A liver lobule chip and its application in analyzing single-cell secretion of spatially heterogeneous liver lobule

Abstract

The present invention belongs to the field of microfluidic chip technology, and discloses a liver lobule chip, including 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 medium channel is provided along the diagonal direction of each vertex of the polygon, and the capture array and the micropore array correspond one to one after assembly. The liver lobule chip overcomes the dependence of the study of spatial heterogeneity of liver cells on precise liver anatomical sections, circumvents the disadvantages of liver sections being difficult to obtain and preserve, and can directly reveal the spatial distribution characteristics of the secretory activities of liver cells in the liver lobule, becoming an ideal tool for the spatial heterogeneity of liver cell secretion. The present invention also discloses the application of the liver lobule chip in the analysis of single-cell secretion of liver lobule spatial heterogeneity, which is simple to operate and does not require large-scale experimental instruments, is low in cost, and is easy to use.

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 its application in single-cell secretion analysis of liver lobule spatial heterogeneity. Background Art

[0002] Cells are the fundamental units of life, and understanding health and disease often relies on cell-based research. However, not all cells within an organism are identical. Even within the same genetic origin and tissue structure, cells near the periphery and those near the center exhibit significant heterogeneity. This phenomenon, in which cells' states, functions, and fates vary significantly due to their spatial location, is referred to in the field as spatial heterogeneity. The hepatic lobule, a regular hexagon, exhibits significant spatial heterogeneity, known as "liver zonation." Hepatocytes within different liver zonations exhibit distinct functional divisions. This "liver zonation" is essentially determined by the spatial location of hepatocytes. Cells near the periphery of a lobule are relatively rich in oxygen and glucose, leading them to primarily engage in energy-consuming metabolic activities such as gluconeogenesis and urea secretion. In contrast, cells in the inner lobule have a higher proportion of glycolysis than peripheral cells and therefore primarily participate in energy-conserving metabolic activities such as xenobiotic metabolism. This spatial heterogeneity of hepatocytes is a result of the highly optimized spatial structure of the hepatic 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 to develop drugs that improve the liver microenvironment and thus regulate liver function.

[0003] Single-cell sequencing is an important tool for analyzing cellular heterogeneity. However, it requires tissue dissociation and subsequent preparation of cell suspensions, which results in a loss of spatial information. Liver tissue sections can be analyzed for functional zonation using various staining techniques (such as RNA in situ hybridization and immunohistochemistry), but these often suffer from limited assay parameters and low throughput. Microdissection or perfusion techniques can be used to isolate target cells from liver sections, but due to limitations in precision and spatial resolution, single-cell resolution analysis is difficult. Recently developed single-cell spatial multi-omics analysis techniques enable single-cell analysis of liver tissue sections, enabling simultaneous measurement of the expression of multiple genes, proteins, and small molecules within a single cell. However, these techniques are unable to simultaneously measure the diverse secretions of a single cell, such as cytokines, exosomes, and microRNAs. Furthermore, these techniques are generally complex and rely on precise liver tissue sections, which are difficult to obtain and preserve, hindering their widespread application. 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:

[0006] 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, with perfusion culture fluid channels arranged along the diagonal directions of each vertex of the polygon; the polygonal vertices 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 are interchangeable); 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.

[0007] 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 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 molding with SU-8 glue.

[0008] 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.

[0009] 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 provided 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 medium channel is provided along the diagonal direction of each vertex of the polygon.

[0010] 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.

[0011] Preferably, the antibody slide is a polylysine slide; the surface of the antibody slide is incubated with any one or more of antibodies, aptamers, and DNA; for example, the antibody is MCP-1 monoclonal antibody and / or CD63 monoclonal antibody.

[0012] 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.

[0013] 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 traditional reliance of 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 hepatocyte secretory activity within the liver lobule, becoming an ideal tool for spatial heterogeneous secretion of hepatocytes.

[0014] 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.

[0015] In the above application, preferably, the application method comprises the following steps:

[0016] (1) Place the single-cell capture array chip on the lower layer with the liquid inlet and outlet facing upward, and 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;

[0017] (2) Inoculating hepatocytes at the liquid inlet of the single-cell capture array chip to form a liver lobule chip;

[0018] (3) Flip the liver lobule chip so that the hepatocytes captured in the capture array fall into the corresponding microwell array, and then perfuse the culture medium and oxygen-rich gas;

[0019] (4) Disassemble the fixture, remove the single-cell capture array chip, and cover it with an antibody slide for incubation;

[0020] (5) Remove the antibody slide and analyze the hepatocyte secretions obtained by incubation on the antibody slide by immunofluorescence.

[0021] Preferably, the external culture medium for 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.

[0022] 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 removing the antibody slide, the detection antibody is added to the slide, imaging is performed using the immune sandwich method, and then fluorescence intensity analysis is performed.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides an in vitro model that can preserve the spatial information of hepatocytes in the liver lobule. It overcomes the reliance on precise liver anatomical sections for the study of hepatocyte spatial heterogeneity and circumvents 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 hepatocyte secretory activity within the liver lobule, becoming an ideal tool for studying the spatial heterogeneity of hepatocyte secretion.

[0025] 2. Compared with the traditional staining analysis of liver tissue sections to analyze liver function zoning, the single-cell chip analysis of spatial heterogeneity of liver lobule secretion has a larger number of detection indicators and a richer variety, such as cytokines, extracellular vesicles, microRNA, etc.; and the detection throughput is higher, reaching tens of thousands or even hundreds of thousands.

[0026] 3. The method for analyzing single-cell secretion of spatial heterogeneity in liver lobules provided by the present invention is simple to operate 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

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0028] Figure 1 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;

[0029] 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;

[0030] 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: a high-throughput microwell array;

[0031] Figure 4Functional 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;

[0032] Figure 5 Figure 1 shows the functional unit 1, the high-throughput single-cell capture array, and the functional unit 2, the high-throughput microwell array, of the liver lobule chip for single-cell secretion analysis of spatially heterogeneous liver lobules in Example 1 of the present invention, assembled using a fixture. The capture structure and the microwell array correspond one-to-one. Figure A shows 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 fixture. Figure B shows the chip after the single-cell capture array chip is removed and covered with an antibody slide for incubation. Figure C shows the effect of the capture array and the upper and lower layers of the microwell array correspond one-to-one, taken under a microscope.

[0033] 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;

[0034] Figure 7 This is a schematic diagram of the experimental process for 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;

[0035] Figure 8 The oxygen content gradient within the liver lobule chip after the periphery of the chip was exposed to 25% oxygen-enriched oxygen in vitro in Example 2 of the present invention. The left figure shows the oxygen content inside the chip measured using Ru(ddp) dye, and the right figure shows the quantitative results of oxygen content from the outer edge to the center of the chip.

[0036] Figure 9 Partial experimental results and statistical analysis of the spatial distribution of MCP-1 secretion by hepatocytes using the Liver Lobular Chip in Example 2 of the present invention. The left figure is a fluorescence image of hepatocyte secretion detected in the chip using immunosandwich technology, and the right figure is a quantitative analysis of the left image.

[0037] Figure 10 Graphs showing the spatial distribution characteristics of MCP-1 and CD81+CD63+ exosomes secreted by hepatocytes in the liver lobule chip according to Example 3 of the present invention; A is a heat map showing the secretion of MCP-1 by hepatocytes in different spatial regions in the liver lobule chip; B is a statistical analysis showing the secretion of MCP-1 by hepatocytes in different spatial regions in the liver lobule chip; and C is a statistical analysis showing the secretion of CD81+CD63+ exosomes by hepatocytes in different spatial regions in the liver lobule chip.

[0038] Figure 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:

[0040] 1. Single-cell capture array core; 2. Microwell array chip; 3. Antibody slide; 1-1. Capture array; 2-1. Microwell array. DETAILED DESCRIPTION

[0041] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0042] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical 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.

[0043] 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.

[0044] Glossary:

[0045] “High throughput” is a technical term in this field, referring to the simultaneous testing of tens of thousands of cells.

[0046] Equipment for fluorescence intensity analysis includes, but is not limited to, gene chip scanners.

[0047] Example 1:

[0048] 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 spatially resolved single-cell secretion map analysis of hepatocytes.

[0049] 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, which is made by using SU-8 glue for soft lithography and then molding. The high-throughput single-cell capture array 1-1 is a regular hexagon as a whole, with an inscribed diameter of 10 mm. The six vertices and the center point of the hexagon are respectively provided with a liquid inlet and a liquid outlet 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.

[0050] like Figure 3The microwell array 2-1 on the high-throughput microwell array chip 2 consists of 18,034 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 of 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.

[0051] like Figure 5 The 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).

[0052] like Figure 4 The multi-color antibody slide is a poly-lysine slide uniformly incubated with MCP-1 monoclonal antibody. The preparation method of the multi-color antibody slide is as follows:

[0053] (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;

[0054] (2) Add the antibody coating solution to one end of the polylysine 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 polylysine glass slide;

[0055] (3) Place the poly-lysine slide in a humidified chamber and incubate at room temperature for 4 h or at 4°C overnight;

[0056] (4) Remove the slides and wash them with gradient washing solution: DPBS, ½DPBS, H2O, H2O, 5 min each time. Centrifuge and store for later use.

[0057] 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 in combination to detect the spatial heterogeneity of MCP-1 secretion in liver single cells in liver lobules.

[0058] Example 2:

[0059] 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 Example 1, comprising the following steps:

[0060] 1. Assembling the Liver Lobule Chip

[0061] Place the single-cell capture array chip 1 on the lower layer with the liquid inlet and outlet facing upward, and the micropore array chip 2 on 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 one to one to the upper and lower layers, and fix them with a clamp.

[0062] 2. Capturing primary human hepatocytes using a single-cell capture array to form a liver lobule chip

[0063] First, prepare a suspension of human primary liver cells. The liver cells are (1) fresh liver biopsy tissue from a clinical patient is taken and rinsed with DPBS (Dulbecco's phosphate buffered saline) containing 1% double antibody on ice under sterile conditions, and fat, connective tissue, blood vessels and other debris are removed; (2) the tissue is cut into 1 mm tissue blocks on ice under sterile conditions and rinsed with DPBS containing 1% double antibody; (3) the tissue is digested with 0.25% trypsin in a 37°C, 5% CO2 incubator, and shaken every 5 minutes. After digestion for 20-40 minutes, William's E complete medium (10% FBS + 1% double antibody + 89% William's E basal medium) and trypsin inhibitor are added to stop digestion, and the tissue is blown in the solution to form a tissue suspension; (4) the tissue suspension is centrifuged at 1000 rpm for 5 minutes. min, discard the supernatant, add type IV collagenase, and place in a 37°C constant temperature shaking shaker to shake until the tissue becomes flocculent; (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.

[0064] 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) Adjust the concentration of the hepatocyte suspension to approximately 2×10 5 / mL, loaded into a 1 mL syringe; (2) The syringe containing the liver 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 minutes, 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 prevent the fluid from flowing. At this point, the human liver lobule chip is captured.

[0065] 3. Flip the chip so that the liver cells fall into the corresponding microwell array and perfuse and culture ( Figure 7)

[0066] The specific steps are as follows: (1) flip the chip over, 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 six inlets at the vertices of the hexagon of the single-cell capture array, with a flow rate of 1 μL / min, and flows out from the outlet at 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.

[0067] 4. Remove the single cell capture array and cover with a multi-color multiplexed antibody slide

[0068] 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.

[0069] 5. Detection of hepatocyte secretions using immunofluorescence

[0070] After incubation, the 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 fixture, remove the slide, and block with 3% BSA for 1 hour; (2) Evenly drip 200 μl of Biotin-MCP-1 detection antibody on the slide and incubate for 1 hour; (3) Rinse the slide with 1% BSA and evenly drip 200 μl of Streptavidin-APC on the slide; (4) Use a gradient washing solution: DPBS, ½ DPBS, H2O, H2O to wash the slide in sequence, each for 5 minutes, and use the MD Gene-Pix 4300A gene chip scanner to obtain the fluorescence results; (5) Analyze the distribution of MCP-1 secretion by hepatocytes at different positions in the liver lobule chip.

[0071] 6. Results Analysis

[0072] The analysis results of this example are as follows Figure 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 showed 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.

[0073] Example 3:

[0074] This example 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 Example 1. The method comprises the following steps:

[0075] 1. Assembling the Liver Lobule Chip

[0076] Place the single-cell capture array chip 1 on the lower layer with the liquid inlet and outlet facing upward, and the micropore array chip 2 on 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 one to one to the upper and lower layers, and fix them with a clamp.

[0077] 2. Capturing primary human hepatocytes using a single-cell capture array to form a liver lobule chip

[0078] HepG2 cells, a liver cancer cell line, were inoculated into the chip. HepG2 cells were introduced into the chip via the central inlet of the regular hexagon using a syringe pump. The specific steps were as follows: (1) Adjust the concentration of the liver cell suspension to approximately 2×10 5 / mL, and 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 minutes, 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 prevent the fluid from flowing. At this point, the human liver lobule chip is captured.

[0079] 3. Flip the chip so that the liver cells fall into the corresponding microwell array and perfuse culture

[0080] The specific steps are as follows: (1) Flip the chip over, and the liver cells 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 liquid inlets at the six vertices of the hexagon at a flow rate of 1 μL / min and flow out from the liquid outlet at 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 hours.

[0081] 4. Remove the single cell capture array and cover with a multi-color multiplexed antibody slide

[0082] 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.

[0083] 5. Detection of hepatocyte secretions using immunofluorescence

[0084] After incubation, the slide has captured MCP-1 and CD63+ exosomes secreted by single hepatocytes in the liver lobule chip. The detection steps are as follows: (1) Remove the clamp, remove the slide, and block with 3% BSA for 1 hour; (2) Evenly add 200 μl of Biotin-MCP-1 / PE-CD81 detection antibody mixture on the slide and incubate for 1 hour; (3) Rinse the slide with 1% BSA and evenly add 200 μl of Streptavidin-APC on the slide; (4) Use gradient washing solution: DPBS, ½DPBS, H2O, H2O to wash the 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 and CD81+CD63+ exosomes secreted by hepatocytes at different positions in the liver lobule chip.

[0085] 6. Results Analysis

[0086] The analysis results of this example are as follows Figure 10 As shown in the results, the secretion distribution characteristics of MCP-1 and CD81+CD63+ exosomes in liver lobules were significant. Hepatocytes had obvious heterogeneity in their ability to secrete MCP-1 and CD81+CD63+ exosomes. Moreover, the MCP-1 secretion zoning showed that cells in the middle area had stronger MCP-1 secretion ability, while cells close to the portal vein or central vein secreted less MCP-1. CD81+CD63+ exosomes did not have this feature.

[0087] In summary, the liver lobule-on-a-chip disclosed in this invention enables the characterization of spatially heterogeneous liver secretion at the single-cell level, overcoming the reliance on precise anatomical sections. Combined with single-cell secretion detection technology, this chip can directly reveal the spatial distribution of hepatocyte secretory activity within the liver lobule, making it an ideal tool for characterizing spatially heterogeneous liver secretion. This chip has important applications in understanding the mechanisms of liver disease and assessing drug metabolism and toxicity.

[0088] Example 4:

[0089] 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 Figure 11The 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, thereby increasing the throughput of simultaneous detection.

Claims

1. A liver lobule chip suitable for performing spatial heterogeneous secretion analysis at the single-cell level, characterized in that: The invention comprises a single-cell capture array chip (1), a micropore array chip (2) and an antibody 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 in the shape of a regular hexagon, and a perfusion culture fluid channel is provided along the diagonal direction of each vertex of the regular hexagon; the capture array (1-1) on the single-cell capture array chip (1) is provided with a liquid inlet and a liquid outlet at the vertex and the center of the regular hexagon, respectively; the micropore array (2-1) on the micropore array chip (2) is provided with a gas channel and its gas inlet and gas outlet at the peripheral edge; 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 after assembly; The capture array (1-1) and the micropore 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 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, 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; the micropores on the micropore array chip (2) include any one or more of circular holes, square holes and long strip 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 provided 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.

2. The liver lobule chip according to claim 1, characterized in that The single cell capture array chip (1) and the microwell array (2-1) chip (2) are PDMS chips, and the capture array (1-1) and the microwell array (2-1) are made by soft lithography and then molding with SU-8 glue.

3. 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 assembly, 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.

4. The liver lobule chip according to any one of claims 1 to 3, characterized in that The antibody slide (3) is a polylysine slide; the surface of the antibody slide (3) is incubated with any one or more of antibodies, aptamers, and DNA; the liver lobule chip also includes a fixture for fixing the single cell capture array chip (1) and the micropore array chip (2), and the fixture is a PMMA fixture.

5. Use of the liver lobule chip according to any one of claims 1 to 4 in single-cell secretion analysis of liver lobule spatial heterogeneity.

6. The use according to claim 5, characterized in that The application method comprises the following steps: (1) The single cell capture array chip (1) is positioned at the lower layer with the liquid inlet and the liquid outlet facing upward, and the microwell array chip (2) is positioned at the upper 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 upper and lower layers, and are fixed with a clamp; (2) Inoculating hepatocytes at the inlet at the center of the regular hexagon of the single-cell capture array chip (1) to form a liver lobule chip; (3) Flip the liver lobule chip so that the liver cells captured in the capture array (1-1) fall into the corresponding microporous array (2-1), connect the culture medium through the liquid inlets at the six vertices of the regular hexagon of the single-cell capture array chip (1), and introduce oxygen-rich gas through the air inlet of the microporous array chip (2) for perfusion culture; (4) Disassemble the fixture, remove the single cell capture array chip (1), cover it with the antibody slide (3) and incubate; (5) The antibody slide (3) is taken out, and the hepatocyte secretions obtained by incubating on the antibody slide (3) are detected and analyzed by immunofluorescence.

7. The use according to claim 6, characterized in that The external culture medium for 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 an incubator at 35-38°C and 3-8% CO2, and the incubation time is 4-24 hours.

8. The use according to claim 6, 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), the detection antibody is dripped on the slide, imaging is performed using the immune sandwich method, and then fluorescence intensity analysis is performed.

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