A non-optically dependent cardiac organ-on-a-chip and detection method

Abstract

The present invention discloses a non-optically dependent cardiac organ-on-chip and a detection method, wherein the cardiac organ-on-chip comprises a culture layer and a monitoring layer, wherein the culture layer is located on the upper side of the monitoring layer; the culture layer comprises a cover plate, on which a plurality of chambers are arranged; the monitoring layer comprises a substrate, on which a plurality of monitoring devices corresponding to the chambers are arranged; the monitoring device comprises two columnar electrodes, a signal acquisition layer and a resistance sensing layer; the resistance sensing layer is arranged on the upper side of the substrate; the signal acquisition layer is arranged on the upper side of the resistance sensing layer, the signal acquisition layer comprises a base, on which two conductive circuits are arranged; the first ends of the two conductive circuits are respectively connected to the two columnar electrodes, and the second ends of the two conductive circuits are respectively connected to the first signal acquisition electrode and the second signal acquisition electrode; the two columnar electrodes are arranged in the chamber of the culture layer at intervals, and the lower ends of the columnar electrodes are fixed on the base of the signal acquisition layer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering organoids and organ chips, and specifically relates to a non-optically dependent cardiac organoid chip and a detection method. Background Art

[0002] The heart is one of the most important organs in the human body. Cardiovascular diseases account for more than one-third of the global deaths each year. The cardiac toxicity of drugs in the development of new drugs is also a must-evaluate content. In order to study the relevant pathophysiological mechanisms of the heart, as well as to screen drugs for the treatment of heart diseases or evaluate the cardiac toxicity of drugs, it is necessary to establish a corresponding heart model. In drug testing, it is usually necessary to clarify the effects of drugs on cardiac function, including: frequency, contractility, action potential, conduction, etc. In particular, due to the mechanism of "excitation-contraction coupling" (also known as "mechanical-electrical coupling") of the heart, the electromechanical signals of the myocardium are closely linked. When this mechanism is weakened, arrhythmias may occur. Therefore, for clarifying the physiological and pathological mechanisms of myocardial tissue, the synchronous measurement of mechanical and electrical signals is crucial. However, the traditional two-dimensional cell culture model is not ideal. The heart chip is an integrated cardiac organoid microphysiological system built based on micromachining, stem cells, biomaterials, tissue engineering and electronic information technologies.

[0003] The current heart chip testing functions are relatively simple, such as testing the contraction frequency and relative contraction force of cardiac organoid tissue, or testing the field potential signal of the tissue, but the synchronous signal test of "mechanical-electrical coupling" is still difficult to achieve. More importantly, most of the currently public heart chips use optical microscopes to observe the beating frequency and amplitude of cardiac organoids, including the subsequent evaluation of contraction force, which is based on the video taken by the optical microscope. Since the number of samples observed by the microscope at the same time scale is extremely limited, these heart chips are difficult to meet high-throughput commercial needs, which greatly limits their application. Summary of the invention

[0004] Purpose of the invention: To address the problems of the inability of cardiac organoid chips in the prior art to synchronously measure the "electromechanical" activity of in vitro myocardial tissue and low flux, a non-optically dependent cardiac organoid chip and a detection method are proposed. The cardiac organoid chip is a non-optically dependent chip and can synchronously measure "mechanical-electrical coupling" signals.

[0005] Technical solution: A non-optically dependent cardiac organoid chip, characterized in that the cardiac organoid chip comprises a culture layer and a monitoring layer, wherein the culture layer is located on the upper side of the monitoring layer; the culture layer comprises a cover plate, on which a plurality of chambers are provided, and the chambers are used to place three-dimensional cardiac organs and cell culture medium liquid; the monitoring layer comprises a substrate, on which a plurality of monitoring devices corresponding to the chambers are provided; the monitoring device comprises two columnar electrodes, a signal acquisition layer and a resistance sensing layer; the resistance sensing layer is arranged on the upper side of the substrate, the resistance sensing layer comprises a base, on which a sensing conductive circuit is provided, and a connection resistance detector of the sensing conductive circuit is used Detect the change in resistance when the base of the resistor sensing layer is bent and deformed; the signal acquisition layer is arranged on the upper side of the resistor sensing layer, the signal acquisition layer includes a base, and two conductive circuits are arranged on the base of the signal acquisition layer; the first ends of the two conductive circuits are respectively connected to the two columnar electrodes, and the second ends of the two conductive circuits are respectively connected to the first signal acquisition electrode and the second signal acquisition electrode; the first signal acquisition electrode and the second signal acquisition electrode are used to connect to the electrical signal collector; the two columnar electrodes are arranged at intervals in the chamber of the culture layer, and the lower ends of the two columnar electrodes are fixed on the base of the signal acquisition layer, and the two columnar electrodes are used to anchor the three-dimensional heart organoid.

[0006] Furthermore, the chamber is a groove-shaped structure with an open upper end; the chamber 9 is rectangular or dumbbell-shaped, with a length-to-diameter ratio greater than 2:1.

[0007] Furthermore, the three-dimensional cardiac organoid is self-assembled by uniformly mixing the gel, cardiomyocytes, fibroblasts and endothelial cells.

[0008] Furthermore, the material of the culture layer is selected from any one of quartz, glass, TPE (thermoplastic elastomer), PMMA (polymethyl methacrylate), and PDMS (polydimethylsiloxane) polymer.

[0009] Furthermore, the material of the substrate is selected from any one of quartz, glass, TPE, PMMA, and PDMS polymer.

[0010] Furthermore, the substrate of the resistive sensing layer is a flexible substrate, and the substrate material of the resistive sensing layer 6 is any one of polyvinyl alcohol (PVA), polyester (PET), polyimide (PI), polyethylene naphthalate (PEN), paper, and PDMS; the material of the sensing conductive circuit is any one of liquid metal gallium, gallium-indium alloy, and conductive PDMS.

[0011] Furthermore, the substrate of the signal acquisition layer is a flexible substrate, and the substrate material of the resistance sensing layer 6 is any one of polyvinyl alcohol (PVA), polyester (PET), polyimide (PI), polyethylene naphthalate (PEN), paper, and PDMS; the material of the first signal acquisition electrode and the second signal acquisition electrode is selected from any one of gold, platinum, silver, copper, and titanium.

[0012] Furthermore, the columnar electrode is a carbon electrode or a conductive silicone electrode.

[0013] The present invention also discloses a high-throughput detection method based on the non-optical dependent cardiac organoid chip, characterized in that it comprises the following steps:

[0014] Step 1), preparing a plurality of non-optically dependent cardiac organ chips, and calibrating the numerical relationship between deformation contraction force and resistance;

[0015] Step 2), injecting a gel suspension containing cardiomyocytes, fibroblasts and endothelial cells into the chamber of the cardiac organ chip culture layer, and injecting a cell culture medium liquid for culture after gelation;

[0016] Step 3), after the three-dimensional cardiac organoid is self-assembled in the chamber, the first signal collection electrode and the second signal collection electrode are connected to the positive and negative electrodes of the electrical signal collector to read out the potential signal in real time;

[0017] Simultaneously, the two ends of the sensing conductive circuit are connected to a resistance detector, and the quantified contractile force of the cardiac organoid is directly displayed according to the calibrated numerical relationship;

[0018] Step 4) detecting the beating frequency and excitation-contraction coupled electromechanical signals of myocardial tissue after drug administration, the specific operation is as follows:

[0019] Adding different reagents or drugs into the chamber, detecting the beating frequency and excitation-contraction coupled electromechanical signals of the myocardial tissue after administration, and obtaining the effect of the reagents or drugs on the cardiac organoid tissue;

[0020] Step 5) To achieve high throughput, multiple cardiac organoid chips are arranged in parallel, and the resistance detector and signal collector are set to multi-channel acquisition, thereby realizing the function of real-time monitoring of electrophysiological signals of cardiac organoids without optical dependence and high throughput.

[0021] Beneficial effects: The present invention overcomes the defects of the prior art such as the inability to synchronously measure the "electrical-mechanical" activity of myocardial tissue in vitro and low flux, and has broad application prospects in the fields of drug screening and personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Schematic diagram of the culture layer structure of the cardiac organ-on-a-chip of the present invention.

[0023] Figure 2 Schematic diagram of the monitoring layer structure of the cardiac organ-on-a-chip of the present invention;

[0024] Numbers in the figure: three-dimensional heart organoid-1; first columnar electrode-2; and second columnar electrode-3; first signal collection electrode-4; second signal collection electrode-5; resistance sensing layer-6; substrate-7; cover-8; chamber-9. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the embodiments.

[0026] A non-optically dependent cardiac organ-on-a-chip, comprising a culture layer and a monitoring layer, wherein the culture layer is located on the upper side of the monitoring layer, and the culture layer and the monitoring layer are closely connected;

[0027] like Figure 1 As shown, the culture layer includes a cover plate 8, and a chamber 9 is provided on the cover plate 8; the chamber 9 is used to place the three-dimensional cardiac organoid and the cell culture medium liquid; the chamber 9 provides space for the three-dimensional cardiac organoid 1 to grow and beat; the chamber 9 is a groove-shaped structure with an upper end opening, and the chamber 9 is rectangular or dumbbell-shaped, with an aspect ratio greater than 2:1. The three-dimensional cardiac organoid is self-assembled by mixing gel, cardiomyocytes, fibroblasts and endothelial cells uniformly; the material of the culture layer is selected from any one of quartz, glass, TPE, PMMA, and PDMS polymers.

[0028] like Figure 2 As shown, the monitoring layer includes a substrate 7, on which a monitoring device corresponding to the chamber 9 is provided; the monitoring device includes two columnar electrodes, a signal acquisition layer, and a resistance sensing layer 6 from top to bottom; the resistance sensing layer 6 is arranged on the upper side of the substrate 7, and the resistance sensing layer 6 and the substrate 7 are tightly connected; the signal acquisition layer and the resistance sensing layer 6 are tightly connected; the two columnar electrodes are a first columnar electrode 2 and a second columnar electrode 3.

[0029] In this embodiment, the material of the substrate 7 is quartz;

[0030] The resistance sensing layer 6 includes a substrate, and a sensing conductive circuit is provided on the substrate of the resistance sensing layer 6; both ends of the sensing conductive circuit are connected to a resistance detector for detecting the change in resistance when the substrate of the resistance sensing layer 6 is bent and deformed; the substrate of the resistance sensing layer 6 is a flexible substrate, the substrate material of the resistance sensing layer 6 is polyvinyl alcohol (PVA), and the material of the sensing conductive circuit is liquid metal gallium.

[0031] The signal collection layer is arranged on the upper side of the resistance sensing layer 6; the signal collection layer includes a substrate, and two conductive circuits are arranged on the substrate of the signal collection layer; the first ends of the two conductive circuits are respectively connected to the lower ends of the two columnar electrodes, and the second ends of the two conductive circuits are respectively connected to the first signal collection electrode 4 and the second signal collection electrode 5, and the first signal collection electrode 4 and the second signal collection electrode 5 are used to connect to the electrical signal collector;

[0032] The substrate of the signal collection layer is a flexible substrate, and the substrate material of the signal collection layer is polyvinyl alcohol; the material of the first signal collection electrode 4 and the second signal collection electrode 5 is copper.

[0033] The upper ends of the two columnar electrodes are spaced apart in the chamber 9 of the culture layer, and the lower ends of the two columnar electrodes are fixed on the base of the signal collection layer; the columnar electrodes are carbon electrodes, and the three-dimensional cardiac organoids are anchored on the two columnar electrodes.

[0034] The cover plate 8 of the cardiac organ-like chip described in the present invention may be provided with 1-10 chambers. When multiple chambers are provided on the cover plate 8, a monitoring device is correspondingly provided above each chamber. The detector and the signal collector may be designed to be multi-channel, for example, 64 channels may be provided, so that the physiological signals of the myocardial strips in 64 chambers can be collected simultaneously. Therefore, multiple chambers can be detected and data collected simultaneously, and data of multiple cardiac organ-like chips can also be collected simultaneously.

[0035] The cardiac organoid chip was used for high-throughput detection as follows:

[0036] Step 1), preparing 10 cardiac organ chips containing one chamber, and calibrating the numerical relationship between deformation contraction force and resistance;

[0037] First, connect the first columnar electrode 2 with a tension instrument and pull it slowly. At the same time, connect the two ends of the sensing conductive circuit to a resistance detector to record the change in resistance during pulling. Finally, based on the size of the pulling force and the resistance value under the corresponding pulling force, the numerical relationship between the deformation contraction force and the resistance can be obtained, and a numerical formula can be obtained.

[0038] Step 2), injecting a gel suspension containing cardiomyocytes, fibroblasts and endothelial cells into the chamber of the cardiac organ chip culture layer, and injecting a cell culture medium liquid for culture after gelation;

[0039] Cardiomyocytes, fibroblasts and endothelial cells were added in a quantitative ratio of 7:1.5:1.5, with a total amount of 3 million / 100 μl of suspension, and the gel suspension was a commercial decellularized extracellular matrix.

[0040] Step 3), after the three-dimensional cardiac organoid is self-assembled in the chamber, the first signal collection electrode 4 and the second signal collection electrode 5 are connected to the positive and negative electrodes of the electrical signal collector to read out the potential signal in real time;

[0041] Simultaneously, the two ends of the sensing conductive circuit are connected to a resistance detector, and the quantified contractile force of the cardiac organoid is directly displayed according to the calibrated numerical relationship; in this experiment, the contractile force of the myocardial strip is about 100uN.

[0042] Step 4), detecting the beating frequency and excitation-contraction coupled electromechanical signals of myocardial tissue after administration, the specific operation is as follows: adding different reagents or drugs into the chamber, detecting the beating frequency and excitation-contraction coupled electromechanical signals of myocardial tissue after administration, and obtaining the effect of the reagent or drug on the cardiac organoid tissue;

[0043] According to the operation of step 3), the synchronously collected contraction force curve and electrical signal curve are associated, that is, isochronously distributed, so as to display the excitation-contraction coupling relationship, wherein the frequency of the peak of the force curve is the beating frequency of the myocardial tissue.

[0044] Commercial drugs with known efficacy, proterenol and verapamil, were added to the chamber; when isoproterenol was added, the contraction frequency of the myocardial strips increased and the contraction force became stronger, indicating the positive chronotropic and mechanotropic effect of the drugs, while the opposite result was obtained when verapamil was added.

[0045] Step 5) To achieve high throughput, 10 of the above cardiac organoid chips are placed in parallel, and the resistance detector and signal collector are set to multi-channel acquisition, so that the function of real-time monitoring of electrophysiological signals of cardiac organoids without optical dependence can be realized.

[0046] Three commercial drugs with known efficacy, isoproterenol, verapamil, and nisoldipine, were tested and consistent drug effects were obtained on each cardiac organ-on-chip.

[0047] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A non-optically dependent cardiac organ-on-a-chip, characterized in that: The cardiac organoid chip includes a culture layer and a monitoring layer, wherein the culture layer is located on the upper side of the monitoring layer; the culture layer includes a cover plate, on which a plurality of chambers are provided, and the chambers are used to place three-dimensional cardiac organs and cell culture medium liquid; the monitoring layer includes a substrate, on which a plurality of monitoring devices corresponding to the chambers are provided; the monitoring device includes two columnar electrodes, a signal acquisition layer and a resistance sensing layer; the resistance sensing layer is arranged on the upper side of the substrate, the resistance sensing layer includes a base, on which a sensing conductive circuit is provided, and both ends of the sensing conductive circuit are connected to a resistance detector for detecting the bending of the base of the resistance sensing layer Changes in resistance during deformation; the signal collection layer is arranged on the upper side of the resistance sensing layer, the signal collection layer includes a substrate, and two conductive circuits are arranged on the substrate of the signal collection layer; the first ends of the two conductive circuits are respectively connected to the two columnar electrodes, and the second ends of the two conductive circuits are respectively connected to the first signal collection electrode and the second signal collection electrode; the first signal collection electrode and the second signal collection electrode are used to connect to an electrical signal collector; the two columnar electrodes are arranged in the chamber of the culture layer at intervals, and the lower ends of the two columnar electrodes are fixed on the substrate of the signal collection layer, and the two columnar electrodes are used to anchor the three-dimensional heart organoid; The base of the resistance sensing layer is a flexible base, and the base material of the resistance sensing layer is any one of polyvinyl alcohol, polyester, polyimide, paper, and PDMS; the material of the sensing conductive circuit is any one of liquid metal gallium, gallium-indium alloy, and conductive PDMS; The columnar electrode is a carbon electrode or a conductive silica gel electrode.

2. The optically independent cardiac organ-on-a-chip according to claim 1, characterized in that: The chamber is a groove-shaped structure with an open upper end. The chamber is rectangular or dumbbell-shaped, and the length-to-diameter ratio is greater than 2:

1.

3. The optically independent cardiac organ-on-a-chip according to claim 1, characterized in that: The three-dimensional cardiac organoid is self-assembled by uniformly mixing gel, cardiomyocytes, fibroblasts and endothelial cells.

4. The optically independent cardiac organ-on-a-chip according to claim 1, characterized in that: The material of the culture layer is selected from any one of quartz, glass, TPE, PMMA and PDMS.

5. The optically independent cardiac organ-on-a-chip according to claim 1, characterized in that: The material of the substrate is selected from any one of quartz, glass, TPE, PMMA and PDMS.

6. The optically independent cardiac organ-on-a-chip according to claim 1, characterized in that: The substrate of the signal collection layer is a flexible substrate, and the substrate material of the signal collection layer is any one of polyvinyl alcohol, polyester, polyimide, paper, and PDMS; the material of the first signal collection electrode and the second signal collection electrode is selected from any one of gold, platinum, silver, copper, and titanium.

7. A high-throughput detection method for a cardiac organ-on-a-chip according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1), preparing a plurality of non-optically dependent cardiac organ chips as described in any one of claims 1 to 6, and calibrating the numerical relationship between deformation contraction force and resistance; Step 2), injecting a gel suspension containing cardiomyocytes, fibroblasts and endothelial cells into the chamber of the cardiac organ chip culture layer, and injecting a cell culture medium liquid for culture after gelation; Step 3), after the three-dimensional cardiac organoid is self-assembled in the chamber, the first signal collection electrode and the second signal collection electrode are connected to the positive and negative electrodes of the electrical signal collector to read out the potential signal in real time; Simultaneously, the two ends of the sensing conductive circuit are connected to a resistance detector, and the quantified contractile force of the cardiac organoid is directly displayed according to the calibrated numerical relationship; Step 4), detecting the beating frequency and excitation-contraction coupled electromechanical signals of myocardial tissue after drug administration, the specific operation is as follows: Adding different reagents or drugs into the chamber, detecting the beating frequency and excitation-contraction coupled electromechanical signals of the myocardial tissue after administration, and obtaining the effect of the reagents or drugs on the cardiac organoid tissue; Step 5) Arrange multiple cardiac organoid chips in parallel, set the resistance detector and signal collector to multi-channel acquisition, and thus realize the function of real-time monitoring of electrophysiological signals of cardiac organoids without optical dependence and high throughput.

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