A heart organoid-pdms microfluidic chip in-vitro circulation culture system and a construction method thereof

Abstract

The present application belongs to the field of regenerative medicine and organ chip technology, and particularly relates to a heart organoid-PDMS microfluidic chip in-vitro circulation culture system and a construction method thereof. The construction method comprises the following steps: (1) extraction of extracellular vesicles and / or peripheral blood mononuclear cells, (2) construction of a heart organoid, (3) construction of a heart organoid-PDMS microfluidic chip in-vitro circulation culture system, and the like. The present application utilizes the circulation culture of extracellular vesicles and / or peripheral blood mononuclear cells in blood plasma, simulates blood circulation in the human body, and places a heart organoid at the cell culture bin position of a multi-channel microfluidic chip, thereby constructing a heart organoid-PDMS microfluidic chip in-vitro circulation culture system. The system can simulate the process of pathological changes of a heart organoid under the stimulation of extracellular vesicles and / or peripheral blood mononuclear cells in different patient blood plasma in-vitro, and lays a foundation for constructing a model more in line with physiological morphology in-vitro.

Description

Technical Field

[0001] This invention belongs to the fields of regenerative medicine and organ-on-a-chip technology, specifically relating to a method for constructing a cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system using extracellular vesicles or peripheral blood mononuclear cells. Background Technology

[0002] Chronic heart failure (CHF) is a clinical syndrome primarily caused by structural or functional abnormalities of the heart leading to impaired ventricular filling or ejection. Its development is a progressive process that can arise from various underlying heart diseases. Myocardial infarction (MI) and myocarditis / sepsis are identified as the two main initiating and contributing factors to chronic heart failure.

[0003] Myocardial infarction (MI) is the leading cause of death and disability worldwide. Its pathological essence is the irreversible necrosis of the myocardium caused by acute occlusion of the coronary arteries, which then leads to ventricular remodeling, heart failure and malignant arrhythmias. According to statistics, there are more than 2.5 million new cases of myocardial infarction in my country every year. Even if reperfusion therapy is successful, 30% of patients still develop chronic heart failure due to myocardial fibrosis. Traditional MI model research relies heavily on "animal models" (such as coronary artery ligation in mice and balloon occlusion in pigs) and "in vitro cell models". This research method has significant defects: (1) Species difference bottleneck: the heart rate (500-600 bpm) and metabolic rate of rodents are 6-8 times that of humans, and they lack the atherosclerotic plaque rupture mechanism unique to humans; although the cardiac anatomy of large animals (such as pigs) is similar to that of humans, the gene regulatory network (such as the expression profile of miRNAs related to post-injury repair) is more than 40% different from that of humans, resulting in a drug conversion failure rate as high as 90%. (2) Insufficient fidelity of in vitro models: Two-dimensional cardiomyocyte culture cannot simulate the three-dimensional hierarchical structure of the heart; commonly used cryo-trauma methods only induce acute cell necrosis, but ignore the dynamic cascade reaction of immune cell infiltration-cytokine storm-fibroblast activation in the in vivo MI process, and cannot reproduce the electromechanical coupling characteristics of the myocardium. The above limitations seriously hinder the analysis of the pathological mechanism of myocardial infarction and the development of treatment strategies.

[0004] Myocarditis / sepsis is an important functional and inflammatory cause of chronic heart failure. It is often caused by autoimmune diseases, drugs, or toxins. The inflammatory response itself can directly damage cardiomyocytes, causing cardiomyocyte necrosis and apoptosis. Even after acute inflammation subsides, the persistent subclinical inflammatory process and inadequate repair mechanisms can lead to myocardial fibrosis and decreased cardiac function. Some cases of myocarditis can progress to dilated cardiomyopathy, becoming the main pathological basis of chronic heart failure. There are two common methods: 1. Virus / pathogen-induced model method: using Coxsackievirus B3 (CVB3) (most common), adenovirus, parvovirus B19, etc. to infect cultured cardiomyocytes. Viral or pathogen components directly damage cardiomyocytes, activate inflammatory pathways (such as TLR4 / NF-κB), leading to apoptosis and necrosis; 2. Immune factor / inflammatory factor stimulation model method: adding exogenous inflammatory factors (such as TNF-α, IL-1β, IL-6, IFN-γ) to the culture medium to simulate an autoimmune response, directly activating inflammatory signaling pathways in cardiomyocytes, inducing oxidative stress and cellular dysfunction.

[0005] For existing chronic heart failure models, the main shortcomings and limitations are the difficulty in simulating a complete immune microenvironment. In vitro models often lack multicellular interactions (such as endothelial cells, fibroblasts, and dendritic cells) and cannot reproduce the complex immune cell infiltration (neutrophils, macrophages, T cells) cascade reactions in myocarditis. More importantly, they lack hemodynamics and cannot simulate the effects of intracardiac pressure load or the systemic circulation of inflammatory factors.

[0006] In recent years, cardiac organoids (COs) have utilized the self-organizing ability of pluripotent stem cells to differentiate into heart-like structures with spontaneous pulsation, atrioventricular cavity differentiation, and electrical conduction functions within a three-dimensional matrix, providing a revolutionary platform for studying core physiological mechanisms. However, existing cardiac organoid pathological models, as well as myocardial infarction modeling methods such as hypoxia, cobalt chloride (CoCl2) chemical simulation, and cryoinjury, have significant limitations in application:

[0007] (1) Insufficient pathological replication: Hypoxia incubator (e.g., 1% O2) only induces global hypoxic stress and cannot simulate the focal ischemic boundary zone after coronary artery occlusion (physiological difference of oxygen partial pressure gradient >15 mmHg between the infarct core area and the penumbra); Cobalt chloride simulates hypoxia by stabilizing HIF-1α, but induces non-specific metal toxicity (cell viability reduction >60%) and mitochondrial metabolic disorders (ATP synthesis inhibition rate more than twice that of real ischemia).

[0008] (2) Distortion of the damage mechanism: Although the cryopreservation probe can cause local necrosis, the instantaneous low temperature leads to acute necrosis dominated by cell membrane rupture (necrosis / apoptosis ratio of 9:1), rather than the calcium overload-reactive oxygen burst-mitochondrial permeability pore (mPTP) cascade reaction of ischemia-reperfusion in vivo, and cannot reproduce the inflammatory microenvironment such as complement activation and neutrophil infiltration.

[0009] (3) Lack of dynamic process: The above methods are all difficult to control the 'progressive fibrosis process' - collagen deposition reaches its peak at 48 hours after hypoxia treatment (accounting for >40% of the area), which is much faster than the evolution of clinical myocardial infarction over several weeks; and cannot simulate key pathological events of reperfusion injury (such as no-reflow phenomenon and microvascular embolism).

[0010] (4) Uncontrollable structural damage: Freezing injury is often accompanied by ice crystal tearing of the extracellular matrix (ECM), which destroys the three-dimensional structural integrity of organoids (elastic modulus decreases by >50%) and causes loss of electromechanical coupling function.

[0011] Therefore, existing technologies cannot accurately simulate the dynamic closed loop of 'reperfusion-inflammatory response-interstitial remodeling' in the spatiotemporal dimensions, which limits the reliability of mechanism analysis and efficacy evaluation. Summary of the Invention

[0012] Abnormal lipids (especially LDL-C), procoagulant substances (platelets / fibrin), and inflammatory mediators in the blood work synergistically through multiple pathways, ultimately leading to the rupture of atherosclerotic plaques and the formation of coronary thrombosis, which is the core pathological basis of myocardial infarction. However, existing in vitro myocardial infarction models are mostly constructed using cryo-trauma or hypoxia-trauma methods. This approach does not accurately reflect the cause of myocardial infarction; it merely creates a physical model of fibrotic damage. Therefore, to further improve the reliability of in vitro models and construct an in vitro myocardial infarction model that more closely resembles the physiological pathological process in vivo, this invention utilizes extracellular vesicles and / or peripheral blood mononuclear cells extracted from the peripheral blood of myocardial infarction patients, along with a system composed of a microfluidic chip and a peristaltic pump. Based on cardiac organoids constructed using IPSC, a cardiac organoid-PDMS microfluidic chip in vitro circulation culture system was constructed, obtaining a patient pathological model and providing a complete circulation and construction system for subsequent cardiac research.

[0013] This invention utilizes the circulating culture of plasma-containing extracellular vesicles and / or peripheral blood mononuclear cells (PBMCs) to simulate human circulating blood, and places cardiac organoids in the cell culture chamber of a multi-channel microfluidic chip, thereby constructing a cardiac organoid-PDMS microfluidic chip in vitro circulating culture system. This system can simulate the pathological changes in cardiac organoids under the stimulation of plasma extracellular vesicles and / or peripheral blood mononuclear cells from different patients, laying the foundation for constructing more physiologically accurate models in vitro.

[0014] To achieve the above objectives, the technical solution created by this invention is implemented as follows:

[0015] A method for constructing a cardiac organoid-PDMS microfluidic chip in vitro circulation culture system includes the following steps:

[0016] (1) Extraction of extracellular vesicles (EVs) and / or extraction of peripheral blood mononuclear cells (PBMCs);

[0017] (2) Construction of cardiac organoids:

[0018] The cell types involved include cardiomyocytes, endocardial cells (or cardiac endothelial cells), epicardial cells, and smooth muscle cells; the specific steps are as follows:

[0019] Induced pluripotent stem cells (iPSCs) were cultured and a cell suspension of the pluripotent stem cells was obtained. The cell suspension of the pluripotent stem cells was placed in a culture plate, centrifuged, and cell clusters were obtained. The cells were then mixed with a basal medium containing inhibitors, placed in a culture plate, and cultured at 37°C for 12-48 hours to obtain stable organoid aggregate precursors.

[0020] The obtained organoid aggregate precursors are then added to differentiation medium A, differentiation medium B or differentiation medium C, and then placed in a culture plate. After culturing at 37°C for 12-190 hours, stable mesodermal cell masses are obtained, which are cardiac organoids (including cardiomyocytes, endothelial cells and cardiac fibroblasts, etc.) with good cavity morphology.

[0021] (3) Construction of a cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system:

[0022] This includes PDMS chip design and fabrication, capture of cardiac organoids in PDMS chips, and construction of a cardiac organoid-PDMS chip in vitro circulation culture system.

[0023] Specifically, in step (1), extracellular vesicles are extracted using an Exoquick kit from plasma samples of patients with myocardial infarction (MI). The obtained extracellular vesicles are analyzed and identified for morphology, particle size and specific markers. The extracellular vesicles are stored at -80°C for later use.

[0024] Specifically, in step (1), the extraction of extracellular vesicles from plasma samples of patients with myocardial infarction (MI) using the Exoquick kit includes steps such as centrifugation for plasma separation, graded centrifugation to remove impurities, and ultracentrifugation to enrich extracellular vesicles.

[0025] Specifically, in step (1), the concentration of extracted extracellular vesicle proteins is 0.5 - 5.0 μg / μL.

[0026] Specifically, in step (1), after obtaining extracellular vesicles through enrichment, the extracellular vesicles are resuspended in sterile PBS and stored at -80°C for later use.

[0027] Specifically, in step (1), the obtained extracellular vesicles are mixed with basal culture medium (or culture medium) to obtain an extracellular vesicle culture medium (or culture medium) with a protein concentration of 0.5-5.0 μg / mL.

[0028] Preferably, in step (1), the basic culture medium is 1640 medium + B27 Minus Insulin; in the extracellular vesicle culture medium (or culture medium), the protein concentration of extracellular vesicles is preferably 25 μg / mL.

[0029] Specifically, in step (1), peripheral blood mononuclear cell (PBMC) extraction includes:

[0030] Human blood samples were collected from patients with myocardial infarction (MI), lymphocyte separation solution was added, centrifuged, the middle cloud layer cells were aspirated, dissolved, and centrifuged again to obtain human PBMC cells.

[0031] Specifically, in step (2), 2D cell culture is used when culturing induced pluripotent stem cells.

[0032] Specifically, in step (2), the culture plate is a low-adhesion 96u-type bottom hole plate or a 96v-type bottom hole plate, preferably a 96u-type bottom hole plate.

[0033] Specifically, in step (2), the volume of the pluripotent stem cell suspension is 50µL-300µL, preferably 200µL.

[0034] Specifically, in step (2), the cell concentration of pluripotent stem cells is 1.0-3.0 × 10⁻⁶. 4 cell / mL, preferably 1.5 × 10⁻⁶ 4 cell / mL.

[0035] Specifically, in step (2), the basal culture medium is preferably 1640 medium + B27 Minus Insulin.

[0036] Specifically, in step (2), the inhibitor is a ROCK inhibitor, which includes, but is not limited to, Y27632 and Thiazovivin; the concentration of the inhibitor in the basal culture medium is controlled at 1μM-18μM, preferably 8μM.

[0037] Specifically, in step (2), when using differentiation medium A for culturing, the WNT pathway activator or GSK-3α / β inhibitor, PI3 kinase inhibitor, and functional components are added to the basic medium (preferably 1640 medium + B27 MinusInsulin) as differentiation medium A. The obtained organoid aggregation precursor is then added to the differentiation medium A and placed in a culture plate for culturing at 37°C for 12-190 h.

[0038] Preferably, in step (2), the WNT pathway activator or GSK-3α / β inhibitor includes, but is not limited to, CHIR99021, Wnt3a (recombinant protein), and WntConditioned Medium (Wnt CM).

[0039] Preferably, the concentration of WNT pathway activator or GSK-3α / β inhibitor in the basal culture medium is controlled between 2 μM and 12 μM.

[0040] Preferably, in step (2), the PI3 kinase inhibitor (i.e., PI3K inhibitor) includes, but is not limited to, LY294002 and Wortmannin.

[0041] Preferably, the concentration of PI3 kinase inhibitor in the basal culture medium is controlled between 1 μM and 10 μM.

[0042] Preferably, in step (2), the functional component in the preparation of differentiation medium A is any one or a combination of FGF2, activator A, and BMP4.

[0043] Preferably, the concentration of FGF2 in the basal culture medium is controlled at 2μM-15μM; the concentration of activin A is controlled at 2μM-12μM; and the concentration of BMP4 is controlled at 3μM-9μM.

[0044] Specifically, in step (2), when using differentiation medium B for culturing, in the absence of WNT pathway activators and / or in the presence of WNT antagonists, the functional components are added to the basal medium (preferably 1640 medium + B27 MinusInsulin) as differentiation medium B, and the obtained organoid aggregation precursor is added to the differentiation medium B, and then placed in a culture plate and cultured at 37°C for 12-190h.

[0045] Preferably, in step (2), a WNT antagonist is used when preparing differentiation medium B, and no WNT pathway activator is added. The WNT antagonist includes, but is not limited to, one or more of XAV939, C59 / IWR-1, and IWP-2.

[0046] Preferably, the concentration of WNT antagonist in the basal culture medium is controlled between 2 μM and 15 μM.

[0047] Preferably, in step (2), the functional component in the preparation of differentiation medium B is any one or a combination of FGF2, BMP4, retinoic acid (RA) and VEGF.

[0048] Preferably, the concentration of FGF2 in the basal culture medium is controlled at 2μM-15μM; the concentration of BMP4 is controlled at 4μM-18μM; the concentration of RA is controlled at 2μM-15μM; and the concentration of VEGF is controlled at 5 ng / mL-50 ng / mL.

[0049] Specifically, in step (2), when using differentiation medium C for culturing, in the absence of WNT pathway activators and WNT antagonists, the functional components are added to the basal medium (preferably 1640 medium + B27 MinusInsulin) as differentiation medium C, and the obtained organoid aggregation precursor is added to the differentiation medium C, and then placed in a culture plate and cultured at 37°C for 12-190h.

[0050] Preferably, in step (2), the functional component is any one or a combination of FGF2, BMP4 and VEGF.

[0051] Preferably, the concentration of FGF2 in the basal culture medium is controlled at 2μM-15μM; the concentration of BMP4 is controlled at 4μM-18μM; and the concentration of VEGF is controlled at 3 ng / mL-50 ng / mL.

[0052] Specifically, in step (3), the PDMS microfluidic chip is a high-throughput micropillar array chip;

[0053] The PDMS microfluidic chip includes 2-6 chambers, an inlet connecting tube, four inlet branch tubes, an outlet connecting tube, four outlet branch tubes, one inlet and one outlet;

[0054] The rear end of the inlet connecting pipe is the inlet, and the front end of the inlet connecting pipe has two branches, namely the first branch and the second branch. The rear ends of the two inlet branch pipes are connected to the first branch, and the rear ends of the other two inlet branch pipes are connected to the second branch. The front ends of the four inlet branch pipes are respectively connected to the inlet ends of the four chambers.

[0055] The front end of the outlet connecting pipe is the outlet. The rear end of the outlet connecting pipe has two branches, namely the third branch and the fourth branch. The front ends of the two outlet branch pipes are connected to the third branch, and the front ends of the other two outlet branch pipes are connected to the fourth branch. The rear ends of the four outlet branch pipes are respectively connected to the outlet ends of the four chambers.

[0056] Specifically, each chamber has 6-8 independent capture areas; each capture area is equipped with multiple arc-shaped columns (i.e., heart organoid culture tanks) with a diameter of 200-350um to support the heart organoids; the role of these columns is to ensure the stability of the heart organoids within the chip and prevent them from being washed away or sticking together during the culture process.

[0057] Specifically, in step (3), the fabrication of the high-throughput micropillar array chip (PDMS microfluidic chip) includes the following steps:

[0058] a. Based on the structure of the PDMS microfluidic chip, design a two-dimensional sketch of the chip using software, and print the photomask of the sketch;

[0059] b. Prepare the SU-8 template using soft etching technology;

[0060] c. The prepared SU-8 template is modified to have low adhesion to ensure that the SU-8 template and PDMS polymer can be easily separated;

[0061] d. Fabrication of high-throughput array micropillar structures for PDMS polymer chips;

[0062] e. Separate the PDMS chip with the high-throughput array micropillar structure from the SU-8 template to obtain the high-throughput micropillar array PDMS chip;

[0063] f. Drill holes in the rear end of the inlet connector and the front end of the outlet connector of the high-throughput micropillar array PDMS chip obtained in step e using a hole punch (usually 0.5-1.0 mm in diameter) to obtain the inlet and outlet; clean and perform oxygen plasma treatment.

[0064] g. Treat the inside of the chip with an alcohol solution for 15-30 minutes to improve its surface properties and make it more suitable for the growth of heart organoids.

[0065] Preferably, step b specifically involves uniformly coating SU-8 material onto a clean 3-inch silicon wafer, then exposing it to ultraviolet light, and successively baking it on a hot plate at 65°C for 10 minutes and on a hot plate at 95°C for 2 hours; afterwards, developing the pattern on the wafer is done using a developer.

[0066] Preferably, step d specifically involves thoroughly mixing a polydimethylsiloxane (PDMS) prepolymer (specifically, component A of Dow Corning Sylgard 184) with a corresponding platinum catalyst initiator (specifically, component B of Dow Corning Sylgard 184) at a volume ratio of 10 to 15:1 to obtain a PDMS mixture, which is then poured onto the SU-8 template prepared in step c, ensuring complete coverage and no bubble accumulation in the microstructure region;

[0067] Place the cast template in a vacuum dryer and vacuum it (usually -0.08 MPa to -0.1 MPa) for about 15-30 minutes (or until all air bubbles are expelled); remove the template and place it horizontally in an oven to cure at 80°C for 60-120 minutes to ensure complete curing.

[0068] Preferably, the cleaning and oxygen plasma treatment steps in step f are as follows: the chip surface is cleaned with anhydrous ethanol or isopropanol and dried with nitrogen or clean air; then the PDMS chip is treated with oxygen plasma together with a clean glass slide (or another flat PDMS layer), and the two are immediately aligned and bonded together, and placed at room temperature for a period of time (or appropriately heated) to achieve stable bonding.

[0069] Parameters for oxygen plasma treatment: power 30-100 W, time 30-60 seconds.

[0070] Specifically, in step (3), the capture of cardiac organoids in the PDMS chip is to collect cardiac organoids using a syringe and inject the cardiac organoids into the prepared PDMS microfluidic chip.

[0071] The preferred specific steps are as follows:

[0072] 1) Place the prepared PDMS microfluidic chip in a flat-panel incubator, and connect two connecting tubes (specifically flexible Tygon tubes) to the inlet and outlet respectively. Use a syringe to inject mTeSR1 culture medium into the chip through the inlet to clean the chip and remove internal air bubbles.

[0073] 2) Then, the heart organoids are injected into the chip using a syringe. During the injection process, the outlet is connected to the connecting tube (specifically a flexible Tygon tube) and the syringe containing mTeSR1 culture medium.

[0074] 3) After injecting the organoids into the chip, remove the syringe and adjust the position of the chip to control the position of the organoids in the chamber, so that they can remain stably in multiple capture areas;

[0075] 4) Using a syringe, replace the normal culture medium with extracellular vesicles and / or peripheral blood mononuclear cells (PBMCs) for culture; then connect the inlet end of the peristaltic pump to the outlet connecting tube (specifically a flexible Tygon tube) through the circulation tubing, and connect the outlet end of the peristaltic pump to the inlet connecting tube (specifically a flexible Tygon tube) through the circulation tubing to construct the chip external circulation culture system.

[0076] Specifically, during in vitro circulation culture, the circulating culture medium (preferably RPMI 1640 medium (Gibco) + extracellular vesicles (EV) and / or peripheral blood mononuclear cells (PBMC)) is replaced every 24-72 hours, and the culture is carried out for a total of 24-240 hours.

[0077] Furthermore, based on a general inventive concept, the present invention also provides a cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system constructed by the above method.

[0078] Furthermore, based on a general inventive concept, the present invention also provides the application of the aforementioned cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system in the diagnosis, prevention, or treatment of myocardial infarction, cardiomyocyte injury, heart failure, and chronic heart disease.

[0079] Specifically, the present invention also provides the application of the cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system in the development, screening or preparation of therapeutic products for myocardial infarction, cardiomyocyte injury or heart failure.

[0080] Compared with the prior art, the advantages of the present invention are:

[0081] To overcome the limitation of lacking systematic humoral factor regulation in in vitro models, this invention proposes a method for constructing cardiac organoids using extracellular vesicles—a PDMS microfluidic chip in vitro circulation culture system. This method constructs an integrated microfluidic cardiac organoid platform based on "patient plasma extracellular vesicles (EVs)," which has the following advantages:

[0082] 1. EVs as pathological signal carriers: EVs (80-200 nm in diameter) isolated from the plasma of myocardial infarction patients have 8-12 times higher levels of pathology-related miRNAs than healthy individuals, and are rich in inflammatory factors such as TNF-α and IL-6, which can well mimic the characteristics of human blood.

[0083] 2. PDMS as a pathological signal carrier: PDMS is isolated from the plasma of patients or healthy individuals, providing an activated pool of immune effector cells (especially T cells and monocytes / macrophages) to drive immune responses against cardiomyocytes (including direct killing, cytokine storm, oxidative stress, etc.), thereby simulating the core pathological process of myocarditis—immune-mediated myocardial injury—in a culture dish.

[0084] 3. Construction of dynamic circulating culture system: A closed-loop perfusion circuit was established in a microfluidic chip to continuously infuse EVs into vascularized cardiac organoids, thereby inducing and reproducing the differentiation of myofibroblasts and the accumulation of pathological collagen in vivo.

[0085] 4. The core innovation of this invention is that it achieves: (a) using "organ-level pathological phenotypes driven by the human body fluid microenvironment" to avoid species bias in animal models; (b) the dynamic delivery of EVs simulates the function of a circulating culture system, simulating the entire process of vascular infiltration-tissue distribution-metabolic clearance; (c) in the later stage, it can be combined with high-throughput drug screening to establish a closed-loop platform of "patient-iPSC organoid-EV model-precision drug use", providing a highly predictive tool for new drug development and personalized treatment. Attached Figure Description

[0086] Figure 1 The results of transmission electron microscopy identification of the morphology of the obtained extracellular vesicles in Example 1 are shown.

[0087] Figure 2 The results of wb analysis of extracellular vesicles (EVs) in healthy individuals and MI patients in Example 1;

[0088] Figure 3 The results of the analysis and identification of exosome particle size and specific markers obtained in Example 1 using ZetaView PMX 110 particle tracker and Western blotting;

[0089] Figure 4 Example 1: Differential analysis of extracellular vesicle (EV) transcriptomes between MI patients and healthy individuals;

[0090] Figure 5 This is a design diagram of the PDMS chip in Example 1; where 1 is the PDMS chip, 2 is the heart organoid culture tank, 3 is the heart organoid, 4 is the inlet, and 5 is the outlet.

[0091] Figure 6 This is an assembly diagram of the external circulation PDMS chip in Example 1; wherein 1, PDMS chip, 2, heart organoid culture tank, 3, heart organoid, 4, inlet, 5, outlet, 6, incubator, 7, circulation tubing (containing circulating culture medium (specifically extracellular vesicles)), 8, circulating peristaltic pump, and 9, extracellular vesicles.

[0092] Figure 7 Immunofluorescence image of vascularized cardiac organoids in Example 1: CD31 represents endothelial cells, TNNT represents cardiomyocyte distribution, and DAPI represents cell nucleus distribution;

[0093] Figure 8 The immunofluorescence results of exosomes (PKH26 labeled) entering the heart organoids after 24 hours of culture using a culture medium containing exosomes (PKH26 labeled) in Example 1;

[0094] Figure 9 The RNA transcriptome sequencing results of the organoids were obtained after culturing cardiac organoids for 72 hours using a culture medium containing EVs from MI patients and EVs from healthy individuals, as described in Example 1.

[0095] Figure 10 The results of transmission electron microscopy identification of PBMC morphology extracted from the blood of healthy individuals and patients are shown in Example 2.

[0096] Figure 11 This is an assembly diagram of the external circulation PDMS chip in Example 2; wherein 1, PDMS chip, 2, cardiac organoid culture tank, 3, cardiac organoid, 4, inlet, 5, outlet, 6, incubator, 7, circulation tubing (containing circulating culture medium (specifically peripheral blood mononuclear cells)), 8, circulating peristaltic pump, and 9, extracellular vesicles.

[0097] Figure 12 KEGG analysis results of RNA transcriptome sequencing of organoids;

[0098] Figure 13 This is an assembly diagram of the external circulation PDMS chip in Example 3; wherein 1, PDMS chip, 2, cardiac organoid culture tank, 3, cardiac organoid, 4, inlet, 5, outlet, 6, incubator, 7, circulation tubing (containing circulating culture medium (specifically extracellular vesicles and peripheral blood mononuclear cells)), 8, circulating peristaltic pump, and 9, extracellular vesicles;

[0099] Figure 14 In Example 3, the accumulation of collagen in cardiac organoids was assessed by immunofluorescence after culturing them in a culture medium containing extracellular vesicles and PBMCs from healthy individuals for 72 hours. Detailed Implementation

[0100] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of the present invention.

[0101] Example 1

[0102] A method for constructing a cardiac organoid-PDMS microfluidic chip in vitro circulation culture system is as follows:

[0103] (I) Experimental Methods:

[0104] To obtain an in vitro model of heart disease using extracellular vesicles from cardiac patients, this invention designs a three-step approach: including the extraction of extracellular vesicles, the construction of cardiac organoids, and the construction of an organoid on-chip in vitro circulation culture system using a peristaltic pump and PDMS.

[0105] 1. Extraction of extracellular vesicles:

[0106] The plasma samples from the healthy control group and myocardial infarction (MI) patients in this invention were obtained from the First Affiliated Hospital of Zhengzhou University. Other reagents or consumables used were commercially available or could be obtained by those skilled in the art through publicly available means.

[0107] Plasma was collected from 14 healthy controls and 18 patients with coronary heart disease and stored promptly at -80°C.

[0108] Extracellular vesicles were extracted from the plasma samples using the Exoquick kit (System Biosciences). The obtained extracellular vesicles were immobilized on a copper grid, stained, and dried. The morphology of the extracellular vesicles was identified using a JEOL-1230 transmission electron microscope. The particle size and specific markers of the obtained extracellular vesicles were analyzed and identified using a ZetaView PMX 110 particle tracker and Western blotting. The specific steps are as follows:

[0109] 1.1 Sample Collection and Preprocessing:

[0110] Collect whole blood (anticoagulated with EDTA or sodium citrate), centrifuge at 2000×g for 20 minutes (4℃) to separate plasma, avoiding hemolysis.

[0111] 1.2 Graded centrifugation to remove impurities:

[0112] 1.2.1 Low-speed centrifugation: 300×g, 10 minutes, discard the precipitate (to remove cells and large debris), and retain the supernatant.

[0113] 1.2.2 Medium-speed centrifugation: 2000×g, 10 minutes (4℃), discard the precipitate (to remove apoptotic bodies and organelle fragments), and retain the supernatant.

[0114] 1.2.3 High-speed centrifugation: 10000×g, 30 minutes (4℃), discard the precipitate (to remove large vesicles and microparticles) and the upper white floating lipid layer, and filter the supernatant through a 0.22μm sterile filter.

[0115] 1.3 Ultracentrifugation to enrich extracellular vesicles:

[0116] Transfer the filtrate to an ultracentrifuge tube, balance it precisely with sterile PBS (accurate to 0.000g), and then centrifuge at 120,000×g for 120 minutes (4°C). Discard the supernatant and retain the precipitate (containing extracellular vesicles).

[0117] To remove contaminating proteins, the precipitate can be resuspended in pre-cooled PBS, centrifuged again at 120,000×g for 90 minutes (4℃), and the supernatant discarded.

[0118] 1.4 Resuspension and preservation of extracellular vesicles:

[0119] The precipitate was gently resuspended in 200 μL of sterile PBS (operated on ice throughout). The NTA was measured using Zeview to ensure the extraction of extracellular vesicles at 30-150 nm. The particle count was confirmed, and the precipitate was appropriately diluted, aliquoted, and stored at -80°C (avoid repeated freeze-thaw cycles).

[0120] The protein concentration of extracellular vesicles was within the reference range of 0.5–5.0 μg / μL. The extracellular vesicles were frozen and stored at -80°C for later use.

[0121] The obtained extracellular vesicles are mixed with basal culture medium (or culture medium) to obtain an extracellular vesicle culture medium (or culture medium) with a protein concentration of 0.5-5.0 μg / mL. The basal culture medium used in this invention is preferably 1640 medium + B27 Minus Insulin. The preferred protein concentration of the extracellular vesicles is 25 μg / mL.

[0122] 2. Construction of cardiac organoids

[0123] This invention relates to cardiac tissue models, including cell types such as cardiomyocytes, endocardial cells (or cardiac endothelial cells), epicardial cells, and smooth muscle cells.

[0124] The present invention further provides a method for constructing a cardiac tissue model, the specific steps of which are as follows:

[0125] 2.1 Cultivating iPSC

[0126] Induced pluripotent stem cells (iPSCs) are cultured using human embryonic stem cell culture medium mTeSR1 (iPSCs originate from various types of somatic cells, ranging from common skin (fibroblasts) and blood (T cells, monocytes, hematopoietic stem cells, etc.), to readily available urine (kidney epithelial cells), and to specific tissue sources such as adipose tissue (mesenchymal stem cells), dental pulp (stem cells), etc.; this invention relates to skin (fibroblasts) and blood (T cells, monocytes, hematopoietic stem cells, etc.), to readily available urine (kidney epithelial cells); and is not limited to healthy individuals and patients). A 2D cell culture method is preferred, with a density of 90% during use.

[0127] 2.2 Preprocessing

[0128] Add 50µL-300µL of cell suspension (200µL in this example) to the well plates (using low-adhesion 96u or 96v bottom plates (96u plates in this example)) and control the cell concentration at 1.0-3.0×10⁻⁶. 4 cell / mL (in this example, the cell concentration was controlled at 1.5 × 10⁻⁶ cells / mL) 4 (cells / mL), then the 96u type bottom plate was centrifuged at 300g-400g for 2-10 min at 4℃ to obtain cell clusters, and then placed in basal culture medium containing ROCK inhibitor.

[0129] Then, the basal culture medium containing cell clusters and ROCK inhibitors was placed in a 96-well plate and cultured at 37°C for 12-48 hours (preferably, the culture time in this example is 12 hours) to obtain stable organoid aggregation precursors;

[0130] ROCK inhibitors include, but are not limited to, Y27632 and Thiazovivin, with Thiazovivin being preferred in this embodiment. The concentration of Thiazovivin is controlled between 1 μM and 18 μM, with 8 μM being preferred in this embodiment.

[0131] In this embodiment, the preferred basal culture medium is 1640 medium + B27 Minus Insulin.

[0132] 2.3 Construction of the first type of circulating heart tissue model

[0133] Based on step 2.2, the WNT pathway activator or GSK-3α / β inhibitor, PI3 kinase inhibitor, and functional components are added to the basal culture medium (preferably 1640 medium + B27 Minus Insulin) as differentiation medium A. Then, the organoid aggregation precursor obtained in step 2.2 is added to the differentiation medium A (10-30 μL of medium per 10,000 cells). The medium is then placed in a 96u bottom plate and cultured at 37°C for 12-48 h (preferably 36 h) to obtain a stable mesodermal cell mass.

[0134] The components added to the differentiation culture medium A are:

[0135] 2.3.1 The added WNT pathway activator or GSK-3α / β inhibitor works by inhibiting GSK-3, which prevents β-catenin from being degraded, thereby stabilizing and activating it to enter the cell nucleus and initiate the transcription of downstream WNT target genes.

[0136] Among them, the WNT pathway activator or GSK-3α / β inhibitor includes, but is not limited to, CHIR99021, Wnt3a (recombinant protein) and Wnt Conditioned Medium (Wnt CM); specifically, in this embodiment, CHIR99021 is preferred, and the concentration of CHIR99021 is controlled at 2μM-12μM, preferably 6μM.

[0137] 2.3.2 The added PI3 kinase inhibitor (i.e., PI3K inhibitor, unlike the absolute dominance of WNT pathway regulation in the early stages of cardiac differentiation, PI3K signaling acts as a "fine tuner," and its main influence occurs during the differentiation and maturation stages after the formation of cardiac progenitor cells) helps guide progenitor cells to differentiate more effectively into cardiomyocytes by adding PI3K inhibitors during the activation of the WNT pathway using CHIR99021.

[0138] Specifically, PI3 kinase inhibitors include, but are not limited to, LY294002 and Wortmannin. In this embodiment, LY294002 is preferred, and the concentration of LY294002 is controlled between 1 μM and 10 μM, preferably 4 μM.

[0139] 2.3.3 Simultaneous use of functional components (any one or a combination of FGF2, activin A, BMP4) can promote the exit of more than 90% of pluripotent stem cells from pluripotency within 24-48 hours after the start of induction, thereby synergistically promoting the generation of mesodermal cell clusters.

[0140] Specifically, the concentration of FGF2 is controlled at 2μM-15μM, preferably 4μM; the concentration of activin A is controlled at 2μM-12μM, preferably 6μM; and the concentration of BMP4 is controlled at 3μM-9μM, preferably 5μM.

[0141] Specifically, in this embodiment, CHIR99021, LY294002, FGF2, activator A, and BMP4 are added to the basal culture medium (preferably 1640 medium + B27 Minus Insulin) as differentiation medium A, wherein the final concentration of CHIR99021 is 6 μM, the final concentration of LY294002 is 4 μM, the final concentration of FGF2 is 4 μM, the final concentration of activator A is 6 μM, and the final concentration of BMP4 is 5 μM.

[0142] 2.4 Construction of the second type of circulating heart tissue model

[0143] Based on step 2.2, in the absence of WNT pathway activators and / or in the presence of WNT antagonists, the functional components are added to the basal culture medium (preferably 1640 medium + B27 Minus Insulin) as differentiation medium B. Then, the organoid aggregation precursor obtained in step 2.2 is added to the differentiation medium B (10-30 μL of medium per 10,000 cells). The mixture is then placed in a 96u type bottom plate and cultured at 37°C for 24-190 h (preferably 120 h) to obtain a stable mesodermal cell mass.

[0144] The components added to the differentiation culture medium B are:

[0145] 2.4.1 Specifically, in this embodiment, a WNT antagonist is used when preparing differentiation medium B, and no WNT pathway activator is added. The WNT antagonist includes, but is not limited to, one or more of XAV939, C59 / IWR-1, and IWP-2, which can effectively block the secretion and activity of endogenous Wnt protein, thereby allowing and promoting cardiac progenitor cells to exit the proliferative state and begin to differentiate into functional cardiomyocytes (expressing markers such as troponin T and α-actin), significantly improving the differentiation efficiency, purity, and maturity of cardiomyocytes. Specifically, in this embodiment, IWP-2 is preferably used, and the concentration of IWP-2 is controlled between 2μM and 15μM, preferably 5μM.

[0146] 2.4.2 Simultaneous use of functional components (any one or a combination of FGF2, BMP4, retinoic acid (RA) and VEGF) can promote the differentiation of mesodermal cell masses into cardiac mesodermal cell masses over the next 24-190 hours, while simultaneously forming the cell mass lumen, which is a cavity formed spontaneously by the combination of cells with each other.

[0147] Specifically, the concentration of FGF2 is controlled at 2μM-15μM, preferably 6μM; the concentration of BMP4 is controlled at 4μM-18μM, preferably 8μM; the concentration of RA is controlled at 2μM-15μM, preferably 5μM; and the concentration of VEGF is controlled at 5 ng / mL - 50 ng / mL, preferably 10 ng / mL.

[0148] Specifically, in this embodiment, IWP-2, FGF2, BMP4, retinoic acid (RA), and VEGF are added to the basal culture medium (preferably 1640 medium + B27 Minus Insulin) as differentiation medium B, wherein the final concentration of IWP-2 is 5 μM, the final concentration of FGF2 is 6 μM, the final concentration of BMP4 is 8 μM, the final concentration of retinoic acid (RA) is 5 μM, and the final concentration of VEGF is 10 ng / mL.

[0149] 2.5 Construction of the Third Type of Circulating Heart Tissue Model

[0150] 2.5.1 Based on step 2.2, in the absence of WNT pathway activators and WNT antagonists, the functional component is added to the basal culture medium (preferably 1640 medium + B27 Minus Insulin) as differentiation medium C. The organoid aggregation precursor obtained in step 2.2 is then added to the differentiation medium C (10-30 μL of medium per 10,000 cells). The mixture is then placed in a 96u type bottom plate and cultured at 37°C for 24-190 h (preferably 72 h) to obtain a stable mesodermal cell mass.

[0151] Using functional components (any one or a combination of FGF2, BMP4 and VEGF) to maintain the differentiation morphology and microvascularization of cardiac mesodermal cell masses; in the mid-to-late stages after the initial establishment of the cardiac lineage, precise introduction of VEGF can drive endothelial differentiation, angiogenesis and maturation.

[0152] Specifically, the concentration of FGF2 is controlled at 2μM-15μM, preferably 6μM; the concentration of BMP4 is controlled at 4μM-18μM, preferably 8μM; and the concentration of VEGF is controlled at 3 ng / mL-50 ng / mL, preferably 8 ng / mL.

[0153] Specifically, in this embodiment, FGF2, BMP4, and VEGF are added to the basal culture medium (preferably 1640 medium + B27 Minus Insulin) as differentiation medium C, wherein the final concentration of FGF2 is 6 μM, the final concentration of BMP4 is 8 μM, and the final concentration of VEGF is 8 ng / mL.

[0154] The cardiac organoids (including cardiomyocytes, endothelial cells, and cardiac fibroblasts) obtained through steps 2.1 to 2.5 have good cavity morphology.

[0155] 3. Peristaltic pump and PDMS chip, cardiac organoid construction: cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system

[0156] 3.1 Chip Design: The high-throughput micropillar array chip (i.e., PDMS chip, hereinafter referred to as: microfluidic chip) used in this invention for culturing cardiac organoids and collecting extracellular vesicles.

[0157] like Figure 5 The figure shown is a top view of the microfluidic chip, which includes 2-6 chambers (specifically, 4 chambers in this embodiment), an inlet connecting pipe (not shown in the figure), four inlet branch pipes (not shown in the figure), an outlet connecting pipe (not shown in the figure), four outlet branch pipes (not shown in the figure), an inlet (4) and an outlet (5);

[0158] Figure 5 In the middle, the rear end of the inlet connecting pipe is the inlet (4), and the front end of the inlet connecting pipe is provided with two branches, namely the first branch on the left and the second branch on the right. The rear ends of the two inlet branch pipes are connected to the first branch, and the rear ends of the other two inlet branch pipes are connected to the second branch. The front ends of the four inlet branch pipes are respectively connected to the inlet ends of the four chambers.

[0159] The front end of the outlet connecting pipe is the outlet (5). The rear end of the outlet connecting pipe is provided with two branches, namely the third branch on the left and the fourth branch on the right. The front ends of the two outlet branch pipes are connected to the third branch, and the front ends of the other two outlet branch pipes are connected to the fourth branch. The rear ends of the four outlet branch pipes are respectively connected to the outlet ends of the four chambers.

[0160] Each chamber has 6-8 independent capture areas; each capture area is equipped with multiple arc-shaped pillars with a diameter of 200-350um (i.e., heart organoid culture tanks (2)) to support heart organoids (3); the role of these pillars is to ensure the stability of heart organoids within the chip and prevent them from being washed away or sticking together during the culture process.

[0161] Specifically, in this embodiment, the chamber width is 700-1000 micrometers, the length is 20-50 millimeters, and the capture area diameter is 2-4 millimeters. The chip height is 400-700 micrometers.

[0162] The fabrication of the aforementioned high-throughput micropillar array chip (PDMS microfluidic chip) is specifically as follows:

[0163] a. Based on the aforementioned PDMS chip structure, design a two-dimensional sketch of the chip using AutoCAD software, and print the photomask of the sketch.

[0164] b. Prepare the SU-8 template using soft etching technology. Specifically, the SU-8 material is uniformly coated onto a clean 3-inch silicon wafer, and then exposed to ultraviolet light. The wafer is then baked on a hot plate at 65°C for 10 minutes and then at 95°C for 2 hours. After that, the pattern on the wafer is developed using a developer.

[0165] c. The prepared SU-8 template is modified to have low adhesion to ensure that the SU-8 template and PDMS polymer can be easily separated.

[0166] d. Fabrication of a high-throughput array micropillar structure PDMS polymer chip: Polydimethylsiloxane (PDMS) prepolymer (specifically, component A of Dow Corning Sylgard 184) and the corresponding platinum catalyst initiator (specifically, component B of Dow Corning Sylgard 184) are thoroughly mixed at a volume ratio of 10-15:1 (preferably 12:1 in this embodiment) to obtain a PDMS mixture. The mixture is then poured onto the SU-8 template prepared in step c, ensuring complete coverage and no air bubbles accumulating in the microstructure region.

[0167] Place the cast template in a vacuum dryer and vacuum it (usually -0.08 MPa to -0.1 MPa) for about 15-30 minutes (or until all air bubbles are expelled); remove the template and place it horizontally in an oven to cure at 80°C for 60-120 minutes (60 minutes is preferred in this embodiment) to ensure complete curing.

[0168] e. Separate the PDMS chip with high-throughput array micropillar structure from the SU-8 template to obtain the high-throughput micropillar array PDMS chip.

[0169] f. Drill holes in the back end of the inlet connector and the front end of the outlet connector of the high-throughput micropillar array PDMS chip obtained in step e using a hole punch (usually 0.5-1.0 mm in diameter) to obtain the inlet (4) and outlet (5).

[0170] Subsequently, the chip surface is cleaned with anhydrous ethanol or isopropanol and dried with nitrogen or clean air. Finally, the PDMS chip is subjected to oxygen plasma treatment (typical parameters: power 30-100 W, time 30-60 seconds) together with a clean glass slide (or another flat PDMS layer), and the two are immediately aligned and bonded, and left at room temperature for a period of time (or appropriately heated) to achieve stable bonding.

[0171] g. Treat the inside of the chip with a 0.5 wt% ethanol solution for 15-30 minutes to improve its surface properties and make it more suitable for the growth of heart organoids.

[0172] 3.2 Capture of cardiac organoids in the microfluidic chip and construction of the extracorporeal circulation culture system: After obtaining mature cardiac organoids in step 2, the cardiac organoids from step 2 were collected using a 1-5 ml syringe and injected into the microfluidic chip prepared in step 3.1. The specific steps are as follows:

[0173] 3.2.1 As Figure 6 As shown, the microfluidic chip prepared in step 3.1 is placed in a flat-panel incubator (6). Two flexible Tygon tubes of 0.5-1.0 mm are connected to the inlet (4) and outlet (5) respectively as connectors. Using a syringe, mTeSR1 culture medium is injected into the chip through the inlet (4). This is repeated three times to clean the chip and remove internal air bubbles.

[0174] 3.2.2 Then, the cardiac organoids were injected into the chip (1) using a syringe. During the injection, the outlet (5) was connected to a flexible Tygon tube with an inner diameter of 0.5-1.0 mm and a 1 ml syringe containing mTeSR1 culture medium.

[0175] 3.2.3 After injecting the organoids into the chip (1), remove the syringe and adjust the position of the chip to control the position of the organoids in the chamber so that they can remain stably in multiple capture areas.

[0176] 3.3 Construction of an in vitro circulating culture system: Using a syringe, the normal culture medium was replaced with the extracellular vesicles from step 1 for culture; then, the inlet end of the peristaltic pump (8) was connected to the flexible Tygon tube at the outlet (5) through the circulating tubing (7), and the outlet end of the peristaltic pump was connected to the flexible Tygon tube at the inlet (4) through the circulating tubing (7), thus constructing an in vitro circulating culture system. The circulating culture medium (i.e., the extracellular vesicles from step 1) was replaced every 24-72 hours (preferably every 24 hours), for a total culture time of 24-240 hours.

[0177] (II) Experimental Results:

[0178] 1. Morphology of extracellular vesicles

[0179] Extracellular vesicles were extracted using the Exoquick kit (System Biosciences). 5 μl of the resulting extracellular vesicle suspension was added to a Formvar-carbon copper grid. After washing the grid with PBS, it was placed in 50 μl of 1% glutaraldehyde solution for 5 min, followed by washing in 100 μl of ddH2O for 2 min. The vesicles were stained with uranyl oxalate and methylcellulose solution, excess liquid was blotted on filter paper, and the cells were air-dried for 5 min. The morphology of the obtained extracellular vesicles was identified using a JEOL-1230 transmission electron microscope.

[0180] TEM images such as Figure 1 As shown, the results indicate that extracellular vesicles (EVs) from both healthy individuals and MI patients exhibit a standard teacup-shaped morphology, demonstrating that the extracted EVs have a standard and complete morphology.

[0181] 2. Western blot results of extracellular vesicles (EVs) in healthy individuals and MI patients.

[0182] Experimental Methods: 20-50 μg of extravesicular protein (BCA quantification) was added to lysis buffer at a 4:1 ratio (specifically, 40 μL lysis buffer + 10 μL sample). Lysis was performed on ice for 30 min, vortexing every 10 min. Centrifugation was carried out at 12,000 × g at 4 °C for 10 min, and the supernatant was collected (precipitate was discarded). The sample was incubated with 5% skim milk (prepared with TBST) at room temperature for 1 h, followed by incubation with primary antibodies against CD9, CD63, TSG101, and Calnexin. Then, the corresponding HRP secondary antibody was incubated at room temperature for 1 h. Finally, development and data analysis were performed.

[0183] The results are as follows Figure 2 As shown, Figure 2 In the diagram, a negative Calnexin result indicates that the sample is uncontaminated. The presence of CD9, CD63, and TSG101 indicates that extracellular vesicles (EVs) in both healthy individuals and MI patients possess these marker proteins. This indicates that the extracellular vesicles extracted in this invention exhibit standard protein expression.

[0184] 3. Extracellular vesicle particle size

[0185] The obtained extracellular vesicle particle size and specific markers were analyzed and identified using a ZetaView PMX 110 particle tracker and Western blotting.

[0186] Nanoparticle tracking analysis (NTA) results are as follows Figure 3 As shown, the results indicate that the size of the EVs is distributed in the range of approximately 100nm-130nm, which conforms to the size distribution standard of EVs.

[0187] Figures 1 to 3The results showed that the extracted vesicles conformed to the characteristics of extracellular vesicles in terms of morphology, particle size distribution, and specific marker labeling.

[0188] 4. Differential analysis of mi-RNA transcriptomes of EVs in MI patients and healthy individuals

[0189] Raw sequencing data: Standardized using ACGT101-miR (v4.2) software: First, adapter dimers, low-complexity sequences, repetitive sequences, and contamination from non-coding RNA families (including rRNA, tRNA, snRNA, and snoRNA) were removed. Then, unique sequences of 18-26 nt in length were screened and annotated using BLAST alignment to the species-specific precursor database in miRBase 22.1. If the sequence matched the hairpin arm region of a known mature miRNA, it was identified as a known miRNA; if it matched the contralateral arm region (5' or 3' arm), it was classified as a novel derived miRNA candidate. Unmatched sequences were further localized to the genome using cross-species precursor BLAST search (excluding the target species), and their secondary structures were predicted using RNAfold.

[0190] Differential expression analysis: When biological replication exists, the differences between groups are analyzed using a two-tailed heteroscedasticity t-test (two groups) or ANOVA (multiple groups); if there is no biological replication, Fisher's exact test, 2×2 chi-square test (two groups), and N×N chi-square test (multiple groups) are applied, with significance thresholds set at (p<0.05) and (p<0.01). In this test, n=3.

[0191] The results of the differential analysis of extracellular vesicle (EV) miRNA between MI patients and healthy individuals are as follows: Figure 4 As shown, it is evident that MI patients have higher levels of miR-142 than healthy individuals, which is a marker of AMI. This demonstrates that the EV extracted in this invention possesses typical characteristics of myocardial infarction patients.

[0192] 5. Immunofluorescence assay results of vascularized cardiac organoids

[0193] The cardiac organoids prepared in step 2 were first washed with DPBS (Gibco), then fixed in 4% paraformaldehyde (PFA) solution at 4°C for 6 to 8 hours, washed again with DPBS, and finally dehydrated in 30% sucrose solution at 4°C until completely infiltrated. Subsequently, the organoids were embedded in the optimal cutting temperature compound (OCT) and cut into 10-micrometer thick sections for immunofluorescence staining. These sections were then treated with 2% Triton X-100 at 0°C for 30 minutes, followed by washing three times with PBS, treatment with antigen retrieval reagent (Beyotime) for 5 minutes, washing three more times with PBS, and blocking with blocking buffer (Beyotime) for 1 hour. The slides were then incubated with primary antibody overnight and secondary antibody for 2 hours. Finally, counterstaining was performed using DAPI (Sigma-Aldrich).

[0194] Immunofluorescence images of vascularized cardiac organoids are shown below. Figure 7 As shown, Figure 7 In the diagram, CD31 represents endothelial cells, TNNT represents cardiomyocyte distribution, and DAPI represents cell nucleus distribution.

[0195] Figure 7 The results showed that the organoids contained a distinct distribution of reticuloendothelial cells. This indicates that the method of the present invention can obtain vascularized cardiac organoids. Positive expression of TNNT indicates that the cardiac organoids possess mature cardiomyocyte types.

[0196] 6. Using the cardiac organoid-PDMS microfluidic chip in vitro circulation culture system constructed in step 3, after culturing cardiac organoids in a culture medium containing extracellular vesicles (PKH26 membrane fluorescent labeling) for 24 hours, the immunofluorescence results of extracellular vesicles (PKH26 labeling) entering the cardiac organoids were detected.

[0197] Methods: Purified extracellular vesicles were suspended in 1 mL of Diluent C solution and mixed with an equal volume of Diluent C solution containing 4 μM PKH26 (Sigma, #MINI26). The mixture was incubated at room temperature in the dark for 20 minutes. The staining reaction was terminated by adding 2 mL of 0.5% BSA / PBS. The mixture was then centrifuged at 100,000 × g for 70 minutes at 4 °C to remove free dye, and the precipitate was resuspended in sterile PBS. The labeled EV-PKH26 was used for co-culturing with microarray organoids using the same EV culture medium prepared in step 1.4 (specifically: 1640 medium + B27 Minus Insulin + EV, with the original EV protein concentration at 0.5–5.0 μg / μL and the concentration in the culture medium at 0.5–5.0 μg / mL). The resulting medium was kept dark throughout the process. After 24 hours, the circulating medium was replaced with 4% paraformaldehyde, the microarray was backflushed, and the organoids were removed. Finally, the organoids were dehydrated in 30% sucrose solution at 4 °C until completely infiltrated. Subsequently, the organoids were embedded in the optimal cutting temperature compound (OCT) and cut into 10-micrometer-thick sections for immunofluorescence staining. These sections were then treated with 2% Triton X-100 at 0.5°C for 30 minutes, followed by washing three times with PBS and counterstaining with DAPI (Sigma Aldrich).

[0198] The results are as follows Figure 8 As shown, the results indicate that EV-PKH 26 can enter cardiac organoids within 24 hours, thereby regulating various physiological activities of the organoids.

[0199] 7. Using the cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system constructed in step 3, cardiac organoids were cultured for 72 hours in a culture medium containing EVs from MI patients and healthy individuals, and then RNA transcriptome sequencing was performed on the organoids.

[0200] The results are as follows Figure 9 As shown, after 72 hours of culture of cardiac organoids from MI patients and healthy individuals, differential gene results indicated that decreased CR2 expression was an early immune abnormality event in myocardial infarction that persisted throughout the disease course. Downregulation of CR2 reflects acquired immunodeficiency in the disease, and humoral immune deficiency may promote the progression of atherosclerosis (e.g., decreased ability to clear apoptotic cells).

[0201] Example 2

[0202] The difference between Example 2 and Example 1 is that EVs are replaced with peripheral blood mononuclear cells (PBMCs).

[0203] Specifically, the peripheral blood mononuclear cell (PBMC) extraction process is as follows:

[0204] 1) Collect human blood using sodium citrate blood collection tubes, and add an equal volume of PBS and mix well.

[0205] 2) Slowly add the product from step 1) into an EP tube containing 4 mL of Ficoll-Hypaque lymphocyte separation medium and centrifuge at 1000 g for 20 min.

[0206] 3) Extract the cells from the middle cloud layer and count them using a cell counting chamber.

[0207] 4) Adjust the volume of PBS to 8 mL, centrifuge at 500 g for 8 min to obtain human PBMCs, and culture them in RPMI 1640 (10% FBS) medium. The final concentration used is 1-6 x 10⁻⁶. 6 cell / mL.

[0208] Figure 10 PBMCs were extracted from the blood of healthy individuals and patients. Morphology of the PBMCs was photographed using an inverted microscope. They contained monocytes, dendritic cells, and lymphocytes.

[0209] Figure 11 This is an assembly diagram of the PDMS chip using PBMC in Example 2.

[0210] Referring to Example 1, the cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system constructed in step 3 (e.g.) Figure 11 As shown in the figure, cardiac organoids were cultured for 72 hours using PBMCs from healthy individuals as the culture medium (the specific setup of the extracorporeal circulation culture system, culture method, and parameter settings are as described in Example 1). RNA transcriptome sequencing results of the organoids were then analyzed using Kegg. The results are as follows: Figure 12 As shown. Figure 12 Using PBMCs from healthy individuals, the final concentration used is 2 x 10⁻⁶. 6 cell / mL.

[0211] Enrichment analysis revealed the presence of immune response, innate immune response, adaptive immune response, defense response to bacteria, complement activation, and the classical pathway. This indicates a strong state of immune activation in the cardiac organoid samples, potentially involving infection, inflammation, or autoimmune responses, with both innate and adaptive immune systems significantly activated. This is consistent with the immune activation state seen in myocarditis.

[0212] Example 3

[0213] The difference between Example 3 and Example 1 is that EV is replaced with EV+PBMC. The PBMC extraction method is the same as in Example 2.

[0214] Referring to Example 1, the cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system constructed in step 3 (e.g.) Figure 13 As shown in the figure, extracellular vesicles and PBMCs from healthy individuals were used as culture media to culture cardiac organoids for 72 hours (the specific setup of the extracellular circulation culture system, culture method, and parameter settings are as described in Example 1), and the accumulation of collagen was assessed by immunofluorescence. The results are as follows: Figure 14 As shown.

[0215] Figure 14 The results showed that collagen III expression was significantly enhanced after culturing in the medium containing external vesicles and PBMCs from healthy individuals, exhibiting a typical heart failure phenotype.

Claims

1. A method for constructing a cardiac organoid-PDMS microfluidic chip in vitro circulation culture system, characterized in that, Includes the following steps: (1) Extraction of extracellular vesicles and / or extraction of peripheral blood mononuclear cells; In step (1), the extraction of extracellular vesicles involves using the Exoquick kit to extract extracellular vesicles from plasma samples of myocardial infarction patients, including: centrifugation to separate plasma, fractional centrifugation to remove impurities, and ultracentrifugation to enrich extracellular vesicles. In step (1), peripheral blood mononuclear cell extraction includes: Collect human blood samples from patients with myocardial infarction, add lymphocyte separation solution, centrifuge, aspirate the middle cloud layer cells, dissolve, centrifuge again, and the result is obtained; (2) Construction of cardiac organoids: Induced pluripotent stem cells were cultured and a cell suspension of pluripotent stem cells was obtained. The cell suspension of pluripotent stem cells was placed in a culture plate, centrifuged, and cell clusters were obtained. The cells were then mixed with a basal medium containing inhibitors, placed in a culture plate, and cultured at 37°C for 12-48 hours to obtain stable organoid aggregate precursors. The obtained organoid aggregate precursors are then added to differentiation medium A, differentiation medium B or differentiation medium C, and then placed in a culture plate and cultured at 37°C for 12-190 hours to obtain stable mesodermal cell masses, which are the heart organoids. In step (2), the basic culture medium is 1640 medium + B27 Minus Insulin; In step (2), the inhibitor is a ROCK inhibitor, and the ROCK inhibitor is Thiazovin; In step (2), when using differentiation medium A for culture, WNT pathway activator or GSK-3α / β inhibitor, PI3 kinase inhibitor, and functional components are added to the basal medium as differentiation medium A; The components added to the differentiation culture medium A are: The WNT pathway activator or GSK-3α / β inhibitor is CHIR99021; The PI3 kinase inhibitor is LY294002; The functional components are FGF2, activator A, and BMP4; In step (2), when using differentiation medium B for culture, in the absence of WNT pathway activators and / or in the presence of WNT antagonists, the functional components are added to the basal medium as differentiation medium B. The components added to the differentiation culture medium B are: The WNT antagonist is IWP-2; The functional components are FGF2, BMP4, RA and VEGF; In step (2), when using differentiation medium C for culture, in the absence of WNT pathway activators and WNT antagonists, the functional components are added to the basal medium as differentiation medium C. The components added to the differentiation medium C are: The functional components are FGF2, BMP4 and VEGF; (3) Construction of a cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system: This includes PDMS chip design and fabrication, capture of cardiac organoids in PDMS chips, and construction of a cardiac organoid-PDMS chip in vitro circulation culture system. In step (3), the capture of cardiac organoids in the PDMS chip involves collecting cardiac organoids using a syringe and injecting them into the prepared PDMS microfluidic chip. The specific steps are as follows: 1) Place the prepared PDMS microfluidic chip in an incubator, connect the two connecting tubes to the inlet and outlet respectively, and use a syringe to inject the culture medium into the chip through the inlet to clean the chip and remove internal air bubbles. 2) Then, the heart organoid is injected into the chip using a syringe. During the injection, the outlet is connected to the connecting tube and the syringe containing the culture medium. 3) After injecting the organoids into the chip, remove the syringe and adjust the position of the chip to control the position of the organoids in the chamber, so that they can remain stably in multiple capture areas; 4) Using a syringe, replace the normal culture medium with extracellular vesicles and / or peripheral blood mononuclear cells for culture; then connect the inlet end of the peristaltic pump to the outlet end through the circulation tubing, and connect the outlet end of the peristaltic pump to the inlet end through the circulation tubing to construct an external circulation culture system for the chip.

2. The construction method according to claim 1, characterized in that, In step (2), 2D cell culture is used when culturing induced pluripotent stem cells; In step (2), the volume of the pluripotent stem cell suspension is 50µL-300µL; In step (2), the cell concentration of pluripotent stem cells is 1.0-3.0 × 10⁻⁶. 4 cell / mL.

3. The construction method according to claim 1, characterized in that, In the differentiation medium A, the concentration of CHIR99021 was controlled at 2μM-12μM; The concentration of LY294002 was controlled at 1μM-10μM; the concentration of FGF2 was 2μM-15μM; the concentration of activin A was 2μM-12μM; and the concentration of BMP4 was 3μM-9μM. In the differentiation medium B, the concentration of IWP-2 was 2 μM-15 μM; the concentration of FGF2 was controlled at 2 μM-15 μM; the concentration of BMP4 was controlled at 4 μM-18 μM; the concentration of RA was controlled at 2 μM-15 μM; and the concentration of VEGF was controlled at 5 ng / mL-50 ng / mL. In the differentiation medium C, the concentration of FGF2 was controlled at 2μM-15μM; the concentration of BMP4 was controlled at 4μM-18μM; and the concentration of VEGF was controlled at 3 ng / mL-50 ng / mL.

4. The construction method according to claim 1, characterized in that, In step (3), the PDMS microfluidic chip is a high-throughput micropillar array chip; The PDMS microfluidic chip includes 2-6 chambers, an inlet connecting tube, four inlet branch tubes, an outlet connecting tube, four outlet branch tubes, one inlet and one outlet; The rear end of the inlet connecting pipe is the inlet, and the front end of the inlet connecting pipe has two branches, namely the first branch and the second branch. The rear ends of the two inlet branch pipes are connected to the first branch, and the rear ends of the other two inlet branch pipes are connected to the second branch. The front ends of the four inlet branch pipes are respectively connected to the inlet ends of the four chambers. The front end of the outlet connecting pipe is the outlet. The rear end of the outlet connecting pipe has two branches, namely the third branch and the fourth branch. The front ends of the two outlet branch pipes are connected to the third branch, and the front ends of the other two outlet branch pipes are connected to the fourth branch. The rear ends of the four outlet branch pipes are respectively connected to the outlet ends of the four chambers.

5. The construction method according to claim 4, characterized in that, Each chamber has 6-8 independent capture zones; each capture zone is equipped with multiple arc-shaped columns with a diameter of 200-350 μm, i.e., heart organoid culture tanks.

6. The construction method according to claim 4, characterized in that, In step (3), the fabrication of the high-throughput micropillar array chip includes the following steps: a. Based on the structure of the PDMS microfluidic chip, design a two-dimensional sketch of the chip using software, and print the photomask of the sketch; b. Prepare the SU-8 template using soft etching technology; c. The prepared SU-8 template is modified to have low adhesion to ensure that the SU-8 template and PDMS polymer can be easily separated; d. Fabrication of high-throughput array micropillar structures for PDMS polymer chips; e. Separate the PDMS chip with the high-throughput array micropillar structure from the SU-8 template to obtain the high-throughput micropillar array PDMS chip; f. Drill holes in the rear end of the inlet connector and the front end of the outlet connector of the high-throughput micropillar array PDMS chip obtained in step e using a hole puncher to obtain the inlet and outlet; clean and perform oxygen plasma treatment. g. Treat the inside of the chip with an alcohol solution for 15-30 minutes to improve its surface properties and make it more suitable for the growth of heart organoids.

7. The cardiac organoid-PDMS microfluidic chip extracorporeal circulation culture system constructed by any of claims 1 to 6.

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