Physical therapeutic apparatus for autoimmune diseases based on microfluidic technology and dendritic cells

Abstract

The invention provides an autoimmune disease physical therapeutic apparatus based on a microfluidic technology and dendritic cells, and belongs to the technical field of biomedical engineering. The therapeutic apparatus provided by the invention comprises an automatic blood sampling system, a target cell separation system, a micro-fluidic chip system, a high-precision liquid flow driving system, a real-time monitoring system, a cell feedback system and an intelligent control system which are connected in sequence. The therapeutic apparatus can integrally complete the whole process from patient blood sampling, target cell separation, physical regulation and control to cell reinfusion, and solves the problem that a safe, effective and controllable autoimmune disease physical therapeutic apparatus is lacked in the prior art.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells. Background Technology

[0002] Autoimmune diseases, such as inflammatory bowel disease (IBD), are caused by abnormal activation of the body's immune system, which attacks its own tissues. Dendritic cells (DCs) are the most potent antigen-presenting cells, playing a central role in initiating and regulating the immune response. Among them, immature dendritic cells (imDCs) have the potential to induce immune tolerance and are an ideal cell type for treating autoimmune diseases.

[0003] Currently, DC-based cell therapy mainly focuses on immune activation therapy for tumors, while research and application of DC-induced immune tolerance therapy for autoimmune diseases are still in their infancy. Existing technologies mostly rely on chemical factors (such as cytokines and drugs) or genetic modifications to regulate DC function. These methods suffer from high costs, safety risks, complex operations, and unstable efficacy. The lack of a safe, controllable, and non-chemically intervened physical method to precisely induce immune tolerance in imDCs has become a technological bottleneck in this field.

[0004] Recent studies have found that fluid shear stress, as a physical factor, can significantly affect the functional state of imDCs (imcocellular dominance cells). For example, appropriate fluid shear stress can inhibit the immune activation capacity of imDCs and promote their mediation of immune tolerance. However, there are currently no integrated medical devices that can translate this scientific discovery into clinical applications.

[0005] Microfluidic chips, also known as biochips, are characterized by their integration and high throughput. They are currently widely used for rapid, convenient, or large-scale sample detection and analysis, disease diagnosis, and drug screening. Furthermore, due to their tiny channels and precise flow control systems, microfluidic chips can achieve precise control of fluid flow velocity and shear force, thus simulating shear force environments under different physiological or pathological conditions. However, there are currently no reports of using microfluidic chips to induce dendritic cells (DCs).

[0006] Based on this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an integrated device that regulates the function of immune cells through physical factors.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells. The device includes an automated blood collection system, a target cell separation system, a microfluidic chip system, a high-precision fluid flow drive system, a real-time monitoring system, a cell reinfusion system, and an intelligent control system, which are connected in sequence.

[0009] Preferably, the microfluidic chip system has a microfluidic chip inside.

[0010] Preferably, the microfluidic chip has a cyclic folded flow channel structure.

[0011] Preferably, the channel cross-sectional depth of the circulating folded channel structure is 40~60 μm.

[0012] Preferably, the microfluidic chip system is configured via a high-precision fluid flow drive system to drive the cell suspension at a perfusion flow rate of 50-100 μL / h, so that the fluid shear force on the cells is 5-10 dy / cm. 2 .

[0013] Preferably, the operating time of the microfluidic chip system is 1 to 3 hours.

[0014] Preferably, the dendritic cells are induced from CD11c dendritic cell precursors.

[0015] The therapeutic device provided by this invention comprises, in sequence, an automated blood collection system, a target cell separation system, a microfluidic chip system, a high-precision fluid flow drive system, a real-time monitoring system, a cell reinfusion system, and an intelligent control system. The automated blood collection system receives whole blood from the patient and mixes it with an anticoagulant. The target cell separation unit separates target cells (such as CD11c dendritic cell precursors) from the mixed whole blood using gradient centrifugation and magnetic bead sorting. The microfluidic chip system is the core of this technology; it contains a microfluidic chip that applies a preset amount of fluid shear force to the target cells. This physical stimulation specifically induces immature dendritic cells to develop immune tolerance characteristics without relying on chemical factors, thus achieving the effect of physical therapy. The high-precision fluid flow drive system provides the power source for the liquid in the microfluidic chip. The real-time monitoring system detects the functional state of the cells using infrared spectroscopy. The cell reinfusion system is used to safely reinfuse the processed cell suspension into the patient. The intelligent control system communicates with the aforementioned systems to set and control the operating parameters (such as flow rate, shear force, temperature, time, etc.) throughout the entire process, ensuring the standardization and repeatability of the operation. The therapeutic device provided by this invention can integrate the entire process from patient blood collection, target cell separation, physical regulation (fluid shear force induction of immune tolerance) to cell reinfusion, solving the problem of the lack of safe, effective, and controllable physical cell tolerance induction devices in the prior art.

[0016] Compared with the prior art, the present invention has the following characteristics: 1. Innovation: For the first time, the biomechanical discovery of "fluid shear force-induced immune tolerance" is applied to integrated medical devices, providing a brand-new physical solution for the treatment of autoimmune diseases.

[0017] 2. High safety: It avoids the potential risks associated with the use of exogenous chemical factors or gene modifications, and utilizes the body's inherent physical signals to regulate cells, resulting in good biocompatibility.

[0018] 3. Highly targeted: By precisely controlling the shear force parameters within the microfluidic chip, the functional state of imDCs can be specifically regulated, with a clear objective and controllable effect.

[0019] 4. Broad application prospects: This platform technology is not only applicable to IBD, but also provides a potential tool for cell therapy of other autoimmune diseases (such as multiple sclerosis and rheumatoid arthritis). Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall structure of the therapeutic device described in this invention; Figure 2 This refers to the overall structure of a microfluidic chip. Figure 3 This is a three-dimensional pressure distribution diagram of the fluid outer wall of a microfluidic chip. Figure 4 This is a two-dimensional interface shear rate diagram of a microfluidic chip. Figure 5 Three-dimensional fluid velocity surface and cross-sectional views of a microfluidic chip; Figure 6 A three-dimensional contour plot of fluid velocity for a microfluidic chip; Figure 7 A three-dimensional distribution diagram of the Reynolds number of the microfluidic chip. Figure 8 S-imDCs represent the expression levels of immunophenotypic molecules in CD11c cell populations before and after FSS loading. resting state Culture under the state DC cells, F-imDCs are DC cells loaded with FSS (the same below). Figure 9 The expression levels of pro-inflammatory cytokines (A), anti-inflammatory cytokines (B), and immunomodulatory molecules (C) in the CD11c cell population after FSS loading; Figure 10 To detect the ability of imDCs to internalize FITC-glucan by flow cytometry; Figure 11 To assess the T cell differentiation capacity after co-culturing DCs and T cells; Figure 12 The proportion of triple-positive cells was determined after co-culturing DCs with spleen-derived naive T cells; Figure 13 A schematic diagram illustrating the construction and treatment of an IBD model; Figure 14 The changes in body weight and disease activity index of mice in each group; Figure 15 The colon length of mice in each group; Figure 16 Representative H&E stained sections from each group; Figure 17 Histological scores of the colon of mice in each group were determined. Figure 18 Count the peripheral blood heme, lymphocytes, monocytes and neutrophils of mice in each group. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] Example 1

[0023] The therapeutic device provided by this invention utilizes microfluidic chip technology to induce immune tolerance in DCs extracted from the patient's body through precisely controlled shear force, without the need for chemical drugs. The processed DCs are then reinfused into the patient to regulate abnormal immune responses, thereby intervening in the progression of autoimmune diseases. The system integrates optical sensors to monitor cell status in real time, enabling controllable physical immunotherapy.

[0024] Taking 7-week-old male C57BL / 6J mice as an example, Figure 1 The diagram below shows the overall structure of the therapeutic device described in this invention. The specific operation of the therapeutic device is as follows: 1. Automated blood collection system: Blood is drawn from the femoral artery of mice and anticoagulated with 15 IU / mL of heparin; 2. Target Cell Separation System: The collected whole blood was sequentially subjected to gradient centrifugation to separate mononuclear cells and to enrich CD11c with magnetic beads using the cell separation system. + Monocytes ( Figure 1 (DCs); specifically: (1) Gradient centrifugation Sample dilution: Dilute whole blood samples 1:1 with sterile PBS buffer.

[0025] Add Ficoll-Paque separation solution: Take an equal volume of Ficoll-Paque PLUS separation solution to the diluted blood sample and add it to a new 1.5 mL sterile centrifuge tube.

[0026] Overlay: Slowly add the diluted blood sample to the surface of the Ficoll-Paque PLUS separation medium to form a clear layer. (Procedure: Use a sterile serum pipette to slowly add the blood sample along the wall of the centrifuge tube to the surface of the Ficoll-Paque PLUS medium, avoiding direct impact of the blood sample on the Ficoll-Paque surface, which may disrupt the layering.)

[0027] Density gradient centrifugation: Carefully place the layered centrifuge tubes into the rotor of a horizontal rotor centrifuge and balance the weight. Centrifugation parameters: Set the centrifugation parameters to 400 g, room temperature (20-25°C), and centrifuge for 30 minutes. Collecting PBMC loops: After centrifugation, carefully remove the centrifuge tube, avoiding shaking as much as possible, and place it vertically in a laminar flow hood. Clear stratification will be visible in the centrifuge tube: the top layer is plasma, the middle turbid ring-shaped layer is the PBMC layer, the bottom clear liquid is the Ficoll-Paque PLUS separation medium, and the bottom layer is a sediment of red blood cells and granulocytes. Using a sterile Pasteur pipette, carefully aspirate the PBMC loops. (Procedure tip: When aspirating, slowly move the pipette tip along the liquid surface above the PBMC loops, aspirating the PBMC layer from top to bottom, avoiding aspirating the upper plasma layer, the lower Ficoll-Paque PLUS separation medium, and the bottom red blood cells and granulocytes. Multiple small aspirations can be performed to improve the purity of the PBMCs). Transfer the collected PBMC suspension to a new sterile centrifuge tube.

[0028] PBMC Washing: Add 2 times the volume of PBS buffer to the centrifuge tube containing the collected PBMCs, gently mix the PBMC suspension, and centrifuge at 1500 rpm for 8 minutes at room temperature. Carefully aspirate the supernatant, removing as much as possible to avoid removing cell pellet. Repeat the washing step twice to further remove residual platelets, plasma proteins, and Ficoll-Paque PLUS separation solution. Aspirate the supernatant after each wash.

[0029] (2) Magnetic bead sorting

[0030] Add CD11c magnetic beads: Centrifuge the above cell suspension at 300 × g for 10 minutes. Completely discard the supernatant. Resuspend 1 × 10⁻⁶ cells in 400 µL buffer. 8 Cell pellet of total cells. Per 1×10 8 Add 100 µL of CD11cMicroBeads UltraPure (Miltenyi Biotec, Order no. 130-125-835) to each total cell. Mix thoroughly and incubate in a refrigerator (2–8°C) in the dark for 10 minutes. For every 1×10⁻⁶ cells, add...8 Add 10 mL of buffer to each cell and centrifuge at 300 × g for 10 minutes. Completely discard the supernatant. Resuspend 1 × 10⁻⁶ cells in 500 µL of buffer. 8 Each cell.

[0031] Magnetic separation involves placing the MS sorting column into the magnetic field of a suitable MACS sorter. The column is then rinsed with 500 µL of buffer solution for preparation. Add the cell suspension to the sorting column. Collect the flow-through, which contains unlabeled cells. Wash the sorting column three times with 500 µL buffer. Collect the unlabeled cells that have flowed through and combine them with the flow-through.

[0032] Remove the sorting column from the sorter and place it on a suitable collection tube. Add 1 mL of buffer to the sorting column. Immediately push the plunger in forcefully to rapidly elute the magnetically labeled CD11c cells.

[0033] 3. Microfluidic Chip System: The enriched CD11c cells were introduced into the microfluidic physical control unit, and parameters were set through the central control unit. The flow rate of the cell suspension was configured through a high-precision fluid drive system, and shear force cycling (FSS) treatment was performed: the cell suspension was driven at a perfusion rate of 100 μL / h, so that the fluid shear force on the cells was 10 dy / cm. 2 The treatment duration was 3 hours, inducing CD11c to develop immune tolerance. During this process, a real-time monitoring system detected the functional state of cells using infrared spectroscopy. Under these conditions, the immune function of DCs was suppressed, and they differentiated into a tolerance phenotype.

[0034] Figures 2-7 This is a schematic diagram of the structure of a microfluidic chip. Figure 2 The overall structure of the microfluidic chip is 6 cm long, 4 cm wide, and 50 μm deep, and is a cyclically folded tubular shape. Figure 3 This is a three-dimensional pressure distribution map of the fluid outer wall of a microfluidic chip. The fluid outer wall pressure is close to the physiological value. Figure 4 This is a two-dimensional interface shear rate diagram of a microfluidic chip. Figure 5 The images show the three-dimensional fluid velocity surface and cross-sectional views of the microfluidic chip. Figure 6 This is a 3D fluid velocity contour plot of a microfluidic chip, with a total of 20 levels. Figure 7 This is a three-dimensional distribution diagram of the Reynolds number of the microfluidic chip. The microfluidic chip used in this embodiment can be fabricated using polydimethylsiloxane (PDMS) with standard soft lithography technology.

[0035] 4. Cell reinfusion system: After collection and processing, cells are intravenously reinfused into the patient via a cell reinfusion system (such as peristaltic pump control).

[0036] Throughout the process, the intelligent control system monitors parameters at each stage to ensure consistency and safety. In this embodiment, the inlet perfusion flow rate is 100 μL / h, and the shear force on the cells in the flow field is 10 dy / cm. 2 It conforms to blood flow characteristics and the flow rate output range of microfluidic peristaltic pumps or syringe pumps.

[0037] The expression levels of immunophenotypic molecules in DCs after FSS loading were detected by flow cytometry: Specifically, cells were labeled for 20 minutes at room temperature with FITC, PE, APC, and BV420-conjugated antibodies against CD80, CD86, CD40, MHC-II, CCR5, CD205, and CD11c (Thermo, USA). After washing twice with PBS, the cells were resuspended in 200 μL of cold (4°C) PBS and measured using a FACS Calibur cytometer (BD Biosciences, USA). CD11c-CD80 / CD86 / CD40 / MHC-II positive cells were analyzed. Results are as follows: Figure 8 As shown, the expression of MHC-II and co-stimulatory molecules CD80, CD86, and CD40 can be significantly reduced through cleavage, indicating that DCs have developed immune tolerance.

[0038] The expression levels of pro-inflammatory cytokines (IL-12a, IL-12b, IFN-γ, TNF-α), anti-inflammatory cytokines (IL-10, TGF-β), and immunomodulatory molecules (ICOS-L, Hmox1) were detected by RT-qPCR. Total RNA was extracted from cleaved (F-imDCs) or uncleaved (S-imDCs) DCs using the TRIzol chloroform method. The total RNA was then reverse transcribed into cDNA using a 5×FastKing RT SuperMix kit (Tiangen Biotech, China) for RT-qPCR analysis. Quantitative real-time amplification was performed on a Bio-Rad quantitative real-time amplification system (Hercules, USA). Two... -ΔΔCt The relative expression of RNA was determined by a method and normalized to GAPDH. The results are as follows: Figure 9 As shown, pro-inflammatory cytokines IL-12a, IL-12b, IFN-γ, and TNF-α were significantly decreased (A), anti-inflammatory cytokines IL-10 and TGF-β were significantly increased (B), and immunomodulatory molecules ICOS-L and Hmox1 were significantly increased (C), indicating that DCs developed immune tolerance. Primer sequences are shown in Table 1.

[0039] Table 1 Primer Sequences

[0040] Endocytosis experiment: To evaluate the endocytosis effect of imDC, 1×106 Immediately incubated direct-acting cells (imDCs) with FITC-dextran particles (final concentration: 1 mg / mL, D1823, Thermo, USA) at 37°C for 2 h. A treatment group was also incubated with an endocytosis inhibitor (a two-site Src tyrosine kinase inhibitor, PP2, 10 μM). The imDCs were then washed three times with PBS to remove unphased particles. After resuspending in cold PBS, the mean fluorescence intensity of the DCs was detected by flow cytometry. Results are as follows: Figure 10 As shown, the ability of F-imDCs to endocytose FITC-glucan was significantly reduced after shearing, indicating a decrease in their antigen "capture" ability.

[0041] T cell activation and apoptosis detection: T cell activation and apoptosis were detected using a mixed lymphocyte reaction (MLR). Allogeneic spleen-derived T cells were isolated using nylon cotton columns (Polysciences, USA) (according to the manufacturer's instructions). DCs and T cells were co-cultured at 37°C and 5% CO2 in an incubator for 48 h at ratios of 1:1, 1:10, and 1:100, respectively. The co-cultured cell suspensions were collected and thoroughly mixed. Then, 100 μL of this mixture was seeded into 96-well plates, followed by the addition of 10 μL of CCK-8 solution (CA1210, Solarbio, China). After 2 h of culture, T cell activation was quantified by measuring the optical density at 450 nm using a microplate reader (Bio-Rad, USA). Figure 11 (As shown). At co-culture ratios of 1:1 and 1:10, F-im DCs significantly reduced the activation capacity of T cells.

[0042] Cells in each co-culture group were labeled with Annexin V Alexa Fluor488 / PI (Solarbio, China) at a 1:10 ratio to induce T cell apoptosis, and the results were analyzed by flow cytometry (BD Biosciences, USA). The results are as follows: Figure 12 As shown, shear force significantly promotes T cell apoptosis compared to S-im DCs cultured under resting conditions. This suggests that F-im DCs cultured under FSS conditions exhibit more pronounced tolerance than S-im DCs cultured under resting conditions.

[0043] The above data were compared between two groups using a paired t-test, and are expressed as mean ± standard deviation. p<0.05, p<0.01, p<0.001, p < 0.0001; ns = no significant difference.

[0044] Example 2

[0045] The therapeutic effect of imDC treated in Example 1 in an IBD model

[0046] Animal Experiment: C57BL / 6J mice were orally administered 3% (w / v) DSS (MB553, Meilunbio, China) in drinking water for 7 days to induce colitis. Forty 6-week-old male mice were randomly divided into four groups: normal control group (control), sick mice + PBS group (PBS), sick mice + S-imDCs group (S-imDCs), and sick mice + F-imDCs group (F-imDCs). The control group was given ordinary drinking water without any treatment. The latter three groups were given 3% DSS in their drinking water for 7 days, and were intraperitoneally injected with 100 μL of PBS or 1×10⁻⁶ PBS on days 3 and 7, respectively. 6 S-imDCs / F-imDCs suspension (e.g.) Figure 13 (As shown). Throughout the experiment, all mice received a standard diet. Symptoms of weight loss, fecal bleeding, and stool consistency were monitored and recorded daily as indicators for calculating the disease activity index (results are shown in...). Figure 14 (As shown). On day 10, blood samples were collected via orbital sinus puncture after anesthesia of the mice. Mice were euthanized by cervical dislocation, and the colon was harvested and its length measured (as shown). Figure 15 (As shown). Histological H&E staining: The colon was fixed in 4% paraformaldehyde and stained with H&E for histopathological analysis (G1120, Solarbio, China). Specifically, after fixation, the colon was longitudinally embedded in paraffin, with sections 5 μm thick, and then processed according to the standard H&E staining protocol. Sections were examined under an orthogonal microscope (Nikon Eclipse E100; Nikon, Japan), and images were captured and histologically scored using an imaging system (Nikon DS-U3; Nikon, Japan) (results shown). Figure 16 , 17 (As shown). Within 1 hour of blood collection, complete blood cell counts were performed using a fully automated hematology analyzer (Mindray, China). The results were analyzed and compared (results are shown in...). Figure 18 (As shown). The above results indicate that the imDC treated with the present invention can effectively alleviate the symptoms of IBD mice.

[0047] In this embodiment, the data were analyzed using one-way ANOVA for comparisons among three or more groups. Mean ± Standard Deviation. p < 0.05 p < 0.01, p < 0.001, p < 0.0001; ns = not significant.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells, characterized in that, The therapeutic device includes an automated blood collection system, a target cell separation system, a microfluidic chip system, a high-precision fluid flow drive system, a real-time monitoring system, a cell reinfusion system, and an intelligent control system, all connected in sequence.

2. The physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells as described in claim 1, characterized in that, The microfluidic chip system contains a microfluidic chip.

3. The physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells as described in claim 2, characterized in that, The microfluidic chip has a cyclic folded flow channel structure.

4. The physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells as described in claim 3, characterized in that, The channel cross-sectional depth of the cyclic folded channel structure is 40~60 μm.

5. The physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells as described in claim 4, characterized in that, The microfluidic chip system is configured via a high-precision fluid flow drive system to drive the cell suspension at a perfusion rate of 50–100 μL / h, so that the fluid shear force on the cells is 5–10 dy / cm. 2 .

6. The physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells as described in claim 5, characterized in that, The microfluidic chip system operates for 1 to 3 hours.

7. The physical therapy device for autoimmune diseases based on microfluidic technology and dendritic cells as described in claim 1, characterized in that, The dendritic cells were induced from CD11c dendritic cell precursors.

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