Application of a functional organ typing system based on human immune organoid in rheumatoid arthritis
By constructing a functional organ typing system based on human immune organoids, the problem of traditional models being unable to simulate the complexity of the human immune system has been solved, enabling accurate in vitro simulation of RA and personalized treatment prediction, and providing an effective tool for RA pathogenesis research and drug screening.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AFFILIATED HOSPITAL OF NANTONG UNIV
- Filing Date
- 2026-05-08
- Publication Date
- 2026-07-10
AI Technical Summary
Traditional cell line cultures or animal models cannot fully simulate the complexity of the human immune system and the individualized pathological characteristics of rheumatoid arthritis, resulting in significant limitations in the study of RA pathological mechanisms and drug development.
A functional organ typing system based on human immune organoids was constructed, including human immune organoid models co-cultured with organoids derived from tonsils and peripheral blood mononuclear cells from RA patients and healthy controls. These models were used to simulate the in vitro state of T and B lymphocytes in RA patients, including germinal center formation, autoantibody secretion, and inflammatory cytokine production.
It successfully reproduces the in vivo immune imbalance characteristics of RA, can simulate the in vitro state of RA patients, provides an accurate disease model, provides a tool for the study of the pathogenesis of RA and personalized treatment, and can predict drug response, supporting personalized drug selection.
Smart Images
Figure CN122369968A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical technology, specifically to the application of a functional organ typing system based on human immune organoids in rheumatoid arthritis. Background Technology
[0002] Rheumatoid arthritis (RA) is a chronic, progressive, systemic autoimmune disease characterized by erosive arthritis. Its course is protracted and prone to relapse, leading not only to synovial hyperplasia and cartilage and bone destruction, but also, in severe cases, affecting multiple internal organs such as the cardiovascular and respiratory systems, significantly impacting patients' quality of life. The key to its core pathological process lies in the imbalance of the immune system, specifically manifested as abnormal activation, proliferation, and dysfunction of autoreactive T lymphocytes (such as Th1 and Th17 cells) and B lymphocytes. Activated T lymphocytes can regulate the immune microenvironment by secreting inflammatory cytokines, while abnormally activated B lymphocytes produce large amounts of autoantibodies such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP). These immune abnormalities collectively drive a persistent inflammatory response and tissue damage in the joints and throughout the body.
[0003] To deepen our understanding of the pathological mechanisms of rheumatoid arthritis (RA), particularly the dynamic regulatory network among immune cells and key molecules in disease progression, constructing in vitro models that can accurately reproduce the core characteristics of the disease has become a core requirement in current research. Traditional cell line cultures or animal models have significant limitations in mechanistic research and drug development because they cannot fully simulate the complexity of the human immune system and the individualized pathological characteristics of RA. Against this backdrop, constructing human immune organoids has become a key research strategy to overcome this bottleneck. Summary of the Invention
[0004] The purpose of this application is to develop a functional organ typing system based on human immune organoids. The key technical point is the development of an in vitro immune organoid model for simulating the pathological features of rheumatoid arthritis (RA). Using this model, the in vivo immune imbalance characteristics of RA were successfully reproduced. This model can be used to simulate the in vitro state of T and B lymphocytes in RA patients, including germinal center formation, autoantibody secretion, inflammatory cytokine production, and immune cell interactions. The immune organoid model involved in this application has great potential for simulating human immune responses and evaluating immunotherapy strategies, providing a powerful tool for studying the pathogenesis of RA and achieving precision treatment of RA. Simultaneously, this type of organoid model can predict clinical drug responses in vitro, and its response to different drugs can reflect the patient's treatment outcome in vivo, highlighting its utility in personalized treatment.
[0005] For the purposes described above, this application provides the following technical solution:
[0006] The application of a functional organ typing system based on human immune organoids in the preparation of models for rheumatoid arthritis research is characterized by the following: the typing system includes human immune organoid models established by co-culturing tonsil-derived organoids with peripheral blood mononuclear cells (PBMCs) from RA patients and healthy controls.
[0007] Preferably, the model used in the rheumatoid arthritis study is used to screen for novel therapeutic targets associated with immune cell populations.
[0008] This application also provides an application of a functional organ typing system based on human immune organoids in establishing drug sensitivity maps, wherein the typing system includes the human immune organoid model described above.
[0009] Preferably, the application includes the following steps:
[0010] S1: Apply the test drug to a human immune organoid model;
[0011] S2: Detect the response of the human immune organoid model to the test drug.
[0012] Preferably, the response of the test drug in S2 is evaluated by one or more of the following indicators: the degree of activation of immune cells, the level of secretion of cytokines, and the level of production of autoantibodies.
[0013] This application also provides an application of a functional organ typing system based on human immune organoids in screening drugs for the treatment of rheumatoid arthritis, wherein the typing system includes the human immune organoid model described above.
[0014] Preferably, the drug is selected from antirheumatic drugs that can improve the disease.
[0015] Preferably, the antirheumatic drugs that can improve the disease include one or more of methotrexate, leflunomide, hydroxychloroquine, tofacitinib, cyclosporine, or tacrolimus.
[0016] This application also provides an application of a functional organ typing system based on human immune organoids in predicting individualized drug responses in patients with rheumatoid arthritis.
[0017] Compared with existing technologies, this application develops a functional organ typing system based on human immune organoids. Using this model, the in vivo immune imbalance characteristics of RA were successfully reproduced. The in vitro state of T and B lymphocytes in RA patients was simulated, including germinal center formation, autoantibody secretion, inflammatory cytokine production, and immune cell interactions. Establishing such organoid models helps to more comprehensively elucidate the pathogenesis of rheumatoid arthritis and identify novel therapeutic targets associated with these immune cell populations.
[0018] Meanwhile, patient-derived organoids can accurately reflect an individual's disease activity. These patient-derived immune organoid models will provide a functional in vitro drug sensitivity testing platform for achieving precision treatment of rheumatoid arthritis (RA). By exposing organoids from specific patients to a range of clinically used RA treatment drugs, healthcare professionals can directly observe and quantify the effects of these drugs on the patient's immunopathological microenvironment, such as whether they can effectively inhibit the secretion of inflammatory factors, reduce the production of autoantibodies, or induce immune cell apoptosis.
[0019] This will allow for the prediction of a patient's sensitivity to different drugs before clinical administration, thereby avoiding ineffective treatment and selecting the most likely individualized treatment plan for the patient. Attached Figure Description
[0020] Figure 1 The images show the morphological representations and morphological-size quantifications of organoids cultured from PBMCs and RA in vitro immunoassays.
[0021] Figure 2 The images show the morphological representations and morphological size quantifications of immune organoids from the HC and RA groups.
[0022] Figure 3 Flow cytometry plots of immune organoids and immune cells in the HC and RA groups.
[0023] Figure 4 The expression of inflammatory factors in the supernatant of immune organoids in the HC and RA groups.
[0024] Figure 5 To analyze the size of immune organoids, expression of inflammatory factors and autoantibodies, and their correlation with disease activity in RA patients with different disease activities.
[0025] Figure 6 A representative image and size quantification of an organoid derived from a drug-resistant patient after exposure to disease-modifying antirheumatic drugs (DMARDs). Detailed Implementation
[0026] I. Experimental Materials and Sources
[0027]
[0028] II. Implementation Methods
[0029] This application provides a functional organ typing system based on human immune organoids and its application in RA.
[0030] Specifically, in one embodiment, the method for constructing the functional organ typing system based on human immune organoids is as follows:
[0031] S1; Tonsil Cell Collection: Healthy tonsil tissue specimens were collected from patients undergoing surgery for tonsillar hypertrophy. Immediately after surgery, fresh tonsil tissue was placed in sterile saline, then transferred to a culture medium containing penicillin-streptomycin antibiotics, and soaked at 4°C for 1 hour to complete tissue decontamination. Subsequently, the tonsil tissue was repeatedly rinsed with sterile phosphate-buffered saline (PBS), sheared into small tissue fragments, and filtered through a 70 μm cell filter to prepare a tonsil single-cell suspension.
[0032] S2: Density gradient centrifugation method for separating PBMCs:
[0033] Collect fresh human peripheral blood. Gently dilute 5-10 mL of peripheral blood with an equal volume of PBS. Slowly spread the diluted peripheral blood onto the tube wall in 3-5 mL of Ficoll separation buffer (density 1.077 ± 0.001 g / mL) at room temperature, forming a clear bilayer interface. Centrifuge at 800 × g for 30-35 min at room temperature using a horizontal rotor centrifuge. Carefully aspirate the intermediate white membrane layer (PBMC) and transfer it to a new centrifuge tube. Add 5-10 mL of PBS, mix well by pipetting, and centrifuge at 300 × g for 10 min. Discard the supernatant. Repeat the washing process twice to obtain the PBMC.
[0034] S3: Cell Culture
[0035] The above-mentioned tonsillar cells and PBMCs were resuspended in 1640 complete medium (containing 10% FBS, 1× non-essential amino acids, 1× sodium pyruvate, 1× penicillin-streptomycin, 1 μg / mL BAFF, and 1× insulin / selenium / transferrin mixture) at specific cell ratios (including 10:1, 2:1, 1:2, and 1:1). They were then seeded onto a mixed collagen and matrix gel (collagen and matrix gel ratios included 1:5, 1:3, 1:2, and 1:1). The optimal cell ratio and collagen / matrix ratio were selected based on the organoid size.
[0036] S4: Organoid observation:
[0037] The size and morphological changes of organoids were observed and recorded daily using an electron microscope. On days 7 and 14, the supernatant of organoid culture was collected and the organoids were stained with immune cells by flow cytometry. The level of inflammatory factors in the supernatant was detected by ELISA.
[0038] S5: Quantitative analysis of organoid morphology, functional parameters, and clinical disease activity in donor patients.
[0039] The organoid morphology includes organoid size and GC-like structure formation; the functional parameters include autoantibody / cytokine secretion and the frequency of specific immune cell subsets.
[0040] S6: Create a personalized drug response prediction platform by exposing organoids to antirheumatic drugs that can improve disease, thereby enabling personalized drug testing. Utilize organoid morphology to correlate in vitro drug responses with clinical indicators such as the DAS28 score. The organoid morphology includes organoid size and GC-like structure formation.
[0041] In one embodiment, the antirheumatic drug is a DMARD, including methotrexate, leflunomide, hydroxychloroquine, tofacitinib, cyclosporine, and tacrolimus.
[0042] This application also provides the application of the aforementioned functional organ typing system based on human immune organoids in the study of the pathogenesis of rheumatoid arthritis, which identifies new therapeutic targets related to immune cell populations through the functional organ typing system based on human immune organoids.
[0043] In one embodiment, the functional organ typing system based on human immune organoids assesses a patient's responsiveness to different drugs in patient-derived immune organoids, the responsiveness including the effects of immune cell activation, cytokine secretion, and autoantibody proliferation. In one embodiment, a functional drug sensitivity profile can be established based on the patient's responsiveness to different drugs.
[0044] III. Experimental Results
[0045] Please see Figure 1 , Figure 1 The images show morphological representations and morphological size quantifications of the PBMC culture system and the in vitro immune organoids for RA constructed in this application. The experimental results show that, compared with the system of simply culturing patient PBMCs, the in vitro immune organoids for RA constructed in this application can more accurately and comprehensively reflect the immune imbalance characteristics in RA patients in terms of morphology, and are more consistent with the actual immune microenvironment state in RA patients.
[0046] Please see Figure 2 , Figure 2This study presents morphological representations and quantitative morphological and size diagrams of immune organoids from the healthy control group (HC group) and the rheumatoid arthritis group (RA group). The experimental results show that, compared with the immune organoids constructed in the HC group, the immune organoids derived from RA patients are significantly larger in size. Furthermore, the immune organoids in the RA group exhibit obvious immune cell aggregation characteristics and clear germinal center (GC)-like structures. The size of the organoids can directly reflect the disease activity of RA patients; the more active the disease activity, the larger the organoid volume.
[0047] Please see Figure 3 , Figure 3 Flow cytometry images of immune cells in immune organoids from the HC and RA groups were obtained. Analysis by flow cytometry showed that, compared to the HC group, the immune organoids derived from RA patients contained more GC-like B cells (CD19). + CD27 + CD38 + ) and antibody-secreting plasma cells (CD19) + CD27 + CD38 ++ Significant amplification was observed, and the frequency changes of specific immune cell subsets were highly consistent with the immunopathological characteristics of RA.
[0048] Please see Figure 4 , Figure 4 The image shows the expression of inflammatory factors and autoantibodies in the supernatant of immune organoids in the HC and RA groups. ELISA detection revealed that, compared with the HC group, the secretion levels of pro-inflammatory cytokines (TNF-α, IL-6) and RA-specific autoantibodies (RF, ACPA) in the cell supernatant of immune organoids in the RA group were significantly increased, which directly reflects the abnormal immune activation characteristics of immune organoids in the RA group.
[0049] Please see Figure 5 , Figure 5This study presents the morphology and size of immune organoids derived from RA patients with different disease activities, the detection results of key inflammatory factors and autoantibody expression levels in the supernatant, and the correlation analysis between the above organoid functional indicators and the patients' clinical disease activity scores. The experimental results show that the overall morphology and size, pro-inflammatory factor secretion levels in the supernatant, and autoantibody production capacity of the successfully constructed RA patient-derived immune organoids exhibit a significant and stable positive correlation with the corresponding RA patients' clinical disease activity. Specifically, the morphology and size of immune organoids derived from RA patients with different disease activities showed a significant positive correlation with the patients' clinical disease activity after in vitro culture. As the DAS28-ESR score and disease activity of RA patients increased, the volume of the constructed immune organoids significantly increased. Furthermore, the correlation between rheumatoid factor (RF) secretion levels and patients' DAS28-ESR disease activity scores was particularly prominent, with a high and statistically significant correlation coefficient. Regarding the expression of inflammatory factors, as the disease activity of patients increased, the secretion levels of core pro-inflammatory factors such as IL-6 and TNF-α in the supernatant of the immune organoids showed a concentration-dependent upregulation. Among them, the expression level of IL-6 was significantly correlated with the DAS28-ESR score. The above results fully demonstrate that the RA immune organoids constructed in this application can simulate the inflammatory microenvironment and immune activation state in vivo in vitro. The morphological and functional parameters of the organoids can serve as intuitive and stable in vitro evaluation indicators for rapidly determining and reflecting the true disease status of RA patients, laying a solid foundation for subsequent disease assessment and personalized drug testing using organoids.
[0050] Please see Figure 6 , Figure 6This study presents representative morphological images and size quantification charts of in vitro immune organoids derived from a methotrexate (MTX)-resistant rheumatoid arthritis (RA) patient. These organoids were exposed to various DMARDs, including methotrexate (MTX), leflunomide, hydroxychloroquine (HCQ), tofacitinib, cyclosporine (CSA), and tacrolimus (FK506). The results showed that treatment with different types and mechanisms of action of DMARDs significantly and drug-dependently altered the volume, morphology, and growth status of the organoids. The inhibitory effects of different drugs on organoid growth varied considerably. Further comparative analysis revealed a high degree of consistency between the in vitro responsiveness and sensitivity of these immune organoids to different DMARDs and the actual treatment response and tolerance observed in the corresponding patient in clinical practice. The above results fully demonstrate that the functional organ typing and drug screening system based on human immune organoids established in this application has good in vitro drug prediction capabilities and can be effectively applied to personalized drug screening and efficacy evaluation for patients with rheumatoid arthritis. It can predict the response and tolerance of patients to different antirheumatic drugs in vitro, providing important experimental evidence and technical support for achieving precise and individualized medication in clinical practice.
[0051] This invention, through model optimization, determined the optimal cell ratio of tonsillar cells to PBMCs to be 2:1 and the collagen to matrix gel ratio to be 1:1 for the model construction, successfully constructing RA patient-specific immune organoids. These organoids can be stably cultured and reproduce adaptive immune responses. Morphologically, the organoids exhibit significant aggregation of immune cells and formation of germinal center (GC)-like structures, and the size of the organoids is positively correlated with clinical disease activity. Functionally, flow cytometry revealed GC-like B cells (CD19) in the RA-derived organoids. + CD27 + CD38 + ) and antibody-secreting plasma cells (CD19) + CD27 + CD38 ++ Significant amplification was observed. ELISA detected increased secretion of pro-inflammatory cytokines (TNF-α, IL-6) and RA-specific autoantibodies (RF, ACPA) in the cell supernatant of the RA group. In summary, these data indicate that the model faithfully reflects the inflammatory and autoimmune microenvironment of RA, and structural and functional indices reflect disease severity. Furthermore, this application demonstrates that this in vitro constructed immune organoid derived from peripheral blood of RA patients can be used for therapeutic drug screening. The in vitro drug responsiveness observed in the organoids was consistent with clinical treatment outcomes in patients, highlighting its potential as a predictive tool for RA treatment selection and personalized treatment strategy optimization.
[0052] In summary, the in vitro immune organoid model for rheumatoid arthritis (RA) constructed through an optimized culture system can stably reproduce the adaptive immune response process and immune imbalance characteristics of RA patients in vitro. Its morphological and functional parameters can accurately reflect the disease activity of patients. At the same time, this model can be effectively used for the study of RA pathogenesis, the screening of new therapeutic targets, and the individualized drug sensitivity testing of RA patients. It provides a brand-new functional in vitro platform for the precision treatment of RA and has important value in the basic research and clinical translational application of rheumatoid arthritis.
Claims
1. The application of a functional organ typing system based on human immune organoids in the preparation of models for rheumatoid arthritis research, characterized by: The typing system includes human immune organoid models established by co-culturing tonsil-derived organoids with peripheral blood mononuclear cells (PBMCs) from RA patients and healthy controls.
2. The application of the functional organ typing system based on human immune organoids according to claim 1 in the preparation of models for rheumatoid arthritis research, characterized in that, The model used in the rheumatoid arthritis study was used to screen for novel therapeutic targets associated with immune cell populations.
3. An application of a functional organ typing system based on human immune organoids in establishing drug sensitivity maps, characterized in that: The typing system includes the human immune organoid model as described in claim 1.
4. The application of the functional organ typing system based on human immune organoids according to claim 3 in establishing drug sensitivity maps, characterized in that, The application includes the following steps: S1: Apply the test drug to a human immune organoid model; S2: Detect the response of the human immune organoid model to the test drug.
5. The application of the functional organ typing system based on human immune organoids according to claim 4 in establishing drug sensitivity maps, characterized in that, The response of the test drug in S2 is assessed by one or more of the following indicators: the degree of activation of immune cells, the level of secretion of cytokines, and the level of production of autoantibodies.
6. The application of a functional organ typing system based on human immune organoids in screening drugs for the treatment of rheumatoid arthritis, characterized in that: The typing system includes the human immune organoid model as described in claim 1.
7. The application of the functional organ typing system based on human immune organoids according to claim 6 in screening drugs for the treatment of rheumatoid arthritis, characterized in that, The drug is selected from antirheumatic drugs that can improve the disease.
8. The application of the functional organ typing system based on human immune organoids according to claim 7 in screening drugs for the treatment of rheumatoid arthritis, characterized in that, The antirheumatic drugs that can improve the disease include one or more of methotrexate, leflunomide, hydroxychloroquine, tofacitinib, cyclosporine, or tacrolimus.
9. Application of a functional organ typing system based on human immune organoids in predicting individualized drug responses in patients with rheumatoid arthritis.