Rheumatoid arthritis-simulated synovitis in-vitro model and construction method thereof

Abstract

The invention provides a rheumatoid arthritis-simulated synovitis in-vitro model and a construction method thereof, and according to the method, a three-channel micro-fluidic chip is used as a carrier to carry out 3D co-culture of synovial fibroblasts, macrophages and vascular endothelial cells. According to the construction method provided by the invention, 3D culture of the three cells can be realized, and a real result closer to that in vivo can be provided for subsequent drug screening and drug efficacy evaluation. According to the construction method provided by the invention, under the condition of not additionally adding stimulating factors, the three cells are cultured at the same time, the effect of simulating synovitis in vitro is achieved through the interaction among the cells, and the construction method has important significance for deeply understanding the effects of different cells in diseases.

Description

Technical Field

[0001] The present invention relates to the field of in vitro culture of inflammation models, and in particular to an in vitro synovitis model simulating rheumatoid arthritis and a construction method thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by erosive and symmetrical joint lesions and joint synovitis. The main clinical symptoms are joint deformity, morning stiffness, and temporomandibular arthritis of the hands, feet, wrists, ankles, etc. The basic pathological changes of RA are synovitis, vasculitis, and the formation of pannus, and the destruction of articular cartilage and bone gradually occurs, eventually leading to joint deformity and loss of function. As the course of the disease prolongs, the incidence of disability and functional limitation in RA patients increases, and the disability rate of patients with a course of ≥15 years is as high as 61.3%. RA not only causes a decline in the patient's physical function and quality of life, but also brings a huge economic burden to the family and society (Reference 1).

[0003] Modern medicine currently believes that RA pathologically originates mainly from synovial inflammation. Synovial fibroblasts are tissue structures that maintain the normal function of joints. They are one of the largest cell groups in the synovial layer and the main lesion site of synovial inflammation. At the same time, fibroblast-like synoviocytes (FLSs), macrophages and vascular endothelial cells resident in the joint microenvironment have also been considered 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 diseases. They lead to the progression of synovitis and bone erosion through cell-to-cell interactions. Therefore, it is an important direction for the treatment of RA to target the proliferation and invasion phenotype of FLSs, restore the dynamic balance of M1 / M2 cells and regulate the proliferation of vascular endothelium, thereby promoting the regression of joint inflammation and tissue repair.

[0004] In the study of many diseases, 2D single-cell models cannot fully simulate the state of human diseases, so their application in basic research has been questioned. In 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 shift from unidirectional to cell-cell interactions and to be converted to synovial tissue-level studies in vitro (Reference 5).

[0005] In the past, synovial organ chips / synovial organoids were mostly stimulated by inflammatory factors (such as TNF-α) (reference 6). In other words, in order to ensure the inflammatory state of the model, various stimulating factors need to be added, and these stimulating factors may become influencing factors in the subsequent research process. For example, in the past, there was a literature that when synovial fibroblasts, macrophages, and endothelial cells were co-cultured, VEGF / bFGF stimulating factors needed to be added (reference 7).

[0006] References cited

[0007] Published references (non-patent literature)

[0008] 1. 2018 Chinese Rheumatoid Arthritis Diagnosis and Treatment Guidelines, Chinese Medical Association Rheumatology Branch [J]. Clinical Medicine 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] In view of the shortcomings of the prior art, the present invention provides an in vitro synovitis model simulating rheumatoid arthritis and a method for constructing the model. A microfluidic chip is used as a carrier to perform 3D co-culture of synovial fibroblasts, macrophages, and endothelial cells, and no additional stimulation factors are required during co-culture. The synovial inflammation state is achieved through interaction between cells.

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

[0017] Step 1, preparing a cell suspension of synovial fibroblasts, a cell suspension of macrophages and a cell suspension of endothelial cells;

[0018] Step 2, mixing the three cell suspensions in step 1 with the extracellular matrix respectively and inoculating them into a microfluidic chip with three channels;

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

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

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

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

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

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

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

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

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

[0028] The in vitro model of synovitis simulating rheumatoid arthritis and the method for constructing it provided by the present invention realize the 3D co-culture of synovial fibroblasts, macrophages and endothelial cells, which is more in line with the synovitis tissue formed in the complex environment of the human body than the traditional 2D single cell culture. In addition, the method provided by the present invention achieves the effect of simulating synovial inflammation through the interaction of the three cells without adding stimulating factors, and the inflammatory state can be maintained for more than 8 days. Thus, a model with small interference and small variables is provided for the subsequent action mechanism of different cells in synovial inflammation. The model provided by the present invention is constructed by the method of the first aspect, which is more in line with the complex physiological conditions in the body than other in vitro models in the prior art, and can provide clear efficacy evaluation indicators, providing a more accurate model for subsequent drug screening and drug evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 1B Confocal microscopy images showing 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 graph shows the data of the FITC-dextran perfusion experiment after mixing three cell suspensions with Matrigel at different ratios.

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

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

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

[0036] Figure 4 Comparative diagram of inflammation evaluation of the in vitro synovitis model constructed in Example 1, Example 11 and Example 12 and the in vitro synovitis model formed by single synovial fibroblast inoculation. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments. The principles and features of the present invention are described below in combination with the accompanying drawings. It should be noted that the embodiments and features in the embodiments in this application can be combined with each other without conflict. The embodiments cited are only used to explain the present invention and are not used to limit the scope of the present invention.

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

[0039] Step 1, preparing a cell suspension of synovial fibroblasts, a cell suspension of macrophages and a cell suspension of endothelial cells; Step 2, mixing the three cell suspensions in step 1 with extracellular matrix respectively and inoculating them into a microfluidic chip with three channels; Step 3, culturing the microfluidic chip in step 2 to form the simulated synovitis model.

[0040] As mentioned above, synovial fibroblasts, macrophages, and endothelial cells resident in the joint microenvironment are considered to be the key tissue components that cause persistent arthritis. However, in various inflammatory tissues currently cultured in vitro, stimulating factors need to be added during the culture process to ensure the continuous state of inflammation, which leads to great interference problems in the subsequent mechanism research process and large variables in the model. This application uses a microfluidic chip as a basis to provide a channel for interaction between cells without adding stimulating factors, and realizes 3D co-culture of synovial fibroblasts, macrophages, and endothelial cells, reducing the interference factors that exist after model construction, and to a large extent fits the complex physiological conditions in the human body. The in vitro model of synovitis simulating rheumatoid arthritis formed thereby provides a more accurate model for subsequent research (such as drug screening and efficacy evaluation).

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

[0042] The method of the present invention uses a microfluidic chip to culture the three cells in a specific structure. Since the microfluidic chip provides a channel for interaction between cells, the three cells can have good permeability. Therefore, without adding stimulating factors, the in vitro synovitis model constructed by the present application can also maintain the 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 the present application, the synovial fibroblasts provided are derived from the synovial tissue of RA patients, the macrophages are formed by stimulating the cell line THP-1 cells, 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] Digest with 0.25% trypsin for 2 to 3 minutes, then add a culture medium containing serum to terminate the digestion, collect the cell fluid and centrifuge, discard the supernatant and add 5 mL of PBS buffer (phosphate buffer) to wash by blowing, centrifuge again and discard the supernatant, add a culture medium containing 85% DMEM culture medium, 15% FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin solution), thereby obtaining cell suspensions of the three cells respectively. It should be explained that before providing synovial fibroblasts, macrophages and endothelial cells to prepare cell suspensions, the present invention also needs to extract (or directly purchase) and pre-culture the synovial fibroblasts, macrophages and endothelial cells. The specific method is as follows:

[0045] Extraction and culture of synovial fibroblasts: The synovial tissue of RA patients was separated and minced, transferred to 0.25% trypsin for pre-digestion, and then digested with 2 mg / mL type II collagenase at 37°C for 60 min, and cultured in an incubator at 37°C and 5% CO2 for use;

[0046] Extraction and culture of macrophages: After the cell line THP-1 cells were stimulated with 100 ng / mL phorbol ester for 48 h, 100 ng / mL LPS + 20 ng / mL IFNγ were added to stimulate the cells, thereby forming M1 macrophages;

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

[0048] Then, the three types of cells obtained above were prepared into cell suspensions respectively and stored for subsequent use.

[0049] Furthermore, in order to optimize the culture rate of the present application and the morphology of simulated arthritis, the amount of culture medium containing 85% DMEM culture medium, 15% FBS and 1% PS can be adjusted in the process of preparing the cell suspension, so that the density of synovial fibroblasts, macrophages and endothelial cells in the cell suspension of synovial fibroblasts, the cell suspension of macrophages and the cell suspension of endothelial cells in each cell suspension reaches 0.5×10 6 / mL~4×10 6 / mL, preferably 1×10 6 / mL~2×10 6 Pieces / mL.

[0050] Furthermore, in a preferred embodiment, the ratio of the cell suspension of the three cells, namely, 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 mentioned here refers to the volume ratio of the cell suspension of the three cells to the extracellular matrix.

[0051] Further, the ratio of the number of inoculated synovial fibroblasts, macrophages and endothelial cells is 1:1:0.5-2, preferably 1:1:1-2, and most preferably 1:1:1. In addition, the extracellular matrix mentioned in the present application can be selected from one of the following: Matrigel, type I rat tail collagen, fibronectin and methacryloyl gelatin, preferably Matrigel, which is most beneficial to the growth of the above three cells. In fact, in the application process, other extracellular matrices suitable for in vitro culture can also be considered.

[0052] Further, after the treated synovial fibroblasts, macrophages and endothelial cells are inoculated into the microfluidic chip, the microfluidic chip is used to form the simulated synovitis model. Specifically, the culture conditions in the above step 3 are: the microfluidic chip in step 2 is inoculated into a culture solution containing 85% DMEM culture medium, 15% FBS and 1% PS, and placed in an incubator at 37°C and 5% CO2 for culture. The culture time is not less than 24 hours.

[0053] The in vitro model of synovitis simulating rheumatoid arthritis was constructed by 3D co-culturing synovial fibroblasts, macrophages and endothelial cells using the above method. No stimulating factors were added, which had little interference in the culture process and subsequent research. Compared with the existing technology, it is more in line with the complex physiological conditions in the human body, and its morphology is consistent with the morphology formed by synovitis in the human joints.

[0054] The following is a further introduction to the method for constructing the above-mentioned in vitro synovitis model simulating rheumatoid arthritis of the present application through specific examples.

[0055] Example 1

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

[0057] Step 1, preparing cell suspensions of synovial fibroblasts, macrophages and endothelial cells: the synovial fibroblasts, macrophages and endothelial cells were treated as follows: digested with 0.25% trypsin for 3 minutes, then the culture medium containing serum was added to terminate the digestion, the cell fluid was collected and centrifuged, the supernatant was discarded and 5 mL of PBS was added for washing, the supernatant was discarded after centrifugation again, and the culture medium containing 85% DMEM culture medium, 15% FBS and 1% PS was added to obtain cell suspensions of the three cells respectively; the density of the synovial fibroblasts, macrophages and endothelial cells was 1×10 6 Pieces / mL;

[0058] Step 2, the synovial fibroblast suspension, macrophage suspension and endothelial cell suspension are respectively mixed with the extracellular matrix Matrigel at a volume ratio of 3:1 and inoculated into a microfluidic chip with three channels, and the number ratio of synovial fibroblasts, macrophages and endothelial cells is 1:1:1; during inoculation, the synovial fibroblasts mixed with Matrigel are inoculated into the middle channel, and the macrophages mixed with Matrigel and the endothelial cells mixed with Matrigel are inoculated into the two side channels respectively;

[0059] Step 3: Inoculate the microfluidic chip in step 2 into a culture solution containing 85% DMEM culture medium, 15% FBS and 1% PS, and culture it in an incubator at 37° C. and 5% CO 2 for 24 hours to form a simulated synovitis model.

[0060] Example 2

[0061] The conditions of this embodiment are consistent with those of embodiment 1, and the only difference from embodiment 1 is that the extracellular matrix in step 2 is replaced with type I rat tail collagen.

[0062] Example 3

[0063] The conditions in this example are consistent with those in Example 1, and the only difference from Example 1 is that the extracellular matrix in step 2 is replaced by fibronectin.

[0064] Example 4

[0065] The conditions of this embodiment are consistent with those of embodiment 1, and the only difference from embodiment 1 is that the extracellular matrix in step 2 is replaced by methacrylated gelatin.

[0066] Example 5

[0067] The conditions in this example are consistent with those in Example 1, and the only difference from Example 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 example are consistent with those in Example 1, and the only difference from Example 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 example are consistent with those in Example 1. The only difference from Example 1 is that the cell density of synovial fibroblasts, macrophages, and endothelial cells is changed to 0.5×10 6 Pieces / mL.

[0072] Example 8

[0073] The conditions in this example are consistent with those in Example 1. The only difference from Example 1 is that the cell density of synovial fibroblasts, macrophages, and endothelial cells is changed to 2×10 6 Pieces / mL.

[0074] Example 9

[0075] The conditions in this example are consistent with those in Example 1. The only difference from Example 1 is that the cell density of synovial fibroblasts, macrophages, and endothelial cells is changed to 4×10 6 Pieces / mL.

[0076] Example 10

[0077] The conditions in this example are consistent with those in Example 1. The only difference from Example 1 is that the cell density of synovial fibroblasts, macrophages, and endothelial cells is changed to 0.5×10 6 Pieces / mL.

[0078] Embodiment 11

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

[0080] Example 12

[0081] The conditions of this embodiment are consistent with those of Embodiment 1, and the only difference from Embodiment 1 is that the number ratio of synovial fibroblasts, macrophages, and endothelial cells is modified to 1:1:2.

[0082] Test Example 1

[0083] Referring to FIG. 1 , in order to demonstrate the survival of synovial fibroblasts, macrophages and endothelial cells provided by the method of the present invention after being mixed with the extracellular matrix, the present application uses a confocal microscope for observation. Figures 1A to 1C The following is a comparison of the survival of the three cells in each extracellular matrix observed under a confocal microscope after mixing synovial fibroblasts (RA-FLS), macrophages (M1), and endothelial cells (HUVEC) with four extracellular matrices: Matrigel, rat tail collagen type I (rat tail type I), fibronectin (FN), and methacryloyl gelatin (GELMA) in Examples 1 to 4 for 3D culture. This test example uses the cell activity and cytotoxicity detection (Calcein / PI) staining method for detection, using calcein (Celcein-AM) to stain live cells, which show green fluorescence under a confocal microscope, and using propidium iodide (PI) to stain dead cells, which show red fluorescence. Merge is a merged image of the two colors.

[0084] from Figures 1A to 1C It can be seen that the survival of the three cells in the above extracellular matrices is good, especially in Matrigel, where the cell morphology is better.

[0085] Test Example 2

[0086] See also Figure 2 In order to prove the material exchange of the three cells in the microfluidic chip during the interaction process in the method of the present application, the present application uses FITC-dextran perfusion experiments with three cell suspensions and Matrigel at the above three different ratios for verification. Figure 2 The schematic diagram of the permeability of different substances in the microfluidic chip after the three cells and the Matrigel extracellular matrix are mixed at different ratios in Examples 1 and 5-7 is shown. The results show that the permeability of the substances is relatively good when the above-mentioned different ratios are mixed, especially when the ratio is 3:1, the permeability of the substances is the best. It can be seen that in the method adopted in this application, the substance exchange of the three cells is very good, from which it can be inferred that the interaction between the three cells is good.

[0087] Test Example 3

[0088] Referring to FIG3 , in order to prove that the three cells can achieve good cell phenotype and functional phenotype when inoculated by the method of the present invention, the present application adopts immunofluorescence method to characterize the above three cells. FIG. 3A to FIG. 3CThe 3D growth of the three cells in Examples 8-10 at different cell densities is shown. The three cells of the present invention are inoculated in a microfluidic chip at four densities of Examples 1 and 8-10, and immunofluorescence staining is performed 24 hours later, wherein synovial fibroblasts are stained with vimentin and cadherin 11 (CDH11), macrophages are stained with platelet endothelial cell adhesion molecule 68 (CD68) and inducible nitric oxide synthase (iNOS), endothelial cells are stained with platelet endothelial cell adhesion molecule-1 (CD31) and VE-cadherin (VE-cadherin), and fluorescence intensity is quantified with CDH11, iNOS, and VE-cadherin, respectively, and cell nuclei are stained (DAPI) to determine that the stain used for each cell is retained on the cell.

[0089] The results showed that the above three cell types had good fluorescence intensity when seeded at various densities, among which synovial fibroblasts had good fluorescence intensity at 2×10 6 The fluorescence intensity of CDH11 was the highest when the density of macrophages was 2×10 / ml. 6 The fluorescence intensity of iNOS was the highest when the density of inoculation was 1 cell / ml, and the fluorescence intensity of VE-Cadherin increased with the increase of cell density.

[0090] Test Example 4

[0091] See also Figure 4 In order to prove that the state of the in vitro synovitis model constructed by the method of the present invention is better than the inflammatory response effect of the in vitro synovitis model of 2D single cell culture, and at the same time, in order to enable the in vitro synovitis model constructed by the method of the present invention to maintain a long-term inflammatory state, the in vitro synovitis model constructed in Example 1, Example 11 and Example 12 and the in vitro synovitis model formed by inoculating a single synovial fibroblast were used to evaluate inflammation. In the experiment, the single synovial fibroblast was cultured using ordinary 2D, and inflammatory TNF-α was added during the culture process for stimulation. In addition, it should be noted that in order to verify that the model constructed by the method of the present invention can maintain a long-term inflammatory state, in this experimental example, the final 3D co-culture time of Example 1, Example 11 and Example 12 was modified to 8 days, and the 2D culture time of the single synovial fibroblast was also modified to 8 days. The inflammation evaluation indicators are IL-1β, IL-6, TNF-α, GM-CSF, Figure 4 A comparison chart of inflammation evaluation of the in vitro synovitis model constructed in Example 1, Example 11 and Example 12 and the in vitro synovitis model formed by inoculation of a single synovial fibroblast is shown.

[0092] As can be seen from the figure, the in vitro model of synovitis cultured with a single synovial fibroblast 2D showed almost no effective inflammatory indicators when evaluating the inflammation of IL-1β, TNF-α and GM-CSF, and only showed an increased inflammatory indicator in IL-6, that is, the synovitis in vitro model cultured in the prior art maintained an inflammatory state for a very short time and had poor effectiveness for inflammatory indicators. In addition, the three cell culture groups of the present invention all had increased inflammatory indicators compared to the single cell group, and the inflammatory state was maintained despite long-term culture. It can be seen that the in vitro model of synovitis cultured by the method of the present invention showed good inflammatory indicators and was able to maintain an inflammatory state for a long time.

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

[0094] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations 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 one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are contradictory.

Claims

1. A method for constructing an in vitro synovitis model simulating rheumatoid arthritis, characterized in that: The method comprises the following steps: Step 1, preparing a cell suspension of synovial fibroblasts, a cell suspension of macrophages and a cell suspension of endothelial cells; Step 2, mixing the three cell suspensions in step 1 with the extracellular matrix respectively and inoculating them into a microfluidic chip with three channels; Step 3: culturing the microfluidic chip in step 2 to form the simulated synovitis model.

2. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 1, characterized in that: In step 2, the microfluidic chip includes a middle channel and two side channels located on both sides of the middle channel. The cell suspension of synovial fibroblasts mixed with the extracellular matrix is ​​inoculated into the middle channel, and the cell suspension of macrophages mixed with the extracellular matrix and the cell suspension of endothelial cells mixed with the extracellular matrix are inoculated into the two side channels respectively.

3. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 1 or 2, characterized in that: The step 1 comprises: treating the synovial fibroblasts, the macrophages and the endothelial cells as follows: digesting them with 0.25% trypsin for 2 to 3 minutes, then adding a culture medium containing serum to terminate the digestion, collecting the cell fluid and centrifuging it, discarding the supernatant and adding 5 mL of PBS to wash by blowing, centrifuging again and discarding the supernatant, adding to a culture medium containing 85% DMEM culture medium, 15% FBS and 1% PS, thereby obtaining cell suspensions of the three cells respectively.

4. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 3, characterized in that: The density of synovial fibroblasts, macrophages and endothelial cells in the cell suspension of synovial fibroblasts, the cell suspension of macrophages and the cell suspension of endothelial cells is 0.5×10 6 / mL~4×10 6 Pieces / mL.

5. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 3, characterized in that: In the step 2, the ratio of the cell suspension of the three types of cells to the extracellular matrix is ​​1 to 3:

1.

6. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 5, characterized in that: In step 2, the ratio of the number of inoculated synovial fibroblasts, macrophages and endothelial cells is 1:1:0.5-2.

7. The method for constructing an in vitro model of synovitis simulating rheumatoid arthritis according to claim 5, characterized in that: The extracellular matrix is ​​selected from one of the following: Matrigel, type I rat tail collagen, fibronectin and methacryloyl gelatin.

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

9. An in vitro synovitis model simulating rheumatoid arthritis, wherein the in vitro synovitis model is constructed by the method according to any one of claims 1 to 8.

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