In vitro model of synovitis simulating rheumatoid arthritis and its construction method

Abstract

This invention provides an in vitro model of synovitis simulating rheumatoid arthritis and its construction method. The method utilizes a three-channel microfluidic chip as a carrier to perform a 3D co-culture model of synovial fibroblasts, macrophages, and vascular endothelial cells. The construction method provided by this invention enables 3D culture of these three cell types, providing results closer to in vivo realities for subsequent drug screening and efficacy evaluation. This method, without adding additional stimuli, simultaneously cultures the three cell types, achieving in vitro simulation of synovial inflammation through cell-cell interactions, which is significant for a deeper understanding of the roles of different cells in the disease.

Description

Technical Field

[0001] This invention relates to the field of in vitro culture of inflammation models, specifically to an in vitro synovitis model simulating rheumatoid arthritis and its construction method. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by erosive and symmetrical joint lesions and synovitis. Clinical symptoms primarily include joint deformities, morning stiffness, and arthritis of the hands, feet, wrists, ankles, and temporomandibular joints. The basic pathological changes in RA involve synovitis, vasculitis, and pannus formation, gradually leading to cartilage and bone destruction, ultimately resulting in joint deformities and loss of function. With prolonged disease progression, the incidence of disability and functional limitations increases, with a disability rate as high as 61.3% in patients with a disease duration of ≥15 years. RA not only causes a decline in patients' physical function and quality of life but also imposes a significant economic burden on families and society (Reference 1).

[0003] Modern medicine currently believes that pathologically, rheumatoid arthritis (RA) mainly originates from synovial inflammation. Synovial fibroblasts are the tissue structures that maintain normal joint function, one of the largest cell groups in the synovial layer, and the main lesion site of synovial inflammation. Meanwhile, fibroblast-like synovial cells (FLSs), macrophages, and vascular endothelial cells residing in the joint microenvironment have also been identified as key tissue components leading to persistent arthritis in various animal studies and in vitro experiments (References 2-4). FLSs, macrophages, and endothelial cells are key factors in the occurrence and development of RA. Through intercellular interactions, they lead to the progression of synovitis and bone erosion. Therefore, targeting and inhibiting the proliferation and invasion phenotype of FLSs, restoring the dynamic balance of M1 / M2 cells, and regulating vascular endothelial proliferation, thereby promoting the resolution of joint inflammation and tissue repair, is an important direction for RA treatment.

[0004] In various disease studies, 2D single-cell models cannot fully simulate the state of human disease, thus their application in basic research has been questioned. Over the past decade, arthritis research has gradually shifted from traditional two-dimensional cell culture to more complex three-dimensional cell culture, which has enabled more physiological process studies to move from unidirectional to intercellular interactions and to be translated into in vitro studies at the synovial tissue level (Reference 5).

[0005] Previous synovial organ microarrays / synovial organoids have mostly used inflammatory factors (such as TNF-α) for stimulation (Reference 6). In other words, in order to maintain the inflammatory state of the model, various stimulating factors need to be added, and these stimulating factors may become influencing factors in subsequent research. For example, previous literature has required the addition of VEGF / bFGF stimulating factors when co-culturing synovial fibroblasts, macrophages, and endothelial cells (Reference 7).

[0006] References cited

[0007] Published references (non-patent documents)

[0008] 1. 2018 Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis, Chinese Rheumatology Branch [J]. Clinical Medical Research and Practice, 2018, 3(12):201.

[0009] 2. Nygaard G, Firestein G S. Restoring synovial homeostasis inrheumatoid arthritis by targeting fibroblast-like synoviocytes[J]. Nature Reviews Rheumatology, 2020, 16(6): 316-333.

[0010] 3.Rose BJ,Kooyman D LA Tale of Two Joints:The Role of MatrixMetalloproteases in Cartilage Biology[J].Disease markers,2016,2016:1-7.

[0011] 4.Leblond A,Allanore Y,Avouac J.Targeting synovial neoangiogenesis inrheumatoid arthritis[J].Autoimmunity reviews,2017,16(6):594-601.

[0012] 5.Pampaloni F,Reynaud EG,Stelzer EH K.The third dimension bridges the gap between cell culture and live tissue[J].Nature reviews Molecular cellbiology,2007,8(10):839-845.

[0013] 6. Rothbauer M, G, Eilenberger C, et al.Monitoring tissue-levelremodelling during inflammatory arthritis using a three-dimensional synovium-on-a-chip with non-invasive light scattering biosensing[J].Lab Chip.2020;20(8):1461-1471.

[0014] 7. Philippon EML, van Rooijen LJE, Khodadust F, van Hamburg JP, van derLaken CJ, Tas SW. A novel 3D spheroid model of rheumatoid arthritis synovialtissue incorporating fibroblasts, endothelial cells, and macrophages[J]. FrontImmunol. 2023; 14:1188835. Summary of the Invention

[0015] To address the shortcomings of existing technologies, this invention provides an in vitro model of synovitis simulating rheumatoid arthritis and its construction method. Using a microfluidic chip as a carrier, synovial fibroblasts, macrophages, and endothelial cells are co-cultured in 3D without the need for additional stimulating factors. The synovial inflammation state is achieved through the interaction between cells.

[0016] A first aspect of the present invention provides a method for constructing an in vitro model of synovitis simulating rheumatoid arthritis, the method comprising the following steps:

[0017] Step 1: Prepare cell suspensions of synovial fibroblasts, macrophages, and endothelial cells;

[0018] Step 2: Mix the three cell suspensions from Step 1 with the extracellular matrix and seed them into a microfluidic chip with three channels;

[0019] Step 3: Cultivate the microfluidic chip described in Step 2 to form the simulated synovitis model.

[0020] Furthermore, in step 2, the microfluidic chip includes a central channel and two side channels located on both sides of the central channel. A cell suspension of synovial fibroblasts mixed with the extracellular matrix is ​​seeded into the central channel, and a cell suspension of macrophages mixed with the extracellular matrix and a cell suspension of endothelial cells mixed with the extracellular matrix are seeded into the two side channels, respectively.

[0021] Further, step 1 includes: treating the synovial fibroblasts, macrophages, and endothelial cells as follows: digesting with 0.25% trypsin for 2-3 minutes, then adding culture medium containing serum to terminate the digestion, collecting the cell suspension and centrifuging, discarding the supernatant, adding 5 mL of PBS for washing, centrifuging again, discarding the supernatant, and adding to a culture medium containing 85% DMEM medium, 15% FBS, and 1% PS, thereby obtaining cell suspensions of the three types of cells respectively.

[0022] Furthermore, the density of synovial fibroblasts, macrophages, and endothelial cells in the cell suspensions of the synovial fibroblasts, macrophages, and endothelial cells is 0.5 × 10⁻⁶. 6 Cells / mL ~ 4 × 10 6 per mL.

[0023] Furthermore, in step 2, the ratio of the cell suspension to the extracellular matrix for the three types of cells is 1 to 3:1.

[0024] Furthermore, in step 2, the ratio of the number of synovial fibroblasts, macrophages, and endothelial cells inoculated is 1:1:0.5 to 2.

[0025] Furthermore, the extracellular matrix is ​​selected from one of the following: Matrigel, type I rat tail collagen, fibronectin, and methacrylamide gelatin.

[0026] Furthermore, the culture conditions in step 3 are as follows: the microfluidic chip from step 2 is seeded into a culture medium containing 85% DMEM medium, 15% FBS and 1% PS, and cultured in an incubator at 37°C and 5% CO2.

[0027] According to a second aspect of the present invention, an in vitro model of synovitis simulating rheumatoid arthritis is provided, the in vitro model of synovitis being constructed by means of the method described in the first aspect.

[0028] The present invention provides an in vitro model of synovitis simulating rheumatoid arthritis and its construction method, achieving 3D co-culture of synovial fibroblasts, macrophages, and endothelial cells. Compared with traditional 2D single-cell culture, this model more closely resembles the complex synovitis tissue formed in the human body under in vitro conditions. Furthermore, the method provided by this invention achieves the effect of simulating synovial inflammation through the interaction of the three cell types without adding any stimulating factors, and the inflammatory state can be maintained for more than 8 days. Therefore, it provides a model with minimal interference and variables for subsequent understanding of the mechanisms of action of different cells in synovial inflammation. The model provided by this invention, constructed using the method described in the first aspect, more closely resembles the complex physiological conditions in vivo compared to other in vitro models in the prior art, and can provide clear efficacy evaluation indicators, offering a more accurate model for subsequent drug screening and evaluation. Attached Figure Description

[0029] Figure 1A Confocal microscopy images showing the survival of synovial fibroblasts in different extracellular matrices.

[0030] Figure 1B Confocal microscopy images showing the survival of endothelial cells in different extracellular matrices.

[0031] Figure 1C Confocal microscopy images showing the survival of macrophages in different extracellular matrices.

[0032] Figure 2 The permeability data of FITC-glucan perfusion experiments after mixing three cell suspensions with Matrigel in different proportions are shown in the figure.

[0033] Figure 3A Confocal microscopy images showing the 3D growth of endothelial cells at different cell densities are presented.

[0034] Figure 3B Confocal microscopy images showing the 3D growth of synovial fibroblasts at different cell densities are presented.

[0035] Figure 3C Confocal microscopy images showing the 3D growth of macrophages at different cell densities are presented.

[0036] Figure 4 Comparison of inflammation evaluation between the in vitro synovitis models constructed in Examples 1, 11, and 12 and the in vitro synovitis model formed by seeding a single synovial fibroblast. Detailed Implementation

[0037] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. The principles and features of the present invention are described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The embodiments given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0038] A first aspect of the present invention provides a method for constructing an in vitro model of synovitis simulating rheumatoid arthritis, the method comprising the following steps:

[0039] Step 1: Prepare cell suspensions of synovial fibroblasts, macrophages, and endothelial cells; Step 2: Mix the three cell suspensions from Step 1 with extracellular matrix and seed them into a microfluidic chip with three channels; Step 3: Cultivate the microfluidic chip from Step 2 to form the simulated synovitis model.

[0040] As mentioned earlier, synovial fibroblasts, macrophages, and endothelial cells residing in the joint microenvironment are considered key tissue components leading to persistent arthritis. However, current in vitro cultured inflammatory tissues require the addition of stimulating factors during the culture process to maintain the inflammatory state, which leads to significant interference in subsequent mechanistic studies and results in large model variables. This application uses microfluidic chips as a foundation to provide channels for cell-to-cell interactions, eliminating the need for added stimulating factors and enabling 3D co-culture of synovial fibroblasts, macrophages, and endothelial cells. This reduces interference factors after model construction and closely resembles the complex physiological conditions in the human body. The resulting in vitro synovitis model simulating rheumatoid arthritis provides a more accurate model for subsequent research (such as drug screening and efficacy evaluation).

[0041] Furthermore, in one embodiment of this application, the microfluidic chip mentioned in this application specifically includes a central channel and two side channels located on both sides of the central channel. In this case, a cell suspension of synovial fibroblasts mixed with the extracellular matrix can be seeded into the central channel, and cell suspensions of macrophages mixed with the extracellular matrix and cell suspensions of endothelial cells mixed with the extracellular matrix can be seeded into the two side channels respectively. This application does not specifically limit which side channel the macrophages and endothelial cells are seeded into.

[0042] The method of the present invention uses a microfluidic chip to culture three types of cells in a specific structure. Since the microfluidic chip provides a channel for interaction between cells, it enables good permeability between the three types of cells. Therefore, without adding stimulating factors, the in vitro synovitis model constructed in this application can maintain an inflammatory state for a long time.

[0043] Furthermore, in step 1 of the method for constructing an in vitro model of synovitis simulating rheumatoid arthritis provided in this application, the synovial fibroblasts are derived from the synovial tissue of RA patients, the macrophages are formed by stimulation with the THP-1 cell line, and the endothelial cells are primary human umbilical vein endothelial cells (purchased from iCell). Specifically, step 1 includes: treating the synovial fibroblasts, macrophages, and endothelial cells as follows:

[0044] Cells were digested with 0.25% trypsin for 2-3 minutes, followed by the addition of serum-containing culture medium to terminate the digestion. The cell suspension was collected and centrifuged. The supernatant was discarded, and 5 mL of PBS buffer (phosphate-buffered saline) was added for washing. After centrifugation again, the supernatant was discarded, and the cell suspension was added to a culture medium containing 85% DMEM, 15% FBS (fetal bovine serum), and 1% PS (penicillin-streptomycin solution). This yielded cell suspensions of the three cell types. It should be noted that before preparing cell suspensions from synovial fibroblasts, macrophages, and endothelial cells, the synovial fibroblasts, macrophages, and endothelial cells require extraction (or direct purchase) and pre-culture. The specific method is as follows:

[0045] Extraction and culture of synovial fibroblasts: Synovial tissue from RA patients was separated, minced, and pre-digested in 0.25% trypsin. Then, it was digested with type II collagenase at 37°C for 60 min and cultured in an incubator at 37°C and 5% CO2 for later use.

[0046] Extraction and culture of macrophages: THP-1 cells were stimulated with 100 ng / mL phorbol ester for 48 h, and then stimulated with 100 ng / mL LPS + 20 ng / mL IFNγ to form M1 macrophages.

[0047] The endothelial cells used were primary human umbilical vein endothelial cells, cultured in ECM endothelial medium (purchased from ScienceCell, 1001).

[0048] The three types of cells obtained were then prepared into cell suspensions and stored for later use.

[0049] Furthermore, to optimize the culture rate and morphology of simulated arthritis, the amount of culture medium containing 85% DMEM, 15% FBS, and 1% PS can be adjusted during the preparation of the cell suspension. This ensures that the densities of synovial fibroblasts, macrophages, and endothelial cells in their respective cell suspensions reach 0.5 × 10⁻⁶. 6 Cells / mL ~ 4 × 10 6 Cells / mL, preferably 1×10⁻⁶ 6 Cells / mL~2×10 6 per mL.

[0050] Furthermore, in a preferred embodiment, the ratio of the cell suspensions of the three cell types—synovial fibroblasts, macrophages, and endothelial cells—to the mixed extracellular matrix is ​​1 to 3:1, preferably 3:1. It should be noted that the ratio referred to here is the volume ratio of the cell suspensions of the three cell types to the extracellular matrix.

[0051] Furthermore, the ratio of inoculated synovial fibroblasts, macrophages, and endothelial cells is 1:1:0.5 to 2, preferably 1:1:1 to 2, and most preferably 1:1:1. Additionally, the extracellular matrix mentioned in this application can be selected from, for example, Matrigel, type I rat tail collagen, fibronectin, and methacrylamide gelatin, with Matrigel being preferred as it is most beneficial to the growth of the aforementioned three cell types. However, in practice, other extracellular matrices suitable for in vitro culture can also be considered.

[0052] Further, after seeding the treated synovial fibroblasts, macrophages, and endothelial cells into the microfluidic chip, the microfluidic chip is used to form the simulated synovitis model. Specifically, the culture conditions in step 3 are as follows: the microfluidic chip from step 2 is seeded into a culture medium containing 85% DMEM, 15% FBS, and 1% PS, and cultured in an incubator at 37°C and 5% CO2 for at least 24 hours.

[0053] The in vitro model of synovitis simulating rheumatoid arthritis, constructed by 3D co-culturing synovial fibroblasts, macrophages, and endothelial cells using the above method, does not involve the addition of stimulating factors, has minimal interference during the culture process and subsequent studies, and is more closely related to the complex physiological conditions in the human body than existing technologies, with morphology consistent with the formation of synovitis in human joints.

[0054] The following specific embodiments further illustrate the method for constructing the above-mentioned in vitro model of synovitis simulating rheumatoid arthritis.

[0055] Example 1

[0056] A method for constructing an in vitro model of synovitis simulating rheumatoid arthritis, the method comprising the following steps:

[0057] Step 1: Preparation of cell suspensions of synovial fibroblasts, macrophages, and endothelial cells: Synovial fibroblasts, macrophages, and endothelial cells were treated as follows: They were digested with 0.25% trypsin for 3 minutes, followed by the addition of serum-containing culture medium to terminate the digestion. The cell suspension was collected and centrifuged. The supernatant was discarded, and 5 mL of PBS was added for washing. After centrifugation again, the supernatant was discarded, and the cells were added to a culture medium containing 85% DMEM, 15% FBS, and 1% PS to obtain cell suspensions of the three cell types. The density of the synovial fibroblasts, macrophages, and endothelial cells was 1 × 10⁻⁶ cells / mL. 6 cells / mL;

[0058] Step 2: Mix the synovial fibroblast suspension, macrophage suspension, and endothelial cell suspension with extracellular matrix Matrigel at a volume ratio of 3:1 and seed them into a microfluidic chip with three channels, ensuring that the ratio of synovial fibroblasts, macrophages, and endothelial cells is 1:1:1. During seeding, the synovial fibroblasts mixed with Matrigel are seeded in the middle channel, and the macrophages and endothelial cells mixed with Matrigel are seeded in the two side channels, respectively.

[0059] Step 3: The microfluidic chip from Step 2 is seeded into a culture medium containing 85% DMEM medium, 15% FBS and 1% PS, and incubated in an incubator at 37°C and 5% CO2 for 24 hours to form a simulated synovitis model.

[0060] Example 2

[0061] The conditions in this embodiment are the same as in Embodiment 1, except that the extracellular matrix in step 2 is replaced with type I rat tail collagen.

[0062] Example 3

[0063] The conditions in this embodiment are the same as in Embodiment 1, except that the extracellular matrix in step 2 is replaced with fibronectin.

[0064] Example 4

[0065] The conditions in this embodiment are the same as in Embodiment 1, except that the extracellular matrix in step 2 is replaced with methacrylamide gelatin.

[0066] Example 5

[0067] The conditions in this embodiment are the same as in Embodiment 1. The only difference from Embodiment 1 is that the ratio of synovial fibroblasts, macrophages, and endothelial cells to Matrigel is modified to 2:1.

[0068] Example 6

[0069] The conditions in this embodiment are the same as in Embodiment 1. The only difference from Embodiment 1 is that the ratio of synovial fibroblasts, macrophages, and endothelial cells to Matrigel is modified to 1:1.

[0070] Example 7

[0071] The conditions in this embodiment are the same as in Embodiment 1, except that the cell density of synovial fibroblasts, macrophages, and endothelial cells is modified to 0.5 × 10⁻⁶. 6 per mL.

[0072] Example 8

[0073] This embodiment follows the same conditions as Embodiment 1, except that the cell density of synovial fibroblasts, macrophages, and endothelial cells is modified to 2 × 10⁻⁶. 6 per mL.

[0074] Example 9

[0075] The conditions in this embodiment are the same as in Embodiment 1, except that the cell density of synovial fibroblasts, macrophages, and endothelial cells is modified to 4 × 10⁻⁶. 6 per mL.

[0076] Example 10

[0077] The conditions in this embodiment are the same as in Embodiment 1, except that the cell density of synovial fibroblasts, macrophages, and endothelial cells is modified to 0.5 × 10⁻⁶. 6 per mL.

[0078] Example 11

[0079] The conditions in this embodiment are the same as in Embodiment 1. The only difference from Embodiment 1 is that the ratio of synovial fibroblasts, macrophages and endothelial cells is modified to 1:1:0.5.

[0080] Example 12

[0081] The conditions in this embodiment are the same as in Embodiment 1. The only difference from Embodiment 1 is that the ratio of synovial fibroblasts, macrophages and endothelial cells is changed to 1:1:2.

[0082] Experimental Example 1

[0083] Referring to Figure 1, in order to demonstrate the survival of synovial fibroblasts, macrophages and endothelial cells provided by the method of the present invention after mixing with the extracellular matrix, this application uses confocal microscopy for observation. Figures 1A to 1C The images show a comparison of the survival of synovial fibroblasts (RA-FLS), macrophages (M1), and endothelial cells (HUVECs) in each extracellular matrix after 3D culture in Examples 1 to 4, where they were mixed with four different extracellular matrices: Matrigel, type I rat tail collagen (type I rat tail), fibronectin (FN), and methacrylamide gelatin (GELMA). Cell viability and cytotoxicity were assessed using the Calcein / PI staining method. Live cells stained with calcein-AM showed green fluorescence under a confocal microscope, while dead cells stained with propidium iodide (PI) showed red fluorescence. The merged image shows a combination of the two colors.

[0084] from Figures 1A to 1C As can be seen, the three cell types all survived well in the extracellular matrixes mentioned above, especially in Matrigel, where their survival was even better and their cell morphology was better.

[0085] Experimental Example 2

[0086] See Figure 2 To demonstrate the material exchange process of the three cell types in the microfluidic chip in the method of this application, this application uses three cell suspensions and Matrigel in three different proportions to perform a FITC dextran perfusion experiment for verification. Figure 2 The diagram illustrates the permeability of different substances in a microfluidic chip after mixing the three types of cells with Matrigel extracellular matrix at different ratios in Examples 1 and 5-7. The results show that the permeability is good at all the different ratios, especially at a ratio of 3:1, where the permeability is optimal. This indicates that the material exchange between the three types of cells is very good in the method used in this application, suggesting that the interaction between the three types of cells is good.

[0087] Experimental Example 3

[0088] Referring to Figure 3, to demonstrate that the method of this invention can achieve good cell phenotype and functional phenotype at the time of inoculation, this application uses immunofluorescence to characterize the above three cell types. Figures 3A to 3CThe 3D growth of the three cell types in Examples 8-10 at different cell densities is shown. The three cell types of the present invention were seeded in a microfluidic chip at four densities as in Examples 1 and 8-10. Immunofluorescence staining was performed 24 hours later. Synovial fibroblasts were stained with vimentin and cadherin 11 (CDH11), macrophages were stained with platelet endothelial cell adhesion molecule 68 (CD68) and inducible nitric oxide synthase (iNOS), and endothelial cells were stained with platelet endothelial cell adhesion molecule-1 (CD31) and VE-cadherin. The fluorescence intensity was quantified by CDH11, iNOS, and VE-cadherin, respectively. In addition, nuclear staining (DAPI) was performed to confirm that the staining agents used for each cell type remained on the cells.

[0089] The results showed that all three cell types exhibited good fluorescence intensity at various seeding densities, with synovial fibroblasts showing the highest fluorescence intensity at 2×10⁻⁶. 6 CDH11 fluorescence intensity was highest when macrophages were seeded at a density of 2 × 10⁶ cells / ml, and the fluorescence intensity was highest at 2 × 10⁶ cells / ml. 6 The iNOS fluorescence intensity was highest when seeded at a density of cells / ml, while the fluorescence intensity of VE-Cadherin increased with increasing cell density.

[0090] Test Example 4

[0091] See Figure 4 To demonstrate that the in vitro synovitis model constructed using the method of this invention exhibits a better inflammatory response compared to the synovitis model constructed using 2D single-cell culture, and to ensure that the in vitro synovitis model constructed using the method of this invention can maintain an inflammatory state for a prolonged period, inflammation was evaluated using the in vitro synovitis models constructed in Examples 1, 11, and 12, and an in vitro synovitis model formed by seeding a single synovial fibroblast. In the experiment, the single synovial fibroblasts were cultured using standard 2D culture, and TNF-α was added for stimulation during the culture process. Furthermore, it should be noted that, to verify that the model constructed using the method of this invention can maintain an inflammatory state for a prolonged period, in this experimental example, the final 3D co-culture time of Examples 1, 11, and 12 was modified to 8 days, and the 2D culture time of the single synovial fibroblasts was also modified to 8 days. Inflammation evaluation indicators included IL-1β, IL-6, TNF-α, and GM-CSF. Figure 4 The diagram shows a comparison of the inflammation evaluation of the in vitro synovitis models constructed in Examples 1, 11, and 12 with the in vitro synovitis model formed by seeding a single synovial fibroblast.

[0092] As can be seen from the figure, the in vitro synovitis model cultured from single synovial fibroblasts showed almost no effective inflammatory markers in evaluating inflammation against IL-1β, TNF-α, and GM-CSF, with only IL-6 showing an increase. This means that the in vitro synovitis models cultured in the prior art maintain an inflammatory state for a very short time and are ineffective in assessing inflammatory markers. Furthermore, all three cell culture groups of this invention showed increased inflammatory markers compared to the single-cell group, and maintained an inflammatory state even after long-term culture. This demonstrates that the in vitro synovitis model cultured using the method of this invention exhibits good inflammatory markers and can maintain an inflammatory state for a long time.

[0093] According to a second aspect of the present invention, an in vitro model of synovitis simulating rheumatoid arthritis is provided, wherein the in vitro model of synovitis simulating rheumatoid arthritis is constructed by the method described in the first aspect of the present invention. The morphology of the in vitro synovitis model provided by the present invention conforms to the complex morphology of synovitis formed in the complex environment of the human body. Compared with the spherical in vitro synovitis models formed in the prior art, the morphology is easier for cell separation, and it provides a more accurate experimental model for subsequent efficacy evaluation indicators and subsequent drug screening experiments.

[0094] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

Claims

1. A method for constructing an in vitro model of synovitis simulating rheumatoid arthritis, characterized in that, The method includes the following steps: Step 1: Prepare cell suspensions of synovial fibroblasts, macrophages, and endothelial cells, wherein the synovial fibroblasts are derived from the synovial tissue of RA patients; Step 2: Mix the three cell suspensions from Step 1 with the extracellular matrix and seed them into a microfluidic chip with three channels; Step 3: Cultivate the microfluidic chip described in Step 2 to form the simulated synovitis model, wherein... The method described above involves constructing an in vitro synovitis model simulating rheumatoid arthritis without adding any stimulating factors. In step 2, the microfluidic chip includes a central channel and two side channels located on either side of the central channel. A cell suspension of synovial fibroblasts mixed with the extracellular matrix is ​​seeded into the central channel, and cell suspensions of macrophages mixed with the extracellular matrix and cell suspensions of endothelial cells mixed with the extracellular matrix are respectively seeded into the two side channels. The ratio of inoculated synovial fibroblasts, macrophages, and endothelial cells is 1:1:0.5~2, and the ratio of the cell suspension of the three types of cells to the extracellular matrix is ​​1~3:

1. The extracellular matrix is ​​selected from one of the following: Matrigel, type I rat tail collagen, fibronectin, and methacrylamide gelatin.

2. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 1, characterized in that, Step 1 includes the following treatment of the synovial fibroblasts, macrophages and endothelial cells: digestion with 0.25% trypsin for 2-3 minutes, followed by adding culture medium containing serum to terminate digestion, collecting cell suspension and centrifuging, discarding the supernatant, adding 5 mL of PBS for washing, centrifuging again and discarding the supernatant, adding to a culture medium containing 85% DMEM, 15% FBS and 1% PS, thereby obtaining cell suspensions of the three types of cells respectively.

3. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 2, characterized in that, The cell suspensions of synovial fibroblasts, macrophages, and endothelial cells all contained a density of 0.5 × 10⁻⁶ synovial fibroblasts, macrophages, and endothelial cells. 6 Cells / mL ~4×10 6 per mL.

4. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 1, characterized in that, The culture conditions in step 3 are as follows: the microfluidic chip from step 2 is seeded into a culture medium containing 85% DMEM medium, 15% FBS and 1% PS, and then cultured in an incubator at 37°C and 5% CO2.

5. An in vitro model of synovitis simulating rheumatoid arthritis, said in vitro model of synovitis being constructed by the method according to any one of claims 1 to 4.

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