Liver lobule chip and application thereof in analysis of spatial heterogeneity single cell secretion of liver lobule

Abstract

The invention belongs to the technical field of micro-fluidic chips, and discloses a hepatic lobule chip which comprises a single cell capture array chip, a micropore array chip and an antibody slide, a capture array on the single cell capture array chip and a micropore array on the micropore array chip are in a polygon shape, perfusion culture solution channels are arranged in the diagonal directions of all vertexes of the polygon, and the capture array and the micropore array are in one-to-one correspondence after being assembled. The liver lobule chip overcomes the dependence of research on spatial heterogeneity of liver cells on precise liver anatomical slices, avoids the defects that the liver slices are difficult to obtain and store, can directly reveal spatial distribution characteristics of secretion activity of the liver cells in the liver lobule, and becomes an ideal tool for spatial heterogeneity secretion of the liver cells. The invention further discloses application of the hepatic lobule chip in hepatic lobule spatial heterogeneity single cell secretion analysis, operation is easy, large experimental instruments are not needed, cost is low, and the hepatic lobule chip is easy to master.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips, and in particular relates to a liver lobule chip and an application thereof in single-cell secretion analysis of liver lobule spatial heterogeneity. Background Art

[0002] Cells are the basic units of life, and the exploration of life health and disease usually needs to be based on cell research. However, the cells in the body are not completely the same. Even in the same genetic origin and the same tissue structure, there is a high degree of heterogeneity between cells close to the edge tissue and cells close to the center tissue. This phenomenon, which is caused by the difference in the spatial position of cells, resulting in the state, function, fate, etc. of cells, is called the spatial heterogeneity of cells. The liver lobule is a regular hexagon, showing extremely obvious spatial heterogeneity, which is called "liver partition". In different liver partitions, liver cells show different functional divisions. "Liver partition" is essentially determined by the different spatial positions of liver cells. The cells near the periphery of the liver lobule are relatively rich in oxygen and glucose, which causes them to mainly participate in energy-consuming metabolic activities, such as gluconeogenesis and urea secretion. On the contrary, the internal cells have a relatively higher proportion of glycolysis than the peripheral cells, and therefore mainly participate in energy-conserving metabolic activities, such as exogenous substance metabolism. This spatial heterogeneity of liver cells is the result of the high spatial optimization of the liver lobule. The analysis of spatial heterogeneity of hepatic lobule cells can not only deepen people's understanding of the liver cell microenvironment, but also help the development of drugs to improve the liver microenvironment and thus regulate liver function.

[0003] Single-cell sequencing is an important tool for analyzing cellular heterogeneity. However, single-cell sequencing requires dissociation of tissues to make cell suspensions, which will lead to the loss of cellular spatial information. Liver tissue sections can be analyzed for liver function zoning using various staining techniques (such as RNA in situ hybridization and immunohistochemistry), but they often have the disadvantages of limited detection indicators and low throughput. Microdissection or perfusion techniques can be used to separate target cells from liver sections, but due to the limitation of accuracy, the spatial resolution is relatively limited, and it is difficult to achieve single-cell resolution analysis. The recently developed single-cell spatial multi-omics analysis technology can realize single-cell analysis of liver tissue sections. It can simultaneously measure the expression of multiple genes, proteins and small molecules in a single cell. However, it cannot simultaneously measure multiple secretions secreted by a single cell, such as cytokines, exosomes, microRNA, etc. Moreover, the above-mentioned technologies are generally complex in experimental operation and rely on precise liver tissue sections. Since liver tissue sections are generally difficult to obtain and preserve, they are difficult to promote and apply. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a liver lobule chip suitable for spatial heterogeneous secretion analysis at the single cell level and its application.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is: A liver lobule chip comprises a single-cell capture array chip, a micropore array chip and an antibody slide; the capture array on the single-cell capture array chip and the micropore array on the micropore array chip are polygonal, and a perfusion culture fluid channel is arranged along the diagonal direction of each vertex of the polygon, and the polygon vertex and center of the capture array on the single-cell capture array chip are respectively provided with a liquid inlet and a liquid outlet (the liquid inlet and the liquid outlet can be used interchangeably); the outer edge of the micropore array on the micropore array chip is provided with a gas channel and its air inlet and air outlet; the capture array on the single-cell capture array chip and the micropore array on the micropore array chip correspond one to one after assembly.

[0006] In the above-mentioned liver lobule chip, preferably, the capture array and micropore array are regular hexagons with an internal diameter of 5-15 mm, and the capture array is provided with liquid inlets at the six vertices of the regular hexagon; the diameters of the liquid inlet and the liquid outlet are 0.5-3 mm; the single-cell capture array chip and micropore array chip are PDMS chips, and their capture array and micropore array are made by soft lithography followed by inverted molding with SU-8 glue.

[0007] Preferably, the capture array is a shuttle-shaped structure array with a large inlet and a small outlet, the shuttle-shaped channel height is 10-30 μm, the channel widths at the inlet and outlet are 5-20 μm and 3-15 μm respectively, and the channel width between two adjacent shuttle-shaped structures is 5-20 μm.

[0008] Preferably, the micropores on the micropore array chip include any one or more of circular holes, square holes and elongated holes; the diameter / side length of the micropores is 20-100 μm, the depth is 10-50 μm, and the distance between two adjacent micropores is 5-50 μm; a liquid outlet is arranged at the center of the micropore array on the micropore array chip, the gas channel is provided with an air inlet and an air outlet, and the width of the gas channel is 5-30 μm; the perfusion culture fluid channel is arranged along the diagonal direction of each vertex of the polygon.

[0009] Preferably, the capture array on the single-cell capture array chip is a single array or multiple arrays are provided at the same time, the micropore array on the micropore array chip is a single array or multiple arrays are provided at the same time, and all capture arrays on the single-cell capture array chip and all micropore arrays on the micropore array chip correspond one to one after assembly.

[0010] Preferably, the antibody slide is a poly-lysine slide; any one or more of antibodies, aptamers and DNA are incubated on the surface of the antibody slide; for example, the antibody is MCP-1 monoclonal antibody and / or CD63 monoclonal antibody.

[0011] Preferably, the liver lobule chip further comprises a fixture for fixing the single cell capture array chip and the microwell array chip, and the fixture is a PMMA fixture.

[0012] The present invention constructs a liver lobule chip suitable for spatial heterogeneous secretion analysis at the single-cell level. The chip reproduces the spatial position relationship of each hepatocyte in the liver lobule, overcoming the dependence of traditional spatial heterogeneity analysis on precise liver anatomical sections. Combined with single-cell secretion detection technology, the chip can directly reveal the spatial distribution characteristics of the secretory activity of hepatocytes in the liver lobule, becoming an ideal tool for spatial heterogeneous secretion of hepatocytes.

[0013] Based on a general inventive concept, the present invention also provides an application of a liver lobule chip in the analysis of single-cell secretion of liver lobule spatial heterogeneity.

[0014] In the above application, preferably, the application method comprises the following steps: (1) Place the single cell capture array chip on the lower layer with the liquid inlet and outlet facing upward, and place the microwell array chip on the upper layer with the air inlet and outlet facing upward, so that the capture array on the single cell capture array chip and the microwell array on the microwell array chip correspond to each other in the upper and lower layers, and fix them with a clamp; (2) Inoculating hepatocytes at the liquid inlet of the single-cell capture array chip to form a liver lobule chip; (3) Flipping the liver lobule chip so that the hepatocytes captured in the capture array fall into the corresponding microwell array, and then connecting the culture medium and oxygen-rich gas for perfusion culture; (4) Disassemble the fixture, remove the single-cell capture array chip, and cover it with an antibody slide for incubation; (5) Remove the antibody slide and analyze the hepatocyte secretions obtained by incubation on the antibody slide by immunofluorescence analysis.

[0015] Preferably, the external culture medium of the perfusion culture is a hepatocyte growth medium, the oxygen concentration in the external oxygen-rich gas is higher than the oxygen concentration in the incubator, and the culture time is 12-60 h; the incubation is carried out in a 5-38°C, 3-8% CO2 incubator, and the incubation time is 4-24 h.

[0016] Preferably, the hepatocyte secretions include any one or more of cytokines, exosomes, and microRNA; the specific operation of the immunofluorescence method is as follows: after taking out the antibody slide, drop the detection antibody on the slide, use the immune sandwich method to image, and then perform fluorescence intensity analysis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides an in vitro model that can retain the spatial information of hepatocytes in the liver lobule, which overcomes the reliance of the study of spatial heterogeneity of hepatocytes on precise liver anatomical sections and avoids the drawbacks of liver sections being difficult to obtain and preserve. Combined with single-cell secretion detection technology, the chip can directly reveal the spatial distribution characteristics of the secretory activity of hepatocytes in the liver lobule, becoming an ideal tool for the spatial heterogeneity of hepatocyte secretion.

[0018] 2. Compared with the traditional method of staining and analyzing liver function zoning using liver tissue sections, the single-cell chip detection indexes for spatial heterogeneity secretion analysis of liver lobule are more numerous and richer in variety, such as cytokines, extracellular vesicles, microRNA, etc.; and the detection throughput is higher, reaching tens of thousands or even hundreds of thousands.

[0019] 3. The method for analyzing single-cell secretion of spatial heterogeneity in liver lobules provided by the present invention has simple experimental operation and does not require large-scale experimental instruments (such as precision tissue slicers). It is low-cost and easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a schematic diagram of the overall structure of the liver lobule chip used for single-cell secretion analysis of liver lobule spatial heterogeneity in Example 1 of the present invention; Figure 2 Functional unit 1 of the liver lobule chip for single-cell secretion analysis of liver lobule spatial heterogeneity in Example 1 of the present invention: a high-throughput single-cell capture array; Figure 3 Functional unit 2 of the liver lobule chip for single-cell secretion analysis of liver lobule spatial heterogeneity in Example 1 of the present invention: high-throughput microwell array; Figure 4 Functional unit 3 of the liver lobule chip for single-cell secretion analysis of liver lobule spatial heterogeneity in Example 1 of the present invention: a multi-color multiplexed antibody slide; Figure 5This is a physical picture of the functional unit 1 high-throughput single-cell capture array and the functional unit 2 high-throughput microwell array of the liver lobule chip used for single-cell secretion analysis of spatial heterogeneity of liver lobules in Example 1 of the present invention after being assembled using a clamp, and the capture structure and the microwell array correspond one to one; A is the physical picture of the chip after the capture array and the upper and lower layers of the microwell array correspond one to one and are fixed with a clamp; B is the physical picture of the chip after the single-cell capture array chip is removed and covered with an antibody slide for incubation; C is the effect of the capture array and the upper and lower layers of the microwell array correspond one to one, taken under a microscope; Figure 6 This is a bright field image of primary hepatocytes obtained after tissue dissociation of a patient's liver biopsy sample in Example 2 of the present invention; Figure 7 This is a schematic diagram of the experimental process of detecting and analyzing the spatial distribution characteristics of MCP-1 secreted by hepatocytes using the liver lobule chip in Example 2 of the present invention; Figure 8 The oxygen content gradient result inside the chip after the periphery of the liver lobule chip is connected to a 25% oxygen-enriched in vitro environment in Example 2 of the present invention; the left figure is the result of measuring the oxygen content inside the chip using Ru (ddp) dye, and the right figure is the quantitative result of the oxygen content inside the chip from the outer edge to the center; Fig. 9 Part of the experimental results and analysis statistics of the spatial distribution analysis of MCP-1 secreted by hepatocytes in the liver lobule chip of Example 2 of the present invention; the left picture is a fluorescence picture of the secretion of hepatocytes in the chip using immune sandwich, and the right picture is a quantitative analysis of the left picture; Fig.10 The results of the spatial distribution characteristics of MCP-1 and CD81+CD63+ exosomes secreted by hepatocytes in the hepatic lobule chip of Example 3 of the present invention are shown; A is a heat map of the secretion of MCP-1 of hepatocytes in different spatial regions in the hepatic lobule chip, B is a statistical analysis of the secretion of MCP-1 of hepatocytes in different spatial regions in the hepatic lobule chip, and C is a statistical analysis of the secretion of CD81+CD63+ exosomes of hepatocytes in different spatial regions in the hepatic lobule chip; Fig.11 Schematic diagram of the overall structure of the high-throughput liver lobule chip used for single-cell secretion analysis of liver lobule spatial heterogeneity in Example 4 of the present invention. Description of the drawings: 1. Single cell capture array core; 2. Microwell array chip; 3. Antibody slide; 1-1. Capture array; 2-1. Microwell array. DETAILED DESCRIPTION

[0023] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0024] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0025] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0026] Glossary: “High throughput” is a technical term in this field, referring to the testing of tens of thousands of cells at the same time.

[0027] Equipment for fluorescence intensity analysis includes, but is not limited to, gene chip scanners.

[0028] Embodiment 1: This embodiment provides a liver lobule chip for analyzing single-cell secretion of liver lobule spatial heterogeneity, the structure of which is shown in FIG. Figure 1-Figure 5 The chip comprises three functional units: a high-throughput single-cell capture array chip 1, which is used for the formation and perfusion culture of liver lobule chips; a high-throughput microwell array chip 2, which is used for single-cell culture and detection of hepatocytes; and a multi-color multiplexed antibody slide 3, which is used for single-cell secretion profiling analysis of hepatocytes with spatial resolution.

[0029] like Figure 2 The capture array 1-1 on the high-throughput single-cell capture array chip 1 is a shuttle-shaped array with a large inlet and a small outlet. The height of the shuttle-shaped channel is 20 μm, and the channel widths at the inlet and outlet are 12 μm and 8 μm, respectively. The high-throughput single-cell capture array chip 1 is a PDMS chip, and the chip is made by using SU-8 glue and then soft lithography and then reverse molding. The high-throughput single-cell capture array 1-1 is a regular hexagon as a whole, and the inscribed diameter of the hexagon is 10 mm. The six vertices and the center point of the hexagon are respectively provided with liquid inlets and liquid outlets with a diameter of 1 mm; six perfusion culture medium channels are provided on the capture array 1-1 along the diagonal direction of the six vertices.

[0030] like Figure 3 The microwell array 2-1 on the high-throughput microwell array chip 2 consists of 18034 circular holes with a diameter of 70 μm and a depth of 40 μm. The distance between two adjacent microwells is 10 μm, and the circular holes are evenly distributed in a hexagon with an inscribed diameter of 10 mm. Six perfusion culture medium channels are arranged along the diagonal directions of the six vertices on the microwell array 2-1. The periphery of the microwell array 2-1 is a regular hexagonal channel coaxial with the microwell array 2-1, and the channel width is 20 μm.

[0031] like Figure 5The high-throughput single-cell capture array 1-1 and the high-throughput microwell array 2-1 were assembled using a PMMA fixture so that the capture structures and microwells in the array corresponded one to one (see figure).

[0032] like Figure 4 The multicolor multiplex antibody slide is a poly-lysine slide, on which the MCP-1 monoclonal antibody is evenly incubated. The preparation method of the multicolor multiplex antibody slide is as follows: (1) Add 2 μL of each MCP-1 monoclonal antibody to 98 μL of antibody diluent to make a total volume of 100 μL of antibody coating solution and mix well; (2) Add antibody coating solution to one end of the poly-lysine glass slide, and use a clean cover glass to slowly press down from the end where the antibody is added to the end where the antibody is not added, so that the coating solution fills the poly-lysine glass slide; (3) Place the poly-L-lysine slide in a humidified chamber and incubate at room temperature for 4 h or at 4°C overnight. (4) Remove the slides and wash them with gradient wash solutions: DPBS, ½DPBS, H2O, H2O, 5 min each time. Centrifuge and store for later use.

[0033] The high-throughput single-cell capture array chip 1, the high-throughput microwell array chip 2 and the multi-color multiplexed antibody slide 3 are used together to complete the detection of the spatial heterogeneity of MCP-1 secretion by liver single cells in the liver lobules.

[0034] Embodiment 2: This embodiment provides an application method for detecting and analyzing the spatial distribution characteristics of MCP-1 secreted by hepatocytes based on the liver lobule chip of embodiment 1, comprising the following steps: 1. Assembling the Liver Lobule Chip The single cell capture array chip 1 is located in the lower layer with the liquid inlet and outlet facing upward, and the micropore array chip 2 is located in the upper layer with the air inlet and outlet facing upward, so that the capture array 1-1 on the single cell capture array chip 1 and the micropore array 2-1 on the micropore array chip 2 correspond to each other in the upper and lower layers, and fixed with a clamp.

[0035] 2. Using single cell capture array to capture human primary hepatocytes to form liver lobule chip First, prepare a suspension of human primary liver cells. Hepatocytes are (1) obtained from fresh liver biopsy tissue from clinical patients, rinsed with DPBS (Dule's phosphate buffered saline) containing 1% double antibody on ice under sterile conditions, and removed fat, connective tissue, blood vessels and other debris; (2) cut the tissue into 1 mm tissue blocks on ice under sterile conditions, and rinsed with DPBS containing 1% double antibody; (3) in a 37°C, 5% CO2 incubator, digest the tissue with 0.25% trypsin, oscillate every 5 min, and after digestion for 20-40 min, add William's E complete medium (10% FBS + 1% double antibody + 89% William's E basal medium) and trypsin inhibitor to terminate digestion, and blow the tissue in the solution to form a tissue suspension; (4) centrifuge the tissue suspension at 1000 rpm for 5 min. min, discard the supernatant, add type IV collagenase, and place in a 37°C constant temperature shaking incubator to shake until the tissue becomes flocs; (5) Place the tissue digested with type I collagenase in a 37°C, 5% CO2 incubator for 5 min, and then evenly place the flocculent precipitate in a culture dish coated with type IV collagenase; (6) Place the dish in a 37°C, 5% CO2 incubator for 1 h, and add complete culture medium to allow the cells to adhere to the wall and grow; (7) Change the medium regularly and use it for experiments when the cells are in good condition.

[0036] Secondly, human primary liver cells were inoculated into the chip. Human primary liver cells were introduced into the chip through the central inlet of the regular hexagon using a syringe pump. The specific steps are as follows: (1) The concentration of the hepatocyte suspension was adjusted to approximately 2×10 5 / mL, loaded into a 1 mL syringe; (2) The syringe containing the hepatocyte suspension is connected to an external injection pump, and the injection pump is turned on. The cell suspension is injected into the chip from the central venous hole at a flow rate of 1 μL / min. When the cells flow through the shuttle-shaped capture structure, they are confined in the shuttle-shaped structure; (3) The cell inoculation is maintained for 30 min, during which the cell capture effect is continuously observed and recorded under a microscope; (4) After the inoculation is completed, the perfusion syringe and injection pump are slowly removed, and the chip is left to stand for 5 minutes to prevent the fluid from flowing. At this point, the human liver lobule chip is captured.

[0037] 3. Flip the chip so that the liver cells fall into the corresponding microwell array and perfuse and culture ( Figure 7 ) The specific steps are as follows: (1) Flip the chip, and the liver cells slowly fall from the single-cell capture array into the microwell array due to gravity; (2) Connect the liver cell perfusion culture pump, so that William's E complete culture medium enters the chip through the inlets at the six vertices of the hexagon of the single-cell capture array, with a flow rate of 1 μL / min, and flows out from the outlet in the center of the hexagon along the perfusion culture channel; (3) Connect the oxygen flow meter to the oxygen channel of the microwell array, and control the oxygen concentration to 25% through the control software FlowDDE32 (the oxygen content gradient results in the chip are shown in Figure 2). Figure 8 (4) Place the entire chip in a CO2 incubator and culture for 48 h.

[0038] 4. Remove the single cell capture array and cover with a multi-color multiplexed antibody slide The specific steps are as follows: (1) After perfusion culture, remove the chip and carefully disassemble the chip fixture; (2) Remove the single-cell capture array layer and replace it with a glass slide incubated with IL-8, MCP-1 and TNF-α monoclonal antibodies to assemble the chip; (3) Continue to incubate the above chip in a 37°C, 5% CO2 incubator for 18 h.

[0039] 5. Detection of hepatocyte secretions using immunofluorescence After incubation, the glass slide has captured IL-8, MCP-1 and TNF-α secreted by a single hepatocyte in the liver lobule chip. The detection steps are as follows: (1) Remove the clamp, take off the glass slide, and block it with 3% BSA for 1 hour; (2) Evenly drip 200 μl Biotin-MCP-1 detection antibody on the glass slide and incubate for 1 hour; (3) Rinse the glass slide with 1% BSA and evenly drip 200 μl Streptavidin-APC on the glass slide; (4) Use gradient cleaning solution: DPBS, ½ DPBS, H2O, H2O to wash the glass slide in sequence, each time for 5 minutes, and use MD Gene-Pix 4300A gene chip scanner to obtain fluorescence results; (5) Analyze the distribution of MCP-1 secretion by hepatocytes at different positions in the liver lobule chip.

[0040] 6. Results Analysis The analysis results of this example are as follows Fig. 9 As shown, the results indicate that this method can display the MCP-1 secretion capacity of hepatocytes in each different spatial position within the liver lobule. Furthermore, the MCP-1 secretion zoning shows that cells in the middle area have a stronger MCP-1 secretion capacity, while cells close to the portal vein or central vein secrete less MCP-1.

[0041] Embodiment 3: This embodiment provides a method for detecting and analyzing the spatial distribution characteristics of MCP-1 and CD81+CD63+ exosomes secreted by hepatocytes based on the liver lobule chip of embodiment 1. The method comprises the following steps: 1. Assembling the Liver Lobular Chip The single cell capture array chip 1 is located in the lower layer with the liquid inlet and outlet facing upward, and the micropore array chip 2 is located in the upper layer with the air inlet and outlet facing upward, so that the capture array 1-1 on the single cell capture array chip 1 and the micropore array 2-1 on the micropore array chip 2 correspond to each other in the upper and lower layers, and fixed with a clamp.

[0042] 2. Using single cell capture array to capture human primary hepatocytes to form liver lobule chip HepG2 cells, a liver cancer cell line, were inoculated into the chip. HepG2 cells were introduced into the chip through the central inlet of the regular hexagon using a syringe pump. The specific steps were as follows: (1) The concentration of the liver cell suspension was adjusted to approximately 2×10 5 / mL, loaded into a 1 mL syringe; (2) The syringe containing HepG2 cell suspension is connected to an external injection pump, and the injection pump is turned on. The cell suspension is injected into the chip from the central venous hole at a flow rate of 1 μL / min. When the cells flow through the shuttle-shaped capture structure, they are confined in the shuttle-shaped structure; (3) The cell inoculation is maintained for 30 min, and the cell capture effect is continuously observed and recorded under a microscope during this period; (4) After the inoculation is completed, the perfusion syringe and injection pump are slowly removed, and the chip is left to stand for 5 minutes to allow no flow of fluid in the chip. At this point, the capture of the human liver lobule chip is completed.

[0043] 3. Flip the chip so that the liver cells fall into the corresponding microwell array and perfuse and culture The specific steps are as follows: (1) Flip the chip over, and the liver cells will slowly fall from the single cell capture layer into the microporous array layer due to gravity; (2) Connect the liver cell perfusion culture pump to allow Gaotang complete culture medium to enter the chip through the inlets at the six vertices of the hexagon at a flow rate of 1 μL / min and flow out from the outlet in the center of the hexagon; (3) Connect the oxygen flow meter to the oxygen channel of the microporous array, and control the oxygen concentration to 25% through the control software FlowDDE32; (4) Place the entire chip in a CO2 incubator and culture for 48 h.

[0044] 4. Remove the single cell capture array and cover with a multi-color multiplexed antibody slide The specific steps are as follows: (1) After perfusion culture, remove the chip and carefully disassemble the chip fixture; (2) Remove the single-cell capture array layer and replace it with a glass slide incubated with MCP-1 and CD63 monoclonal antibodies to assemble the chip; (3) Continue to incubate the above chip in a 37°C, 5% CO2 incubator for 18 h.

[0045] 5. Detection of hepatocyte secretions using immunofluorescence After incubation, the glass slide has captured MCP-1 and CD63+ exosomes secreted by a single hepatocyte in the liver lobule chip. The detection steps are as follows: (1) Remove the clamp, take off the glass slide, and block it with 3% BSA for 1 hour; (2) Evenly drip 200 μl of Biotin-MCP-1 / PE-CD81 detection antibody mixture on the glass slide and incubate for 1 hour; (3) Rinse the glass slide with 1% BSA and evenly drip 200 μl of Streptavidin-APC on the glass slide; (4) Use gradient washing solution: DPBS, ½DPBS, H2O, H2O to wash the glass slide in sequence, each time for 5 minutes, and use MD Gene-Pix 4300A gene chip scanner to obtain fluorescence results; (5) Analyze the secretion distribution of MCP-1 and CD81+CD63+ exosomes by hepatocytes at different positions in the liver lobule chip.

[0046] 6. Results Analysis The analysis results of this example are as follows Fig.10 As shown in the results, the secretion distribution characteristics of MCP-1 and CD81+CD63+ exosomes in the liver lobules, the ability of hepatocytes to secrete MCP-1 and CD81+CD63+ exosomes has obvious heterogeneity, and the MCP-1 secretion zoning shows that cells in the middle area have stronger MCP-1 secretion ability, while cells close to the portal vein or central vein secrete less MCP-1, while CD81+CD63+ exosomes do not have this feature.

[0047] In summary, the liver lobule chip disclosed in the present invention enables the spatial heterogeneity of liver secretion at the single-cell level to be realized through the liver lobule chip, overcoming its dependence on precise anatomical sections. Combined with single-cell secretion detection technology, the chip can directly reveal the spatial distribution characteristics of hepatocyte secretion activity in the liver lobule, becoming an ideal tool for hepatocyte spatial heterogeneity secretion. The chip has important application value in understanding the mechanism of liver disease, evaluating drug metabolism and toxic effects, etc.

[0048] Embodiment 4: This embodiment is a high-throughput liver lobule chip for single-cell secretion analysis of liver lobule spatial heterogeneity based on the embodiment 1. The structure is shown in Fig.11 The chip parameters of this embodiment are consistent with those of Example 1, except that the regular hexagonal capture array and microwell array in the chip of Example 1 are expanded according to the benzene ring structure in this embodiment, thereby increasing the throughput of simultaneous detection.

Claims

1. A liver lobule chip, characterized in that: The invention comprises a single cell capture array chip (1), a micropore array chip (2) and an antibody glass slide (3); the capture array (1-1) on the single cell capture array chip (1) and the micropore array (2-1) on the micropore array chip (2) are polygonal, and a perfusion culture fluid channel is arranged along the diagonal direction of each vertex of the polygon; the polygon vertex and the center of the capture array (1-1) on the single cell capture array chip (1) are respectively provided with a liquid inlet and a liquid outlet; the outer edge of the micropore array (2-1) on the micropore array chip (2) is provided with a gas channel and an air inlet and an air outlet; the capture array (1-1) on the single cell capture array chip (1) and the micropore array (2-1) on the micropore array chip (2) correspond to each other one by one after assembly.

2. The liver lobule chip according to claim 1, characterized in that: The capture array (1-1) and microwell array (2-1) are regular hexagons with an inscribed diameter of 5-15 mm. The capture array (1-1) is provided with liquid inlets at the six vertices of the regular hexagon. The diameters of the liquid inlets and liquid outlets are 0.5-3 mm. The single cell capture array chip (1) and microwell array (2-1) chip (2) are PDMS chips. The capture array (1-1) and microwell array (2-1) are made by soft lithography and then reverse molding using SU-8 glue.

3. The liver lobule chip according to claim 1, characterized in that: The capture array (1-1) is a shuttle-shaped structure array with a large inlet and a small outlet. The height of the shuttle-shaped channel is 10-30 μm, and the channel widths at the inlet and outlet are 5-20 μm and 3-15 μm respectively. The channel width between two adjacent shuttle-shaped structures is 5-20 μm.

4. The liver lobule chip according to claim 1, characterized in that: The micropores on the micropore array chip (2) include any one or more of circular holes, square holes and elongated holes; the diameter / side length of the micropores is 20-100 μm, the depth is 10-50 μm, and the distance between two adjacent micropores is 5-50 μm; a liquid outlet is arranged at the center of the micropore array (2-1) on the micropore array chip (2), the gas channel is provided with an air inlet and an air outlet, and the width of the gas channel is 5-30 μm.

5. The liver lobule chip according to claim 1, characterized in that: The capture array (1-1) on the single-cell capture array chip (1) is a single array or is provided with multiple arrays at the same time, and the micropore array (2-1) on the micropore array chip (2) is a single array or is provided with multiple arrays at the same time. After being assembled, all the capture arrays (1-1) on the single-cell capture array chip (1) and all the micropore arrays (2-1) on the micropore array chip (2) correspond to each other one by one.

6. The liver lobule chip according to any one of claims 1 to 5, characterized in that: The antibody slide (3) is a poly-lysine slide; any one or more of antibodies, aptamers, and DNA are incubated on the surface of the antibody slide (3); the liver lobule chip also includes a fixture for fixing the single cell capture array chip (1) and the microwell array chip (2), and the fixture is a PMMA fixture.

7. An application of the liver lobule chip according to any one of claims 1 to 6 in single-cell secretion analysis of liver lobule spatial heterogeneity.

8. The use according to claim 7, characterized in that: The application method comprises the following steps: (1) placing the single cell capture array chip (1) at the bottom layer with the liquid inlet and the liquid outlet facing upward, and placing the microwell array chip (2) at the top layer with the air inlet and the air outlet facing upward, so that the capture array (1-1) on the single cell capture array chip (1) and the microwell array (2-1) on the microwell array chip (2) correspond to each other in the top and bottom layers, and fixing them with a clamp; (2) Inoculating hepatocytes at the inlet at the center of the hexagon of the single cell capture array chip (1) to form a liver lobule chip; (3) Flipping the liver lobule chip so that the liver cells captured in the capture array (1-1) fall into the corresponding microporous array (2-1), connecting the culture fluid to the liquid inlets at the six vertices of the hexagon of the single cell capture array chip (1), and introducing oxygen-rich gas through the air inlet of the microporous array chip (2) to perform perfusion culture; (4) disassemble the fixture, remove the single cell capture array chip (1), cover it with an antibody slide (3) and incubate; (5) The antibody slide (3) is taken out, and the hepatocyte secretions obtained by incubation on the antibody slide (3) are detected and analyzed by immunofluorescence method.

9. The use according to claim 8, characterized in that The external culture medium of the perfusion culture is a hepatocyte growth medium, the oxygen concentration in the external oxygen-rich gas is higher than the oxygen concentration in the incubator, and the culture time is 12-60 hours; the incubation is carried out in a 35-38°C, 3-8% CO2 incubator, and the incubation time is 4-24 hours.

10. The use according to claim 8, characterized in that: The hepatocyte secretions include any one or more of cytokines, exosomes, and microRNAs; the specific operation of the immunofluorescence method is as follows: after taking out the antibody slide (3), a detection antibody is dripped onto the slide, imaging is performed using the immunosandwich method, and then fluorescence intensity analysis is performed.

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