Detachable plug-in microfluidic organ-on-a-chip

Abstract

The application provides a detachable plug-in type micro-fluid organ chip, and relates to the field of micro-fluid chips.The application comprises a main chip and two external plugs which are detachably connected with two sides of the main chip.The main chip comprises two splicable half-assembled chip groups, each of which comprises a first layer chip, a second layer chip, a third layer chip, a fourth layer chip and a fifth layer chip which can be stacked and detached in sequence;the second layer chip is further provided with a liquid flow channel;the third layer chip is provided with a liquid flow through hole and an arc surface culture flow channel;in the same half-assembled chip group, the liquid flow channel is connected with the arc surface culture flow channel through the liquid flow through hole.The cell culture flow channel is formed between the two arc surface culture flow channels;each external plug is provided with a total liquid injection hole which is connected with the cell culture flow channel.The micro-fluid organ chip has a flexible structure and an optimized cell culture space.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic chips, in particular to a detachable plug-in type microfluidic organ chip. BACKGROUND

[0002] Microfluidic organ chip is one of the core research directions in the field of biomedical engineering in recent years, which simulates the microenvironment of human organs to provide an in vitro research platform for drug screening, toxicity testing and disease modeling. In the prior art, the typical organ chip design includes: 1) multi-layer microfluidic structure: a PDMS chip prepared by soft lithography technology, integrating microchannels, cell culture chambers and fluid control modules. 2) Luer joint or inverted taper joint sealing scheme: using standard Luer interface to seal and connect the fluid channel with the external conduit. 3) Static or simple dynamic culture mode: periodic perfusion of culture solution is achieved by gravity-driven or pump-controlled system.

[0003] In the prior art, microfluidic organ chips usually adopt a fixed structure to realize cell culture and liquid flow through microfluidic channels. Common microfluidic organ chips include liver chips, lung chips, heart chips, etc. These chips are usually stacked by multiple layers of materials, with microfluidic channels and cell culture units designed inside. Therefore, the existing microfluidic organ chip has the following significant limitations:

[0004] 1. Fixed structure limits flexibility: The existing microfluidic organ chip is mostly of fixed structure, which cannot be flexibly adjusted and assembled according to experimental requirements, limiting its application in various experimental scenarios.

[0005] 2. Limited cell culture space: The cell culture space of the existing chip is small, which is difficult to meet the needs of three-dimensional cell culture, and the liquid flow channel design is not optimized, affecting the cell culture efficiency and the stability of the growth environment.

[0006] 3. Sealing structure occupies large space: The existing chip usually adopts fixed sealing structures such as Luer joint, which occupies a large area and space, limiting the compactness and portability of the chip.

[0007] 4. Insufficient ability to simulate the physiological structure of hollow structure of tissues and organs: The existing chip has insufficient ability to simulate the physiological structure of hollow tubular structure of human body, which is difficult to provide flexible culture space, limiting its application in three-dimensional cell culture.

[0008] In summary, there is an urgent need to provide a microfluidic organ chip with flexible structure and optimized cell culture space. SUMMARY

[0009] To solve the above problems, the present application provides a detachable plug-in type microfluidic organ chip, which has flexible structure and optimized cell culture space.

[0010] The detachable plug-in microfluidic organ chip provided by the application comprises a main chip and two external plug-ins detachably connected with two sides of the main chip, the main chip comprises two splicable half-assembly chip sets, each half-assembly chip set comprises a first layer chip, a second layer chip, a third layer chip, a fourth layer chip and a fifth layer chip which can be stacked and detached in sequence, the first layer chip is provided with a first through hole, the second layer chip is provided with a second through hole and a positive convex positioning unit, and the second layer chip is further provided with a liquid flow channel, the third layer chip is provided with a third through hole, a liquid flow through hole and a positive convex positioning hole, and the third layer chip is further provided with an arc surface culture flow channel, the fourth layer chip is provided with a fourth through hole and a positive convex positioning hole, the fifth layer chip is provided with a fifth through hole and a positive convex positioning hole, in the same half-assembly chip set, the liquid flow channel is communicated with the arc surface culture flow channel through the liquid flow through hole, the liquid flow channel is further communicated with the second through hole and the first through hole in another half-assembly chip set through the third through hole, the fourth through hole and the fifth through hole in the same half-assembly chip set, the positive convex positioning unit is detachably connected with each positive convex positioning hole in the other half-assembly chip set, and the arc surface culture flow channels between the two arc surface culture flow channels form a cell culture flow channel.

[0011] In an implementable embodiment, the arc angle α of the arc surface culture flow channel is 60-150°.

[0012] In an implementable embodiment, each external plug-in is provided with a side convex positioning unit, both sides of the second layer chip, the third layer chip, the fourth layer chip and the fifth layer chip are provided with side convex positioning grooves, the side convex positioning grooves on the same side form a side convex positioning hole, and the side convex positioning unit is detachably connected with the side convex positioning hole.

[0013] In an implementable embodiment, each external plug-in comprises a plug-in chip and a plurality of variable-diameter chips connected with the plug-in chip in sequence, the side convex positioning unit is arranged on the plug-in chip, the plug-in chip is further provided with a first liquid injection hole, each variable-diameter chip is provided with a second liquid injection hole, and the first liquid injection hole and the plurality of second liquid injection holes form a total liquid injection hole.

[0014] In an implementable embodiment, the closer to the second liquid injection hole of the plug-in chip, the smaller the diameter, and the diameter of the first liquid injection hole is smaller than the diameter of the smallest second liquid injection hole.

[0015] In an implementable embodiment, the number of the variable-diameter chips is 3, the diameter of the second liquid injection hole farthest from the plug-in chip is 5000 mu m, the diameter of the second liquid injection hole at the middle position is 4000 mu m, the diameter of the second liquid injection hole closest to the plug-in chip is 2360 mu m, the diameter of the first liquid injection hole is 1600 mu m; and / or, the thickness of the variable-diameter chip farthest from the plug-in chip is greater than or equal to 1350 mu m, and the sum of the thicknesses of the remaining variable-diameter chips and the plug-in chip is 2570 mu m.

[0016] In an implementable embodiment, the diameters of the third through hole and the fourth through hole are the same, the diameters of the first through hole, the second through hole and the fifth through hole gradually decrease, and the diameters of the third through hole and the fourth through hole are smaller than the diameter of the fifth through hole.

[0017] In an implementable embodiment, the number of the liquid flow holes is 4, and the intervals between adjacent liquid flow holes are the same.

[0018] The application also provides an assembly method of the detachable plug-in microfluidic organ chip, characterized in that:

[0019] Step S1) two half-chip assemblies are assembled respectively;

[0020] Step S2) the two half-chip assemblies are assembled into a main chip in a central symmetric manner;

[0021] Step S3) two external plug-ins are installed on the two sides of the main chip.

[0022] The application also provides a cell culture method of the detachable plug-in microfluidic organ chip, characterized in that:

[0023] Step T1) cells not subjected to static culture are inoculated into the arc-shaped culture flow channel and subjected to static culture until the cell fusion degree reaches the experimental requirement; or, three-dimensional cells subjected to static culture are directly inoculated into the arc-shaped culture flow channel.

[0024] Step T2) the main chip and the external plug-in are assembled;

[0025] Step T3) the microfluidic peripheral device is used to push the culture liquid to flow in the cell culture flow channel, so as to perform dynamic culture of the cells.

[0026] The detachable plug-in microfluidic organ chip provided by the application has the following beneficial effects:

[0027] 1) The main chip and the external plug-in of the present application are splicing structures, and the main chip and the external plug-in can be detachably connected through multiple layers of interlaced hierarchical structure. Thus, it can be flexibly assembled according to experimental requirements, or form a sealed cell culture functional unit, or be used alone. This design not only improves the convenience of operation, but also enhances the multifunctionality of the microfluidic organ chip, facilitates the connection of other downstream chips or functional structure units, improves the overall performance of the microfluidic organ chip, and provides great convenience for experimental expansion, so that the same microfluidic organ chip can be used in various experimental scenarios.

[0028] 2) The main chip of the present application is provided with a splicing type cell culture flow channel, which can meet the requirements of planar culture and adapt to the placement of three-dimensional cells, and is designed with two different liquid flow modes. The combination of space and function enables the present application to meet the needs of cell culture and nutrition supply, improves the culture efficiency and the stability of the cell growth environment, and can adapt to two-dimensional and three-dimensional cell models such as liver organ, lung organ, heart organ, cartilage ball, midbrain micro-cluster, tumor cell ball, etc., and can also be compatible with two-dimensional cells cultured on biological membranes.

[0029] (3) Further, the present application is designed with multiple through holes corresponding to the external sealing unit, and the sealing unit is not directly designed in the microfluidic organ chip. Thus, the external sealing functional unit can be integrated on a small and limited microfluidic organ chip area, reducing the area and space occupied by the conventional luer fitting fixation and sealing. It is small, flexible and easy to assemble, and can achieve equivalent sealing.

[0030] (4) Further, the arc surface culture flow channel in the present application has an arc, which is easier to assemble into a hollow structure and can better simulate the hollow tubular structure in the body. When used for culturing three-dimensional cells, the hollow cell culture flow channel can provide a more flexible culture space, so that multiple diameter or depth cell culture area units do not need to be set. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a schematic diagram of the overall structure of the present application.

[0032] Figure 2 It is an explosion diagram of the overall structure of the present application.

[0033] Figure 3 It is an explosion diagram of the semi-assembled chip set of the present application.

[0034] Figure 4 It is an explosion diagram of the external plug-in of the present application.

[0035] Figure 5 It is a top view of the present application.

[0036] Figure 6 A side view of the present application. Figure 2 A sectional view in the direction of B-B of the present application.

[0037] Figure 7 A sectional view in the direction of C-C of the present application. Figure 2

[0038] Figure 8 A side view of the present application.

[0039] Figure 9 A sectional view in the direction of A-A of the present application. Figure 5

[0040] Figure 10 A schematic view of the curvature of the arc culture flow channel in the present application.

[0041] Reference numerals

[0042] Main chip 100

[0043] Semi-assembly chip set 1

[0044] First layer chip 11

[0045] Second layer chip 12

[0046] Liquid flow channel 12.1

[0047] Third layer chip 13

[0048] Liquid flow through hole 13.1

[0049] Arc culture flow channel 13.2

[0050] Fourth layer chip 14

[0051] Fifth layer chip 15

[0052] Total through hole 16

[0053] First through hole 16.1

[0054] Second through hole 16.2

[0055] Third through hole 16.3

[0056] Fourth through hole 16.4

[0057] Fifth through hole 16.5

[0058] External connector 2

[0059] Plug-in chip 21

[0060] Variable diameter chip 22

[0061] ​​total liquid inlet hole 23

[0062] first liquid inlet hole 23.1

[0063] second liquid inlet hole 23.2

[0064] positive convex positioning unit 3

[0065] positive convex positioning hole 4

[0066] cell culture flow channel 5

[0067] lateral convex positioning unit 6

[0068] lateral convex positioning hole 7

[0069] lateral convex positioning groove 71 DETAILED DESCRIPTION

[0070] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application. In the description of the present application, it should be noted that the terms "left side", "right side", "upper side", "lower side", "upper", "lower" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first" and "second" are only for the purpose of description, and cannot be understood as indicating or implying relative importance.

[0071] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0072] In addition, in the description of the present application, unless otherwise specified, the meaning of "multiple" is two or more.

[0073] The embodiments of the present application provide a detachable plug-in type microfluidic organ chip, which is described with reference to Figure 1, including a main chip 100 and two external plug-in units 2 respectively detachably connected with two sides of the main chip 100, the main chip 100 including two splicable half-assembly chip sets 1. Refer to Figure 2 or Figure 3 Each of the half-assembly chip sets 1 includes first-layer chips 11, second-layer chips 12, third-layer chips 13, fourth-layer chips 14 and fifth-layer chips 15 which can be stacked and detached in sequence, the overall shape of the half-assembly chip set 1 after stacking all the layer chips in the half-assembly chip set 1 being L-shaped, and one half-assembly chip set 1 being right-side-up and the other half-assembly chip set 1 being upside-down when assembling the two half-assembly chip sets 1 into the main chip 100. Refer to Figure 3 The first-layer chips 11 are provided with first through holes 16.1, the second-layer chips 12 are provided with second through holes 16.2 and right convex positioning units 3, the second-layer chips 12 are further provided with liquid flow channels 12.1, the third-layer chips 13 are provided with third through holes 16.3, liquid flow through holes 13.1 and right convex positioning holes 4, the third-layer chips 13 are further provided with arc surface culture flow channels 13.2, the fourth-layer chips 14 are provided with fourth through holes 16.4 and right convex positioning holes 4, and the fifth-layer chips 15 are provided with fifth through holes 16.5 and right convex positioning holes 4.

[0074] Next, in some examples, half-assembly chip set one and half-assembly chip set two will be mentioned, which are the aliases of the two half-assembly chip sets 1, only different in names, and completely consistent in structures, which is hereby stated. Continue to refer to Figure 3 Meanwhile, refer to Figure 9 In the same group of the half-assembly chip sets 1, the liquid flow channels 12.1 are communicated with the arc surface culture flow channels 13.2 through the liquid flow through holes 13.1, for example, in the half-assembly chip set one, when the second-layer chips 12 and the third-layer chips 13 are stacked together, the liquid flow through holes 13.1 are aligned with the liquid flow channels 12.1, and the bottoms of the third-layer chips 13 can seal the liquid flow channels 12.1, and the arc surface culture flow channels 13.2 are communicated with the liquid flow channels 12.1 only through the liquid flow through holes 13.1. Continue to refer to Figure 3 Meanwhile, refer to Figure 6 and Figure 7, the liquid flow channel 12.1 is also connected with the second through hole 16.2 and the first through hole 16.1 in the other group of semi-assembled chip sets 1 through the third through hole 16.3, the fourth through hole 16.4 and the fifth through hole 16.5 in the same group of semi-assembled chip sets 1, for example, the third through hole 16.3, the fourth through hole 16.4 and the fifth through hole 16.5 in the first group of semi-assembled chips and the second through hole 16.2 and the first through hole 16.1 in the second group of semi-assembled chips are aligned, so that the liquid flow channel 12.1 can be connected with the second through hole 16.2 and the first through hole 16.1 in the second group of semi-assembled chips through the third through hole 16.3, the fourth through hole 16.4 and the fifth through hole 16.5 to form a complete total through hole 16, the function of the total through hole 16 is to enable the external sealing joint to enter the main chip 100, thereby avoiding the design of a sealing unit directly in the chip, and the liquid flowing out of the external sealing joint can be directly injected into the liquid flow channel 12.1, and then enters the arc surface culture flow channel 13.2 through the liquid flow hole 13.1, that is, enters the cell culture flow channel 5, the formation of the cell culture flow channel 5 will be described below. Continue to refer to Figure 3 , and refer to Figure 6 , the positive convex positioning unit 3 is detachably connected with each positive convex positioning hole 4 in the other group of semi-assembled chip sets 1, for example, the positive convex positioning unit 3 in the first group of semi-assembled chips can pass through each positive convex positioning hole 4 in the second group of semi-assembled chips in turn, and vice versa, the positive convex positioning unit 3 is in the shape of a column, preferably an elliptical column, the diameters of each positive convex positioning hole 4 are the same as the diameter of the positive convex positioning unit 3, so as to generate sufficient friction force to firmly splice the positive convex positioning unit 3 with each positive convex positioning hole 4, thereby realizing the splicing between the first group of semi-assembled chips and the second group of semi-assembled chips except for the first layer of chips 11 (the level chips refer to the first layer of chips 11, the second layer of chips 12, the third layer of chips 13, the fourth layer of chips 14 and the fifth layer of chips 15), and then the first layer of chips 11 is connected with the corresponding second layer of chips 12 by adhesion or other equivalent mounting methods. Refer to Figure 7 , the cell culture flow channel 5 is formed between the two arc surface culture flow channels 13.2, specifically, the width of the third layer of chips 13 is greater than that of the fourth layer of chips 14 and the fifth layer of chips 15, and the part with the greater width is provided with the arc surface culture flow channel 13.2, after the two groups of semi-assembled chips 1 are spliced into the main chip 100, the two arc surface culture flow channels 13.2 are oppositely arranged, and the side walls between the two arc surface culture flow channels 13.2 are ingeniously used as the side walls of the fourth layer of chips 14, so that the closed space between the two arc surface culture flow channels 13.2 and the two side walls of the fourth layer of chips 14 can form a hollow cell culture flow channel 5, in some cases, the size of the cell culture flow channel 5 can also be expanded by increasing the number of the fourth layer of chips 14. Continue to refer toFigure 1 With reference to Figure 2 and Figure 4 Each of the external plug-in 2 is provided with a total liquid injection hole 23, which can communicate with the cell culture flow channel 5. After the half-chip group and the half-chip group are assembled into the main chip 100, the cell culture flow channel 5 is aligned with the total liquid injection hole 23, so that the total liquid injection hole 23 is in communication with the cell culture flow channel 5. In detail, the cell culture flow channel 5 penetrates the main chip 100, and the two ports thereof are respectively located on the two sides of the main chip 100. The total liquid injection hole 23 in the two external plug-ins 2 is respectively aligned with the two ports of the cell culture flow channel 5, so that the total liquid injection hole 23 is in communication with the cell culture flow channel 5.

[0075] In the detachable plug-in microfluidic organ chip provided by the embodiment of the present application, with reference to Figure 7 , the curvature α of the curved surface culture flow channel 13.2 is 60-150°, and the angle of the curved surface is measured by the included angle of the tangent vectors of the two curves, which can be seen from Figure 10 .

[0076] In the detachable plug-in microfluidic organ chip provided by the embodiment of the present application, with reference to Figure 4 , and with reference to Figure 2 and Figure 3 Each of the external plug-in 2 is provided with a side protruding positioning unit 6, and the second layer chip 12, the third layer chip 13, the fourth layer chip 14 and the fifth layer chip 15 are respectively provided with a side protruding positioning groove 71 on the two sides. The side protruding positioning grooves 71 on the same side form a side protruding positioning hole 7, and the side protruding positioning unit 6 is detachably connected with the side protruding positioning hole 7. Preferably, the shape of the side protruding positioning unit 6 is an oval column, and correspondingly, the side protruding positioning groove 71 on the second layer chip 12 is a curved groove. In a specific implementation, with reference to Figure 4 , and with reference to Figure 6 , the external plug-in 2 is provided with two side protruding positioning units 6, and correspondingly, the second layer chip 12, the third layer chip 13, the fourth layer chip 14 and the fifth layer chip 15 are respectively provided with two side protruding positioning grooves 71 on the two sides, that is, each side of the main chip 100 can form two side protruding positioning holes 7. The two side protruding positioning holes 7 on the same side cooperate with the two side protruding positioning units 6 on the same external plug-in 2, which is similar to the relationship between a two-hole socket and a two-head plug. Generally, the size of the corresponding side protruding positioning unit 6 matches the size of the side protruding positioning hole 7, so as to generate sufficient friction force to splice the external plug-in 2 and the main chip 100 together.

[0077] Further, with reference to Figure 4Each of the external connectors 2 comprises a plug-in chip 21 and a plurality of variable-diameter chips 22 connected in sequence with the plug-in chip 21, the side convex positioning unit 6 is arranged on the plug-in chip 21, and the plug-in chip 21 is further provided with a first liquid injection hole 23.1, each of the variable-diameter chips 22 is provided with a second liquid injection hole 23.2, when the plug-in chip 21 and the variable-diameter chip 22 are assembled, the first liquid injection hole 23.1 is aligned with the second liquid injection hole 23.2, so that the first liquid injection hole 23.1 and the plurality of second liquid injection holes 23.2 form a total liquid injection hole 23, the function of the total liquid injection hole 23 is to enable the external sealing connector to enter the external connector 2, so that the liquid flowing out of the external sealing connector can be directly injected into the liquid flow channel 12.1 from both sides of the main chip 100. Further, referring to Figure 9 , the closer to the second liquid injection hole 23.2 of the plug-in chip 21, the smaller the diameter, and the diameter of the first liquid injection hole 23.1 is smaller than the diameter of the smallest second liquid injection hole 23.2. In a specific embodiment, the number of variable-diameter chips 22 is 3, which are a first variable-diameter chip 22, a second variable-diameter chip 22 and a third variable-diameter chip 22, the third variable-diameter chip 22 is farthest from the plug-in chip 21, the first variable-diameter chip 22 is closest to the plug-in chip 21, and the second variable-diameter chip 22 is between the first variable-diameter chip 22 and the third variable-diameter chip 22. In order to correspond to the external sealing connector, taking the inverted taper compression ring sealing connector with a market common inner diameter of 1600 μm as an example, the outer diameter of the narrowest part of the lowermost edge of the inverted taper compression ring is 2360 μm, the outer diameter of the widest part of the uppermost edge of the inverted taper compression ring is 5000 μm, the thickness is 1350 μm, the outer diameter of the middle section of the inverted taper compression ring is 4000 μm, and the total height is 3920 μm. Therefore, the diameter of the second liquid injection hole 23.2 of the third variable-diameter chip 22 is 5000 μm (corresponding to the widest part of the uppermost edge of the inverted taper compression ring), the diameter of the second liquid injection hole 23.2 in the second variable-diameter chip 22 is 4000 μm (corresponding to the middle section of the inverted taper compression ring), the diameter of the second liquid injection hole 23.2 in the first variable-diameter chip 22 is 2360 μm (corresponding to the narrowest part of the lowermost edge of the inverted taper compression ring), the diameter of the first liquid injection hole 23.1 is 1600 μm (corresponding to the inner diameter of the inverted taper compression ring), the thickness of the third variable-diameter chip 22 is ≥ 1350 μm (corresponding to the thickness of the widest part of the uppermost edge of the inverted taper compression ring), and the sum of the thicknesses of the remaining variable-diameter chips 22 and the plug-in chip 21 is 2570 μm, that is, the sum of the thicknesses of the second variable-diameter chip 22, the first variable-diameter chip 22 and the plug-in chip 21 is 2570 μm (corresponding to the sum of the thicknesses of the middle section of the inverted taper compression ring and the lowermost edge of the inverted taper compression ring), so that the external sealing function unit can be integrated on a smaller and limited microfluidic organ chip area under the premise of ensuring sealing, the area and space occupied by the conventional luer connector fixation and sealing are reduced, it is small, flexible, easy to assemble, and can equivalently realize sealing.

[0078] As an illustration, generally, the adjacent variable-diameter chips 22 are connected by adhesion or other equivalent mounting means, and the plug-in chip 21 is also connected with the variable-diameter chips 22 by adhesion or other equivalent mounting means. In some specific embodiments, the side protruding positioning units 6 are arranged on the variable-diameter chips 22 farthest from the plug-in chip 21, and the through holes corresponding to the side protruding positioning units 6 are arranged on the other variable-diameter chips 22 and the plug-in chip 21, so that all the variable-diameter chips 22 and the plug-in chip 21 can be connected together directly through the side protruding positioning units 6.

[0079] In a specific embodiment, referring to Figure 7 , the diameters of the third through hole 16.3 and the fourth through hole 16.4 are the same, the diameters of the first through hole 16.1, the second through hole 16.2, and the fifth through hole 16.5 gradually decrease, and the diameters of the third through hole 16.3 and the fourth through hole 16.4 are smaller than the diameter of the fifth through hole 16.5. In order to seal the liquid flow channel 12.1 in the main chip 100, the diameters of the through holes in each level chip need to correspond to the external sealing joint and the capillary. Taking the commercially available inverted taper compression ring sealing joint as an example, the diameter of the first through hole 16.1 is the outer diameter of the uppermost edge of the inverted taper compression ring (the outer diameter of the widest part of the inverted taper compression ring), the diameter of the second through hole 16.2 is not less than the outer diameter of the widest part of the middle section of the inverted taper compression ring, the diameter of the fifth through hole 16.5 is the outer diameter of the lowermost edge of the inverted taper compression ring (the outer diameter of the narrowest part of the inverted taper compression ring), and the diameters of the third through hole 16.3 and the fourth through hole 16.4 are the inner diameters of the inverted taper compression ring (equivalent to the outer diameter of the capillary used in the experiment).

[0080] In the detachable plug-in microfluidic organ chip provided by the embodiment of the present application, the number of the liquid flow through holes 13.1 is four, and the intervals between adjacent liquid flow through holes 13.1 are the same. In an improved embodiment, the liquid flow channel 12.1 includes four branch flow channels, and the ends of each branch flow channel are respectively connected with one liquid flow through hole 13.1.

[0081] The present application also provides an assembly method of the detachable plug-in microfluidic organ chip, which includes the following steps:

[0082] Step S1) Assemble two half-chip sets 1 respectively; further, stack all the level chips in the two half-chip sets 1 in sequence.

[0083] Step S2) Assembling two half-chip sets 1 into a main chip 100 in a central symmetry; further, positioning the male convex positioning units 3 in one half-chip set 1 through the female convex positioning holes 4 in the other half-chip set 1, thereby plugging all the level chips except the first layer chip 11 together, and connecting the first layer chip 11 with the corresponding second layer chip 12 by adhesion or the like.

[0084] Step S3) Installing two external plug-in units 2 on the two sides of the main chip 100; further, connecting a plurality of variable-diameter chips 22 by adhesion or the like, connecting the variable-diameter chips 22 with the plug-in chips 21 in the same manner, and finally inserting the side protrusion positioning units into the side protrusion positioning holes on the two sides of the main chip 100.

[0085] The application also provides a cell culture method of the detachable plug-in microfluidic organ chip, which comprises the following steps:

[0086] Step T1) inoculating cells not in static culture into the arc surface culture flow channel 13.2 and culturing statically until the cell fusion degree reaches the experimental requirement; or directly inoculating three-dimensional cells in static culture into the arc surface culture flow channel 13.2.

[0087] Step T2) assembling the main chip 100 and the external plug-in unit 2;

[0088] Step T3) using the microfluidic peripheral device to drive the culture liquid to flow in the cell culture flow channel 5 for dynamic culture of the cells.

[0089] The above cell culture method provided by the application can be summarized into two schemes:

[0090] Scheme I: the cells can be directly inoculated into the arc surface region of the third chip layer, and when the cell fusion degree reaches the experimental requirement, the main body and the plug-in unit are assembled, and the culture liquid is further driven to flow by the microfluidic peripheral device for dynamic culture.

[0091] Scheme II: the three-dimensional cells (such as cell spheres, tumor organoids, etc.) in culture can be directly placed into the arc surface region of the third chip layer, and after the main body and the plug-in unit are assembled, the culture liquid is further driven to flow by the microfluidic peripheral device for dynamic culture.

[0092] Application of Example I in the study of biological barrier function

[0093] In this example, the cells are first cultured with the biofilm and then placed on the curved surface culture flow channel 13.2, and then the two half core chip sets 1 are assembled so that the cells are located in the cell culture flow channel 5. When the cell density reaches the experimental research requirements, the cell culture flow channel 5 can simulate the barrier function of hollow biological structures such as blood vessels, airways, and lymphatic vessels. When the microfluidic peripheral device is used to drive the flow of the culture solution, there are two flow modes in total, namely flow mode one and flow mode two.

[0094] Flow mode one: the inverted tapered compression ring sealing joint is inserted into the first through hole 16.1, the second through hole 16.2, and the fifth through hole 16.5. The culture solution flowing out of the inverted tapered compression ring sealing joint flows into the liquid flow channel 12.1 through the fourth through hole 16.4 and the third through hole 16.3 (the diameters of the fourth through hole 16.4 and the third through hole 16.3 are the same as the inner diameter of the inverted tapered compression ring sealing joint), and then the culture solution flows into the cell culture flow channel 5 through the liquid flow hole 13.1.

[0095] Flow mode two: the inverted tapered compression ring sealing joint is inserted into the plurality of second liquid injection holes 23.2. The culture solution flowing out of the inverted tapered compression ring sealing joint flows into the cell culture flow channel 5 through the first liquid injection hole 23.1 (the first liquid injection hole 23.1 is the same as the inner diameter of the inverted tapered compression ring sealing joint), and then flows out in the same way through the external plug-in device 2 on the other side of the main body chip 100.

[0096] When flow mode one and flow mode two are started at the same time, the liquids on both sides of the cells located in the cell culture flow channel 5 are in a balanced state, which can be used to study the integrity or protective effect of the barrier structure.

[0097] Further, when the barrier structure is destroyed under any kind of physical and chemical factor treatment, in the case that flow mode one is opened and flow mode two is closed: if the liquid volume flowing in from the inverted tapered compression ring sealing joint of one external plug-in device 2 and flowing out from the other external plug-in device 2 gradually increases, or specific additives (such as fluorescent dyes, particulate matter, etc.) appear. At this time, it can be prompted that the barrier structure composed of cells is destroyed from the outside.

[0098] Similarly, in the case that flow mode one is closed and flow mode two is opened, the liquid volume in the fourth through hole 16.4, the third through hole 16.3, the liquid flow channel 12.1, and the liquid flow hole 13.1 can be detected to gradually increase, or specific additives (such as fluorescent dyes, particulate matter, etc.) appear. At this time, it can be prompted that the barrier structure composed of cells is destroyed from the inside.

[0099] Similarly, when the first flow mode is connected to the culture solution and the second flow mode is connected to the air, the barrier function of the "air-liquid" interface can be simulated. When the barrier structure is destroyed under any kind of physical and chemical factor, the bubbles increase or specific additives (such as fluorescent dyes, particulate matter, etc.) appear in the fourth through hole 16.4, the third through hole 16.3, the liquid flow channel 12.1 and the liquid flow hole 13.1 of the chip main body. At this time, it can be prompted that the "air-liquid" interface barrier structure is abnormal.

[0100] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the technical principles of the present application, a number of improvements and substitutions can be made, and these improvements and substitutions should also be considered as the protection scope of the present application.

Claims

1. A detachable plug-in microfluidic organ-on-a-chip, characterized in that: It includes a main chip (100) and two external plugs (2) that are detachably connected to both sides of the main chip (100). The main chip (100) includes two splicable half-chip groups (1). Each half-chip group (1) includes a first layer chip (11), a second layer chip (12), a third layer chip (13), a fourth layer chip (14), and a fifth layer chip (15) that can be stacked and detached in sequence. The first layer chip (11) is provided with a first through hole (16.1); the second layer chip (12) is provided with a second through hole (16.2) and a positive convex positioning unit (3), and the second layer chip (12) is also provided with a liquid flow channel (12.1); the third layer chip (13) is provided with a third through hole (16.3), a liquid flow hole (13.1) and a positive convex positioning hole (4), and the third layer chip (13) is also provided with an arc-shaped culture flow channel (13.2); the fourth layer chip (14) is provided with a fourth through hole (16.4) and a positive convex positioning hole (4); the fifth layer chip (15) is provided with a fifth through hole (16.5) and a positive convex positioning hole (4). In the same group of half-mounted chip sets (1), the liquid flow channel (12.1) is connected to the arc-shaped culture channel (13.2) only through the liquid flow hole (13.1); the liquid flow channel (12.1) is also connected to the second through hole (16.2) and the first through hole (16.1) in another group of half-mounted chip sets (1) through the third through hole (16.3), the fourth through hole (16.4) and the fifth through hole (16.5) in the same group of half-mounted chip sets (1); the positive convex positioning unit ( 3) It can be detachably connected to each positive convex positioning hole (4) in another set of half-assembled chip groups (1); after the two half-assembled chip groups (1) are spliced ​​into the main chip (100), the two arc-shaped culture channels (13.2) are arranged opposite to each other, and the sidewall between the two arc-shaped culture channels (13.2) is the sidewall of the fourth layer chip (14). The closed space between the two arc-shaped culture channels (13.2) and the sidewall of the two fourth layer chips (14) can form a hollow cell culture channel (5). Each of the external plugs (2) is provided with a total injection hole (23), which is connected to the cell culture channel (5); the arc α of the arc culture channel (13.2) is 60~150°, and the arc is measured by the angle between the tangent vectors of the two curves.

2. The detachable plug-in microfluidic organ-on-a-chip according to claim 1, characterized in that: Each of the external plug-in (2) is provided with a side protrusion positioning unit (6). The second layer chip (12), the third layer chip (13), the fourth layer chip (14) and the fifth layer chip (15) are provided with side protrusion positioning grooves (71) on both sides. The side protrusion positioning grooves (71) located on the same side form a side protrusion positioning hole (7). The side protrusion positioning unit (6) and the side protrusion positioning hole (7) are detachably connected.

3. The detachable plug-in microfluidic organ-on-a-chip according to claim 2, characterized in that: Each of the external plugs (2) includes a plug-in chip (21) and a plurality of variable diameter chips (22) connected in sequence to the plug-in chip (21). The side protrusion positioning unit (6) is disposed on the plug-in chip (21). The plug-in chip (21) is also provided with a first injection hole (23.1). Each of the variable diameter chips (22) is provided with a second injection hole (23.2). The first injection hole (23.1) and the plurality of second injection holes (23.2) form a total injection hole (23).

4. The detachable plug-in microfluidic organ-on-a-chip according to claim 3, characterized in that: The diameter of the second injection hole (23.2) closer to the plug-in chip (21) is smaller, and the diameter of the first injection hole (23.1) is smaller than the diameter of the smallest second injection hole (23.2).

5. The detachable plug-in microfluidic organ-on-a-chip according to claim 4, characterized in that: The number of variable diameter chips 22 is 3. The diameter of the second injection hole (23.2) furthest from the plug-in chip (21) is 5000µm, the diameter of the second injection hole (23.2) located in the middle is 4000µm, the diameter of the second injection hole (23.2) closest to the plug-in chip (21) is 2360µm, and the diameter of the first injection hole (23.1) is 1600µm; and / or, the thickness of the variable diameter chip (22) furthest from the plug-in chip (21) is ≥1350µm, and the sum of the thicknesses of the remaining variable diameter chips (22) and plug-in chips (21) is 2570µm.

6. The detachable plug-in microfluidic organ-on-a-chip according to claim 1, characterized in that: The third through hole (16.3) and the fourth through hole (16.4) have the same diameter. The diameters of the first through hole (16.1), the second through hole (16.2), and the fifth through hole (16.5) gradually decrease. The diameters of the third through hole (16.3) and the fourth through hole (16.4) are both smaller than the diameter of the fifth through hole (16.5).

7. The detachable plug-in microfluidic organ-on-a-chip according to claim 1, characterized in that: The number of liquid flow holes (13.1) is 4, and the spacing between adjacent liquid flow holes (13.1) is the same.

8. The assembly method of the detachable plug-in microfluidic organ-on-a-chip according to any one of claims 1 to 7, characterized in that: Step S1) First, assemble the two half-assembled chipsets (1) respectively; Step S2) Assemble the two half-chipsets (1) into a main chip (100) in a centrally symmetrical manner. Step S3) Install the two external plugs (2) on both sides of the main chip (100).

9. The cell culture method for the detachable plug-in microfluidic organ-on-a-chip according to any one of claims 1 to 7, characterized in that: Step T1) Seed the uncultured cells into the curved culture channel (13.2) and culture them statically until the cell confluence meets the experimental requirements; or, directly seed the statically cultured three-dimensional cells into the curved culture channel (13.2). Step T2) Assemble the main chip (100) and the external plug-in (2); Step T3) Use microfluidic peripherals to push the culture liquid to flow in the cell culture channel (5) to carry out dynamic cell culture.

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