A microfluidic device and method for evaluating idiosyncratic liver toxicity of drugs

Abstract

The application discloses a microfluidic device and method for evaluating drug idiosyncratic hepatotoxicity, wherein the microfluidic device comprises a microfluidic chip, the microfluidic chip comprises a lower substrate and an upper substrate, the upper substrate is provided with a first culture channel and a second culture channel, the first culture channel and the second culture channel are communicated through a micro fence, and the first culture channel and the second culture channel are respectively provided with corresponding fluid interfaces; the upper substrate is further provided with a liquid storage pool, the liquid storage pool is communicated with the second culture channel; wherein the first culture channel is used for culturing liver cells, the second culture channel is used for culturing immune cells, and the liquid storage pool is used for storing culture medium; and the micro fence is configured to allow the culture medium and the immune cells to pass through, but not allow the liver cells to pass through. The microfluidic device provided by the application can realize evaluation of drug idiosyncratic hepatotoxicity.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and in particular to a microfluidic device and evaluation method for evaluating drug-specific hepatotoxicity. Background Technology

[0002] Drug-induced hepatotoxicity is a common adverse drug reaction that threatens people's health. Some clinical studies and case reports indicate that long-term or high-dose drug use may be associated with liver damage. Drug-induced hepatotoxicity generally includes intrinsic hepatotoxicity and idiosyncratic hepatotoxicity (DILI). Intrinsic hepatotoxicity is liver damage directly caused by the drug and is dose-dependent. Idiosyncratic hepatotoxicity (DILI) refers to the difference in liver toxicity responses to the same drug among different individuals. This difference may be due to individual genetic variations, metabolic capacity, immune responses, and other factors. Idiosyncratic hepatotoxicity may lead to drug-induced liver injury exhibiting different severity and types in different individuals.

[0003] Idiogeneous hepatotoxicity is often unrelated to drug dosage but is related to individual differences, and can be further divided into immune-specific hepatotoxicity, metabolic-specific hepatotoxicity, and genetic-specific hepatotoxicity. Among them, immune-specific hepatotoxicity is hepatotoxicity induced by an individual's immune response. Assessing the immune-specific hepatotoxicity of drugs is very important because: (1) if a drug has immune-specific hepatotoxicity, it may interact with other drugs, leading to abnormal drug metabolism and increasing the drug's toxic side effects; (2) some people may be more sensitive to drugs and more prone to liver damage. Therefore, assessing the immune-specific hepatotoxicity of drugs can help identify high-risk individuals, thereby avoiding possible adverse reactions.

[0004] Drug hepatotoxicity models are experimental models used to assess the potential hepatotoxicity of drugs. Traditional models typically use mice or rats as experimental animals. After administration of a certain dose of the drug, the hepatotoxicity is assessed by detecting biochemical indicators (such as serum enzymes, endogenous antioxidants, and liver injury markers) and histopathological changes; or by using well plate models to assess the direct toxicity of drugs to hepatocytes. However, there is currently a lack of assessment models for drug-specific hepatotoxicity. The problems with traditional hepatotoxicity models are: the cell types are limited, resulting in significant species differences; while drug components are complex, and their pharmacological effects are multicellular and multi-target, especially with immune factors having a significant impact on their hepatotoxicity. Therefore, traditional hepatotoxicity models are insufficient to meet the needs of assessing drug-specific hepatotoxicity.

[0005] Therefore, there is an urgent need to develop an assessment model for evaluating drug-specific hepatotoxicity. Summary of the Invention

[0006] The purpose of this invention is to overcome the problem that existing models are difficult to assess drug-specific hepatotoxicity, and to provide an immune cell-liver microarray model that can evaluate drug-specific hepatotoxicity.

[0007] In a first aspect, the present invention provides a microfluidic device for evaluating drug-specific hepatotoxicity, the microfluidic device comprising a microfluidic chip, the microfluidic chip comprising:

[0008] Lower substrate; and

[0009] An upper substrate is attached and fixed to the lower substrate; the upper substrate is provided with a first culture channel and at least one second culture channel, the first culture channel and at least one second culture channel are connected by a microgrid, and the first culture channel and at least one second culture channel are respectively provided with corresponding fluid interfaces; the upper substrate is also provided with at least one liquid storage tank, the liquid storage tank is connected to the at least one second culture channel;

[0010] The first culture channel is used to culture hepatocytes, the second culture channel is used to culture immune cells, and the reservoir is used to store culture medium. The microgrid is configured to allow the culture medium and the immune cells to pass through, but not the hepatocytes.

[0011] In this invention, the upper and lower substrates can be made of the same or different materials, including but not limited to rigid plastics, elastic plastics, glass, quartz, silicon, ceramics, or metals. Further, rigid plastics include, but are not limited to, polymethyl methacrylate (PMMA), polycarbonate (PC), and polystyrene (PS); elastic plastics include, but are not limited to, polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), high-density polyethylene (HDPE), and polyvinyl chloride (PVC). The first and second substrates can be bonded together and sealed in any way, including but not limited to adhesive bonding and welding. For example, when both the upper and lower substrates are made of plastic, the bonding surfaces of the upper and lower substrates can be plasma-treated to break the chemical bonds on the bonding surfaces, forming polar or reactive groups. Then, the upper and lower substrates are pressed together, causing the broken bonds on the bonding surfaces to chemically bond, thus achieving a sealed connection between the first and second substrates.

[0012] In this invention, the purpose of setting up microbarriers is to allow the culture medium and immune cells in the second culture channel to enter the first culture channel, while preventing hepatocytes in the first culture channel from entering the second culture channel. Since hepatocytes are adherent cells and immune cells are suspension cells, the gap width in the microbarriers can be set according to the different sizes of the immune cells to allow their passage. Further, the gap width in the microbarriers is 0.1–100 μm, for example, any value or a range of any two values ​​from 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 8 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 80 μm, and 100 μm. In some embodiments, the microbarrier is rectangular in shape, and its width (i.e., the projected length of the microbarrier on the first or second culture channel) can be 0.1–10000 μm.

[0013] In some embodiments, the upper substrate is provided with a first culture channel and a pair of second culture channels, the pair of second culture channels are respectively disposed on both sides of the first culture channel and are respectively connected to the first culture channel through micro-grids; each end of each second culture channel is connected to a liquid storage tank.

[0014] In some embodiments, a pair of fluid interfaces are provided for the first culture channel, and the pair of fluid interfaces are respectively connected to both ends of the first culture channel; a fluid interface is provided for the second culture channel, and the fluid interface is connected to the middle of the second culture channel.

[0015] In this invention, the first culture channel, the second culture channel, the micro-fence, the liquid storage tank, and the fluid interface can be fabricated using micromachining processes, including but not limited to surface etching and MEMS fabrication.

[0016] In this invention, the first culture channel is used to culture one or more hepatocytes, which may be derived from primary hepatocytes or hepatocytes differentiated from stem cells. These hepatocytes include, but are not limited to, one or more of HepG2 cells, HepRG cells, LO-2 cells, Huh-7 cells, and LX-2 cells.

[0017] In this invention, the second culture channel is used to culture one or more immune cells, which may be derived from immortalized cell lines, stem cell-differentiated cells, or primary extracted cells. The immune cells include, but are not limited to, one or more of macrophages, NK cells, monocytes, neutrophils, T lymphocytes, and B lymphocytes.

[0018] Furthermore, the microfluidic device may also include a shaker in which the microfluidic chip is placed. This shaker allows for the dynamic culture of hepatocytes and immune cells, better simulating the in vivo growth environment.

[0019] Secondly, the present invention provides a method for evaluating drug-specific hepatotoxicity, the method using the aforementioned microfluidic device and comprising the following steps:

[0020] S1. Hepatocytes are seeded into the first culture channel through a fluid interface, and after static culture for 0.2 to 1 hour, culture medium is added to the storage tank, and then the microfluidic chip is placed in a shaker for dynamic culture for 12 to 24 hours.

[0021] S2. Aspirate the culture medium from the microfluidic chip, inoculate the first immune cells into the second culture channel through the fluid interface, and then place the microfluidic chip in a shaker for dynamic culture for 1 to 6 hours;

[0022] S3. Aspirate the culture medium from the microfluidic chip, add the culture medium containing the drug to the reservoir, and then place the microfluidic chip in a shaker for dynamic culture for 48-96 hours;

[0023] S4. Evaluate the hepatocytes and / or the first immune cells to determine the drug-specific hepatotoxicity of the drug.

[0024] Thirdly, the present invention provides a method for evaluating drug-specific hepatotoxicity, the method using the aforementioned microfluidic device and comprising the following steps:

[0025] S1. Hepatocytes are seeded into the first culture channel through a fluid interface, and after static culture for 0.2 to 1 hour, culture medium is added to the storage tank, and then the microfluidic chip is placed in a shaker for dynamic culture for 12 to 24 hours.

[0026] S2. Aspirate the culture medium from the microfluidic chip, add culture medium containing drugs and second immune cells to the reservoir, and then place the microfluidic chip in a shaker for dynamic culture for 48-96 hours;

[0027] S3. Evaluate the hepatocytes and / or the second immune cells to determine the drug-specific hepatotoxicity of the drug.

[0028] Fourthly, the present invention provides a method for evaluating drug-specific hepatotoxicity, the method using the microfluidic device described above, and comprising the following steps:

[0029] S1. Hepatocytes are seeded into the first culture channel through a fluid interface, and after static culture for 0.2 to 1 hour, culture medium is added to the storage tank, and then the microfluidic chip is placed in a shaker for dynamic culture for 12 to 24 hours.

[0030] S2. Aspirate the culture medium from the microfluidic chip, inoculate the first immune cells into the second culture channel through the fluid interface, and then place the microfluidic chip in a shaker for dynamic culture for 1 to 6 hours;

[0031] S3. Aspirate the culture medium from the microfluidic chip, add culture medium containing drugs and second immune cells to the reservoir, and then place the microfluidic chip in a shaker for dynamic culture for 48-96 hours;

[0032] S4. Evaluate the hepatocytes and / or the first immune cell and the second immune cell to determine the drug-specific hepatotoxicity of the drug.

[0033] The principle of this invention for evaluating drug-specific hepatotoxicity is:

[0034] The immune cell-liver chip structure of this invention can simulate the structure of hepatic sinusoids, where hepatocytes are fixed in one region to simulate hepatic plates, and fluid channels on both sides can simulate blood. Immune cells exist in the blood. During the interaction between drugs and hepatocytes, immune cells can chemotactically move to the liver region with the flow of fluid to exert their effects, thus enabling the evaluation of immune cell-mediated idiosyncratic hepatotoxicity. The advantages of this microfluidic chip structure are: it allows observation of the chemotactic adhesion behavior of immune cells to evaluate drug toxicity; furthermore, this method can obtain not only hepatocyte toxicity indicators but also multi-dimensional indicators including immune cell infiltration behavior and changes in inflammatory factors, thereby providing a more comprehensive evaluation of drug-specific hepatotoxicity.

[0035] In existing well plate models, hepatocytes and immune cells are mixed in the well plate. This makes it impossible to distinguish between hepatocytes and immune cells, making it difficult to detect the toxicity of hepatocytes and observe whether immune cells have undergone chemotaxis.

[0036] In step S1 of this invention, the microfluidic device is sterilized by high temperature and high pressure before inoculating hepatocytes.

[0037] Furthermore, in step S2, before inoculating the first or second immune cells into the chip, a culture medium containing the drug and [the drug] can be added to the chip and cultured for a period of time.

[0038] Furthermore, in steps S1 to S3, the shaking table is a shaking table that swings up and down, with an amplitude range of 1° to 90° and a swing speed of 1 to 99 r / s.

[0039] During dynamic culture, a shaker equipped with a microfluidic device was placed in a cell culture incubator, and the environment inside the cell culture incubator was controlled at 37°C and 5% CO2.

[0040] Furthermore, the first immune cell is an immune cell found in the liver, including macrophages. Macrophages and other immune cells found in the liver are inoculated into the second culture channel. During the interaction between the drug and hepatocytes, these immune cells can rapidly chemotact with the hepatocyte region through the microbarrier, thus more accurately mimicking the action of immune cells.

[0041] Furthermore, the second immune cell is an immune cell present in the blood or lymph, including at least one of NK cells, monocytes, neutrophils, T lymphocytes, and B lymphocytes.

[0042] In this invention, immune cells present in the blood or lymph, such as monocytes, neutrophils, T lymphocytes, and B lymphocytes, are added to the reservoir along with the culture medium. During the interaction between the drug and hepatocytes, these immune cells move with the culture medium (used to simulate blood) and chemotactically migrate to the hepatocyte region. Their immune response lags behind that of macrophages and other immune cells already present in the liver. Therefore, this method more accurately simulates the role of different types of immune cells in the process of hepatotoxicity, improving the accuracy of drug-specific hepatotoxicity evaluation.

[0043] Furthermore, the drug includes at least one of small molecule drugs, macromolecule drugs, gene drugs, and cell drugs; and / or,

[0044] The small molecule drugs include abacavir, troglitazone, sulfamethoxazole, acetaminophen, and emodin; and / or,

[0045] The macromolecular drugs include insulin, interferon, tuximab, and Herceptin; and / or,

[0046] The gene therapy drugs include viral vector-based in vivo gene therapy drugs, in vitro gene therapy drugs, naked plasmid drugs, antisense oligonucleotide drugs, siRNA drugs, and mRNA gene therapy drugs; and / or,

[0047] The cell-based drugs include CAR-T cells, NK cells, and stem cells.

[0048] Furthermore, conventional biological methods can be used to assess hepatotoxicity, including but not limited to immunofluorescence, flow cytometry, ELISA, and biochemical analyzers.

[0049] Through one or more embodiments of the above embodiments of the present invention, at least the following technical effects can be achieved:

[0050] 1. This invention provides a microfluidic device and corresponding evaluation method for evaluating drug-specific hepatotoxicity, thereby overcoming the deficiency of existing technologies in the lack of assessment models for drug-specific hepatotoxicity.

[0051] 2. The microfluidic device for evaluating drug-specific hepatotoxicity provided by this invention can accurately simulate the process of immune cells chemotaxis to the hepatocyte region to exert their effects during the interaction between drugs and hepatocytes. Furthermore, this method can not only observe hepatocyte toxicity indicators, but also observe multi-dimensional evaluation indicators, including immune cell infiltration behavior and changes in inflammatory factors, thereby improving the accuracy of drug-specific hepatotoxicity evaluation. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the microfluidic device in one embodiment of the present invention;

[0053] Figure 2 for Figure 1 Top view of the microfluidic device in the image;

[0054] Figure 3 The image shows the fluorescence of GFP-HepG2 cells from Example 1.

[0055] Figure 4 for Figure 3 A statistical graph of the fluorescent area in the middle;

[0056] Figure 5 This is a diagram showing the changes in neutrophil-related genes in Example 1;

[0057] Figure 6 The level of reactive oxygen species within the chip in Example 1;

[0058] Figure 7 for Figure 6 Fluorescence intensity analysis diagram;

[0059] Figure 8 A diagram showing the infiltration of neutrophils (red) into the liver region in the chip when different concentrations of troglitazone are added;

[0060] Figure 9 Fluorescence area analysis of the liver region in the chip when different concentrations of troglitazone were added;

[0061] Figure 10 Here is a fluorescence image of HepG2 cells from Example 2;

[0062] Figure 11 This is a statistical chart showing the survival rate of HepG2 cells in Example 2;

[0063] Figure 12 Here is a fluorescence image of HepG2 cells from Example 3;

[0064] Figure 13 This is a statistical chart showing the survival rate of HepG2 cells in Example 3;

[0065] Figure 14 Here is a fluorescence image of HepG2 cells from Example 4;

[0066] Figure 15 This is a statistical chart showing the survival rate of HepG2 cells in Example 4;

[0067] The components include: 1. Upper substrate; 2. Lower substrate; 3. First culture channel; 4. Second culture channel; 5. Microgrid; 6. Liquid reservoir; 7. Fluid interface;

[0068] Control indicates that the culture medium does not contain the drug troglitazone; 15μM TGZ indicates that the culture medium contains 15μM of the drug troglitazone; -dHL-60 indicates that the culture medium does not contain dHL-60 cells; +dHL-60 indicates that the culture medium contains dHL-60 cells; -NK-92 indicates that the culture medium does not contain NK-92 cells; +NK-92 indicates that the culture medium contains NK-92 cells. Detailed Implementation

[0069] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] In the following embodiments and comparative examples, all raw materials and equipment, unless otherwise specified, can be obtained by purchasing.

[0071] Example 1: Hepatotoxicity of troglitazone (TGZ) in the presence of neutrophils

[0072] For the first time, this embodiment provides a microfluidic device for evaluating drug-specific hepatotoxicity, including a shaker and a microfluidic chip.

[0073] As attached Figure 1-2As shown, the microfluidic chip consists of two layers: a lower substrate 2 and an upper substrate 1. The upper substrate 1 is fabricated using microfabrication processes to create microstructures such as a first culture channel 3, a second culture channel 4, a micro-gate 5, a reservoir 6, and a fluid interface 7. Specifically, the upper substrate 1 has a first culture channel 3 and a pair of second culture channels 4, both of which are rectangular channels. The pair of second culture channels 4 are located on opposite sides of the first culture channel 3 and connected to it via micro-gates 5. Each second culture channel 4 has a reservoir 6 connected to both ends. The first culture channel 3 has a pair of fluid interfaces 7 connected to both ends; each second culture channel 4 has a single fluid interface 7 connected to the middle of the second culture channel 4.

[0074] Secondly, the aforementioned microfluidic chip was used to test the hepatotoxic effects of neutrophils on troglitazone.

[0075] Neutrophils (dHL-60) are the most abundant white blood cells in the blood and are specialized phagocytes. They are produced by hematopoietic stem cells in the bone marrow and released into the bloodstream. They are rapidly recruited to sites of inflammation, where they release proteolytic enzymes stored in the granules and produce reactive oxygen species (ROS). According to reports, acetaminophen toxicity induces strong neutrophil recruitment into the liver.

[0076] In this embodiment, the method for evaluating the drug-specific hepatotoxicity of troglitazone is as follows:

[0077] S1. GFP-HepG2 cells were prepared at a density of 1.3 × 10⁻⁶. 7 cells·mL -1 The cell suspension was mixed with Matrigel at a volume ratio of 3:1. 24 microfluidic chips were divided into 4 groups of 6. 5 μL of the cell mixture was added to the first culture channel of each microfluidic chip, and the chips were placed in an incubator and allowed to stand for 0.5 h. Then, 200 μL of culture medium was added to the reservoir at one end of the second culture channel until the chip was full. Another 200 μL of culture medium was then added, and the microfluidic chip was placed on a shaker in the incubator for further culture.

[0078] S2. On the second day, aspirate the culture medium from the chip, then add 600 μL of culture medium to the reservoir of the first set of chips, and add 600 μL of culture medium containing dHL-60 cells (density 1.5 × 10⁻⁶) to the reservoir of the second set of chips. 6 cells·mL -1600 μL of culture medium containing troglitazone was added to the reservoir of the third chip, and 600 μL of culture medium containing troglitazone and dHL-60 cells (density 1.5 × 10⁻⁶) was added to the reservoir of the fourth chip. 6 cells·mL -1 The chip was placed in a shaker and cultured dynamically for 24 hours, then 200 μL of culture medium was added; after another 24 hours of culture, another 200 μL of culture medium was added, and the culture continued for another 24 hours.

[0079] The state of GFP-HepG2 cells in the first culture channel was detected using an inverted fluorescence microscope, and the area of ​​green fluorescence in the first culture channel was counted using Imagej software.

[0080] Figure 3 This is a fluorescence image of GFP-HepG2 cells. Figure 4 The graph shows the fluorescence area, which represents the number of GFP-HepG2 cells. As can be seen from the graph, in both standalone hepatocyte microarrays (LOCs, i.e., groups one and three) and immuno-hepatocyte microarrays (i-LOCs, i.e., groups two and four), the fluorescence area decreases with the addition of troglitazone, indicating a decrease in the number of GFP-HepG2 cells, i.e., a gradual increase in toxicity. Therefore, troglitazone is significantly more toxic in immuno-hepatocyte microarrays than in standalone hepatocyte microarrays.

[0081] Figure 5 This diagram shows the changes in neutrophil-related genes. As can be seen from the diagram, neutrophil inflammatory factors were upregulated in the treatment groups (i.e., groups three and four), indicating that neutrophils were activated during the drug's hepatotoxicity process and upregulated inflammation-related genes to exert their effects.

[0082] Figure 6 The level of reactive oxygen species within the chip. Figure 7 Yes Figure 6 Fluorescence intensity analysis was performed. The figure shows that the fluorescence intensity was highest in the group receiving simultaneous drug administration and the neutrophil group (i.e., group four), indicating the strongest level of reactive oxygen species, which is the main cause of hepatotoxicity.

[0083] Figure 8 This image shows the infiltration of neutrophils (red) into the liver region when different concentrations of troglitazone are added. Figure 9 The graph shows the fluorescence area analysis of the liver region. As can be seen from the graph, the number of infiltrating neutrophils in the drug-treated groups (i.e., groups three and four) is greater than that in the drug-free groups (i.e., groups one and two), indicating that neutrophil changes not only occurred at the genetic level but also involved infiltration.

[0084] Example 2: Hepatotoxicity of troglitazone in the presence of NK-92 cells

[0085] Natural killer (NK) cells are important members of the immune system, playing a crucial role in recognizing and eliminating foreign invaders. During inflammatory damage, NK cells infiltrate in large numbers and begin their function.

[0086] In this embodiment, the method for evaluating the drug-specific hepatotoxicity of troglitazone is as follows:

[0087] (1) Prepare GFP-HepG2 cells at a density of 1.3 × 10⁻⁶. 7 cells·mL -1 The cell suspension was mixed with Matrigel at a volume ratio of 3:1. 24 microfluidic chips were divided into 4 groups of 6. 5 μL of the cell mixture was added to the first culture channel of each microfluidic chip, and the chips were placed in an incubator and allowed to stand for 0.5 h. Then, 200 μL of culture medium was added to the reservoir at one end of the second culture channel until the chip was full. Another 200 μL of culture medium was then added, and all microfluidic chips were placed on a shaker in the incubator for further culture.

[0088] (2) On the second day, aspirate the culture medium from the chip, then add 600 μL of culture medium to the reservoir of the first set of chips, and add 600 μL of NK-92 cells (density 1.5 × 10⁻⁶) to the reservoir of the second set of chips. 6 cells·mL -1 600 μL of culture medium containing troglitazone was added to the reservoir of the third chip, and 600 μL of culture medium containing troglitazone and NK-92 cells (density 1.5 × 10⁻⁶) was added to the reservoir of the fourth chip. 6 cells·mL -1 The chip was placed in a shaker and cultured dynamically for 24 hours, then 200 μL of culture medium was added; after another 24 hours of culture, another 200 μL of culture medium was added, and the culture continued for another 24 hours.

[0089] The culture medium inside the chip was aspirated, and a culture medium containing calcein AM and PI was added. The chip was placed in an incubator and allowed to stand for 20 minutes. After rinsing the chip three times with the culture medium, the chip was photographed using an inverted fluorescence microscope and finally processed using Imagej software.

[0090] Figure 10 Here is a fluorescence image of HepG2 cells. Figure 11The graph shows the survival rate of HepG2 cells. As can be seen, NK-92 cells can induce cytotoxicity against HepG2 cells independently, consistent with the characteristics of NK-92 cells. Surprisingly, this cytotoxicity did not increase or remain constant with the addition of troglitazone, but rather decreased, indicating that NK-92 cells have no significant effect on the hepatotoxicity of troglitazone.

[0091] Example 3

[0092] Kupffer cells are macrophages that reside in the liver. The number and state of Kupffer cells change during physiological and pathological processes in the liver, which is crucial for studying drug-induced liver damage. In this embodiment, a series of liver microarrays with varying numbers of Kupffer cells were fabricated to investigate the association between troglitazone and Kupffer cell count and liver damage.

[0093] The specific experimental steps are as follows:

[0094] (1) Prepare GFP-HepG2 cells into 1×10 7 cells·mL -1 Cell suspension was prepared. Sixty microfluidic chips were divided into 10 groups of 6. 5 μL of the cell suspension was added to the first culture channel of each microfluidic chip. After standing in an incubator for 1 hour, 200 μL of culture medium was added to the reservoirs of the second culture channels on both sides until the chip was filled. Then, another 200 μL of culture medium was added. All microfluidic chips were then placed on a shaker in an incubator for culture.

[0095] (2) On the second day, the culture medium inside the chip was aspirated, and then 600 μL of culture medium was added to the chips of groups 1, 3, 5, 7 and 9, and 600 μL of culture medium containing troglitazone (15 μM) was added to the chips of groups 2, 4, 6, 8 and 10. The chips were placed on a shaker for dynamic culture at a speed of 0.1 r / min. On the third day, 200 μL of culture medium was added to each chip.

[0096] (3) On the fourth day, the culture medium inside the chip was aspirated, and the induced THP-1 macrophages were digested and prepared into cells with densities of 2.5 × 10⁻⁶. 4 cells·mL -1 12.5×10 4 cells·mL -1 62.5×10 4 cells·mL -1 125×10 4 cells·mL -1400 μL of cell suspension was added to the second culture channel of chips in groups 3 to 10, respectively. After culturing for 4 hours, the culture medium in the chip was aspirated. Then, 600 μL of culture medium was added to chips in groups 1, 3, 5, 7, and 9, and 600 μL of culture medium containing troglitazone (15 μM) was added to chips in groups 2, 4, 6, 8, and 10. The chips were then placed in an incubator for dynamic culture.

[0097] After culturing for 24 hours, the culture medium was placed in a -20℃ refrigerator for later use. 200 μL of fresh culture medium was added to each group of chips. The status of GFP-HepG2 cells in the liver channel was detected using an inverted fluorescence microscope, and the green fluorescence area of ​​the liver channel was counted using Imagej software.

[0098] like Figures 12-13 As shown, when the number of THP-1 macrophages is 0-25 × 10⁻⁶ 4 At time 1 hour, there was no significant change in the number of hepatocytes in the chip between the drug-treated group and the drug-free group; when THP-1 macrophages were at 50 × 10⁻⁶... 4 At that time, the number of hepatocytes in the chip containing 15 μM troglitazone was significantly reduced. Figure 12 The same result was obtained by statistically analyzing the fluorescence area. Figure 13 ).

[0099] Example 4

[0100] Interactions exist between immune cells. For example, macrophages can release pro-inflammatory or anti-inflammatory factors that promote or inhibit inflammatory responses. For instance, macrophages recruit neutrophils by releasing MIP-2, thus influencing neutrophil recruitment and activation. Therefore, this study investigated whether macrophages and neutrophils have a synergistic effect on the hepatotoxicity of troglitazone.

[0101] The specific experimental steps are as follows:

[0102] (1) GFP-HepG2 cells were prepared at a density of 1×10⁻⁶. 7 cells·mL -1 Cell suspension was prepared. Forty-eight microfluidic chips were divided into eight groups of six. 5 μL of the cell suspension was added to the first culture channel of each microfluidic chip. After standing in an incubator for 1 hour, 200 μL of culture medium was added to the reservoirs of the second culture channels on both sides until the chip was filled. Then, another 200 μL of culture medium was added. All microfluidic chips were then placed on a shaker in an incubator for culture.

[0103] (2) On the second day, the culture medium inside the chip was aspirated, and then 600 μL of culture medium was added to the chips of groups 1, 3, 5 and 7, and 600 μL of culture medium containing troglitazone (15 μM) was added to the chips of groups 2, 4, 6 and 8. The chips were placed on a shaker for dynamic culture at a speed of 0.1 r / min. On the third day, 200 μL of culture medium was added to each chip.

[0104] (3) On the fourth day, the culture medium inside the chip was aspirated, and the induced THP-1 macrophages were digested and prepared to a density of 62.5 × 10⁶ cells / cm². 4 cells mL -1 The cell suspension was added to the second culture channel of the 3rd to 4th and 7th to 8th groups of chips and then placed in an incubator for culture.

[0105] (4) After culturing for 4 hours, the culture medium in each chip was aspirated. Then, 600 μL of culture medium was added to the chips in groups 1 and 3, 600 μL of culture medium containing troglitazone (15 μM) was added to the chips in groups 2 and 4, and 600 μL of culture medium containing dHL-60 cells (1.5 × 10⁻⁶) was added to the chips in groups 5 and 7. 6 cells mL -1 In a culture medium containing 15 μM troglitazone and 1.5 × 10⁶ dHL-60 cells, 600 μL of the medium was added to the 6th and 8th groups of chips. 6 cells·mL -1 The culture medium is placed in an incubator for incubation.

[0106] After culturing for 24 hours, the culture medium was removed, centrifuged at 1000 rpm for 5 min, and the supernatant was collected and placed in a -20℃ refrigerator for later use. 200 μL of fresh culture medium was added to the chip, and the status of GFP-HepG2 cells in the liver channel was detected using an inverted fluorescence microscope. The green fluorescence area of ​​the liver channel was counted using Imagej software.

[0107] This embodiment sets up separate liver microarrays (only HepG2, i.e., groups 1-2), macrophage liver microarrays (+THP-1, i.e., groups 3-4), neutrophil liver microarrays (+dHL-60, i.e., groups 5-6), and macrophage-neutrophil liver microarrays (+THP-1 and dHL-60, i.e., groups 7-8). Figure 14 As shown in the image, the green fluorescence plot indicates the remaining number of hepatocytes in different groups. It is clearly visible from the figure that the macrophage-neutrophil liver microarray treatment group had the fewest remaining hepatocytes, showing a significant difference compared to the control group. See also... Figure 15 The same result was obtained by statistically analyzing the area of ​​green fluorescence. This indicates that the combined effect of macrophages and neutrophils increases the hepatotoxicity of troglitazone.

[0108] In summary, although the present invention has been disclosed above with reference to preferred embodiments, the above preferred embodiments are not intended to limit the present invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope defined in the claims.

Claims

1. A method for evaluating drug-specific hepatotoxicity, characterized in that, The method uses a microfluidic device, the microfluidic device including a microfluidic chip, the microfluidic chip including: Lower substrate; and An upper substrate is attached and fixed to the lower substrate; the upper substrate is provided with a first culture channel and at least one second culture channel, the first culture channel and at least one second culture channel are connected by a microgrid, and the first culture channel and at least one second culture channel are respectively provided with corresponding fluid interfaces; the upper substrate is also provided with at least one liquid storage tank, the liquid storage tank is connected to the at least one second culture channel; The first culture channel is used to culture liver-derived cells, the second culture channel is used to culture immune cells, and the reservoir is used to store culture medium; the microbarrier is configured to allow the culture medium and the immune cells to pass through, but not the liver-derived cells. The method includes method one, method two, or method three; method one includes the following steps: S1. Liver-derived cells are seeded into the first culture channel through a fluid interface, and after static culture for 0.2-1 h, culture medium is added to the reservoir, and then the microfluidic chip is placed in a shaker for dynamic culture for 12-24 h; S2. Aspirate the culture medium from the microfluidic chip, inoculate the first immune cells into the second culture channel through the fluid interface, and then place the microfluidic chip in a shaker for dynamic culture for 1-6 hours; S3. Aspirate the culture medium from the microfluidic chip, add the culture medium containing the drug to the reservoir, and then place the microfluidic chip in a shaker for dynamic culture for 48~96h; S4. Evaluate the liver-derived cells and / or the first immune cells to determine the drug-specific hepatotoxicity of the drug; The second method includes the following steps: S1. Liver-derived cells are seeded into the first culture channel through a fluid interface, and after static culture for 0.2-1 h, culture medium is added to the reservoir, and then the microfluidic chip is placed in a shaker for dynamic culture for 12-24 h; S2. Aspirate the culture medium from the microfluidic chip, add culture medium containing drugs and second immune cells to the reservoir, and then place the microfluidic chip in a shaker for dynamic culture for 48~96h; S3. Evaluate the liver-derived cells and / or the second immune cells to determine the drug-specific hepatotoxicity of the drug; Method 3 includes the following steps: S1. Liver-derived cells are seeded into the first culture channel through a fluid interface, and after static culture for 0.2-1 h, culture medium is added to the reservoir, and then the microfluidic chip is placed in a shaker for dynamic culture for 12-24 h; S2. Aspirate the culture medium from the microfluidic chip, inoculate the first immune cells into the second culture channel through the fluid interface, and then place the microfluidic chip in a shaker for dynamic culture for 1-6 hours; S3. Aspirate the culture medium from the microfluidic chip, add culture medium containing drugs and second immune cells to the reservoir, and then place the microfluidic chip in a shaker for dynamic culture for 48~96h; S4. Evaluate the liver-derived cells and / or the first and second immune cells to determine the drug-specific hepatotoxicity of the drug; Wherein, the first immune cell is an immune cell present in the liver, including macrophages; and / or, The second immune cell is an immune cell present in the blood or lymph, including at least one of NK cells, monocytes, neutrophils, T lymphocytes, and B lymphocytes; and / or, The liver-derived cells include one or more of HepG2, HepRG, LO-2, Huh-7, and LX-2.

2. The method for evaluating drug-specific hepatotoxicity as described in claim 1, characterized in that, The gap width in the micro fence is 0.1~100 μm.

3. The method for evaluating drug-specific hepatotoxicity as described in claim 1, characterized in that, The upper substrate is provided with a first culture channel and a pair of second culture channels. The pair of second culture channels are respectively located on both sides of the first culture channel and are connected to the first culture channel through micro-grids. Each end of the second culture channel is connected to a liquid storage tank.

4. The method for evaluating drug-specific hepatotoxicity as described in claim 1, characterized in that, The drug includes at least one of small molecule drugs, macromolecule drugs, gene drugs, and cell drugs; The small molecule drugs include abacavir, troglitazone, sulfamethoxazole, acetaminophen, and emodin; and / or, The macromolecular drugs include insulin, interferon, rituximab, and trastuzumab; and / or, The gene therapy drugs include viral vector-based in vivo gene therapy drugs, in vitro gene therapy drugs, naked plasmid drugs, antisense oligonucleotide drugs, siRNA drugs, and mRNA gene therapy drugs; and / or, The cell-based drugs include CAR-T cell drugs, NK cell drugs, and stem cell drugs.

Images