A high-throughput organ chip and its application

Abstract

The present application relates to a high-throughput organ chip and its application. The chip includes: multiple culture modules; a single culture module includes: a first culture layer, a second culture layer, a porous diaphragm layer and an electrode layer; the first culture layer is an open culture layer; the second culture layer is a closed culture layer; the first culture layer is provided with a cell culture hole and at least two fluid holes; the cell culture hole and the fluid hole are arranged adjacently; the second culture layer is provided with a cell culture flow channel; the cell culture flow channel is connected to the fluid hole; the porous diaphragm layer is arranged between the cell culture flow channel and the cell culture hole; the electrode layer is arranged on the side of the second culture layer away from the first culture layer. The organ chip of the present application is easy to implement automated operation; it can also be measured by immersing the insertable electrode rod into the cell culture hole, and then connecting the electrode layer to the electrochemical workstation respectively, so as to provide effective experimental data for physiological and pathological model research and drug evaluation.

Description

Technical Field

[0001] The present application relates to the technical field of microfluidic organ chips, and in particular to a high-throughput organ chip and its application. Background Art

[0002] Organ chip is the abbreviation of microfluidic organ chip, which is a three-dimensional cell culture device, including the microenvironment composed of cells, secretions and physical forces around the corresponding organ. Compared with traditional drug screening models, organ chips can better simulate the functions of real organs and avoid the species differences existing in animal models.

[0003] At present, although many organ chips can achieve high-throughput structures with multiple culture units, experiments still rely on manual injection and sampling, because the closed chip structure only has entrances and exits connected to the outside world, which is difficult to be compatible with existing automated devices. In addition, for the study of epithelial or endothelial barrier function, transmembrane resistance is an important functional indicator, but the current measurement methods have certain problems. For chips without integrated electrode structures, the measuring electrodes are applied at the entrances and exits of the chip, far away from the location of the epithelial or endothelial barrier, resulting in low measurement accuracy. For chips with integrated electrode structures, the chip structure is closed and cannot be automatically sampled and sampled. Therefore, it is necessary to provide a new type of organ chip to solve the technical problems existing in the prior art. Summary of the invention

[0004] In order to solve the problems of the prior art, the present application provides a high-throughput organ chip and its application. The technical solution is as follows:

[0005] In one aspect, a high-throughput organ chip is provided, the chip comprising: a plurality of culture modules;

[0006] A single culture module includes: a first culture layer, a second culture layer, a porous diaphragm layer and an electrode layer;

[0007] The first culture layer is an open culture layer;

[0008] The second culture layer is a closed culture layer;

[0009] The first culture layer is provided with a cell culture hole and at least two fluid holes; the cell culture hole and the fluid hole are arranged adjacent to each other;

[0010] The second culture layer is provided with a cell culture flow channel;

[0011] The cell culture flow channel is connected to the fluid hole;

[0012] The porous diaphragm layer is disposed between the cell culture channel and the cell culture hole;

[0013] The electrode layer is arranged on a side of the second culture layer away from the first culture layer.

[0014] Further, the material of the porous diaphragm layer includes at least one of polyethylene terephthalate, polydimethylsiloxane, polycarbonate and glass;

[0015] The pore size of the porous diaphragm layer is 0.4um to 3um.

[0016] Furthermore, an electrode structure is provided on the electrode layer; the electrode structure is a two-circle annular structure; one end of the electrode structure is provided at the cell culture channel; the electrode structure is in contact with the liquid in the cell culture channel; when measuring transmembrane resistance, the electrode structure cooperates with the electrode inserted in the cell culture hole.

[0017] Furthermore, the cell culture well is used to culture at least one of organoids, cell spheroids or monolayer adherent cells, and to store cell culture medium or drug dilution solution.

[0018] Further, the cell culture flow channel is used for static or dynamic culture of a biological barrier cell layer composed of epithelial or endothelial barrier cells;

[0019] The dynamic culture is to set the parameters of the fluid in the cell culture channel based on the parameters of the blood flow of the target object; during dynamic culture, the fluid hole is connected to a peristaltic pump or a syringe pump, and the peristaltic pump or the syringe pump controls the flow rate of the solution in the cell culture channel.

[0020] Furthermore, the organ chip includes a plurality of culture modules arranged in an array.

[0021] Furthermore, the cell culture well has a pore size of 5 to 20 mm;

[0022] The diameter of the fluid hole is 0.8-3 mm.

[0023] Furthermore, the processing method of the organ chip is at least one of integrated molding and layered processing; the micro-processing method of the organ chip includes at least one of glass inner engraving method, CNC milling method, laser etching method, chemical corrosion method, soft etching method, injection molding, hot pressing method, metal sputtering and metal electroplating.

[0024] Further, the material of the first culture layer and the second culture layer includes at least one of polymethyl methacrylate, polystyrene, polydimethylsiloxane and glass;

[0025] Furthermore, the high-throughput organ chip also includes a pipetting workstation;

[0026] The liquid transfer workstation is used to add liquid, replace liquid and perform sampling on the first culture layer;

[0027] The xyz three-axis displacement arm of the pipetting workstation immerses the insertable electrode rod into the cell culture hole and cooperates with the electrode structure to measure the transmembrane resistance.

[0028] On the other hand, this application also provides a high-throughput organ chip detection platform, please refer to Figure 5 , Figure 5 It is a structural schematic diagram of a high-throughput organ chip and detection platform provided in an embodiment of the present application; a high-throughput organ chip detection platform includes a robotic arm for measuring the transmembrane resistance of a barrier structure, and a first electrode is arranged on the robotic arm; it also includes a second electrode matching the electrode structure of the above-mentioned high-throughput organ chip, and electrochemical impedance spectroscopy testing and barrier structure transmembrane impedance measurement can be realized through the first electrode and the second electrode.

[0029] On the other hand, the present application provides an application of a high-throughput organ chip, which is used to simulate a preset physiological environment structure according to the application of the high-throughput organ chip structure, and provide effective experimental data for physiological and pathological model research and drug evaluation.

[0030] The high-throughput organ chip and its application provided in this application have the following technical effects:

[0031] The organ chip of the present application is convenient for implementing automated operations, such as adding liquid, changing liquid, and sampling the first culture layer through an xyz three-axis translation stage and an electric pipette; the insertable electrode rod can also be immersed in the cell culture well, and then the electrode layer is respectively connected to the electrochemical workstation to measure the transmembrane resistance of the barrier structure, thereby providing effective experimental data for physiological and pathological model research and drug evaluation.

[0032] The organ chip of the present application sets the first culture layer as an open culture layer and the second culture layer as a closed culture layer, and realizes the construction of a vascular-organ model in the organ chip by combining the open and closed culture systems; or at least two organ cells are cultured in the organ chip to construct a multi-organ co-culture model, which can provide a platform for large-scale metabolic drug screening and related mechanism research. The present application immerses the insertable electrode rod into the cell culture hole of the first culture layer, and connects the insertable electrode rod and the electrode layer to the electrochemical workstation respectively, so as to measure the transmembrane resistance of the barrier structure, providing effective experimental data for physiological and pathological model research and drug evaluation.

[0033] The organ chip of the present application is conducive to interaction with drugs through the open first culture layer, and the drugs are applied efficiently. The organ chip of the present application simulates the structure and function of human organs, is a miniaturized, automated and visualized tool, and can provide effective experimental data for physiological and pathological model research and drug evaluation.

[0034] The organ chip of the present application adopts a two-circle annular electrode structure so that the generated electrical signals can be evenly distributed in the cell co-culture area without affecting the imaging observation of the cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 is a schematic diagram of the structure of a high-throughput organ chip provided in an embodiment of the present application;

[0037] Figure 2 is a top view of a high-throughput organ chip provided in an embodiment of the present application;

[0038] Figure 3 This is a schematic diagram of the structure layering of a high-throughput organ chip provided in an embodiment of the present application;

[0039] Figure 4 is a top view of an electrode structure of a culture module provided in an embodiment of the present application;

[0040] Figure 5 It is a schematic diagram of the structure of a high-throughput organ chip and detection platform provided in an embodiment of the present application;

[0041] Figure 6 It is a schematic diagram of electrochemical impedance spectroscopy measurement after the culture module of the high-throughput organ chip provided in the embodiment of the present application is inserted into the electrode rod;

[0042] Figure 7 This is an immunofluorescence image of Ve-Cadherin expression of hCMEC / d3 cells cultured in a chip on day 3 provided in the examples of the present application;

[0043] Figure 8 It is a schematic diagram of the electrochemical impedance spectroscopy results when hCMEC / d3 cells are cultured in an organ chip provided in an example of the present application.

[0044] Among them, the corresponding reference numerals in the figure are: 1-first culture layer; 2-second culture layer; 3-porous diaphragm layer; 4-electrode layer; 11-cell culture hole; 12-fluid hole; 21-cell culture flow channel; 41-electrode structure. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0046] For the following defined terms, these definitions shall apply unless a different definition is given in the claims or elsewhere in this specification. All numerical values, whether or not explicitly indicated, are defined herein as modified by the term "about". The term "about" generally refers to a numerical range that a person of ordinary skill in the art considers to be equivalent to the stated values ​​to produce substantially the same properties, functions, results, etc. A numerical range indicated by a lower value and a higher value is defined to include all numerical values ​​included in the numerical range and all subranges included in the numerical range.

[0047] It should be noted that the terms "include" and "have" in the specification and claims of the present disclosure and the above drawings and any variations thereof are intended to cover non-exclusive inclusions.

[0048] Before further describing the embodiments of the present application in detail, the nouns and terms involved in the embodiments of the present application are explained. The nouns and terms involved in the embodiments of the present application are subject to the following interpretations.

[0049] Epithelial tissue (epithelium): It is the first tissue to form during individual development. It is composed of densely packed epithelial cells, which are generally connected to each other in a membrane-like sheet. It covers the body surface or lines the cavity of hollow organs in the body, as well as the cavity surface of the body. The epithelial cells that make up the skin protect the underlying tissue from damage, bacterial invasion, and harmful chemicals, and prevent excessive loss of water. When necessary, the epithelial cells of the skin also secrete sweat to regulate body temperature. The epithelial cells covering the pancreas secrete enzymes to promote digestion. The epithelial cells on the surface of the small intestine absorb nutrients from digested food. The epithelial cells on the surface of the respiratory tract form mucous membranes, which can secrete mucus to prevent inhaled bacteria and viruses from entering the lungs. The skin, nose, tongue, eyes and other organs that come into contact with nerve endings also secrete specialized epithelial cells that can recognize sensory stimuli. The main functions of epithelial cells involve secretion, absorption, and protection. Epithelial tissue has obvious polarity. The two sides of epithelial cells are different in structure and function. One side faces the body surface or the cavity of the tube, cavity, or sac in the body, which is called the free surface; the other side opposite to the free surface is called the basal surface. Tight junctions (zonulae occludens) separate the serous membrane of epithelial cells into a free surface and a basal surface. The free surfaces of epithelial tissue cells distributed in different parts often have different specialized structures to adapt to their specific functions. For example, the intestinal epithelium has dense microvilli that expand the absorption surface; the respiratory epithelium has cilia that can swing and can expel invading dust and other foreign matter. The basal surface is generally connected to its deep connective tissue by a layer of basement membrane. Considering the wide distribution and the importance of function, the research on epithelial cells (including endothelial cells) has involved multiple fields such as physiology, pathology, pharmacology and toxicology.

[0050] Endothelial tissue (endothelium): It is a type of epithelial tissue. Endothelial cells constitute endothelial tissue. Endothelial cells are distributed on the inner surface of blood vessels. In the lumen, endothelial tissue constitutes the interface between the blood circulatory system or lymphatic system and other parts of the blood vessels. Endothelial cells that are in direct contact with blood are called vascular endothelial cells, while endothelial cells that are in direct contact with lymph are called lymphatic endothelial cells. From the heart to the smallest capillaries, vascular endothelial cells are distributed throughout the circulatory system. Endothelial cells covering the walls of blood vessels can regulate blood flow in blood vessels. These cells can release NO, which is a vasodilator factor that is related to vasoconstriction and vasodilation, promotes blood circulation, and helps control blood pressure. Endothelial cells can also secrete a variety of proteins that cause blood disorders, but they also stop bleeding. Endothelial cells are contained inside the glomerulus, and their function is to filter blood.

[0051] Combine the following Figure 1 The organ chip structure diagram shown is used to explain in detail the technical solution provided by the implementation of this application.

[0052] Specifically, Figure 1 , Figure 2 and Figure 3 As shown, a high-throughput organ chip, the chip includes: multiple culture modules; a single culture module includes: a first culture layer 1, a second culture layer 2, a porous diaphragm layer 3 and an electrode layer 4; the first culture layer 1 is an open culture layer; the second culture layer 2 is a closed culture layer; the first culture layer 1 is provided with a cell culture hole 11 and at least two fluid holes 12; the cell culture hole 11 and the fluid hole 12 are arranged adjacently; the second culture layer 2 is provided with a cell culture channel 21; the cell culture channel 21 is connected to the fluid hole 12; the porous diaphragm layer 3 is arranged between the cell culture channel 21 and the cell culture hole 11; the electrode layer 4 is arranged on the side of the second culture layer 2 away from the first culture layer 1. It should be noted that the organ chip includes not only a culture module, but also a perfusion system for dynamic cell culture. The perfusion system refers to the entire process of the culture fluid entering from the fluid hole 12, passing through the cell culture channel 21, and being discharged from another fluid hole 12. It should be noted that the desired drug can also be added to the culture fluid.

[0053] In some embodiments, the closed culture layer is used to simulate blood vessels; the open culture layer is used to simulate solid organs, which is convenient for automatic pipetting operations and application of upper electrodes.

[0054] The organ chip of the present application sets the first culture layer as an open culture layer and the second culture layer as a closed culture layer, and realizes the construction of a vascular-organ model in the organ chip by combining the open and closed culture systems; or at least two organ cells are cultured in the organ chip to construct a multi-organ co-culture model, which can provide a platform for large-scale metabolic drug screening and related mechanism research.

[0055] The organ chip of the present application is conducive to interaction with drugs through the open first culture layer, and the drugs are applied efficiently. The organ chip of the present application simulates the structure and function of human organs, is a miniaturized, automated and visualized tool, and can provide effective experimental data for physiological and pathological model research and drug evaluation.

[0056] In some embodiments, the cell culture well 11 is used to culture at least one of organoids, cell spheroids or monolayer adherent cells, and to store cell culture medium or drug diluent. The cell culture well can be used to insert electrodes to measure the transmembrane impedance of the barrier structure. It should be noted that parenchymal cells can be added to the cell culture well 11 to construct a vascular-organ chip model; endothelial / epithelial barrier support cells can also be added to construct a barrier model that simulates the in vivo cell growth environment; cell culture medium or drug diluent can also be added to provide the entire system with the nutrients necessary for growth or the drugs that need to be tested; a robotic arm can also be used to insert electrodes for measuring the transmembrane impedance of the barrier structure.

[0057] In some embodiments, the cell culture hole 11 is a straight hole, and the pore size of the cell culture hole 11 is 5 to 20 mm; specifically, the at least two fluid holes 12 of the first culture layer 1, one is a liquid inlet, and the other is a liquid outlet, the fluid hole 12 can be connected to the pipeline of an external pump such as a syringe pump or a peristaltic pump through a connector (such as a Luer connector), and is used to transport the liquid in the cell culture channel 21 of the second culture layer 2, and the pore size of the fluid hole 12 is 0.8 to 3 mm. It should be noted that, under environmental conditions, multiple fluid holes can be set, specifically, there can be one liquid inlet hole and two liquid inlet and outlet holes; there can also be two liquid inlet holes and one liquid inlet and outlet hole; the number of fluid holes is not limited here.

[0058] In some embodiments, the cell culture well 11 may have a pore diameter of 5 to 15 mm.

[0059] In some embodiments, the cell culture well 11 may have a pore diameter of 10 to 15 mm.

[0060] In some embodiments, the material of the porous diaphragm layer 3 includes at least one of polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polycarbonate (PC) and glass. Among them, PET film is also called PET porous film, which is a packaging film with relatively comprehensive performance. Its toughness is the best among all thermoplastics, and its tensile strength and impact strength are much higher than those of general films. It has good transparency, gloss, good air tightness and other properties as a reliable basis for selecting the intermediate film.

[0061] In some embodiments, the porous membrane layer 3 is a porous membrane with a relatively small pore size and a relatively high porosity. The porous membrane layer 3 is used to separate the cell culture flow channel and the cell culture hole and provide a channel for the culture systems of the first culture layer and the second culture layer to interact. The pore size of the porous membrane layer 3 is smaller than the cell size to avoid the movement of cells in the culture areas of the first culture layer and the second culture layer. Specifically, the pore size of the porous membrane layer 3 is 0.4um to 3um. It should be noted that the size and density of the holes on the porous membrane layer 3 can be selected as needed.

[0062] In some embodiments, an electrode structure 41 is disposed on the electrode layer 4. Figure 4 The electrode structure 41 adopts a two-circle annular electrode design, so that the generated electrical signal can be evenly distributed in the cell co-culture area without affecting the imaging observation of the cells.

[0063] One end of the electrode structure 41 is disposed at the cell culture channel 21 ; the electrode structure 41 is in contact with the liquid in the cell culture channel 21 ; when measuring transmembrane resistance, the electrode structure 41 cooperates with the electrode inserted into the cell culture hole 11 .

[0064] The electrode structure is used to measure the barrier structure transmembrane impedance of cells in cell culture wells and fluid wells.

[0065] In some embodiments, the electrode material of the electrode structure 41 may be at least one of gold and platinum, and the electrode structure 41 may be formed on the surface of the second culture layer 2 away from the porous membrane layer 3 by at least one of metal sputtering and metal etching.

[0066] The present application immerses the insertable electrode rod into the cell culture hole of the first culture layer, and simultaneously connects the insertable electrode rod and the electrode structure provided on the electrode layer to the electrochemical workstation, thereby being able to measure the transmembrane resistance of the barrier structure, and providing effective experimental data for physiological and pathological model research and drug evaluation. Among them, the insertable electrode rod can use a commercial concentric axis electrode, the upper electrode material of the insertable electrode rod can be selected from platinum or gold, and the diameter of the upper electrode of the insertable electrode rod is smaller than the diameter of the cell culture hole 11 of the first culture layer 1.

[0067] In some embodiments, the cell culture channel 21 is used for static or dynamic culture of a biological barrier cell layer composed of epithelial or endothelial barrier cells; dynamic culture is to set the parameters of the fluid in the cell culture channel 21 based on the parameters of the blood flow of the target object; during dynamic culture, the fluid hole 21 is connected to a peristaltic pump or a syringe pump through a connector (such as a Luer connector) and a hose, and the peristaltic pump or the syringe pump controls the flow rate of the solution in the cell culture channel 21. The cell culture channel 21 is used to simulate blood flow in a blood vessel. Specifically, the average linear velocity of the fluid in the cell culture channel 21 ranges from 1 to 3000 microliters / minute.

[0068] In some embodiments, the size and shape of the cell culture flow channel 21 are limited, and it can be a long straight channel or a channel with a gradually changing width. Preferably, the size of the two ends of the cell culture flow channel 21 connected to the two fluid holes 12 is the same as the size of the fluid holes 12, and the width of the cell culture flow channel 21 gradually widens from the two ends to the middle. Specifically, the flow channel width of the cell culture flow channel 21 can be 5-20mm, the flow channel length of the cell culture flow channel 21 can be 10-30mm, and the flow channel height of the cell culture flow channel 21 can be 0.1-5mm.

[0069] In some embodiments, the flow channel width of the cell culture flow channel 21 may be 5-15 mm, the flow channel length of the cell culture flow channel 21 may be 10-20 mm, and the flow channel height of the cell culture flow channel 21 may be 0.1-4 mm.

[0070] In some embodiments, the flow channel width of the cell culture flow channel 21 may be 10-15 mm, the flow channel length of the cell culture flow channel 21 may be 15-20 mm, and the flow channel height of the cell culture flow channel 21 may be 1-4 mm.

[0071] In some embodiments, the organ chip includes a plurality of culture modules arranged in an array. It should be noted that each culture module has the same size, so as to meet the high-throughput application and perform high-throughput cell culture functions. High-throughput culture technology has the characteristics of trace, rapid, sensitive and accurate, and can obtain a large amount of valuable information through one experiment, making the results of cell culture more accurate and reliable.

[0072] In the embodiments of the present application, the size of the organ chip of the present application is the same as that of a conventional 96-well plate, so the organ chip can be directly placed in an instrument such as an ELISA reader for direct reading, eliminating the transfer step, which is both convenient and quick.

[0073] In some embodiments, the material of the first culture layer 1 and the second culture layer 2 is at least one of polymethyl methacrylate, polystyrene, polydimethylsiloxane and glass. It should be noted that the material of the first culture layer 1 and the second culture layer 2 is not limited to the listed materials.

[0074] In some embodiments, the processing method of the organ chip is at least one of one-piece molding and layered processing; the micro-processing method of the organ chip includes at least one of glass inner engraving, CNC milling, laser etching, chemical corrosion, soft etching, injection molding, hot pressing, metal sputtering and metal electroplating. It should be noted that the formation method of the chip is not limited, wherein the one-piece forming means can be laser etching or one-time injection molding to form an integrated culture system. The formation means of layered processing is specifically to form corresponding cell culture holes, fluid holes and cell culture channels on each layer to form an upper and lower culture system, and stack each layer in sequence. The layers of chips can be bonded and assembled together by sealing processes such as double-sided tape, ultrasound, thermal bonding, plasma and hot pressing. Exemplarily, the chip bonding method is glue bonding, using a solid adhesive as the adhesive of the organ chip, and the three layers are bonded together by heating the adhesive and bonding at room temperature, and finally a chip with a complete structure is obtained. Please refer to Figure 1 , Figure 3 , Figure 3 This is a schematic diagram of the layered structure of the organ chip before bonding. Figure 1 The schematic diagram of the structure of the organ chip after three layers are bonded. In the organ chip bonding process, the adhesive bonding method is selected to make the porous diaphragm layer 3 fit better with the first culture layer 1 and the second culture layer 2 to avoid leakage and other problems.

[0075] In a specific embodiment, an in vitro 3D liver model is cultured in the cell culture wells 11 of the first culture layer 1 of the organ chip, and a small intestinal absorption model is cultured in the cell culture channel 21 of the second culture layer 2, to establish an intestinal-liver co-culture model, thereby realizing the construction of an in vitro model that can predict drug intestinal absorption and liver toxicity.

[0076] In another specific embodiment, the coating liquid type I rat tail collagen is introduced into the cell culture channel 21 of the second culture layer 2 of the organ chip through the first fluid hole 12 for coating treatment, and then the brain microvascular endothelial cells hCMEC / D3 are added to the cell culture channel 21 of the second culture layer 2 of the organ chip through the first fluid hole 12, and the organ chip is turned upside down, with the first culture layer placed at the bottom and the second culture layer placed at the top, so that the newly added brain microvascular endothelial cells hCMEC / D3 are settled on the upper surface of the cell culture channel 21 That is, the porous membrane layer 3 is close to the side of the cell culture channel 21. After the brain microvascular endothelial cells hCMEC / D3 are attached and grown on the porous membrane layer 3, the organ chip is flipped again to make it upright, and astrocytes are added to the cell culture wells 11 of the first culture layer 1 to achieve the co-culture of brain microvascular endothelial cells and astrocytes in the chip. The first fluid hole 12 and the second fluid hole 12 are connected to the peristaltic pump, and the culture fluid is passed to realize the flow of the culture medium in the cell culture channel 21, realizing dynamic co-culture and constructing a blood-brain barrier model. Please refer to Figure 7 , Figure 7 This is an immunofluorescence image of Ve-Cadherin expression in hCMEC / d3 cells cultured in the chip on the third day provided in the embodiment of the present application. It should be noted that the scale of the figure is 50 microns. When testing the physiological indicators of the blood-brain barrier organ chip, whether the cells are tightly arranged is crucial to the model construction. At the same time, vascular endothelial cell cadherin (VE-cadherin) is a key molecule for adhesion connection between vascular endothelial cells (EC). This application performed VE-cadherin immunofluorescence characterization on the organ chip model cultured on the third day, and the results are as follows Figure 7 As shown in the figure, it can be clearly seen that the brain microvascular endothelial cells hCMEC / D3 in the organ chip are closely arranged and VE-cadherin is detected in the cells, indicating that the blood-brain barrier model constructed in this application is reliable.

[0077] The transmembrane resistance of the blood-brain barrier model constructed above was measured:

[0078] The insertable electrode rod is immersed under the liquid surface of the cell culture hole 11 of the first culture layer 1, and the insertable electrode rod and the electrode structure in the cell culture flow channel 21 of the second culture layer 2 are respectively connected to the electrochemical workstation to perform an electrochemical impedance spectroscopy (EIS) test to obtain the transmembrane impedance (TEER) of the barrier model. It should be noted that the impedance technology is used to measure the transmembrane impedance as an indicator of cell barrier integrity and permeability. This method can be used to monitor the cell barrier state in the chip in real time and quantify the changes in barrier properties over time. Figure 8 Schematic diagram of the electrochemical impedance spectroscopy results when the organ chip cultured hCMEC / d3 cells provided in the embodiment of the present application. The blood-brain barrier model was subjected to equivalent circuit analysis. It should be noted that the equivalent circuit analysis of the electrochemical impedance spectroscopy can obtain electrical parameters that can characterize the cell barrier characteristics, such as the dielectric resistance (R medium ), the capacitance component of the cell (C cell ) and cell resistance (R cell ), which can help understand the model barrier formation status. Figure 8 This is a typical EIS result. It can be seen from the figure that at high frequencies (>10kHz), the impedance amplitude |Z| remains unchanged, which is related to the medium resistance. At low frequencies (<10Hz), the impedance amplitude |Z| is determined by the cell resistance and the medium resistance. The experimental EIS data are fitted to the equivalent circuit model to estimate the cell capacitance component introduced by the current through the transcellular pathway to obtain the best fitting parameters.

[0079] In some embodiments, the high-throughput organ chip further comprises a pipetting workstation; the pipetting workstation is used to add liquid, replace liquid and perform sampling on the first culture layer 1; the xyz three-axis displacement arm of the pipetting workstation immerses the insertable electrode rod into the cell culture hole 11 and cooperates with the electrode structure 41 to measure the transmembrane resistance, please refer to Figure 6 .

[0080] In some embodiments, the high-throughput organ chip also includes an electrochemical workstation; the transmembrane resistance is measured using a constant current electrochemical impedance spectroscopy test mode of the electrochemical workstation to obtain an impedance spectrum at a frequency of 10 Hz to 1 MHz, and transmembrane impedance information is obtained, and the measurement process lasts about 5-20 seconds.

[0081] On the other hand, this application also provides a high-throughput organ chip detection platform, please refer to Figure 4 , Figure 4It is a structural schematic diagram of a high-throughput organ chip and detection platform provided in an embodiment of the present application; a high-throughput organ chip detection platform includes a robotic arm for measuring the transmembrane resistance of a barrier structure, and a first electrode is arranged on the robotic arm; it also includes a second electrode matching the electrode structure of the above-mentioned high-throughput organ chip, and electrochemical impedance spectroscopy testing and barrier structure transmembrane impedance measurement can be realized through the first electrode and the second electrode.

[0082] On the other hand, the present application also provides an application of a high-throughput organ chip, which is used to simulate a preset physiological environment structure according to the application of the above-mentioned high-throughput organ chip structure, and provide effective experimental data for physiological and pathological model research and drug evaluation.

[0083] It can be seen from the above schemes of the embodiments of the present application that the high-throughput organ chip and its application provided by the present application have the following technical effects:

[0084] The organ chip of the present application sets the first culture layer as an open culture layer and the second culture layer as a closed culture layer, and realizes the construction of a vascular-organ model in the organ chip by combining the open and closed culture systems; or at least two organ cells are cultured in the organ chip to construct a multi-organ co-culture model, which can provide a platform for large-scale metabolic drug screening and related mechanism research.

[0085] The present application immerses the insertable electrode rod into the cell culture hole of the first culture layer, and simultaneously connects the insertable electrode rod and the electrode structure provided on the electrode layer to an electrochemical workstation respectively, thereby being able to measure the transmembrane resistance of the barrier structure, thereby providing effective experimental data for physiological and pathological model research and drug evaluation.

[0086] The organ chip of the present application is conducive to interaction with drugs through the open first culture layer, and the drugs are applied efficiently. The organ chip of the present application simulates the structure and function of human organs, is a miniaturized, automated and visualized tool, and can provide effective experimental data for physiological and pathological model research and drug evaluation.

[0087] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A high-throughput organ chip, characterized in that: The chip comprises: a plurality of culture modules; A single culture module comprises: a first culture layer (1), a second culture layer (2), a porous diaphragm layer (3) and an electrode layer (4); The first culture layer (1) is an open culture layer; The second culture layer (2) is a closed culture layer; The first culture layer (1) is provided with a cell culture hole (11) and at least two fluid holes (12); the cell culture hole (11) and the fluid hole (12) are arranged adjacent to each other; the pore diameter of the cell culture hole (11) is 5 to 20 mm; the pore diameter of the fluid hole (12) is 0.8 to 3 mm; during dynamic culture, the cell culture flow channel (21) is used to simulate blood flow in a blood vessel, the fluid hole (12) is connected to a peristaltic pump or a syringe pump, and the peristaltic pump or the syringe pump controls the flow rate of the solution in the cell culture flow channel (21); The second culture layer (2) is provided with a cell culture flow channel (21); The cell culture flow channel (21) is connected to the fluid holes (12); the size of the end of the cell culture flow channel (21) connected to the at least two fluid holes (12) is the same as the size of the fluid holes (12), and the width of the cell culture flow channel (21) gradually widens from the connected end to the middle; The porous diaphragm layer (3) is arranged between the cell culture flow channel (21) and the cell culture hole (11); The electrode layer (4) is arranged on a side of the second culture layer (2) away from the first culture layer (1); an electrode structure (41) is arranged on the electrode layer (4); and the electrode structure (41) is a two-ring annular structure.

2. The high-throughput organ chip according to claim 1, characterized in that: The material of the porous diaphragm layer (3) includes at least one of polyethylene terephthalate, polydimethylsiloxane, polycarbonate and glass; The pore diameter of the porous diaphragm layer (3) is 0.4 um to 3 um.

3. The high-throughput organ chip according to claim 1, characterized in that: One end of the electrode structure (41) is disposed at the cell culture flow channel (21); the electrode structure (41) is in contact with the liquid in the cell culture flow channel (21); When measuring transmembrane resistance, the electrode structure (41) cooperates with the electrode inserted into the cell culture well (11).

4. The high-throughput organ chip according to claim 1, characterized in that: The cell culture well (11) is used to culture at least one of organoids, cell spheroids or monolayer adherent cells, and to store cell culture medium or drug dilution solution.

5. The high-throughput organ chip according to claim 1, characterized in that: The cell culture flow channel (21) is used for static or dynamic culture of a biological barrier cell layer composed of epithelial or endothelial barrier cells; The dynamic culture is to set the parameters of the fluid in the cell culture channel (21) based on the parameters of the blood flow of the target object.

6. The high-throughput organ chip according to claim 1, characterized in that: The organ chip includes a plurality of culture modules arranged in an array.

7. The high-throughput organ chip according to claim 1, characterized in that: The processing method of the organ chip is at least one of integrated molding and layered processing; The micro-machining method of the organ chip includes at least one of glass inner engraving, CNC milling, laser etching, chemical corrosion, soft etching, injection molding, hot pressing, metal sputtering and metal electroplating.

8. The high-throughput organ chip according to claim 3, characterized in that: The high-throughput organ chip also includes a pipetting workstation; The liquid transfer workstation is used to perform liquid addition, liquid replacement and sampling processing on the first culture layer (1); The xyz three-axis displacement arm of the liquid transfer workstation immerses the insertable electrode rod into the cell culture hole (11) and cooperates with the electrode structure (41) to measure the transmembrane resistance.

9. An application of a high-throughput organ chip, characterized in that: The use of the high-throughput organ chip structure according to any one of claims 1 to 8 is used to simulate a preset physiological environment structure.

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