Bionic liver lobule, preparation method therefor, and use thereof

Abstract

A bionic liver lobule, a preparation method therefor, and a use thereof. A 3D stamping mold is used to carve a cultured liver cell sheet into a liver lobule membrane having a hexagonal hollow cross-section. The 3D stamping mold comprises a stamp body, a push-pull apparatus, and a push rod head. The 3D stamping mold is used to stamp several carved liver lobule membranes layer by layer, and vascular endothelial cells infiltrate same to form a bionic liver lobule having a certain height, cell density, and bionic function. The bionic liver lobule can be used to prepare liver chips for drug screening and can also be used to treat acute liver failure.

Description

A bionic liver lobule and its preparation method and application Technical Field

[0001] The present invention belongs to the field of medical bionics, and in particular relates to a bionic liver lobule and a preparation method and application thereof. Background Art

[0002] Engineered livers have long been considered promising for the treatment of liver diseases. Generally speaking, the human liver is composed of tiny radially shaped hepatic lobules combined with a network of blood vessels and biliary ducts. To mimic the liver structure, many strategies have been developed to create three-dimensional (3D) liver models. Generally, by applying geometric patterning of material cues, human parenchymal cells, stromal cells, and even vascular cells can be arranged into specific grid structures to mimic the interstitial microstructure and achieve cell distribution and some liver functions. In particular, bioprinting and other tissue engineering technologies can construct structures with three-dimensional structures and further realize vascular networks and microcirculatory systems. Despite many successes, most current studies are based on simple imitation of geometric structures, making it difficult to construct high-density compositions with cell stacking and multi-cellular structural interactions, resulting in unsatisfactory results in liver tissue engineering. Therefore, achieving the simultaneous construction of cell types, density, and distribution remains an important direction in liver tissue engineering research.

[0003] Summary of the Invention

[0004] The purpose of the present invention is to provide a bionic liver lobule and its preparation method and application, which can be used to prepare a liver chip for drug screening and can also be used to treat acute liver failure.

[0005] To achieve the above object, the technical solution provided by the present invention is:

[0006] A method for preparing a bionic liver lobule comprises using a 3D imprinting mold to carve cultured liver cell sheets into hexagonal liver lobule membrane sheets with a hollow cross-section. The 3D imprinting mold is used to imprint several carved liver lobule membrane sheets layer by layer, and vascular endothelial cells are infiltrated into them to form a bionic liver lobule with a certain height, cell density and bionic function.

[0007] The hepatocytes in the hepatocyte sheet are hiPSC-Heps. The hepatocytes are seeded onto a cell culture dish grafted with a temperature-responsive polymer and cultured under conventional adherent conditions to generate a hepatocyte sheet.

[0008] When the temperature was lowered from 37°C to 20°C, the cultured hepatocyte sheets detached from the temperature-responsive polymer-grafted cell culture dish.

[0009] After being cultured in vitro for 1-4 hours, the layers of the hepatic lobule membrane sheets printed layer by layer gradually fuse together; human umbilical vein endothelial cells are perfused into the hollow middle lumen of the bionic hepatic lobule.

[0010] Macrophages and hepatic stellate cells were also added during the hepatocyte culture, and hiPSC-Heps, macrophages and hepatic stellate cells were mixed and cultured in proportion in a cell culture dish grafted with a temperature-responsive polymer; the mixing ratios of hiPSC-Heps, macrophages and hepatic stellate cells were 67.86%, 30.16% and 1.98% respectively in terms of cell number percentage.

[0011] The 3D imprinting mold includes a mold body, a push-pull device and a push rod head; the mold body includes a cylinder with a regular hexagonal cross-section and a hexagonal solid column built into the cylinder, one end of the solid column is fixedly connected to the cylinder through a mounting seat, and the mounting seat is provided with three holes; the push-pull device includes a push-pull disk and three cylindrical rods fixed on the push-pull disk, and the push-pull disk has a hexagonal hollow structure; the push rod head is provided with three circular grooves; when the push-pull device and the push rod head are assembled, the cylindrical rods of the push-pull device pass through the hexagonal solid column of the mold body at intervals, the hexagonal hollow structure of the push-pull disk passes through the hexagonal solid column of the mold body, and the cylindrical rods of the push-pull disk pass through the holes on the mounting seat and then are inserted into the grooves on the push rod head; the preparation material of the 3D imprinting mold is biocompatible light-curing resin.

[0012] When preparing the hepatic lobule membrane sheet, pull up the movable unit assembled from the push-pull device and the push rod head, press the mold body onto the cultured hepatocyte sheet, carve the hepatocyte sheet into a hexagonal hepatic lobule membrane sheet with a hollow cross-section, and then push the movable unit from one end of the mounting seat of the mold body to the other end to push out the hepatic lobule membrane sheet; when several hepatic lobule membrane sheets are imprinted layer by layer, stack the carved hepatic lobule membrane sheets in the mold body, and use the movable unit to push and squeeze them in the mold body.

[0013] The present invention also protects the bionic liver lobule prepared by the method.

[0014] The present invention also protects the application of the bionic liver lobule in drug screening, wherein drugs are delivered to the bionic liver lobule, and drug screening is performed based on the response of the bionic liver lobule.

[0015] The present invention also protects the application of the bionic liver lobule in drug screening. The prepared bionic liver lobule is connected to a concentration gradient module. The concentration gradient module has multiple channels, and the drug outlet of each channel is connected to an independent bionic liver lobule to construct a bionic liver lobule chip; drug screening is carried out based on the reaction after the drug solution flowing out of the channel is perfused into the bionic liver lobule.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The research of the present invention includes constructing a liver lobule structural functional unit through 3D imprinting technology, preparing a bionic liver lobule with a multi-layer, multi-scale bionic liver lobule membrane, functional verification of the multi-layer bionic liver lobule structure, construction of a bionic liver lobule chip, and studying the role of the constructed bionic liver lobule structure in the treatment of acute liver failure.

[0018] The present invention uses a developed 3D imprinting mold to carve normally cultured liver cell material into a hexagonal hollow cross-section. Through layer-by-layer imprinting, the device ultimately produces a 3D liver lobule assembly with a certain height, high cell density, and complete cell interactions in the vertical direction. Endothelial cells are infiltrated into the hexagonal hollow lumen of the 3D liver lobule assembly, creating a biomimetic liver lobule with vascularized channels for nutrient diffusion and drug perfusion. Based on this vascularized biomimetic liver lobule, it is integrated into a microfluidic chip to form a liver-on-a-chip, demonstrating its functionality for drug screening.

[0019] The biomimetic liver lobule fabricated by this invention has a similar structure and function to a real liver lobule (centered around a central vein, with hepatic plates and sinusoids radiating outwards). The invention also innovatively mimics the cellular composition of the human liver, assembling a liver lobule membrane sheet composed of 22.23% hiPSC-Heps, 9.88% macrophages, and 0.65% hepatic stellate cells (HSCs), respectively. To simulate the spatial structure and composition of a single three-dimensional liver lobule, the biomimetic liver lobule fabricated by this invention can also be macroscopically magnified.

[0020] The research results show that the 3D bionic liver lobule of the present invention can provide a new method for drug screening and shows significant potential in clinical liver tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a diagram of a liver lobule membrane sheet constructed using 3D imprinting technology; in Figure 1, a is a schematic diagram of the assembly and operation process of the imprinting mold; b and c are imprinting operation images; d is a confocal image of a cell sheet (scale bar is 25 μm); e is a diagram of a single-layer cell membrane sheet, wherein i, ii, iii, and iv are respectively a schematic diagram of the liver lobule membrane sheet structure, a bright field image, a calcein AM staining fluorescence image, and a propidium iodide (PI) staining fluorescence image (scale bars are 200 μm respectively); f is a diagram of a green-red double-layer liver lobule membrane sheet, wherein i, ii, iii, and iv are respectively a schematic diagram of the double-layer liver lobule membrane sheet structure, a green layer liver lobule membrane sheet image, a red layer liver lobule membrane sheet image, and a double-layer merged liver lobule membrane sheet image (scale bars are 200 μm respectively).

[0022] Figure 2 is a diagram of the preparation of multi-layer, multi-scale bionic liver lobule; Figure 2 a is a diagram of a 5-layer liver lobule membrane, wherein i, ii, and iii are schematic diagrams of the structure of the 5-layer liver lobule membrane, ii is a laser confocal three-dimensional reconstruction image of the 5-layer green-red interval-stained liver lobule membrane, and iii is a Z-axis depth display after the laser confocal three-dimensional reconstruction of the 5-layer green-red interval-stained liver lobule membrane; b is a diagram of a 10-layer liver lobule membrane, wherein i, ii, and iii are schematic diagrams of the structure of the 10-layer liver lobule membrane, ii is a laser confocal three-dimensional reconstruction image of the 10-layer green-red interval-stained liver lobule membrane, and iii is a Z-axis depth display after the laser confocal three-dimensional reconstruction of the 10-layer green-red interval-stained liver lobule membrane; c is a diagram of a 20-layer liver lobule membrane, wherein i, ii, and iii are schematic diagrams of the structure of the 15-layer liver lobule membrane, Schematic diagram of the sheet structure; ii is a laser confocal three-dimensional reconstruction image of a 20-layer green-red interval-stained liver lobule sheet; iii is a Z-axis depth display after laser confocal three-dimensional reconstruction of a 20-layer green-red interval-stained liver lobule sheet; d is the adhesion of HUVECs in the lumen and outside the sheet, with scale bars of 200 μm (top) and 20 μm (bottom); e is a fluorescence image of a bile duct sheet stained with fluorescein diacetate (FDA), with a scale bar of 50 μm; f is a schematic diagram of the self-assembly of a 20-layer liver lobule sheet after 0, 1, and 4 h of in vitro culture; i, ii, and iii in g are hematoxylin-eosin-stained images of the self-assembly of a 20-layer liver lobule sheet after 0, 1, and 4 h of in vitro culture, respectively; h is an image of a cell sheet stained with phalloidin (F-actin, red) and DAPI (blue), with a scale bar of 50 μm.

[0023] Figure 3 shows the construction and performance of a liver chip based on a biomimetic liver lobule. Figure 1 (a) shows a 50-layer liver lobule membrane sheet, where i, ii, iii, and iv are a schematic diagram of the 50-layer liver lobule membrane sheet structure, a fluorescence image (scale bar, 200 μm), a three-dimensional reconstruction of albumin (ALB) staining (green) and DAPI staining (blue) (scale bar, 50 μm), and a periodic acid-Schiff (PAS) staining image (scale bar, 200 μm), respectively. b shows a schematic diagram of the liver chip structure using a multichannel concentration gradient, c shows a photograph of the multichannel concentration gradient chip, and d shows a concentration gradient simulation image. e shows a quantitative comparison of the expression levels of genes related to drug metabolism, and f shows a quantitative comparison of the expression levels of genes related to urea synthesis. Data are means ± SEMs from three independent experiments. *p < 0.05, **p < 0.001.

[0024] Figure 4 is a diagram showing the application effect of the constructed bionic liver lobule in the treatment of acute liver failure. In Figure 1, a is a schematic diagram of bionic liver lobule transplantation; b is a survival rate diagram; c is an ALT level diagram; d is an AST level diagram; e is an in vivo image of the bionic liver lobule construct in ALF rats; f is a staining diagram of human albumin (ALB), α-smooth muscle actin (a-SMA) and CD31 in the liver lobule construct of the ALF rats; and g is a diagram of inflammatory factor levels.

[0025] FIG5 is a schematic diagram of the structure of a real liver lobule.

[0026] FIG6 is a schematic diagram of the printed surface area and lumen size of the liver lobule membrane. DETAILED DESCRIPTION

[0027] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0029] Example 1

[0030] 3D printing technology constructs liver lobule structural functional units:

[0031] 1) Materials and methods

[0032] A 3D imprinting mold was fabricated using 3D printing equipment. The 3D model of the imprinting device was designed using C4D software, and three imprinting mold components (imprint body, push-pull mechanism, and push rod head) with a resolution of 2 μm were produced using a projection microstereolithography (PμSL) 3D printing system (nanoArch S140, BMF Materials Technology Co., Ltd., China). These three imprinting mold components were assembled into a 3D imprinting mold. The 3D imprinting mold was prepared using a biocompatible light-curable resin.

[0033] The stamp body includes a cylinder with a regular hexagonal cross-section and a hexagonal solid column built into the cylinder. One end of the solid column is fixedly connected to the cylinder through a mounting seat with three holes distributed on the mounting seat; the push-pull device includes a push-pull disk and three cylindrical rods fixed on the push-pull disk, and the push-pull disk has a hexagonal hollow structure; three circular grooves are distributed on the push rod head; when the push-pull device and the push rod head are assembled, the cylindrical rods of the push-pull device pass through the hexagonal solid column of the stamp body at intervals, the hexagonal hollow structure of the push-pull disk passes through the hexagonal solid column of the stamp body, and the cylindrical rods of the push-pull disk pass through the holes on the mounting seat and then are inserted into the grooves on the push rod head; the preparation material of the 3D imprint mold is biocompatible light-curing resin.

[0034] When preparing a hepatic lobule sheet, the movable unit composed of the push-pull device and the push rod head is pulled up (i.e., pulled toward the mounting seat), and then the main body of the stamp is pressed against the cultured hepatocyte sheet. Because the space between the cylindrical body and the solid column of the stamp body is hollowed out, the hepatocyte sheet is carved into a hexagonal hepatic lobule sheet with a hollow cross-section. Finally, the movable unit composed of the push-pull device and the push rod head is pushed from one end of the mounting seat of the stamp body to the other end, thereby pushing out the carved hepatic lobule sheet. When several hepatic lobule sheets are stamped layer by layer, the carved hepatic lobule sheets are retained in the stamp body (not pushed out), and the movable unit is used to push and squeeze them inside the stamp body, and finally pushed out together.

[0035] 2) Results

[0036] In order to mimic the structure of human liver lobule, a new imprinting mold was designed in this example for 3D assembly of liver cell sheets (a, b, c in FIG1 ).

[0037] The hepatic lobule is a component of the liver parenchyma, consisting of hepatocytes arranged in the hepatic sinusoidal network and the central vein within the hepatic cord. In order to design this complex hepatic lobule structure, the main body of the impression is designed to have a hexagonal star-shaped solid column with a hollow cavity formed around it. Specifically, the central solid part is a column with 6 slender columns radiating outward to simulate the circulation of the hepatic lobule duct.

[0038] Because the edges of the main body of the stamp are sharp, the hexagonal, hollow structure of the liver lobule can be etched out. The assembled 3D stamping mold can be used to obtain a biomimetic liver lobule membrane by moving the push-pull device up and down. By pushing the push-pull device to the bottom, the etched liver lobule membrane is pushed out.

[0039] By imprinting a liver cell sheet and then pressing it onto another liver cell sheet, the shape of the liver lobule can be imprinted layer by layer. After stacking layer by layer, the push rod device will move up to obtain a bionic liver lobule of a certain height, realizing the imprinting of the spatial height of the liver lobule structure.

[0040] Finally, the imprinted bionic liver lobule is pushed out, revealing a complete hexagonal hollow structure that maintains close connections between cells and preserves the extracellular matrix intact. This is a method of obtaining the shape of a liver lobule while preserving the integrity of the cells and their microenvironment.

[0041] In the embodiment, in order to prepare a cell tissue with complete extracellular matrix (ECM) proteins and cell-cell connections, hiPSC-Heps were seeded onto a temperature-responsive polymer-grafted cell culture dish (TRCD) and cultured under conventional adherent conditions to generate hepatocyte sheets; when the temperature was reduced from 37°C to 20°C, the TRCD surface became hydrophilic, which helped the cells to separate from the adherent culture tension, thereby constructing a 3D cell assembly by contraction; subsequently, the imprinting mold carved the hepatocyte sheet into a liver lobule membrane sheet with a hollow lumen, as shown in Figure 1d; calcein AM / propidium iodide (PI) staining was used to determine the viability of the lobule-like hiPSC-Heps sheet, as shown in Figure 1e; because the imprinting mold has a push-pull device, multiple hepatocyte sheets can be imprinted and stacked at the same time, and the stacked hepatocyte sheets can be squeezed by the push-pull device; further attempts to cover two hepatocyte sheet layers revealed a clear green-red fluorescent structure, as shown in Figure 1f.

[0042] Figure 6 is a schematic diagram of the surface area and lumen size of the printed liver lobule membrane. The surface area is (0.6587±0.0172) mm 2 The lumen area is (0.2304±0.0090) mm 2 .

[0043] Example 2

[0044] Fabrication and functional verification of multi-layer, multi-scale bionic liver lobule:

[0045] In order to simulate the height of the liver lobule and illustrate the feasibility of using 3D printing technology to construct three-dimensional multilayer tissue assemblies for tissue engineering applications, this example attempts to assemble more layers of liver lobule membrane sheets: first, a 5-layer liver lobule model was assembled, presented in the form of a hexagonal prism with a central and radial void structure to simulate a real liver lobule, as shown in Figure 2a; inspired by the successful construction of the 5-layer liver lobule model, 10-layer and 20-layer liver lobule models were further produced, as shown in Figure 2b and c; in addition, the liver lobule membrane sheets stained with green fluorescent protein (GFP, green) and mCherry (red) were alternately superimposed, allowing people to intuitively and clearly observe the liver cell membrane sheets. The self-assembly process of the layer; the middle lumen of the prepared liver lobule model was perfused with human umbilical vein endothelial cells (HUVECs) at a concentration of 10^6 cells / mL and cultured in an incubator overnight; the three-dimensional structure with a hexagonal star-shaped hollow lumen significantly increased the interaction between cells and the environment, promoted the transfer of nutrients and waste, and enhanced the cell viability of the central cells in the structure; studies have shown that adjacent HUVECs located in the hollow lumen or surface of the hexagonal star column can form a single layer and at the same time connect with HUVECs located between the hexagonal star column hollow lumen and the outside, as shown in Figure 2d, which contributes to vascular circulation in tissue engineering and enables the delivery of substances to the host.

[0046] In this example, the bile excretion function of a multilayered hepatic lobule sheet with blood vessels was studied by incubating it with fluorescein diacetate (FDA). After FDA entered the bionic hepatic lobule cells through passive diffusion, it was subsequently hydrolyzed into fluorescein by intracellular esterases and then excreted through the bile ducts (bile ducts are tiny tubes formed between adjacent hepatocytes by local depressions of the plasma membrane into grooves that dock and seal with each other. When the density of hepatocytes reaches a certain range, they will squeeze each other), as shown in Figure 2e. This shows that the hepatic lobule sheet, as a tissue engineering with a complete extracellular matrix, has certain advantages in forming blood vessels and bile duct circulation, providing prospects for further assembly of biomimetic applications.

[0047] Hematoxylin-eosin (HE) staining can detect the adhesion ability of cells after self-assembly and is used for printing customizable liver lobule membranes. In the embodiment, when the 20-layer hepatic lobule membrane sheet was just assembled, the cells in each hepatic lobule membrane sheet were individually and tightly connected together without interruption or interruption, confirming the robustness of the hepatic lobule membrane sheet, as shown in FIG2 f and g(i), but there were large cavity gaps between the layers, and a few gaps were large enough to cause direct separation between the cell layers. The assembled hepatic lobule membrane sheet was cultured in vitro (cell culture medium was cultured in a cell culture incubator at 37 degrees) for 1 hour and 4 hours. As shown in FIG2 g(ii) and (iii), the layers gradually merged, the interlayer gaps narrowed, and the intercellular vacuoles gradually disappeared; after 4 hours of in vitro culture, the 20 layers of cells were completely integrated, and only the boundaries between the layers were vaguely visible; the three-dimensional cytoskeleton arrangement of the hepatic lobule membrane sheet was observed by fluorescent staining of phalloidin (F-actin), as shown in FIG2 h, which was a random multi-directional arrangement, suggesting that unlike previous studies, the self-assembly culture process of the imprinted multilayer hepatic lobule membrane sheet of the present invention can be integrated into the overall three-dimensional structure of the bionic hepatic lobule, thereby realizing a truly engineered cell tissue.

[0048] Example 3

[0049] Building a liver chip based on a bionic liver lobule:

[0050] In order to simulate the cellular composition of the human liver, hiPSC-Heps, macrophages and hepatic stellate cells (HSCs) were mixed in proportion when culturing liver lobule membranes in this embodiment, with the proportions being 67.86%, 30.16% and 1.98% (percentage of cell number), respectively. In order to simulate the space and composition of a single three-dimensional liver lobule, hiPSC-Heps, macrophages and HSCs were mixed in the above proportions in this embodiment to form a multi-cell monolayer. These cell sheets were then imprinted and assembled to create a 50-layer cell mixed structure, as shown in a(i) and (ii) in Figure 3. In order to evaluate the liver function of this structure, albumin (ALB) and glycogen were stained in this embodiment, as shown in a(iii) and (iv) in Figure 3; these results indicate that the 3D assembled cell mixed imprinted structure has good albumin and glycogen synthesis functions.

[0051] Liver-on-a-chip technology has become a biomimetic integration technology for miniature organs. By precisely controlling biochemical factors and mechanical stress, it can simulate the function of the smallest liver unit in vitro. To achieve this concept, this example utilizes microfluidics to integrate a biomimetic liver lobule with a graded hydrogel (concentration gradient module) to construct a biomimetic liver lobule chip, as shown in Figure 3 (bd). The biomimetic liver lobule chip designed in this example includes two modules: a concentration gradient module and a drug response module. The concentration gradient module has a classic Christmas tree structure and can screen seven different drug concentrations. The drug response module is composed of seven independent biomimetic liver lobules, each containing a 50-layer, 7-unit liver lobule structure (i.e., composed of seven 50-layer liver lobule structures). Therefore, each biomimetic liver lobule can react with different drug concentration levels without series interference between them. Through microfluidics, the culture medium (red) and drug solution (blue) are pumped into the two inlets of the concentration gradient module respectively. The colors of the seven channels change significantly in a gradient manner, and through up and down perfusion, they fully contact and react with the bionic liver lobule in the drug reaction module, which can be used as an effective drug screening platform.

[0052] In addition, this embodiment also uses a bionic liver lobule to simulate the function of the smallest unit of the liver. Compared with equivalent cells cultured in 2D, the bionic liver lobule chip of the present invention can better play the role of the liver and show stronger drug metabolism and urea synthesis functions, as shown in Figure 3 (ef); therefore, the bionic liver lobule chip can be used as a powerful tool for drug screening and disease simulation.

[0053] Example 4

[0054] The role of bionic liver lobule in the treatment of acute liver failure:

[0055] To further expand the application of bionic liver lobule in liver regeneration, this example uses d-galactose (D-Gal) to establish a rat ALF model and transplants the bionic liver lobule construct in situ to observe whether it can treat ALF and promote regeneration of failing livers. As shown in Figure 4a, 24 hours after inducing liver failure, the liver lobule is transplanted into the rat liver by laparotomy and sutured. Since ALF progresses very rapidly, the disease model deteriorates to a peak on the 3rd day. By the 7th day, the recovery of liver function represents the disease outcome. Therefore, the ultimate therapeutic effect of the bionic lobule can be observed.

[0056] Sixty rats with ALF were randomly divided into three groups, 20 in each, to observe the regenerative effects of the biomimetic liver lobule: Group 1 underwent sham surgery and served as the ALF group; Group 2 received a biomimetic liver lobule construct transplant (BLLC group), with a total cell volume of 107 cells; and Group 3 received a cell transplant (Cells group), with the same cell number and cell ratio as Group 2. After treatment, the patients' survival rates, liver function indicators, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured, as shown in Figure 4 (b,d). The survival rate and liver function of rats in the BLLC group were significantly better than those in the ALF group, indicating that liver failure was controlled and liver damage was restored after treatment. Furthermore, the BLLC group showed significantly less liver damage and faster recovery than the other two groups.

[0057] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent replacement and improvement made by any technician familiar with the profession to the above embodiment without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a bionic liver lobule, characterized in that: The cultured liver cell sheets are carved into liver lobule membrane sheets with a hexagonal hollow cross-section using a 3D imprinting mold. Several carved liver lobule membrane sheets are imprinted layer by layer through the 3D imprinting mold, and vascular endothelial cells are infiltrated therein to form bionic liver lobules with a certain height, cell density and bionic function.

2. The method for preparing the bionic liver lobule according to claim 1, characterized in that: The hepatocytes in the hepatocyte sheet are hiPSC-Heps. The hepatocytes are seeded on a cell culture dish grafted with a temperature-responsive polymer and cultured under conventional adherent conditions to generate a hepatocyte sheet.

3. The method for preparing the bionic liver lobule according to claim 2, characterized in that: When the temperature was decreased from 37°C to 20°C, the cultured hepatocyte sheets detached from the temperature-responsive polymer-grafted cell culture dish.

4. The method for preparing the bionic liver lobule according to claim 1, characterized in that: After being cultured in vitro for 1-4 hours, the layers of the hepatic lobule membrane sheets printed layer by layer gradually fuse together; human umbilical vein endothelial cells are perfused into the hollow middle lumen of the bionic hepatic lobule.

5. The method for preparing the bionic liver lobule according to claim 2, characterized in that: Macrophages and hepatic stellate cells were also added during the hepatocyte culture, and hiPSC-Heps, macrophages and hepatic stellate cells were mixed and cultured in proportion in a cell culture dish grafted with a temperature-responsive polymer; the mixing ratios of hiPSC-Heps, macrophages and hepatic stellate cells were 67.86%, 30.16% and 1.98% respectively in terms of cell number percentage.

6. The method for preparing a bionic liver lobule according to claim 1, characterized in that: The 3D imprinting mold comprises a mold body, a push-pull device and a push rod head; the mold body comprises a cylinder with a regular hexagonal cross-section and a hexagonal star-shaped solid column built into the cylinder, one end of the solid column is fixedly connected to the cylinder through a mounting seat, and the mounting seat is provided with three holes; the push-pull device comprises a push-pull disk and three cylindrical rods fixed on the push-pull disk, and the push-pull disk has a hexagonal star-shaped hollow structure; three circular grooves are provided on the push rod head; when the push-pull device and the push rod head are assembled, the cylindrical rods of the push-pull device respectively pass through the hexagonal star-shaped solid cylinder of the mold body at intervals, the hexagonal star-shaped hollow structure of the push-pull disk passes through the hexagonal star-shaped solid cylinder of the mold body, and the cylindrical rods of the push-pull disk pass through the holes on the mounting seat and then are inserted into the grooves on the push rod head; the preparation material of the 3D imprinting mold is a biocompatible photocurable resin.

7. The method for preparing the bionic liver lobule according to claim 6, characterized in that: When preparing the hepatic lobule membrane sheet, pull up the movable unit assembled from the push-pull device and the push rod head, press the mold body onto the cultured hepatocyte sheet, carve the hepatocyte sheet into a hexagonal hepatic lobule membrane sheet with a hollow cross-section, and then push the movable unit from one end of the mounting seat of the mold body to the other end to push out the hepatic lobule membrane sheet; when several hepatic lobule membrane sheets are printed layer by layer, stack the carved hepatic lobule membrane sheets in the mold body, and use the movable unit to push and squeeze them in the mold body.

8. The bionic liver lobule prepared by the method according to any one of claims 1 to 7.

9. The use of the bionic liver lobule according to claim 8 in drug screening, characterized in that: The drugs are delivered to the bionic liver lobule, and drug screening is performed based on the response of the bionic liver lobule.

10. The use of the bionic liver lobule in drug screening according to claim 9, characterized in that: The prepared bionic liver lobule is connected to a concentration gradient module having multiple channels. The drug outlet of each channel is respectively connected to an independent bionic liver lobule to construct a bionic liver lobule chip. Drug screening is performed based on the reaction of the drug solution flowing out of the channel after being perfused into the bionic liver lobule.

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