Bionic liver chip containing bile duct and application thereof
By adding bile duct cells and bile duct simulation structures to the liver chip, the interaction between hepatobiliary cells is achieved, and the problem of ignoring bile duct structure in existing liver chips is solved, which improves the authenticity and accuracy of the in vitro liver model, and is suitable for a variety of biomedical research.
Patent Information
- Application Number
- CN202411753303.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-02
- Publication Date
- 2025-03-28
AI Technical Summary
The existing liver chip design ignores the interaction between bile duct cells and liver cells and the secretion and collection of bile, resulting in insufficient authenticity and accuracy of the in vitro liver model, making it difficult to effectively simulate liver diseases and drug metabolism processes.
A bionic liver chip containing bile ducts was designed, and the bile duct simulated structure was added to the bile duct cells and the bile duct simulated structure to achieve the interaction between hepatobiliary cells through the bile duct/hepatocyte U-type channel to simulate the secretion and collection of bile.
It improves the authenticity and accuracy of the in vitro liver model, can better simulate the interaction between tissues in the liver, and is suitable for research such as liver disease modeling, drug toxicity detection and drug efficacy evaluation.
Smart Images
Figure CN119709399A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of microfluidics technology, and in particular to a bionic liver chip comprising a bile duct and applications thereof. Background Art
[0002] Organ-on-a-chip is a micro-culture device based on microfluidic chips that simulates the specific structure and physiological function of complex human tissues or organs on a chip of a few square centimeters. This technology combines microfluidic platforms with in vitro cell culture technology to create advanced in vitro models of multi-cell / tissue co-culture, which can highly reproduce the physiological or pathological microenvironment at the organ level and is used for disease modeling, drug safety evaluation and drug efficacy screening, etc., providing a bionic platform for promoting basic research and transformation in biomedicine.
[0003] The liver is the central hub of exogenous metabolism in the human body and is particularly susceptible to drug toxicity. Drug-induced liver injury is reported to be the most common reason for the withdrawal of approved drugs. Traditional preclinical and clinical testing stages rely on 2D cell culture and animal experiments. The former has a simple structure and cannot reproduce the complex microenvironment under physiological conditions. As an in vitro liver culture system, the liver chip shows great potential in drug efficacy evaluation and safety prediction. Traditional 2D cell culture lacks cell-cell interactions and is usually unable to effectively predict physiologically relevant drug efficacy and toxicity characteristics. In contrast, microfluidic cell culture tools that can load multiple cells at the same time solve this problem well and provide a more accurate in vitro model in terms of physiology. Animals metabolize and respond to drugs differently from humans, and cannot truly represent the physiological, pathological and genetic characteristics of humans. The results of their preclinical experimental predictions are often inconsistent with the actual clinical application effects. Compared with animal models, liver chips also have the advantages of high-throughput bioassays and large-scale drug screening.
[0004] The currently disclosed liver chip platform is mainly used in the fields of toxicology research and drug development. For example, a disclosed 3D liver chip is used to test the effects of chemicals on the liver, so as to evaluate the toxicity, metabolic process and mechanism of action of these substances in the liver. The design and construction of existing liver chips mainly focus on simulating the microenvironment of hepatic cord-stellate cell gap-hepatic sinusoids. Few bile duct cells and bile duct structures are taken into account, ignoring the interaction between hepatocytes and bile duct cells in the liver and the secretion and collection of bile. Biliary cells are highly specialized cells in the liver. Although they only account for 5% of liver cells, they play a key role in liver regeneration, especially when hepatocyte regeneration is impaired. Biliary cells are involved in the metabolism and secretion of bile, and are related to tissue damage and repair. In addition, they can secrete proinflammatory factors and chemokines and have immune barrier function. Biliary cells actively participate in bile duct mucosal immunity by interacting with lymphocytes and Kupffer cells in the liver, and constitute part of the overall immunity of the liver. In chronic liver diseases induced by multiple factors such as alcoholic fatty liver or viral infection, bile duct cells can protect the liver by enhancing liver regeneration. In addition, the occurrence of liver disease can affect the structure and function of bile duct cells, leading to cholestasis, which in turn induces liver inflammation, fibrosis, and liver dysfunction. Therefore, we believe that it is necessary to add bile duct cells and construct a bile duct structure that conforms to physiological characteristics in the design of liver chips, which is of great significance for research such as liver disease modeling and drug screening.
[0005] In summary, there are few studies on the interaction between bile duct cells and hepatocytes in the currently published liver chips, but bile secretion plays a vital role in the fat decomposition and toxic metabolism of the liver. Adding bile duct structures to the liver chip can further improve the authenticity and accuracy of the liver in vitro model. Therefore, the technical field urgently needs a bile duct liver chip that contains a bile duct structure and can reproduce the interaction between the internal tissues of the liver, providing a more bionic human liver model for toxicity testing and drug efficacy evaluation. Summary of the invention
[0006] In order to solve the above technical problems, the present invention provides a bionic liver chip containing bile duct and its application. The liver chip of the present invention simulates the physiological structure of the liver and adds bile duct cells and a bile duct simulation structure for collecting bile. The bile duct simulation structure can realize the interaction between hepatobiliary cells, making the in vitro liver system more bionic, so as to better serve biomedical related research, including liver disease modeling, drug toxicity detection and drug efficacy evaluation.
[0007] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a bionic liver chip including a bile duct, which includes a substrate; a surface of the substrate is provided with: Central venous pool; The portal vein channel and the proper hepatic artery channel are connected to the central venous pool respectively; A bile duct / hepatocyte U-shaped channel located between the hepatic portal vein channel and the hepatic artery channel; the bile duct / hepatocyte U-shaped channel includes a hepatocyte channel A and a hepatocyte channel B constituting both ends of the U-shaped channel and a bile duct cell channel constituting the middle section of the U-shaped channel; both ends of the bile duct cell channel are connected to the two hepatocyte channels through a plurality of connecting channels; A stellate cell channel A is located between the portal vein channel and the hepatocyte channel A; the two sides of the stellate cell channel A are respectively connected with the portal vein channel and the hepatocyte channel A through a plurality of connecting channels; A stellate cell channel B is located between the proper hepatic artery channel and the hepatocyte channel B; the two sides of the stellate cell channel B are connected to the proper hepatic artery channel and the hepatocyte channel B respectively through a number of connecting channels.
[0008] The liver is the largest gland in the human body, with multiple functions such as detoxification, metabolism and bile secretion. The hepatic lobule is the basic unit of liver structure and function. It is a polygonal prism with a typical hexagonal cross section. There are about 500,000 to 1 million hepatic lobules in an adult liver. Hepatocytes are arranged in a cord-like manner from the center of the hepatic lobule to the outside to form hepatic cords. Adjacent hepatic cords are separated by hepatic sinusoids composed of endothelial cells. Hepatic stellate cells are distributed in the gaps between hepatocytes and hepatic sinusoids. The blood from the portal vein (~75%) and the oxygenated blood from the hepatic artery (~25%) merge at the vertices of the hexagon to form hepatic sinusoids. After exchanging substances with hepatocytes, they flow into the central vein in the center of the hepatic lobule and then into the inferior vena cava to leave the liver. The tight junctions of the apical membranes of two adjacent hepatocytes are concave downward to form bile ducts, which collect bile secreted by hepatocytes. The bile flows in the opposite direction of the blood flow and eventually flows into the bile duct to leave the liver.
[0009] As described in the background technology section of this application, the design and construction of existing liver chips mainly focuses on simulating the microenvironment of hepatic cords-stellate cell gaps-hepatic sinusoids, and rarely takes bile duct cells and bile duct structures into consideration, ignoring the interaction between hepatocytes and bile duct cells in the liver and the secretion and collection of bile. However, bile secretion plays a vital role in the fat decomposition and toxic metabolism of the liver, so adding bile duct structures to the liver chip can further improve the authenticity and accuracy of the liver in vitro model.
[0010] To this end, the present invention provides a liver chip with a bile duct cell channel as described above. The liver chip of the present invention adds bile duct cells and a bile duct simulation structure for collecting bile on the basis of simulating the interaction structure of vascular endothelial cells-hepatic stellate cells-hepatocytes. The bile duct simulation structure is a bile duct / hepatocyte U-shaped channel, which includes a hepatocyte channel A, a hepatocyte channel B and a bile duct cell channel constituting the middle section of the U-shaped channel at both ends of the U-shaped channel; the two ends of the bile duct cell channel are connected to the two hepatocyte channels through a number of connecting channels. The above-mentioned bile duct / hepatocyte U-shaped channel can receive bile secreted by hepatocytes, and the bile collection direction in the liver chip of the present invention is opposite to the direction of arterial and venous blood, which is consistent with the physiological conditions of the human body. Therefore, the bile duct / hepatocyte U-shaped channel structure can realize the interaction between hepatobiliary cells, can highly simulate the physiological microenvironment of human bile ducts and hepatocytes, make the in vitro liver system more biomimetic, and thus can better serve biomedical related research, including liver disease modeling, drug toxicity detection and drug efficacy evaluation.
[0011] Preferably, the cross-sectional area of the portal vein channel is 2.5-3.5 times that of the proper hepatic artery channel.
[0012] Limiting the cross-sectional area ratio of the portal vein channel and the proper hepatic artery channel (i.e., the fluid flow ratio) within the above range is used to highly simulate the blood flow ratio and blood flow direction of the inlet and outlet hepatic arteries and veins.
[0013] Preferably, the cross-sectional area of each connecting channel is smaller than the size of the hepatocytes and bile duct cells (ie, the hepatocytes and bile duct cells cannot flow through the connecting channel, thereby achieving zoned culture). Further preferably, the width of each adjacent connecting channel is 5-10 μm.
[0014] Designing the cross-sectional area of the connecting channel to be smaller than the size of hepatocytes and bile duct cells can ensure that after the injection of hepatocyte and bile duct cell suspensions, the two will grow adherently in their respective channels, and small molecules such as cell secretions can freely pass through the connecting channel, thereby achieving independent growth and interaction between cells with a higher degree of simulation.
[0015] Preferably, the middle position of the bile duct cell channel is the bile duct cell channel entrance, and the two ends are the bile duct cell channel exit / hepatocyte channel A entrance and the bile duct cell channel exit / hepatocyte channel B entrance, respectively.
[0016] Preferably, the end of the hepatocyte channel A (ie, the end away from the bile duct cell channel) is the hepatocyte channel A outlet.
[0017] Preferably, the end of the hepatocyte channel B (ie, the end away from the bile duct cell channel) is the hepatocyte channel B outlet.
[0018] Preferably, the end of the portal vein channel away from the central venous pool is the entrance of the portal vein channel.
[0019] Preferably, the end of the proper hepatic artery channel away from the central venous pool is the entrance of the proper hepatic artery channel.
[0020] Preferably, the two ends of the stellate cell channel A and the stellate cell channel B are their outlet and inlet respectively, and their flow directions are the same as those of the two hepatocyte channels, the portal vein channel and the proper hepatic artery channel.
[0021] Preferably, the bionic liver chip further comprises a cover plate; the cover plate is bonded to a surface of a side of the base plate where the channel is provided.
[0022] Further preferably, the cover plate is provided with through holes which are respectively connected to the portal vein channel entrance, the hepatic artery channel entrance, the bile duct cell channel entrance, the bile duct cell channel A exit / hepatocyte channel A entrance, the hepatocyte channel A exit, the bile duct cell channel B exit / hepatocyte channel B entrance, the hepatocyte channel B exit, the stellate cell channel A entrance, the stellate cell channel A exit, the stellate cell channel B entrance and the stellate cell channel B exit.
[0023] Further preferably, the area on the cover plate corresponding to the central venous pool is a hollow structure.
[0024] Preferably, the material of each component in the bionic liver chip is PDMS.
[0025] In a second aspect, the present invention provides the use of the bionic liver chip in simulating the liver to conduct drug toxicity testing.
[0026] In a third aspect, the present invention provides the use of the bionic liver chip in simulating the liver for drug efficacy evaluation.
[0027] In a fourth aspect, the present invention provides the use of the bionic liver chip in simulating the liver for liver disease modeling.
[0028] Compared with the prior art, the beneficial effects of the present invention are: (1) The bionic liver chip of the present invention simulates the physiological structure of the liver and adds bile duct cells and a bile duct simulation structure for collecting bile. The bile duct simulation structure can realize the interaction between hepatobiliary cells, making the in vitro liver system more bionic, so as to better serve biomedical related research, including liver disease modeling, drug toxicity detection and efficacy evaluation.
[0029] (2) In the bionic liver chip of the present invention, the cross-sectional area of the portal vein channel is designed to be 2.5-3.5 times that of the hepatic artery channel, which can highly simulate the blood flow ratio and blood flow direction of the inlet and outlet hepatic arteries and veins.
[0030] (3) In the bionic liver chip of the present invention, the cross-sectional area of the connecting channel is designed to be smaller than the size of hepatocytes and bile duct cells, which can ensure that after the injection of hepatocyte and bile duct cell suspensions, the two cells grow adherently in their respective channels, and small molecules such as cell secretions can freely pass through the connecting channel, thereby achieving independent growth and interaction of the two cells with a higher degree of simulation. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 The diagram is a schematic diagram of the explosion structure of the liver bionic chip of the present invention.
[0032] Figure 2 for Figure 1 A plan view of the substrate structure is shown.
[0033] Figure 3 This is a diagram of the basic unit of the liver's physiological structure.
[0034] Figure 4 Schematic diagram of the substrate structure in Test Example 1.
[0035] Figure 5 Schematic diagram of the substrate structure in Test Example 2.
[0036] Figure 6 Schematic diagram of the substrate structure in Example 1.
[0037] Figure 7 This is a schematic diagram of the complete structure of the base plate and the cover plate after being assembled up and down in Example 1.
[0038] The figures are marked as follows: 1 substrate; 2 cover plate; 3 portal vein channel entrance; 4 bile duct cell channel entrance; 5 bile duct cell channel exit / hepatocyte channel A entrance; 6 hepatocyte channel A exit; 7 bile duct cell channel exit / hepatocyte channel B entrance; 8 hepatocyte channel B exit; 9 stellate cell channel A entrance; 10 stellate cell channel A exit; 11 stellate cell channel B entrance; 12 stellate cell channel B exit; 13 proper hepatic artery channel entrance; 14 central venous cistern; 15 portal vein channel; 16 proper hepatic artery channel; 171 hepatocyte channel A; 172 hepatocyte channel B; 18 bile duct cell channel; 19 stellate cell channel; 191 stellate cell channel A; 192 stellate cell channel B; 20 bile duct / hepatocyte U-shaped channel. DETAILED DESCRIPTION
[0039] The present invention will be further described below in conjunction with the embodiments.
[0040] Overall embodiment In a first aspect, the present invention provides a bionic liver chip comprising a bile duct, such as Figure 1 As shown, it includes a substrate 1; the surface of the substrate 1 is provided with: Central venous pool 14; a hepatic portal vein channel 15 and a hepatic artery channel 16 respectively connected to the central venous pool 14; A bile duct / hepatocyte U-shaped channel 20 located between the portal vein channel 15 and the proper hepatic artery channel 16; the bile duct / hepatocyte U-shaped channel 20 includes a hepatocyte channel A 171 and a hepatocyte channel B 172 constituting two ends of the U-shaped channel and a bile duct cell channel 18 constituting a middle section of the U-shaped channel; two ends of the bile duct cell channel 18 are respectively connected to the two hepatocyte channels through a plurality of connecting channels; A stellate cell channel A 191 located between the portal vein channel 15 and the hepatocyte channel A 171; the stellate cell channel A 191 is connected to the portal vein channel 15 and the hepatocyte channel A 171 on both sides through a plurality of connecting channels; A stellate cell channel B 192 is located between the proper hepatic artery channel 16 and the hepatocyte channel B 172; the two sides of the stellate cell channel B 192 are respectively connected with the proper hepatic artery channel 16 and the hepatocyte channel B 172 through a plurality of connecting channels.
[0041] In some preferred implementation cases, the cross-sectional area of the portal vein channel 15 is 2.5-3.5 times that of the proper hepatic artery channel 16 .
[0042] In some preferred embodiments, the cross-sectional area of each connecting channel is smaller than the size of hepatocytes and bile duct cells (ie, hepatocytes and bile duct cells cannot flow through the connecting channel).
[0043] In some more preferred embodiments, the width of each adjacent connecting channel is 5-10 μm.
[0044] In some preferred implementation cases, the middle position of the bile duct cell channel 18 is the bile duct cell channel entrance 4, and the two ends are the bile duct cell channel exit / hepatocyte channel A entrance 5 and the bile duct cell channel exit / hepatocyte channel B entrance 7 respectively.
[0045] In some preferred implementation cases, the end of the hepatocyte channel A 171 (ie, the end away from the bile duct cell channel 18 ) is the hepatocyte channel A outlet 6 .
[0046] In some preferred implementation cases, the end of the hepatocyte channel B 172 (i.e., the end away from the bile duct cell channel 18) is the outlet of the hepatocyte channel B 8. In some preferred implementation cases, the end of the portal vein channel 15 away from the central venous pool 14 is the portal vein channel entrance 3.
[0047] In some preferred implementation cases, the end of the proper hepatic artery channel 16 away from the central venous pool 14 is the proper hepatic artery channel entrance 13 .
[0048] In some preferred implementation cases, the two ends of the stellate cell channel A 191 and the stellate cell channel B 192 are their outlet and inlet, respectively, and their flow directions are the same as those of the two hepatocyte channels, the portal vein channel 15 and the proper hepatic artery channel 16.
[0049] In some preferred implementation cases, the bionic liver chip further includes a cover plate 2; the cover plate 2 is bonded to the surface of one side of the substrate 1 having the channel; in some more preferred implementation cases, the cover plate 2 is provided with through holes respectively connected to the portal vein channel inlet 3, the proper hepatic artery channel inlet 13, the bile duct cell channel inlet 4, the bile duct cell channel A outlet / hepatocyte channel A inlet 5, the hepatocyte channel A outlet 6, the bile duct cell channel B outlet / hepatocyte channel B inlet 7, the hepatocyte channel B outlet 8, the stellate cell channel A inlet 9, the stellate cell channel A outlet 10, the stellate cell channel B inlet 11 and the stellate cell channel B outlet 12. In some more preferred implementation cases, the area on the cover plate 2 corresponding to the central venous pool 14 is a hollow structure.
[0050] In some preferred implementation cases, the material of each component in the bionic liver chip is PDMS.
[0051] In a second aspect, the present invention provides the use of the bionic liver chip in simulating the liver to conduct drug toxicity testing.
[0052] In a third aspect, the present invention provides the use of the bionic liver chip in simulating the liver for drug efficacy evaluation.
[0053] In a fourth aspect, the present invention provides the use of the bionic liver chip in simulating the liver for liver disease modeling.
[0054] Specific test examples and implementation examples Test Example 1 A bionic liver chip containing a bile duct, wherein the channel structure on the substrate is based on Figure 3 The physiological structure of the basic unit of the liver is preliminarily designed. Figure 4 As shown, the substrate surface includes a bile duct / hepatocyte U-shaped channel 20, a stellate cell channel 19, a hepatic portal vein channel 15, a hepatic artery channel 16, and a central vein pool 14. However, during the application of the bionic liver chip, it was found that the hepatic artery channel 16 could not interact with the bile duct / hepatocyte U-shaped channel 20.
[0055] Test Example 2 In the bionic liver chip of test example 1, the hepatic artery channel 16 cannot interact with the bile duct / hepatocyte U-shaped channel 20. Therefore, the present invention provides an optimized bionic liver chip after optimization on the basis. Figure 5 As shown, the present invention adds an additional bile duct / hepatocyte U-shaped channel 20 and a stellate cell channel 19 on one side of the hepatic artery channel 16. In the bionic liver chip, the interaction between the three tissues of artery and vein, hepatocyte and bile duct cell can be realized.
[0056] Example 1 In order to further simplify the structure of the bionic liver chip for better practical application, the present invention further optimizes the design of the bionic liver chip in Test Example 2 while ensuring the integrity of the basic structure and functions. Specifically, the position of the bile duct / hepatocyte U-shaped channel 20 is optimized, thereby obtaining the following: Figure 1 as well as Figure 6-7 The structure of the bionic liver chip shown is as follows: A bionic liver chip containing a bile duct comprises a substrate 1 and a cover plate 2. Wherein: The surface of the substrate 1 is grooved with: a central vein pool 14, a portal vein channel 15, a proper hepatic artery channel 16, a bile duct / hepatocyte U-shaped channel 20, a stellate cell channel A 191 and a stellate cell channel B 192. Specifically: The portal vein channel 15 and the hepatic artery channel 16 are respectively connected to the central venous pool 14; the end of the portal vein channel 15 away from the central venous pool 14 is the portal vein channel entrance 3; the end of the hepatic artery channel 16 away from the central venous pool 14 is the hepatic artery channel entrance 13.
[0057] The bile duct / hepatocyte U-shaped channel 20 is located between the hepatic portal vein channel 15 and the hepatic artery channel 16; the bile duct / hepatocyte U-shaped channel 20 includes a hepatocyte channel A 171 and a hepatocyte channel B 172 constituting the two ends of the U-shaped channel and a bile duct cell channel 18 constituting the middle section of the U-shaped channel; the two ends of the bile duct cell channel 18 are connected to the hepatocyte channel A 171 and the hepatocyte channel B 172 through a plurality of connecting channels. In the bile duct / hepatocyte U-shaped channel 20, the middle position of the bile duct cell channel 18 is the bile duct cell channel entrance 4, and the two ends are the bile duct cell channel exit / hepatocyte channel A entrance 5 and the bile duct cell channel exit / hepatocyte channel B entrance 7; the end of the hepatocyte channel A 171 (i.e., the end away from the bile duct cell channel 18) is the hepatocyte channel A exit 6; the end of the hepatocyte channel B 172 (i.e., the end away from the bile duct cell channel 18) is the hepatocyte channel B exit 8.
[0058] The stellate cell channel A 191 is located between the portal vein channel 15 and the hepatocyte channel A 171; the two sides of the stellate cell channel A 191 are respectively connected to the portal vein channel 15 and the hepatocyte channel A 171 through a plurality of connecting channels; the stellate cell channel B 192 is located between the proper hepatic artery channel 16 and the hepatocyte channel B 172; the two sides of the stellate cell channel B 192 are respectively connected to the proper hepatic artery channel 16 and the hepatocyte channel B 172 through a plurality of connecting channels. The two ends of the stellate cell channel A 191 are respectively the stellate cell channel A outlet 9 and the stellate cell channel A inlet 10, and the two ends of the stellate cell channel B 192 are respectively the stellate cell channel B outlet 11 and the stellate cell channel B inlet 12; and the flow directions of the stellate cell channel A 191 and the stellate cell channel B 192 are the same as those of the hepatocyte channel A 171, the hepatocyte channel B 172, the portal vein channel 15 and the proper hepatic artery channel 16.
[0059] The cover plate 2 is attached to the side surface of the base plate 1 where the channel is provided. Figure 2 As shown, the cover plate 2 is provided with through holes which are respectively connected to the portal vein channel inlet 3, the hepatic artery channel inlet 13, the bile duct cell channel inlet 4, the bile duct cell channel outlet / hepatocyte channel A inlet 5, the hepatocyte channel A outlet 6, the bile duct cell channel outlet / hepatocyte channel B inlet 7, the hepatocyte channel B outlet 8, the stellate cell channel A inlet 9, the stellate cell channel A outlet 10, the stellate cell channel B inlet 11 and the stellate cell channel B outlet 12, and at the same time, the area on the cover plate 2 corresponding to the central venous pool 14 is a hollow structure.
[0060] The bionic liver chip with bile duct cell channel finally provided in Example 1 of the present invention. The bionic liver chip of the present invention adds bile duct cells and a bile duct simulation structure for collecting bile on the basis of simulating the interaction structure of vascular endothelial cells-hepatic stellate cells-hepatocytes. The bile duct simulation structure is a bile duct / hepatocyte U-shaped channel, which includes a hepatocyte channel A, a hepatocyte channel B and a bile duct cell channel constituting the middle section of the U-shaped channel at both ends of the U-shaped channel; the two ends of the bile duct cell channel are connected to the two hepatocyte channels through a number of connecting channels. The above-mentioned bile duct / hepatocyte U-shaped channel can receive bile secreted by hepatocytes, and the bile collection direction in the bionic liver chip of the present invention is opposite to the direction of arterial and venous blood, which is consistent with the physiological conditions of the human body. Therefore, the bile duct / hepatocyte U-shaped channel structure can realize the interaction between hepatobiliary cells, can highly simulate the physiological microenvironment of human bile ducts and hepatocytes, make the in vitro liver system more bionic, and thus can better serve biomedical related research, including liver disease modeling, drug toxicity detection and efficacy evaluation.
[0061] Regarding the dimensions of the above-mentioned substrate 1 and cover plate 2: The base plate and the cover plate are both rectangular, with dimensions of 30 mm*50 mm*1 mm. The depth of various channels on the surface of the base plate 1 is 0.1 mm. The length of the portal vein channel 15 is 20 mm and the width is 3 mm. The length of the two hepatocyte channels and the stellate cell channel are 10 mm and the width is 1.5 mm. The bile duct cell channel 18 is a U-shaped channel composed of concentric semicircles with a diameter of 4 mm and a diameter of 2.5 mm, with a central arc length of about 10 mm and a width of 1.5 mm. The length of the proper hepatic artery channel 16 is 20 mm and the width is 1 mm. The length of a single connecting channel is 0.05 mm, the width is 0.01 mm, and the depth is 0.1 mm.
[0062] The central venous pool 14 is a cylindrical cavity with a diameter of 8 mm, the hepatic artery channel entrance 13 is a cylinder with a diameter of 1 mm, the portal vein channel entrance 3 is a cylinder with a diameter of 3 mm, the bile duct cell channel entrance 4 is a cylinder with a diameter of 1.5 mm, the bile duct cell channel outlet / hepatocyte channel A entrance 5, the bile duct cell channel outlet / hepatocyte channel B entrance 7, the stellate cell channel A entrance 9, the stellate cell channel A outlet 10, the stellate cell channel B entrance 11, and the stellate cell channel B outlet 12 are all cylinders with a diameter of 1.5 mm.
[0063] Regarding the materials and preparation methods of the bionic liver chip containing the bile duct: The material of each component in the bionic liver chip is PDMS. It is only necessary to use soft lithography technology to make the templates of the substrate 1 and the cover plate 2, and then use PDMS to reverse the mold. Punch holes in the corresponding positions of the PDMS of the cover plate 2, and then use oxygen plasma cleaner to perform surface treatment with the substrate 1 to complete irreversible bonding. The PDMS material has good light transmittance, allowing in situ monitoring on live cell imaging platforms such as inverted fluorescence microscopes and even confocal microscopes. In addition, compared with the classic sandwich model organ chip, the design of the bionic liver chip containing the bile duct of the present invention does not require a porous membrane as an interlayer, which simplifies the bionic chip production steps.
[0064] Application Examples After the bionic liver chip containing the bile duct of Example 1 is irreversibly bonded by oxygen plasma treatment, it is first sterilized by ultraviolet for more than 40 min, and then a collagen solution is introduced into each channel for surface modification to promote better cell adhesion and growth. Specifically, the collagen solution is slowly injected into the channel through the channel entrance until a droplet is formed at the outlet, and then the entrance is also sealed with a collagen droplet, and the droplet size at both ends of the channel is ensured to be similar, and placed in an incubator overnight. On the second day, the channel is slowly rinsed with the corresponding cell culture medium to discharge excess collagen solution.
[0065] Cell inoculation: At the entrance of the portal vein channel and the entrance of the hepatic artery, slowly introduce the vascular endothelial cell (HUVEC) suspension using a syringe pump to fill the entire channel. Be careful to avoid the generation of bubbles. The cell density is 1×10 7 LX-2 cell suspension was introduced into the two stellate cell channels from the entrance of stellate cell channel A and stellate cell channel B, with a density of 5×10 6 Since the bile duct cell channel and the two hepatocyte channels are connected, when inoculating bile duct cell BIBECs in the bile duct cell channel, the outlets of the two hepatocyte channels need to be sealed first so that the bile duct cell suspension (5×10 6 cells / mL) flowed out from the bile duct cell channel outlet / hepatocyte channel A entrance and bile duct cell channel outlet / hepatocyte channel B entrance. After the inoculation was completed, the bionic liver chip was placed in an incubator for 2 h to allow the cells to adhere to the wall. Then, after the bile duct cell channel entrance was sealed, the hepatocyte (QSG7701) suspension (1×10 7 After inoculating the liver cells, the bionic liver chip was placed in the incubator again for overnight culture to allow all cells to adhere to the wall and grow.
[0066] Fluid perfusion: Before introducing fluid into the channel, observe whether the cell adhesion state in the bionic liver chip is good under a microscope. After confirming that it is good, connect the peristaltic pump to the bionic liver chip through a hose, and adjust the flow rate and perfusion time of each channel to achieve dynamic cell culture. In order to ensure that the flow ratio of the portal vein and the hepatic artery is 3:1 and minimize the number of peristaltic pumps used, a 5-channel peristaltic pump is used to provide the same flow rate to achieve dynamic culture of different cells.
[0067] Depending on the application example, hepatic macrophages (Kupffer cells) can be introduced into the portal vein channel and the hepatic artery channel to construct a viral or inflammatory liver pathology model.
[0068] Example 2 The structure of the bionic liver chip including the bile duct in Example 2 is the same as that in Example 1, except that the size is different, as follows: The base plate and the cover plate are both rectangular, with dimensions of 30 mm*50 mm*1 mm. The depth of various channels on the surface of the base plate 1 is 0.1 mm. The length of the portal vein channel 15 is 20 mm and the width is 3 mm. The length of the two hepatocyte channels and the two stellate cell channels are 10 mm and the width is 1.5 mm. The bile duct cell channel 18 is a U-shaped channel composed of concentric semicircles with a diameter of 4 mm and a diameter of 2.5 mm, with a central arc length of about 10 mm and a width of 1.5 mm. The length of the proper hepatic artery channel 16 is 20 mm and the width is 1 mm. The length of a single connecting channel is 0.05 mm, the width is 0.01 mm, and the depth is 0.05 mm.
[0069] The central venous pool 14 is a cylindrical cavity with a diameter of 8 mm, the hepatic artery channel entrance 13 is a cylinder with a diameter of 1 mm, the portal vein channel entrance 3 is a cylinder with a diameter of 3 mm, the bile duct cell channel entrance 4 is a cylinder with a diameter of 1.5 mm, the bile duct cell channel outlet / hepatocyte channel A entrance 5, the bile duct cell channel outlet / hepatocyte channel B entrance 7, the stellate cell channel A entrance 9, the stellate cell channel A outlet 10, the stellate cell channel B entrance 11, and the stellate cell channel B outlet 12 are all cylinders with a diameter of 1.5 mm.
[0070] Example 3 The structure of the bionic liver chip including the bile duct in Example 3 is the same as that in Example 1, except that the size is different, as follows: The base plate and the cover plate are both rectangular, with dimensions of 30 mm*50 mm*1 mm. The depth of various channels on the surface of the base plate 1 is 0.1 mm. The length of the portal vein channel 15 is 20 mm and the width is 3 mm. The length of the two hepatocyte channels and the two stellate cell channels are 10 mm and the width is 1.5 mm. The bile duct cell channel 18 is a U-shaped channel composed of concentric semicircles with a diameter of 4 mm and a diameter of 2.5 mm, with a central arc length of about 10 mm and a width of 1.5 mm. The length of the proper hepatic artery channel 16 is 20 mm and the width is 1 mm. The length of a single connecting channel is 0.05 mm, the width is 0.005 mm, and the depth is 0.05 mm.
[0071] The central venous pool 14 is a cylindrical cavity with a diameter of 8 mm, the hepatic artery channel entrance 13 is a cylinder with a diameter of 1 mm, the portal vein channel entrance 3 is a cylinder with a diameter of 3 mm, the bile duct cell channel entrance 4 is a cylinder with a diameter of 1.5 mm, the bile duct cell channel outlet / hepatocyte channel A entrance 5, the bile duct cell channel outlet / hepatocyte channel B entrance 7, the stellate cell channel A entrance 9, the stellate cell channel A outlet 10, the stellate cell channel B entrance 11, and the stellate cell channel B outlet 12 are all cylinders with a diameter of 1.5 mm.
[0072] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.
[0073] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.
Claims
1. A bionic liver chip comprising a bile duct, characterized in that: The invention comprises a substrate, the surface of which is provided with: Central venous pool; The portal vein channel and the proper hepatic artery channel are connected to the central venous pool respectively; A bile duct / hepatocyte U-shaped channel, comprising a hepatocyte channel A, a hepatocyte channel B and a bile duct cell channel; both ends of the bile duct cell channel are connected to the two hepatocyte channels through connecting channels respectively; The stellate cell channel A is connected to the portal vein channel and the hepatocyte channel A via connecting channels on both sides; The stellate cell channel B is connected to the hepatic artery channel and the hepatocyte channel B via connecting channels on both sides.
2. The bionic liver chip according to claim 1, characterized in that: The cross-sectional area of the portal vein channel is 2.5-3.5 times that of the proper hepatic artery channel.
3. The bionic liver chip according to claim 1, characterized in that: The cross-sectional area of each connecting channel is smaller than the size of hepatocytes and cholangiocytes.
4. The bionic liver chip according to claim 1, characterized in that: The middle position of the bile duct cell channel is the bile duct cell channel entrance, and the two ends are the bile duct cell channel exit / hepatocyte channel A entrance and the bile duct cell channel exit / hepatocyte channel B entrance respectively; The end of the hepatocyte channel A is the hepatocyte channel A outlet; The end of the hepatocyte channel B is the hepatocyte channel B outlet.
5. The bionic liver chip according to claim 1 or 4, characterized in that: The bile duct / hepatocyte U-shaped channel is located between the portal vein channel and the proper hepatic artery channel; The stellate cell channel A is located between the portal vein channel and the hepatocyte channel A; The stellate cell channel B is located between the proper hepatic artery channel and the hepatocyte channel B.
6. The bionic liver chip according to claim 4, characterized in that: The two ends of the stellate cell channel A and the stellate cell channel B are their outlet and inlet respectively, and their flow directions are the same as those of the two hepatocyte channels, the portal vein channel and the proper hepatic artery channel.
7. The bionic liver chip according to claim 6, characterized in that: It also includes a cover plate; the cover plate is bonded to a surface of one side of the base plate where the channel is provided.
8. The bionic liver chip according to claim 7, characterized in that: The cover plate is provided with through holes which are respectively connected to the portal vein channel entrance, the hepatic artery channel entrance, the bile duct cell channel A exit / hepatocyte channel A entrance, the hepatocyte channel A exit, the bile duct cell channel B exit / hepatocyte channel B entrance, the hepatocyte channel B exit, the stellate cell channel A entrance, the stellate cell channel A exit, the stellate cell channel B entrance and the stellate cell channel B exit.
9. The bionic liver chip according to claim 7, characterized in that: The area on the cover plate corresponding to the central venous pool is a hollow structure.
10. Use of the bionic liver chip according to any one of claims 1 to 9 in simulating the liver for liver disease modeling, drug toxicity testing or drug efficacy evaluation.