A method of constructing an endometriosis organoid model

Abstract

The application discloses a method for constructing an ovarian endometriosis gland epithelium organoid and stromal cell co-culture model, which comprises the following steps: obtaining an ovarian endometriosis cyst tissue sample, releasing and enriching gland epithelium cell clusters through two-step enzymatic digestion treatment; mixing the enriched gland epithelium cell clusters with Matrigel for three-dimensional culture and subculturing to the fourth generation to obtain high-purity gland epithelium organoids; collecting stromal cells for expansion culture and inoculating the stromal cells in a low-adhesion culture vessel to form three-dimensional stromal cell spheres; digesting the high-purity gland epithelium organoids into cell clusters containing 3-5 cells, mixing the stromal cells after dissociation of the three-dimensional stromal cell spheres at a ratio of 1:2, embedding the mixture in Matrigel, and using co-culture medium for three-dimensional co-culture to obtain a co-culture model. The method can effectively obtain gland epithelium cells from fibrotic tissue and remove stromal cell contamination, and the constructed model highly simulates the characteristics of in-vivo lesions in terms of cell composition and molecular phenotype.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a method for constructing organoid models of endometriosis. Background Technology

[0002] In endometriosis research, constructing organoid models that accurately simulate the pathological features of the disease is both a challenge and a key focus. Traditional animal models and in vitro cell culture methods have limitations in replicating the complexity of human diseases, failing to fully reproduce the structure, function, and microenvironment characteristics of diseased tissues, thus affecting the accuracy and effectiveness of related drug screening and mechanism studies. Therefore, a more precise and efficient method for constructing endometriosis organoid models is urgently needed to improve the success rate of model construction and more realistically reflect the pathological characteristics of the disease. Summary of the Invention

[0003] In view of this, the present disclosure provides a method for constructing an organoid model of endometriosis, which at least partially solves the problems existing in the prior art.

[0004] A method for constructing a co-culture model of glandular epithelial organoids and stromal cells in patients with ovarian endometriosis, comprising the following steps:

[0005] S1, Obtain ovarian endometriotic cyst tissue specimens and perform two-step enzymatic digestion to release and enrich glandular epithelial cell clusters from fibrotic tissue; the two-step enzymatic digestion includes: firstly, using a first enzymatic digestion solution to digest the tissue to degrade the fibrotic matrix and release glandular structures, and then using a second enzymatic digestion solution to digest the released glandular structures to achieve intercellular dissociation.

[0006] S2, enriched glandular epithelial cell clusters are mixed with matrix gel for three-dimensional culture to form glandular epithelial organoids, and passaged to the fourth generation. Differential sedimentation is used to remove the interstitial cells mixed in during passage to obtain high-purity glandular epithelial organoids.

[0007] S3, collect mesenchymal cells from step S1 and / or step S2, expand and culture them, and then seed them in a low-adhesion culture dish to form three-dimensional mesenchymal cell spheres;

[0008] S4, the high-purity glandular epithelial organoids obtained in step S2 are digested and decomposed into cell clusters containing 3-5 cells. The cell clusters are mixed with the mesenchymal cells after the three-dimensional mesenchymal cell spheres obtained in step S3 are dissociated at a ratio of 1:2, embedded in matrix gel, and three-dimensional co-culture is performed using co-culture medium to obtain a co-culture model of glandular epithelial organoids and mesenchymal cells in ovarian endometriosis.

[0009] In one specific embodiment, the two-step enzymatic digestion process in S1 is specifically performed as follows:

[0010] Step 1: Cut the ovarian endometriotic cyst tissue specimen into 1-2 mm³ tissue fragments, add the first enzymatic digestion solution, and digest in a shaker at 37°C for 40-120 minutes, blowing and oscillating every 15 minutes.

[0011] Step 2: When loose tissue and obvious release of glandular structures are observed under an inverted phase-contrast microscope, the degree of digestion is determined to meet the following conditions: if the glandular release rate is ≥80%, the first enzymatic digestion is terminated; otherwise, digestion continues and is evaluated every 10 minutes.

[0012] Step 3: Add the second enzymatic hydrolysate, TrypLE, to the digestion system and continue digestion for 10-15 minutes to achieve cell dissociation.

[0013] Step 4: Dilute the digestion product with 5 times the volume of organoid washing solution, filter through a 100 μm sieve, collect the filtrate and centrifuge to obtain epithelial cell clusters;

[0014] The first enzymatic hydrolysate is enzymatic hydrolysate D001-1 containing additive I, and the second enzymatic hydrolysate is TrypLE.

[0015] In one specific embodiment, the passage purification of the glandular epithelial organoids in S2 specifically includes:

[0016] Step 1: Use a pipette to aspirate the mixture of glandular epithelial organoids and matrix gel and transfer it into a centrifuge tube;

[0017] Step 2: Add organoid recovery solution, repeatedly blow and agitate to fully dissolve the matrix gel, and let stand for 1-2 minutes to allow the organoids to settle naturally;

[0018] Step 3: If the total number of cells in the supernatant after sedimentation is ≥1×10⁻⁶ 5 If the number of cells is less than 1, it indicates the presence of interstitial cell contamination, and the differential sedimentation process needs to be repeated until the total number of cells in the supernatant is <1×10⁻⁶. 4 indivual;

[0019] Step four: The purified organoids are re-embedded in fresh matrix gel and cultured to the fourth generation to obtain high-purity glandular epithelial organoids.

[0020] In one specific embodiment, the collection and expansion of the S3 intermediate cells specifically includes:

[0021] Step 1: Collect the undigested tissue fragments that were filtered through a 100 μm sieve in step S1, resuspend them in DMEM / F-12 medium containing 10% FBS, and inoculate them into culture dishes for 5-7 days.

[0022] Step 2: Collect the mesenchymal cells that adhere to the bottom of the glandular epithelial organoid culture system in step S2 and combine them with the cells obtained in step 1;

[0023] Step 3: If the total number of cells after merging is ≥5×10 5 If the number of cells is ≥90%, the conditions for 3D culture inoculation are met; otherwise, continue expansion culture for 1-2 generations.

[0024] Step 4: Take the expanded mesenchymal cells from passages 3-5, and use 5 × 10⁻⁶ cells per cell line. 4 -1×10 5 Seeds were placed at a density of cells / well in CSwell 600 low-adhesion culture dishes and cultured for 3-5 days to form three-dimensional mesenchymal cell spheroids.

[0025] In one specific implementation, the construction of the S4 co-cultivation model specifically includes:

[0026] Step 1: Collect the high-purity glandular epithelial organoids obtained in step S2 using organoid recovery solution, and digest them with TrypLE for 5-10 minutes.

[0027] Step 2: If the proportion of clusters of 3-5 cells in the cell clusters formed after digestion is ≥70%, then the degree of digestion is considered to meet the requirements for co-culture; otherwise, adjust the digestion time and reassess.

[0028] Step 3: The three-dimensional mesenchymal cell spheres obtained in step S3 are dissociated into single-cell suspensions using TrypLE, counted, and then mixed with the glandular epithelial cell clusters obtained in step 2 at a ratio of 1:2.

[0029] Step 4: Inoculate the mixed cell-matrix mixture into a culture plate and culture it in a co-culture medium containing organoid culture medium and mesenchymal cell culture medium at a 1:1 volume ratio for 9 days.

[0030] In one specific embodiment, the preparation and usage conditions of the co-culture medium are as follows:

[0031] Step 1: Prepare complete organoid culture medium, which includes basal culture medium for endometriosis organoids and additive I;

[0032] Step 2: Prepare complete culture medium for mesenchymal cells, wherein the culture medium is DMEM / F-12 medium containing 10% FBS;

[0033] Step 3: If the co-culture ratio of glandular epithelial organoids and mesenchymal cells deviates from the range of 1:2±10%, the ratio is deemed substandard, and the cell count needs to be readjusted and the mixture mixed.

[0034] Step 4: Mix the culture media obtained in Step 1 and Step 2 at a volume ratio of 1:1 to form a co-culture medium. Change the medium every 2 days. After culturing for 9 days, collect the co-culture model for identification.

[0035] In one specific embodiment, the criterion for determining the digestion endpoint of the first enzymatic hydrolysate in the two-step enzymatic digestion process S1 is as follows:

[0036] Step 1: When digestion has progressed to 40 minutes, the tissue morphology is observed for the first time under an inverted phase-contrast microscope.

[0037] Step 2: If the number of glandular structures released in each field of view is ≥50 and the area of ​​fibrotic matrix fragments is ≤30% of the total field of view, then digestion is considered complete.

[0038] Step 3: If the above standards are not met, continue digestion and reassess every 15 minutes, with a total digestion time not exceeding 120 minutes;

[0039] Step four: After digestion is complete, immediately add the second enzymatic hydrolysate for the second digestion step to ensure maximum yield of glandular epithelial cells.

[0040] In one specific embodiment, the specific conditions for removing interstitial cells by differential sedimentation in S2 are as follows:

[0041] Step 1: The glandular epithelial organoids passaged to the fourth generation were released from the matrix gel and resuspended in the organoid washing solution.

[0042] Step 2: Let the cell suspension stand for 1-2 minutes to allow the glandular epithelial organoids to settle naturally, and collect the precipitate.

[0043] Step 3: If the proportion of vimentin-positive cells in the precipitate is ≤5%, the removal of interstitial cells is considered to have met the standard; otherwise, repeat the sedimentation process 1-2 times.

[0044] Step four: The precipitate that has met the standards is re-embedded in the matrix gel for subsequent co-culture experiments.

[0045] In one specific embodiment, the method for culturing and identifying the three-dimensional mesenchymal cell spheres in S3 is as follows:

[0046] Step 1: The expanded 3rd-5th generation mesenchymal cells are divided into groups of 5 × 10⁻⁵ cells. 4 -1×10 5 Seeds were placed at a density of cells / well in CSwell600 low-adhesion culture dishes;

[0047] Step 2: Incubate at 37℃ in a 5% CO2 incubator for 3-5 days, and observe the formation of cell spheroids daily.

[0048] Step 3: If the diameter of the formed three-dimensional mesenchymal cell spheres is ≥100 μm and the proportion of spherical structures is ≥80%, the culture is considered successful; otherwise, adjust the seeding density or extend the culture time.

[0049] Step four: Collect successfully cultured three-dimensional mesenchymal cell spheres, perform immunohistochemical staining, and detect the expression of vimentin, ITGB3, ICAM1, MMP2, MMP9, MMP12, TIMP1, and TIMP2 to confirm the mesenchymal cell phenotype.

[0050] In one specific implementation, the method for identifying the co-culture model is as follows:

[0051] Step 1: Collect model samples from the 9th day of co-culture, and perform paraffin embedding and sectioning.

[0052] Step 2: Perform H&E staining and PAS staining on the sections to observe the morphological characteristics of glandular epithelial organoids and stromal cells and their mucus secretion.

[0053] Step 3: If the Ki67 positive cell rate in the glandular epithelial organoids is ≥30% and the stromal cell invasion depth is ≥50 μm, the model is considered to have been successfully constructed.

[0054] Step four: IHC staining was performed on the successfully constructed model to detect the expression of ER, PR, CK7, E-cadherin, CD44, ICAM1, ITGB3, MMP2, MMP9, TIMP1 and TIMP2, in order to verify the homology between the model and the primary lesion.

[0055] Compared with existing technologies, this invention has the following advantages: First, addressing the highly fibrotic characteristics of ovarian endometriotic cyst tissue, a two-step enzymatic digestion strategy is employed (first, the fibrotic matrix is ​​degraded and glandular structures are released using an enzymatic digestion solution D001-1 containing additive I, and then cell dissociation is achieved using TrypLE), effectively improving the efficiency of obtaining glandular epithelial cell clusters and cell viability, overcoming the deficiency of traditional single-step digestion methods in releasing glandular epithelial cells from fibrotic tissue. Second, differential sedimentation is introduced during organoid passage, utilizing the difference in sedimentation rates between glandular epithelial organoids and individual stromal cells for purification. Combined with vimentin staining verification, the proportion of stromal cell contamination can be controlled below 5%, significantly improving the purity of glandular epithelial organoids. Third, by co-culturing glandular epithelial organoids with pre-formed three-dimensional stromal cell spheres at an optimal ratio of 1:2, the spatial interactions between cells are re-established in the three-dimensional matrix gel. The constructed model highly mimics the characteristics of in vivo lesions in terms of cell composition, tissue structure, proliferative activity (Ki67 positive cell rate ≥30%), and molecular expression profile (expression of markers such as ER, PR, CK7, and E-cadherin is consistent with the primary lesion), providing a reliable in vitro platform for the study of the pathogenesis of endometriosis and drug screening. Fourth, the method of this invention has a complete operation procedure, clear parameters, and good reproducibility. It can successfully construct models from tissue specimens from multiple patients, demonstrating good universality and stability. Attached Figure Description

[0056] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0057] Figure 1 This is a flowchart of a method for constructing a co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis according to the present invention. Detailed Implementation

[0058] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0059] This invention relates to the field of biomedical engineering technology, specifically to a method for constructing a model of glandular epithelial organoids and stromal cells co-cultured in ovarian endometriosis. More specifically, it relates to a method for constructing a model by enriching glandular epithelial cell clusters from ovarian endometriotic cyst tissue through two-step enzymatic digestion, obtaining high-purity glandular epithelial organoids through three-dimensional culture and differential sedimentation purification, simultaneously culturing three-dimensional stromal cell spheres independently, and then co-culturing the two in a specific ratio to simulate the in vivo microenvironment of endometriosis.

[0060] This invention constructs a co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis, with ovarian endometriotic cyst tissue as the target material. Ovarian endometriotic cysts are one of the most common pathological types of endometriosis, characterized histologically by a cyst wall composed of fibrotic matrix, with clustered endometrioid glandular epithelial cells visible in the inner layer of the cyst wall, surrounded by stromal cells. Under in vivo physiological conditions, there is a close paracrine and direct contact interaction between glandular epithelial cells and stromal cells, which together constitute the cellular basis of endometriosis lesions. Glandular epithelial cells are mainly responsible for epithelial barrier function and secretory activity, while stromal cells participate in the invasion, angiogenesis, and fibrosis processes of lesions by secreting extracellular matrix components and expressing adhesion molecules and matrix metalloproteinases. However, in traditional two-dimensional cell culture systems, glandular epithelial cells and stromal cells grow in a monolayer adherent manner, resulting in loss of cell polarity and disruption of the spatial organization of cell-cell and cell-matrix interactions. This makes it impossible to reproduce the complex dialogue between glandular epithelium and stroma in the three-dimensional microenvironment in vivo. The position and role of this invention in the entire organoid construction process is as follows: First, glandular epithelial cell clusters and stromal cells are isolated from patient-derived ovarian endometriotic cyst tissue, and three-dimensional glandular epithelial organoids and three-dimensional stromal cell spheres are constructed respectively. Then, both are co-cultured in a three-dimensional matrix gel, thereby reconstructing an endometriosis lesion model with histological structure and cellular composition in vitro. This model can retain the histological characteristics, cell phenotype, and molecular expression profile of the primary lesion, providing a reliable in vitro platform for studying the pathogenesis of endometriosis and drug screening.

[0061] The infrastructure required to implement this invention includes the following: For tissue collection, an operating room or standard surgical environment equipped with sterile surgical instruments (including surgical scissors, tissue forceps, sterile containers, etc.) is needed to collect cyst tissue specimens during ovarian endometriotic cyst removal surgery. After collection, tissue specimens must be placed in phosphate-buffered saline (PBS) or organoid washing solution containing antibiotics and transported to the cell culture laboratory within 1 hour at 4°C. For cell processing, a Class II Type A2 biosafety cabinet is required for aseptic operations such as tissue mincing, enzymatic digestion, and cell seeding. A constant-temperature shaker (e.g., Thermo Scientific MaxQ 4000 or equivalent) with a constant temperature of 37°C and adjustable oscillation function, covering an oscillation speed range of 50-200 rpm, is required for the tissue enzymatic digestion process. An inverted phase-contrast microscope (e.g., Olympus CKX53 or equivalent) equipped with 10×, 20×, and 40× objectives is required for real-time observation of the degree of tissue digestion and organoid morphology. A benchtop centrifuge (e.g., Eppendorf 5810 R or equivalent) is required, equipped with both horizontal and angle rotors, with a centrifugal force range of 100-1000×g and a 4°C cryogenic control function for cell washing and collection. A CO2 cell culture incubator (e.g., Thermo Scientific FormaSteri-Cult or equivalent) is also required, with a constant temperature of 37°C, 5% CO2 concentration control, and saturated humidity environment for cell expansion and organoid culture. Regarding reagents and consumables, a dedicated organoid construction kit is required, containing components such as Enzyme Digestion Buffer I (catalog number D001-1), Organoid Washing Buffer (catalog number B004), Organoid Recovery Buffer, Matrigel (catalog number M001), Additive I (catalog number S001), and Endometriosis Organoid Culture Medium (catalog number ODM-001). TrypLE™ Express enzyme (1×, phenol red-free, Thermo Fisher Scientific, catalog number 12605028 or 12604013) is required. This is a non-animal-derived recombinant enzyme that cleaves the peptide bonds at the C-terminus of lysine and arginine, directly replacing trypsin for cell dissociation. CSwell 600 low-adhesion culture dishes (Jiyan Biotechnology, catalog number KIT000-0001) are required. These are specialized micro-culture chambers for preparing three-dimensional cell spheroids, containing a standard array of micropores for high-throughput preparation of uniformly sized three-dimensional cell spheroids. The inoculation volume of cell suspension in each CSwell 600 culture chamber is 1 mL. DMEM / F-12 medium (Gibco, catalog number 11320033) and fetal bovine serum (FBS, Gibco, catalog number 10099141) are required.A 24-well cell culture plate (Corning, catalog number 3524) and a 100 μm cell sieve (Corning, catalog number 352360) are required. For data recording and quality control, a cell counter (e.g., Countess II FL Automated CellCounter, Thermo Fisher Scientific) or a hemocytometer is needed for cell counting and viability assessment. A paraffin embedding machine, microtome (e.g., Leica RM2255), and primary antibodies (including anti-vimentin, anti-ITGB3, anti-ICAM1, anti-MMP2, anti-MMP9, anti-MMP12, anti-TIMP1, anti-TIMP2, anti-Ki67, anti-ER, anti-PR, anti-CK7, anti-E-cadherin, anti-CD44, etc.) and secondary antibodies, DAB chromogenic solution, hematoxylin-eosin (H&E) staining solution, and periodic acid-Schiff (PAS) staining solution are also required. In terms of data storage and processing, a computer with an image acquisition and analysis system is required, equipped with cell image analysis software (such as ImageJ or CellSens) for organoid morphology measurement, immunohistochemical staining positivity rate statistics, and image archiving.

[0062] The applicable operating conditions and boundary conditions for this invention are as follows. The conditions for initiating the method process of this invention are: obtaining a fresh tissue specimen confirmed by postoperative pathology as an ovarian endometriotic cyst, with a tissue specimen volume of not less than 50 mm³, an in vitro time of not more than 1 hour, and no significant signs of necrosis or infection observed by the naked eye. During tissue digestion, if the glandular release rate of the tissue specimen is still less than 80% after 120 minutes of digestion with the first enzymatic digestion solution, the tissue specimen is deemed to have excessive fibrosis and is unsuitable for subsequent organoid construction; the process is terminated and recorded as a non-compliant sample. In the cell acquisition stage, if the total number of epithelial cell clusters obtained after two enzymatic digestions is less than 1 × 10⁻⁶, the method is deemed ineffective. 5 If the number of cells is less than 100 or the cell viability is less than 70%, the cell count or viability is deemed insufficient for three-dimensional culture, and the process is terminated. In the organoid passage and purification stage, if the total cell count in the differential sedimentation supernatant remains ≥1×10⁻⁶ after four passages... 5 If the number of cells is less than 5 × 10⁶, it indicates that interstitial cell contamination cannot be effectively removed, and the purification process for that sample should be terminated. In the interstitial cell expansion stage, if the total number of cells after expansion is less than 5 × 10⁶, the purification process should be terminated. 5If the number of cells or cell viability is below 90%, the conditions for seeding three-dimensional mesenchymal cell spheres are not met, and further expansion culture is required. If the conditions are still not met after expansion to the 5th generation, the process is terminated. During co-culture, if the ratio of glandular epithelial cell clusters to mesenchymal cells deviates from 1:2 ± 10% (i.e., a ratio lower than 1:2.2 or higher than 1:1.8), the cell count must be readjusted before mixing. If the ratio still does not meet the standard after three adjustments, the co-culture experiment for that batch is terminated. When collecting the model for identification on day 9 of co-culture, if the Ki67 positive cell rate in the glandular epithelial organoids is lower than 30% or the mesenchymal cell invasion depth is lower than 50 μm, the model construction is considered substandard, and this batch of models will not be used in subsequent experiments.

[0063] The following provides a detailed description of the acquisition methods and preprocessing steps for various types of basic data used in this invention.

[0064] First, the collection and recording of tissue specimens. Tissue specimens from ovarian endometriotic cysts were obtained from patients who underwent ovarian endometriotic cyst removal surgery. The surgery was performed under sterile conditions, with a gynecologist harvesting cyst wall tissue intraoperatively. The tissue block size was approximately 5mm×5mm×5mm to 10mm×10mm×10mm. The harvested tissue specimen was immediately placed in organoid washing solution (catalog number B004) containing 1% penicillin-streptomycin (100U / mL penicillin and 100μg / mL streptomycin), placed in a 4°C insulated container, and transferred to the cell culture laboratory within 1 hour. The following information was recorded simultaneously during tissue collection: patient age, menstrual cycle stage, cyst diameter, cyst location (left or right ovary), intraoperative gross observation of cyst wall characteristics (cyst wall thickness, presence or absence of solid components, characteristics of intracystic fluid, etc.), and the histological diagnosis results from the postoperative pathology report (glandular epithelial type, stromal cell distribution, presence or absence of hemosiderin deposition, etc.). This information is recorded in a dedicated experimental record sheet, serving as the basis for subsequent experimental data traceability and quality control.

[0065] Second, the preparation of the enzyme hydrolysate and culture medium. The first enzyme hydrolysate is enzyme hydrolysate D001-1 containing additive I. Enzyme hydrolysate D001-1 is a ready-to-use solution, with a shelf life of 6 months when stored at -20℃. After thawing, it can be stored at 2-8℃ for 1 week. Before use, remove enzyme hydrolysate D001-1 from -20℃ and thaw slowly at 4℃. After thawing, gently invert and mix to avoid generating air bubbles. Additive I (product number S001) is a ready-to-use additive, stored at -20℃. When preparing the first enzyme hydrolysate, add additive I to enzyme hydrolysate D001-1 at a ratio of 1:100 (i.e., add 1 mL of additive I per 100 mL of enzyme hydrolysate D001-1), gently invert and mix, and then place on ice for later use. The function of additive I is to block apoptosis of cultured cells that have already dissociated after treatment, thereby improving cell viability and cloning efficiency. The second enzymatic hydrolysate is TrypLE™ Express enzyme (1×, phenol red-free), a ready-to-use liquid, stored at 2-8°C. Before use, remove TrypLE from 2-8°C, preheat in a 37°C water bath for 5-10 minutes, and then transfer to a biosafety cabinet for later use. Organoid washing solution (catalog number B004) is a ready-to-use solution, stored at 2-8°C, with a shelf life of 12 months. The function of the organoid washing solution is to remove impurities from the surface or surrounding areas of organoids to maintain their cleanliness and stability. Organoid recovery solution is a ready-to-use solution, stored at 2-8°C, with a shelf life of 12 months. The organoid recovery solution can rapidly dissolve matrix gel at 2-8°C, allowing for the non-destructive recovery of intact organoids. Matrix gel (catalog number M001) is a soluble basement membrane extract isolated from mouse sarcoma, rich in extracellular matrix proteins such as laminin (the main component), type IV collagen, heparan sulfate proteoglycans, nestin, and various growth factors. The substrate gel has a shelf life of 12 months when stored at -20°C, and can be maintained for 1 month when stored at 2-8°C. Before use, remove the substrate gel from -20°C and thaw it slowly at 4°C (usually requiring 12-24 hours). After thawing, aliquot it into small portions and store at -20°C, avoiding repeated freeze-thaw cycles. The substrate gel will solidify rapidly at room temperature; it should be kept on ice at all times during handling. The preparation method for the complete organoid culture medium for endometriosis is as follows: Take the basal culture medium for endometriosis organoids (catalog number ODM-001), add the appropriate proportion of additive I (usually 1:100) according to the instructions, and gently invert to mix to obtain the complete organoid culture medium. The complete stromal cell culture medium is DMEM / F-12 medium containing 10% fetal bovine serum (FBS). The preparation method is as follows: Take 500 mL of DMEM / F-12 medium, add 55 mL of FBS (final concentration 10%), add 5 mL of penicillin-streptomycin (100×, final concentration 1×), gently invert to mix, and store at 4℃ for later use. The shelf life is 4 weeks. The co-culture medium is a 1:1 volume ratio mixture of organoid complete medium and mesenchymal cell complete medium.When mixing, take equal volumes of the two culture media, gently invert them in a sterile container to mix, and use immediately. Do not store for a long time.

[0066] Third, pretreatment and quality control of reagents and consumables. All cell culture-related reagents must be batch-recorded before use, including reagent name, catalog number, batch number, expiration date, and opening date. Before each use, the appearance of the reagent must be observed; if turbidity, precipitation, or abnormal color is found, it should be discarded. Matrix gel must be validated for gel-forming ability before use: a small amount of matrix gel (approximately 50 μL) is dropped into the bottom of a pre-cooled 24-well plate and incubated at 37°C for 30 minutes. Observe whether a stable gel structure forms. If no gel forms after 30 minutes or the gel is loose and brittle, this batch of matrix gel is not suitable for organoid culture. Before use, the CSwell 600 low-adhesion culture dish must be checked for packaging integrity and sterility. This dish is suitable for three-dimensional culture of most types of stem cells and tumor cells; however, for cells with strong adhesion, it must be used in conjunction with a low-adhesion surface modification solution. The seeding volume of cell suspension in each CSwell 600 culture chamber is 1 mL. When preparing cell suspensions, multi-step dilution methods should be avoided as much as possible to ensure the accuracy of cell concentration.

[0067] Fourth, data recording and handling of missing data. Throughout the experiment, all key data must be recorded in a standardized experimental record form, including but not limited to: tissue specimen number, collection time, digestion start and end times, cell count results for each step, cell viability assessment results, digestion endpoint determination results, cell count results in differential sedimentation supernatant, organoid morphology observation results, and immunohistochemical staining results. If data from any step is missing due to operational errors or equipment malfunction, that batch of experiments will be marked as incomplete and will not be included in subsequent analysis and validation. For cell count data, if accurate counting is impossible due to the presence of large cell clumps in the cell suspension, a short digestion (2-3 minutes) using TrypLE should be performed followed by recounting. If accurate counting is still not possible, the sample will be marked as difficult to count, and the estimated range will be recorded.

[0068] The following details the theoretical basis and parameter determination method of the core construction method upon which this invention is based.

[0069] In the method of this invention, the core model is the construction of a three-dimensional model of co-culture of glandular epithelial organoids and stromal cells in ovarian endometriosis. The input variables of this model include the following categories: The first category of input variables is tissue origin variables: volume of ovarian endometriotic cyst tissue specimens (unit: mm³, range ≥50 mm³), time of in vitro tissue specimen extubation (unit: minutes, range ≤60 minutes), and degree of fibrosis of the tissue specimens (assessed by observation under an inverted phase-contrast microscope, classified as mild, moderate, and severe). The second category of input variables is enzymatic digestion parameters: digestion time of the first enzymatic hydrolysate (unit: minutes, range 40-120 minutes), digestion temperature of the first enzymatic hydrolysate (fixed at 37℃), shaking speed of the shaker (unit: revolutions / minute, range 50-150 revolutions / minute), and digestion time of the second enzymatic hydrolysate TrypLE (unit: minutes, range 10-15 minutes). The third type of input variables are cell culture parameters: passage number of glandular epithelial organoids (value is 4 passages), differential sedimentation time (unit: minutes, value range is 1-2 minutes), mesenchymal cell expansion passage number (value range is 3-5 passages), and mesenchymal cell seeding density (unit: cells / well, value range is 5×10⁻⁶). 4 -1×10 5 The input variables include: * **[number of cells / well]** * **Culturing time of three-dimensional mesenchymal cell spheres (unit: days, range 3-5 days)** * **Co-culture ratio of glandular epithelial cell clusters to mesenchymal cells (fixed at 1:2)** * **Co-culture time (fixed at 9 days)**. The fourth type of input variable is the culture medium composition: the ratio of basal medium to supplement I in the organoid complete culture medium (fixed at 100:1), and the volume ratio of organoid complete culture medium to mesenchymal cell complete culture medium in the co-culture medium (fixed at 1:1). The model's output variables include the following categories: * **Purity index of glandular epithelial organoids:** the percentage of vimentin-positive cells in the sediment after differential sedimentation (unit: %, threshold ≤5%). * **Quality index of three-dimensional mesenchymal cell sphere formation:** cell sphere diameter (unit: μm, threshold ≥100 μm), and the percentage of spherical structures (unit: %, threshold ≥80%). The third type of output variable is the success indicator of the co-culture model: the percentage of Ki67-positive cells in glandular epithelial organoids (unit: %, threshold: ≥30%) and the depth of stromal cell invasion (unit: μm, threshold: ≥50μm). The fourth type of output variable is the homology validation indicator between the model and the primary lesion: the positive expression of immunohistochemical staining for ER, PR, CK7, E-cadherin, CD44, ICAM1, ITGB3, MMP2, MMP9, TIMP1, and TIMP2.

[0070] The theoretical basis for the construction method of this invention is organoid three-dimensional culture technology and tissue engineering principles. Organoids refer to cells with stem cell potential derived from human or animal tissues placed in a hydrogel (such as matrix gel) in a three-dimensional culture system, with the addition of appropriate exogenous factors, thereby forming a tissue structure similar to the corresponding organ source, capable of simulating or reproducing a series of in vivo physiological and pathological processes in vitro. The endometriosis organoid model can retain the histological, physiological, and genetic characteristics of the original in vivo tissue. This invention makes the following improvements based on the classic organoid culture method. First, considering the highly fibrotic characteristics of ovarian endometriotic cyst tissue, a two-step enzymatic digestion strategy is adopted: the first step uses an enzymatic digestion solution D001-1 containing additive I to degrade the fibrotic matrix and release glandular structures; the second step uses TrypLE to further digest the released glandular structures to achieve intercellular dissociation, thereby maximizing the preservation of the integrity of glandular epithelial cell clusters while increasing cell yield. Second, to address the issue of mesenchymal cell contamination during the passage of glandular epithelial organoids, a differential sedimentation method is introduced for purification. This method utilizes the physical difference between glandular epithelial organoids (larger volume, faster sedimentation rate) and individual mesenchymal cells (smaller volume, slower sedimentation rate), allowing the glandular epithelial organoids to settle preferentially after a 1-2 minute settling period, thus achieving separation from the mesenchymal cells. Third, to address the problem that traditional two-dimensional co-culture cannot simulate a three-dimensional microenvironment, glandular epithelial organoids and mesenchymal cells are constructed as three-dimensional structures before co-culturing, allowing the two cell types to re-establish cell-cell and cell-matrix interactions in three-dimensional space.

[0071] The key parameters involved in this invention and the basis for their determination are as follows.

[0072] The digestion time for the first enzymatic hydrolysate was set between 40 and 120 minutes, determined based on the following criteria. Preliminary experiments were conducted on 10 ovarian endometriotic cyst tissue specimens, sampled at 30, 40, 60, 90, 120, and 150 minutes. The results showed that within 40 minutes, the glandular release rate in most specimens was below 50%, and the tissue remained as relatively intact fibrotic clumps. Between 40 and 90 minutes, the glandular release rate showed a linear upward trend, with most specimens reaching a release rate of over 80% within 60-90 minutes. Between 90 and 120 minutes, the release rate plateaued. After 120 minutes, the release rate no longer increased significantly, but the released glandular structures began to show signs of over-digestion, such as blurred edges and cell dissociation. Therefore, the digestion time for the first enzymatic hydrolysate was set at 40-120 minutes, with 40 minutes as the initial assessment time and 120 minutes as the maximum upper limit for digestion time.

[0073] The digestion time for the second enzymatic hydrolysate, TrypLE, was set within the range of 10-15 minutes. This range was determined based on the following: Preliminary experiments were conducted on the released glandular structures, digesting them with TrypLE for 5, 10, 15, and 20 minutes respectively, observing the degree of cell dissociation and cell viability. The results showed that after 5 minutes of digestion, most glandular structures remained intact, and cell dissociation was insufficient. After 10-15 minutes of digestion, the proportion of glandular structures dissociated into cell clusters containing 3-5 cells reached over 70%, and cell viability remained above 90%. After 20 minutes of digestion, although dissociation was more complete, the proportion of single cells increased significantly, and cell viability decreased to below 80%. Therefore, the TrypLE digestion time was set at 10-15 minutes to obtain cell clusters of suitable size while maintaining high cell viability.

[0074] The settling time for differential sedimentation is set within the range of 1-2 minutes, determined based on the following: After passage, glandular epithelial organoids typically present as spherical structures with a diameter of 50-200 μm, while mesenchymal cells are single cells (approximately 10-20 μm in diameter). Under gravity, the settling velocity of organoids is much greater than that of single cells. Preliminary experiments comparing the sedimentation effects after settling for 0.5, 1, 2, 3, and 5 minutes revealed that after 1-2 minutes of settling, the recovery rate of glandular epithelial organoids in the precipitate reached over 85%, while the proportion of single cells (mainly mesenchymal cells) in the supernatant reached over 90%. After settling for more than 3 minutes, some mesenchymal cells also began to settle into the precipitate, leading to a decrease in purification efficiency. Therefore, the differential sedimentation settling time was set at 1-2 minutes.

[0075] The range of mesenchymal cell seeding density is 5 × 10⁻⁶. 4 -1×10 5 The recommended cell number per well for CSwell 600 is 100-10,000 cells. Each CSwell 600 culture chamber contains 580 wells. Therefore, the total cell seeding density per CSwell 600 culture chamber should be between 100 × 580 = 58,000 and 10,000 × 580 = 5,800,000 cells. Considering the optimal cell density for mesenchymal cell spheroid formation, preliminary experiments compared a density of 3 × 10⁶ cells per well. 4 5×10 4 8×10 4 1×10 5 1.5×10 5 The sphericity of individual cells was observed at 5×10⁻⁶. 4 -1×10 5 At a seeding density of cells / well, the diameter of the formed three-dimensional mesenchymal cell spheroids can reach over 100 μm, and the proportion of spherical structures can reach over 80%; below 5 × 10⁻⁶ cells / well, the diameter of the formed spherical cell spheroids can reach over 100 μm. 4When the number of cells / well is higher than 1×10, the resulting cell spheroids are smaller (diameter <80μm); when ... 5 When the number of cells per well is low, cell spheroids are prone to fusion or central necrosis. Therefore, the mesenchymal cell seeding density is set at 5 × 10⁶ cells / well. 4 -1×10 5 One hole / hole.

[0076] The determination of the 1:2 co-culture ratio of glandular epithelial cell clusters to stromal cells was based on the following. Preliminary experiments compared the model quality after co-culturing glandular epithelial cell clusters and stromal cells at different ratios of 1:1, 1:2, 1:3, 1:4, and 2:1, using the Ki67 positive cell rate and stromal cell invasion depth as evaluation indicators. The results showed that at a 1:1 ratio, the number of stromal cells was insufficient, with an invasion depth of only about 20-30 μm; at a 1:2 ratio, the Ki67 positive cell rate reached over 30%, and the invasion depth reached over 50 μm; at 1:3 and 1:4 ratios, there were too many stromal cells, inhibiting the growth of glandular epithelial organoids, and the Ki67 positive cell rate decreased to below 20%. Therefore, the co-culture ratio was determined to be 1:2 (glandular epithelial cell clusters:stromal cells), with a permissible deviation range of ±10% (i.e., a ratio between 1:1.8 and 1:2.2 is acceptable).

[0077] The co-culture time was determined to be 9 days. Preliminary experiments compared the model status after co-culture for 3, 6, 9, 12, and 15 days. It was found that at 3 days, the glandular epithelial organoids and stromal cells had not yet established a stable interaction; at 6 days, stromal cells began to migrate into the matrix gel; at 9 days, the glandular epithelial organoids were structurally intact and proliferating actively (Ki67 positive cell rate ≥30%), and the stromal cell invasion depth reached over 50 μm, indicating optimal model maturity; after 12 days, the matrix gel began to degrade, and the organoid structure collapsed. Therefore, the co-culture time was determined to be 9 days.

[0078] The following are the quantification rules for discrete variables. In the method of this invention, the following discrete variables need to be quantified into numerical values ​​for use in the judgment steps. First, the determination of the degree of tissue digestion involves the continuous variable "glandular release rate" (unit: %), which is obtained by the ratio of the number of released glandular structures in the counting field under an inverted phase-contrast microscope to the total number of glandular structures. This is a measurable continuous variable and can be directly used for threshold comparison without discrete-to-continuous conversion. Second, the degree of tissue fibrosis is divided into three levels—mild, moderate, and severe—in the initiation judgment. However, this classification is only used for qualitative description of sample screening and does not participate in subsequent mathematical calculations, so it does not need to be quantified. Third, the "yes" and "no" of the judgment result are binary logical variables, which are directly used to determine the subsequent branch path in the process control and do not participate in numerical calculations. Fourth, the positive expression judgment of the immunohistochemical staining result is a binary variable (positive / negative), which is used for qualitative verification in model identification and does not participate in the calculation of the continuous variable formula. The above methods of handling discrete variables all meet the requirements of subsequent logical judgments and process control, and there is no situation where ordinal variables are directly substituted into the continuous variable formula.

[0079] The following is for reference Figure 1 The various steps of the method flow of the present invention are described in detail in chronological order.

[0080] S1. Obtain ovarian endometriotic cyst tissue specimens and perform two-step enzymatic digestion to release and enrich glandular epithelial cell clusters from fibrotic tissue.

[0081] The specific steps for S1 are as follows.

[0082] Step S1-1: Receiving and Pre-processing of Tissue Specimens. Remove the centrifuge tube containing the ovarian endometriotic cyst tissue specimen from the 4°C transport container and open it in a biosafety cabinet. Use sterile forceps to transfer the tissue specimen to a sterile culture dish, and add 5-10 mL of pre-cooled organoid washing solution (catalog number B004) to clean the tissue surface of blood. If there are many blood clots or mucus on the surface of the tissue specimen, wash repeatedly with organoid washing solution 2-3 times until the washing solution is mostly clear. Transfer the cleaned tissue specimen to a new sterile culture dish and use sterile surgical scissors to mince the tissue into 1-2 mm³ fragments. Keep the tissue moist during mincing; add small amounts of organoid washing solution occasionally to prevent drying. After mincing, transfer the tissue fragments to a 15 mL sterile centrifuge tube.

[0083] Steps S1-2: Preparation and addition of the first enzymatic hydrolysate. Take enzymatic hydrolysate D001-1 (product number D001-1) from -20°C and thaw slowly at 4°C. Take additive I (product number S001) from -20°C and thaw at 4°C. Add additive I to enzymatic hydrolysate D001-1 at a volume ratio of 1:100 (e.g., add 100 μL of additive I to 10 mL of enzymatic hydrolysate D001-1), gently invert to mix, and you will obtain the first enzymatic hydrolysate. Add the first enzymatic hydrolysate to a 15 mL centrifuge tube containing tissue fragments. The volume of the first enzymatic hydrolysate should be 5-10 times the volume of the tissue fragments (e.g., if the volume of the tissue fragment precipitate is approximately 1 mL, add 5-10 mL of the first enzymatic hydrolysate). Tightly cap the centrifuge tube and seal the opening with sealing film to prevent leakage.

[0084] Steps S1-3: Shaking digestion of the first enzymatic hydrolysis. Place the centrifuge tube containing tissue fragments and the first enzymatic hydrolysate in a constant-temperature shaker at 37°C and a shaking speed of 100-150 rpm. During digestion, remove the centrifuge tube from the shaker every 15 minutes, open the tube cap in a biosafety cabinet, and gently pipette 10-15 times to promote sufficient contact between the tissue fragments and the enzymatic hydrolysate and the release of glandular structures. After pipetting, tighten the tube cap again and return it to the shaker to continue digestion.

[0085] Steps S1-4: Determination of the endpoint of the first enzymatic digestion. At 40 minutes into digestion, a sample is taken for the first assessment of the degree of digestion. The sampling method is as follows: In a biosafety cabinet, open the centrifuge tube cap, use a pipette to aspirate approximately 100 μL of the digestion mixture, drop it onto a glass slide, cover with a coverslip, and observe under an inverted phase-contrast microscope. Observation indicators include: (1) the number of glandular structures released per field of view (10× objective lens, field diameter approximately 2 mm); (2) the proportion of the area of ​​fibrotic matrix fragments to the total area of ​​the field of view; and (3) the morphological integrity of the released glandular structures. The judgment criteria are as follows: If the number of glandular structures released per field of view is ≥50, and the area of ​​fibrotic matrix fragments is ≤30% of the total area of ​​the field of view, then the first enzymatic digestion is considered complete. If the above criteria are not met, digestion continues and is reassessed every 15 minutes, with a total digestion time not exceeding 120 minutes. If the above criteria are not met after 120 minutes of digestion, the tissue specimen is deemed to have excessive fibrosis and is unsuitable for subsequent organoid construction. Step S1 is terminated, and the sample is recorded as unqualified. Furthermore, the morphology of the glandular structures must be observed during digestion: if loose tissue and significant release of glandular structures are observed under an inverted phase-contrast microscope, the glandular release rate is further quantitatively assessed. The formula for calculating the glandular release rate is: Glandular release rate (%) = (Number of glandular structures released from the fibrotic matrix ÷ Total number of glandular structures in the field of view) × 100%. If the glandular release rate is ≥80%, the first enzymatic digestion is terminated; otherwise, digestion continues, with assessment every 10 minutes. The first enzymatic digestion is terminated if either of the two criteria (number of glandular structures ≥50 and fibrotic matrix fragment area ≤30%, or glandular release rate ≥80%) is met, whichever comes first.

[0086] Steps S1-5: Addition and digestion of the second enzymatic hydrolysate. Immediately after the first enzymatic digestion reaches its endpoint, add the second enzymatic hydrolysate, TrypLE™ Express enzyme (1×, phenol red-free), to the digestion system. The amount of TrypLE added should be 1 / 10 to 1 / 5 of the total volume of the digestion system (e.g., if the total volume of the digestion system is 10 mL, add 1-2 mL of TrypLE). Continue digestion in a shaker at 37°C for 10-15 minutes. The function of TrypLE is to cleave the peptide bonds at the C-terminus of lysine and arginine residues at intercellular junctions, thereby achieving cell dissociation within the glandular structure and breaking it down into cell clusters containing 3-5 cells.

[0087] Steps S1-6: Dilution and Filtration of Digestion Products. Add 5 times the volume of pre-chilled organoid washing solution (catalog number B004) to the digestion system to dilute the digestion products and terminate the enzymatic reaction. For example, if the total volume of the current digestion system is 12 mL, add 60 mL of organoid washing solution. Gently mix by pipetting. Filter the diluted digestion products through a 100 μm cell sieve to remove incompletely digested tissue fragments and fibrotic matrix residue. During filtration, gently push the tissue residue on the sieve with a sterile syringe plunger or sterile scraper to facilitate liquid passage, but avoid excessive force that could damage the sieve. Collect the filtrate in a new 50 mL sterile centrifuge tube.

[0088] Steps S1-7: Centrifugation collection of epithelial cell clusters. Place the centrifuge tube containing the filtrate in a centrifuge and centrifuge at 300×g for 5 minutes (4℃). Discard the supernatant after centrifugation; the precipitate is the enriched epithelial cell clusters. Resuspend the precipitate in 5 mL of organoid washing buffer and centrifuge again at 300×g for 5 minutes (4℃) for one wash. Discard the supernatant after washing, resuspend the precipitate in an appropriate amount of organoid washing buffer, and take 10 μL of the suspension for cell counting and viability assessment using a cell counter or hemocytometer (trypan blue staining exclusion method). Record the total number of epithelial cell clusters and cell viability. If the total number of epithelial cell clusters is ≥1×10⁻⁶, the cell count is considered positive. 5 If the cell viability is ≥70%, then step S1 is considered successful, and the process proceeds to step S2. If the condition is not met, the process terminates and is recorded as cell acquisition failure.

[0089] S2 involves mixing enriched glandular epithelial cell clusters with matrix gel for three-dimensional culture to form glandular epithelial organoids. These organoids are then passaged to the fourth generation. Differential sedimentation is used to remove interstitial cells mixed in during passage to obtain high-purity glandular epithelial organoids.

[0090] The specific steps for S2 are as follows.

[0091] Step S2-1, Initial 3D Culture of Glandular Epithelial Organoids (Passage 0). The epithelial cell cluster pellet obtained in step S1 is taken, resuspended in pre-chilled organoid complete culture medium, and the cell density is adjusted to 1×10⁻⁶. 5 -5×10 5Cells / mL. Mix the cell suspension with pre-chilled matrix gel (catalog number M001) at a volume ratio of 1:1 to 1:2 (e.g., mix 100 μL of cell suspension with 100-200 μL of matrix gel), resulting in a final matrix gel concentration of approximately 8-13 mg / mL. Mixing should be performed on ice using pre-chilled pipette tips, gently blowing to mix and avoiding air bubbles. Seed the cell-matrix mixture at a rate of 50 μL / well into preheated 24-well cell culture plates. After seeding, incubate the plates at 37°C for 30 minutes to allow the matrix gel to solidify. After gelation, add 500 μL of preheated organoid complete culture medium to each well and incubate at 37°C with 5% CO2. Change the culture medium every 2-3 days and observe organoid formation. Typically, glandular epithelial organoids with a diameter of 50-200 μm will form after 7-14 days of culture.

[0092] Step S2-2, Subculturing of Glandular Epithelial Organoids (Generations 1-4). When the glandular epithelial organoids reach a diameter of 100-200 μm or have been cultured for 10-14 days, subculturing is performed. The subculturing procedure is as follows: Aspirate the culture medium from each well of a 24-well plate using a pipette, and add 500 μL of pre-chilled organoid recovery solution to each well. Incubate the plate at 4°C for 15-30 minutes to allow the matrix gel to fully dissolve. During incubation, gently pipette every 5-10 minutes to promote matrix gel dissolution. Once microscopic observation confirms complete matrix gel dissolution, transfer the contents of each well (including organoids and recovery solution) to a 15 mL centrifuge tube. Wash each well with an appropriate amount of organoid washing solution, combining the washings in the same centrifuge tube. Centrifuge the tube at 200 × g for 3 minutes (4°C). Discard the supernatant after centrifugation; the precipitate is the recovered glandular epithelial organoid. Resuspend the pellet with TrypLE™ Express enzyme and digest at 37°C for 3-5 minutes, gently pipetting during digestion to promote organoid dissociation into small cell clusters. Stop digestion by adding 5 volumes of organoid washing buffer, centrifuge at 200×g for 3 minutes (4°C), and discard the supernatant. Resuspend the pellet in pre-chilled organoid complete medium and mix with pre-chilled matrix gel at a volume ratio of 1:1 to 1:2. Seed at 50 μL / well in new 24-well plates, gel at 37°C for 30 minutes, then add 500 μL of organoid complete medium and continue culturing. Repeat the above passage procedure until the 4th generation.

[0093] Steps S2-3: Differential sedimentation to remove mesenchymal cells. During each passage, differential sedimentation is used to remove contaminated mesenchymal cells. Specifically, after dissolving the matrix gel and recovering the organoids (i.e., after adding the organoid recovery solution and incubating to dissolve the matrix gel), the mixture containing the organoids and recovery solution is transferred to a centrifuge tube. The matrix gel is thoroughly dissolved by repeatedly pipetting. The centrifuge tube is placed at room temperature for 1-2 minutes to allow the glandular epithelial organoids to settle naturally under gravity. Avoid shaking the centrifuge tube during this period. After settling, the supernatant (containing unsettled single cells, mainly mesenchymal cells) is carefully aspirated by pipette and transferred to another centrifuge tube for later use (for mesenchymal cell collection in step S3). The precipitate is the enriched glandular epithelial organoids. 10 μL of the supernatant is used to count the total number of cells using a cell counter. If the total number of cells in the supernatant is ≥1 × 10⁻⁶... 5 If the number of cells is less than 1, it indicates significant interstitial cell contamination, and the differential sedimentation step needs to be repeated. When repeating the procedure, resuspend the precipitate in organoid washing fluid, let it stand for 1-2 minutes again, and then aspirate the supernatant and count the cells again. Repeat differential sedimentation until the total number of cells in the supernatant is <1×10⁻⁶. 4 If the total number of cells in the supernatant is <1×10⁻⁶. 4 If a certain number of cells are removed, the removal of interstitial cells is considered to have met the standard.

[0094] Steps S2-4: Verification after differential sedimentation reaches the target. The precipitate after reaching the target is resuspended in organoid washing solution. A small amount of cell suspension is taken to prepare a cell smear for vimentin immunocytochemical staining. Vimentin is a marker protein of mesenchymal cells; glandular epithelial cells do not express vimentin. The proportion of vimentin-positive cells to total cells is counted under a microscope. If the proportion of vimentin-positive cells is ≤5%, mesenchymal cell removal is considered successful; if the proportion of vimentin-positive cells is >5%, differential sedimentation needs to be repeated 1-2 times. After repetition, vimentin staining is performed again for verification until the proportion of vimentin-positive cells is ≤5%. The precipitate after reaching the target is re-embedded in fresh matrix gel and cultured to the fourth generation. After the fourth generation culture is completed, differential sedimentation purification is performed again to obtain high-purity glandular epithelial organoids. High-purity glandular epithelial organoids, after vimentin staining verification showing a vimentin-positive cell proportion of ≤5%, are used for the co-culture experiment in subsequent step S4.

[0095] S3. Collect mesenchymal cells from step S1 and / or step S2, expand and culture them, and then seed them in a low-adhesion culture dish to form three-dimensional mesenchymal cell spheres.

[0096] The specific steps for S3 are as follows.

[0097] Step S3-1: Collect mesenchymal cells from step S1. During the filtration process in step S1, undigested tissue fragments retained on the 100μm sieve are collected into a 15mL centrifuge tube using a pipette or sterile scraper. Add 5mL of DMEM / F-12 medium (complete mesenchymal cell culture medium) containing 10% FBS, and repeatedly pipette to disperse the tissue fragments. Seed the cell suspension into a T25 cell culture flask and incubate at 37℃ with 5% CO2 for 5-7 days. Change the culture medium every 2-3 days during culture. During culture, mesenchymal cells migrate from the tissue fragments and adhere to the culture vessel. After 5-7 days of culture, when the adherent cells reach 80-90% confluence, collect the cells using TrypLE digestion and perform cell counting.

[0098] Step S3-2: Collect mesenchymal cells from step S2. During the differential sedimentation process in step S2, collect the supernatant (containing unsedimented single cells, mainly mesenchymal cells) from each collection into a 15 mL centrifuge tube. Combine the supernatants from each collection, centrifuge at 300×g for 5 minutes (4℃), discard the supernatant, and the precipitate is the mesenchymal cells collected from the organoid culture system. Resuspend the precipitate in complete mesenchymal cell culture medium, seed it into T25 cell culture flasks, and incubate at 37℃ in a 5% CO2 incubator. Change the culture medium every 2-3 days during the culture period.

[0099] Step S3-3: Merging and Expanding Mesenchymal Cells. Merge the cells obtained in Step S3-1 (mesenchymal cells migrating from tissue fragments) and the cells obtained in Step S3-2 (mesenchymal cells collected from the bottom of the organoid culture system). Resuspend the merged cells in complete mesenchymal cell culture medium and seed them into T25 or T75 culture flasks for further expansion. Change the culture medium every 2-3 days. When the cells reach 80-90% confluence, passage them. For passage, digest with TrypLE for 3-5 minutes, add an equal volume of complete mesenchymal cell culture medium to stop digestion, centrifuge at 300×g for 5 minutes, discard the supernatant, resuspend the pellet in fresh complete mesenchymal cell culture medium, and divide into flasks at a 1:2 or 1:3 ratio for further culture.

[0100] Steps S3-4: Assessment of mesenchymal cell number and viability. At each passage, 10 μL of cell suspension is stained with trypan blue for cell counting and viability assessment. The total cell count and cell viability are recorded (trypan blue exclusion method: live cells do not stain, dead cells stain blue). When the total cell count after confluence is ≥5 × 10⁻⁶, the cell count is considered viable. 5 When the number of cells is ≥90%, the seeding conditions for three-dimensional mesenchymal cell culture are considered met. If the conditions are not met, continue expansion and culture for 1-2 generations and then reassess. If the conditions are still not met after expansion to the 5th generation, the batch of mesenchymal cells is deemed unsuitable for three-dimensional mesenchymal cell culture, and the process is terminated.

[0101] Steps S3-5: Formation and culture of three-dimensional mesenchymal cell spheres. Take the expanded mesenchymal cells from passages 3-5, prepare cell suspensions using complete mesenchymal cell culture medium, and adjust the cell density to 5 × 10⁻⁵. 4 -1×10 5 Cells / mL. Take a CSwell 600 low-adhesion culture dish (Jiyan Biotechnology, catalog number KIT000-0001) and add 1 mL of cell suspension to each culture chamber. The CSwell 600 culture dish contains a standard array of microwells, each well can hold 100-10000 cells. Seeding density: 5 × 10⁻⁶ cells / mL. 4 -1×110 5 Cells / well corresponds to approximately 86-172 cells per microwell (based on 580 microwells per well). Incubate the seeded CSwell 600 culture dishes at 37°C in a 5% CO2 incubator for 3-5 days. Observe cell spheroid formation daily under an inverted phase-contrast microscope.

[0102] Steps S3-6: Quality assessment of three-dimensional mesenchymal cell spheres. After culturing for 3-5 days, observe and assess the formation quality of three-dimensional mesenchymal cell spheres under an inverted phase-contrast microscope. Assessment indicators include: (1) Cell sphere diameter—randomly select 10 cell spheres to measure their diameter and calculate the average value; (2) Spherical structure percentage—count the proportion of spherical structures (defined as cell aggregates with clear boundaries and regular spherical shapes) to the total cell aggregates in 10 fields of view. If the diameter of the formed three-dimensional mesenchymal cell spheres is ≥100μm and the spherical structure percentage is ≥80%, the three-dimensional mesenchymal cell spheres are considered to have been successfully cultured. If the above criteria are not met, adjust the seeding density (increase to 8×10⁻⁶). 4 -1.5×10 5 Alternatively, extend the culture time to 5-7 days and reassess. If the standard is still not met after adjustment, the batch of three-dimensional mesenchymal cell spheroids is considered a failure and will not be used for subsequent co-culture experiments.

[0103] Steps S3-7: Phenotypic identification of three-dimensional mesenchymal cell spheres. Collect successfully cultured three-dimensional mesenchymal cell spheres and perform immunohistochemical staining to confirm the mesenchymal cell phenotype. The identification method is as follows: fix the three-dimensional mesenchymal cell spheres with 4% paraformaldehyde, embed them in paraffin, section them (4-5 μm thick), and perform immunohistochemical staining to detect the expression of the following markers: vimentin (mesenchymal cell marker), ITGB3 (integrin β3, involved in cell adhesion and migration), ICAM1 (intercellular adhesion molecule 1, involved in inflammation and adhesion), MMP2 (matrix metalloproteinase 2, involved in matrix degradation), MMP9 (matrix metalloproteinase 9, involved in matrix degradation), MMP12 (matrix metalloproteinase 12, involved in matrix remodeling), TIMP1 (tissue inhibitor of matrix metalloproteinase 1), and TIMP2 (tissue inhibitor of matrix metalloproteinase 2). If vimentin is positively expressed, and at least two of MMP2, MMP9, and MMP12 are positively expressed, the mesenchymal cell phenotype is confirmed. The qualified three-dimensional mesenchymal cell spheres were used for the co-culture experiment in the subsequent S4 step.

[0104] S4, the high-purity glandular epithelial organoids obtained in step S2 are digested and decomposed into cell clusters containing 3-5 cells. The cell clusters are mixed with the mesenchymal cells after the three-dimensional mesenchymal cell spheres obtained in step S3 are dissociated at a ratio of 1:2, embedded in matrix gel, and three-dimensional co-culture is performed using co-culture medium to obtain a co-culture model of glandular epithelial organoids and mesenchymal cells in ovarian endometriosis.

[0105] The specific steps for S4 are as follows.

[0106] Step S4-1: Recovery and digestion of high-purity glandular epithelial organoids. Take the fourth-generation high-purity glandular epithelial organoids obtained in step S2 (purified by differential sedimentation and verified by vimentin staining, with vimentin-positive cells ≤5%) and aspirate the culture medium from each well of a 24-well plate. Add 500 μL of pre-chilled organoid recovery solution to each well and incubate the plate at 4°C for 15-30 minutes to allow the matrix gel to fully dissolve. During incubation, gently pipette every 5-10 minutes to promote matrix gel dissolution. Once the matrix gel is confirmed to be completely dissolved under a microscope, transfer the contents of each well to a 15 mL centrifuge tube. Wash each well with organoid washing buffer, combining the washes into the same centrifuge tube. Centrifuge at 200×g for 3 minutes (4°C) and discard the supernatant. Add 1-2 mL of TrypLE™ Express enzyme (1×) to the precipitate and digest at 37°C for 5-10 minutes. During digestion, gently pipette every 2-3 minutes to promote organoid dissociation.

[0107] Step S4-2: Determining the degree of glandular epithelial cell digestion. After digestion for 5-10 minutes, take 10 μL of the digestion product and observe the size distribution of cell clusters under an inverted phase-contrast microscope. Count the number of clusters containing 3-5 cells in 100 cell aggregates. If the proportion of clusters containing 3-5 cells is ≥70%, the degree of digestion is considered to meet the co-culture requirements. If the proportion is <70%, continue digestion for 2-3 minutes and reassess. If the total digestion time exceeds 15 minutes and still does not meet the requirements, stop digestion and record the degree of digestion as substandard. After digestion is considered satisfactory, add 5 times the volume of organoid washing solution to terminate digestion, centrifuge at 200×g for 3 minutes (4℃), and discard the supernatant. The precipitate is the digested glandular epithelial cell cluster (containing 3-5 cells), resuspend it in pre-chilled organoid complete culture medium, and keep it on ice for later use. Take 10 μL for cell counting and record the total number of glandular epithelial cell clusters.

[0108] Step S4-3: Dissociation of three-dimensional mesenchymal cell spheroids. Take the successfully cultured three-dimensional mesenchymal cell spheroids obtained in step S3 (diameter ≥100μm, spherical structure ratio ≥80%, phenotypic identification qualified), and transfer the cell spheroids from the CSwell 600 culture dish to a 15mL centrifuge tube using a pipette. Centrifuge at 200×g for 3 minutes (4℃), and discard the supernatant. Add 1-2mL of TrypLE™ Express enzyme (1×) to the pellet, and digest at 37℃ for 5-10 minutes, gently pipetting during digestion to promote dissociation of the cell spheroids into a single-cell suspension. After confirming complete dissociation of the cell spheroids into single cells under an inverted phase-contrast microscope, add 5 times the volume of complete mesenchymal cell culture medium to stop the digestion. Centrifuge at 300×g for 5 minutes (4℃), and discard the supernatant. The pellet is the dissociated mesenchymal cell single-cell suspension, which should be resuspended in complete mesenchymal cell culture medium. Take 10 μL of the sample and count the cells using a cell counter. Record the total number of mesenchymal cells and cell viability (trypan blue staining). Cell viability must be ≥90% before it can be used for subsequent co-culture.

[0109] Step S4-4: Adjustment and mixing of cell ratios. Based on the glandular epithelial cell cluster count results from Step S4-2 and the stromal cell count results from Step S4-3, mix the cells at a ratio of glandular epithelial cell clusters:stromal cells = 1:2. Specifically, take a suspension containing N glandular epithelial cell clusters and a suspension containing 2N stromal cells, and combine them in a sterile centrifuge tube. If the ratio deviates from 1:2 ± 10% (i.e., the actual ratio is lower than 1:2.2 or higher than 1:1.8), the ratio is considered substandard, and the cell count needs to be readjusted and the cells mixed. The adjustment method is as follows: calculate the number of cells needed to be added based on the actual ratio, add the corresponding number of cells, and then count again to confirm the ratio. If the ratio is still substandard after three adjustments, terminate the co-culture experiment for that batch.

[0110] Steps S4-5: Preparation and inoculation of the cell-Matrix gel mixture. Centrifuge the mixed glandular epithelial cell clusters and mesenchymal cells at 300×g for 5 minutes (4°C), and discard the supernatant. Resuspend the pellet in pre-chilled organoid complete culture medium, adjusting the total volume to an appropriate level (e.g., 100-200 μL). Mix the cell suspension with pre-chilled matrix gel at a volume ratio of 1:1 to 1:2, resulting in a final cell density of approximately 1×10⁻⁶ cells / year. 5 -5×10 5 Cells / mL (approximately one-third of which are glandular epithelial cells and two-thirds are stromal cells). Mix on ice, gently pipetting with a pre-chilled pipette tip to avoid air bubbles. Seed the cell-Matrix gel mixture at 50 μL / well into preheated 24-well cell culture plates. After seeding, incubate the plates at 37°C for 30 minutes to allow the matrix gel to solidify.

[0111] Steps S4-6: Addition and incubation of co-culture medium. After gel formation, add 500 μL of co-culture medium to each well. The co-culture medium is a 1:1 volume mixture of organoid complete medium and mesenchymal cell complete medium. Incubate the culture plates at 37°C in a 5% CO2 incubator, changing the co-culture medium every 2 days. The total co-culture time is 9 days.

[0112] Steps S4-7: Collection and preliminary observation of the co-culture model. On day 9 of co-culture, observe the morphological characteristics of the co-culture model under an inverted phase-contrast microscope, including the structural integrity of glandular epithelial organoids and the distribution and migration of stromal cells in the matrix gel. Collect samples from the co-culture model after observation for subsequent identification.

[0113] Identification method for co-culture models.

[0114] The identification of the co-culture model was carried out according to the following steps.

[0115] Step M1: Sample Fixation and Embedding. Collect model samples from day 9 of co-culture and carefully aspirate the culture medium from each well of a 24-well plate using a pipette. Add 500 μL of 4% paraformaldehyde fixative (tissue fixative, catalog number B003) to each well and fix at room temperature for 24 hours. After fixation, wash three times with PBS for 10 minutes each time. Embed the fixed samples in paraffin: sequentially dehydrate with a gradient of ethanol (70% ethanol for 30 minutes, 80% ethanol for 30 minutes, 90% ethanol for 30 minutes, 95% ethanol for 30 minutes, 100% ethanol for 45 minutes × 2 times), clear with xylene (15 minutes × 2 times), and then embed using a paraffin embedding machine. Store the embedded paraffin blocks at 4°C for later use.

[0116] Step M2: Sectioning and Staining. Continuously section the paraffin block using a microtome to a thickness of 4-5 μm. Mount the sections onto a detachable glass slide and bake at 60°C for 2 hours. After dewaxing to water, perform H&E staining and PAS staining. The H&E staining procedure is as follows: hematoxylin staining for 5 minutes, rinsing with running water for 5 minutes, differentiation with 1% hydrochloric acid ethanol for 3 seconds, rinsing with running water for 5 minutes, eosin staining for 2 minutes, dehydration with graded ethanol, clearing with xylene, and mounting with neutral resin. The PAS staining procedure is as follows: periodic acid oxidation for 10 minutes, Schiff's reagent staining for 15 minutes, rinsing with running water for 10 minutes, counterstaining with hematoxylin for 1 minute, rinsing with running water for 5 minutes, dehydration with graded ethanol, clearing with xylene, and mounting with neutral resin. After staining, observe the morphological characteristics and mucus secretion of glandular epithelial organoids and stromal cells under a microscope. Under H&E staining, glandular epithelial organoids should appear as glandular lumen-like structures surrounded by columnar or cuboidal epithelial cells, with round or oval nuclei located at the cell base; stromal cells should be spindle-shaped or stellate, distributed in the stroma surrounding the glandular epithelial organoids. Under PAS staining, the glandular lumen of the glandular epithelial organoids should show a purplish-red positive staining, indicating the presence of mucus secretion.

[0117] Step M3: Ki67 proliferation activity assessment and invasion depth measurement. Ki67 immunohistochemical staining was performed on the co-culture model sections. The Ki67 staining procedure was as follows: dewaxing the sections to water, antigen retrieval (citrate buffer, microwave retrieval for 15 minutes), blocking endogenous peroxidase with 3% H2O2 for 10 minutes, blocking with 5% goat serum for 30 minutes, adding anti-Ki67 primary antibody (1:200 dilution), incubating overnight at 4°C, washing three times with PBS, adding HRP-labeled secondary antibody, incubating at room temperature for 30 minutes, washing three times with PBS, developing with DAB, counterstaining with hematoxylin, and mounting. The proportion of Ki67-positive cells (nuclei stained brown) in the total number of glandular epithelial organoids was counted under a microscope. If the Ki67-positive cell rate was ≥30%, the glandular epithelial organoid proliferation activity was considered satisfactory. Simultaneously, the maximum distance (in μm) of stromal cells invading the stroma from the stromaglio-glandular organoid interface was measured under a microscope. If the depth of mesenchymal cell invasion is ≥50μm, the mesenchymal cell invasion activity is considered to have met the standard. If the Ki67 positive cell rate is ≥30% and the depth of mesenchymal cell invasion is ≥50μm, the model is considered to have been successfully constructed.

[0118] Step M4: Homology verification of primary lesions. Immunohistochemical staining was performed on the successfully constructed model to detect the expression of the following markers: ER (estrogen receptor), PR (progesterone receptor), CK7 (cytokeratin 7, glandular epithelial marker), E-cadherin (E-cadherin, epithelial marker), CD44 (cell adhesion molecule), ICAM1 (intercellular adhesion molecule 1), ITGB3 (integrin β3), MMP2 (matrix metalloproteinase 2), MMP9 (matrix metalloproteinase 9), TIMP1 (tissue inhibitor of metalloproteinases 1), and TIMP2 (tissue inhibitor of metalloproteinases 2). The immunohistochemical staining procedure was the same as in step M3. After staining, the immunohistochemical expression profile of the model was compared with that of primary ovarian endometriotic cyst tissue. If the model expression profile is basically consistent with that of the primary tissue (ER, PR, CK7, and E-cadherin are positively expressed in glandular epithelium, vimentin is positively expressed in stromal cells, MMP2 and MMP9 are positively expressed in stromal cells, and TIMP1 and TIMP2 are positively expressed in stromal cells), then the model is verified to have homology with the primary lesion.

[0119] Example 1

[0120] The method of the present invention will be described in detail below through a specific embodiment.

[0121] The ovarian endometriotic cyst tissue specimen used in this embodiment came from a 32-year-old female patient who underwent laparoscopic ovarian cystectomy at a tertiary hospital for bilateral ovarian endometriotic cysts. During the operation, a piece of tissue from the right ovarian cyst wall, approximately 8mm × 8mm × 6mm in size, was excised. Macroscopically, the cyst wall was about 3-5mm thick, with a dark red inner wall and visible chocolate-like cyst contents. Immediately after excision, the tissue was placed in organoid washing solution (catalog number B004) containing 1% penicillin-streptomycin, in a 4°C insulated container, and transported to the cell culture laboratory within 45 minutes.

[0122] In a biosafety cabinet, tissue specimens were transferred to sterile culture dishes and washed three times with 10 mL of pre-cooled organoid washing solution to remove surface blood. The tissue was then minced using sterile surgical scissors to approximately 1-2 mm³ fragments, yielding approximately 1.2 mL of tissue fragment precipitate. The tissue fragments were transferred to 15 mL centrifuge tubes, and 10 mL of the first enzymatic digestion solution (enzymatic digestion solution D001-1 and additive I mixed at a 100:1 volume ratio) was added. The centrifuge tubes were placed in a constant-temperature shaker at 37°C and 120 rpm for digestion. At 40 minutes of digestion, the first sample was observed. Microscopically, approximately 35 glandular structures were released per field of view, and fibrotic matrix fragments occupied approximately 45% of the field of view, indicating that digestion had not yet reached its endpoint. Digestion continued, with assessment every 15 minutes. At 75 minutes of digestion, samples were taken again for observation. Microscopically, approximately 62 glandular structures were released per field of view, with fibrotic matrix fragments occupying about 25% of the field, reaching the digestion endpoint (≥50 glandular structures and ≤30% fibrotic matrix fragments). Immediately, 1.5 mL of TrypLE™ Express enzyme (1×) was added, and digestion continued at 37°C with shaking for 12 minutes. After digestion, 60 mL of pre-chilled organoid washing buffer was added for dilution, and the mixture was filtered through a 100 μm sieve. The filtrate was collected and centrifuged at 300×g for 5 minutes. The precipitate was resuspended and washed with 5 mL of organoid washing buffer, and centrifuged again. The final product was an epithelial cell cluster pellet, counted at 2.3 × 10⁻⁶. 5 The cells were tested, and trypan blue staining showed that the cell viability was 92%.

[0123] Epithelial cell clusters were resuspended in pre-chilled organoid complete medium to a final volume of 200 μL, mixed with 200 μL of pre-chilled matrix gel, and seeded at 50 μL / well in 24-well plates (8 wells total). After gelation at 37°C for 30 minutes, 500 μL of organoid complete medium was added, and the plates were incubated at 37°C in a 5% CO2 incubator. Small cell clusters were observed to form on day 3, organoids with a diameter of approximately 80-120 μm were observed on day 7, and the organoids reached a diameter of 120-180 μm on day 10, at which point the first passage was performed.

[0124] During subculturing, the culture medium was aspirated, and 500 μL of pre-chilled organoid recovery solution was added to each well. The mixture was incubated at 4°C for 20 minutes to dissolve the matrix gel. The contents of each well were transferred to centrifuge tubes and centrifuged at 200 × g for 3 minutes. The precipitate was digested with 1 mL of TrypLE at 37°C for 4 minutes, and digestion was terminated by adding 5 mL of organoid washing solution. After centrifugation, the precipitate was resuspended in complete organoid culture medium, mixed with matrix gel, and re-inoculated. After matrix gel dissolution and organoid recovery, differential sedimentation was performed: the centrifuge tubes were allowed to stand for 1.5 minutes, and the supernatant was aspirated into another centrifuge tube. The precipitate was used for subculturing. The supernatant count was 8.7 × 10⁻⁶. 4 Cells, less than 1×10 5Therefore, repeated differential sedimentation is unnecessary. However, according to standard procedures, vimentin staining verification is still required. A small amount of the precipitate is used to prepare a cell smear, which is then stained with vimentin immunocytochemistry and counted. The percentage of vimentin-positive cells is approximately 3.8%, ≤5%, which meets the standard. The precipitate that meets the standard is then re-embedded in Matrigel and cultured again. The above passage operation is repeated, with differential sedimentation and vimentin verification performed at each passage, until the fourth generation is reached. After the fourth generation is completed, differential sedimentation purification is performed again, reducing the percentage of vimentin-positive cells to 1.2%, resulting in high-purity glandular epithelial organoids.

[0125] Meanwhile, mesenchymal cells were collected from the 100 μm sieve residue in step S1. The tissue fragments on the sieve were resuspended in 5 mL of DMEM / F-12 medium containing 10% FBS and seeded into T25 culture flasks. After 6 days of culture, when the adherent cells reached 85% confluence, they were collected by TrypLE digestion and counted at 1.8 × 10⁻⁶ cells / mL. 5 2.5 × 10⁶ cells. Mesenchymal cells were collected from the supernatant of each differential sedimentation in step S2, combined, centrifuged, and the pellet resuspended in complete mesenchymal cell culture medium. The pellets were then seeded into T25 culture flasks and cultured for 5 days before collection, with a cell count of 2.5 × 10⁶ cells. 5 The total number of cells after merging the two sources was 4.3 × 10⁻⁶ cells. 5 1, not reaching 5×10 5 The cell count was determined based on the required number of cells. The cell culture was expanded for one more generation, and the cells were counted again after 5 days. The total number of cells was 7.1 × 10⁻⁶. 5 The cells were counted, and trypan blue staining showed a cell viability of 94%, meeting the seeding conditions for three-dimensional mesenchymal cell culture. Fourth-generation mesenchymal cells were collected, and a cell suspension was prepared using complete mesenchymal cell culture medium, adjusted to a density of 8 × 10⁶ cells / mL. 4 Cells / mL. Take a CSwell 600 low-adhesion culture dish and add 1 mL of cell suspension to each chamber (equivalent to 8 × 10⁶ cells per chamber). 4 Approximately 138 cells per well were incubated at 37°C in a 5% CO2 incubator. On day 4, observation under an inverted phase-contrast microscope revealed the formation of numerous well-defined, regularly spherical three-dimensional cell spheroids. Ten cell spheroids were randomly selected and their diameters measured; the average diameter was 135 μm. Counting was performed in 10 fields of view, and the proportion of spherical structures was 88%, meeting the criteria of a diameter ≥100 μm and a spherical structure proportion ≥80%. Immunohistochemical staining of the three-dimensional mesenchymal cell spheroids showed strong positive results for vimentin, MMP2, MMP9, and MMP12, and positive results for TIMP1 and TIMP2, confirming the mesenchymal cell phenotype.

[0126] Fourth-generation high-purity glandular epithelial organoids were collected, recovered with organoid recovery buffer, and then digested at 37°C for 8 minutes with 1.5 mL of TrypLE. The digested products were observed under a microscope; clusters containing 3-5 cells accounted for 76%, meeting the ≥70% digestion requirement. Digestion was terminated by adding organoid washing buffer, and the tissues were centrifuged and resuspended, with a count of 4.2 × 10⁻⁶ cells. 4 A cluster of glandular epithelial cells. Three-dimensional stromal cell spheroids were collected and digested with TrypLE at 37°C for 8 minutes until completely dissociated into single cells; the cell count was 8.6 × 10⁻⁶. 4 The number of mesenchymal cells was 4.2 × 10⁶, with a cell viability of 93%. The ratio of glandular epithelial cell clusters to mesenchymal cells was 4.2 × 10⁶. 4 8.6×10 4 The ratio was approximately 1:2.05, within the range of 1:2 ± 10% (i.e., 1:1.8 to 1:2.2), meeting the target ratio. The two cell lines were combined, centrifuged at 300×g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in pre-chilled organoid complete medium to 150 μL. This was mixed with 150 μL of pre-chilled matrix gel and seeded at 50 μL / well in a 24-well plate (6 wells total). After gelation at 37°C for 30 minutes, 500 μL of co-culture medium (a 1:1 mixture of organoid complete medium and mesenchymal cell complete medium) was added. The plates were incubated at 37°C in a 5% CO2 incubator, with the co-culture medium changed every 2 days. Model samples were collected on day 9 of co-culture.

[0127] On day 9 of co-culture, observation under an inverted phase-contrast microscope revealed that the glandular epithelial organoids exhibited a glandular lumen-like structure with intact morphology. Mesenchymal cells migrated radially from the periphery of the organoids into the stroma. Samples were collected, embedded in paraffin, and sectioned. H&E staining showed that the glandular epithelial organoids consisted of columnar epithelial cells surrounding a glandular lumen, with the cell nuclei located at the base. Mesenchymal cells were distributed in a spindle shape within the surrounding stroma. PAS staining showed a purplish-red positive staining within the lumen. Ki67 immunohistochemical staining showed that the Ki67-positive cell rate in the glandular epithelial organoids was 38%, ≥30%. The maximum invasion depth of the mesenchymal cells was measured to be 62 μm, ≥50 μm. The model was successfully constructed. Further immunohistochemical staining showed that ER, PR, CK7, and E-cadherin were positive in glandular epithelium, vimentin was positive in stromal cells, MMP2 and MMP9 were positive in stromal cells, TIMP1 and TIMP2 were positive in stromal cells, and CD44, ICAM1, and ITGB3 were also positively expressed. These findings were consistent with the immunohistochemical expression profile of primary ovarian endometriotic cyst tissue, verifying the homology between the model and the primary lesion.

[0128] Example 2

[0129] This embodiment illustrates the effect of different first enzymatic digestion times on the efficiency of glandular epithelial cell acquisition in the method of the present invention.

[0130] Three ovarian endometriotic cyst tissue specimens (numbered EM-01, EM-02, and EM-03) were collected. Each specimen was processed according to step S1 of this invention, but the first enzymatic digestion time was set to 60 minutes (EM-01), 90 minutes (EM-02), and 120 minutes (EM-03), respectively, with all other conditions identical (the second enzymatic digestion, TrypLE digestion time, was 12 minutes for all specimens, and the shaking speed was 120 rpm for all specimens). Results showed that after 60 minutes of digestion, the glandular release rate of EM-01 was 72%, which did not reach the 80% release rate standard. Digestion was continued for 85 minutes to reach the endpoint, ultimately yielding 1.7 × 10⁻⁶ epithelial cell clusters. 5 The activity rate was 89%; after 90 minutes of digestion, EM-02 achieved a glandular release rate of 87%, reaching the endpoint, yielding 2.5 × 10⁶ epithelial cell clusters. 5 One cell was obtained, with a viability of 91%; after 120 minutes of digestion, EM-03 showed a glandular release rate of 92%, yielding 2.8 × 10⁶ epithelial cell clusters. 5 However, the viability decreased to 84%. The first enzymatic digestion time for all three specimens was within the 40-120 minutes range specified in this invention, and all successfully yielded epithelial cell clusters (≥1×10⁶) that met the requirements for subsequent culture. 5 (and the cell viability ≥ 70%). However, the cell viability of EM-03 is relatively low, suggesting that the digestion time should not be prolonged too much, and about 90 minutes is the better choice.

[0131] Example 3

[0132] This embodiment verifies the effectiveness of the differential sedimentation method for removing interstitial cells in this invention.

[0133] Cell suspensions of fourth-generation glandular epithelial organoids (without differential sedimentation purification) were collected and subjected to differential sedimentation as described in step S2 of this invention. After allowing the cell suspension to stand for 1.5 minutes, the precipitate and supernatant were collected separately. Cell counting and vimentin immunocytochemical staining were performed on the precipitate and supernatant, respectively. The results showed that the total number of cells in the precipitate was 1.4 × 10⁻⁶. 6 The percentage of vimentin-positive cells was 18.5%; the total number of cells in the supernatant was 3.2 × 10⁻⁶. 5The percentage of vimentin-positive cells was 92.3%, indicating that differential sedimentation can remove approximately 92% of the mesenchymal cells by allowing them to enter the supernatant. After resuspending the precipitate, differential sedimentation was repeated once. After the second sedimentation, the percentage of vimentin-positive cells in the precipitate decreased to 4.2%, meeting the ≤5% standard. Differential sedimentation purification was performed on three consecutive batches of fourth-generation glandular epithelial organoids, and the average percentage of vimentin-positive cells decreased from 21.3% before purification to 3.6% after purification (after two sedimentation cycles), demonstrating that differential sedimentation can effectively remove mesenchymal cells contaminated during passage.

[0134] Example 4

[0135] This embodiment illustrates the morphological and functional changes of the co-culture model of the present invention at different culture time points.

[0136] A co-culture model was constructed according to step S4 of this invention. Samples were collected on days 3, 6, 9, and 12 of co-culture for H&E staining, Ki67 immunohistochemical staining, and measurement of stromal cell invasion depth. Results showed that on day 3, the glandular epithelial organoid structure was incomplete, with some organoid edges blurred. Stromal cells mainly clustered around the glandular epithelial organoids, with no obvious intrastromal migration observed. The Ki67-positive cell rate was 12%, and the invasion depth was 8 μm. On day 6, the glandular epithelial organoid structure became more complete, the glandular lumen was clearly formed, and stromal cells began to migrate into the surrounding stroma. The Ki67-positive cell rate was 24%, and the invasion depth was 32 μm. On day 9, the glandular epithelial organoid structure was complete, the glandular lumen was clear, and stromal cells migrated radially into the stroma. The Ki67-positive cell rate was 36%, and the invasion depth was 58 μm. On day 12, some of the matrix gel degraded, the glandular epithelial organoid structure began to collapse, the Ki67 positive cell rate decreased to 28%, and the invasion depth was 65 μm (migration had reached the edge of the matrix gel). These results validate the rationality of determining the co-culture time to be 9 days in this invention; day 9 is the optimal time point for model maturity.

[0137] Implementation Results and Effect Verification

[0138] The co-culture model of glandular epithelial organoids and stromal cells for ovarian endometriosis constructed in this invention was compared with the following three control models: (1) glandular epithelial organoids cultured alone (without stromal cells); (2) two-dimensional co-culture of glandular epithelial organoids and stromal cells (the two cell types were mixed and cultured on the surface of a culture plate); (3) three-dimensional co-culture of glandular epithelial organoids and single-cell suspensions of stromal cells that had not formed three-dimensional cell spheres (stromal cells were not pre-cultured into spheres using CSwell 600). Three replicate samples were set for each group, and identification was performed after 9 days of co-culture.

[0139] The results showed that the Ki67 positive cell rate of the model of this invention (co-culture of glandular epithelial organoids and three-dimensional mesenchymal cell spheres) was 36.7%±4.2% (mean±standard deviation, n=3), which was significantly higher than that of glandular epithelial organoids alone (18.3%±3.1%, p<0.01), two-dimensional co-culture (22.5%±3.8%, p<0.05), and three-dimensional co-culture of mesenchymal cell single-cell suspensions (26.1%±4.0%, p<0.05). The mesenchymal cell invasion depth of the model of this invention was 61.3±8.7 μm, which was significantly higher than that of two-dimensional co-culture (18.5±5.2 μm, p<0.001) and three-dimensional co-culture of mesenchymal cell single-cell suspensions (35.7±6.9 μm, p<0.01). The positive expression rates of ER, PR, CK7, and E-cadherin in the glandular epithelial organoids of this invention were all ≥80%, showing the highest consistency (92%) with the expression profile of primary tissues, while the consistency of the two-dimensional co-culture model was only 58%. These validation data demonstrate that this invention, by co-culturing glandular epithelial organoids with pre-formed three-dimensional stromal cell spheres at a 1:2 ratio, can better simulate the microenvironment of endometriosis lesions in vivo, promote the proliferation of glandular epithelial organoids and the invasion of stromal cells, while better preserving the molecular phenotypic characteristics of the primary lesions.

[0140] Regarding the efficiency of the method, the total time from receiving tissue specimens to obtaining the co-culture model is approximately 35-42 days (including 1 day for tissue digestion, approximately 28 days for passage of glandular epithelial organoids to the fourth generation, approximately 10-14 days for stromal cell expansion, 3-5 days for three-dimensional stromal cell spheroid culture, and 9 days for co-culture). If only the time from the start of co-culture of the fourth-generation glandular epithelial organoids and expanded stromal cells to model identification is considered, the co-culture and identification phase takes approximately 12-14 days. Regarding stability, the method of this invention was used to construct models from ovarian endometriotic cyst tissue specimens from 5 different patients. All 5 cases successfully obtained co-culture models (100% success rate). The Ki67 positive cell rate ranged from 32% to 41% in each model, and the stromal cell invasion depth ranged from 53 to 72 μm, showing good consistency.

[0141] The applicable scope of this invention is as follows: For ovarian endometriotic cyst tissue specimens, the model construction effect is optimal when the cyst wall thickness is moderate (3-8 mm) and the degree of fibrosis is moderate (the glandular release rate can reach more than 80% within 40-120 minutes). For specimens with excessively thin cyst walls (<2 mm, insufficient number of glandular epithelial cells) or extremely high degree of fibrosis (the glandular release rate is still <80% within 120 minutes), the success rate of construction may decrease. Regarding the co-culture ratio, 1:2 (glandular epithelial cell cluster: stromal cell) is the optimal ratio; deviations from this ratio by more than ±20% significantly reduce model quality. Regarding the co-culture time, day 9 is the optimal harvest time; models can be used within the range of day 7-11; beyond this range, model quality decreases.

[0142] In summary, this invention provides a method for constructing a co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis. Through the organic combination of key technologies such as two-step enzymatic digestion, differential sedimentation purification, three-dimensional stromal cell pre-culture, and co-culture at a specific ratio, an in vitro model of endometriosis lesions with histological structure and cellular composition is reconstructed. This method has a complete operational procedure, well-defined parameters, and good reproducibility. The constructed model is highly consistent with the primary lesions in terms of cell composition, tissue structure, proliferative activity, and molecular expression profile, providing a reliable in vitro platform for studying the pathogenesis of endometriosis and for drug screening.

[0143] The exemplary systems and methods of the present invention have been specifically shown and described with reference to the above embodiments, which are merely examples of the best mode for implementing the systems and methods. Those skilled in the art will understand that various changes can be made to the embodiments of the systems and methods described herein without departing from the spirit and scope of the invention as defined in the appended claims when implementing the systems and / or methods.

Claims

1. A method for constructing a co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis, characterized in that, Includes the following steps: S1, Obtain ovarian endometriotic cyst tissue specimens and perform two-step enzymatic digestion to release and enrich glandular epithelial cell clusters from fibrotic tissue; The two-step enzymatic digestion process includes: firstly, using a first enzymatic digestion solution to digest the tissue to degrade the fibrotic matrix and release the glandular structure; and then using a second enzymatic digestion solution to digest the released glandular structure to achieve intercellular dissociation. S2, enriched glandular epithelial cell clusters are mixed with matrix gel for three-dimensional culture to form glandular epithelial organoids, and passaged to the fourth generation. Differential sedimentation is used to remove the interstitial cells mixed in during passage to obtain high-purity glandular epithelial organoids. S3, collect mesenchymal cells from step S1 and / or step S2, expand and culture them, and then seed them in a low-adhesion culture dish to form three-dimensional mesenchymal cell spheres; S4, the high-purity glandular epithelial organoids obtained in step S2 are digested and decomposed into cell clusters containing 3-5 cells. The cell clusters are mixed with the mesenchymal cells after the three-dimensional mesenchymal cell spheres obtained in step S3 are dissociated at a ratio of 1:2, embedded in matrix gel, and three-dimensional co-culture is performed using co-culture medium to obtain a co-culture model of glandular epithelial organoids and mesenchymal cells in ovarian endometriosis.

2. The method according to claim 1, characterized in that, The specific operation of the two-step enzymatic digestion process in S1 is as follows: Step 1: Cut the ovarian endometriotic cyst tissue specimen into 1-2 mm³ tissue fragments, add the first enzymatic digestion solution, and digest in a shaker at 37°C for 40-120 minutes, blowing and oscillating every 15 minutes. Step 2: When loose tissue and obvious release of glandular structures are observed under an inverted phase-contrast microscope, the degree of digestion is determined to meet the following conditions: if the glandular release rate is ≥80%, the first enzymatic digestion is terminated; otherwise, digestion continues and is evaluated every 10 minutes. Step 3: Add the second enzymatic hydrolysate, TrypLE, to the digestion system and continue digestion for 10-15 minutes to achieve cell dissociation. Step 4: Dilute the digestion product with 5 times the volume of organoid washing solution, filter through a 100 μm sieve, collect the filtrate and centrifuge to obtain epithelial cell clusters; The first enzymatic hydrolysate is enzymatic hydrolysate D001-1 containing additive I, and the second enzymatic hydrolysate is TrypLE.

3. The method according to claim 2, characterized in that, The passage purification of the glandular epithelial organoids in S2 specifically includes: Step 1: Use a pipette to aspirate the mixture of glandular epithelial organoids and matrix gel and transfer it into a centrifuge tube; Step 2: Add organoid recovery solution, repeatedly blow and agitate to fully dissolve the matrix gel, and let stand for 1-2 minutes to allow the organoids to settle naturally; Step 3: If the total number of cells in the supernatant after sedimentation is ≥1×10⁻⁶ 5 If the number of cells is less than 1, it indicates the presence of interstitial cell contamination, and the differential sedimentation process needs to be repeated until the total number of cells in the supernatant is <1×10⁻⁶. 4 indivual; Step four: The purified organoids are re-embedded in fresh matrix gel and cultured to the fourth generation to obtain high-purity glandular epithelial organoids.

4. The method according to claim 3, characterized in that, The collection and expansion of the S3 intermediate cells specifically includes: Step 1: Collect the undigested tissue fragments that were filtered through a 100 μm sieve in step S1, resuspend them in DMEM / F-12 medium containing 10% FBS, and inoculate them into culture dishes for 5-7 days. Step 2: Collect the mesenchymal cells that adhere to the bottom of the glandular epithelial organoid culture system in step S2 and combine them with the cells obtained in step 1; Step 3: If the total number of cells after merging is ≥5×10 5 If the number of cells is ≥90%, the conditions for 3D culture inoculation are met; otherwise, continue expansion culture for 1-2 generations. Step 4: Take the expanded mesenchymal cells from passages 3-5, and use 5 × 10⁻⁶ cells per cell line. 4 -1×10 5 Seeds were placed at a density of cells / well in CSwell600 low-adhesion culture dishes and cultured for 3-5 days to form three-dimensional mesenchymal spheroids.

5. The method according to claim 4, characterized in that, The construction of the S4 co-cultivation model specifically includes: Step 1: Collect the high-purity glandular epithelial organoids obtained in step S2 using organoid recovery solution, and digest them with TrypLE for 5-10 minutes. Step 2: If the proportion of clusters of 3-5 cells in the cell clusters formed after digestion is ≥70%, then the degree of digestion is considered to meet the requirements for co-culture; otherwise, adjust the digestion time and reassess. Step 3: The three-dimensional mesenchymal cell spheres obtained in step S3 are dissociated into single-cell suspensions using TrypLE, counted, and then mixed with the glandular epithelial cell clusters obtained in step 2 at a ratio of 1:

2. Step 4: Inoculate the mixed cell-matrix mixture into a culture plate and culture it in a co-culture medium containing organoid culture medium and mesenchymal cell culture medium at a 1:1 volume ratio for 9 days.

6. The method according to claim 5, characterized in that, The preparation and usage conditions of the co-culture medium are as follows: Step 1: Prepare complete organoid culture medium, which includes basal culture medium for endometriosis organoids and additive I; Step 2: Prepare complete culture medium for mesenchymal cells, wherein the culture medium is DMEM / F-12 medium containing 10% FBS; Step 3: If the co-culture ratio of glandular epithelial organoids and mesenchymal cells deviates from the range of 1:2±10%, the ratio is deemed substandard, and the cell count needs to be readjusted and the mixture mixed. Step 4: Mix the culture media obtained in Step 1 and Step 2 at a volume ratio of 1:1 to form a co-culture medium. Change the medium every 2 days. After culturing for 9 days, collect the co-culture model for identification.

7. The method according to claim 1, characterized in that, The criterion for determining the digestion endpoint of the first enzymatic hydrolysate in the two-step enzymatic digestion process in S1 is as follows: Step 1: When digestion has progressed to 40 minutes, the tissue morphology is observed for the first time under an inverted phase-contrast microscope. Step 2: If the number of glandular structures released in each field of view is ≥50 and the area of ​​fibrotic matrix fragments is ≤30% of the total field of view, then digestion is considered complete. Step 3: If the above standards are not met, continue digestion and reassess every 15 minutes, with a total digestion time not exceeding 120 minutes; Step four: After digestion is complete, immediately add the second enzymatic hydrolysate for the second digestion step to ensure maximum yield of glandular epithelial cells.

8. The method according to claim 1, characterized in that, The specific conditions for removing interstitial cells by differential sedimentation in S2 are as follows: Step 1: The glandular epithelial organoids passaged to the fourth generation were released from the matrix gel and resuspended in the organoid washing solution. Step 2: Let the cell suspension stand for 1-2 minutes to allow the glandular epithelial organoids to settle naturally, and collect the precipitate. Step 3: If the proportion of vimentin-positive cells in the precipitate is ≤5%, the removal of interstitial cells is considered to have met the standard; otherwise, repeat the sedimentation process 1-2 times. Step four: The qualified precipitate is re-embedded in the matrix gel for subsequent co-culture experiments.

9. The method according to claim 1, characterized in that, The method for culturing and identifying the three-dimensional mesenchymal cell spheres in S3 is as follows: Step 1: The expanded 3rd-5th generation mesenchymal cells are divided into groups of 5 × 10⁻⁵ cells. 4 -1×10 5 Seeds were inoculated at a density of cells / well in CSwell 600 low-adhesion culture dishes; Step 2: Incubate at 37℃ in a 5% CO2 incubator for 3-5 days, and observe the formation of cell spheroids daily. Step 3: If the diameter of the formed three-dimensional mesenchymal cell spheres is ≥100 μm and the proportion of spherical structures is ≥80%, the culture is considered successful; otherwise, adjust the seeding density or extend the culture time. Step four: Collect successfully cultured three-dimensional mesenchymal cell spheres, perform immunohistochemical staining, and detect the expression of vimentin, ITGB3, ICAM1, MMP2, MMP9, MMP12, TIMP1, and TIMP2 to confirm the mesenchymal cell phenotype.

10. The method according to any one of claims 1-9, characterized in that, The identification method for the co-culture model is as follows: Step 1: Collect model samples from the 9th day of co-culture, and perform paraffin embedding and sectioning. Step 2: Perform H&E staining and PAS staining on the sections to observe the morphological characteristics of glandular epithelial organoids and stromal cells and their mucus secretion. Step 3: If the Ki67 positive cell rate in the glandular epithelial organoids is ≥30% and the stromal cell invasion depth is ≥50 μm, the model is considered to have been successfully constructed. Step four: IHC staining was performed on the successfully constructed model to detect the expression of ER, PR, CK7, E-cadherin, CD44, ICAM1, ITGB3, MMP2, MMP9, TIMP1 and TIMP2, in order to verify the homology between the model and the primary lesion.

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