A liver organ chip model and its use

Abstract

The present invention relates to the fields of biomedical engineering and microfluidics, and in particular to a liver organ chip model and its use, wherein the liver organ chip model comprises a top chip, an intermediate chip and a bottom chip, wherein the intermediate chip is arranged between the top chip and the bottom chip, and the liver organ chip model is provided with a sample addition hole, a collection hole and a cell culture chamber, wherein the cell culture chamber is arranged on the intermediate chip, and the sample addition hole and the collection hole are both connected to the cell culture chamber. The liver organ chip model can be used for drug development, toxicology, nutrition and other research. The liver organ chip model adds a bile flow function, realistically reproduces the physiological structure of the liver lobule, and then reproduces the systemic function of the liver organ, more realistically simulates the interaction between drugs or other substances and the liver; and can realize the co-culture of liver non-parenchymal cells such as sinusoidal endothelial cells, bile duct endothelial cells, fibroblasts and other immune cells.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and microfluidics, and in particular to a liver organ chip model and applications thereof. Background Art

[0002] The liver is a crucial site for the oxidative metabolism and detoxification of drugs and various exogenous substances in the body. The hepatic lobule is considered the smallest functional unit of the liver and is polygonal in shape. The center of the polygonal hepatic lobule is a central vein, and the periphery contains the portal vein, hepatic artery, and bile duct. The substance of each hepatic lobule is composed of hepatocytes radiating from the central vein and separated by vascular endothelial cells. The apical surfaces of adjacent hepatocytes form channels for the bile duct. Bile acids produced from cholesterol in hepatocytes are secreted into the bile duct. Bile flows from the common bile duct of the hepatic lobule into the hepatic duct, then into the gallbladder through the cystic duct, and finally into the intestine through the common bile duct.

[0003] The Liver Organ Chip is a specialized chip technology based on 3D culture and paired with a microfluidic system. It utilizes microfluidics to control fluid flow, combining cell-cell interactions, matrix properties, and biochemical and biomechanical properties to construct a three-dimensional physiological microsystem of the human liver organ on a chip. Studies have found that the Liver Organ Chip's microfluidic system can simulate the flow environment of body fluids and concentration changes in the extracellular environment in vitro. By generating shear stress to trigger the development of liver cell polarity, it is more conducive to improving liver cell CYP activity and albumin secretion, bringing it closer to the physiological environment in vivo.

[0004] Although mammalian experiments are commonly used in drug research to study the effects of drugs on liver function, research data indicate that some animal experimental results are questionable. Because background differences between mammals and humans are difficult to eliminate, animal experiments cannot accurately reflect the actual effects of drugs in the human body. Therefore, it is urgent and necessary to establish more accurate and reliable in vitro liver models to more realistically study the physiological and pathological development of the liver and the effects of drugs on the liver. In recent years, basic research on liver organ chips has become increasingly popular, and their application in drug toxicity screening and pharmacology research has also been increasingly valued. Liver organ chip technology can more accurately and rapidly detect the potential toxicity and efficacy of drugs in humans, reducing the time and economic costs of animal experiments, and will become one of the important methods for drug screening in the future.

[0005] However, current liver organ-on-a-chip research focuses on simulating the hepatocyte-sinusoidal cross-section, neglecting the role of bile duct flow and the systemic physiological functions of the hepatic lobule. While a few studies have explored bile duct design within organ-on-a-chips, these studies have been limited to recreating a single, simple liver plate, preventing the study of inter-plate signaling. Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a liver organ chip model and its use to solve the problems in the prior art.

[0007] To achieve the above objectives and other related objectives, the present invention provides a liver organ chip model, which includes a top chip, a middle chip, and a bottom chip. The middle chip is arranged between the top chip and the bottom chip. The liver organ chip model is provided with a sample addition well, a collection well, and a cell culture chamber. The cell culture chamber is arranged on the middle chip, and the sample addition well and the collection well are both connected to the cell culture chamber.

[0008] The intermediate layer chip is provided with a culture fluid peripheral channel, a culture fluid perfusion channel, and a bile fluid perfusion channel. The culture fluid peripheral channel is connected to each culture fluid perfusion channel. Multiple culture fluid perfusion channels and bile fluid perfusion channels are provided. The culture fluid peripheral channel is connected to the culture fluid perfusion channel, and the bile fluid perfusion channel is connected to the cell culture chamber.

[0009] The sample addition holes extend from the top chip to the middle chip.

[0010] The sample injection hole includes any one or more of a cell injection hole, a culture fluid injection hole, and a bile fluid injection hole. The cell injection hole is directly connected to the cell culture chamber. The culture fluid injection hole is connected to the cell culture chamber via a culture fluid peripheral channel and a culture fluid perfusion channel in sequence. The bile fluid injection hole is connected to the cell culture chamber via a bile fluid perfusion channel.

[0011] The collection holes include a culture fluid collection hole and a bile fluid collection hole. The culture fluid collection hole extends from the top layer chip to the middle layer chip, and the culture fluid collection hole is connected to the culture fluid injection hole through each culture fluid perfusion channel and the culture fluid peripheral channel; the bile fluid collection hole extends from the top layer chip to the bottom layer chip, and the bile fluid collection hole is connected to the bile fluid injection hole.

[0012] Each cell culture chamber is adjacent to each other, and each cell culture chamber is separated from adjacent cell culture chambers by a culture fluid perfusion channel and a bile fluid perfusion channel on both sides. The culture fluid perfusion channel and the bile fluid perfusion channel are both permeable membrane structures.

[0013] A bile drainage hole is provided at the end of the bile perfusion channel, and the bile drainage hole extends from the middle layer chip to the bottom layer chip.

[0014] A bile collection channel is provided on the bottom chip. One end of the bile collection channel is communicated with the bile collection hole, and the other end is communicated with each bile drainage hole.

[0015] The present invention also provides uses of the liver organ chip model in drug development, toxicology, nutrition and other research.

[0016] As described above, the liver organ chip model of the present invention and its uses have the following beneficial effects:

[0017] 1) The system has been enhanced with a culture medium perfusion system to simulate the liver sinusoidal flow and bile flow, which can realistically reproduce the physiological structure of the liver lobule. This system can also reproduce the systemic function of the liver organ and more realistically simulate the interaction between drugs or other substances and the liver.

[0018] 2) Co-culture of liver non-parenchymal cells such as sinusoidal endothelial cells, bile duct endothelial cells, fibroblasts, and other immune cells can be achieved, which is closer to the real physiological environment of the liver lobule;

[0019] 3) Connecting to oxygen monitoring equipment can realize the regional cultivation of oxygen partial pressure of liver lobule;

[0020] 4) It can provide multiple cell culture chambers and liquid flow channels, and ensure that the liquid flow environments of the culture fluid and bile fluid do not affect each other while multiple cells are co-cultured, and realize the recovery and detection of cell culture fluid and bile fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an exploded view of the liver organ chip model of the present invention;

[0022] Figure 2 This is a top view of the top chip of the liver organ chip model of the present invention;

[0023] Figure 3 A top view of the middle layer chip of the liver organ chip model of the present invention;

[0024] Figure 4 This is a bottom view of the middle layer chip of the liver organ chip model of the present invention;

[0025] Figure 5 This is a top view of the bottom chip of the liver organ chip model of the present invention;

[0026] Figure 6 A three-dimensional diagram of the culture medium perfusion channel of the middle layer chip of the liver organ chip model of the present invention;

[0027] Figure 7 This is a three-dimensional diagram of the bile perfusion channel of the middle chip of the liver organ chip model of the present invention;

[0028] Figure 8 Schematic diagram of the bile flow pathway of the liver organ chip model of the present invention. The arrows indicate the direction of bile flow.

[0029] Figure 9Schematic diagram of the flow pathway of the culture medium in the liver organ chip model of the present invention. The direction indicated by the arrow is the flow direction of the culture medium.

[0030] Component number description

[0031] 1 Top chip

[0032] 2 Intermediate chip

[0033] 3 underlying chip

[0034] 11 sample wells

[0035] 111 Cell injection hole

[0036] 112 Culture medium injection hole

[0037] 113 Bile injection hole

[0038] 12 collection holes

[0039] 121 Culture medium collection hole

[0040] 122 Bile collection hole

[0041] 21 Cell culture chamber

[0042] 22 Culture medium peripheral channel

[0043] 23 Culture medium perfusion channel

[0044] 24 Bile perfusion channel

[0045] 25 Bile drainage hole

[0046] 26 Bile collecting duct DETAILED DESCRIPTION

[0047] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0048] See also Figures 1 to 9. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no substantive technical significance. Any modification of the structure, change in the proportional relationship or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0049] like Figure 1 As shown, the present invention provides a liver organ chip model, which includes a top chip 1, a middle chip 2 and a bottom chip 3. The middle chip 2 is arranged between the top chip 1 and the bottom chip 3. The liver organ chip model is provided with a sample addition well 11, a collection well 12 and a cell culture chamber 21. The cell culture chamber 21 is arranged on the middle chip 2, and the sample addition well 11 and the collection well 12 are both connected to the cell culture chamber 21.

[0050] The top chip 1 , the middle chip 2 and the bottom chip 3 form a sandwich structure.

[0051] The top chip 1 is a sample addition and collection layer, the middle chip 2 is a perfusion culture layer, and the bottom chip 3 is a bile fluid collection layer.

[0052] like Figure 3 As shown, the intermediate layer chip 2 is provided with a culture fluid peripheral channel 22, a culture fluid perfusion channel 23, and a bile fluid perfusion channel 24. There are multiple culture fluid perfusion channels 23 and bile fluid perfusion channels 24. The culture fluid peripheral channel 22 is connected to the culture fluid perfusion channel 23, and the bile fluid perfusion channel 24 is connected to the cell culture chamber 21.

[0053] like Figure 1 As shown, the sample addition hole 11 extends from the top layer chip 1 to the middle layer chip 2 .

[0054] like Figure 2 As shown, the sample injection hole 11 includes any one or more of a cell injection hole 111, a culture fluid injection hole 112, and a bile fluid injection hole 113. Figure 3As shown, the cell injection hole 111 is directly connected to the cell culture chamber 21, the culture fluid injection hole 112 is connected to the cell culture chamber 21 through the culture fluid peripheral channel 22 and the culture fluid perfusion channel 23 in sequence, and the bile fluid injection hole 113 is connected to the cell culture chamber 21 through the bile fluid perfusion channel 24.

[0055] The cell injection hole 111 is provided with one or more. Figure 2 In the illustrated embodiment, a plurality of cell injection wells 111 are provided. Preferably, the number of the cell injection wells 111 is equal to the number of the cell culture chambers 21. Different cell injection wells 111 can be used to add different cell types, such as normal liver cells, liver cancer cells, or cell mixtures, and the sample addition process of multiple wells does not affect each other, and there is no cross contamination. The number of the cell injection wells 111 is 6 to 16, for example, 6 to 8, 8 to 10, 10 to 12, 12 to 14, or 14 to 16.

[0056] The arrangement of the cell injection holes 111 matches the cell culture chamber 21. Figure 2 and 3 As shown, the position of the cell injection hole 111 corresponds to the position of the cell culture chamber 21 with a larger cross-sectional area, that is, corresponds to the outer portion of the cell culture chamber 21.

[0057] The culture solution injection hole 112 is provided with one or more. Figure 2 In the embodiment shown, there are a plurality of culture fluid injection holes 112 . There are two culture fluid injection holes 112 . One culture fluid injection hole 112 can be connected to three culture fluid perfusion channels 23 .

[0058] The bile injection hole 113 is provided with one or more. Figure 2 In the embodiment shown, there are multiple bile injection holes 113. The number of bile injection holes 113 matches the number of cell culture chambers 21. Preferably, one bile injection hole 113 is provided for each two adjacent cell culture chambers 21. Figure 3 In the illustrated embodiment, six bile injection holes 113 are provided. In a preferred embodiment, the plurality of bile injection holes 113 are distributed in a circular pattern. This circular pattern facilitates connection to an annular bile injection component, allowing the plurality of bile injection holes 113 to be simultaneously injected with bile, thereby ensuring a relatively uniform amount of bile in each hole.

[0059] like Figure 1 and 2As shown, the collection hole 12 includes a culture fluid collection hole 121 and a bile fluid collection hole 122. The culture fluid collection hole 121 extends from the top layer chip 1 to the middle layer chip 2, and the culture fluid collection hole 121 is connected to the culture fluid injection hole 112 through each culture fluid perfusion channel 23 and the culture fluid peripheral channel 22; the bile fluid collection hole 122 extends from the top layer chip 1 to the bottom layer chip 3, and the bile fluid collection hole 122 is connected to the bile fluid injection hole 113.

[0060] like Figure 1-3 As shown, the culture fluid collecting hole 121 is provided with one or more. Figure 2 In the embodiment shown, there is one culture fluid collecting hole 121 . One culture fluid collecting hole 121 is connected to all the culture fluid perfusion channels 23 .

[0061] The culture fluid injected into the culture fluid injection hole 112 flows through the culture fluid peripheral channel 22 into each culture fluid perfusion channel 23 to exchange substances with the corresponding cell culture chamber 21, and then converges into the culture fluid collection hole 121. Since the culture fluid collection hole 121 extends from the top chip 1 to the middle chip 2, the culture fluid at the perfusion end can be collected on the top chip 1.

[0062] The bile collection hole 122 is provided with one or more. Figure 2 In the embodiment shown, there are two bile collection holes 122 , and one bile collection hole 122 can be connected to multiple bile drainage holes 25 .

[0063] The cell culture chamber 21 is provided with one or more. Figure 3 In the embodiment shown, the cell culture chamber 21 is provided with a plurality of cells. Preferably, the cell culture chamber 21 is provided with 12 cells. Figure 3 In the illustrated embodiment, twelve cell culture chambers 21 are adjacent to each other to form a regular hexagonal prism.

[0064] like Figure 3 As shown, each cell culture chamber 21 is adjacent to each other, and each cell culture chamber 21 is separated from the adjacent cell culture chamber 21 by a culture fluid perfusion channel 23 and a bile fluid perfusion channel 24 on both sides. The culture fluid perfusion channel 23 and the bile fluid perfusion channel 24 are both permeable membrane structures.

[0065] The culture fluid perfusion channel 23 with a permeable membrane structure can allow the culture fluid in the culture fluid perfusion channel 23 to permeate into each cell culture chamber 21 , and the cell culture chambers 21 can also exchange substances and information.

[0066] In one embodiment, the permeable membrane structure of the culture fluid perfusion channel 23 is composed of a PC membrane and a PDMS membrane. The pore sizes of the PC and PDMS membranes can be selected based on the type and size of the co-cultured cells and the composition of the culture fluid. For example, the pore size of the PC membrane is 0.2-20 μm, and the pore size of the PDMS membrane is 0.2-20 μm.

[0067] The cell culture chamber 21 is suitable for 2D and 3D culture of cells in a gel-like cell mixture such as matrix gel or hydrogel, or cell microspheres or ordinary small suspensions.

[0068] In one embodiment, the width of the culture fluid perfusion channel 23 is non-uniform. Figure 3 In the illustrated embodiment, the culture fluid perfusion channel 23 is wider near the culture fluid collection hole and narrower distal to the culture fluid collection hole. The uneven width of the culture fluid perfusion channel 23 more closely resembles the actual shape of the hepatic sinusoidal vessels of the hepatic lobule, thereby more realistically simulating the hepatic sinusoidal vessels of the hepatic lobule.

[0069] Culture medium perfusion can simulate hepatic sinusoidal flow.

[0070] The bile perfusion channel 24, with a permeable membrane structure, allows bile acids and other metabolites from the culture medium in the cell culture chamber 21 to permeate through the bile perfusion channel 24 and be discharged through the perfusion system. In one embodiment, the bile perfusion channel 24, with a permeable membrane structure, is composed of a PDMS membrane. The pore size of the permeable membrane of the bile perfusion channel 24 is, for example, 0.2-20 μM.

[0071] The width of the bile perfusion channel 24 is not uniform. Figure 3 In the embodiment shown, the bile perfusion channel 24 is narrower near the bile injection hole and wider far from the bile injection hole. The bile perfusion channel 24 with uneven width is closer to the actual shape of the bile duct, thereby more realistically simulating the liver lobule.

[0072] The widest part of the bile perfusion channel 24 is thinner than the narrowest part of the culture medium perfusion channel 23. The main reason for this design is that the hepatic sinusoidal vessels are physiologically wider than the bile duct.

[0073] The bile fluid injected into the bile fluid injection hole 113 exchanges substances with the cell culture chamber 21 through the bile fluid perfusion channel 24 and then flows to the bottom chip 3 .

[0074] like Figure 3-5 As shown, a bile drainage hole 25 is provided at the end of the bile perfusion channel 24 , and the bile drainage hole 25 extends from the middle layer chip 2 to the bottom layer chip 3 .

[0075] like Figure 6As shown, the bottom chip 3 is provided with a bile collection channel 26, one end of which is connected to the bile collection hole 122 and the other end is connected to each bile drainage hole 25. That is, the bile injection hole 113 is connected to the bile collection hole 122 via the bile perfusion channel 24, the bile drainage hole 25, and the bile collection channel 26. The number of bile drainage holes 25 is equal to the number of bile injection holes 113 and the number of bile perfusion channels 24. That is, each bile injection hole 113 has a bile perfusion channel 24 connected to it, and each bile perfusion channel 24 is provided with a bile drainage hole 25 at its end. One bile collection hole 122 is connected to three bile drainage holes 25.

[0076] The bile injected into each bile injection hole 113 exchanges substances with each cell culture chamber 21 through the corresponding bile perfusion channel 24, flows to the bile drainage hole 25, and then converges into the bile collection hole 122 through the bile collection channel 26. Because the bile collection hole 122 extends from the top chip 1 to the bottom chip 3, bile can be collected at the top chip 1.

[0077] The lengths of the top chip 1, the middle layer chip 2, and the bottom chip 3 are equal, and the widths of the top chip 1, the middle layer chip 2, and the bottom chip 3 are equal. Equal length and width facilitate the formation of a regular shape after assembly. The height of the top chip 1 is the smallest, the height of the bottom chip 3 is the second largest, and the height of the middle layer chip 2 is the highest. The middle layer chip 2 is the highest for the convenience of cell culture. The present invention does not specifically limit the values ​​of the length, width, and height of the top chip 1, the middle layer chip 2, and the bottom chip 3, as well as the dimensions of the sample addition holes 11, the collection holes 12, the cell culture chamber 21, and the various liquid flow channels. Those skilled in the art can customize them according to actual needs. In certain embodiments of the present invention, the lengths of the top chip 1, the middle chip 2, and the bottom chip 3 are 4-8 cm; the widths of the top chip 1, the middle chip 2, and the bottom chip 3 are 2-3.5 cm; the height of the top chip 1 is 0.2-1 cm; the height of the bottom chip 3 is 0.2-1 cm; the height of the middle chip 2 is 0.4-1.5 cm; the diameter of the cell injection hole 111 is 0.3-0.5 cm; the diameter of the injection hole of the culture medium injection hole 112 is 0.5 The diameter of the bile injection hole 113 is 0.2-0.3 cm; the diameter of the culture fluid collection hole 121 is 0.5-1.0 cm; the diameter of the bile collection hole 122 is 0.5-0.7 cm; the length of the culture fluid perfusion channel 23 is 0.5-1 cm and the width is 0.2-1 mm; the length of the bile perfusion channel 24 is 0.3-0.8 cm and the width is 0.2-1 mm; the height of the cell culture chamber 21 is equal to the height of the intermediate layer chip 2. The above values ​​are only illustrative of the order of magnitude of the liver organ chip model and do not limit the size of the liver organ chip model.

[0078] The liver organ chip model is made of a material suitable for cell culture. In a preferred embodiment, it is made of a transparent material suitable for cell culture. The transparent material ensures that the liquid level and cells in the liver organ chip model can be observed by the naked eye.

[0079] The top chip 1, middle chip 2, and bottom chip 3 are sealed and fixedly connected by a reversibly restorable material. The reversibly restorable material facilitates assembly and disassembly of the chip and facilitates the recovery of cell cultures within the cell culture chamber. Examples of the reversibly restorable material include hydrogels made from polyhydroxyethyl methacrylate (pHEMA) polymers, thermoreversible gelatin, and light-controlled reversible adhesives such as azobenzene derivatives.

[0080] Each sample addition hole and collection hole of the liver organ chip model can be connected to a catheter and used in conjunction with a power system such as microfluidics. The present invention does not specifically limit the source and flow rate of the fluid power.

[0081] The present invention also provides uses of the liver organ chip model in drug development, toxicology, nutrition and other research.

[0082] Specifically, the use is in studying the interaction between drugs or other substances and the liver. The interaction between drugs or other substances and the liver includes, for example, studies on drug metabolism in the liver, the effect of the liver on drug metabolism, the effect of drugs on liver physiological functions, drug toxicology, and nutrition.

[0083] One method of using the liver organ chip model of the present invention is as follows:

[0084] The overall process is: first reverse perfusion to expel the air, and then forward injection of various liquids.

[0085] Specifically, the culture medium is first injected from the culture medium collection hole 121, i.e., reverse perfusion, to expel the air in the culture medium peripheral channel 22, and then the cell suspension of vascular endothelial cells is slowly injected into the culture medium perfusion channel 23 to simulate the physiological environment inside the blood vessel. After the vascular endothelial cells are cultured and attached to the wall, the vascular endothelial cells are cultured through the culture medium injection hole 112 in the forward direction.

[0086] First, bile is injected from the bile collection hole 122, i.e., reverse perfusion, to expel the air in the bile collection channel 26, and then the cell suspension of bile duct endothelial cells is slowly injected in a forward direction from the bile injection hole 113 until the cell suspension fills the bile perfusion channel 24 to simulate the physiological environment of the bile duct. After the cells are cultured and adhere to the wall, bile is injected from the bile injection hole 113 again to culture the bile duct endothelial cells in a forward perfusion direction.

[0087] A gelatinous cell mixture such as matrix gel or hydrogel or cell microspheres is injected into the cell culture chamber 21 through the cell injection hole 111 . After solidification, an appropriate volume of culture fluid is added. The level of the culture fluid is slightly lower than the level of the culture fluid perfusion channel 23 .

[0088] The culture fluid perfusion channel 23 and the bile fluid perfusion channel 24 are not limited to injecting only endothelial cells, and other immune cells such as Kupffer cells, PBMCs, etc. can also be injected according to actual experimental requirements.

[0089] The culture medium of the liver organ chip model of the present invention can be perfused using an external catheter and power pump as is conventionally done. Specifically, fresh culture medium is introduced through the culture medium inlet 112, which is connected to the catheter and power pump. The fresh culture medium then flows through the culture medium peripheral channel 22 and the various culture medium perfusion channels 23 before being collected at the culture medium collection hole 121. This collection hole 121 is also connected to a catheter or power pump to collect the culture medium.

[0090] The bile in the liver organ-on-a-chip model of the present invention can be perfused using an external catheter and power pump, as is conventionally done. Bile infusion port 113 is connected to the catheter and power pump. Fresh bile is introduced through bile infusion port 113, flows through the corresponding bile perfusion channel 24 to the bile drainage port 25, and then through the bile collection channel 26 to the bile collection port 122. Bile collection port 122 is also connected to a catheter or power pump to collect bile.

[0091] In summary, the present invention effectively overcomes various shortcomings of the prior art and has high industrial utilization value.

[0092] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A liver organ chip model, characterized in that: The liver organ chip model comprises a top chip (1), an intermediate chip (2) and a bottom chip (3), wherein the intermediate chip (2) is arranged between the top chip (1) and the bottom chip (3), and the liver organ chip model is provided with a sample addition hole (11), a collection hole (12) and a cell culture chamber (21), wherein the cell culture chamber (21) is arranged on the intermediate chip (2), wherein the sample addition hole (11) and the collection hole (12) are both connected to the cell culture chamber (21), and wherein the intermediate chip (2) is provided with a culture fluid peripheral channel (22), a culture fluid perfusion channel (23) and a bile fluid perfusion channel (24), wherein the culture fluid perfusion channel (23) and the bile fluid perfusion channel (24) are both provided with The cell culture chamber (21) is connected to the culture fluid perfusion channel (23), and the bile perfusion channel (24) is connected to the cell culture chamber (21); each cell culture chamber (21) is adjacent to each other, and each cell culture chamber (21) is separated from the adjacent cell culture chamber (21) on both sides by the culture fluid perfusion channel (23) and the bile perfusion channel (24), respectively, and the culture fluid perfusion channel (23) and the bile perfusion channel (24) are both permeable membrane structures; the width of the culture fluid perfusion channel (23) is uneven, and the width of the bile perfusion channel (24) is uneven; the widest part of the bile perfusion channel (24) is thinner than the narrowest part of the culture fluid perfusion channel (23).

2. The liver organ chip model according to claim 1, characterized in that The sample addition hole (11) extends from the top layer chip (1) to the middle layer chip (2).

3. The liver organ chip model according to claim 1, characterized in that The sample injection hole (11) includes any one or more of a cell injection hole (111), a culture fluid injection hole (112), and a bile fluid injection hole (113); the cell injection hole (111) is directly connected to the cell culture chamber (21); the culture fluid injection hole (112) is connected to the cell culture chamber (21) via a culture fluid peripheral channel (22) and a culture fluid perfusion channel (23) in sequence; and the bile fluid injection hole (113) is connected to the cell culture chamber (21) via a bile fluid perfusion channel (24).

4. The liver organ chip model according to claim 3, characterized in that The cell injection hole (111) has one or more of the following characteristics: 1) The number of the cell injection holes (111) is equal to the number of the cell culture chambers (21); 2) The arrangement of the cell injection holes (111) matches that of the cell culture chamber (21); 3) The position of the cell injection hole (111) corresponds to the periphery of the cell culture chamber (21).

5. The liver organ chip model according to claim 3, characterized in that There are multiple culture solution injection holes (112).

6. The liver organ chip model according to claim 3, characterized in that There are two culture solution injection holes (112).

7. The liver organ chip model according to claim 5, characterized in that Each of the culture fluid injection holes (112) is connected to a plurality of culture fluid perfusion channels (23).

8. The liver organ chip model according to claim 3, characterized in that The number of the bile injection holes (113) matches the number of the cell culture chambers (21).

9. The liver organ chip model according to claim 3, characterized in that A bile injection hole (113) is provided between every two adjacent cell culture chambers (21).

10. The liver organ chip model according to claim 1, characterized in that The collection hole (12) includes a culture fluid collection hole (121) and a bile fluid collection hole (122). The culture fluid collection hole (121) extends from the top chip (1) to the middle chip (2), and the culture fluid collection hole (121) is connected to the culture fluid injection hole (112) via the culture fluid perfusion channel (23) and the culture fluid peripheral channel (22); the bile fluid collection hole (122) extends from the top chip (1) to the bottom chip (3), and the bile fluid collection hole (122) is connected to the bile fluid injection hole (113).

11. The liver organ chip model according to claim 10, characterized in that The culture fluid collection hole (121) is provided with one; and / or the bile fluid collection hole (122) is provided with multiple.

12. The liver organ chip model according to claim 10, characterized in that There are two bile collection holes (122).

13. The liver organ chip model according to claim 1, characterized in that The cell culture chambers (21) are provided in plurality.

14. The liver organ chip model according to claim 1, characterized in that There are 12 cell culture chambers (21).

15. The liver organ chip model according to claim 14, characterized in that: Twelve cell culture chambers (21) are adjacent to each other to form a regular hexagonal prism shape.

16. The liver organ chip model according to claim 1, characterized in that The culture fluid perfusion channel (23) is composed of a PC membrane and a PDMS membrane; and / or the bile fluid perfusion channel (24) is composed of a PDMS membrane.

17. The liver organ chip model according to claim 1, characterized in that The culture fluid perfusion channel (23) is composed of a PC membrane and a PDMS membrane, wherein the pore size of the PDMS membrane is 0.2-20 μm; the pore size of the PC membrane is 0.2-20 μm.

18. The liver organ chip model according to claim 1, characterized in that The bile perfusion channel (24) is composed of a PDMS membrane, and the pore size of the PDMS membrane is 0.2-20 μm.

19. The liver organ chip model according to claim 1, characterized in that The culture fluid perfusion channel (23) is wider near the culture fluid collection hole, and narrower far from the culture fluid collection hole; and / or, The bile perfusion channel (24) is narrower near the bile injection hole and wider far from the bile injection hole.

20. The liver organ chip model according to claim 10, characterized in that A bile drainage hole (25) is provided at the end of the bile perfusion channel (24), and the bile drainage hole (25) extends from the middle layer chip (2) to the bottom layer chip (3).

21. The liver organ chip model according to claim 20, characterized in that A bile liquid collection channel (26) is provided on the bottom chip (3), one end of the bile liquid collection channel (26) is connected to the bile liquid collection hole (122), and the other end is connected to each bile liquid drainage hole (25).

22. The liver organ chip model according to claim 21, characterized in that The plurality of bile drainage holes (25) are connected to the same bile collection hole (122).

23. The liver organ chip model according to claim 1, characterized in that The top layer chip (1), the middle layer chip (2), and the bottom layer chip (3) have the same lengths, and the top layer chip (1), the middle layer chip (2), and the bottom layer chip (3) have the same widths.

24. Use of the liver organ chip model according to any one of claims 1 to 23 in drug development or nutritional research.

25. The use according to claim 24, characterized in that The use is in studying the interaction between drugs and liver.

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