Microfluidic organ chip for three-dimensional cell culture

Abstract

The invention provides a micro-fluidic organ chip for three-dimensional cell culture. The micro-fluidic organ chip comprises a cell model loading and culturing unit, a liquid inflow-outflow unit with height difference, and an embedded inverted cone compression ring sealing unit, the cell model loading and culturing unit is constructed by overlapping at least three layers of chips up and down; and the liquid inflow-outflow unit with the height difference is constructed and formed by respectively forming liquid circulation holes which are hermetically connected through the embedded inverted conical compression ring sealing unit on at least three layers of chips. According to the invention, the cell culture space is expanded through the cell model loading and culture unit, high compatibility of cell culture is realized, and the problem of non-uniform concentration of a test substance is eliminated through the liquid inflow-outflow unit with height difference; and high-reliability miniaturized sealing design is realized through the embedded inverted cone pressing ring sealing unit, and the device can be widely applied to culture of cell models and test substance treatment and effect index detection taking toxicological and pharmacological researches as target scenes.

Description

Technical Field

[0001] The present invention belongs to the cross - field of microfluidic chip design, microfabrication, toxicology, pharmacology and cell biology, and relates to the design and manufacture of cell culture devices and toxicity testing devices. Specifically, it is a microfluidic - driven organ chip that integrates different functional units, is suitable for three - dimensional cell culture, and has different liquid flow modes. Background Art

[0002] The microfluidic organ - on - a - chip (OoC) is one of the core research directions in the field of biomedical engineering in recent years. By simulating the microenvironment of human organs, it provides an in - vitro research platform for drug screening, toxicity testing and disease modeling.

[0003] In the prior art, the typical design of microfluidic organ chips mainly includes: (1) Multilayer microfluidic structure: A PDMS chip prepared by soft lithography technology, integrating microchannels, cell culture chambers and fluid control modules; (2) Luer connector or inverted cone connector sealing scheme: Using standard Luer interfaces to achieve the sealing of fluid channels and connection with external catheters; (3) Static or simple dynamic culture mode: Periodic perfusion of culture medium is achieved through gravity drive or pump control systems.

[0004] Currently, existing microfluidic organ-on-a-chip still have significant limitations in the following aspects: (1) Insufficient compatibility and culture efficiency of three-dimensional cell models: First, the cell culture chamber volume of most chips is limited, making it difficult to adapt to large-size three-dimensional models such as liver organoids and tumor spheroids, and unable to accommodate the co-culture of two-dimensional cells and three-dimensional models. Second, the connectivity between the chamber and the fluid channel is insufficient, resulting in uneven distribution of nutrient and oxygen gradients and affecting cell viability. (2) Insufficient fluid control precision and uneven distribution of test substances: Although the traditional flat laminar flow channel design is suitable for the supply of soluble test substances, when studying insoluble solid powders, suspensions, etc. in the field of toxicology / pharmacology, solid test substances (such as nanoparticles and poorly soluble compounds) are prone to local accumulation, interfering with the results of toxicology / pharmacodynamics tests. (3) The sealing structure occupies a large space and has low integration: First, external sealing components such as Luer connectors and inverted cone connectors occupy a large amount of space at the edge of the chip, restricting the miniaturization of the chip and the parallel design of high-throughput multi-organ chips. Second, the sealing reliability depends on external fixtures, and leakage or contamination is likely to occur during long-term perfusion, or the chip structure may be damaged and deformed. (4) Difficulty in post-processing of cell models: Most existing chips have a closed structure and require destructive disassembly to remove three-dimensional cells, resulting in the inability to perform in-situ detection (such as immunofluorescence staining) or low sample recovery rate, affecting downstream molecular biology analysis (RNA / protein extraction). At the same time, in actual experimental operations, there are also the following technical operation difficulties that may affect the experimental results: a) Chips with a fully enclosed structure can only be tested once and cannot be reused; b) Flexible materials such as PDMS have strong adsorption of chemicals in the flow channel, which may affect the effective concentration of test substances; c) Chips with a fully enclosed structure have poor flexibility when loading three-dimensional cell models of different sizes; d) The liquid flow channels of chips usually suitable for two-dimensional cell culture are 100-500 μm wide, which highly coincides with the diameter of three-dimensional cell models such as organoids or cell spheres. When the microfluidic device drives the liquid to flow, non-adherent three-dimensional cell models are prone to block the channels (organoids or cell spheres may roll to the entrances and exits of the liquid flow channels), affecting the renewal and flow of the culture medium and may cause the three-dimensional cell models to rupture or their structures to be damaged. Summary of the Invention

[0005] The object of the present invention is to provide a microfluidic organ chip for three-dimensional cell culture through functional unit design. The microfluidic organ chip includes a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone press ring sealing unit; wherein, the cell model loading and culturing unit is constructed by stacking at least three chips up and down; the liquid inflow-outflow unit with a height difference is constructed by respectively opening liquid circulation holes sealed and connected through the embedded inverted cone press ring sealing unit on at least three chips. The microfluidic organ chip of the present invention not only expands the cell culture space and realizes high compatibility of cell culture through the cell model loading and culturing unit, but also eliminates the problem of uneven concentration of the test substance through the liquid inflow-outflow unit with a height difference, and also realizes a highly reliable miniaturized sealing design through the embedded inverted cone press ring sealing unit, and can be widely used for the culture of cell models and the detection of the treatment of test substances and effect indexes with toxicology and pharmacology research as the target scenarios.

[0006] To this end, the present invention provides the following technical solutions:

[0007] A microfluidic organ chip for three-dimensional cell culture, including a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone press ring sealing unit; the cell model loading and culturing unit is constructed by stacking at least three chips up and down; the liquid inflow-outflow unit with a height difference is constructed by respectively opening liquid circulation holes sealed and connected through the embedded inverted cone press ring sealing unit on the at least three chips.

[0008] In some embodiments, corresponding to the same area in the up-down direction, the lowest chip layer is in a concave form, all intermediate chip layers are in a hollowed-out form, and the highest chip layer is in a convex or concave form to jointly construct the cell model loading and culturing unit.

[0009] In some embodiments, the liquid inflow-outflow unit with a height difference includes a first vertical flow channel; corresponding to the first same position in the up-down direction, the lowest chip layer is in a concave form, and the other chip layers are in a hollowed-out form to respectively open first liquid circulation holes to form the first vertical flow channel; the embedded inverted cone press ring sealing unit includes a first inverted cone press ring; the first inverted cone press ring is adapted to be sequentially embedded into the first liquid circulation holes from top to bottom, and the embedded first liquid circulation holes are sealed and connected.

[0010] In some embodiments, the first vertical flow channel communicates with the cell model loading and culturing unit through the first fluid circulation hole of the lowest chip layer.

[0011] In some embodiments, the liquid inflow-outflow unit with a height difference includes a second vertical flow channel; at the second same positions corresponding to the up-down direction, second liquid circulation holes are respectively formed in a hollowed-out manner from any intermediate chip layer to the highest chip layer to form the second vertical flow channel; the embedded inverted cone press ring sealing unit includes a second inverted cone press ring; the second inverted cone press ring is adapted to be sequentially embedded into the second liquid circulation holes from top to bottom, and the embedded second liquid circulation holes are hermetically connected.

[0012] In some embodiments, the second vertical flow channel communicates with the cell model loading and culturing unit through the second fluid circulation holes in its lowest circulation layer.

[0013] In some embodiments, it includes six chip layers; second liquid circulation holes are respectively formed in a hollowed-out manner from the third chip layer to the sixth chip layer to form the second vertical flow channel through the third chip layer to the sixth chip layer.

[0014] In some embodiments, the highest chip layer is detachable.

[0015] In some embodiments, the embedded inverted cone press ring sealing unit is adapted to be externally connected to a go-no-go fitting.

[0016] In some embodiments, it includes at least one microfluidic organ chip module; each microfluidic organ chip module includes the cell model loading and culturing unit, the liquid inflow-outflow unit with a height difference, and the embedded inverted cone press ring sealing unit.

[0017] The present invention adopts the above technical solutions to design the functional units of the microfluidic organ chip, and constructs a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone press ring sealing unit. Among them:

[0018] (1) The cell model loading and culturing unit is constructed by stacking multiple chip layers, and has a large cell culture space. It has good compatibility with three-dimensional cell models of different sizes and shapes, such as liver organoids, lung organoids, heart organoids, cartilage balls, midbrain microaggregates, tumor cell balls, etc., and can also be compatible with the culture of two-dimensional cells growing on a biofilm or microcarriers.

[0019] (2) The liquid inflow-outflow unit with a height difference forms a height difference by separating the liquid inflow and outflow channels on different chip layers, realizes different culture medium inflow / outflow modes, eliminates problems such as uneven concentration of test substances, such as dead volume or local accumulation of solid test substances, and is suitable for accurate analysis and detection of the toxicity or efficacy of soluble or solid test substances.

[0020] (3) The embedded inverted cone compression ring sealing unit can integrate external sealing functional units on a relatively small and limited chip area, which can not only equivalently achieve the function of sealing liquid flow channels, but also reduce the area and space occupied during conventional joint fixing and sealing. At the same time, its structural design can match commercially available general gauge fittings such as inverted cone compression rings and capillaries, thus supporting the expansion requirements and scenarios such as multi-chip series connection or combination with sensors.

[0021] (4) The uppermost chip layer, i.e., the upper cover, can also be disassembled to facilitate the extraction of loaded or cultured cells, so as to further obtain index information that cannot be detected in the culture medium. For example, immunofluorescence staining, pathological index staining, RNA extraction, protein extraction, etc. can be carried out in three-dimensional cells.

[0022] The microfluidic organ chip of the present invention has achieved remarkable breakthroughs in terms of technical performance, application scenarios and operation convenience through the innovative design and systematic integration of functional units. Compared with the prior art, it has at least the following beneficial effects:

[0023] For example, the microfluidic organ chip provided by the present invention improves the compatibility and culture efficiency for diverse cell models through the enlarged chamber volume, and can be compatible with three-dimensional cell models and the efficient culture of two-dimensional cells grown on biofilms or microcarriers. It can simultaneously be compatible with the co-culture of three-dimensional cell models such as large-sized and irregularly shaped organoids and tumor spheroids of the liver, lung, heart, etc., as well as two-dimensional cells, and can cover the requirements of mainstream in vitro toxicology / pharmacology research models.

[0024] For another example, the microfluidic organ chip provided by the present invention realizes different inflow / outflow modes of the culture medium, eliminating problems such as uneven concentration of the test substance, such as dead volume or local accumulation of solid test substances, and is suitable for the precise analysis and detection of the toxicity or efficacy of soluble or solid test substances.

[0025] For another example, the microfluidic organ chip provided by the present invention has a highly reliable miniaturized sealing design. It adopts an embedded inverted cone compression ring sealing unit, which can set more detection units in a smaller chip area and can ensure the sealed environment of the liquid channel. It not only optimizes the sealing structure, reduces the chip volume, but also improves the reliability.

[0026] For another example, the microfluidic organ chip provided by the present invention realizes non-destructive recovery of non-damaged samples of three-dimensional cells and two-dimensional cells through the detachable upper cover design. It is not only easy to operate, but also can be seamlessly docked with downstream effect observation and detection analysis, such as live / dead cell staining, pathological sectioning, RNA or protein extraction, etc.

[0027] For another example, the microfluidic organ chip provided by the present invention also has the functions of modular expansion and compatibility, and can be paired with commercially available general-purpose accessories such as capillary tubes and inverted cone connectors, so as to expand application scenarios and requirements. For example, it can be extended to scenarios where a micropump is used in combination with a detection instrument or chips are connected in series, and has good compatibility, expandability, and adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a schematic diagram of each chip layer of the microfluidic organ chip provided by the embodiment of the present invention;

[0029] Figure 2 is a partial side view of the highest chip layer of the microfluidic organ chip provided by the embodiment of the present invention;

[0030] Figure 3 is a schematic diagram of the embedded inverted cone compression ring sealing unit of the microfluidic organ chip provided by the embodiment of the present invention;

[0031] Figure 4 is an actual assembly diagram of the microfluidic organ chip provided by the embodiment of the present invention; among them, sub- Figure 4 A is a schematic diagram of the actual object of the cell model loading and culturing unit; sub- Figure 4 B is a schematic diagram of the operation on the machine after the embedded inverted cone compression ring sealing unit is assembled on the cell model loading and culturing unit; the blue part is a commercially available general-purpose inverted cone compression ring with an inner diameter of 1600 μm; the pink liquid is the culture medium of the three-dimensional cell model, and it flows in through the K1 hole and out through the K2 hole by using a capillary tube with an outer diameter of 1600 μm. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The present invention provides a microfluidic organ chip for three-dimensional cell culture.

[0033] Specifically, through the design of functional modules, the microfluidic organ chip constructs a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone compression ring sealing unit, and drives the circulation of the culture medium by a microfluidic driving peripheral device.

[0034] In a specific implementation, the cell model loading and culturing unit is constructed by stacking at least three chips up and down; the liquid inflow-outflow unit with a height difference is constructed by respectively opening liquid circulation holes on at least three chips that are suitable for being sealed and connected by the embedded inverted cone compression ring sealing unit.

[0035] In some embodiments, the microfluidic organ chip may include at least one microfluidic organ chip module. Moreover, each microfluidic organ chip module includes a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone compression ring sealing unit.

[0036] For example, the microfluidic organ-on-a-chip may include four microfluidic organ-on-a-chip modules. Moreover, each microfluidic organ-on-a-chip module includes a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone pressing ring sealing unit.

[0037] In some embodiments, corresponding to the same region in the up-down direction, for example, the vertical direction, the lowest chip layer is in a concave form, all intermediate chip layers are in a hollowed-out form, and the highest chip layer is in a convex or concave-up form to jointly construct the cell model loading and culturing unit.

[0038] By way of example and not limitation, in some embodiments, the microfluidic organ-on-a-chip includes six chip layers stacked in sequence from bottom to top. In this case, corresponding to the same region in the vertical direction, the first chip layer at the lowest layer is in a concave form, the second to fifth chip layers in the intermediate layers are in a hollowed-out form, and the sixth chip layer at the highest layer is in a convex or concave-up form to jointly construct the cell model loading and culturing unit.

[0039] In some embodiments, the liquid inflow-outflow unit with a height difference includes a first vertical flow channel. Correspondingly, at the first same position in the up-down direction, for example, the vertical direction, the first chip layer is in a concave form, and the other chip layers are in a hollowed-out form to respectively form first liquid circulation holes suitable for constructing the first vertical flow channel. At the same time, the embedded inverted cone pressing ring sealing unit includes a first inverted cone pressing ring. Moreover, the first inverted cone pressing ring is adapted to be sequentially embedded into the first liquid circulation holes from top to bottom and make the embedded first liquid circulation holes in sealed connection.

[0040] By way of example and not limitation, in some embodiments, the microfluidic organ-on-a-chip includes six chip layers stacked in sequence from bottom to top. In this case, at the first same position in the vertical direction, the first chip layer is in a concave form, and the second to sixth chip layers are in a hollowed-out form to respectively form first liquid circulation holes suitable for constructing the first vertical flow channel. Correspondingly, the embedded inverted cone pressing ring sealing unit includes a first inverted cone pressing ring. Moreover, the first inverted cone pressing ring is adapted to be sequentially embedded into the first liquid circulation holes from top to bottom and make the embedded first liquid circulation holes in sealed connection.

[0041] In some embodiments, the first inverted cone pressing ring is adapted to be sequentially embedded into all the first liquid circulation holes from top to bottom and make all the first liquid circulation holes in sealed connection to be suitable for constructing the first vertical flow channel.

[0042] In a further embodiment, the outer diameter of the first inverted conical pressing ring decreases layer by layer from top to bottom. Correspondingly, the aperture diameters of the first liquid flow holes of the highest to the lowest chip layers, for example, the sixth to the first chip layers, also decrease layer by layer and match the outer diameter of the first inverted conical pressing ring. In this way, when the first inverted conical pressing ring is sequentially inserted into all the first liquid flow holes from top to bottom, it is suitable for sealingly connecting all the first liquid flow holes to construct the first vertical flow channel.

[0043] In some other embodiments, the first inverted conical pressing ring is also suitable for being sequentially inserted into the first liquid flow holes of the highest chip layer to any intermediate chip layer, for example, the sixth chip layer to the fourth chip layer, from top to bottom, and for sealingly connecting the first liquid flow holes embedded along the vertical direction, for example, sealingly connecting the first liquid flow holes of the sixth chip layer to the fourth chip layer embedded.

[0044] In a further embodiment, the outer diameter of the first inverted conical pressing ring decreases layer by layer from top to bottom. Correspondingly, the aperture diameters of the first liquid flow holes of the highest chip layer to any intermediate chip layer, for example, the sixth chip layer to the fourth chip layer, into which the first inverted conical pressing ring is inserted, also decrease layer by layer and match the outer diameter of the first inverted conical pressing ring. In this way, when the first inverted conical pressing ring is sequentially inserted into the first liquid flow holes of the highest chip layer to any intermediate chip layer, for example, the sixth chip layer to the fourth chip layer, from top to bottom, it is suitable for sealingly connecting these first liquid flow holes into which it is inserted.

[0045] In a still further embodiment, in the experiment, a capillary tube passes through the middle of the first inverted conical pressing ring and spans from the highest chip layer to the second chip layer. In this case, the aperture diameter of the first fluid flow hole of the first chip layer is larger than the inner diameter of the capillary tube used in the experiment and smaller than its outer diameter, while the aperture diameters of the first liquid flow holes of the other chip layers not inserted with the first inverted conical pressing ring, for example, the third to the second chip layers, are equal to the inner diameter of the first inverted conical pressing ring and equivalent to the outer diameter of the capillary tube used in the experiment.

[0046] In this way, it is suitable for jointly constructing the first vertical flow channel through the first liquid flow holes of all the chip layers provided with the first liquid flow holes, for example, the sixth to the first chip layers.

[0047] In some embodiments, the first vertical flow channel communicates with the cell model loading and culturing unit through the first fluid flow hole of the first chip layer.

[0048] In some embodiments, the first chip layer is also provided with a first chip layer horizontal flow channel in a concave form, and the first chip layer horizontal flow channel communicates the cell model loading and culturing unit and the first vertical flow channel of the liquid inflow-outflow unit with a height difference.

[0049] In some embodiments, the liquid inflow-outflow unit with a height difference further includes a second vertical flow channel, and the lowest flow-through layer of the second vertical flow channel is lower than that of the first vertical flow channel to form a height difference between the first vertical flow channel and the second vertical flow channel. Correspondingly, at the second same position corresponding to the up-down direction, for example, the vertical direction, second liquid flow holes are respectively formed in a hollowed-out manner from any intermediate chip layer to the highest chip layer to be suitable for constructing the second vertical flow channel. At the same time, the embedded inverted cone pressing ring sealing unit further includes a second inverted cone pressing ring. And the second inverted cone pressing ring is suitable for being sequentially embedded into the second liquid flow holes from top to bottom and making the embedded second liquid flow holes be in sealed connection.

[0050] In specific implementation, any intermediate chip layer includes any one of all chip layers except the lowest chip layer and the highest chip layer.

[0051] By way of example and not limitation, in some embodiments, the microfluidic organ chip includes six chip layers stacked in sequence from bottom to top. In this case, at the second same position corresponding to the vertical direction, second liquid flow holes are respectively formed in a hollowed-out manner from any intermediate chip layer in the second to fifth chip layers to the sixth chip layer. At the same time, the second inverted cone pressing ring is sequentially embedded into the second liquid flow holes from top to bottom and makes the embedded second liquid flow holes be in sealed connection.

[0052] In specific implementation, any intermediate chip layer in the second to fifth chip layers includes the second chip layer, or the third chip layer, or the fourth chip layer, or the fifth chip layer. That is, second liquid flow holes can be respectively formed in a hollowed-out manner from the second chip layer to the sixth chip layer, or from the third chip layer to the sixth chip layer, or from the fourth chip layer to the sixth chip layer, or from the fifth chip layer to the sixth chip layer.

[0053] In some embodiments, the second inverted cone pressing ring is suitable for being sequentially embedded into all the second liquid flow holes from top to bottom and making all the second liquid flow holes be in sealed connection to be suitable for constructing the second vertical flow channel.

[0054] In a further embodiment, the outer diameter of the second inverted cone pressing ring decreases layer by layer from top to bottom. Correspondingly, the pore diameters of the second liquid flow holes of all chip layers with the second liquid flow holes formed therein, for example, the second to sixth chip layers, also decrease layer by layer and match the outer diameter of the second inverted cone pressing ring. In this way, when the second inverted cone pressing ring is sequentially embedded into the second liquid flow holes of all chip layers with the second liquid flow holes formed therein from top to bottom, it is suitable for sealingly connecting these second liquid flow holes to construct the second vertical flow channel.

[0055] In some other embodiments, the second inverted conical pressing ring is further adapted to be sequentially embedded into the second liquid circulation holes of all the chip layers provided with the second liquid circulation holes, excluding the lowest chip layer, from top to bottom, and to seal-connect the second liquid circulation holes of these chip layers embedded in the vertical direction. For example, the microfluidic organ chip includes six chip layers stacked in sequence from bottom to top, and the second to sixth chip layers are all provided with second liquid circulation holes in a hollowed-out form. The second inverted conical pressing ring is adapted to be sequentially embedded into the second liquid circulation holes of the sixth to fourth chip layers from top to bottom, and to seal-connect the second liquid circulation holes of the sixth to fourth chip layers embedded.

[0056] In a further embodiment, the outer diameter of the second inverted conical pressing ring decreases layer by layer from top to bottom. Correspondingly, the diameters of the second liquid circulation holes of these chip layers embedded with the second inverted conical pressing ring, such as the second to fourth chip layers, also decrease layer by layer from top to bottom and match the outer diameter of the second inverted conical pressing ring. In this way, when the second inverted conical pressing ring is sequentially embedded into the second liquid circulation holes of these chip layers, such as the sixth to fourth chip layers, from top to bottom, it is adapted to seal-connect the second liquid circulation holes it is embedded in.

[0057] In a still further embodiment, during the experiment, a capillary tube passes through the middle of the second inverted conical pressing ring and spans all the chip layers provided with the second liquid circulation holes, excluding the lowest chip layer, such as spanning the sixth to fourth chip layers. In this case, the diameter of the second fluid circulation hole of the lowest chip layer provided with the second liquid circulation hole, such as the third chip layer, is larger than the inner diameter of the capillary tube used in the experiment and smaller than its outer diameter.

[0058] In this way, it is adapted to jointly construct a second vertical flow channel through the second liquid circulation holes of all the chip layers provided with the second liquid circulation holes, such as the sixth to third chip layers.

[0059] In some embodiments, the second vertical flow channel communicates with the cell model loading and culturing unit through the second fluid circulation hole of its lowest circulation layer.

[0060] By way of example and not limitation, in some embodiments, the microfluidic organ chip includes six chip layers stacked in sequence from bottom to top, and corresponding to the second same position in the vertical direction, the second to sixth chip layers are all provided with second liquid circulation holes in a hollowed-out form respectively. In this case, the second vertical flow channel can communicate with the cell model loading and culturing unit through the second fluid circulation hole of the third chip layer.

[0061] In some embodiments, the lowest flow layer with the second liquid flow holes, such as the third chip layer, is also provided with a third chip layer horizontal flow channel in a concave form, and the third chip layer horizontal flow channel communicates with the cell model loading and culturing unit and the second vertical flow channel of the liquid inflow-outflow unit with a height difference.

[0062] In some embodiments, the embedded inverted cone pressing ring sealing unit includes a first inverted cone pressing ring and a second inverted cone pressing ring, both of which are adapted to be externally connected to a gauge fitting such as an inverted cone pressing ring, or a capillary tube, etc. Thus, it can support the expansion requirements and scenarios such as multi-chip series connection, or combination with a sensor, etc., for example, it can be extended to the scenario of combination with a micropump and a detection instrument.

[0063] In some embodiments, the top chip layer is detachable. Thus, it is convenient to take out the loaded or cultured cells, so as to further obtain the index information that cannot be detected in the culture solution, and seamlessly dock with the downstream effect observation and detection analysis, such as immunofluorescence staining, pathological index staining, RNA extraction, protein extraction, etc. in three-dimensional cells.

[0064] To make the objectives, features, and beneficial effects of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It can be understood that the specific embodiments described below are only used to explain the present invention, rather than to limit the present invention. And, in the drawings, the same or similar reference numerals may be used to refer to the same or similar elements in different embodiments, and the description of the same or similar elements in different embodiments and the description of the prior art elements, features, effects, etc. may also be omitted.

[0065] Moreover, in the embodiments of the present invention, the flow channel refers to a strip-shaped groove on the chip surface for fluid flow, and its width, length, internal depth, and slope can be specifically designed according to needs. The upper and lower surfaces can also be adhered by means of buckling, pressing, auxiliary fixing, sealing after chemical treatment, etc. between multiple chip layers. The culturing unit refers to a groove structure formed between chip layers, and its shape, width, internal depth, and slope can also be designed and selected according to needs.

[0066] Referring to Figures 1 to 4 , an embodiment of the present invention provides a microfluidic organ chip for three-dimensional cell culture.

[0067] Referring to Figure 1 , the microfluidic organ chip constructs a cell model loading and culturing unit through six chips. Among them, the first chip layer 101, the second chip layer 102, the third chip layer 103, the fourth chip layer 104, the fifth chip layer 105, and the sixth chip layer 106 are stacked in sequence from bottom to top.

[0068] In the embodiments of the present invention, for the microfluidic organ chip, except for the sixth chip layer 106, i.e., the upper cover, the other layers can be combined with conventional bonding and sealing methods according to their respective requirements to achieve the sealing of the cell model loading and culturing units and ensure the smooth flow of the culture medium.

[0069] The sixth chip layer 106 of the microfluidic organ chip designed in the embodiments of the present invention, i.e., the upper cover, can be disassembled by using conventional technical means, so as to facilitate cell recovery and the reuse of the chip body.

[0070] Each chip layer of the microfluidic organ chip designed in the embodiments of the present invention can be manufactured and processed by using raw materials such as transparent or semi-transparent PC, PMMA, PS, etc. Moreover, when the microfluidic organ chip is used for pharmacological or toxicological research, it is not recommended to use PDMS as the raw material for processing, because PDMS has a strong adsorption capacity for compounds, making it difficult to ensure the consistency of the given concentration of compounds in toxicity tests.

[0071] In the embodiments of the present invention, the microfluidic organ chip includes four microfluidic organ chip modules. Moreover, each microfluidic organ chip module includes a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone compression ring sealing unit.

[0072] In specific implementation, each of the four microfluidic organ chip modules includes a cell model loading and culturing unit, namely the first cell model loading and culturing unit A1, the second cell model loading and culturing unit A2, the third cell model loading and culturing unit A3, and the fourth cell model loading and culturing unit A4.

[0073] The first cell model loading and culturing unit A1, the second cell model loading and culturing unit A2, the third cell model loading and culturing unit A3, and the fourth cell model loading and culturing unit A4 are all constructed by stacking the first chip layer 101, the second chip layer 102, the third chip layer 103, the fourth chip layer 104, the fifth chip layer 105, and the sixth chip layer 106 in sequence from bottom to top, and the sixth chip layer 106 can be independently used as the upper cover of each cell model loading and culturing unit A1 - A4.

[0074] The first chip layer 101 is the bottom layer of each cell model loading and culturing unit A1 - A4, and it constructs the bottom regions of each cell model loading and culturing unit A1 - A4 in a concave form.

[0075] The first chip layer 101 also constructs four first-chip liquid circulation holes a1-a4 and four first-chip liquid flow channels a5-a8 in a concave form respectively. Among them, the four first-chip liquid circulation holes a1-a4 belong to four microfluidic organ chip modules respectively, and are all liquid circulation holes suitable for vertically connecting to the upper chip layer, i.e., the second chip layer 102. The four first-chip liquid flow channels a5-a8 also belong to four microfluidic organ chip modules respectively, and are all liquid flow channels suitable for horizontally connecting to the corresponding first-chip liquid circulation holes a1-a4.

[0076] In a specific implementation, the bottom regions of the respective cell model loading and culturing units A1-A4 are respectively communicated with the corresponding first-chip liquid circulation holes a1-a4 through the corresponding first-chip liquid flow channels a5-a8. That is, the bottom region of the first cell model loading and culturing unit A1 is communicated with the first-chip liquid circulation hole a1 through the first-chip liquid flow channel a5, the bottom region of the second cell model loading and culturing unit A2 is communicated with the first-chip liquid circulation hole a2 through the first-chip liquid flow channel a6, the bottom region of the third cell model loading and culturing unit A3 is communicated with the first-chip liquid circulation hole a3 through the first-chip liquid flow channel a7, and the bottom region of the fourth cell model loading and culturing unit A4 is communicated with the first-chip liquid circulation hole a4 through the first-chip liquid flow channel a8.

[0077] In some embodiments, the thickness of the first chip layer 101 can be in the range of 500μm - 5000μm.

[0078] In some embodiments, the bottom regions of the respective cell model loading and culturing units A1-A4, the respective first-chip liquid circulation holes a1-a4, and the respective first-chip liquid flow channels a5-a8 can all be formed by being recessed by 1 / 5 - 1 / 2 of the overall thickness of the first chip layer 101.

[0079] In some embodiments, the length of each of the first-chip liquid flow channels a5-a8 can be in the range of 500μm - 100000μm.

[0080] In some embodiments, the aperture of each of the first-chip liquid circulation holes a1-a4 is larger than the inner diameter of the capillary used in the experiment and smaller than its outer diameter. For example, when using a capillary with an outer diameter of 1600μm and an inner diameter of 500μm, the aperture of each of the first-chip liquid circulation holes a1-a4 can be between 500μm - 1600μm.

[0081] The second chip layer 102 is respectively hollowed out at the parts vertically corresponding to the bottom regions of the respective cell model loading and culturing units A1-A4 to locally define the respective cell model loading and culturing units A1-A4.

[0082] The second chip layer 102 is also provided with four second-chip liquid circulation holes b1-b4 in the form of a hollowed-out treatment. The four second-chip liquid circulation holes b1-b4 respectively correspond to the respective first-chip liquid circulation holes a1-a4 in the vertical direction, belong to four microfluidic organ chip modules, and are all liquid circulation holes suitable for vertically connecting to the upper chip layer, i.e., the third chip layer 103.

[0083] In some embodiments, the aperture diameters of the respective second-chip liquid circulation holes b1-b4 can be the same as the outer diameter of the capillary used in the experiment. For example, when using a capillary with an outer diameter of 1600 μm and an inner diameter of 500 μm, the aperture diameters of the respective second-chip liquid circulation holes b1-b4 can be 1600 μm.

[0084] The third chip layer 103 is also subjected to a hollowed-out treatment at the parts corresponding vertically to the bottom regions of the respective cell model loading and culturing units A1-A4, so as to locally define the respective cell model loading and culturing units A1-A4.

[0085] The third chip layer 103 is also provided with four third-chip liquid circulation holes d1-d4 in the form of a hollowed-out treatment. The four third-chip liquid circulation holes d1-d4 respectively correspond to the respective first-chip liquid circulation holes a1-a4 and the respective second-chip liquid circulation holes b1-b4 in the vertical direction, belong to four microfluidic organ chip modules, and are also all liquid circulation holes suitable for vertically connecting to the upper chip layer, i.e., the fourth chip layer 104.

[0086] In some embodiments, the aperture diameters of the respective third-chip liquid circulation holes d1-d4 can be the same as the outer diameter of the capillary used in the experiment. For example, when using a capillary with an outer diameter of 1600 μm and an inner diameter of 500 μm, the aperture diameters of the respective third-chip liquid circulation holes d1-d4 can be 1600 μm.

[0087] The third chip layer 103 is also provided with another four third-chip liquid circulation holes c1-c4 and four third-chip liquid flow channels c5-c8 in the form of a hollowed-out treatment. Among them, the other four third-chip liquid circulation holes c1-c4 are respectively offset from the respective first-chip liquid circulation holes a1-a4 and the respective second-chip liquid circulation holes b1-b4 in the vertical direction, belong to four microfluidic organ chip modules, and are also all liquid circulation holes suitable for vertically connecting to the upper chip layer, i.e., the fourth chip layer 104. The four third-chip liquid flow channels c5-c8 are also respectively offset from the respective first-chip liquid flow channels a5-a8 in the vertical direction, belong to four microfluidic organ chip modules, and are all liquid flow channels suitable for horizontally connecting to the corresponding third-chip liquid circulation holes c1-c4.

[0088] In a specific implementation, the third chip layer 103 is respectively communicated with the corresponding third chip liquid circulation holes c1 - c4 through the corresponding third chip liquid flow channels c5 - c8 in the corresponding areas of each cell model loading and culturing unit A1 - A4. That is, the corresponding area of the first cell model loading and culturing unit A1 is communicated with the third chip liquid circulation hole c1 through the third chip liquid flow channel c5, the corresponding area of the second cell model loading and culturing unit A2 is communicated with the third chip liquid circulation hole c2 through the third chip liquid flow channel c6, the corresponding area of the third cell model loading and culturing unit A3 is communicated with the third chip liquid circulation hole c3 through the third chip liquid flow channel c7, and the bottom area of the fourth cell model loading and culturing unit A4 is communicated with the third chip liquid circulation hole c4 through the third chip liquid flow channel c8.

[0089] In some embodiments, the length of each of the third chip liquid flow channels c5 - c8 can be in the range of 500μm - 100000μm.

[0090] In some embodiments, the pore diameters of the other four third chip liquid circulation holes c1 - c4 can be the same as the outer diameter of the capillary used in the experiment. For example, when using a capillary with an outer diameter of 1600μm and an inner diameter of 500μm, the pore diameters of the other four third chip liquid circulation holes c1 - c4 can be 1600μm.

[0091] The fourth chip layer 104 is also respectively hollowed out at the parts corresponding vertically to the bottom areas of each cell model loading and culturing unit A1 - A4, so as to locally define each cell model loading and culturing unit A1 - A4 respectively.

[0092] The fourth chip layer 104 also opens four fourth chip liquid circulation holes e1 - e4 and another four fourth chip liquid circulation holes f1 - f4 in the form of hollowing out. Among them, the four fourth chip liquid circulation holes e1 - e4 are respectively corresponding to the respective first chip liquid circulation holes a1 - a4, the respective second chip liquid circulation holes b1 - b4, and the respective third chip liquid circulation holes d1 - d4 in the vertical direction, belong to four microfluidic organ chip modules, and are all liquid circulation holes suitable for vertically connecting to the upper chip layer, that is, the fifth chip layer 105. The other four fourth chip liquid circulation holes f1 - f4 are respectively corresponding to the other four third chip liquid circulation holes c1 - c4 in the vertical direction one by one, belong to four microfluidic organ chip modules, and are all liquid circulation holes suitable for vertically connecting to the upper chip layer, that is, the fifth chip layer 105.

[0093] The fifth chip layer 105 is also respectively hollowed out at the parts corresponding vertically to the bottom areas of each cell model loading and culturing unit A1 - A4, so as to locally define each cell model loading and culturing unit A1 - A4 respectively.

[0094] The fifth chip layer 105 is also provided with four fifth-chip liquid circulation holes g1-g4 and another four fifth-chip liquid circulation holes h1-h4 in the form of a hollowed-out process. Among them, the four fifth-chip liquid circulation holes g1-g4 correspond vertically to the respective first-chip liquid circulation holes a1-a4, the respective second-chip liquid circulation holes b1-b4, the respective third-chip liquid circulation holes d1-d4, and the respective fourth-chip liquid circulation holes e1-e4, and belong to four microfluidic organ chip modules, and are all liquid circulation holes suitable for vertically connecting to the upper chip layer, i.e., the sixth chip layer 106. The other four fifth-chip liquid circulation holes h1-h4 correspond vertically to the other four third-chip liquid circulation holes c1-c4 and the other four fourth-chip liquid circulation holes f1-f4, and belong to four microfluidic organ chip modules, and are all liquid circulation holes suitable for vertically connecting to the upper chip layer, i.e., the sixth chip layer 106.

[0095] Refer to Figure 2 , in the sixth chip layer 106, upper covers B1-B4 corresponding to the respective cell model loading and culturing units A1-A4 are respectively constructed in a downwardly protruding form at the parts vertically corresponding to the bottom regions of the respective cell model loading and culturing units A1-A4.

[0096] The sixth chip layer 106 is also provided with four sixth-chip liquid circulation holes i1-i4 and another four sixth-chip liquid circulation holes j1-j4 in the form of a hollowed-out process. Among them, the four sixth-chip liquid circulation holes i1-i4 correspond vertically to the respective first-chip liquid circulation holes a1-a4, the respective second-chip liquid circulation holes b1-b4, the respective third-chip liquid circulation holes d1-d4, the respective fourth-chip liquid circulation holes e1-e4, and the respective fifth-chip liquid circulation holes g1-g4, and belong to four microfluidic organ chip modules. The other four sixth-chip liquid circulation holes j1-j4 correspond vertically to the other four third-chip liquid circulation holes c1-c4, the other four fourth-chip liquid circulation holes f1-f4, and the other four fifth-chip liquid circulation holes h1-h4, and belong to four microfluidic organ chip modules.

[0097] Refer to Figure 3, four first inverted conical pressing rings 201 are respectively embedded from top to bottom into the corresponding sixth chip liquid circulation holes i1 - i4, fifth chip liquid circulation holes g1 - g4, and fourth chip liquid circulation holes e1 - e4 in the vertical direction, and the liquid circulation holes embedded in the vertical direction are sequentially and hermetically connected (that is, the sixth chip liquid circulation hole i1, the fifth chip liquid circulation hole g1, and the fourth chip liquid circulation hole e1 are sequentially and closely connected, the sixth chip liquid circulation hole i2, the fifth chip liquid circulation hole g2, and the fourth chip liquid circulation hole e2 are sequentially and closely connected, the sixth chip liquid circulation hole i3, the fifth chip liquid circulation hole g3, and the fourth chip liquid circulation hole e3 are sequentially and closely connected, the sixth chip liquid circulation hole i4, the fifth chip liquid circulation hole g4, and the fourth chip liquid circulation hole e4 are sequentially and closely connected). Then, through the corresponding sixth chip liquid circulation holes i1 - i4, fifth chip liquid circulation holes g1 - g4, fourth chip liquid circulation holes e1 - e4, third chip liquid circulation holes d1 - d4, second chip liquid circulation holes b1 - b4, and first chip liquid circulation holes a1 - a4 in the vertical direction, four first vertical flow channels K1 are jointly formed. The four first vertical flow channels K1 belong to four liquid inflow - outflow units with a height difference.

[0098] , four second inverted conical pressing rings 202 are respectively embedded from top to bottom into the corresponding sixth chip liquid circulation holes j1 - j4, fifth chip liquid circulation holes h1 - h4, and fourth chip liquid circulation holes f1 - f4 in the vertical direction, and the liquid circulation holes embedded in the vertical direction are sequentially and hermetically connected (that is, the sixth chip liquid circulation hole j1, the fifth chip liquid circulation hole h1, and the fourth chip liquid circulation hole f1 are sequentially and closely connected, the sixth chip liquid circulation hole j2, the fifth chip liquid circulation hole h2, and the fourth chip liquid circulation hole f2 are sequentially and closely connected, the sixth chip liquid circulation hole j3, the fifth chip liquid circulation hole h3, and the fourth chip liquid circulation hole f3 are sequentially and closely connected, the sixth chip liquid circulation hole j4, the fifth chip liquid circulation hole h4, and the fourth chip liquid circulation hole f4 are sequentially and closely connected). Then, through the corresponding sixth chip liquid circulation holes j1 - j4, fifth chip liquid circulation holes h1 - h4, fourth chip liquid circulation holes f1 - f4, and third chip liquid circulation holes c1 - c4 in the vertical direction, four second vertical flow channels K2 are jointly formed. The four second vertical flow channels K2 belong to four liquid inflow - outflow units with a height difference.

[0099] In an embodiment of the present invention, a liquid inflow-outflow unit with a height difference is constructed through a first vertical flow channel K1 and a second vertical flow channel K2. Since the first vertical flow channel K1 and the second vertical flow channel K2 are designed through different chip layers and have different chip layer heights, a height difference is thus achieved.

[0100] Referring to Figure 1 and Figure 3 , the pore diameters of the first vertical flow channel K1 and the second vertical flow channel K2 of each liquid inflow-outflow unit with a height difference are adapted to the inner and outer diameters of the corresponding inverted cone compression ring, and both can be changed layer by layer.

[0101] Taking the first vertical flow channel K1 of the liquid inflow-outflow unit with a height difference corresponding to the first cell model loading and culturing unit A1 as an example. The pore diameters of the sixth chip liquid circulation hole i1, the fifth chip liquid circulation hole g1, the fourth chip liquid circulation hole e1, the third chip liquid circulation hole d1, the second chip liquid circulation hole b1, and the first chip liquid circulation hole a1 can decrease in the direction from top to bottom. Among them, the pore diameter of the sixth chip liquid circulation hole i1 is the outer diameter of the uppermost edge of the first inverted cone compression ring 201, that is, the outer diameter of the widest part of the first inverted cone compression ring 201, the pore diameter of the fifth chip liquid circulation hole g1 is not less than the outer diameter of the widest part of the middle section of the first inverted cone compression ring 201, the pore diameter of the fourth chip liquid circulation hole e1 is the outer diameter of the lowermost edge of the first inverted cone compression ring 201, that is, the outer diameter of the narrowest part of the first inverted cone compression ring 201, the pore diameters of the third chip liquid circulation hole d1 and the second chip liquid circulation hole b1 are the inner diameter of the first inverted cone compression ring 201 and can be equal to the outer diameter of the capillary used in the experiment; and the pore diameter of the first chip liquid circulation hole a1 is greater than the inner diameter of the capillary used in the experiment and less than its outer diameter, so that the end of the capillary does not enter the first chip layer, and only liquid can flow into the first chip layer.

[0102] Furthermore, the total thickness of the fourth to sixth chip layers 104-106 is not less than the total height of the first inverted cone compression ring 201.

[0103] Taking the second vertical flow channel K2 of the liquid inflow-outflow unit with a height difference corresponding to the first cell model loading and culturing unit A1 as an example. The pore diameters of the sixth chip liquid circulation hole j1, the fifth chip liquid circulation hole h1, the fourth chip liquid circulation hole f1, and the third chip liquid circulation hole c1 can decrease in the direction from top to bottom. Among them, the pore diameter of the sixth chip liquid circulation hole j1 is the outer diameter of the uppermost edge of the second inverted cone pressing ring 202, that is, the outer diameter at the widest part of the second inverted cone pressing ring 202. The pore diameter of the fifth chip liquid circulation hole h1 is not less than the outer diameter at the widest part of the middle section of the second inverted cone pressing ring 202. The pore diameter of the fourth chip liquid circulation hole f1 is the outer diameter of the lowermost edge of the second inverted cone pressing ring 202, that is, the outer diameter at the narrowest part of the second inverted cone pressing ring 202. The pore diameter of the third chip liquid circulation hole c1 is greater than the inner diameter of the capillary used in the experiment and less than its outer diameter.

[0104] Furthermore, the total thickness of the fourth to sixth chip layers 104-106 is not less than the total height of the second inverted cone pressing ring 202.

[0105] Taking the first inverted cone pressing ring 201 and the second inverted cone pressing ring 202 as examples of commercially available inverted cone pressing rings with a nominal inner diameter of 1600 μm. When the outer diameter at the widest part of the inverted cone pressing ring is 5000 μm, the outer diameter at the widest part of the middle section is 4000 μm, the outer diameter at the narrowest part is 2360 μm, the thickness of the annular region at the widest part is 1350 μm, and the total height is 3920 μm, the pore diameters of the sixth chip liquid circulation holes i1 and j1 can be 5000 μm, the pore diameters of the fifth chip liquid circulation holes g1 and h1 can be 4000 μm, the pore diameters of the fourth chip liquid circulation holes e1 and f1 can be 2360 μm, the pore diameters of the third chip liquid circulation hole d1 and the second chip liquid circulation hole b1 can be 1600 μm, and the pore diameters of the first chip liquid circulation hole a1 and the third chip liquid circulation hole c1 can be between 500-1600 μm. At the same time, the total thickness of the fourth, fifth, and sixth chip layers 104-106 can be determined to be 4500 μm according to the total height of the inverted cone pressing ring. First, the thickness of the sixth chip layer 106 can be determined to be 1500 μm according to the thickness of the annular region at the widest part of the inverted cone pressing ring, and then the sum of the thicknesses of the fourth chip layer 104 and the fifth chip layer 105 can be 3000 μm.

[0106] Since the microfluidic organ chip provided by the embodiment of the present invention forms a height difference by separating the liquid inflow and outflow channels into different chip layers, realizing different culture medium inflow / outflow modes and eliminating the problem of uneven concentration of the test substance, this microfluidic organ chip can be applied to the accurate analysis and detection of the toxicity or efficacy of soluble or solid test substances.

[0107] The following will respectively describe the liquid circulation usage methods when different states of test substances are added to the culture medium.

[0108] When the test substance added to the culture medium is an insoluble solid or solid state (suspension), in order to avoid the gradual increase in the concentration of the test substance due to the local deposition of the insoluble solid substance, the culture medium can flow into the second vertical flow channel K2 through the capillary and flow out from the first vertical flow channel K1.

[0109] When the test substance added to the culture medium is a soluble substance, the culture medium can flow into the first vertical flow channel K1 through the capillary and flow out from the second vertical flow channel K2, or flow into the second vertical flow channel K2 and flow out from the first vertical flow channel K1.

[0110] Although specific embodiments have been described above, these embodiments are not intended to limit the scope of the present disclosure, even when a single embodiment is described only with respect to specific features. The feature examples provided in the present disclosure are intended for illustration rather than limitation, unless otherwise stated. In specific implementations, according to actual needs and when technically feasible, the technical features of one or more dependent claims can be combined with the technical features of the independent claim, and the technical features from the corresponding independent claims can be combined in any appropriate way rather than only through the specific combinations listed in the claims.

[0111] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. A microfluidic organ-on-a-chip for three-dimensional cell culture, characterized in that, It includes a cell model loading and culturing unit, a liquid inflow-outflow unit with a height difference, and an embedded inverted cone pressing ring sealing unit; the cell model loading and culturing unit is constructed by stacking at least three layers of chips vertically; the liquid inflow-outflow unit with a height difference is constructed by respectively opening liquid circulation holes sealed and connected through the embedded inverted cone pressing ring sealing unit on the at least three layers of chips.

2. The microfluidic organ-on-a-chip according to claim 1, characterized in that, Corresponding to the same area in the up-down direction, the lowest chip layer is in a concave form, all intermediate chip layers are in a hollowed-out form, and the highest chip layer is in a convex or concave form to jointly construct the cell model loading and culturing unit.

3. The microfluidic organ-on-a-chip according to claim 1, wherein The liquid inflow-outflow unit with a height difference includes a first vertical flow channel; corresponding to the first same position in the up-down direction, the lowest chip layer is in a concave form, and the other chip layers are in a hollowed-out form to respectively open first liquid circulation holes to form the first vertical flow channel; the embedded inverted cone pressing ring sealing unit includes a first inverted cone pressing ring; the first inverted cone pressing ring is adapted to be sequentially embedded into the first liquid circulation holes from top to bottom and seal the connected first liquid circulation holes.

4. The microfluidic organ-on-a-chip according to claim 3, wherein The first vertical flow channel communicates with the cell model loading and culturing unit through the first fluid circulation hole of the lowest chip layer.

5. The microfluidic organ-on-a-chip according to claim 1, characterized in that The liquid inflow-outflow unit with a height difference includes a second vertical flow channel; corresponding to the second same position in the up-down direction, from any intermediate chip layer to the highest chip layer, second liquid circulation holes are respectively opened in a hollowed-out form to form the second vertical flow channel; the embedded inverted cone pressing ring sealing unit includes a second inverted cone pressing ring; the second inverted cone pressing ring is adapted to be sequentially embedded into the second liquid circulation holes from top to bottom and seal the connected second liquid circulation holes.

6. The microfluidic organ-on-a-chip according to claim 5, characterized in that, The second vertical flow channel communicates with the cell model loading and culturing unit through the second fluid circulation hole of its lowest circulation layer.

7. The microfluidic organ-on-a-chip according to claim 5, characterized in that It includes six layers of chips; from the third chip layer to the sixth chip layer, second liquid circulation holes are respectively opened in a hollowed-out form to form the second vertical flow channel through the third chip layer to the sixth chip layer.

8. The microfluidic organ-on-a-chip according to any one of claims 1 or 7, characterized in that The highest chip layer is detachable.

9. The microfluidic organ-on-a-chip according to any one of claims 1 or 7, characterized in that, The embedded inverted cone pressing ring sealing unit is adapted to be externally connected to a go-no-go gauge fitting.

10. The microfluidic organ-on-a-chip according to any one of claims 1 or 7, characterized in that, It includes at least one microfluidic organ chip module; each microfluidic organ chip module includes the cell model loading and culturing unit, the liquid inflow-outflow unit with a height difference, and the embedded inverted cone pressing ring sealing unit.

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