An organ-chip-based simulated model of the maternal-fetal interface in systemic lupus erythematosus and a construction method thereof

Abstract

The application provides a trophoblast organ-on-chip system for simulating a maternal-fetal interface of a systemic lupus erythematosus model based on an organ chip and a construction method thereof, the system taking an organ chip as a carrier and comprising three core parts of a trophoblast organ, an endometrial chip and an SLE disease model. The application completes separation culture and characterization of the trophoblast organ, multi-cell co-culture construction and characterization of the endometrial chip, and then constructs a pathological model, and the three parts cooperatively form a three-dimensional maternal-fetal interface. The system can clearly observe the dynamic process of the trophoblast organ invading the endometrial chip, can detect the continuous secretion of hCG and SLE-related inflammatory factors, and breaks through the defects of the prior art model, such as low authenticity, inability to dynamically observe, single detection dimension and lack of standardization, and has both physiological and pathological adhesion and experimental repeatability, can multi-dimensionally characterize the cell phenotype and function of the maternal-fetal interface, and provides an in-vitro research platform for the pathological mechanism research of the SLE pregnancy-related abnormal maternal-fetal interface.

Description

Technical Field

[0001] This application relates to the design of organ-on-a-chip, and more particularly to a trophoblast organoid-endometrial chip system based on an organ-on-a-chip model of the maternal-fetal interface of systemic lupus erythematosus and its construction method. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple organ damage. Its pathogenesis is closely related to adaptive immune dysfunction and antigen-antibody complex deposition, leading to chronic autoimmune reactions that can cause dysfunction in multiple systems, including the skin, kidneys, and joints. Clinically, SLE can occur at any age, but it is particularly common in women of childbearing age; epidemiological data shows that the disease affects approximately 3.4 million people worldwide. Although the exact cause of SLE is not fully understood, current research generally suggests that its pathogenesis is the result of the interaction of multiple factors, including genetic susceptibility, epigenetic regulation, environmental exposure, and hormonal changes. This complex network of causes collectively promotes abnormal activation of the immune system, leading to systemic inflammation and tissue damage—which partly explains why the clinical manifestations of SLE are so diverse, ranging from vasculitis and rashes to nephritis, arthritis, and even neuropsychiatric symptoms.

[0003] In pregnancies of patients with systemic lupus erythematosus (SLE), the risk of adverse outcomes is significantly increased, particularly preterm birth, preeclampsia, and miscarriage. Clinical observations suggest that these complications are often closely related to increased disease activity, decreased complement levels, positive anti-dsDNA antibodies, prior kidney involvement, and the presence of antiphospholipid antibodies (aPL). These factors collectively constitute the high-risk characteristics specific to SLE pregnancies. In fact, SLE-related chronic inflammation and immune complex deposition can lead to vascular endothelial dysfunction, thereby interfering with the normal placental vascular remodeling process. In healthy pregnancies, this process supports development by increasing uterine blood flow and expanding fetal growth space; however, in SLE patients, it can lead to a series of complications such as thrombotic tendency, fetal growth restriction, and even oligohydramnios.

[0004] Pregnancy-related complications in women of reproductive age with systemic lupus erythematosus (SLE) are closely related to pathological changes at the maternal-fetal interface. Constructing accurate in vitro simulation models is crucial for elucidating its pathological mechanisms. However, existing technologies suffer from several key limitations: traditional two-dimensional culture models lack a three-dimensional physiological microenvironment, failing to replicate maternal-fetal interface cell interactions and introduce the SLE pathological environment; single organoid systems cannot achieve co-culture of the trophoblast and endometrium, making it difficult to reflect their pathological interactions; SLE animal models exhibit species differences, failing to accurately replicate human maternal-fetal interface pathological characteristics and unable to observe trophoblast invasion dynamics in real time; conventional in vitro SLE models do not incorporate maternal-fetal interface cell systems, failing to simulate the specific effects of SLE; and related organ-on-a-chip models often only simulate normal endometrial conditions, lacking core SLE pathological factors, three-dimensional cell co-culture, and the ability to detect pregnancy-related hormones and SLE-related factors. Systematic detection of inflammatory factors and limited functional verification dimensions; at the same time, the lack of standardized in vitro research systems in this field, inconsistent experimental methods leading to poor reproducibility of results, and existing research methods are unable to simultaneously achieve multi-dimensional research such as maternal-fetal interface cell phenotypic characterization, dynamic observation of invasion, and factor secretion detection. The lack of model authenticity and research comprehensiveness seriously restricts the in-depth exploration of the pathological mechanisms of SLE pregnancy-related maternal-fetal interface abnormalities. Summary of the Invention

[0005] The purpose of this application is to provide a method for establishing a trophoblast organoid-endometrial chip system based on organ-on-chip to simulate a systemic lupus erythematosus model of the maternal-fetal interface. This method can be used to observe in depth the process of placental invasion of the endometrial layer. This method has the feature of real-time monitoring of cell movement and can monitor the movement trajectory of trophoblast organoids in real time.

[0006] A trophoblast organoid-endometrial chip system for simulating a systemic lupus erythematosus (SLE) model at the maternal-fetal interface, wherein the system uses an organ-chip as a carrier and includes a trophoblast organoid, an endometrial chip, and a SLE model.

[0007] Preferably, the organ-on-a-chip includes a cell inlet pool, a cell culture chamber, and an outlet pool. The cell culture chamber is connected to the cell inlet pool at the top and the outlet pool at the bottom. The trophoblast organoids are used to simulate the placenta invading the endometrium. The endometrial chip is a chip system based on organ-on-a-chip that simulates the three-dimensional structure of the endometrial layer. The remaining trophoblast organoids together construct a three-dimensional maternal-fetal interface. The systemic lupus erythematosus (SLE) model is constructed with the patient's plasma IgG as the core and is used to simulate the disease state of SLE patients.

[0008] Preferably, the system construction method is as follows:

[0009] S1: Trophoblast organoid construction: First, trophoblast cells were isolated and cultured in a microwell culture array.

[0010] S2: Construction of endometrial microarray: Endometrial cells were mixed with matrix gel and aOH solution and added to the microarray through the cell inlet pool. When the cells were observed to be evenly distributed in the cell culture chamber under an optical microscope, the microarray was immediately transferred to a carbon dioxide incubator for further culture. After the cell-matrix gel suspension in the cell culture pool solidified, the culture medium of the three cell types was added to the four channels through the cell inlet pool and cultured for a period of time, so that the cells were in the same growth phase and fully extended.

[0011] S3: Construction of a systemic lupus erythematosus (SLE) disease model: Plasma IgG was extracted and added to the aforementioned endometrial microarray to complete the construction of the SLE disease model.

[0012] Preferably, the analysis of trophoblast cells in S1 uses placental villus tissue. After washing, the placental villus is physically cut into pieces, and then mixed digestive enzymes are added to the placental villus tissue to separate single cells. The trophoblast cells are obtained by centrifugation.

[0013] Preferably, the mixed digestive enzymes comprise 0.25% pancreatin-EDTA and collagenase IV.

[0014] Preferably, the trophoblast organoid culture medium in S1 includes a basal culture medium and additives. The basal culture medium is Advanced DMEM / F12, and the additives include N2 additive, B27 additive, 1X penicillin-streptomycin, 1.25 mM N-acetyl-L-cysteine, 2 mM L-glutamine, 50 ng / mL EGF, 1.5 μM CHIR99021, 80 ng / mL R-spondin-1, 100 ng / mL FGF-2, 50 ng / mL HGF, 500 nM A83-01, 2.5 μM prostaglandin E2, and 5 μM Y-27632.

[0015] Preferably, the endometrial cells in S2 include three types of cells: endometrial stromal cells (HESC), endometrial epithelial cells (HEEC), and endometrial endothelial cells (HUVEC).

[0016] This application also provides the above-described organ-on-a-chip system for simulating systemic lupus erythematosus at the maternal-fetal interface trophoblast organoid-endometrial chip as a model for in-depth observation of the process of placental invasion of the endometrial layer.

[0017] Preferably, this system allows for clear observation of the dynamic process of trophoblast organoids invading the endometrial microarray. During the culture process, it can continuously secrete detectable levels of human chorionic gonadotropin (hCG). After the application of plasma IgG from SLE patients, at least one inflammatory factor selected from TNF-α, IFN-γ, IL-1β, and IFN-β can be detected in the cell culture supernatant.

[0018] Compared with the prior art, this application has at least the following beneficial effects:

[0019] 1. This application constructs an endometrial microarray by co-culturing endometrial stromal cells (HESC), epithelial cells (HEEC), and endothelial cells (HUVEC) on an organ-on-a-chip. Simultaneously, trophoblast organoids are directionally cultured using a proprietary culture medium. The co-culture of these two components on the organ-on-a-chip constructs a three-dimensional maternal-fetal interface. Plasma IgG from SLE patients is then introduced to build an SLE pathological model. This organically combines the pathological environment with the three-dimensional cell interaction of the maternal-fetal interface, overcoming the deficiency of traditional two-dimensional culture models that lack a three-dimensional physiological microenvironment in vivo. It also solves the technical problem that traditional SLE models can only simulate immune abnormalities and cannot simultaneously reproduce the spatial interaction of cells at the maternal-fetal interface. This significantly improves the physiological and pathological realism of the in vitro model and achieves accurate replication of the maternal-fetal interface during SLE pregnancy.

[0020] 2. This application utilizes the interconnected structure and visualized culture conditions of organ-on-a-chip to co-culture trophoblast organoids and endometrial microarrays in the cell culture chamber of the microarray. This enables continuous observation and real-time monitoring of the dynamic process of trophoblast organoid invasion of the endometrium microarray, overcoming the limitations of traditional in vivo experiments that cannot directly observe the placental invasion process and traditional in vitro experiments that require fixation and staining and cannot capture dynamic changes. It can clearly record the invasion trajectory and behavioral characteristics of trophoblast organoids, providing intuitive and continuous experimental data support for exploring the molecular mechanisms of abnormal placental invasion into the endometrium under the pathological environment of SLE.

[0021] 3. The system constructed in this application can continuously secrete detectable levels of human chorionic gonadotropin (hCG) under the pathological environment of SLE. It not only retains the core physiological functions of the trophoblast organoid placenta, but also presents the influence of the SLE pathological environment on the secretion of pregnancy-related hormones through quantitative detection. At the same time, after the application of plasma IgG from SLE patients, the system can specifically secrete SLE-related inflammatory factors such as TNF-α, IFN-γ, IL-1β, and IFN-β, and can achieve quantitative statistics of inflammatory factors. It accurately restores the immune disorder characteristics of the maternal-fetal interface under the pathological environment of SLE, and solves the problem that traditional models cannot simultaneously detect hormone secretion and inflammatory factor release at the maternal-fetal interface under pathological conditions. It provides direct in vitro experimental evidence for exploring the pathological mechanisms of abnormal hormone secretion and immune disorder during SLE pregnancy.

[0022] 4. This application establishes standardized construction procedures for trophoblast organoids, endometrial microarrays, and SLE disease models. Simultaneously, it uniformly employs immunofluorescence staining to characterize core biomarkers of each component under normal and SLE pathological conditions. It also develops qualitative and quantitative detection methods for hCG and inflammatory factors. All construction and detection operations are conducted on a controllable organ-on-a-chip platform, solving the problems of large individual differences in in vivo models and inconsistent in vitro model procedures in traditional SLE pregnancy research. This ensures a high degree of standardization and reproducibility in the construction and detection of the system, enabling stable reproduction of experimental results and effectively reducing experimental errors during the research process. It provides a standardized in vitro research model for SLE pregnancy-related studies.

[0023] 5. The system constructed in this application enables multi-dimensional detection and analysis of the structural phenotype and functional characteristics of the SLE maternal-fetal interface. It can characterize the expression changes of core biomarkers of trophoblast organoids and endometrial microarrays under the pathological environment of SLE through immunofluorescence staining, clarifying the impact of the pathological environment on the cell phenotype of the maternal-fetal interface; it can also dynamically observe the invasive behavior of trophoblast organoids, exploring abnormal cell interactions at the maternal-fetal interface under the pathological environment; and it can quantitatively detect hormone secretion and inflammatory factor release, analyzing the impact of the pathological environment on the physiological and immune functions of the maternal-fetal interface. It breaks through the limitations of traditional models with single detection dimensions, and can simultaneously explore the mechanisms of SLE pregnancy-related maternal-fetal interface abnormalities from multiple perspectives, including cell phenotype, cell interaction, hormone secretion, and immune factor release, providing a comprehensive experimental platform for multi-mechanism integrated research in this field.

[0024] 6. This application uses organ-on-a-chip as the system carrier, eliminating the need for additional independent chip construction work, which significantly reduces the technical threshold for model construction. Moreover, the construction steps and detection methods of the system are clear and the conditions are controllable, making it easy to repeat in the laboratory. It can be widely used in research fields such as exploring the pathological mechanisms of SLE pregnancy-related maternal-fetal interface abnormalities and in vitro screening and efficacy evaluation of SLE pregnancy-related candidate drugs, and has high practical application value and promotion value.

[0025] 7. Relationship between Trophoblast Organoids and Organ-on-a-Chip: This invention relates to the fields of biomedical engineering and tissue engineering, specifically to a model for co-culturing trophoblast organoids with endometrial microarrays, its construction method, and applications, particularly an in vitro functional system for implanting trophoblast organoids in the microenvironment of an endometrial microarray. This invention integrates trophoblast organoids with an endometrial microarray to construct a dynamic maternal-fetal interface model. This model overcomes the shortcomings of traditional organoids, which lack blood flow, mechanical forces, and maternal-fetal interaction, and also improves upon the current situation where endometrial microarrays only use simple cell lines. The fluid shear forces and hormonal microenvironment provided by the microarray can induce organoids to transform from a proliferative to an invasive phenotype; the factors secreted by the organoids then feedback-regulate the endometrial receptivity. The two communicate dynamically through microchannels, simulating the embryo implantation process. Experiments show that the gene expression of organoids in the microarray environment is closer to the early pregnancy state. Therefore, this model is a model that combines both approaches. Attached Figure Description

[0026] Figure 1 The overall structure diagram and line drawing of the organ-on-a-chip of this invention are shown. The chip source is Shenzhen Xirui Biotechnology XR-MESL-TriC.

[0027] Attached diagram labels: 1. Cell inlet pool; 2. Cell culture chamber; 3. Outlet pool.

[0028] Figure 2 Trophoblast organoid characterization

[0029] Figure 3 Schematic diagram of qualitative results of hCG-β in the supernatant of trophoblast organoid culture medium.

[0030] Figure 4 Characterization of trophoblast organoids treated with IgG from patients with systemic lupus erythematosus

[0031] Figure 5 Endometrial microarray characterization

[0032] Figure 6 Endometrial microarray characterization after IgG treatment in patients with systemic lupus erythematosus

[0033] Figure 7 Endometrial microarray of trophoblast organoids treated with IgG from patients with systemic lupus erythematosus.

[0034] Figure 8 Dynamic process of trophoblastic organoid invasion into the endometrial microarray

[0035] Figure 9 Statistical chart of human chorionic gonadotropin (hCG) secreted by the trophoblast organoid-endometrial microarray system.

[0036] Figure 10Statistical chart of inflammatory factors produced by the maternal-fetal interface trophoblast organoid-endometrial microarray system in systemic lupus erythematosus Detailed Implementation

[0037] This application provides an organ-on-a-chip system for simulating systemic lupus erythematosus (SLE) at the maternal-fetal interface, comprising a trophoblast organoid, an endometrial chip, and a SLE model. The organ-on-a-chip is purchased from Shenzhen Xirui Biotechnology XR-MESL-TriC. The organ-on-a-chip includes a cell inlet pool, a cell culture chamber, and an outlet pool. The cell culture chamber is connected to the cell inlet pool at the top and the outlet pool at the bottom.

[0038] The specific structure is as follows: Figure 1 As shown, Figure 1 A is a physical image of the endometrial microarray used in this study, with a standard one-yuan coin with a diameter of 25 mm placed next to it as a scale. Figure 1 B is a top view of the chip. A single chip contains three units. Each unit consists of one main channel, two side channels, a cell inlet pool 1, a cell culture chamber 2, and an outlet pool 3. The upstream of the main channel is the sample inlet, used to inject biological samples or reagent solutions, and the downstream is the sample outlet, used to discharge the liquid after culture, processing, or reaction. The side channel inlet is near the confluence of the two side channels, and the outer port of the confluence point is the side channel outlet. Figure 1 C is a line drawing of the chip.

[0039] The trophoblast organoids are used to simulate the placenta invading the endometrium. The endometrial chip is a chip system based on organ-on-a-chip that simulates the three-dimensional structure of the endometrial layer. The remaining trophoblast organoids together construct a three-dimensional maternal-fetal interface. The systemic lupus erythematosus (SLE) model is constructed with the patient's plasma IgG as the core and is used to simulate the disease state of SLE patients.

[0040] In one embodiment, the construction and characterization of the trophoblast organoids are as follows:

[0041] The method for constructing the trophoblast organoids is as follows:

[0042] S1: Separation of trophoblast cells:

[0043] Placental villus tissue was aseptically isolated, washed, and then physically sheared. Mixed digestive enzymes were then added to the placental villus tissue to separate single cells, which were then centrifuged to obtain trophoblast cells.

[0044] In one embodiment, the mixed digestive enzymes comprise 0.25% pancreatin-EDTA and collagenase IV.

[0045] S2: Trophoblast organoid formation:

[0046] The isolated trophoblast cells were seeded in a microwell culture array and cultured. Trophoblast organoids were obtained using trophoblast organoid culture medium.

[0047] The Tropoblastic Organoid Medium (TOM) comprises a basal medium and additives. The basal medium is Advanced DMEM / F12. The TOM additives are formulated in the following proportions: 1X N2 additive, 1X B27 additive (vitamin A-free), 1X penicillin-streptomycin, 1.25 mM N-acetyl-L-cysteine, 2 mM L-glutamine, 50 ng / mL EGF, 1.5 μM CHIR99021, 80 ng / mL R-spondin-1, 100 ng / mL FGF-2, 50 ng / mL HGF, 500 nM A83-01, 2.5 μM prostaglandin E2, and 5 μM Y-27632.

[0048] After establishing the trophoblast organoid-endometrial microarray system, the trophoblast organoid culture medium was replaced with EVTM medium (with the following formulation: 1X Advanced DMEM / F12, 0.1 mM 2-mercaptoethanol, 0.5% penicillin-streptomycin, 0.3% BSA, 1% ITS-X supplement, 100 ng mL−1 NRG1, 7.5 μM A83-01, and 4% knockout serum replacement).

[0049] In one embodiment, the expression of CGB, CK7, HLA-G, and E-cadherin in trophoblast organoids is characterized by conventional immunofluorescence staining, thereby characterizing the establishment of trophoblast organoids. At the same time, this method is used to characterize the expression of CGB, CK7, and HLA-G in trophoblast organoids after IgG treatment in patients, in order to characterize trophoblast organoids in a systemic lupus erythematosus disease model.

[0050] The construction and characterization of the endometrial microarray are as follows:

[0051] S1: Seeding and culture of endometrial microarray cells

[0052] Endometrial stromal cells (HESC), endometrial epithelial cells (HEEC), and endothelial cells (HUVEC) were mixed with matrix gel and NaOH solution in a 1:1:1 ratio and added to the chip through the cell inlet pool. When the cells were observed to be evenly distributed in the cell culture chamber under an optical microscope, the chip was immediately transferred to a carbon dioxide incubator for further culture.

[0053] S2: Establishment of endometrial microarray

[0054] After the cell-matrix gel suspension of the three cell types in the cell culture pool solidifies, the culture medium of the three cell types is added to the four channels through the cell inlet pool and cultured for a period of time, so that the cells are in the same growth phase and fully extended.

[0055] In one embodiment, the expression of CK18, Vimentin, and CD31 in the endometrial microarray is characterized by the conventional immunofluorescence staining method, thereby characterizing the establishment of the endometrial microarray. At the same time, this method is used to characterize the expression of CK18, Vimentin, and CD31 in the endometrial microarray after the patient's IgG treatment, thus characterizing the endometrial microarray in the systemic lupus erythematosus disease model.

[0056] The construction and characterization of the systemic lupus erythematosus disease model are as follows:

[0057] The method for constructing the systemic lupus erythematosus disease model is as follows:

[0058] S1: Extract plasma IgG: Extract IgG from the patient's plasma using the appropriate kit.

[0059] S2: Construction of a systemic lupus erythematosus disease model: The extracted IgG was added to the above-mentioned endometrial chip to complete the construction of the systemic lupus erythematosus disease model.

[0060] In one embodiment, the binding of IgG to cells is characterized by a conventional immunofluorescence staining method, thereby characterizing the establishment of a systemic lupus erythematosus disease model.

[0061] Based on the organ-on-a-chip system for simulating systemic lupus erythematosus at the maternal-fetal interface trophoblast organoid-endometrial chip obtained above, this application also provides the application of the above-mentioned system as a model in the in-depth observation of the process changes of placental invasion of the endometrial layer.

[0062] The above content will be elaborated below with specific verification experiments:

[0063] I. Experimental Materials and Sources

[0064] Table 1 Experimental materials and their sources

[0065] Serial Number Experimental materials source 1 Organ-on-a-chip XR-MESL-TriC Shenzhen Xirui Biotechnology 2 CAwell600 micro-pore array chip Shenzhen Xirui Biotechnology 3 Phosphate-buffered saline (PBS) Anhui White Shark Biology 4 4% paraformaldehyde (PFA) fixative Anhui White Shark Biology 5 Human endometrial stromal cell culture medium Xiamen Yimo Biotechnology 6 Culture medium for human endometrial epithelial cells Xiamen Yimo Biotechnology 7 Endothelial cell culture medium ScienCell 8 trypsin Shanghai Beyotime Biotechnology 9 Immunostaining permeabilization solution (Triton X-100) Shanghai Beyotime Biotechnology 10 <![CDATA[QuickBlock TM Immunostaining blocking solution Shanghai Beyotime Biotechnology 11 Universal antibody diluent Suzhou Xinsaimei Biotechnology 12 Type I collagen Corning 13 matrix adhesive Corning 14 Anti-CD31 antibody Abcam 15 Vimentin Polyclonal Antibody Proteintech 16 Cytokeratin 18 Rabbit mAb Abclonal 17 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) Abcam 18 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) Abcam 19 DAPI Abcam 20 ADVANCE DMEM / F12 Gibco 21 N-2 supplement 1X 22 B27 supplement minus vitamin A 1X 23 N-Acety-L-cyteine 1.25 mM 24 L-glulamine 2 mM 25 EGF 50 ng / ml 26 FGF-2 100 ng / mL 27 HGF 50 ng / ml 28 R-spondin-1 80ng / ml 29 CHIR 99021 1.5 μM 30 Prostaglandin E2 2.5 μM 31 A83-01 500 nM 32 Y-27632 2 μM 33 Human interferon-gamma (IFN-γ) ELISA research kit Jiangsu Jingmei Biotechnology 34 Human Tumor Necrosis Factor α (TNF-α) ELISA Research Kit Jiangsu Jingmei Biotechnology 35 Human interleukin-1β (IL-1β) ELISA research kit Jiangsu Jingmei Biotechnology 36 Human interferon-gamma (IFN-β) ELISA research kit Jiangsu Jingmei Biotechnology 37 Human Chorionic Gonadotropin (HCG) ELIS Research Kit Jiangsu Jingmei Biotechnology 38 E-cadherin Monoclonal antibody Proteintech 39 Goat Anti-Human IgG H&L (DyLight® 488) Abcam 40 2-mercaptoethanol Sigma-Aldrich 41 BSA Sigma-Aldrich 42 ITS-X supplement Gibco 43 NRG1 Novoprotein 44 Knockout serum replacement Thermo 45 CGB Polyclonal antibody Proteintech 46 Anti-HLA G Abcam 47 Anti-Cytokeratin 7 Abcam 48 Anti-Syndecan-1 (SDC-1) Abcam

[0066] II. Verification Experiment

[0067] Example 1: Basic characterization verification of trophoblast organoids

[0068] Collect trophoblast organoids into centrifuge tubes and discard the supernatant. Add 4% PFA to fix the organoids and centrifuge after 10 minutes. Discard the 4% PFA supernatant and wash three times with PBS. Then permeabilize with immunofluorescence permeabilization buffer for 10 minutes, centrifuge, and wash three times with PBS. Add immunofluorescence blocking buffer and block for 30 minutes. Add different dilutions of primary antibody (CGB, CK7, HLA-G, SDC-1, and E-cad) and incubate overnight at 4°C. Aspirate the primary antibody and wash three times with PBS. Add diluted secondary antibody and incubate at room temperature in the dark for 1 hour. DAPI counterstaining: Remove the secondary antibody, wash three times with PBS, add diluted DAPI to counterstain cell nuclei, and incubate at room temperature in the dark for 10 minutes.

[0069] Image acquisition: Immunofluorescence expression was observed using a laser confocal microscope, and images were acquired.

[0070] Please see Figure 2 Chorionic gonadotropin subunit beta (CGB), cytokeratin 7 (CK7), human leukocyte antigen-G (HLA-G), and E-cadherin (E-cadherin) are molecules expressed in trophoblast organoids and are commonly used as biomarkers for trophoblast organoid identification. After successful construction of the trophoblast organoid model, immunofluorescence staining was used to identify the above four biomarkers, and multi-slice scanning images of the cells were acquired using a microscope. Microscopic observation confirmed the simultaneous presence of syncytiotrophoblast (ST) cells and undifferentiated trophoblast cell populations within the constructed trophoblast organoids.

[0071] Example 2: Qualitative Detection Experiment of hCG-β Secretion in Trophoblast Organoids

[0072] The hCG level in the cell supernatant was detected using a commercially available ELISA kit according to the instructions. The prepared supernatant and diluted standard solution were added to the wells of a plate and incubated at 37°C for 30 minutes. After washing the plate, enzyme-labeled reagent was added, and incubation continued at 37°C for 30 minutes. After washing again, chromogenic solution was added, and incubation was continued at 37°C for 10 minutes. Finally, stop solution was added to terminate the reaction. The optical density was measured using an ELISA reader.

[0073] Please see Figure 3Human chorionic gonadotropin (hCG) test strips were used to detect the supernatant of cultured trophoblast organoids. The results showed that the cultured trophoblast organoids were positive for hCG, indicating that they had secretory activity.

[0074] Example 3: Characterization experiment of trophoblast organoids after SLE-IgG treatment

[0075] For trophoblast organoids cultured solely with IgG, three groups of IgG antibodies from different sources were incubated with the isolated trophoblast organoids at room temperature for 1 hour. After incubation, the supernatant was discarded, and the organoids were washed three times with PBS. Subsequent fixation, permeabilization, blocking, and antibody staining (CGB, CK7, HLA-G) followed the universal immunofluorescence protocol described herein. The bound IgG was labeled with the fluorescent secondary antibody goat anti-human IgG H&L according to the manufacturer's instructions. Images were acquired after staining.

[0076] Please see Figure 4 To clarify the specific role of systemic lupus erythematosus (SLE)-derived IgG in trophoblast organoids, this application exposed trophoblast organoids to IgG derived from healthy controls (HC) and SLE patients, respectively. Immunofluorescence was performed on the IgG-treated trophoblast organoids, using co-staining with anti-human IgG secondary antibody (green) and maternal-fetal interface-specific marker antibody to assess the binding of patient-derived IgG to trophoblast organoids.

[0077] Example 4: Basic Characterization Experiments of Endometrial Microarray

[0078] First, aspirate the old culture medium from the microarray channels using a pipette. Then, wash the cells three times with PBS, add 4% paraformaldehyde (PFA), and fix for 15 minutes. Remove the 4% paraformaldehyde (PFA), and wash the cells three more times with PBS. Add immunostaining permeabilization buffer and permeabilize for 10 minutes. Discard the immunostaining permeabilization buffer and wash the cells three times with PBS. Add immunostaining blocking buffer and block at room temperature for 30 minutes.

[0079] Primary antibody incubation: Dilute the primary antibody with antibody diluent according to the ratio, add it to the chip channel, and incubate overnight at 4°C.

[0080] Adding secondary antibody: On the second day, first use a pipette to remove the primary antibody from the chip channel, wash the cells with PBS, and then add the diluted secondary antibody. Incubate at room temperature in the dark for 1 hour.

[0081] DAPI counterstaining: Remove the secondary antibody, wash 3 times with PBS, add diluted DAPI staining solution to counterstain the cell nuclei, and incubate at room temperature in the dark for 10 min.

[0082] Image acquisition: Immunofluorescence expression was observed using a microscope, and images were collected.

[0083] Please see Figure 5 Vimentin, Cytokeratin 18 (CK18), and Cluster of Differentiation 31 (CD31) are characteristic markers of HESC, HEEC, and HUVEC, respectively, and can be used for cell identification. This application characterized the endometrial microarray using immunofluorescence staining and confocal microscopy with multi-scan imaging. The results showed that the constructed microarray could support three-dimensional co-culture of endometrial stromal cells, epithelial cells, and endothelial cells. Cells were evenly distributed and grew well within the scaffold, spontaneously forming an in vivo network structure. Immunofluorescence labeling provided a direct and dynamic view of cell distribution and growth in the three-dimensional microenvironment, providing morphological evidence for successful model construction and laying the foundation for subsequent research on cell function and mechanisms.

[0084] Example 5: Characterization experiment of endometrial microarray after SLE-IgG treatment

[0085] The endometrial microarrays were collected after treatment with IgG from SLE patients, following the same procedure as in Example 4 of this article.

[0086] Please refer to the figure. Figure 6 To investigate the effects of IgG from SLE patients on endometrial cells, this application utilized an endometrial cell microarray model, administered SLE-derived IgG as an intervention, and subsequently acquired images. The results showed that IgG from SLE patients could cause significant damage to endometrial tissue.

[0087] Example 6: Characterization experiment of SLE-IgG treated trophoblast organoid-endometrial microarray co-culture system

[0088] The methods and steps in this section are the same as in Example 1.

[0089] Please see Figure 7 To more accurately simulate the maternal-fetal interface microenvironment, this application integrates trophoblast organoids with an endometrial microarray. After the organoids attach to the matrix, extravillous trophoblasts (EVTs) migrate towards the endometrial side, a process that recreates the key links in maternal-fetal communication and pregnancy maintenance during placental development. The results show that this trophoblast organoid-endometrial microarray co-culture platform can effectively simulate the migration and invasion behavior of trophoblast cells at the maternal-fetal interface.

[0090] Example 7: Dynamic observation experiment on the invasion of trophoblast organoids into the endometrial microarray

[0091] The trophoblast organoids were seeded into the constructed endometrial microarray. After the organoids adhered to the endometrial side, the culture medium was replaced with EVTM medium and the microarray was incubated. The constructed trophoblast organoid-endometrial microarray microsystem was observed under a microscope daily to monitor its invasion and photographed for record-keeping.

[0092] Please see Figure 8 To further investigate functional changes, this application conducted dynamic observations of the invasive ability of trophoblast cells. Bright-field imaging results indicated that after 4 days of culture, SLE-IgG significantly inhibited the proliferation and invasive behavior of EVT organoids in the endometrial stroma.

[0093] Example 8: Quantitative Detection Experiment of Secretory Function of SLE Maternal-Fetal Interface System

[0094] The levels of TNF-α, IFN-γ, IFN-β, and IL-1β in cell supernatant were detected using a commercially available ELISA kit according to the manufacturer's instructions. The prepared supernatant and diluted standard solutions were added to wells of a plate and incubated at 37°C for 30 minutes. After washing the plate, enzyme-labeled reagent was added, and incubation continued at 37°C for 30 minutes. After washing again, chromogenic solution was added, and incubation was continued at 37°C for 10 minutes. Finally, stop solution was added to terminate the reaction. The optical density was measured using an ELISA reader.

[0095] Please see Figure 9 Given the important role of human chorionic gonadotropin (hCG) in embryo implantation and placental development, this application uses ELISA to quantitatively detect the hCG secretion level in the supernatant of the microculture system. The results showed that after treatment with SLE-derived IgG, the hCG level was significantly lower than that of the healthy control (HC) group.

[0096] Please see Figure 10 The level of inflammation in the maternal-fetal interface microsystem was detected using ELISA. Compared with the healthy control group (HC), the expression of pro-inflammatory cytokines (IFN-γ, IL-1β, IFN-β) in each SLE-IgG group was significantly upregulated, suggesting that SLE-IgG can aggravate the local inflammatory response at the maternal-fetal interface.

[0097] Statistical Analysis and Conclusions

[0098] Statistical analysis was performed using GraphPad Prism 10.0 software. All quantitative data are presented as mean ± standard deviation (SD). Independent samples t-test (Student's t-test) was used for comparisons between two groups. Statistical differences were indicated as *p<0.05, **p<0.01, and ***p<0.001.

[0099] In summary, this application constructs a three-dimensional maternal-fetal interface simulation system that uses organ-on-a-chip as a single carrier and integrates trophoblast organoids, endometrial microarrays, and a systemic lupus erythematosus (SLE) disease model. It also establishes standardized methods for constructing trophoblast organoids and endometrial microarrays, SLE pathological models, and multi-dimensional characterization and functional detection techniques. This system organically integrates the creation of the SLE pathological environment, maternal-fetal interface cell interactions, and functional detection under pathological conditions. It overcomes the technical limitations of traditional SLE pregnancy-related research, such as insufficient model physiological and pathological realism, limited detection methods, and single research dimensions. It successfully achieves accurate in vitro simulation and multi-dimensional research of the maternal-fetal interface in SLE patients during pregnancy, providing a standardized, reproducible, and visualized in vitro research platform for basic research on SLE pregnancy-related maternal-fetal interface abnormalities.

Claims

1. A trophoblast organoid-endometrial chip system for simulating a systemic lupus erythematosus (SLE) model at the maternal-fetal interface, characterized in that: The system uses organ-on-a-chip as a carrier and includes trophoblast organoids, endometrial chips, and a systemic lupus erythematosus model.

2. The organ-on-a-chip system for trophoblast organoids-endometrial membranes at the maternal-fetal interface in a simulated systemic lupus erythematosus model based on organ-on-a-chip, as described in claim 1, is characterized in that: The organ-on-a-chip includes a cell inlet pool, a cell culture chamber, and an outlet pool. The cell culture chamber is connected to the cell inlet pool at the top and the outlet pool at the bottom. The trophoblast organoids are used to simulate the placenta invading the endometrium. The endometrial chip is a chip system based on organ-on-a-chip that simulates the three-dimensional structure of the endometrial layer. The remaining trophoblast organoids together construct a three-dimensional maternal-fetal interface. The systemic lupus erythematosus (SLE) model is constructed with the patient's plasma IgG as the core and is used to simulate the disease state of SLE patients.

3. The organ-on-a-chip system for trophoblast organoids-endometrial membranes at the maternal-fetal interface in a simulated systemic lupus erythematosus model based on organ-on-a-chip, as described in claim 2, is characterized in that: The system construction method is as follows: S1: Trophoblast organoid construction: First, trophoblast cells are isolated and cultured in a microporous culture array. S2: Construction of endometrial microarray: Endometrial cells were mixed with matrix gel and aOH solution and added to the microarray through the cell inlet pool. When the cells were observed to be evenly distributed in the cell culture chamber under an optical microscope, the microarray was immediately transferred to a carbon dioxide incubator for further culture. After the cell-matrix gel suspension in the cell culture pool solidified, the culture medium of the three cell types was added to the four channels through the cell inlet pool and cultured for a period of time, so that the cells were in the same growth phase and fully extended. S3: Construction of a systemic lupus erythematosus disease model: Plasma IgG was extracted and added to the endometrial chip to complete the construction of the systemic lupus erythematosus disease model.

4. The organ-on-a-chip system for trophoblast organoids-endometrial membranes at the maternal-fetal interface in a simulated systemic lupus erythematosus model based on organ-on-a-chip, as described in claim 2, is characterized in that: The analysis of trophoblast cells in S1 uses placental villus tissue. After washing, the placental villus is physically cut into pieces. Then, mixed digestive enzymes are added to the placental villus tissue to separate single cells, and trophoblast cells are obtained by centrifugation.

5. The organ-on-a-chip system for trophoblast organoids-endometrial membranes at the maternal-fetal interface in a simulated systemic lupus erythematosus model based on organ-on-a-chip, as described in claim 4, is characterized in that: The mixed digestive enzymes comprise 0.25% pancreatin-EDTA and collagenase IV.

6. The organ-on-a-chip system for trophoblast organoids-endometrial membranes at the maternal-fetal interface in a simulated systemic lupus erythematosus model based on organ-on-a-chip, as described in claim 2, is characterized in that: The trophoblast organoid culture medium in S1 includes a basal culture medium and additives. The basal culture medium is Advanced DMEM / F12, and the additives include N2 additive, B27 additive, 1X penicillin-streptomycin, 1.25 mM N-acetyl-L-cysteine, 2 mM L-glutamine, 50 ng / mL EGF, 1.5 μM CHIR99021, 80 ng / mL R-spondin-1, 100 ng / mL FGF-2, 50 ng / mL HGF, 500 nM A83-01, 2.5 μM prostaglandin E2, and 5 μM Y-27632.

7. The organ-on-a-chip system for trophoblast organoids-endometrial membranes at the maternal-fetal interface in a simulated systemic lupus erythematosus model based on organ-on-a-chip, as described in claim 2, is characterized in that: The endometrial cells in S2 include three types of cells: endometrial stromal cells (HESC), endometrial epithelial cells (HEEC), and endothelial cells (HUVEC).

8. The application of the organ-on-a-chip system for simulating systemic lupus erythematosus at the maternal-fetal interface trophoblast organoid-endometrial chip as described in any one of claims 1-7 as a model in the in-depth observation of the process changes in placental invasion of the endometrial layer.

9. The application as described in claim 8, characterized in that: This system allows for clear observation of the dynamic process of trophoblast organoids invading the endometrial microarray. During the culture process, it can continuously secrete detectable levels of human chorionic gonadotropin (hCG). After the application of plasma IgG from SLE patients, at least one inflammatory factor selected from TNF-α, IFN-γ, IL-1β, and IFN-β can be detected in the cell culture supernatant.

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