Ovarian endometriosis glandular epithelial organ and interstitial cell co-culture model and construction method thereof
By constructing a co-culture model of glandular epithelial organoids and stromal cells for ovarian endometriosis, the shortcomings of existing models in simulating the invasion and colonization process of lesions were addressed, the optimal ratio was determined, and a highly biomimetic cell culture and personalized drug screening platform was realized.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-07
- Publication Date
- 2026-04-10
AI Technical Summary
Existing in vitro models of ovarian endometriosis cannot fully reproduce the process of lesion invasion and colonization, and the optimal co-culture ratio of glandular epithelial cells and stromal cells is unclear, affecting the stability and biological relevance of the model.
A co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis was constructed. Ovarian endometriotic cyst tissue was digested and processed, and glandular epithelial organoids and stromal cells were isolated and cultured. They were mixed at a ratio of 1:2 to 1:4 and embedded in a matrix gel for three-dimensional co-culture. Organoid culture medium and stromal cell culture medium were mixed at a volume ratio of 1:1.
A highly biomimetic patient-derived model was established, clarifying the optimal ratio of glandular epithelial organoids to stromal cells, improving cell viability, solving the problems of ambiguous proportions and difficulty in maintaining cell activity in existing models, and providing a personalized drug screening platform.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of organoid technology, and more particularly to a co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis and its construction method. Background Technology
[0002] Endometriosis is a common chronic gynecological disease in women of reproductive age, with ovarian endometriotic cysts being the most common. Its pathogenesis is not yet fully understood, and clinical treatment faces significant challenges such as heterogeneity in efficacy, inability to achieve a complete cure, and prominent adverse drug reactions. In terms of basic research, existing in vitro models for ovarian endometriosis have significant limitations: traditional two-dimensional culture systems struggle to simulate the three-dimensional microenvironment and intercellular interactions in vivo; single epithelial organoid models lack stromal cell components, failing to fully reproduce key pathological processes such as lesion invasion and colonization; and existing co-culture models often lack a clearly defined optimal ratio of glandular epithelium to stromal cells, affecting model stability and biological relevance.
[0003] Therefore, constructing a 3D co-culture model that integrates two cell types, glandular epithelium and stromal, clarifies the optimal co-culture ratio, and reflects patient specificity is of great significance for elucidating disease mechanisms and realizing personalized drug screening. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis and its construction method.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect is to provide a method for constructing a co-culture model of glandular epithelial organoids and stromal cells in ovarian endometriosis, including the following steps: S1, constructing glandular epithelial organoids of ovarian endometriotic cysts; S2, constructing a 3D culture of stromal cells from ovarian endometriotic cysts; S3, Co-culture model construction: The glandular epithelial organoids obtained in step S1 are digested and processed, mixed with the mesenchymal cells obtained in step S2 in a certain proportion, and embedded in matrix gel for three-dimensional co-culture. The ratio of the cell clusters formed after digestion of the glandular epithelial organoids to the mixed number of the interstitial cells is 1:2 to 1:4.
[0006] Furthermore, the cells used in constructing the glandular epithelial organoids in S1 and the 3D culture bodies in S2 were both derived from ovarian endometriotic cyst tissue specimens from the same patient.
[0007] Furthermore, the method for constructing the ovarian endometriotic cyst glandular epithelial organoid specifically includes: S11, Ovarian endometriotic cyst tissue was taken, cleaned and minced to obtain tissue fragments; S12, the tissue fragments are subjected to a first enzymatic hydrolysis treatment to release glandular structures; then the released glandular structures are subjected to a second enzymatic hydrolysis treatment to obtain epithelial cell clusters; S13, the mixed suspension containing epithelial cell clusters after enzymatic digestion is filtered to remove undigested tissue fragments; and the filtered cell suspension is centrifuged and purified to obtain enriched epithelial cell clusters. S14 involves mixing and solidifying enriched epithelial cell clusters with matrix gel to form a three-dimensional culture system, which is then cultured using organoid culture medium to form and passage amplify glandular epithelial organoids. S15: Take the glandular epithelial organoids obtained by passage expansion, and use differential sedimentation to separate and remove mixed stromal cells to obtain high-purity glandular epithelial organoids.
[0008] Furthermore, the two-step enzymatic digestion in S12 specifically involves: First, the tissue fragments were digested using enzymatic hydrolysate I to degrade the fibrotic matrix and release the glandular structures; then, the released glandular structures were digested using TrypLE to achieve intercellular dissociation, thereby obtaining the epithelial cell clusters. The two-step enzymatic digestion is designed to address the pathological characteristics of severe fibrosis and low glandular content in ovarian endometriotic cysts, in order to maximize the yield of glandular epithelial cells.
[0009] Furthermore, in S15, the glandular epithelial organoids are passaged to the fourth generation to achieve complete removal of interstitial cells.
[0010] Furthermore, the method for constructing the 3D culture of stromal cells from ovarian endometriotic cysts specifically includes: S21, mesenchymal cells were isolated from undigested tissue fragments and cells that migrated from the glandular epithelial organoid culture system after enzymatic digestion and were then expanded and cultured. S22, the expanded 3rd-5th generation mesenchymal cells were seeded in a low-adhesion culture dish and cultured to form 3D mesenchymal cell spheres.
[0011] Furthermore, the low-adhesion culture dish is a CSwell 600 culture dish.
[0012] Furthermore, the method for constructing the co-cultivation model specifically includes: S31, which digests and breaks down purified glandular epithelial organoids with a diameter of 150-200μm into cell clusters of 20-30μm containing 3-5 cells; S32, The cell clusters are mixed with mesenchymal cells of the same origin in a preset ratio and then embedded in matrix gel; S33 was cultured in a co-culture medium containing organoid culture medium and mesenchymal cell culture medium at a volume ratio of 1:1 for 9 days.
[0013] Furthermore, the ratio of the mixed number of cell clusters to mesenchymal cells is 1:2.
[0014] The second aspect is to provide a co-culture model of glandular epithelial organoids and stromal cells for ovarian endometriosis obtained by the construction method described above.
[0015] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention establishes for the first time a co-culture system of glandular epithelial organoids and stromal cells derived from patients with ovarian endometriosis. The model and the primary lesion are highly homologous in terms of tissue morphology and biomarker expression, exhibiting high biomimicry. It clarifies that the glandular epithelial organoids are most abundant when the ratio of glandular epithelial cell clusters to stromal cells from patients with ovarian endometriosis is 1:2, and the viability of glandular epithelial organoids is significantly increased in the 1:2 and 1:4 ratio groups, thus solving the technical bottleneck of ambiguous ratios and difficulty in maintaining cell viability in existing co-culture models.
[0016] The co-culture model of glandular epithelial organoids and stromal cells for ovarian endometriosis constructed in this invention can be used to study cell-to-cell interactions and pathogenesis of ovarian endometriosis; it can provide a customized platform for individualized drug screening for ovarian endometriosis; and the construction method is simple to operate, requires no complex instruments, all reagents are commercial products with clear catalog numbers, have strong reproducibility, and are easy to promote in scientific research and clinical translation scenarios. Attached Figure Description
[0017] Figure 1 To establish and identify glandular epithelial organoids from patients with ovarian endometriosis; where A is a photomicrograph of primary glandular epithelial organoids at different times; B is a photomicrograph of different generations of epithelial organoids at different times; and C is a scanning electron microscope (SEM) image of the glandular epithelial organoids.
[0018] Figure 2This study aimed to culture and identify stromal cells from patients with ovarian endometriosis. A shows the formation of three-dimensional spherical structures in stromal cells after 3 days of culture; B shows the surface morphology of these structures using SEM after 3 days of culture; and C shows the expression of vimentin, ITGB3, ICAM1, MMP2, MMP9, MMP12, TIMP1, and TIMP2 in the spherical structures of stromal cells after 3 days of culture using immunohistochemistry (IHC).
[0019] Figure 3 Phenotypic comparison of glandular epithelial organoids and ovarian endometriotic cysts (lesions); where A is the result of H&E staining; B is the result of PAS staining; and C is the result of IHC staining.
[0020] Figure 4 The diagram shows the construction of the co-culture model and the viability comparison of co-culture systems with different ratios. A represents the observation results of the co-culture systems of glandular epithelial organoids and stromal cells at different ratios on days 3 and 9; B represents the statistical results of the number of glandular epithelial organoids on days 3 and 9 under different co-culture ratios; C represents the diameter measurement results of glandular epithelial organoids on days 3 and 9 under different co-culture ratios; and D represents the expression level and positive rate analysis of Ki67 in glandular epithelial organoids under different co-culture ratios. Detailed Implementation
[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.
[0022] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.
[0023] Example 1: Establishment and identification of glandular epithelial organoids from patients with ovarian endometriosis 1.1 Specimen processing Specimens were selected from premenopausal women who underwent laparoscopic surgery for ovarian endometriotic cysts, with ethical approval and informed consent obtained from the patients. Specimens were stored at 4°C in tissue preservation solution (B001, Suzhou Jiyan Biotechnology Co., Ltd.) for 6–48 hours, rinsed twice with tissue washing buffer (B002, Suzhou Jiyan Biotechnology Co., Ltd.), and aseptically minced to 1 mm. 3 Collect tissue fragments by centrifuging at 500 rpm for 5 minutes in small pieces.
[0024] 1.2 Two-step enzymatic hydrolysis Transfer tissue fragments to a 60mm culture dish and add 3-5mL of enzymatic hydrolysis solution I (D001-1, Suzhou Jiyan Biotechnology Co., Ltd.) containing additive I (S001, Suzhou Jiyan Biotechnology Co., Ltd.). Shake at 37℃ for 40-120 minutes, with pipetting every 15 minutes. Once the tissue is loose and epithelial glands are clearly released under an inverted phase-contrast microscope, add TrypLE for digestion for 10-15 minutes. Dilute with 5 times the volume of organoid washing solution (B004, Suzhou Jiyan Biotechnology Co., Ltd.), filter through a 100μm sieve, collect the filtrate, centrifuge, and remove red blood cells with erythrocyte lysis buffer (B005, Suzhou Jiyan Biotechnology Co., Ltd.).
[0025] 1.3 Organoid Culture Resuspend cells in organoid culture medium for endometriosis (ODM001, Suzhou Jiyan Biotechnology Co., Ltd.), mix with an equal volume of matrix gel (356231, Kangning), and add 50 μL to each well of a 24-well plate. Incubate at 37°C for 5 minutes, then invert the plate and continue incubating at 37°C for 15 minutes. Add 0.5 mL of organoid complete culture medium containing additive I (S001, Suzhou Jiyan Biotechnology Co., Ltd.). After culturing for 48 hours, replace with culture medium without additive I. Change the medium every 2 days. Obvious glandular epithelial organoids can be seen after 10-20 days.
[0026] 1.4 Organoid Purification Organoids were aspirated from the matrix gel using a pipette, and interstitial cells were separated by differential sedimentation. After passage to the fourth generation, there was no obvious interstitial cell contamination in the glandular epithelial organoids, and the maximum diameter reached 500 μm.
[0027] This invention uses a special organoid culture medium for endometriosis to culture for 10-20 days. Irregular glandular structures in the matrix gel gradually enlarge, while most stromal cells adhere to the bottom of the culture plate. SEM results ( Figure 1 The study showed that the organoids had a spherical structure with a rough surface and were rich in cellular protrusions.
[0028] Example 2: Culture and identification of stromal cells from patients with ovarian endometriosis 2.1 Isolation of mesenchymal cells Mesenchymal tissue fragments were collected from a 100 μm sieve, resuspended in DMEM / F-12 medium containing 10% FBS, and seeded into culture dishes for 5-7 days until mesenchymal cells migrated from the tissue blocks. At the same time, mesenchymal cells adhering to the bottom of the glandular epithelial organoid culture system were collected, merged, and expanded.
[0029] 2.2 Construction of 3D Cell Spheroids Mesenchymal cells from passages 3 to 5 were seeded into CSwell 600 culture dishes (KIT000-0001, Suzhou Jiyan Biotechnology Co., Ltd.) to construct uniform 3D mesenchymal cell spheres.
[0030] 2.3 Detection of 3D mesenchymal spheroid markers The immunohistochemical (IHC) procedure was as follows: Sections were sequentially immersed in xylene and a series of ethanol concentrations (100%, 95%, 70%) for dewaxing and rehydration. Subsequently, the sections were placed in EDTA solution (pH 9.0) or sodium citrate buffer (10 mmol / L, pH 6.0) and autoclaved for 2 minutes to complete antigen retrieval. Endogenous peroxidase activity was inactivated, and the sections were blocked with 5% BSA. The sections were then mixed with diluted specific primary antibody and incubated overnight at 4°C. After washing with PBS to remove unbound primary antibody, the corresponding secondary antibody was added to the sample, and the mixture was incubated at room temperature for 1 hour. The cell nuclei were then counterstained with hematoxylin. The IHC assay was used to detect the expression of vimentin, ITGB3, ICAM1, MMP2, MMP9, MMP12, TIMP1, and TIMP2 in spherical structures of mesenchymal cells cultured for 3 days.
[0031] 3D mesenchymal cell spheres constructed using CSwell 600 culture dishes can be stably cultured for more than 10 days, and SEM analysis shows that... Figure 2 AB) Its surface is smoother than that of epithelial organoids, IHC results ( Figure 2 (C) confirmed that vimentin, ITGB3, ICAM1, MMP2, MMP9, TIMP1, TIMP2, and MMP12 were all positive in 3D mesenchymal spheroids.
[0032] Example 3: Phenotypic comparison between glandular epithelial organoids and ovarian endometriotic cysts (lesions) 3.1 H&E staining ( Figure 3 A) Perform the procedure according to the kit instructions (C0105M, Beyotime). The morphology of the glandular epithelial organoids is similar to the glandular structure of the lesion, and cavities surrounded by tightly packed columnar epithelial cells can be seen.
[0033] 3.2 PAS staining ( Figure 3 B) According to the kit instructions (C0142M, Beyotime), mucus secretion was detected in both the cavities of glandular organoids and the glandular tissues of lesions.
[0034] 3.3 IHC staining ( Figure 3 C) IHC results showed that both glandular epithelial organoids and lesions expressed sex hormone-related receptors (ERs, PRs), intercellular / cell-matrix interaction molecules (E-cadherin, CD44, ICAM1, ITGB3, CK7), and invasion-related molecules (MMP2, MMP9, TIMP1, TIMP2). Furthermore, the mesenchymal cell marker vimentin was also detected in glandular epithelial organoids.
[0035] Example 4: Construction of the co-culture model and comparison of the viability of co-culture systems with different ratios. 4.1 Cell Preparation The glandular epithelial organoids from Example 1 were collected using organoid recovery solution (B008, Suzhou Jiyan Biotechnology Co., Ltd.), and digested with TrypLE for 5-10 minutes to break them down into cell clusters of 20-30 μm; the glandular epithelial cell clusters and the mesenchymal cells from Example 2 were counted.
[0036] 4.2 Gradient Proportion Co-culture Glandular epithelial cell clusters were mixed with mesenchymal cells at ratios of 1:2, 1:4, 1:10, 1:50, 1:100, and 1:200, embedded in matrix gel, and then co-cultured with organoid culture medium and mesenchymal cell culture medium at a ratio of 1:1 for 9 days.
[0037] 4.3 Comparison of viability of co-culture systems with different ratios The diameters of glandular epithelial organoids with different co-culture ratios were counted and measured on days 3 and 9. The expression levels of Ki67 in the glandular epithelial organoids were also detected at different co-culture ratios. The results are as follows: Figure 4 As shown.
[0038] A controllable and biomimetic in vitro model was constructed by co-culturing glandular epithelial organoids with stromal cells at different ratios. After 9 days of co-culture, stromal cells invaded and proliferated rapidly in the matrix gel, resulting in a significant increase in the volume of the glandular epithelial organoids. The number of glandular epithelial organoids peaked in the group with a 1:2 ratio of glandular epithelial cell clusters to stromal cells, significantly higher than in other groups. Furthermore, the survival rate of glandular epithelial organoids was significantly higher in the 1:2 and 1:4 ratio groups than in other groups. The expression level of Ki67 in the glandular epithelial organoids decreased in a dose-dependent manner with increasing stromal cell ratio.
[0039] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.
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, constructing glandular epithelial organoids of ovarian endometriotic cysts; S2, constructing a 3D culture of stromal cells from ovarian endometriotic cysts; S3, Co-culture model construction: The glandular epithelial organoids obtained in step S1 are digested and processed, mixed with the mesenchymal cells obtained in step S2 in a certain proportion, and embedded in matrix gel for three-dimensional co-culture. The ratio of the cell clusters formed after digestion of the glandular epithelial organoids to the mixed number of the interstitial cells is 1:2 to 1:
4.
2. The construction method according to claim 1, characterized in that, The cells used in constructing the glandular epithelial organoids in S1 and the 3D culture bodies in S2 were both derived from ovarian endometriotic cyst tissue specimens from the same patient.
3. The construction method according to claim 1, characterized in that, The method for constructing glandular epithelial organoids from ovarian endometriotic cysts specifically includes: S11, Ovarian endometriotic cyst tissue was taken, cleaned and minced to obtain tissue fragments; S12, the tissue fragments are subjected to a first enzymatic hydrolysis treatment to release glandular structures; then the released glandular structures are subjected to a second enzymatic hydrolysis treatment to obtain epithelial cell clusters; S13, the mixed suspension containing epithelial cell clusters after enzymatic digestion is filtered to remove undigested tissue fragments; and the filtered cell suspension is centrifuged and purified to obtain enriched epithelial cell clusters. S14 involves mixing and solidifying enriched epithelial cell clusters with matrix gel to form a three-dimensional culture system, which is then cultured using organoid culture medium to form and passage amplify glandular epithelial organoids. S15: Take the glandular epithelial organoids obtained by passage expansion, and use differential sedimentation to separate and remove mixed stromal cells to obtain high-purity glandular epithelial organoids.
4. The construction method according to claim 3, characterized in that, The two-step enzymatic digestion in S12 is as follows: First, the tissue fragments were digested using enzymatic hydrolysate I to degrade the fibrotic matrix and release the glandular structures; then, the released glandular structures were digested using TrypLE to achieve intercellular dissociation, thereby obtaining the epithelial cell clusters. The two-step enzymatic digestion is designed to address the pathological characteristics of severe fibrosis and low glandular content in ovarian endometriotic cysts, in order to maximize the yield of glandular epithelial cells.
5. The construction method according to claim 3, characterized in that, In S15, the glandular epithelial organoids are passaged to the fourth generation to achieve complete removal of interstitial cells.
6. The construction method according to claim 3, characterized in that, The method for constructing a 3D culture of stromal cells from ovarian endometriotic cysts specifically includes: S21, mesenchymal cells were isolated from undigested tissue fragments and cells that migrated from the glandular epithelial organoid culture system after enzymatic digestion and were then expanded and cultured. S22, the expanded 3rd-5th generation mesenchymal cells were seeded in a low-adhesion culture dish and cultured to form 3D mesenchymal cell spheres.
7. The construction method according to claim 6, characterized in that, The low-adhesion culture dish is a CSwell 600 culture dish.
8. The construction method according to claim 1, characterized in that, The method for constructing the co-cultivation model specifically includes: S31, which digests and breaks down purified glandular epithelial organoids with a diameter of 150-200μm into cell clusters of 20-30μm containing 3-5 cells; S32, The cell clusters are mixed with mesenchymal cells of the same origin in a preset ratio and then embedded in matrix gel; S33 was cultured in a co-culture medium containing organoid culture medium and mesenchymal cell culture medium at a volume ratio of 1:1 for 9 days.
9. The construction method according to claim 8, characterized in that, The ratio of the mixed number of cell clusters to mesenchymal cells is 1:
2.
10. A co-culture model of glandular epithelial organoids and stromal cells for ovarian endometriosis obtained by the construction method as described in any one of claims 1-9.