A method for constructing an inflammation-ischemia synergistic injury model based on a biomimetic physiological chip and application thereof

Abstract

The application discloses a kind of based on inflammation-ischemia synergistic damage model of bionic chip and its application, the method includes: bionic physiology substrate is placed in microfluidic chip, cell is inoculated to carry out three-dimensional dynamic culture, obtain bionic organ chip;Patient serum is introduced into chip to carry out inflammation stimulation, then accurate controllable ischemia reperfusion treatment is carried out, to construct inflammation-ischemia synergistic damage model.The application combines patient-derived complex inflammation spectrum with ischemia reperfusion injury under bionic microenvironment, successfully reproduces the synergistic damage state of double pathological stimulation coexisting and mutually exacerbating in clinical complex disease for the first time in vitro.The model highly simulates pathological microenvironment in vivo, retains individualized inflammation characteristics, and can be used for pathological mechanism research, drug screening and precision medicine, with advantages such as high bionics and strong clinical relevance.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and biomimetic chip technology, specifically relating to a method for constructing an inflammation-ischemia synergistic injury model based on a physiological chip and its application. Background Technology

[0002] In the pathological processes of various complex clinical diseases (such as systemic sclerosis, systemic lupus erythematosus, myocarditis, vasculitis, vasospastic diseases, sepsis, and post-organ transplant injury), systemic inflammation and ischemia-reperfusion injury often coexist and exacerbate each other, forming a unique "inflammatory-ischemic synergistic injury microenvironment." This synergistic effect accelerates tissue dysfunction and irreversible damage. For example, in patients with systemic sclerosis, microvascular lesions and high levels of pro-inflammatory factors in the circulation can lead to repeated ischemia-reperfusion injury and chronic inflammatory infiltration in the heart, eventually developing into myocardial fibrosis and even heart failure.

[0003] However, existing in vitro models struggle to accurately reproduce this complex, synergistic pathological state. Traditional two-dimensional cell culture models have significant drawbacks: firstly, their planar culture method lacks the three-dimensional framework of tissues and organs and the intercellular matrix interactions, failing to simulate the real microenvironment of cells in vivo; secondly, they typically use only a single cell type, neglecting intercellular communication and collaboration. Regarding inflammation induction, existing models mostly rely on single cytokines such as tumor necrosis factor-α and interleukin-1β, which cannot reflect the complex, individualized, and heterogeneous inflammatory spectrum within patients, encompassing multiple immune effector molecules (such as autoantibodies, complement, and chemokines). While animal models can reflect the overall pathological response to some extent, existing models rarely and accurately introduce both systemic inflammation and ischemia simultaneously; furthermore, species differences pose significant challenges to the clinical translation of animal experimental results, resulting in high costs and low throughput.

[0004] Microfluidic chip technology offers a novel solution to these problems. Leveraging precise fluid control capabilities, this technology enables three-dimensional dynamic cell culture at the micrometer scale, simulating the perfusion microenvironment, shear forces, and mechanical stresses at the organ level. Simultaneously, the microfluidic system can precisely regulate biochemical factors in time and space, accurately reproducing key pathological events such as the accumulation of metabolic products during ischemia and the burst of oxygen free radicals during reperfusion. Notably, the serum of clinical patients contains abundant, individualized, heterogeneous immune effector molecules, serving as an ideal "repository of inflammatory factors" that can directly reflect the true characteristics of systemic inflammation in patients.

[0005] Therefore, there is an urgent need to develop a novel in vitro model that can integrate a biomimetic microenvironment, complex patient-derived inflammatory stimulation, and controllable ischemia-reperfusion injury in order to overcome the bottlenecks of existing technologies. Summary of the Invention

[0006] Purpose of the invention: The present invention aims to solve the technical problem that existing in vitro models cannot realistically reproduce the synergistic pathological state of inflammation and ischemia-reperfusion injury that coexist and aggravate each other in complex clinical diseases, and provides a method for constructing an inflammation-ischemia synergistic injury model that can simulate the complex pathological microenvironment in vivo.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for constructing an inflammation-ischemia synergistic injury model based on a biomimetic chip, comprising the following steps:

[0009] (1) Place the biomimetic substrate in a microfluidic chip, seed cells or tissues, and perform three-dimensional dynamic culture to obtain a biomimetic organ chip.

[0010] (2) The patient's serum was introduced into the biomimetic organ chip obtained in step (1) for inflammatory stimulation, and then the biomimetic organ chip after inflammatory stimulation was subjected to ischemia-reperfusion treatment to construct an inflammation-ischemia synergistic injury model.

[0011] Furthermore, the biomimetic substrate described in step (1) is made from one or more of gelatin, base glue, chitosan, acrylamide, methacrylamide hyaluronic acid (HAMA), and methacrylamide gelatin (GelMA). These materials have good biocompatibility and adjustable mechanical properties, and can mimic the physicochemical properties of the natural extracellular matrix.

[0012] Further, the cells or tissues mentioned in step (1) are co-cultures of functional cells from mice or humans with fibroblasts. Preferably, the functional cells are selected from at least one of heart cells, liver cells, lung cells, vascular cells, and skin cells. Co-culture can simulate the interaction between parenchymal cells and stromal cells in organ tissues.

[0013] Furthermore, the inflammatory stimulation described in step (2) is performed using a complete culture medium containing patient serum, wherein the patient serum has a volume percentage concentration of 1%-20% in the complete culture medium, and the inflammatory stimulation time is 1-100 hours. By adjusting the serum concentration and stimulation time, systemic inflammatory states of different severity can be simulated.

[0014] Further, the ischemia-reperfusion treatment in step (2) includes: replacing the culture medium in the chip with serum-free culture medium for a first culture (starvation treatment); then replacing the culture medium after the first culture with serum-free and glucose-free culture medium, and performing a second culture (simulated ischemia) under hypoxic conditions (e.g., 1% O2, 5% CO2, 94% N2); finally replacing the culture medium after the second culture with complete culture medium containing patient serum, restoring normal oxygen partial pressure conditions (e.g., 21% O2, 5% CO2), and performing a third culture (simulated reperfusion).

[0015] Preferably, the first culture is an overnight culture (approximately 12-16 hours); the second culture is for 1-24 hours; and the third culture is for 1-24 hours. The time for each step can be adjusted according to the specific cell type and the simulated disease state.

[0016] Furthermore, the patient serum was derived from subjects suffering from systemic sclerosis, systemic lupus erythematosus, myocarditis, vasculitis, vasospastic disorders, sepsis, or post-organ transplant ischemia-reperfusion injury. It could also be derived from other patients with significant inflammatory-ischemic synergistic pathological features.

[0017] Secondly, the present invention provides an inflammation-ischemia synergistic injury model constructed by any of the above-mentioned construction methods.

[0018] Thirdly, the present invention provides the application of the above-mentioned inflammation-ischemia synergistic injury model in the preparation of a kit for screening candidate drugs for treating inflammation-ischemia synergistic injury-related diseases, and in the preparation of a kit for studying the pathological mechanisms of inflammation-ischemia synergistic injury-related diseases.

[0019] Beneficial effects:

[0020] Compared with the prior art, the present invention has the following outstanding advantages:

[0021] (1) Realistic simulation of complex synergistic pathology: This invention is the first to combine "complex systemic inflammation induced by patient serum" with "precisely controllable ischemia-reperfusion injury on microfluidic chip" to successfully construct an inflammation-ischemia synergistic injury model. This breaks through the limitation of traditional models that can only simulate a single stimulus and can realistically reflect the complex pathological state of coexistence and interaction of two injury factors in clinical practice.

[0022] (2) Preservation of individualized patient characteristics: This invention directly uses the serum of clinical patients, which contains molecular profiles such as cytokines, chemokines, and autoantibodies, completely preserving the systemic inflammatory characteristics unique to each individual patient. This enables the model to be used for personalized drug sensitivity testing, biomarker discovery, and precision medicine research, and has extremely high clinical translational value.

[0023] (3) Highly biomimetic microenvironment: This invention combines biomimetic substrates prepared by micro-nano fabrication technology with microfluidic chip technology to achieve three-dimensional dynamic cell culture. Compared with traditional two-dimensional static culture, this model can more realistically simulate the extracellular matrix environment, fluid shear force and material exchange in vivo, making the cell function and response closer to the in vivo state.

[0024] (4) High throughput and standardization potential: Microfluidic chips are easy to miniaturize, array, and automate, and have the potential for high-throughput drug screening and standardized production. This model can replace or significantly reduce corresponding animal experiments in the early stages of drug development, reduce R&D costs, improve screening efficiency, and avoid misjudgments of efficacy due to species differences. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a flowchart illustrating the construction process of an inflammation-ischemia synergistic injury model based on a biomimetic chip according to the present invention.

[0027] Figure 2 To characterize cell structure, apoptosis, and proliferation in a systemic sclerosis (SSc) cardiac involvement model constructed using the method of this invention.

[0028] Figure 3 To detect and analyze calcium transient signals in a systemic sclerosis (SSc) cardiac involvement model constructed using the method of this invention. Detailed Implementation

[0029] The present invention can be better understood from the following embodiments.

[0030] Example 1: Fabrication of a Physiological Heart-Inspired Chip

[0031] (1) Preparation of physiologically similar substrate: Methacrylamide gelatin (GelMA) was weighed and dissolved in cell culture medium preheated to 37°C to prepare a 0.15 g / mL solution. A photoinitiator (e.g., 0.25% w / v lithium phenyl-2,4,6-trimethylbenzoylphosphonite, LAP) was added. The mixed solution was dropped onto a silicon template with a microgroove structure and exposed to ultraviolet light (365 nm, 10 mW / cm²). 2 Crosslink for 30 seconds. Peel the crosslinked GelMA hydrogel film off the template to obtain a biomimetic substrate with a biomimetic microstructure on the surface.

[0032] (2) Assembly of the microfluidic chip: A microfluidic chip with a cell culture chamber and fluid channels was prepared using polydimethylsiloxane (PDMS) through soft photolithography. The above-mentioned physiologically inspired substrate was cut into appropriate sizes and placed at the bottom of the culture chamber of the chip. The PDMS chip layer was irreversibly bonded to the glass substrate by oxygen plasma treatment to complete the chip packaging.

[0033] (3) Cell inoculation and culture: Neonatal rat cardiomyocytes and cardiac fibroblasts were mixed at a ratio of 3:1 and cultured at 2×10⁻⁶ cells / year. 6 Cells were resuspended in cardiomyocyte culture medium at a density of cells / mL. The cell suspension was slowly injected into the chip culture chamber through a microfluidic channel. The chip was placed in a cell culture incubator (37°C, 5% CO2) and incubated for 4 hours to allow the cells to adhere to the physiologically similar substrate. Subsequently, perfusion culture was initiated using the microfluidic system at a rate of 10 μL / min for 48 hours. During this process, the spontaneous beating function of cardiomyocytes was observed to be restored, forming a functionalized physiologically similar cardiac chip, such as... Figure 1 As shown.

[0034] Example 2: Fabrication of a physiologically biomimetic cardiac chip

[0035] (1) Preparation of the physiological substrate: Methacrylamide hyaluronic acid (HAMA) was weighed and dissolved in cell culture medium preheated to 37°C to prepare a 0.1 g / mL solution. A photoinitiator (e.g., 0.25% w / v lithium phenyl-2,4,6-trimethylbenzoylphosphonite, LAP) was added. The mixed solution was dropped onto a silicon template with a microgroove structure and exposed to ultraviolet light (365 nm, 10 mW / cm²). 2 Crosslink for 30 seconds. Peel the crosslinked HAMA hydrogel film off the template to obtain a biomimetic substrate with a biomimetic microstructure on the surface.

[0036] (2) Assembly of the microfluidic chip: A microfluidic chip with a cell culture chamber and fluid channels was prepared using polydimethylsiloxane (PDMS) through soft photolithography. The above-mentioned physiologically inspired substrate was cut into appropriate sizes and placed at the bottom of the culture chamber of the chip. The PDMS chip layer was irreversibly bonded to the glass substrate by oxygen plasma treatment to complete the chip packaging.

[0037] (3) Cell seeding and culture: Human cardiomyocytes, human cardiac microvascular endothelial cells, and human cardiac fibroblasts were mixed in a ratio of 6:5:1 and cultured at 2×10⁻⁶. 6Cells were resuspended in cardiomyocyte culture medium at a density of cells / mL. The cell suspension was slowly injected into the chip culture chamber through a microfluidic channel. The chip was placed in a cell culture incubator (37°C, 5% CO2) and incubated for 4 hours to allow the cells to adhere to the physiologically similar substrate. Subsequently, perfusion culture was initiated using the microfluidic system at a rate of 10 μL / min for 48 hours to form a physiologically similar vascularized heart chip.

[0038] Example 3: Construction and characterization of a cardiac involvement model in systemic sclerosis

[0039] (1) Serum collection and grouping of patients: Based on the clinical manifestations of cardiac involvement in systemic sclerosis (SSc), patients were divided into the SSc occult cardiac involvement (SSc-OHI) group and the SSc severe cardiac involvement (SSc-SHI) group. Serum from healthy volunteers was also collected as a healthy control group. The serum from each group was mixed separately and stored at -80℃ for later use.

[0040] (2) Model building:

[0041] Inflammatory stimulation phase: The biomimetic cardiac chip prepared in Example 1 was used. On day 3 of culture, serum from each group was added to the basal medium (e.g., DMEM / F12) at a final concentration of 5% (v / v) to prepare a complete medium. The medium containing healthy control serum, SSc-OHI serum, or SSc-SHI serum was injected into the corresponding chip, and continuous perfusion stimulation was performed at a flow rate of 10 μL / min for 48 hours.

[0042] Ischemia-reperfusion phase: After the inflammatory stimulation ended, the chip was washed three times with HBSS buffer. First, the perfusion medium was replaced with serum-free medium (DMEM / F12 + 1% Pen-Strep), and perfusion continued overnight (approximately 14 hours) to induce starvation. Second, the perfusion medium was replaced with glucose-free serum-free medium (glucose-free DMEM + 1% Pen-Strep), and the chip was transferred to a hypoxic incubator (1% O2, 5% CO2, 94% N2), and perfused at a flow rate of 5 μL / min for 4 hours to simulate the ischemic period. Finally, the perfusion medium was replaced back with the original complete medium containing patient serum, and the chip was transferred back to a normal incubator (21% O2, 5% CO2), and perfused at a flow rate of 10 μL / min for 12 hours to simulate the reperfusion period.

[0043] (3) Model representation:

[0044] Immunofluorescence staining: After model construction, the cells in the chip were fixed, permeabilized, and blocked. Cardiac sarcomeres were labeled with anti-α-actinin antibody, fibroblasts with anti-Vimentin antibody, and apoptotic cells were labeled using the TUNEL assay kit. Results are as follows: Figure 2 As shown, (a) is an immunofluorescence image of sarcomere structures (α-actinin staining) in cardiomyocytes of each group; (b) is a statistical graph of the number of cells with sarcomere structure disintegration based on (a); (c) is an immunofluorescence image of apoptotic cells (TUNEL staining) and fibroblasts (Vimentin staining) in each group; (d) is a statistical graph of the proportion of TUNEL-positive cells in (c); and (e) is a statistical graph of Vimentin fluorescence intensity in (c). The scale bar in the figures is 50 μm. The results showed that compared with the healthy control group, both the SSc-OHI group and the SSc-SHI group showed significant α-actinin sarcomere structure disorder and breakage, with the SSc-SHI group showing extensive sarcomere loss areas in the cytoplasm. Figure 2 a, 2b); Simultaneously, the model group showed a significant increase in TUNEL-positive apoptotic cells and Vimentin-positive fibroblasts (a, 2b). Figure 2 c, 2d, 2e).

[0045] Functional assays: Calcium transients in cardiomyocytes were detected using a calcium ion fluorescent probe (such as Fluo-4 AM). Results are as follows: Figure 3 As shown in the figures, (a) is the calcium mapping of cardiomyocytes in each group; (b) is the calcium transient waveform of cardiomyocytes in each group; (c) is the calcium transient amplitude of cardiomyocytes in each group; and (d) is the peak-to-peak value of calcium transients in cardiomyocytes in each group. The scale bar in the figures is 25 μm. The results showed that, compared with the healthy control group, the model group (especially the SSc-SHI group) exhibited significant rhythmic irregularities and significantly reduced calcium transient amplitude.

[0046] The above results demonstrate that the model constructed in this invention successfully reproduces the main pathological and functional characteristics of SSc cardiac involvement.

[0047] This invention provides a method for constructing an inflammation-ischemia synergistic injury model based on a biomimetic chip, along with its application ideas and methods. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing an inflammation-ischemia co-injury model based on a biomimetic chip, characterized in that, Includes the following steps: (1) Place the biomimetic substrate in a microfluidic chip, seed cells or tissues, and perform three-dimensional dynamic culture to obtain a biomimetic organ chip. (2) The patient's serum was introduced into the biomimetic organ chip obtained in step (1) for inflammatory stimulation, and then the biomimetic organ chip after inflammatory stimulation was subjected to ischemia-reperfusion treatment to construct an inflammation-ischemia synergistic injury model.

2. The construction method according to claim 1, characterized in that, The biomimetic substrate mentioned in step (1) is made from one or more of gelatin, base glue, chitosan, acrylamide, methacrylamide hyaluronic acid, and methacrylamide gelatin.

3. The construction method according to claim 1, characterized in that, The cells or tissues mentioned in step (1) are co-cultures of functional cells and fibroblasts of mouse or human origin; preferably, the functional cells are selected from at least one of heart cells, liver cells, lung cells, vascular cells, and skin cells.

4. The construction method according to claim 1, characterized in that, The inflammatory stimulation in step (2) is performed using a complete culture medium containing patient serum, wherein the patient serum has a volume percentage concentration of 1%-20% in the complete culture medium, and the inflammatory stimulation time is 1-100 hours.

5. The construction method according to claim 1, characterized in that, The ischemia-reperfusion treatment described in step (2) includes: replacing the culture medium in the chip with serum-free culture medium for the first culture; then replacing the culture medium after the first culture with serum-free and glucose-free culture medium and performing a second culture under hypoxic conditions; and finally replacing the culture medium after the second culture with complete culture medium containing patient serum for the third culture.

6. The construction method according to claim 5, characterized in that, The first culture is an overnight culture; the second culture lasts for 1-24 hours; and the third culture lasts for 1-24 hours.

7. The construction method according to any one of claims 1-6, characterized in that, The patient serum was obtained from subjects suffering from systemic sclerosis, systemic lupus erythematosus, myocarditis, vasculitis, vasospastic diseases, sepsis, or ischemia-reperfusion injury after organ transplantation.

8. An inflammation-ischemia synergistic injury model constructed by the construction method of any one of claims 1-7.

9. The use of the inflammation-ischemia synergistic injury model of claim 8 in the preparation of a kit for screening candidate drugs for treating diseases related to inflammation-ischemia synergistic injury.

10. The application of the inflammation-ischemia synergistic injury model of claim 8 in the preparation of a kit for studying the pathological mechanisms of diseases related to inflammation-ischemia synergistic injury.

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