An organoid model of pelvic fibrosis based on organoid chip, its fabrication method and application

Abstract

This invention discloses an organoid model of pelvic inflammatory disease (PID) fibrosis based on organoid microarrays, its fabrication method, and its applications. The fabrication method includes: isolating endometrial epithelial cells and fibroblasts from pathological endometrial tissue; separately culturing and expanding endometrial organoids and fibroblasts; mixing the cells obtained from organoid digestion with fibroblasts at a cell ratio of 5:1 to 10:1, seeding the mixture onto an organoid microarray, and co-culturing it under dynamic perfusion of 5–20 μL / min in a culture medium containing multiple factors such as nicotinamide, RSPO1, Wnt-3a, Noggin, bFGF, EGF, and HRG-β1. The resulting model has a complete structure and uniform size (CV < 15%), and Vimentin staining confirms the presence of a large number of fibroblasts. Utilizing the multi-parallel units of the microarray, different traditional Chinese medicine components can be processed in parallel, fibrosis indicators can be detected, and high-throughput anti-fibrotic efficacy evaluation can be achieved. This invention provides a new platform for research and drug screening in PID fibrosis.

Description

Technical Field

[0001] This invention relates to the field of organoid model technology, and in particular to an organoid model of pelvic fibrosis based on organoid chip, its fabrication method and application. Background Technology

[0002] Pelvic inflammatory disease (PID) is an infectious inflammation of the female upper reproductive tract, including endometritis, salpingitis, tubo-ovarian abscess, and pelvic peritonitis. It has a high global prevalence. If left untreated, severe PID can develop into chronic PID, leading to serious complications such as fallopian tube obstruction, pelvic adhesions, and tissue fibrosis. This can result in infertility and ectopic pregnancy, and is associated with an increased risk of gynecological cancers.

[0003] Traditional Chinese medicine has a long history of clinical application and proven efficacy in the treatment of pelvic inflammatory disease and its sequelae of fibrosis. Danshen (Salvia miltiorrhiza) is a representative Chinese herb for promoting blood circulation and removing blood stasis; the Pharmacopoeia of the People's Republic of China records its effects of "promoting blood circulation, removing blood stasis, regulating menstruation, and relieving pain." Sun Ningyuan et al., in their research paper "Research Progress on the Anti-fibrotic Pharmacological Effects and Mechanisms of Danshen Chemical Components," systematically reviewed the anti-fibrotic effects and molecular mechanisms of the main active components of Danshen, such as tanshinone IIA and salvianolic acid B, in multi-organ fibrosis models involving the liver, lungs, kidneys, and heart. They confirmed that multiple components of Danshen can exert anti-fibrotic activity by inhibiting signaling pathways such as TGF-β / Smad and NF-κB.

[0004] A novel organoid model of endometrial inflammation, published in CN 115125187 A, along with its establishment method and applications, was constructed by adding components that induce inflammation in organoids to a culture system. However, serious complications of pelvic inflammatory disease (PID) (such as infertility and fallopian tube obstruction) are often closely related to tissue fibrosis. Existing inflammatory models struggle to simulate the pathological process of fibrosis and cannot meet the requirements for model uniformity and high throughput in anti-fibrotic drug screening. Currently, no fibrotic organoid models for PID have been reported. Summary of the Invention

[0005] In view of the above situation and to overcome the defects of the prior art, the present invention provides an organoid model of pelvic fibrosis based on organoid chip, its manufacturing method and application, and specifically provides the following technical solutions.

[0006] In a first aspect, the present invention provides a method for fabricating a pelvic fibrosis organoid model based on organoid microarrays, the method comprising the following steps:

[0007] S1 isolates endometrial epithelial cells and fibroblasts from pathological endometrial tissue;

[0008] S2 cultured and expanded the endometrial epithelial cells and the fibroblasts respectively to obtain endometrial organoids and expanded fibroblasts;

[0009] S3 digests and disperses the endometrial organoids and the expanded fibroblasts respectively to obtain cell suspensions containing endometrial organoids and expanded fibroblasts respectively.

[0010] S4. The cell suspension is mixed to obtain a mixed cell suspension. The mixed cell suspension is seeded onto an organoid chip equipped with multiple parallel culture units for co-culture to form a pelvic fibrosis organoid model.

[0011] In S4, the mixing ratio of the number of endometrial organoid cells and the expanded fibroblasts in the mixed cell suspension is 5:1 to 10:1.

[0012] The culture medium used for the co-culture was based on DMEM / F12 and contained the following components at final concentrations:

[0013] 5–20 mM nicotinamide, 2.5–10 ng / mL bFGF, 0.1–5 mM N-acetylcysteine, 1× B27 additive, 1× N2 additive, 20–100 ng / mL EGF, 0.1–2 μM PGE2, 50–200 ng / mL RSPO1, 50–200 ng / mL Wnt-3a, 50–200 ng / mL Noggin, 10–50 nM 17β-estradiol, 5–20 ng / mL FGF10, 0.1–0.5 μM SB202190, 0.1–1 μM A83-01, 10–50 ng / mL HRG-β1, 1–5 μM Forskolin.

[0014] In a further embodiment, the co-culture in step S4 is carried out under dynamic fluid perfusion conditions at a perfusion rate of 5–20 μL / min.

[0015] In a further embodiment, step S1 includes:

[0016] The pathological endometrial tissue was washed with HBSS, cut into small pieces to form tissue fragments, and Dispase II and type IV collagenase tissue hydrolysate was added until the tissue fragments were completely covered.

[0017] The enzymatic hydrolysate containing the tissue fragments was evenly distributed into a low-adsorption plate and incubated at 37±0.5℃ for 40–60 min. The solution was then ground through a 70 μm sieve to obtain a tissue suspension through enzymatic hydrolysis.

[0018] Add high-glucose culture medium containing 5% FBS to the tissue suspension to terminate the enzymatic reaction, centrifuge for 3-5 minutes, and collect the endometrial cell precipitate.

[0019] In a further embodiment, the method for producing the endometrial organoid in step S2 includes the following steps:

[0020] The endometrial cell pellet was resuspended in endometrial organoid culture medium, and matrix gel was added to a final concentration of 5% for 3D culture. The culture was incubated at 37±0.5℃ for 25-30 min. After the matrix gel solidified, endometrial organoid culture medium was added, and the culture was carried out for 10-20 days to obtain the endometrial organoid.

[0021] In a further embodiment, the method for preparing the amplified fibroblasts in step S2 includes the following steps:

[0022] Take the enzymatic hydrolysate containing the tissue fragments from step S1 after enzymatic incubation and before sieving, centrifuge to remove the hydrolysate, resuspend the cells in DMEM / F12 fibroblast culture medium containing 5%–10% FBS, seed them in cell culture plates, and culture them in a two-dimensional adherent culture at 37°C and 5% CO2 for 3–5 days. When the cells grow to 80%–95% confluence, perform passage culture to obtain the expanded fibroblasts.

[0023] In a further embodiment, the co-cultivation in step S4 includes the following steps:

[0024] The number of cells in the two cell suspensions obtained in step S3 were counted respectively.

[0025] Based on the counting results, the endometrial organoid cells and the expanded fibroblasts were mixed at a cell ratio of 5:1 to 10:1, and matrix gel was added to a final concentration of 2%. After mixing, the mixture was evenly seeded onto the organoid chip and cultured for 3 to 7 days to form a pelvic inflammatory disease fibrosis organoid model.

[0026] In a further embodiment, the culture medium used for the co-culture is based on DMEM / F12 and contains the following components at final concentrations:

[0027] 15 mM nicotinamide, 5 ng / mL bFGF, 1.25 mM N-acetylcysteine, 1× B27 additive, 1× N2 additive, 50 ng / mL EGF, 0.5 μM PGE2, 100 ng / mL RSPO1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 10 nM 17β-estradiol, 10 ng / mL FGF10, 0.5 μM SB202190, 0.5 μM A83-01, 25 ng / mL HRG-β1, 5 μM Forskolin.

[0028] In a further embodiment, the mixing ratio of the endometrial organoid cells and the expanded fibroblasts in the mixed cell suspension in S4 is 5:1.

[0029] A second aspect of the present invention provides a pelvic fibrosis organoid model obtained according to the aforementioned method.

[0030] A third aspect of the present invention provides an application of the aforementioned pelvic inflammatory disease fibrosis organoid model in the efficacy evaluation of traditional Chinese medicine for anti-fibrosis.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] This invention employs a specific mixing ratio of endometrial organoid cells and expanded fibroblasts of 5:1 to 10:1, an optimized co-culture medium containing A83-01, HRG-β1, Forskolin, etc., and microfluidic dynamic perfusion at a rate of 5–20 μL / min. The synergistic effect of these three factors reduces the coefficient of variation (CV) of pelvic fibrosis organoids from 25.7% in Control Example 12 to 12.0% in Example 1. The controlled ratio ensures the reproducibility of cell composition, the optimized medium maintains the proliferation balance between the two cell types, and the dynamic perfusion provides a suitable shear stress microenvironment; all three are indispensable. This invention solves the technical problems of incomplete organoid structure, uneven size, and unsatisfactory fibroblast distribution in existing technologies, providing a more reliable and efficient technical platform for evaluating the anti-fibrotic effects of traditional Chinese medicine. Attached Figure Description

[0033] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0034] Figure 1 This is a light micrograph of the fibrotic organoids in Example 1, caused by pelvic inflammatory disease.

[0035] Figure 2This is an immunofluorescence staining image of α-SMA organoids in pelvic fibrosis, as described in Example 1.

[0036] Figure 3 This is a representative image of dual immunofluorescence of α-SMA and CK7 in organoids of pelvic fibrosis in Example 1. Detailed Implementation

[0037] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0038] The above technical solution will be described in detail below with reference to specific embodiments.

[0039] Terminology Explanation:

[0040] Final concentration: refers to the final concentration that a solution reaches after reaction or dilution.

[0041] ×: This refers to the dilution factor relative to the supplier's recommended working concentration. For example, 1× means dilution to the supplier's recommended final concentration.

[0042] Endometrial organoid cells: In this invention, "endometrial organoid cells" and "endometrial epithelial cells" refer to the same cell population.

[0043] Specifically:

[0044] In the context of steps S1 and S2, since the cells have not yet been cultured in 3D to form organoid structures, the term "endometrial epithelial cells" is used to refer to the epithelial cells and their amplification products isolated from pathological endometrial tissue.

[0045] In the context of steps S3 and S4, the aforementioned endometrial epithelial cells have been cultured in 3D to form endometrial organoids, and then digested to lose their organoid structure and become a single-cell suspension. At this time, the term "endometrial organoid cell suspension" or "endometrial organoid cells" is used to reflect that they are derived from the formed organoids and are closer to the in vivo state in terms of function and phenotype.

[0046] Both are essentially epithelial cells derived from the endometrium, differing only in their manufacturing process. In the claims of this invention, "endometrial organoid cell suspension," "endometrial epithelial cells," "endometrial organoid cells," and "digested organoid cells" all refer to the same population of endometrial epithelial cells.

[0047] Unless otherwise specified, the experimental methods used in this application are all conventional methods. Unless otherwise specified, all materials and reagents used in this application are commercially available. The experimental materials involved in this application and their sources are as follows:

[0048] Organoid chip: Black Jade Star Rock Science and Technology Co., Ltd., DSO Nebula B, (design disclosed in patent number 202430655931X);

[0049] Pathological endometrial tissue: This procedure was conducted in collaboration with a top-tier hospital and underwent rigorous medical ethics review. Based on the intraoperative findings, the attending physician immediately harvested a sufficient quantity of viable pathological tissue (approximately 1 cm) after the surgically removed uterine specimen. 3 (Size), placed in sterile preservation solution, and rapidly transported to the laboratory at low temperature for further processing.

[0050] Nicotinamide: MCE, catalog number HY-B0150;

[0051] bFGF (basic fibroblast growth factor): Thermo Fisher Scientific, catalog number 100-18B;

[0052] N-acetylcysteine: Sigma, catalog number A0737;

[0053] B27 Additive: Invitrogen, 50× stock solution, diluted to a final working concentration of 1×;

[0054] N2 additive: Thermo Fisher Scientific, 200× stock solution, diluted to a final working concentration of 1×;

[0055] EGF (Epidermal Growth Factor): Thermo Fisher Scientific, catalog number E3477;

[0056] PGE2 (prostaglandin E2): Peprotech, catalog number 3632464;

[0057] RSPO1 (R-spondin-1 protein): Peprotech, catalog number SRP3292-20UG;

[0058] Wnt-3a (recombinant human Wnt-3a protein): Merck, catalog number GF154;

[0059] Noggin (recombinant Noggin protein): Merck, catalog number GF173;

[0060] 17β-Estradiol: Merck, catalog number E1024;

[0061] FGF10 (fibroblast growth factor 10): Merck, catalog number ABN44;

[0062] SB202190: MCE, Part No. HY-10295;

[0063] A83-01 (Receptor Inhibitor): Merck, catalog number A8301;

[0064] HRG-β1 (NRG1-beta 1 protein): STEMCELL Technologies, product Heregulin-β1 (hereinafter referred to as HRG-β1), catalog number 78071;

[0065] Forskolin: STEMCELL Technologies, catalog number 72114;

[0066] Hanks' Balanced Salt Solution (HBSS): Thermo Fisher Scientific, catalog number 13150016;

[0067] Type IV collagenase tissue hydrolysate: Sigma-Aldrich, catalog number C5138;

[0068] High glucose culture medium: Servicebio, catalog number G4203-500mL;

[0069] Y27632: MCE, part number HY-10071;

[0070] IGF-1: MCE, product number HY-P7018;

[0071] GlutaMax: Thermo Fisher Scientific, product number 35050061;

[0072] Endometrial organoid culture medium: DMEM / F12 as the basal medium, supplemented with the following final concentrations: 1×GlutaMax, 100 ng / mL R-Spondin-1; 1×B27 additive, 1×N2 additive, 100 ng / mL Noggin, 2 mM Nicotinamide, 10 μM Y27632, 0.5 μM A83-01, 50 ng / mL EGF, 25 ng / mL bFGF, 20 ng / mL LIFG-1, 0.1 μM SB202190, 10 nM 17β-estradiol.

[0073] Matrix adhesive: Corning, part number 356231;

[0074] Cell culture plate: Nest, catalog number 702001;

[0075] Fetal bovine serum (FBS): Pronosai, catalog number 164210;

[0076] Phosphate-buffered saline (PBS): Servicebio, catalog number G4250-500ML;

[0077] DMEM / F12 basal culture medium (DMEM / F12): source culture, catalog number L330KJ;

[0078] Tryple digestive solution: Thermo-Fisher, catalog number 12604021;

[0079] Paraformaldehyde fixative: Merck, product number 158127;

[0080] Polyethylene glycol octylphenyl ether (Triton X-100): Merck, product number 2315025;

[0081] Alexa Fluor 488-tagged goat anti-mouse IgG: Beyotime Biotechnology, catalog number A0428;

[0082] Goat serum: Beyotime Biotechnology, catalog number C0265;

[0083] Mouse anti-human Vimentin monoclonal antibody: Beyotime Biotechnology, catalog number AF0318;

[0084] 4′,6-Diamidinyl-2-phenylindole dihydrochloride (DAPI): Merck, catalog number D9542;

[0085] Anti-fluorescence quenching mounting medium: SouthernBiotech, catalog number 0100-01;

[0086] TGF-β receptor type I inhibitor (SB431542): Yisheng Biotechnology, catalog number 53002ES03;

[0087] Dimethyl sulfoxide (DMSO): Thermo Fisher Scientific, product number BP231;

[0088] Salvia miltiorrhiza extract: Shanghai Yuanye Biotechnology Co., Ltd., product number S27206;

[0089] Guizhi Fuling Wan: a traditional Chinese medicine formula developed by Tsumura.

[0090] Red vine extract: Huaguang Biotechnology;

[0091] Astragaloside IV: Merck, product number Y0001171;

[0092] Calcein-AM: MedChemExpress, catalog number HY-D0041;

[0093] Propidium iodide (PI): Merck, product number P4170;

[0094] Mouse anti-human α-SMA monoclonal antibody: Shanghai Jiyi Biotechnology Co., Ltd., catalog number ABM0052;

[0095] Rabbit anti-human Collagen I polyclonal antibody: novus, catalog number NBP1-30054;

[0096] Alexa Fluor 488-tagged goat anti-rabbit IgG: Beyotime Biotechnology, catalog number A0423;

[0097] Alexa Fluor 594-tagged goat anti-mouse IgG: Zhongshan Jinqiao, catalog number ZF-0513.

[0098] Example 1

[0099] This embodiment provides a method for creating a pelvic fibrosis organoid model, including the following steps:

[0100] S1: Isolation of endometrial epithelial cells and fibroblasts from pathological endometrial tissue.

[0101] The pathological endometrial tissue was washed with HBSS, minced to form tissue fragments, and Dispase II and type IV collagenase tissue hydrolysate was added until the tissue fragments were completely covered. The hydrolysate containing the tissue fragments was evenly distributed into a low-absorption plate and incubated at 37±0.5℃ for 40 min. The tissue fragments were then ground through a 70 μm sieve to obtain a tissue suspension. A high-glucose medium containing 5% FBS was added to the tissue suspension to terminate the enzymatic reaction. The suspension was centrifuged for 3 min to collect the endometrial cell pellet.

[0102] S2: The endometrial epithelial cells and fibroblasts are cultured and expanded separately to obtain endometrial organoids and expanded fibroblasts.

[0103] Culture and expansion of endometrial organoids: The endometrial cell pellet obtained in step S1 was resuspended in endometrial organoid culture medium, and matrix gel was added to a final concentration of 5% for 3D culture. The culture was incubated at 37±0.5℃ for 25 min. After the matrix gel solidified, endometrial organoid culture medium was added, and the culture was carried out for 10 days to obtain endometrial organoids.

[0104] Culture and expansion of fibroblasts: Take the enzymatic hydrolysate containing the tissue fragments from step S1 after enzymatic incubation and before sieving, centrifuge to remove the hydrolysate, resuspend the cells in DMEM / F12 fibroblast culture medium containing 5% FBS, seed them in cell culture plates, and perform two-dimensional adherent culture at 37℃ and 5% CO2. When the cells grow to 90% confluence, perform passage culture to obtain expanded fibroblasts.

[0105] S3: The endometrial organoids and the expanded fibroblasts are digested and dispersed separately to obtain cell suspensions containing endometrial organoids and expanded fibroblasts, respectively. Specifically,

[0106] The endometrial organoids obtained in step S2, along with the culture medium, were transferred to a 15 mL centrifuge tube. After centrifugation, cell pellets were obtained. Tryple digestion solution was added and the pellets were dispersed. The pellets were then digested at 37°C for 3 min. Digestion was terminated with a high-glucose culture medium containing 5% FBS. After centrifugation, the pellets were resuspended in the culture medium used for co-culture to obtain an endometrial organoid cell suspension. The number of cells in the cell suspension was counted.

[0107] Discard the supernatant of the amplified fibroblasts obtained in step S2, wash with PBS, add Accutase digestion solution, digest at 37°C for 30 seconds, pipette to detach the cells, add DMEM / F12 fibroblast culture medium containing 5% FBS to terminate digestion, centrifuge, add the culture medium used for co-culture to resuspend to obtain a fibroblast suspension, and count the number of cells in the cell suspension.

[0108] S4: Mix the two cell suspensions to obtain a mixed cell suspension, and seed it onto an organoid chip equipped with multiple parallel culture units for co-culture to form a pelvic inflammatory disease fibrosis organoid model.

[0109] Based on the counting results, the endometrial organoid cell suspension and the fibroblast suspension were mixed at a cell ratio of 5:1, and matrix gel was added to a final concentration of 2%. After mixing, the mixture was evenly seeded onto the organoid chip and co-cultured under dynamic fluid perfusion conditions at a perfusion rate of 10 μL / min for 3 days to form a pelvic inflammatory disease fibrosis organoid model.

[0110] The culture medium used for the co-culture is based on DMEM / F12 and contains the following components at final concentrations:

[0111] 15 mM nicotinamide, 5 ng / mL bFGF, 1.25 mM N-acetylcysteine, 1×B27 additive, 1×N2 additive, 50 ng / mL EGF, 0.5 μM PGE2, 100 ng / mL RSPO1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 10 nM 17β-estradiol, 10 ng / mL FGF10, 0.5 μM SB202190, 0.5 μM A83-01, 25 ng / mL HRG-β1, 5 μM Forskolin.

[0112] Example 2

[0113] This embodiment provides a method for creating a pelvic fibrosis organoid model:

[0114] The difference between this example and Example 1 lies in the composition of the co-culture medium, specifically:

[0115] 10 mM nicotinamide, 7 ng / mL bFGF, 3 mM N-acetylcysteine, 1× B27 additive, 1× N2 additive, 20 ng / mL EGF, 0.1 μM PGE2, 200 ng / mL RSPO1, 200 ng / mL Wnt-3a, 150 ng / mL Noggin, 20 nM 17β-estradiol, 15 ng / mL FGF10, 0.3 μM SB202190, 0.1 μM A83-01, 10 ng / mL HRG-β1, 3 μM Forskolin.

[0116] The remaining steps are the same as in Example 1.

[0117] Example 3

[0118] This embodiment provides a method for creating a pelvic fibrosis organoid model:

[0119] The difference between this example and Example 1 lies in the composition of the co-culture medium, specifically:

[0120] 5 mM nicotinamide, 10 ng / mL bFGF, 5 mM N-acetylcysteine, 1×B27 additive, 1×N2 additive, 70 ng / mL EGF, 1 μM PGE2, 50 ng / mL RSPO1, 70 ng / mL Wnt-3a, 50 ng / mL Noggin, 50 nM 17β-estradiol, 5 ng / mL FGF10, 0.1 μM SB202190, 0.7 μM A83-01, 35 ng / mL HRG-β1, 1 μM Forskolin.

[0121] The remaining steps are the same as in Example 1.

[0122] Example 4

[0123] This embodiment provides a method for creating a pelvic fibrosis organoid model:

[0124] The difference between this example and Example 1 lies in the composition of the co-culture medium, specifically:

[0125] 20 mM nicotinamide, 2.5 ng / mL bFGF, 0.1 mM N-acetylcysteine, 1×B27 additive, 1×N2 additive, 100 ng / mL EGF, 2 μM PGE2, 150 ng / mL RSPO1, 200 ng / mL Wnt-3a, 200 ng / mL Noggin, 35 nM 17β-estradiol, 20 ng / mL FGF10, 0.2 μM SB202190, 1 μM A83-01, 50 ng / mL HRG-β1, 2.5 μM Forskolin.

[0126] The remaining steps are the same as in Example 1.

[0127] Example 5

[0128] This embodiment provides a method for creating a pelvic fibrosis organoid model:

[0129] The incubation time in S1 is 50 min, and the enzymatic hydrolysis reaction is terminated by centrifugation for 4 min.

[0130] The incubation time for endometrial organoid culture in S2 was 30 min, and the culture time was 15 days; during the culture and expansion of fibroblasts, the fibroblast culture medium used was DMEM / F12 containing 10% FBS.

[0131] The digestion conditions for endometrial organoids in S3 are 37.5℃ for 5 minutes;

[0132] In S4, the ratio of endometrial organoid cells to expanded fibroblasts is 7:1; the perfusion rate of the dynamic fluid perfusion is 15 μL / min.

[0133] The remaining steps are the same as in Example 1.

[0134] Example 6

[0135] This embodiment provides a method for creating a pelvic fibrosis organoid model:

[0136] The difference between it and Example 1 is that:

[0137] The incubation time in S1 is 60 min, and the enzymatic hydrolysis reaction is terminated by centrifugation for 5 min.

[0138] The culture time for endometrial organoids in S2 was 17 days; during the culture and expansion of fibroblasts, the fibroblast culture medium used was DMEM / F12 containing 8% FBS.

[0139] The digestion conditions for endometrial organoids in S3 were 37.5℃ for 4 minutes;

[0140] In S4, the ratio of endometrial organoid cells to expanded fibroblasts is 8:1; the perfusion rate of the dynamic fluid perfusion is 17 μL / min.

[0141] The remaining steps are the same as in Example 1.

[0142] Example 7

[0143] This embodiment provides a method for creating a pelvic fibrosis organoid model:

[0144] The difference between it and Example 1 is that:

[0145] The incubation time in S1 is 45 minutes;

[0146] The culture time for endometrial organoids in S2 was 13 days; during the culture and expansion of fibroblasts, the fibroblast culture medium used was DMEM / F12 containing 7% FBS.

[0147] The ratio of S4 endometrial organoid cells to expanded fibroblasts is 10:1; the perfusion rate of the dynamic fluid perfusion is 20 μL / min.

[0148] The remaining steps are the same as in Example 1.

[0149] Example 8

[0150] This embodiment provides a method for creating a pelvic fibrosis organoid model:

[0151] The difference between it and Example 1 is that:

[0152] The incubation time in S1 is 45 minutes;

[0153] The culture time for endometrial organoids in S2 was 12 days; during the culture and expansion of fibroblasts, the fibroblast culture medium used was DMEM / F12 containing 9% FBS.

[0154] The digestion conditions for endometrial organoids in S3 are 36.5℃ for 5 minutes;

[0155] The ratio of S4 endometrial organoid cells to expanded fibroblasts is 10:1; the perfusion rate of the dynamic fluid perfusion is 5 μL / min.

[0156] The remaining steps are the same as in Example 1.

[0157] Compare with Example 1

[0158] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0159] The difference between this and Example 1 is that in S4, the ratio of endometrial organoid cells to expanded fibroblasts is 11:1.

[0160] The remaining steps are the same as in Example 1.

[0161] Compare with Example 2

[0162] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0163] The difference between this example and Example 1 is that in S4, the ratio of endometrial organoid cells to expanded fibroblasts is 4:1. The remaining steps are the same as in Example 1.

[0164] Compare with Example 3

[0165] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0166] The difference between this example and Example 1 is that in S4, the mixing ratio of endometrial organoid cells to expanded fibroblasts is 5:2. The remaining steps are the same as in Example 1.

[0167] Compare with Example 4

[0168] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0169] The difference between this example and Example 1 is that in S4, the ratio of endometrial organoid cells to expanded fibroblasts is 1:5. The remaining steps are the same as in Example 1.

[0170] Compare with Example 5

[0171] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0172] The difference between this example and Example 1 lies in the composition of the co-culture medium, specifically:

[0173] 15 mM nicotinamide, 5 ng / mL bFGF, 1.25 mM N-acetylcysteine, B27 additive, N2 additive, 50 ng / mL EGF, 0.5 μM PGE2, 100 ng / mL RSPO1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 10 nM 17β-estradiol, 10 ng / mL FGF10, 0.5 μM SB202190.

[0174] Compare with Example 6

[0175] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0176] The difference between this example and Example 1 lies in the composition of the co-culture medium, specifically:

[0177] 15 mM nicotinamide, 5 ng / mL bFGF, 1.25 mM N-acetylcysteine, B27 additive, N2 additive, 50 ng / mL EGF, 0.5 μM PGE2, 100 ng / mL RSPO1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 10 nM 17β-estradiol, 10 ng / mL FGF10, 0.5 μM SB202190, 0.5 μM A83-01, 25 ng / mL HRG-β1.

[0178] Compare with Example 7

[0179] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0180] The difference between this example and Example 1 lies in the composition of the co-culture medium, specifically:

[0181] 15 mM nicotinamide, 5 ng / mL bFGF, 1.25 mM N-acetylcysteine, B27 additive, N2 additive, 50 ng / mL EGF, 0.5 μM PGE2, 100 ng / mL RSPO1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 10 nM 17β-estradiol, 10 ng / mL FGF10, 0.5 μM SB202190, 0.5 μM A83-01.

[0182] Compare with Example 8

[0183] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0184] The difference between this example and Example 1 lies in the composition of the co-culture medium, specifically:

[0185] 15 mM nicotinamide, 5 ng / mL bFGF, 1.25 mM N-acetylcysteine, B27 additive, N2 additive, 50 ng / mL EGF, 0.5 μM PGE2, 100 ng / mL RSPO1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 10 nM 17β-estradiol, 10 ng / mL FGF10, 0.5 μM SB202190, 25 ng / mL HRG-β1, 5 μM Forskolin.

[0186] Compare with Example 9

[0187] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0188] The difference between this example and Example 1 is that the infusion rate in S4 is 25 μL / min. The remaining steps are the same as in Example 1.

[0189] Compare with Example 10

[0190] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0191] The difference between this example and Example 1 is that the infusion rate in S4 is 3 μL / min. The remaining steps are the same as in Example 1.

[0192] Compare with Example 11

[0193] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0194] The difference between it and Example 1 is that the culture medium used for co-culturing in S4 is DMEM / F12 medium.

[0195] The remaining steps are the same as in Example 1.

[0196] Compare with Example 12

[0197] This comparative example provides a method for creating a pelvic fibrosis organoid model:

[0198] The difference from Example 1 is that this comparative example refers to the method for constructing a breast epithelial organoid model disclosed in CN202210656281A and applies it to endometrial tissue. The specific steps are as follows:

[0199] The collected endometrial tissue was washed thoroughly with PBS, minced, and added to digestion buffer (DMEM / F12 medium containing type I, II, and IV collagenases). The mixture was then incubated at 37°C with shaking for 1 hour. After digestion, the cell suspension was filtered through a 100μm filter, centrifuged at 300g for 5 minutes, and the supernatant was discarded. Red blood cell lysis buffer was added, and the cells were lysed at room temperature for 5 minutes. The cells were then centrifuged again to collect the cell pellet. 2 mL of pre-warmed blank DMEM / F12 medium was added to the pellet for resuspending. The pellet was then evenly seeded into six-well plates and incubated at 37°C with 5% CO2 for 20 minutes. The supernatant was aspirated and seeded into new wells. After incubation for another 20 minutes, the supernatant was aspirated. The adherent cells in the first and second wells were fibroblasts; the supernatant from the second well was collected, centrifuged, and endometrial epithelial cells were obtained.

[0200] Endometrial epithelial cell culture: Collected endometrial epithelial cells were centrifuged at 500g for 10 minutes at 4℃, the supernatant was discarded, and the cells were resuspended in pre-cooled matrix gel and seeded into 24-well plates at 40μL per well. After the matrix gel solidified, 400μL of endometrial epithelial organoid culture medium was added, and the cells were cultured in a 37℃, 5% CO2 incubator for 7 days to obtain endometrial epithelial organoids.

[0201] Fibroblast culture: Adherent fibroblasts were added to DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin and cultured for 7 days at 37°C in a 5% CO2 incubator.

[0202] After 7 days of culture, epithelial organoids and fibroblasts were collected separately. Epithelial organoids were digested with Tryple enzyme for 5 minutes, centrifuged, resuspended, and counted. Fibroblasts were digested with trypsin for 3 minutes, centrifuged, resuspended, and counted. Fibroblasts and digested epithelial organoids were mixed at a 1:1 ratio and added to endometrial epithelial organoid culture medium containing 5% Matrigel. The mixture was then seeded into 24-well plates and cultured routinely for 48 hours.

[0203] Experimental Example 1

[0204] Experimental Objective: This experiment aims to verify whether the method of the present invention successfully constructed an organoid model of pelvic inflammatory disease fibrosis with interstitial components by observing the optical morphology and detecting immunofluorescence in the organoid models constructed in the negative control group, Examples 1-8, and Control Examples 1-12. The negative control group also confirms that fibroblasts are the source of the fibrotic features of the model.

[0205] Test method:

[0206] 1. Grouping: The following experimental groups were set up, with 3 replicates in each group, and 10 fields of view were randomly selected from each replicate. The negative control group was cultured according to the preparation method of Example 1, but without the addition of fibroblasts, containing only endometrial epithelial cell organoids; Examples 1-8 and Control Examples 1-12.

[0207] 2. Observation under an optical microscope

[0208] After co-culture, the chip was observed under an inverted phase-contrast microscope (Nikon, Ts2-FL, constant temperature 37℃, 5% CO2). Ten fields of view were randomly selected from the sample for photographing. Fields of view selection avoided the chip's edge area and areas prone to interference such as air bubbles and impurities, ensuring that each field of view contained at least five organoids. The morphology of the organoids and the distribution of stromal cells were recorded. Endometrial epithelial cells formed typical spherical or ellipsoidal organoids, surrounded by fibroblasts.

[0209] 3. Immunofluorescence staining

[0210] After co-culture, the culture medium in each culture unit of the organoid microarray was aspirated, and the organoids were gently washed twice with PBS preheated to 37°C for 5 minutes each time. 4% paraformaldehyde fixative (prepared with PBS, pH 7.4) was added to each culture unit, ensuring complete coverage of the organoids, and fixed at room temperature for 30 minutes. After fixation, the fixative was aspirated, and the microarray was placed on a shaker and washed three times with PBS for 10 minutes each time at 60 rpm. Then, permeabilization buffer (PBST containing 0.2% Triton X-100) was added to each culture unit, and incubated at room temperature for 15 minutes to allow antibodies to enter the cells and bind to the antigen. The permeabilization buffer was aspirated, and the microarray was washed three times with PBS for 5 minutes each time. Finally, blocking buffer (prepared with PBS) containing 5% normal goat serum was added to each culture unit, and incubated at room temperature for 1 hour to block non-specific binding sites. Remove the blocking buffer, add mouse anti-human Vimentin monoclonal antibody diluted with the blocking buffer (1:200 dilution), ensuring complete coverage of the sample, and incubate overnight at 4°C. Recover the primary antibody, wash three times with PBST for 10 minutes each time on a shaker. Then add Alexa Fluor 488-labeled goat anti-mouse IgG diluted with the blocking buffer (1:400 dilution), and incubate at room temperature in the dark for 1 hour. Remove the secondary antibody, wash three times with PBST for 10 minutes each time in the dark. Next, add PBS containing DAPI (final concentration 1 μg / mL) to each culture unit and incubate at room temperature in the dark for 5 minutes for nuclear counterstaining. Finally, wash twice rapidly with PBS for 2 minutes each time to remove excess DAPI. Add a small amount of anti-fluorescence quenching mounting medium to the culture chamber of the chip, and observe and acquire images using a fluorescence microscope (Nikon, Ts2-FL). Calculate the Vimentin-positive cell rate.

[0211] All data are expressed as mean ± standard deviation (mean ± SD).

[0212] Table 1. Laser confocal microscope settings parameters

[0213] DAPI Channel 405nm 420~480nm Cell nucleus (blue) FITC channel 488nm 500-550nm Vimentin-positive cells (green)

[0214] For each sample, 5 to 10 fields of view were randomly selected for image acquisition. The DAPI and FITC channel images were recorded separately and then overlaid.

[0215] Image analysis was performed using ImageJ software to calculate the rate of Vimentin-positive cells.

[0216] Vimentin positive cell rate (%) = (Number of Vimentin positive cells / Total number of DAPI positive cells) × 100%.

[0217] Experimental results:

[0218] Table 2. Results of optical microscopy observations

[0219] Group form negative control It is typically spherical with clear and smooth boundaries, and no long spindle-shaped fibroblasts are observed around the organoid. Examples 1-8 The organoids are mainly spherical or near-spherical structures with clear boundaries. Numerous long spindle-shaped fibroblasts are distributed around and within the spaces between the organoids. The cell processes interconnect to form a network structure that is in close contact with the organoids. In Examples 1 and 2, the proportion of typical spherical organoids exceeds 85%. Compare with Example 1 The organoids have relatively complete morphology, but the distribution of fibroblasts is sparse. Compare with Example 2 The organoids have irregular shapes and are densely distributed with fibroblasts. Compare with Example 3 The organoids have irregular shapes and are densely distributed with fibroblasts. Compare with Example 4 Organoid formation is inhibited, and fibroblasts overgrow to form cell layers. Compare with Example 5 The organoids are irregular in shape, vary significantly in size, and have indistinct boundaries; the number of surrounding fibroblasts is sparse and they are scattered, with no network structure formed by long spindle-shaped cell processes. Compare with Example 6 The organoids are spherical, but multiple organoids are deformed by pressure and have irregular boundaries; fibroblasts are excessively aggregated in some areas, forming dense cell clumps, and are unevenly distributed in different regions. Compare with Example 7 The organoid body is roughly spherical with discernible boundaries, but the overall structure is relatively loose. The number of surrounding fibroblasts is moderate, but their distribution is uneven. In some areas, the cells are sparse and the connections between the processes are loose, failing to form a complete encapsulating network. Compare with Example 8 Most organoids are irregular in shape and have indistinct boundaries. Some organoids are deformed by fibroblast clusters. Fibroblasts often aggregate in large numbers on one side or in a localized area, forming messy clumps, and the interstitial structure of organoids is disordered. Compare with Example 9 The organoids are incomplete in shape, and the cells are scattered. Compare with Example 10 Organoids are small in size and their interstitial cells adhere poorly to the substrate. Compare with Example 11 The organoids are spherical in shape with relatively clear boundaries, but the fibroblasts are unevenly distributed, with some organoids clustered together and others sparsely distributed. Compare with Example 12 Some organoids are spherical in shape with clear boundaries; most organoids are irregular in shape, with some organoids having excessively dense fibroblasts around them and others having sparse fibroblasts.

[0220] Table 3. Statistics on Vimentin-positive cell rates in each group

[0221] Group Vimentin-positive cell rate (%) Positive degree negative control group 2.3±1.2 Negative Example 1 86.5±5.3 Strong positive Example 2 83.7±6.0 Strong positive Example 3 82.1±5.6 Strong positive Example 4 80.4±6.8 Strong positive Example 5 84.2±6.5 Strong positive Example 6 76.8±7.2 Strong positive Example 7 68.3±6.1 Positive Example 8 71.5±6.5 Strong positive Compare with Example 1 35.7±8.3 Positive Compare with Example 2 91.2±4.5 Strong positive Compare with Example 3 88.6±7.1 Strong positive Compare with Example 4 96.3±3.2 Strong positive Compare with Example 5 57.8±9.2 Positive Compare with Example 6 72.6±8.1 Strong positive Compare with Example 7 74.7±7.6 Strong positive Compare with Example 8 78.5±8.6 Strong positive Compare with Example 9 18.4±6.2 Weak positive Compare with Example 10 45.6±9.3 Positive Compare with Example 11 52.3±7.8 Positive Compare with Example 12 78.2±8.5 Strong positive

[0222] Table 4. Observation results of distribution characteristics by immunofluorescence staining.

[0223] Group Distribution characteristics observation results negative control Occasionally, individual Vimentin-positive cells (possibly residual stromal cells) were observed, and the main body of the organoids consisted of Vimentin-negative endometrial organoid cells. Examples 1-8 Numerous Vimentin-positive cells were distributed around the organoid body. The cells were elongated spindle-shaped, with interconnected processes forming a network structure, and were in close contact with the endometrial organoid cells. The positive cells were evenly distributed, with no localized excessive aggregation observed. Compare with Example 1 The number of Vimentin-positive cells was small, their distribution sparse, and they did not form a complete network. Compare with Example 2 The excessive number and dense distribution of Vimentin-positive cells, with some areas having positive cells covering the surface of the organoids, caused the organoid structures to be compressed and deformed. Compare with Example 3 The excessive number and dense distribution of Vimentin-positive cells, with some areas having positive cells covering the surface of the organoids, caused the organoid structures to be compressed and deformed. Compare with Example 4 The field of view was mostly composed of Vimentin-positive cells, forming a continuous cell layer, with a relatively small number of organoids. Compare with Example 5 The number of Vimentin-positive cells was small, their distribution sparse, and they did not form a complete network, resulting in irregular organoid morphology. Compare with Example 6 The number of Vimentin-positive cells was moderate, but their distribution was uneven, with fibroblasts excessively aggregated in some areas. Compare with Example 7 The number of Vimentin-positive cells is moderate, distributed around the organoids, with a relatively loose network structure and sparse fibroblasts in some areas. Compare with Example 8 Vimentin-positive cells are numerous but unevenly distributed, often clustered in clumps, with disordered structures and irregular organoid morphology. Compare with Example 9 Vimentin-positive cells were few in number, scattered, and did not form a stable structure. Compare with Example 10 Vimentin-positive cells adhere poorly to the culture vessel, exhibit weak staining intensity, and are unevenly distributed. Compare with Example 11 The number of Vimentin-positive cells was moderate, but their distribution was uneven. Some organoids were surrounded by fibroblasts, while others were sparse. The organoid structure was acceptable, but the uniformity was poor. Compare with Example 12 Vimentin-positive cells are distributed around the organoids, with some organoids having an overly dense distribution of fibroblasts and others having a sparse distribution.

[0224] Results analysis:

[0225] Optical microscopy and immunofluorescence staining results (Tables 2-4) showed that the positive rate of Vimentin in the negative control group was only 2.3%, and no fibroblasts were observed under light microscopy, confirming that there were no stromal components under pure epithelial organoid culture conditions, providing a control for verifying the fibrotic characteristics of the co-culture model.

[0226] Examples 1-8 used cell ratios of 5:1 to 10:1, achieving Vimentin positivity rates of 68.3% to 86.5%. Positive cells were evenly distributed in a network around the organoids, with intact organoid structures and clear boundaries. Examples 1 (a 5:1 ratio of endometrial organoid cells to expanded fibroblasts) and 5 (a 7:1 ratio of endometrial organoid cells to expanded fibroblasts) showed the best performance, with typical spherical organoid proportions exceeding 85%. A representative light micrograph of Example 1 is shown below. Figure 1 In Control Example 1 (11:1), the Vimentin positivity rate was only 35.7%, with insufficient fibroblast numbers, sparse distribution, and no complete network. This indicates that when the proportion of endometrial organoid cells is too high, the number of fibroblasts is insufficient to construct a fibrosis model with stromal components. In Control Example 2 (4:1), the Vimentin positivity rate reached 91.2%, but the positive cells were too densely distributed, covering part of the surface of the pelvic fibrotic organoids, causing compression and deformation of the pelvic fibrotic organoid structure. This indicates that when the proportion of fibroblasts is too high, their excessive growth destroys the integrity of the organoid structure. In Control Example 3 (5:2), the Vimentin positivity rate was 88.6%, also showing excessive fibroblast distribution and compression of the pelvic fibrotic organoid structure. In Control Example 4 (1:5), the Vimentin positivity rate was 96.3%, with the vast majority of cells in the field of view being positive, forming a continuous cell layer, and the number of pelvic fibrotic organoids was sparse. This indicates that when fibroblasts are absolutely dominant, endometrial organoid cells cannot effectively self-assemble to form pelvic fibrotic organoids.

[0227] By comparing Example 1 with Control Examples 1-4, it was found that a ratio of 5:1 to 10:1 of endometrial organoid cells to fibroblasts is a key parameter for achieving "moderate number of fibroblasts, uniform distribution, and intact pelvic fibrotic organoid structure".

[0228] Comparing the data of Example 1 with those of Controls 5-8 (Tables 2-4), it was found that Control 5 lacked all three components: A83-01, HRG-β1, and Forskolin. The Vimentin positivity rate decreased from 86.5% in Example 1 to 57.8%. Under light microscopy, the pelvic fibrotic organoids exhibited irregular morphology and blurred boundaries, with sparse and scattered fibroblasts. Immunofluorescence showed sparse distribution and failure to form a complete network, making it the worst performing control among Controls 5-8. This indicates that these three components play an important role in the construction of pelvic fibrotic organoids. Control 7 contained only A83-01 and lacked HRG-β1 and Forskolin, resulting in a positivity rate of 74.7%. Under light microscopy, the structure was relatively loose, and immunofluorescence showed an incomplete network structure and sparse fibroblasts in local areas, indicating insufficient fibroblast viability and quantity, making it difficult to form a dense encapsulating network. Control Example 8, lacking only A83-01, had a positive rate of 78.5%. Although the value was lower, the pelvic fibrotic organoids under light microscopy showed irregular morphology and indistinct boundaries. Fibroblasts were abundantly aggregated on one side in disordered clumps. Immunofluorescence showed that positive cells were unevenly aggregated in clumps with disordered structure, indicating that the lack of A83-01 would disrupt the structural regularity of the pelvic fibrotic organoid model. Control Example 6, lacking only Forskolin, had a positive rate of 72.6%. Under light microscopy, multiple pelvic fibrotic organoids were deformed by pressure, and fibroblasts were excessively aggregated locally to form dense clumps. Immunofluorescence also showed uneven distribution, indicating that the lack of Forskolin in the system would lead to local overgrowth.

[0229] In summary, the three components HRG-β1, A83-01, and Forskolin in the co-culture medium of this invention have a synergistic effect. All three are indispensable, and without any one component, it is impossible to obtain a structurally complete and morphologically uniform organoid model of pelvic inflammatory disease fibrosis.

[0230] Examples 1-8 all used perfusion rates of 5-20 μL / min, with Vimentin positivity rates of 68.3%-86.5%. Light microscopy and immunofluorescence showed uniform cell distribution and intact structure (Tables 2-4). In Control Example 9, the perfusion rate was increased to 25 μL / min, and the Vimentin positivity rate decreased to 18.4%. Light microscopy revealed incomplete organoid morphology and cell dispersion. Immunofluorescence showed a small number of positive cells, scattered distribution, and failure to form stable structures, indicating that excessively high flow rates dispersed the co-cultured cells, disrupting cell aggregation and the conditions for pelvic fibrosis organoid formation. Control Example 10 reduced the perfusion rate to 3 μL / min, and the Vimentin positivity rate decreased to 45.6%. Light microscopy showed small pelvic fibrosis organoids with poor stromal cell adhesion. Immunofluorescence showed poor adhesion and weak staining intensity of positive cells, indicating that excessively low flow rates could not provide sufficient nutrient exchange and appropriate fluid shear forces, which was detrimental to cell survival and normal distribution. Therefore, it is evident that simply adjusting the ratio of endometrial organoid cells and fibroblasts, as well as the composition of the co-culture medium, is insufficient to obtain morphologically intact pelvic fibrotic organoids; an appropriate perfusion rate is also necessary.

[0231] Control Example 11 used commercially available conventional culture medium (DMEM / F12). Although the optimized ratio and flow rate were maintained, the Vimentin positivity rate was only 52.3%, and the distribution of positive cells was uneven (Tables 2-4), further confirming the importance of optimizing the culture medium composition of this invention. Control Example 12 used a conventional breast co-culture method for transplantation. The results showed that the Vimentin positivity rate was 78.2%, a decrease of approximately 8% compared to Example 1. Although the positivity rate was still at a high level, the distribution was uneven, with some organoids having excessively dense fibroblasts around them and sparse fibroblasts in other areas (Tables 2-4). This indicates that simply transplanting the breast co-culture method to endometrial organoid culture, without optimization for pelvic inflammatory disease fibrosis (specific ratio 5:1-10:1, optimized culture medium, and dynamic perfusion on a microarray), cannot obtain a structurally intact and uniformly distributed pelvic inflammatory disease fibrosis organoid model.

[0232] In summary, this invention has constructed a pelvic inflammatory disease fibrosis organoid model containing a large number of Vimentin-positive fibroblasts by comprehensively optimizing the ratio of endometrial organoid cells to fibroblasts (5:1 to 10:1), establishing a culture medium synergistically regulated by HRG-β1, A83-01, and Forskolin, and combining it with microfluidic dynamic perfusion of 5 to 20 μL / min. The cells are evenly distributed and structurally intact, providing a model basis for subsequent evaluation of the anti-fibrotic effects of traditional Chinese medicine.

[0233] Example 2: Evaluation of the homogeneity of the organoid model of pelvic inflammatory disease and fibrosis

[0234] Experimental Objective: This experiment analyzes the pelvic inflammatory disease fibrosis organoid models constructed by the negative control group, Examples 1-8, and Control Examples 1-12 to evaluate the performance of the pelvic inflammatory disease fibrosis organoid models constructed by the method of this invention in terms of size uniformity and organoid density, and to verify its technical advantages as a high-throughput drug screening platform.

[0235] Experimental Method: The experimental method is the same as the optical microscope observation method in Experimental Example 1. The following quantitative analysis was performed using ImageJ software:

[0236] Organoid density: Measured the area (mm²) of each field of view. 2 ), count the number of organoids with a diameter > 50 μm, organoid density (number / mm²) 2 = Number of organoids / Field of view area

[0237] Organoid diameter and homogeneity: The maximum and minimum diameters of at least 50 organoids per sample were measured, and the average was taken as the diameter of that organoid. The mean diameter, standard deviation, and coefficient of variation (CV) were calculated. CV (%) = (Standard deviation / Mean diameter) × 100%

[0238] The smaller the CV value, the more uniform the organoid size. The evaluation criteria are: CV < 15% is highly uniform, CV 15% to 25% is moderately uniform, and CV > 25% is heterogeneous.

[0239] Experimental results:

[0240] Table 5. Statistics on the uniformity of organoid density and size in each group

[0241] Group <![CDATA[Organoid density (number / mm 2 ).]]> Average diameter (μm) Coefficient of variation (CV) (%) negative control group 25.3±3.2 82.6±10.3 12.5 Example 1 42.6±3.8 105.4±12.7 12.0 Example 2 40.3±4.1 103.8±13.2 12.7 Example 3 39.8±4.3 99.6±13.6 13.6 Example 4 37.2±3.8 96.3±12.7 13.2 Example 5 40.2±4.1 102.8±13.1 12.7 Example 6 38.5±4.3 95.6±14.2 14.8 Example 7 32.9±3.5 91.4±13.4 14.7 Example 8 34.1±4.0 92.7±13.8 14.9 Compare with Example 1 28.4±3.3 88.8±16.1 18.1 Compare with Example 2 30.5±4.9 86.5±22.1 25.5 Compare with Example 3 29.7±4.6 85.2±21.3 25.0 Compare with Example 4 8.2±2.9 Unable to count Unable to count Compare with Example 5 19.1±4.6 76.4±17.8 23.3 Compare with Example 6 24.6±5.1 84.7±18.2 21.5 Compare with Example 7 28.3±3.7 86.9±18.4 21.2 Compare with Example 8 29.6±6.1 85.8±20.3 23.6 Compare with Example 9 12.5±3.4 70.3±17.7 25.2 Compare with Example 10 18.7±3.7 75.8±16.9 22.3 Compare with Example 11 30.2±4.5 90.3±18.5 20.5 Compare with Example 12 32.5±6.8 92.5±23.8 25.7

[0242] Results Analysis

[0243] The results in Table 5 show that the density of fibrotic organoids in Examples 1–8 of pelvic inflammatory disease was high (32.9–42.6 organs / mm²). 2 The organoids exhibited good size uniformity (CV value 12.0%–14.9%), significantly better than the control groups. Examples 1–8 all met the high uniformity standard (CV < 15%). Among them, Examples 1 and 5 performed best, with organoid densities reaching 42.6 organs / mm². 2 and 40.2 pieces / mm 2 The proportion of typical spherical organoids reached over 85%. This indicates that within the cell ratio of 5:1 to 10:1, the co-culture medium composition, and the perfusion rate of 5 to 20 μL / min specified in this invention, a fibrotic model with intact organoid structure and uniform size can be obtained. The organoid density in the negative control group was 25.3 organs / mm². 2 The CV was 12.5%. Although the size was uniform, there was no interstitial component, so it could not be used for fibrosis research.

[0244] Examples 1-8 used cell ratios of 5:1 to 10:1, resulting in high density of pelvic inflammatory disease (PID) fibrotic organoids with CV values ​​below 15%. Examples 1 (5:1) and 5 (7:1) showed the best performance. When the cell ratio exceeded the limits defined in this invention, the homogeneity of the model decreased. In Control Example 1, the ratio was increased to 11:1, and the CV value increased to 18.1%, while homogeneity decreased, indicating that when the proportion of endometrial organoid cells was too high, the number of fibroblasts was insufficient, leading to reduced efficiency of PID fibrotic organoid formation and increased size variation. In Control Example 2, the ratio was reduced to 4:1, and in Control Example 3, it was reduced to 5:2, with CV values ​​of 25.5% and 25.0%, respectively, indicating that when the proportion of fibroblasts was too high, their excessive growth led to a significant increase in the size variation of PID fibrotic organoids. In Control Example 4, the ratio was further reduced to 1:5, resulting in a density of only 8.2 organs / mm². 2 The scarcity of endometrial organoid cells to fibroblasts, making it impossible to statistically analyze their CV values, indicates that endometrial organoid cells cannot effectively self-assemble into organoids when fibroblasts are dominant. Therefore, a cell ratio of 5:1 to 10:1 between endometrial organoid cells and fibroblasts is a key parameter for obtaining a high-density, highly homogeneous pelvic inflammatory disease fibrosis organoid model. Exceeding this range significantly reduces homogeneity (CV > 18%) or leads to model construction failure.

[0245] Example 1 was compared with Control Examples 5-8. Control Example 5 lacked A83-01, HRG-β1, and Forskolin, its CV value increased to 23.3%, and its pelvic fibrotic organoid density decreased to 19.1 / mm². 2 Among the controls 5–8, the homogeneity was the worst. Control 7, containing only A83-01 but lacking HRG-β1 and Forskolin, showed a CV value of 21.2%, indicating a significant decrease in homogeneity. Control 8, lacking only A83-01, showed a CV value of 23.6%, indicating that the absence of A83-01 disrupts the size uniformity of organoids in pelvic fibrosis. Control 6, lacking only Forskolin, showed a CV value of 21.5%. In summary, HRG-β1, A83-01, and Forskolin work synergistically to maintain the orderly distribution of fibroblasts in the co-culture system.

[0246] Examples 1-8 used dynamic perfusion rates of 5-20 μL / min, with CV values ​​of 12.0%-14.9%, achieving a high degree of homogeneity. In contrast, control example 9 (25 μL / min) showed an organoid density reduced to 12.5 organs / mm². 2The CV value increased to 25.2% (non-uniform). This is because excessively high perfusion rates can dissipate cells and disrupt cell aggregation, resulting in low efficiency and significant size variation in pelvic fibrosis organoid formation; in control case 10 (3 μL / min), the density of pelvic fibrosis organoids was 18.7 / mm². 2 The CV value was 22.3% (moderately homogeneous). This is because the low perfusion rate cannot provide sufficient nutrient exchange and appropriate fluid shear force, resulting in restricted growth of pelvic fibrotic organoids, smaller size (average diameter 75.8 μm), and decreased homogeneity.

[0247] Although control example 11 (commercially available conventional endometrial organoid culture medium) maintained a 5:1 ratio and a perfusion rate of 10 μL / min, the density of pelvic fibrosis organoids was only 30.2 / mm². 2 The CV value was 20.5% (moderately uniform), significantly lower than that of Examples 1-5. This indicates that proportions and perfusion conditions alone are insufficient to obtain a highly uniform pelvic inflammatory disease fibrosis organoid model; optimization of the culture medium composition is equally important. The optimized culture medium of this invention can promote the uniform development of pelvic inflammatory disease fibrosis organoids while maintaining the growth balance between the two cell types.

[0248] Control Example 12 (referring to existing breast co-culture methods, transplanted to the endometrium): Conventional static plate culture was used at a 1:1 ratio. Table 5 shows that the density of pelvic fibrotic organoids was 32.5 organs / mm². 2 The CV value was 25.7% (non-uniform) lower than that of Examples 1-5, and significantly higher than that of Examples 1-5 (12.0%-14.9%). The standard deviation (23.8 μm) was much larger than that of Example 1 (12.7 μm), indicating a significant difference in size. This suggests that simply transplanting the breast co-culture method to the endometrium without optimization for pelvic inflammatory disease fibrosis (specific ratio 5:1-10:1, optimized culture medium, and dynamic perfusion on the chip) cannot obtain a fibrosis model with highly uniform size.

[0249] As shown in Table 5, the CV values ​​of Examples 1-8 ranged from 12.0% to 14.9%, which was 10.8% to 13.7% lower than the CV value of Control Example 12 (25.7%). The density of pelvic fibrotic organoids in Examples 1-8 was 32.9 to 42.6 per mm². 2The density of the pelvic fibrosis organoids was higher than that of Control Example 12, with Example 1 showing the highest density. Furthermore, the standard deviation of the pelvic fibrosis organoid size in Example 1 was reduced by 11.1 μm compared to Control Example 12, significantly reducing the size difference between pelvic fibrosis organoids and resulting in a more uniformly sized pelvic fibrosis organoid model. This indicates that this application successfully constructed high-density and uniformly sized pelvic fibrosis organoids by further optimizing the ratio of endometrial organoid cells to fibroblasts, optimizing the co-culture medium, and employing a dynamic culture technique with a specific perfusion rate.

[0250] The above results confirm that the pelvic fibrosis organoid model constructed by the method of the present invention has the characteristics of high density and high size uniformity. A single 5×10 chip can simultaneously construct 50 parallel culture units, which meets the requirements of high-throughput drug screening for model uniformity and parallelism.

[0251] Experimental Example 3

[0252] Experimental Objective: This experiment utilizes the pelvic inflammatory disease (PID) fibrosis organoid model constructed in Example 1 of this invention. Using live-cell fluorescent dye staining, the effects of different concentrations of *Salvia miltiorrhiza* extract on cell viability and fibrosis indices in this model are evaluated. This verifies the feasibility and effectiveness of the PID fibrosis organoid model constructed in this invention as an evaluation platform for traditional Chinese medicine's anti-fibrotic properties. Simultaneously, through dual immunofluorescence staining with α-SMA and CK7, the myofibroblast activation marker α-SMA and the epithelial cell marker CK7 are simultaneously labeled to identify the epithelial origin of the organoid and observe the spatial relationship between myofibroblasts and epithelial cells.

[0253] Test method:

[0254] Danshen (Salvia miltiorrhiza) is a representative drug for promoting blood circulation and removing blood stasis, listed in the Pharmacopoeia of the People's Republic of China. Clinically, it is widely used in the treatment of gynecological blood stasis syndrome and pelvic inflammatory disease. Its active components, tanshinone IIA and salvianolic acid B, have been systematically reviewed and confirmed to have significant anti-fibrotic activity. In this experimental case, Danshen extract was selected as the model drug.

[0255] The extract of Salvia miltiorrhiza (Shanghai Yuanye Biotechnology Co., Ltd., catalog number S27206) was prepared into a series of concentrations using DMEM / F12 basal medium and sterilized by filtration through a 0.22 μm filter membrane. The positive control drug SB431542 was prepared as a 10 mM stock solution using DMSO and diluted to 10 μM with culture medium before use (final concentration DMSO < 0.1%).

[0256] The negative control group used pure epithelial cell organoids without fibroblasts as the experimental subjects; the model control group, positive control group and each concentration group of Danshen (Danshen 1-5) all used the pelvic inflammatory disease fibrosis organoid model constructed by the method in Example 1 as the experimental subjects.

[0257] After co-culture (pelvic fibrosis organoids have initially formed), the old culture medium in each culture unit of the organoid microarray was aspirated. Fresh culture medium containing different concentrations of the tested traditional Chinese medicine, positive control drug, or an equal volume of PBS was added to each well (100 μL). Three replicates were set for each group. The microarray was incubated at 37°C in a 5% CO2 incubator for 48 hours. 48 hours after drug administration, a mixed dye mixture of Calcein-AM and propidium iodide (PI) was added to each culture unit to final concentrations of 2 μM and 4 μM, respectively. The microarray was then returned to the incubator and incubated in the dark for 30 minutes.

[0258] Observation and image acquisition were performed using a fluorescence microscope (Nikon, Ts2-FL). The following parameters were set:

[0259] Table 6. Laser confocal microscope parameter settings

[0260] FITC channel 488nm 500-550nm Live cells (calcein, green) Cy3 channel 561nm 570~620nm Dead cells (PI, red)

[0261] Five fields of view were randomly selected for each sample for photography.

[0262] After live cell staining, some samples were subjected to dual immunofluorescence staining with α-SMA and Collagen I. The specific procedure was the same as the immunofluorescence staining method in Experiment 1, with the following differences:

[0263] (1) Primary antibody incubation: Rabbit anti-human α-SMA polyclonal antibody (1:100 dilution) and mouse anti-human Collagen I monoclonal antibody (1:200 dilution) were added simultaneously and incubated overnight at 4°C;

[0264] (2) Secondary antibody incubation: Alexa Fluor 488-labeled goat anti-rabbit IgG (1:400 dilution) and Alexa Fluor 594-labeled goat anti-mouse IgG (1:400 dilution) were added simultaneously and incubated at room temperature in the dark for 1 hour.

[0265] The remaining steps are the same as the immunofluorescence staining method in Experiment Example 1.

[0266] A separate sample was subjected to dual immunofluorescence staining for α-SMA and CK7 to simultaneously visualize the spatial relationship between myofibroblast activation and organoid epithelial cells. Primary antibodies included rabbit anti-human α-SMA polyclonal antibody (1:100 dilution) and mouse anti-human CK7 monoclonal antibody (1:100 dilution), incubated overnight at 4°C. Secondary antibodies included Alexa Fluor 594-labeled goat anti-rabbit IgG (1:400 dilution) and Alexa Fluor 488-labeled goat anti-mouse IgG (1:400 dilution), incubated at room temperature in the dark for 1 hour. Under this staining protocol, α-SMA showed red fluorescence, CK7 showed green fluorescence, and cell nuclei showed blue fluorescence after DAPI counterstaining. The remaining fixation, permeabilization, blocking, and DAPI counterstaining procedures were the same as in Experiment 1. Images were observed and acquired using a fluorescence microscope.

[0267] Image analysis was performed using ImageJ software: viable cell rate, relative cell viability, α-SMA positive area ratio, Collagen I positive area ratio, and inhibition rate.

[0268] Viable cell percentage (%) = (Green fluorescent area / (Green fluorescent area + Red fluorescent area)) × 100%

[0269] Relative cell viability = (Viable cell rate in the treatment group / Viable cell rate in the model control group) × 100%

[0270] α-SMA positive area ratio (%) = α-SMA positive area / total visual field area × 100%

[0271] Collagen I positive area ratio (%) = Collagen I positive area / total visual field area × 100%

[0272] Fibrosis inhibition rate (%) = (Positive area ratio of model control group - Positive area ratio of treatment group) / Positive area ratio of model control group × 100%.

[0273] Experimental results:

[0274] Table 7. Statistics on viable cell rate and relative cell viability for each group.

[0275] Group Drugs and Concentrations viable cell rate (%) Relative cell viability (%) negative control Equal volume PBS 94.8±2.5 124.4±3.3 Model comparison Equal volume PBS 76.2±3.8 100.0±5.0 Positive control (SB431542) SB431542 10μM 91.5±3.1 120.1±4.1 Group 1 of Danshen Salvia miltiorrhiza extract 12.5 μg / mL 79.3±3.6 104.1±4.5 Group 2 of Danshen 25 μg / mL of tanshinone extract 82.7±3.3 108.5±3.9 Danshen Group 3 50 μg / mL of tanshinone extract 85.2±3.9 111.8±5.1 Group 4 of Danshen 100 μg / mL of tanshinone extract 84.6±3.5 111.0±4.2 Group 5 of Danshen 200 μg / mL of tanshinone extract 75.5±4.6 99.1±5.4

[0276] Table 8. Statistics of Fibrinization Indicators for Each Group

[0277] Group α-SMA positive area ratio (%) α-SMA inhibition rate (%) Collagen I positive area ratio (%) Collagen I inhibition rate (%) negative control 3.2±1.1 - 2.8±0.9 - Model comparison 28.5±3.2 0 24.6±2.8 0 Positive control (SB431542) 8.6±1.8 69.8 6.5±1.4 73.6 Group 1 of Danshen 24.6±2.9 13.7 23.3±2.4 5.3 Group 2 of Danshen 21.3±2.5 25.3 18.2±2.1 26.0 Danshen Group 3 16.8±2.4 41.1 14.5±2.0 41.1 Group 4 of Danshen 13.3±2.7 53.3 11.6±2.3 52.8 Group 5 of Danshen 11.8±2.8 58.6 10.2±2.5 58.5

[0278] Results analysis:

[0279] As shown in Table 7, the negative control group (pure epithelial cell organoids, without fibroblasts) had the highest cell viability (94.8%) and a relative cell viability of 124.4%, indicating that the epithelial cell organoids were in good condition without the involvement of fibroblasts and were not affected by the stress of the fibrotic microenvironment. The model control group had a cell viability of 76.2%, significantly lower than the negative control group, consistent with the characteristics of some cells undergoing stress or apoptosis under fibrotic pathological conditions. After treatment with the positive control drug SB431542, the cell viability increased to 91.5%, and the relative cell viability recovered to 120.1%, close to that of the negative control group, confirming that this model responds well to known antifibrotic drugs and validating the model's effectiveness.

[0280] Among the various groups treated with Danshen extract, the viable cell rate in Danshen 1 group (12.5 μg / mL) was 79.3%, only slightly higher than that in the model control group (76.2%), with a relative cell viability of 104.1%, showing no significant difference. The viable cell rates in Danshen 2 group (25 μg / mL) and Danshen 3 group (50 μg / mL) increased to 82.7% and 85.2%, respectively, with relative cell viability reaching 108.5% and 111.8%, respectively, showing a concentration-dependent increasing trend. This indicates that Danshen extract can effectively alleviate cellular stress caused by the fibrotic microenvironment within this concentration range. The viable cell rate in Danshen 4 group (100 μg / mL) was 84.6%, with a relative cell viability of 111.0%, maintaining a relatively high level. The viable cell rate in Danshen 5 group (200 μg / mL) decreased to 75.5%, and the relative cell viability dropped to 99.1%, comparable to the model control group, indicating that the excessively high drug concentration weakened the cell-protective effect.

[0281] As shown in Table 8, the positive area ratios of α-SMA and Collagen I in the negative control group were both below 5% (3.2% and 2.8%, respectively), confirming that there was almost no myofibroblast activation and collagen deposition in the pure epithelial organoids. In the model control group, the positive area ratios of α-SMA and Collagen I reached 28.5% and 24.6%, respectively, which were higher than those in the negative control group. The positive control drug SB431542 (a type I TGF-β receptor inhibitor) showed inhibition rates of 69.8% and 73.6% on α-SMA and Collagen I expression, respectively, further validating the sensitivity of this pelvic inflammatory disease fibrosis organoid model to anti-fibrotic drugs.

[0282] Within the concentration range of 12.5–200 μg / mL, the fibrosis indicators showed a significant concentration-dependent inhibition of the extracts. The inhibition rates of α-SMA and Collagen I in group 1 (12.5 μg / mL) were only 13.7% and 5.3%, respectively, indicating a weak anti-fibrotic effect. The inhibition rates in group 2 (25 μg / mL) increased to 25.3% and 26.0%, respectively; in group 3 (50 μg / mL), they further increased to 41.1% and 41.1%; in group 4 (100 μg / mL), they reached 53.3% and 52.8%; and in group 5 (200 μg / mL), they reached 58.6% and 58.5%. These results indicate that the extracts can inhibit the expression of α-SMA and Collagen I in a concentration-dependent manner in a pelvic inflammatory disease fibrosis organoid model, with the inhibition rate increasing with increasing concentration, reaching near the maximum effect at 200 μg / mL.

[0283] Representative α-SMA immunofluorescence staining images of the model control group are shown below. Figure 2 (Single-channel pseudocolor) shows that a large number of myofibroblasts are activated in the model control group.

[0284] Results of dual immunofluorescence staining for α-SMA and CK7: Both the negative control group and the model control group showed strong CK7 positive expression in their organoids, with the signal localized in the cytoplasm of epithelial cells, indicating that the organoids were composed of endometrial epithelial cells with a clear epithelial phenotype. In the model control group, α-SMA positive myofibroblasts (red fluorescence) surrounded CK7 positive epithelial cells, demonstrating the spatial relationship between myofibroblast activation and epithelial cells. The superimposed image of α-SMA and CK7 dual immunofluorescence staining in the model control group is shown below. Figure 3 .

[0285] Comprehensive analysis of cell viability and fibrosis indicators showed that *Salvia miltiorrhiza* extract could simultaneously enhance cell viability and inhibit fibrosis within a concentration range of 25–100 μg / mL, with the best effect observed at 50 μg / mL (cell viability 111.8%, α-SMA inhibition rate 41.1%, Collagen I inhibition rate 41.1%). While the fibrosis inhibition rate was highest at 200 μg / mL (α-SMA inhibition rate 58.6%, Collagen I inhibition rate 58.5%), cell viability had already returned to the model control level. These results indicate that the pelvic inflammatory disease fibrosis organoid model constructed in this invention can effectively evaluate the anti-fibrotic effect of *Salvia miltiorrhiza* extract, preliminarily confirming the application potential of this model as an evaluation platform for traditional Chinese medicine anti-fibrotic drugs, and providing a reliable tool for the subsequent evaluation of more traditional Chinese medicines and compound formulas.

[0286] The above results indicate that the pelvic fibrosis organoid model constructed in this invention can serve as an effective platform for evaluating the anti-fibrotic effects of traditional Chinese medicine, possessing advantages such as high throughput and quantification, and providing a new tool for evaluating the efficacy of traditional Chinese medicine in pelvic fibrosis organoids.

[0287] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. The above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for fabricating a pelvic fibrosis organoid model based on organoid microarrays, characterized in that, The manufacturing method includes the following steps: S1 isolates endometrial epithelial cells and fibroblasts from pathological endometrial tissue; S2 cultured and expanded the endometrial epithelial cells and the fibroblasts respectively to obtain endometrial organoids and expanded fibroblasts; S3 digests and disperses the endometrial organoids and the expanded fibroblasts respectively to obtain cell suspensions containing endometrial organoids and expanded fibroblasts respectively. S4. The cell suspension is mixed to obtain a mixed cell suspension. The mixed cell suspension is seeded onto an organoid chip equipped with multiple parallel culture units for co-culture to form a pelvic fibrosis organoid model. In S4, the mixing ratio of the number of endometrial organoid cells and the expanded fibroblasts in the mixed cell suspension is 5:1 to 10:

1. The culture medium used for the co-culture was based on DMEM / F12 and contained the following components at final concentrations: 5–20 mM nicotinamide, 2.5–10 ng / mL bFGF, 0.1–5 mM N-acetylcysteine, 1× B27 additive, 1× N2 additive, 20–100 ng / mL EGF, 0.1–2 μM PGE2, 50–200 ng / mL RSPO1, 50–200 ng / mL Wnt-3a, 50–200 ng / mL Noggin, 10–50 nM 17β-estradiol, 5–20 ng / mL FGF10, 0.1–0.5 μM MSB202190, 0.1–1 μM A83-01, 10–50 ng / mL HRG-β1, 1–5 μM Forskolin.

2. The manufacturing method according to claim 1, characterized in that, The co-culture in step S4 was carried out under dynamic fluid perfusion conditions at a perfusion rate of 5–20 μL / min.

3. The manufacturing method according to claim 1, characterized in that, Step S1 includes: The pathological endometrial tissue was washed with HBSS, cut into small pieces to form tissue fragments, and Dispase II and type IV collagenase tissue hydrolysate was added until the tissue fragments were completely covered. The enzymatic hydrolysate containing the tissue fragments was evenly distributed into a low-adsorption plate and incubated at 37±0.5℃ for 40–60 min. The solution was then ground through a 70 μm sieve to obtain a tissue suspension through enzymatic hydrolysis. Add high-glucose culture medium containing 5% FBS to the tissue suspension to terminate the enzymatic reaction, centrifuge for 3-5 minutes, and collect the endometrial cell precipitate.

4. The manufacturing method according to claim 3, characterized in that, The method for producing the endometrial organoid in step S2 includes the following steps: The endometrial cell pellet was resuspended in endometrial organoid culture medium, and matrix gel was added to a final concentration of 5% for 3D culture. The culture was incubated at 37±0.5℃ for 25-30 min. After the matrix gel solidified, endometrial organoid culture medium was added, and the culture was carried out for 10-20 days to obtain the endometrial organoid.

5. The manufacturing method according to claim 3, characterized in that, The method for preparing the expanded fibroblasts in step S2 includes the following steps: Take the enzymatic hydrolysate containing the tissue fragments from step S1 after enzymatic incubation and before sieving, centrifuge to remove the hydrolysate, resuspend the cells in DMEM / F12 fibroblast culture medium containing 5%–10% FBS, seed them in cell culture plates, and culture them in a two-dimensional adherent culture at 37°C and 5% CO2 for 3–5 days. When the cells grow to 80%–95% confluence, perform passage culture to obtain the expanded fibroblasts.

6. The manufacturing method according to claim 1, characterized in that, The co-cultivation described in step S4 includes the following steps: The number of cells in the two cell suspensions obtained in step S3 were counted respectively. Based on the counting results, the endometrial organoid cells and the expanded fibroblasts were mixed at a cell ratio of 5:1 to 10:1, and matrix gel was added to a final concentration of 2%. After mixing, the mixture was evenly seeded onto the organoid chip and cultured for 3 to 7 days to form a pelvic inflammatory disease fibrosis organoid model.

7. The manufacturing method according to claim 1, characterized in that, The culture medium used for the co-culture was based on DMEM / F12 and contained the following components at final concentrations: 15 mM nicotinamide, 5 ng / mL bFGF, 1.25 mM N-acetylcysteine, 1× B27 additive, 1× N2 additive, 50 ng / mL EGF, 0.5 μM PGE2, 100 ng / mL RSPO1, 100 ng / mL Wnt-3a, 100 ng / mL Noggin, 10 nM 17β-estradiol, 10 ng / mL FGF10, 0.5 μM SB202190, 0.5 μM A83-01, 25 ng / mL HRG-β1, 5 μM Forskolin.

8. The manufacturing method according to claim 1 or 6, characterized in that, The mixing ratio of the endometrial organoid cells and the expanded fibroblasts in the mixed cell suspension in S4 is 5:

1.

9. A pelvic inflammatory disease fibrosis organoid model, characterized in that, The organoid model is established using the fabrication method described in any one of claims 1 to 8.

10. The application of the pelvic inflammatory disease fibrosis organoid model according to claim 9 in the efficacy evaluation of traditional Chinese medicine for anti-fibrosis.

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