are usually large and full of round, cytoplasm-rich.

[0068] Spheroid diameter measurement: Photographs are taken using a microscope and analyzed using software such as ImageJ to evaluate spheroid size. (4) Functional / toxicological detection

[0069] Effect of environmental toxicants (such as bisphenol A) on endometrial decidualization: For example, by adding bisphenol A to the co-culture system, it is detected whether the expression of decidualization genes is inhibited, thereby verifying the sensitivity of the system to hormone signals.

[0070] The present application utilizes agarose micromold to construct a 3D human endometrial stromal cell organoid based on primary human endometrial stromal cells (HESCs) for the first time, and co-cultures with 3D follicle organoid to develop a system integrating dynamic hormone secretion of ovary and decidualization process of endometrium. The research results show that in the 3D co-culture system, the decidualization markers of endometrium (such as IGFBP1 、 PRL 、 LEFTY1 、 FOXO1 ) are significantly up-regulated compared with the E2+P4+cAMP induction group; further immunofluorescence and H&E staining verify that the endometrial stromal cells present a more typical large and round decidualization morphology under co-culture conditions; the intervention experiment of the classic environmental pollutant BPA suggests that the system can be used for detection and screening of environmental toxins and drugs.

[0071] The existing 2D endometrial model and traditional endometrial organoid system have deficiencies in simulating the interaction between ovary and uterus, while the "3D follicle organoid and endometrial organoid co-culture system" proposed by the present application can more realistically reproduce the in vivo physiological conditions, which can be used to explore the molecular mechanism of decidualization, and also has the potential to construct a high-throughput detection platform for drugs and environmental toxins.

[0072] Through the above technical solutions, compared with the prior art, the present application provides a co-culture model based on endometrial stromal cell organoids and follicle organoids, and a construction method and application thereof. Compared with the existing two-dimensional adherent culture model and single organoid culture method, the present application has the following beneficial effects: (1) Construction of primary human endometrial stromal cell organoids: the endometrial stromal cells are cultured into three-dimensional cell spheres through agarose micromold, which avoids the problems of insufficient cell polarity and intercellular interaction in traditional two-dimensional adherent culture, and is closer to the in vivo tissue structure.

[0073] (2) Introduction of follicle organoids to provide a dynamic hormone secretion environment: follicle organoids can gradually secrete estrogen and progesterone during culture, and simulate the ovulation and luteinization process under hCG stimulation, forming an endocrine environment that is more consistent with in vivo rules, overcoming the limitations of traditional models that can only induce decidualization through static single concentration of exogenous hormones added at a time.

[0074] (3) Significantly enhancing the decidualization level of endometrial stromal cells: the experimental results show that in the co-culture system, the expression of decidualization marker genes of endometrial stromal cells (such as IGFBP1 、 PRL 、 LEFTY1 、 FOXO1 ) is significantly up-regulated, and the cell morphology is closer to the in vivo decidualization characteristics.

[0075] (4) Possessing the application potential of drug and environmental toxicant detection: The system shows detectable response to drugs such as bisphenol A, which indicates that the application can be used as an evaluation platform for drug screening and the influence of environmental toxicants on endometrial function, and has high throughput and repeatability.

[0076] (5) Promoting mechanism research and clinical transformation: The application provides a new tool for in-depth analysis of the molecular mechanism of the ovary-uterus axis in regulating the decidualization process, and has broad application prospects in the research of infertility etiology, drug development and reproductive toxicology detection. BRIEF DESCRIPTION OF DRAWINGS

[0077] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0078] Figure 1 Flow chart for isolation, embedding and culture of mouse secondary follicles.

[0079] Figure 2 Microscope image for follicle development process; wherein, culture day 0 (D0): freshly isolated secondary follicles; culture day 2 (D2): developing healthy secondary follicles; culture day 6 (D6): secondary follicles gradually enlarged in sodium alginate capsules; culture day 8 (D8): formation of sinusoidal follicles and appearance of obvious sinus cavity structure; scale: 100 µm.

[0080] Figure 3 Typical picture of rupture of mouse secondary follicles after addition of hCG.

[0081] Figure 4 Flow chart for making endometrial organoids; wherein, left picture: spindle-shaped morphology of endometrial stromal cells in two-dimensional adherent state; middle picture: agarose 3D culture mold; right picture: three-dimensional spheres (spherical, tight intercellular junctions) formed by endometrial stromal cells in agarose mold.

[0082] Figure 5 Flow chart for co-culture experiment.

[0083] Figure 6 Design diagram for experimental grouping; wherein, three groups of controls are shown: control group: endometrial organoids + cAMP; EPC group: endometrial organoids + cAMP + E2 + P4; co-culture group: endometrial organoids + follicle organoids + cAMP (no exogenous E2 / P4).

[0084] Figure 7Figure 2 shows the effect of co-culture with EPC on decidualization of endometrial organoids; A: typical image showing that the co-culture group of endometrial organoids had increased volume, closely arranged cells, and a morphology closer to in vivo decidualization; scale bar: 100 pm; B: a statistical diagram of the diameter of endometrial organoids in each group.

[0085] Figure 8 Figure 3 shows the qPCR detection results.

[0086] Figure 9 Figure 4 shows the immunofluorescence staining results; DAPI (red) shows the cell nucleus, and green shows decidualization proteins; scale bar: 100 pm.

[0087] Figure 10 Figure 5 shows the H&E staining section (A) and diameter statistical diagram (B); scale bar: 100 pm.

[0088] Figure 11 Figure 6 shows the experimental process flowchart; the whole process of establishing a follicle-endometrial organoid co-culture system on D6, adding different doses of BPA (1 µM, 10 µM, 50 µM) at the same time, adding hCG on D8, and detecting on D12 is shown.

[0089] Figure 12 Figure 7 shows the grouping design schematic; blank control group: endometrial organoids + cAMP; EPC group: endometrial organoids + cAMP + E2+ P4; EPC low-dose group: endometrial organoids + cAMP + E2+ P4+ BPA (1 µM); EPC medium-dose group: endometrial organoids + cAMP + E2+ P4+ BPA (10 µM); EPC high-dose group: endometrial organoids + cAMP + E2+ P4+ BPA (50 µM); co-culture group: follicle organoids (D6) + cAMP + DMSO; co-culture low-dose group: endometrial organoids + follicle organoids (D6) + cAMP + BPA (1 µM); co-culture medium-dose group: endometrial organoids + follicle organoids (D6) + cAMP + BPA (10 µM); co-culture high-dose group: endometrial organoids + follicle organoids (D6) + cAMP + BPA (50 µM).

[0090] Figure 13 Figure 8 shows the qPCR detection results; A: IGFBP1 ; B: FOXO1 ; C: PRL ; D: LEFTY1 .

[0091] Figure 14 Figure 9 shows the H&E staining results (A) and diameter statistical diagram (B); scale bar: 100 pm. Detailed Implementation

[0092] 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.

[0093] The α-MEM (containing GlutaMAX) base solution was purchased from ThermoFisher Scientific, catalog number: A1048901.

[0094] Example 1: Preparation and culture of mouse 3D follicular organoids 1.1 Follicle separation

[0095] Female mice aged 3–4 weeks were selected, and their ovaries were isolated under sterile conditions after euthanasia.

[0096] The ovaries were placed in a culture dish containing L15 medium (supplemented with 1% penicillin and streptomycin), and secondary follicles with a diameter of approximately 100-130 μm were separated by enzymatic digestion (Liberase 25 μg / mL + DNAse I 200 μg / mL).

[0097] The separated follicles were transferred to fresh PBS and rinsed twice. 1.2 Preparation of sodium alginate capsules

[0098] To prepare a 0.5% (w / v) sodium alginate solution: Weigh 0.5g of sodium alginate and add it to 100mL of sterile PBS. Stir and dissolve at 65℃, then filter through a 0.22μm filter membrane for sterilization.

[0099] A single follicle was embedded in 5 μL of sodium alginate solution and dropped into a CaCl2 cross-linking solution containing 50 mM CaCl2 + 140 mM NaCl. After cross-linking for 2 minutes, microcapsules with a diameter of about 300 μm were formed.

[0100] Rinse the capsules three times with sterile PBS to remove residual CaCl2. 1.3 Follicle Culture

[0101] Transfer the capsules to a low-absorption 96-well plate and add 100 μL of follicle culture medium to each well.

[0102] Follicle culture medium (for in vitro three-dimensional culture and development of follicle organoids) components: α-MEM (containing GlutaMAX) base solution: 100 mL; FBS: 10 mL (10%) ITS: 1 mL (1 x); BSA: 300 mg (3 mg / mL); Fetuin: 1 g (10 mg / mL); FSH: 50 mIU / mL; E2: 10 nM; Penicillin: 100 IU / mL.

[0103] Culture condition: 37 °C, 5% CO2, 95% humidity.

[0104] Replace 50% of culture medium every 48 h until day 8. 1.4 Ovulation induction and luteinization

[0105] When follicles grow to antral stage (day 8), remove sodium alginate capsules or digest by Alginate Lyase.

[0106] Switch to mature medium (Mature Medium): a-MEM + 10% FBS + FSH (10 mIU / mL) + EGF (10 ng / mL) + hCG (1.5-10 IU / mL).

[0107] Incubate for 12-16 h to induce ovulation, continue for 48 h to induce luteinization and secretion of progesterone.

[0108] The flow chart of mouse secondary follicle isolation, embedding and culture is shown in Figure 1 . The microscope images of follicle development are shown in Figure 2 .

[0109] Figure 3 Typical image of rupture after adding hCG to mouse secondary follicle. After adding hCG on day 8, follicle ruptured to release cumulus-oocyte complex (COC); follicle residual tissue transformed into lutein-like structure and secreted progesterone.

[0110] Example 2. Construction of primary human endometrial stromal cell spheroids 2.1 Tissue acquisition and processing

[0111] Endometrial tissue was derived from clinical curettage or hysterectomy surgical specimens, with ethical approval and informed consent from patients.

[0112] Fresh tissue was immediately placed in cold PBS solution containing 1% penicillin and 1% amphotericin B, and transported to the laboratory at 4 °C. 2.2 Tissue mincing and enzyme digestion

[0113] Under sterile conditions, the tissue was cut into approximately 1 mmSmall pieces of 3

[0114] Digestion solution: Collagenase I 1 mg / mL + DNase I 0.1 mg / mL, dissolved in serum-free DMEM / F12.

[0115] Add tissue pieces into the digestion solution, and place in a 37 °C shaker at 80 rpm for 40 min.

[0116] Gently mix every 10 min. 2.3 Cell isolation

[0117] After digestion, filter the solution through a 70-μm cell strainer. The filtrate contains mainly endometrial stromal cells, while the precipitate (containing glandular epithelial pieces) is discarded.

[0118] Centrifuge the filtrate (300 g, 5 min), discard the supernatant, and resuspend the precipitate in DMEM / F12 complete medium to obtain primary human endometrial stromal cells (HESCs). 2.4 Cell cryopreservation and recovery

[0119] Resuspend the cells in cryopreservation solution (90% FBS + 10% DMSO), and store in liquid nitrogen after programmed cooling.

[0120] Recover the cryopreserved cells when needed, quickly thaw them in a 37 °C water bath, and immediately wash them with DMEM / F12 complete medium. After subculture, use the cells for experiments. 2.5 Agarose micromould preparation

[0121] Prepare a 2% (w / v) agarose solution (dissolved in PBS, heated to clarity at 95 °C, and cooled to 60 °C).

[0122] Pour the molten agarose into a sterile 48-well mould, and place the silicone micropost array template on top. Cool at room temperature for 20 min.

[0123] Remove the silicone template to form regular hemispherical microwells (about 400 μm in diameter) in the agarose.

[0124] Store the mould in sterile PBS after treatment with 70% ethanol for 30 min. 2.6 Spheroid culture

[0125] Prepare a single-cell suspension of HESCs at a density of 2-5 × 10 3 cells / 50 μL.

[0126] Add 50 μL of the cell suspension to each well, and allow the cells to settle at the bottom of the agarose microwells.

[0127] ​Incubate at 37°C, 5% CO2 incubator for 24-48h, cells will aggregate under gravity and form spheroids naturally.

[0128] Spheroid formation medium composition: TM MammoCult TM Proliferation Supplement: 50 ml; Heparin Stock: 1 mL, final concentration 4 µg / ml; Hydrocortisone Stock: 2.5 ml, final concentration 0.48 μg / mL; Penicillin-Streptomycin: 100 IU / mL.

[0129] Culture condition: Temperature: 37 ℃; Gas environment: 5% CO2, 95% humidity.

[0130] DMEM / F12 + 10% FBS is the basal medium for spheroid formation and co-culture, any chemical or environmental toxicant or drug testing, add corresponding culture additives.

[0131] Figure 4 Flow chart for endometrial organoid preparation.

[0132] Example 3 Co-culture of 3D follicle and endometrial organoids 3.1 Materials and base solution

[0133] Follicle organoids: prepared according to Example 1, cultured to day 6 (D6).

[0134] Endometrial organoids: spheroid formation in agarose micromolds according to Example 2, stable for 24-48h before use.

[0135] Co-culture base solution: DMEM / F12 (100 mL) + FBS 10% (10 mL) + ITS 1x (1 mL) + Penicillin-Streptomycin (100 IU / mL). 3.2 Establish co-culture (D6→D8, first stage)

[0136] D6: Transfer D6 follicles into agarose micromold wells containing endometrial organoids (1 endometrial organoid + 4 follicles per well).

[0137] Start on the same day and divide into three groups with different reagents: Control group: only add cAMP (0.5 mM), no follicles.

[0138] EPC group: add cAMP (0.5 mM), no follicles.

[0139] Co-culture group: cAMP (0.5 mM) + follicle, without E2 / P4.

[0140] Incubation condition: 37℃, 5% CO2, 48h.

[0141] hCG induction and prolonged co-culture (D8→D14, second phase) D8: hCG (5 IU / mL) was added to the follicle-containing groups to induce follicle rupture and luteinization.

[0142] EPC group: cAMP (0.5 mM) + P4 (1 μM) and E2 (10 nM) were added on D8.

[0143] Co-culture group: without exogenous P4 / E2, cAMP (0.5 mM) and hCG (5 IU / mL) were added on D8, and the endogenous estrogen and progesterone environment was provided by the follicle / luteinization residual tissue. hCG (5 IU / mL): maintain luteinization.

[0144] Control group: only cAMP (0.5 mM) was added from D8, without follicle and E2 / P4.

[0145] The culture was maintained until D12 (4 days after D8), and the medium was replaced every 48h, and the components of each group were maintained.

[0146] Figure 5 Flow chart of co-culture experiment; shows the whole process of follicle culture in sodium alginate capsules to D6→ transfer to agarose micro-mold holes containing endometrial organoids→ co-culture from D6 to D8→ add hCG on D8→ continue to culture to D12.

[0147] Figure 6 Design of experimental grouping. 3.3 Sampling and subsequent detection time points

[0148] End of co-culture, take each group of endometrial organoids for subsequent detection (qPCR, immunofluorescence, H&E). Key operation points

[0149] Follicles enter the co-culture system from D6, and can experience the hCG induction window of D8 synchronously with endometrial organoids.

[0150] The co-culture group relies on the endogenous P4 of follicle / luteinization tissue to form a physiologically relevant progesterone background after D8.

[0151] The half-volume replacement strategy (every 48h) not only maintains nutritional stability, but also retains the dynamic accumulation of paracrine factors and hormones.

[0152] Example 4 Decidualization verification of endometrial stromal cell spheroids (D12 detection) 4.1 Sample collection

[0153] Endometrial stromal cell spheroids of the control group, EPC group and co-culture group were collected respectively after the culture period of Example 3 (D12).

[0154] The spheroids were taken out from the agarose molds, gently rinsed with sterile PBS for 2 times, and prepared for use.

[0155] Figure 7 Schematic diagram of decidualization effect comparison between co-culture and EPC group. 4.2 Real-time quantitative PCR (qPCR) detection

[0156] RNA extraction: PicoPure™ RNA Isolation Kit was used to extract total RNA according to the instructions.

[0157] Reverse transcription: 1 μg of RNA was taken and reverse transcription was performed using a cDNA synthesis kit (Thermofisher Scientific).

[0158] qPCR detection: SYBR Green Master Mix was used for detection on ABI7500 instrument.

[0159] Primer sequence (example): IGFBP1 -F: 5'-tctgatggccccttctgaag-3'; SEQ ID NO. 1.

[0160] IGFBP1 -R: 5'-tgtctcacactgtctgctgt-3'; SEQ ID NO. 2.

[0161] PRL -F: 5'-caacagctgccacacttctt-3'; SEQ ID NO. 3.

[0162] PRL -R: 5'-ccgtttggtttgctcctcaa-3'; SEQ ID NO. 4.

[0163] LEFTY1 -F: 5'-ctgaagcaccaatgaccgag-3'; SEQ ID NO. 5.

[0164] LEFTY1- R: 5'-cagaaacggccacttgaagg-3'; SEQ ID NO. 6.

[0165] FOXO1 - F: 5'-ccgagctgccaagaagaaag-3'; SEQ ID NO. 7.

[0166] FOXO1 - R: 5'-atgcacatccccttctccaa-3'; SEQ ID NO. 8.

[0167] Internal reference gene GAPDH - F: 5'-aggtcggagtcaacggattt-3'; SEQ ID NO. 9.

[0168] GAPDH - R: 5'-gacggtgccatggaatttg-3'; SEQ ID NO. 10.

[0169] Result determination: the relative expression was calculated by the method of ΔΔCt, and the control group was used as the control to compare the differences among the three groups.

[0170] Figure 8 The bar chart of qPCR detection results shows IGFBP1 , FOXO1 , PRL , LEFTY1 The relative mRNA expression levels in the three groups (control group, EPC group, and co-culture group). It is shown that the co-culture group is significantly higher than the EPC group and the control group. 4.3 Immunofluorescence staining

[0171] Fixation: the spheroids were fixed with 4% paraformaldehyde for 15 min and washed with PBS for 3 times.

[0172] Permeabilization: 0.2% Triton X-100 treatment for 15 min.

[0173] Blocking: 5% BSA blocking for 1 h.

[0174] Primary antibody incubation: anti-IGFBP1 antibody (company: abcam; item number: ab228741; 1:500 dilution); 4°C overnight.

[0175] Secondary antibody incubation: Alexa Fluor 488 / 594 labeled secondary antibody (1:1000 dilution), room temperature incubation for 1 h.

[0176] Nuclear staining: DAPI (1 μg / mL, 5 min).

[0177] Imaging: Protein localization and expression level were observed under laser confocal microscope (Leica SP8, Germany).

[0178] Figure 9 Figure 4.4 Immunofluorescence staining results. Immunofluorescence comparison of three groups of endometrial stromal cells: control group: weak IGFBP1 signal; EPC group: enhanced signal; co-culture group: strongest signal and uniform distribution. DAPI (red) in the figure shows the nucleus, and green shows decidualization proteins. Scale bar: 100 pm. 4.4 H&E Staining

[0179] The spheroids were embedded in low-melting-point agarose to prepare paraffin sections (5 pm thick).

[0180] The sections were deparaffinized with xylene, rehydrated with gradient alcohol, stained with hematoxylin-eosin, and mounted after dehydration.

[0181] Cell morphology was observed under a light microscope: decidualized endometrial stromal cells were round, with abundant cytoplasm and tightly arranged nuclei.

[0182] Figure 10 Figure 4.4 H&E staining section and diameter statistics. Control group: endometrial stromal cells are spindle-shaped and arranged loosely; EPC group: some cells are round and have slightly abundant cytoplasm; co-culture group: cells are typically decidualized, with round cell bodies, abundant cytoplasm, and dense nuclei. Scale bar: 100 pm.

[0183] Example 5 Detection of the effect of environmental toxins on the decidualization of endometrial stromal cells 5.1 Experimental materials

[0184] Follicle organoids: prepared according to Example 1 and cultured for 6 days.

[0185] Endometrial stromal cell spheroids: prepared according to Example 2 and stabilized into spheroids.

[0186] Co-culture system: according to the method of Example 3, follicles were co-cultured with endometrial organoids on the 6th day.

[0187] Test substance: bisphenol A (BPA, Sigma-Aldrich, purity > 99%).

[0188] Solvent control: 0.1% DMSO. 5.2 Treatment groups

[0189] On the basis of EPC and co-culture systems, the following groups were divided (n > 3 wells per group): Control group: cAMP (0.5 mM) + DMSO (0.1%), without BPA.

[0190] EPC group: Endometrial organoids + cAMP + E2 + P4; EPC low-dose group: endometrial organoids + cAMP + E2 + P4 + BPA (1 μM); EPC medium-dose group: endometrial organoids + cAMP + E2 + P4 + BPA (10 μM). High-dose EPC group: Endometrial organoids + cAMP + E2 + P4 + BPA (50 μM); Co-culture group: Follicular organoids (D6) + cAMP + DMSO; Co-culture low-dose group: endometrial organoids + follicular organoids (D6) + cAMP + BPA (1 μM); Co-culture medium-dose group: endometrial organoids + follicular organoids (D6) + cAMP + BPA (10 μM); High-dose co-culture group: endometrial organoids + follicular organoids (D6) + cAMP + BPA (50 μM).

[0191] All groups started receiving medication on day 8 and continued until day 12; half of the culture medium was replaced every 48 hours, and the corresponding dose of BPA was added. 5.3 hCG induction and luteinization

[0192] On day 8, hCG (5 IU / mL) was added to all groups containing follicles to induce ovulation and luteinization of residual tissue.

[0193] BPA treatment was continued until day 12 following hCG stimulation.

[0194] Figure 11 This is a flowchart of the experimental process; it illustrates the entire process from establishing the follicle-endometrial organoid co-culture system on day 6 → simultaneously adding different doses of BPA (1µM, 10µM, 50µM) to different groups on day 8 → adding hCG on day 12 → detection on day 12.

[0195] Figure 12 A schematic diagram for group design. 5.4 Detection indicators (D12 sampling)

[0196] qPCR detection: Endometrial organoids were collected, RNA was extracted, and decidualization-related genes were detected. IGFBP1 , PRL , LEFTY1 , FOXO1 The expression of ).

[0197] Figure 13Figure 5. qPCR detection results. The relative expression levels of decidualization related genes in different BPA treatment groups were shown. There was a clear trend: with the increase of BPA concentration, the expression of genes gradually decreased. But in EPC treatment group, after adding BPA, the decidualization related genes had a significant decrease, but there was no obvious concentration gradient change. IGFBP1 、 PRL 、 LEFTY1 、 FOXO1 There was a clear trend: with the increase of BPA concentration, the expression of genes gradually decreased. But in EPC treatment group, after adding BPA, the decidualization related genes had a significant decrease, but there was no obvious concentration gradient change. IGFBP1 、 PRL 、 LEFTY1 、 FOXO1 There was a clear trend: with the increase of BPA concentration, the expression of genes gradually decreased. But in EPC treatment group, after adding BPA, the decidualization related genes had a significant decrease, but there was no obvious concentration gradient change.

[0198] H&E staining: paraffin section was used to observe the morphological changes of spheroids, and to evaluate whether the cell arrangement was loose and whether the decidualization characteristics were weakened.

[0199] Figure 14 H&E staining results and diameter statistics. Control group: endometrial stromal cells were round, with abundant cytoplasm, and typical decidualization morphology; BPA treatment group: cell arrangement was loose, cytoplasm was reduced, and under high dose, it was closer to fibrous morphology. Scale bar: 100 μm. 5.5 Results determination

[0200] Because the BPA treatment group showed a decrease in the expression of decidualization marker genes and a trend of fibrous cell morphology compared to the control group, it was determined that BPA had an inhibitory effect on the decidualization of endometrial stromal cells.

[0201] The above results prove that the follicle-endometrial organoid co-culture system of the present application can be used for functional screening of environmental toxins and reproductive toxicology research, and can significantly produce a concentration gradient response, which is superior to the traditional EPC treatment group response to environmental toxins.

[0202] The above description of disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a co-culture model based on endometrial stromal cell organoids and follicular organoids, characterized in that, Includes the following steps: 1) Culture methods for follicular organoids (1) Use mechanical or enzymatic digestion to separate intact follicles. After separation, the follicles are rinsed with PBS and then used for later use. (2) The follicles were suspended in a 0.5% sodium alginate solution and cross-linked with CaCl2 cross-linking solution to form capsules with a diameter of 300-400 μm; (3) Transfer the capsules into follicle culture medium for culture; culture temperature: 37℃; gas environment: 5% CO2, 95% humidity; replace half of the culture medium every 48 hours; (4) Cultured until day 6 to obtain follicle organoids; 2) Culture methods for endometrial stromal cell organoids (1) Under sterile conditions, the endometrial tissue was cut into pieces and digested with digestive solution at 37 °C for 30-60 min; the digestive solution was filtered through a 70 μm cell sieve, the filtrate was collected, centrifuged and resuspended in DMEM / F12 complete culture medium to obtain primary human endometrial stromal cells. (2) Primary human endometrial stromal cell suspension was inoculated into the micropores of a 2% agarose micromold and cultured in spheroidizing medium for 24-48 hours to form stable three-dimensional spheres; 3) Co-culture of follicular organoids and endometrial stromal cell organoids (1) Transfer the follicle organoids obtained in step 1) into the agarose micromold wells containing endometrial stromal cell organoids obtained in step 2), and culture them in a co-culture basal medium containing 0.5 mM cAMP for 48 h, and then culture them in a co-culture basal medium containing 0.5 mM cAMP and 5 IU / mL hCG for 4 days. During this period, half of the culture medium is replaced every 48 h.

2. The construction method according to claim 1, characterized in that, The composition of the follicle culture medium is as follows: α-MEM or DMEM / F12 containing GlutaMAX as the basal medium, 10% fetal bovine serum, 1× insulin-transferrin-selenium complex, 3 mg / mL BSA, 10 mg / mL Fetuin, 5-100 mIU / mL FSH, 10 nME2, and 100 IU / mL penicillin-streptomycin.

3. The construction method according to claim 1, characterized in that, Replace the follicular organoid culture method described in step 1) with the hanging drop method.

4. The construction method according to claim 1, characterized in that, The pelleting culture medium has the following composition: MammoCult TM medium: 450 ml; MammoCult TM Proliferation Supplement: 50 ml; Heparin storage solution: 1 mL, final concentration 4 µg / ml; Hydrocortisone storage solution: 2.5 ml, final concentration 0.48 μg / mL; Penicillin and Streptomycin: 100 IU / mL.

5. The construction method according to claim 1, characterized in that, Replace the endometrial stromal cell organoid culture method described in step 2) with the self-aggregation of ultra-low adhesion round-bottomed plates into spheres.

6. The construction method according to claim 1, characterized in that, The co-culture basal medium composition is as follows: DMEM / F12 as the basal culture medium, 10% FBS, 1×ITS, and 100 IU / mL penicillin and streptomycin.

7. The co-culture model of endometrial stromal cell organoids and follicle organoids prepared by the method according to any one of claims 1-6.

8. The application of the co-culture model of endometrial stromal cell organoids and follicular organoids as described in claim 7 in environmental toxicant screening.

9. The application according to claim 8, characterized in that, The environmental toxin is bisphenol A.

10. The application of the co-culture model of endometrial stromal cell organoids and follicular organoids as described in claim 7 in simulating endometrial decidualization.

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