Light response liver chip system for drug immunotoxicity evaluation and application thereof

Abstract

The invention belongs to the technical field of toxicological evaluation, and discloses a photoresponsive liver chip system for evaluating drug immunotoxicity, which comprises a liver chip with a multilayer structure, a photoresponsive immune cell elimination reagent and an illumination device. The invention also discloses an application of the photoresponsive liver chip system in drug immunotoxicity evaluation, which comprises the following steps: firstly, in a state that the photoresponsive immune cell elimination reagent is not activated, performing a first round of toxicity detection on a to-be-detected drug to obtain baseline toxicity data; then, activating a reagent through illumination, and specifically eliminating immune cells in the chip; then carrying out a second round of toxicity detection; the immunotoxicity contribution of the to-be-detected medicine is accurately evaluated by comparing the difference of two rounds of toxicity detection data. The method realizes sequential resolution evaluation on the direct toxicity and the immune-mediated toxicity of the drug on the same biological sample, overcomes the defects that a traditional method is tedious in operation and cannot distinguish toxicity sources, and has the advantages of rapidness, accuracy and high throughput.

Description

Technical Field

[0001] This invention belongs to the field of toxicology evaluation technology, specifically relating to an organ-on-a-chip technology, particularly a liver-on-a-chip system integrating photoresponsive immune cell elimination reagents and its application in rapid evaluation of drug immunotoxicity. Background Technology

[0002] Drug-induced liver injury ( Drug-Induced Liver Injury Drug-induced liver injury (DILI) is one of the leading causes of drug clinical trial failures and post-marketing withdrawals. Its pathogenesis is complex and can generally be categorized into two types: first, the direct toxic effects of drugs and their metabolites on hepatocytes; and second, indirect toxic reactions mediated by the immune system, where drugs or their metabolites act as haptens triggering abnormal immune responses, or stressed hepatocytes release damage-related molecular patterns that activate immune cells, initiating a cytokine storm and amplifying liver damage. Accurately distinguishing between these two mechanisms is crucial for understanding the nature of drug toxicity, assessing clinical risks, and guiding the design of safe drugs.

[0003] However, current preclinical toxicity assessment systems have significant limitations. Traditional two-dimensional static hepatocyte culture models cannot simulate the complex in vivo immune microenvironment, thus completely ignoring the contribution of immune-mediated toxicity. While animal models possess a complete immune system, their predictive value is limited due to significant species differences from humans in drug metabolism and immune response, and they also incur high ethical costs. In recent years, advanced liver-on-a-chip technology has simulated the physiological microenvironment of the liver through microfluidic engineering and achieved the co-culture of hepatocytes and immune cells, providing a better platform for in vitro studies of immune-mediated hepatotoxicity. However, existing co-culture models still rely on setting up multiple parallel experimental groups (such as hepatocyte-only groups and co-culture groups) for comparison, which is not only cumbersome and has low throughput, but also introduces huge variables due to differences in samples between groups, making it impossible to achieve temporal and dynamic separation of the two toxicity contributions on the same biological sample. Developing an innovative assessment tool that can dynamically and precisely regulate immune cell function and non-destructively distinguish between direct drug toxicity and immune-mediated toxicity in the same system has become a key breakthrough for improving the accuracy and efficiency of drug safety assessment. Summary of the Invention

[0004] This invention aims to solve the technical problems of traditional methods, such as the need to set up multiple parallel control groups, cumbersome operation, and large variability between samples. It provides a new, reliable and efficient solution for drug safety evaluation, and provides a compact and functionally integrated photoresponsive liver chip system and its application.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A photoresponsive liver chip system for evaluating drug immunotoxicity includes: a multilayer liver chip, a photoresponsive immune cell elimination reagent, and a light irradiation device capable of activating the activity of the photoresponsive immune cell elimination reagent. The liver chip comprises, from top to bottom, an upper substrate, an upper porous membrane, a middle substrate, a lower porous membrane, and a lower substrate; The upper substrate is provided with a perfusion main channel, an immune cell culture chamber, and a reagent preloading chamber. The immune cell culture chamber and the reagent preloading chamber are respectively connected to the perfusion main channel through microchannels. The microchannels can achieve fluid communication, ensuring that signal molecules such as cytokines and metabolites can be freely exchanged, simulating intercellular communication in vivo. Through this structural design, the culture of immune cells and the preloading of reagents are integrated on the same layer of the chip, and the functions are linked through the microenvironment, which greatly improves the integration of operation and the accuracy of timing control. The intermediate substrate and the lower substrate are respectively provided with an intermediate cell culture chamber and a lower cell culture chamber. The shapes of the intermediate cell culture chamber and the lower cell culture chamber are consistent with the shape of the perfusion main channel and their positions correspond one-to-one. The upper porous membrane and the lower porous membrane are respectively disposed on the upper and lower sides of the middle cell culture chamber, and separate the main perfusion channel, the middle cell culture chamber and the lower cell culture chamber to form three chambers: upper, middle and lower.

[0006] This system, through its unique chamber layout and pre-loaded design of photoresponsive immune cell elimination reagents, combined with time-sequential light control technology, can achieve precise and specific elimination of immune cells within a single chip unit. This allows for seamless differentiation and quantitative evaluation of the direct toxic effects of drugs and their contribution to immune-mediated toxicity on the same biological sample.

[0007] Furthermore, in the aforementioned photoresponsive liver chip system, the liver chip also includes an upper cover plate and a lower cover plate, which are respectively disposed on the upper layer of the upper substrate and the lower layer of the lower substrate; the main perfusion channel, the immune cell culture chamber, the reagent preloading chamber and the lower cell culture chamber are all provided with inlet and outlet ports, as well as corresponding sealing plugs.

[0008] Furthermore, the main perfusion channel, the immune cell culture chamber, and the reagent preloading chamber are located on the same horizontal plane; the width of the main perfusion channel is 0.5-2 mm; the microchannel is formed by several rectangular protruding microstructures arranged at vertical intervals to form multiple channels 5-30 µm wide, with the left or right corners of the rectangular protruding microstructures having a 15-45° chamfer with a width of 1-2 mm, so that the chamfers of adjacent rectangular protruding microstructures form a micro-trumpet-shaped channel with the opening end facing the main perfusion channel, and the opening ends of the micro-trumpet-shaped channels on both sides of the main perfusion channel 2-1 correspond one-to-one.

[0009] The aforementioned micro-trumpet-shaped channels effectively improve the stability of fluids within the chip, reduce the formation of eddies at the junctions of the immune cell culture chamber, reagent preloading chamber, and main perfusion channel, and ensure the smooth flow of immune cells and preloaded reagents, allowing them to function effectively in the main perfusion channel.

[0010] Furthermore, the upper and lower porous membranes are PC porous membranes with pore sizes of 1-10 µm. One function of the porous membrane is to seed cells onto it, ensuring cell growth; another function is to divide the chambers and ensure sufficient space for the exchange of nutrients and metabolic substances. If the pore size is too large, cells cannot be seeded properly; if the pore size is too small, substance exchange will be affected.

[0011] Furthermore, the photoresponsive immune cell elimination agent is photocage-encapsulated cyclophosphamide; the photocage-encapsulated cyclophosphamide is obtained by reacting cyclophosphamide with nitroaminochloroformate; the light source of the light irradiation device is a UV-LED light source with a wavelength of 330-385 nm. When not exposed to light of a specific wavelength, the photocage-encapsulated cyclophosphamide remains inert; once exposed to light of a specific wavelength, it is activated and diffuses, specifically inducing the death or inactivation of immune cells in adjacent chambers.

[0012] Based on a general inventive concept, the present invention also provides an application of a photoresponsive liver chip system in the rapid evaluation of the immunotoxicity of drugs for non-diagnostic purposes.

[0013] The above application, further, the method of application includes the following steps: (1) Some non-parenchymal liver cells are introduced into the main perfusion channel of the upper substrate, immune cells are introduced into the immune cell culture chamber of the upper substrate, liver parenchymal cells and some non-parenchymal liver cells are introduced into the intermediate cell culture chamber of the middle substrate, the remaining non-parenchymal liver cells are introduced into the lower cell culture chamber of the lower substrate, and the unactivated photoresponsive immune cell elimination reagent is introduced into the reagent preloading chamber of the upper substrate. Then, the drug to be tested is introduced into the main perfusion channel of the upper substrate for the first round of fluid perfusion culture, and then the first round of toxicity detection is performed to obtain the first toxicity data. The first toxicity data reflects the total toxicity effect of the drug. (2) The liver chip after the first round of toxicity testing in step (1) is irradiated with light using an irradiation device to activate the activity of the photoresponsive immune cell elimination reagent therein, so that the immune cells in the liver chip are eliminated or inactivated. (3) After the immune cells described in step (2) are eliminated or inactivated, a second round of fluid perfusion culture is performed, followed by a second round of toxicity testing to obtain second toxicity data. Since the immune cells have been eliminated, the second toxicity data only characterizes the direct toxicity of the drug to liver parenchymal cells without the participation of immune cells. (4) Compare the first toxicity data and the second toxicity data, and evaluate the immunotoxicity contribution of the test drug based on the difference or ratio.

[0014] Furthermore, the immune cells are selected from at least one of macrophages, natural killer cells, T cells, neutrophils, and peripheral blood mononuclear cells; the liver parenchymal cells are selected from at least one of hepatocyte lines, hepatocytes derived from induced pluripotent stem cells, and primary hepatocytes (such as HepG2 cells); the non-parenchymal cells include at least one of endothelial cells (such as EA.hy926 cells), stellate cells (such as LX-2 cells), Kupffer cells (such as U937 cells), and bile duct epithelial cells (such as TFK-1 cells). The endothelial cells and Kupffer cells are introduced into the main perfusion channel, the stellate cells are introduced into the intermediate cell culture chamber, and the bile duct epithelial cells are introduced into the lower cell culture chamber.

[0015] Furthermore, the fluid perfusion culture steps are as follows: using a syringe pump, complete culture medium for hepatocyte culture is perfused into the inlet of the main perfusion channel and the inlet of the lower cell culture chamber in opposite directions, so that the nutrients in the complete culture medium diffuse through the upper and lower porous membranes to the main perfusion channel, the middle cell culture chamber and the lower cell culture chamber to provide the nutrients required for cell growth in each layer, while removing metabolic products; the flow rate of the fluid perfusion culture is 0.1-5 μL / min; the light source of the illumination device is a UV-LED light source with a wavelength of 330-385 nm, and the illumination duration is 2-15 min.

[0016] Furthermore, the toxicity test specifically includes the following steps: after fluid perfusion culture for 8-48 h, the effluent from each outlet in the upper substrate (2) is collected for toxicity testing; the indicators of the toxicity test include at least one of cell viability, cell apoptosis rate, lactate dehydrogenase (LDH) release, expression or release level of inflammatory factors (such as TNF-α, IL-6), and liver-specific functional indicators.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The photoresponsive liver chip system of the present invention integrates the immune cell culture chamber and the photoresponsive reagent preload chamber in adjacent positions on the same chip level and connects them through the microenvironment, thereby achieving extreme simplification and precise control of the immune cell elimination operation, reducing disturbances caused by external intervention, and has the advantages of compact structure, high integration and precise control.

[0018] 2. The photoresponsive liver chip system of the present invention can realize that all key data come from the same physical chip and the same batch of cell samples. By completing the toxicity detection of "immune" and "non-immune" states in sequence on the timeline, the individual differences between different samples are completely eliminated, and the quantitative calculation of immune contribution reaches an unprecedented level of accuracy and reliability.

[0019] 3. The photoresponsive liver chip system and its application method of the present invention eliminate the need for cumbersome preparation and processing of multiple parallel control groups. A single chip can complete the entire experimental process, which not only significantly reduces cell and reagent consumption and operation time and simplifies the operation process, but is also suitable for high-throughput screening in the early stages of drug development.

[0020] 4. The photoresponsive liver chip system of the present invention can be easily packaged into a standardized reagent kit, which includes a liver chip and a photoresponsive reagent, making it easy to promote and use, and has clear commercial prospects and broad industrial application potential. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the structure of the photoresponsive liver chip used for drug immunotoxicity evaluation in Example 1; Figure 2 This is a breakdown diagram of each layer of the photoresponsive liver chip used for drug immunotoxicity evaluation in Example 1; Figure 3 This is a schematic diagram of the chambers and locations in which immune cells, liver parenchymal cells and non-parenchymal cells are introduced into the liver chip in Example 2, as well as the changes in the morphology of cells and cyclophosphamide in the chip before and after activating photocage-encapsulated cyclophosphamide with 365 nm UV. Figure 4 In Example 2, the toxicity curves of different concentrations of allopurinol on the hepatotoxicity of photoactivated pre-loaded photocage cyclophosphamide before and after photoactivation were compared using LDH release (first toxicity data vs. second toxicity data). Figure 5 The area under the curve for comparing the first and second toxicity data of allopurinol using a photoresponsive immune liver chip in Example 2; Figure 6 In Example 3, the toxicity curves of different concentrations of ethinyl estradiol and abacavir on the hepatotoxicity of photoactivated pre-loaded photocage-treated cyclophosphamide before and after treatment were compared using LDH release data (first toxicity data vs. second toxicity data). Figure 7 Example 3 uses a photoresponsive immune liver chip to compare the area under the curve of the first and second toxicity data of ethinyl estradiol and abacavir; Figure 8 Example 4 compares the toxicity curves (first toxicity data vs. second toxicity data) of different concentrations of ethinyl estradiol and abacavir on the hepatotoxicity of photoactivated pre-loaded photocage-cageed cyclophosphamide before and after activating ALT release using ALT release data. Figure 9 Example 4 uses a photoresponsive immune liver chip to compare the area under the curve of the first and second toxicity data of ethinyl estradiol and abacavir; Figure 1 , Figure 2 Legend of the diagram: 1. Top cover plate; 2. Top substrate; 3. Top porous membrane; 4. Middle substrate; 5. Bottom porous membrane; 6. Bottom substrate; 7. Bottom cover plate; 2-1. Main perfusion channel; 2-2. Immune cell culture chamber; 2-3. Reagent preloading chamber; 2-4. Microchannel; 4-1. Intermediate cell culture chamber; 6-1. Lower cell culture chamber; 2-1-1, Inlet of main perfusion channel; 2-1-2, Outlet of main perfusion channel; 2-2-1, Inlet of immune cell culture chamber; 2-2-2, Outlet of immune cell culture chamber; 2-3-1, Inlet of reagent preloading chamber; 2-3-2, Outlet of reagent preloading chamber; 6-1-1, Inlet of lower cell culture chamber; 6-1-2, Outlet of lower cell culture chamber. Detailed Implementation

[0023] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0024] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

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

[0026] In the following embodiments of the present invention, the upper substrate, middle substrate and lower substrate of the photoresponsive liver chip are made of polydimethylsiloxane (PDMS), the upper cover plate and lower cover plate are made of polymethyl methacrylate (PMMA), and the upper porous membrane and lower porous membrane are made of PC porous membrane with a pore size of 1 µm.

[0027] In the following embodiments of the present invention, photocage-encapsulated cyclophosphamide is obtained by reacting cyclophosphamide with nitroaminochloroformate, and its preparation specifically includes the following steps: Step 1: Synthesize nitromannol chloroformate. The synthesis reaction formula is as follows: ; Nitrobenzyl alcohol (also known as 4,5-dimethoxy-2-nitrobenzyl alcohol, CAS number: 1016-58-6, molecular formula: C9H) 11 NO5 (1.0 eq) was dissolved in anhydrous dichloromethane (DCM) and cooled to 0°C in an ice bath; triphosgene (0.33 eq, as triphosgene is equivalent to 3 equivalents of phosgene) was slowly added; N,N-diisopropylethylamine (also known as N-ethyldiisopropylamine, DIPEA, molecular formula: (CH3)2CHN(C2H5)CH(CH3)2, CAS number: 7087-68-5) (2.0 eq) was added dropwise, and the reaction was maintained at 0°C for 1 h; the temperature was gradually increased to room temperature, and the reaction was continued for 2 h (the reaction was monitored by TLC to indicate completion); the reaction solution was washed with water, saturated NaHCO3, and brine, and dried over anhydrous Na2SO4. The solvent was removed by rotary evaporation to obtain nitromannol chloroformate (note that this reaction product should be prepared and used immediately).

[0028] Step 2: Synthesis of photocage-encapsulated cyclophosphamide. The synthetic reaction formula is as follows: ; Cyclophosphamide (CTX) (containing a primary amino group) (1.0 eq) was dissolved in anhydrous DCM and cooled in an ice bath. N,N-diisopropylethylamine (DIPEA) (2.0 eq) was added and stirred for 10 min. A DCM solution of nitroaminochloroformate (1.2 eq) was slowly added dropwise. The reaction was maintained at 0°C for 1 h, then raised to room temperature and reacted overnight (monitored by TLC). After the reaction was complete, the mixture was washed with water, dilute hydrochloric acid, saturated NaHCO3, and brine. The organic phase was dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (eluent: DCM / MeOH = 20:1 v / v) to obtain photocage-treated cyclophosphamide.

[0029] In the following embodiments of the present invention, the steps of fluid perfusion culture are as follows: using an injection pump, complete culture medium for hepatocyte culture is perfused into the inlet of the main perfusion channel 2-1 and the inlet of the lower cell culture chamber 6-1 in opposite directions, so that the nutrients in the complete culture medium diffuse through the upper porous membrane 3 and the lower porous membrane 5 into the main perfusion channel 2-1, the middle cell culture chamber 4-1 and the lower cell culture chamber 6-1 to provide the nutrients required for the growth of cells in each layer, while removing metabolic products.

[0030] The complete culture medium was DMEM-HG (Hyclone) medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1% non-essential amino acid solution (NEAA, Gibco), and 1% penicillin-streptomycin.

[0031] Example 1: A photoresponsive liver-on-a-chip system for evaluating drug immunotoxicity, such as Figure 1 , 2 It includes: a multi-layered liver chip, photocage cyclophosphamide, and a UV-LED light source with a wavelength of 365 nm.

[0032] The liver chip consists of, from top to bottom, an upper cover plate 1, an upper substrate 2, an upper porous membrane 3, an intermediate substrate 4, a lower porous membrane 5, a lower substrate 6, and a lower cover plate 7. The upper substrate 2 is provided with a perfusion main channel 2-1, an immune cell culture chamber 2-2, and a reagent preloading chamber 2-3 located on the same horizontal plane. The immune cell culture chamber 2-2 and the reagent preloading chamber 2-3 are connected to the perfusion main channel 2-1 through microchannels 2-4 respectively. The width of the perfusion main channel 2-1 is 1 mm. The microchannel 2-4 is formed by several rectangular protruding microstructures arranged in an alternating manner to form multiple channels with a width of 20 µm (there is a row of rectangular protruding microstructures on each side of the perfusion main channel 2-1). The two corners on the left or right side of the rectangular protruding microstructures are chamfered at 30° with a width of 1 mm, so that the chamfers of the adjacent rectangular protruding microstructures form a micro-trumpet-shaped channel with the opening end facing the perfusion main channel 2-1, and the opening ends of the micro-trumpet-shaped channels on both sides of the perfusion main channel 2-1 correspond one-to-one. The intermediate layer substrate 4 and the lower layer substrate 6 are respectively provided with an intermediate layer cell culture chamber 4-1 and a lower layer cell culture chamber 6-1. The shapes of the intermediate layer cell culture chamber 4-1 and the lower layer cell culture chamber 6-1 are consistent with the shape of the perfusion main channel 2-1 and their positions correspond one-to-one. The upper porous membrane 3 and the lower porous membrane 5 are respectively located on the upper and lower sides of the middle cell culture chamber 4-1, and separate the main perfusion channel 2-1, the middle cell culture chamber 4-1 and the lower cell culture chamber 6-1 to form three chambers: upper, middle and lower. The main perfusion channel 2-1, the immune cell culture chamber 2-2, the reagent preloading chamber 2-3, and the lower cell culture chamber 6-1 are all equipped with inlets and outlets, as well as corresponding sealing plugs.

[0033] Example 2: Evaluation of allopurinol hepatotoxicity based on a photoresponsive liver chip system An application of a photoresponsive liver chip system in the rapid evaluation of the immunotoxicity of drugs for non-diagnostic purposes: The photoresponsive liver chip system of Example 1 was used to accurately evaluate the hepatotoxicity characteristics of allopurinol.

[0034] The application method includes the following steps: (1) The immunohepatic microarray consists of six cell lines (HepG2, LX-2, EA.hy926, U937, HuT-78, and HL-60 cells). EA.hy926 and U937 cells are introduced into the main perfusion channel 2-1 of the upper substrate 2. A mixture of HuT-78 and HL-60 cells is introduced into the immunocellular culture chamber 2-2 of the upper substrate 2. HepG2 liver parenchymal cells and LX-2 non-parenchymal cells are introduced into the intermediate cell culture chamber 4-1 of the middle substrate 4. No cells are introduced into the lower cell culture chamber 6-1 of the lower substrate 6. Unactivated photocage-cageed cyclophosphamide prodrug (100 μM) is introduced into the reagent preloading chamber 2-3 of the upper substrate 2. After the microarray has been running normally for 24 h, allopurinol (0 μM, 50 μM, 100 μM, 150 μM, 200 μM, etc.) is introduced into the microarray. The LDH was injected into the main perfusion channel 2-1 of the upper substrate 2 with a fluid perfusion of 1 μM. The first round of fluid perfusion culture was carried out for 24 h under fluid perfusion (1 μL / min). Then the effluent from the liquid outlet of the upper substrate was collected and the LDH release in the effluent was detected (first round of toxicity detection) to obtain the first toxicity data. (2) A 365 nm UV-LED light source (8 mW / cm²) is used. 2 Irradiate the reagent preload chamber for 2-3 minutes to activate the activity of the photocage-encapsulated cyclophosphamide prodrug, thereby eliminating or inactivating the immune cells in the liver chip. (3) After the immune cells in step (2) are eliminated or inactivated, a second round of fluid perfusion culture is carried out for 24 h under fluid perfusion (1 μL / min). Then, the effluent from the liquid outlet of the upper substrate is collected and the amount of LDH released in the effluent is detected (second round of toxicity detection) to obtain the second toxicity data. (4) Plot the toxicity curves of allopurinol on the chip before and after activation by the photoinducible reagent, compare the area under the curve, and determine the characteristics of allopurinol hepatotoxicity.

[0035] Figure 3 The study shows the compartments and locations into which immune cells (HuT-78 and HL-60 cells), hepatocytes (HepG2), and non-parenchymal cells (EAhy926, U937, and LX-2 cells) are placed within a liver microarray, as well as the changes in cell and cyclophosphamide morphology before and after photocage-encapsulation of cyclophosphamide using 365 nm UV activation. It is evident that the cage-encapsulation effect of photocage-encapsulated cyclophosphamide disappears after photoactivation, releasing cyclophosphamide, which can inhibit immune cell function within the microarray.

[0036] Figure 4 The toxicity curves of allopurinol on the chip before and after activation by the photoinducible reagent are shown, and the areas under the curves of the toxicity curves are further compared. Figure 5The results showed that the second toxicity data of allopurinol was significantly lower than the first toxicity data by 8.65% (p<0.05), indicating that the immune system significantly mediated the hepatotoxicity of allopurinol.

[0037] Example 3: Evaluation of hepatotoxicity of ethinyl estradiol and abacavir based on a photoresponsive liver chip system An application of a photoresponsive liver chip system in the rapid evaluation of the immunotoxicity of drugs for non-diagnostic purposes: The photoresponsive liver chip system of Example 1 was used to accurately evaluate the hepatotoxicity characteristics of ethinyl estradiol (EE) and abacavir (ABC).

[0038] The application method includes the following steps: (1) The immunohepatic microarray consists of seven cell lines (HepG2, LX-2, EA.hy926, U937, HuT-78, HL-60, and TFK-1 cells). EA.hy926 and U937 cells are introduced into the main perfusion channel 2-1 of the upper substrate 2. A mixture of HuT-78 and HL-60 cells is introduced into the immunocellular culture chamber 2-2 of the upper substrate 2. HepG2 liver parenchymal cells and LX-2 non-parenchymal cells are introduced into the intermediate cell culture chamber 4-1 of the middle substrate 4. TFK-1 cells are introduced into the lower cell culture chamber 6-1 of the lower substrate 6. Unactivated photocage-cageed cyclophosphamide prodrug (100 μM) is introduced into the reagent preloading chamber 2-3 of the upper substrate 2. After the microarray has been running normally for 24 h, ethinyl estradiol (0 μM, 50 μM, 100 μM, 250 μM, 500 μM) is introduced into the microarray. LDH (0 μM, 20 μM, 40 μM, 60 μM, 80 μM) and abacavir (0 μM, 20 μM, 40 μM, 60 μM, 80 μM) were added to the main perfusion channel 2-1 of the upper substrate 2. The first round of fluid perfusion culture was carried out for 24 h under fluid perfusion (1 μL / min). Then, the effluent from the liquid outlet of the upper substrate was collected and the LDH release in the effluent was detected (first round of toxicity detection) to obtain the first toxicity data. (2) A 365 nm UV-LED light source (8 mW / cm²) is used. 2 Irradiate the reagent preload chamber for 2-3 minutes to activate the activity of the photocage-encapsulated cyclophosphamide prodrug, thereby eliminating or inactivating the immune cells in the liver chip. (3) After the immune cells in step (2) are eliminated or inactivated, a second round of fluid perfusion culture is carried out for 24 h under fluid perfusion (1 μL / min). Then, the effluent from the liquid outlet of the upper substrate is collected and the amount of LDH released in the effluent is detected (second round of toxicity detection) to obtain the second toxicity data. (4) Plot the toxicity curves of ethinyl estradiol and abacavir before and after activation of the chip by the photoinducible reagent, compare the area under the curve, and determine the characteristics of the hepatotoxicity of ethinyl estradiol and abacavir.

[0039] Figure 6 The toxicity curves of ethinyl estradiol and abacavir before and after activation of the chip by the photoinducible reagent were displayed. Further comparison of the areas under the toxicity curves was conducted. Figure 7 The data on the second toxicity of ethinyl estradiol showed no significant difference from the data on the first toxicity (p>0.05), indicating that the hepatotoxicity of ethinyl estradiol was not mediated by immune cells. In contrast, the data on the second toxicity of abacavir was significantly lower than the data on the first toxicity by 7.7% (p<0.001), indicating that the immune system significantly mediated the hepatotoxicity of allopurinol.

[0040] Example 4: Evaluation of hepatotoxicity of ethinyl estradiol and abacavir based on a photoresponsive liver chip system An application of a photoresponsive liver chip system in the rapid evaluation of the immunotoxicity of drugs for non-diagnostic purposes: The photoresponsive liver chip system of Example 1 was used to accurately evaluate the hepatotoxicity characteristics of ethinyl estradiol (EE) and abacavir (ABC).

[0041] The application method includes the following steps: (1) The immunohepatic microarray consists of seven cell lines (HepG2, LX-2, EA.hy926, U937, HuT-78, HL-60, and TFK-1 cells). EA.hy926 and U937 cells are introduced into the main perfusion channel 2-1 of the upper substrate 2. A mixture of HuT-78 and HL-60 cells is introduced into the immunocellular culture chamber 2-2 of the upper substrate 2. HepG2 liver parenchymal cells and LX-2 non-parenchymal cells are introduced into the intermediate cell culture chamber 4-1 of the middle substrate 4. TKF-1 cells are introduced into the main perfusion channel 6-1 of the lower substrate 6. Unactivated photocage-cageed cyclophosphamide prodrug (100 μM) is introduced into the reagent preloading chamber 2-3 of the upper substrate 2. After the microarray has been running normally for 24 h, ethinyl estradiol (0 μM, 50 μM, 100 μM, 250 μM, 500 μM) is introduced into the microarray. ALT (0 μM), abacavir (0 μM, 20 μM, 40 μM, 60 μM, 80 μM) and abacavir (0 μM, 20 μM, 40 μM, 60 μM, 80 μM) were added to the main perfusion channel 2-1 of the upper substrate 2. The first round of fluid perfusion culture was carried out for 24 h under fluid perfusion (1 μL / min). Then the effluent from the liquid outlet of the upper substrate was collected and the ALT release in the effluent was detected (first round of toxicity detection) to obtain the first toxicity data. (2) A 365 nm UV-LED light source (8 mW / cm²) is used.2 Irradiate the reagent preload chamber for 2-3 minutes to activate the activity of the photocage-encapsulated cyclophosphamide prodrug, thereby eliminating or inactivating the immune cells in the liver chip. (3) After the immune cells in step (2) are eliminated or inactivated, a second round of fluid perfusion culture is carried out for 24 h under fluid perfusion (1 μL / min). Then, the effluent from the liquid outlet of the upper substrate is collected and the ALT release in the effluent is detected (second round of toxicity detection) to obtain the second toxicity data. (4) Plot the toxicity curves of ethinyl estradiol and abacavir before and after activation of the chip by the photoinducible reagent, compare the area under the curve, and determine the characteristics of the hepatotoxicity of ethinyl estradiol and abacavir.

[0042] Figure 8 The toxicity curves of ethinyl estradiol and abacavir before and after activation of the chip by the photoinducible reagent were displayed. Further comparison of the areas under the toxicity curves was conducted. Figure 9 The data on the second toxicity of ethinyl estradiol showed no significant difference from the data on the first toxicity (p>0.05), indicating that the hepatotoxicity of ethinyl estradiol was not mediated by immune cells. In contrast, the data on the second toxicity of abacavir was significantly reduced by 21.8% compared with the data on the first toxicity (p<0.001), indicating that the immune system significantly mediated the hepatotoxicity of allopurinol.

Claims

1. A photoresponsive liver chip system for evaluating drug immunotoxicity, characterized in that, include: A multi-layer liver chip, a photoresponsive immune cell elimination reagent, and a light irradiation device capable of activating the activity of the photoresponsive immune cell elimination reagent; The liver chip comprises, from top to bottom, an upper substrate (2), an upper porous membrane (3), an intermediate substrate (4), a lower porous membrane (5), and a lower substrate (6). The upper substrate (2) is provided with a perfusion main channel (2-1), an immune cell culture chamber (2-2) and a reagent preloading chamber (2-3). The immune cell culture chamber (2-2) and the reagent preloading chamber (2-3) are connected to the perfusion main channel (2-1) through microchannels (2-4). The intermediate substrate (4) and the lower substrate (6) are respectively provided with an intermediate cell culture chamber (4-1) and a lower cell culture chamber (6-1). The shapes of the intermediate cell culture chamber (4-1) and the lower cell culture chamber (6-1) are consistent with the shape of the perfusion main channel (2-1) and their positions correspond one-to-one. The upper porous membrane (3) and the lower porous membrane (5) are respectively located on the upper and lower sides of the middle cell culture chamber (4-1), and separate the main perfusion channel (2-1), the middle cell culture chamber (4-1) and the lower cell culture chamber (6-1) to form upper, middle and lower chambers.

2. The photoresponsive liver chip system according to claim 1, characterized in that, The liver chip also includes an upper cover plate (1) and a lower cover plate (7), which are respectively located on the upper layer of the upper substrate (2) and the lower layer of the lower substrate (6); the main perfusion channel (2-1), the immune cell culture chamber (2-2), the reagent preloading chamber (2-3) and the lower cell culture chamber (6-1) are all provided with inlet and outlet ports, as well as corresponding sealing plugs.

3. The photoresponsive liver chip system according to claim 1, characterized in that, The main perfusion channel (2-1), the immune cell culture chamber (2-2), and the reagent preloading chamber (2-3) are located on the same horizontal plane; the width of the main perfusion channel (2-1) is 0.5-2 mm; the microchannel (2-4) is formed by several rectangular protruding microstructures arranged in an alternating manner to form multiple channels 5-30 µm wide. The left or right corners of the rectangular protruding microstructures are chamfered at 15-45° with a width of 1-2 mm, so that the chamfers of the adjacent rectangular protruding microstructures form a micro-trumpet-shaped channel with the opening end facing the main perfusion channel (2-1), and the opening ends of the micro-trumpet-shaped channels on both sides of the main perfusion channel (2-1) correspond one-to-one.

4. The photoresponsive liver chip system according to claim 1, characterized in that, The upper porous membrane (3) and the lower porous membrane (5) are PC porous membranes with a pore size of 1-10 µm.

5. The photoresponsive liver chip system according to any one of claims 1-4, characterized in that, The photoresponsive immune cell elimination reagent is photocage cyclophosphamide; the photocage cyclophosphamide is obtained by reacting cyclophosphamide with nitrocarbamate; the light source of the illumination device is a UV-LED light source with a wavelength of 330-385 nm.

6. The application of a photoresponsive liver chip system as described in any one of claims 1-5 in the rapid evaluation of the immunotoxicity of drugs for non-diagnostic purposes.

7. The application according to claim 6, characterized in that, The method of application includes the following steps: (1) Some non-parenchymal liver cells are introduced into the perfusion main channel (2-1) of the upper substrate (2), immune cells are introduced into the immune cell culture chamber (2-2) of the upper substrate (2), liver parenchymal cells and some non-parenchymal liver cells are introduced into the intermediate cell culture chamber (4-1) of the middle substrate (4), the remaining non-parenchymal liver cells are introduced into the lower cell culture chamber (6-1) of the lower substrate (6), and the unactivated photoresponsive immune cell elimination reagent is introduced into the reagent preloading chamber (2-3) of the upper substrate (2). Then the drug to be tested is introduced into the perfusion main channel (2-1) of the upper substrate (2) for the first round of fluid perfusion culture, and then the first round of toxicity detection is performed to obtain the first toxicity data. (2) The liver chip after the first round of toxicity testing in step (1) is irradiated with light using an irradiation device to activate the activity of the photoresponsive immune cell elimination reagent therein, so that the immune cells in the liver chip are eliminated or inactivated. (3) After the immune cells described in step (2) are eliminated or inactivated, a second round of fluid perfusion culture is performed, followed by a second round of toxicity testing to obtain second toxicity data; (4) Compare the first toxicity data and the second toxicity data, and evaluate the immunotoxicity contribution of the test drug based on the difference or ratio.

8. The application according to claim 7, characterized in that, The immune cells are selected from at least one of macrophages, natural killer cells, T cells, neutrophils, and peripheral blood mononuclear cells; the liver parenchymal cells are selected from at least one of hepatocyte lines, hepatocytes derived from induced pluripotent stem cells, and primary hepatocytes; the non-parenchymal cells include at least one of endothelial cells, stellate cells, Kupffer cells, and bile duct epithelial cells. The endothelial cells and Kupffer cells are placed in the main perfusion channel (2-1), the stellate cells are placed in the intermediate cell culture chamber (4-1), and the bile duct epithelial cells are placed in the lower cell culture chamber (6-1).

9. The application according to claim 7, characterized in that, The steps of the fluid perfusion culture are as follows: using a syringe pump, complete culture medium for hepatocyte culture is perfused into the inlet of the main perfusion channel (2-1) and the inlet of the lower cell culture chamber (6-1) in opposite directions, so that the nutrients in the complete culture medium diffuse through the upper porous membrane (3) and the lower porous membrane (5) into the main perfusion channel (2-1), the middle cell culture chamber (4-1) and the lower cell culture chamber (6-1); the flow rate of the fluid perfusion culture is 0.1-5 μL / min; the light source of the illumination device is a UV-LED light source with a wavelength of 330-385 nm, and the illumination time is 2-15 min.

10. The application according to any one of claims 7-9, characterized in that, The toxicity test specifically includes the following steps: after fluid perfusion culture for 8-48 h, the effluent from each outlet in the upper substrate (2) is collected and toxicity is tested; the indicators of the toxicity test include at least one of cell viability, cell apoptosis rate, lactate dehydrogenase release, inflammatory factor expression or release level, and liver-specific functional indicators.

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