Organ microfluidic chip and drug safety evaluation system

Abstract

The present invention belongs to the field of drug research and development, and relates to an organ microfluidic chip and a drug safety evaluation system. The organ microfluidic chip includes a chip substrate, a cell culture matrix network, a cell injection loading port, an organ chip perfusate output port, and a drug perfusion port; a groove is provided on the chip substrate; the cell culture matrix network is embedded in the groove on the chip substrate; holes are provided on the cell culture matrix network; the cell injection loading port is provided on the top of the cell culture matrix network and is connected to the cell culture matrix network; the cell injection loading port injects cells required by the organ chip into the cell culture matrix network, and the cells eventually gather in the holes; the drug perfusion port is connected to the organ chip perfusate output port through the cell culture matrix network. The present invention provides an organ microfluidic chip and a drug safety evaluation system that can directly and conveniently evaluate the safety or effectiveness of drugs, has a short detection time, and simplifies processing.

Description

Technical Field

[0001] The present invention belongs to the field of drug research and development, and relates to a safety evaluation system, in particular to an organ microfluidic chip and a drug safety evaluation system. Background Art

[0002] As an important part of the new drug development process, the difficulty of drug safety evaluation research lies in how to judge the risks and harms that candidate drugs may cause to human organs based on the results of animal safety evaluation tests and human cell tests. Although the results of the interaction between cells cultured in vitro and drugs in culture dishes are a simple and high-throughput basic drug screening and testing method, these cell models lack the microstructure and physiological functions of in vivo tissues. To date, animal testing remains the gold standard for preclinical safety and efficacy evaluation and verification of drugs. Animal model studies of drugs can relatively simulate the effects of drugs on human organs and multi-organ level functions. However, they are costly, long-term, and affected by many factors, making it difficult to completely simulate the metabolic mechanism of drugs in the human body. In addition, due to the huge differences between model animals and humans in comparative physiology and metabolic systems, when the results of safety and efficacy evaluation that have been obtained in preclinical studies are converted to clinical studies, especially when conducting phase I clinical trials, the "first entry of drugs into the human body" or increased doses can easily put clinical volunteers in a highly risky situation. The results obtained in animal experiments are also challenged in terms of accuracy and repeatability in clinical research experiments. Some drugs did not show adverse effects on animals in preclinical research, but showed damage to the liver, heart, and kidney functions of patients in clinical trials. There is no doubt that animal testing has promoted the clinical application of safe and effective drugs and made a significant contribution to medical progress. However, continuing to use it as a mandatory process for drug screening may also have adverse effects.

[0003] With the rapid development of life science and technology, researchers are constantly exploring alternatives to animal experiments. Animal models are holistic, but they differ from humans in species; human cells do not differ in species, but they lack integrity. Organ-on-a-Chip technology combines the advantages of both by culturing human cells in a tissue-specific three-dimensional environment. Organ-on-a-Chip is usually composed of key components such as microfluidics, living cell tissues, and stimulation / drug delivery equipment. It has two significant features: (1) The 3D properties and arrangement of tissues on the chip platform, presenting and integrating multiple cell types to reflect the physiological balance of cells and presenting biomechanics related to modeled tissues. (2) Organ-on-a-Chip has precise dimensions and microchannels, which can accurately achieve cell ordering, regulate various fluid and chemical parameters to provide controllable culture conditions, and effectively reflect the in vivo microstructure and functional characteristics of human tissues and organs. Organ-on-a-Chip can be divided into single organ-on-a-Chip and multi-organ-on-a-Chip. At present, researchers have successfully constructed single organ-on-a-Chip and multi-organ-on-a-Chip for lungs, liver, kidneys, intestines, hearts, brains, blood, bones, skin, nerves, pancreatic islets, tumors, etc. Many countries and organizations are actively promoting research on organ chips. Organ chip technology can be used in toxicology testing platforms, drug screening, organ reconstruction and manufacturing, research on the safety of COVID-19 therapies and vaccines, early diagnosis and precise treatment of diseases, identification of species-specific toxicity, space experiments, and other fields. It is believed that with the further promotion and use of organ chip technology, organ chip technology will release a huge application space in drug discovery, aerospace medicine, personalized medicine, and environmental assessment. Due to the advantages of convenience, sensitivity, and rapidity, organ chips have higher specificity and selectivity in predicting human drug toxicity than existing models.

[0004] There are important application prospects in the evaluation of drug efficacy and toxicity. Although organ chips have outstanding advantages in drug toxicity assessment, their development and application still have certain limitations and challenges. The first is the source of living cells. Living cells commonly used to construct organ chips include primary cells, cell lines, and induced pluripotent stem cells (iPSC). Primary cells are difficult to obtain, limited in number, have low growth potential, and must be collected through invasive operations. The unavailability and unreliability of patient-derived cells are also a major obstacle. Cell lines are relatively easy to culture and expand, but they usually lack the phenotypic functional characteristics of the target organ. Although improvements can be achieved using perfusion and / or co-culture, cell lines are usually used in organ chips when there is no better choice. iPSCs are derived from small cells or tissue samples (such as blood), are patient-specific, can expand and selectively differentiate into multiple lineages, and provide unlimited patient-specific cell sources for multi-organ organ chips. iPSCs can generate autologous target organs or tissues, enabling the construction of patient-specific organ chips for personalized disease modeling and drug screening. Using a single iPSC to generate all tissue units in an organ chip will be able to express genotypic and phenotypic effects. However, not all cell lineages can be derived from the same iPSC line, and effective iPSC differentiation and maturation remains one of the challenges in this field. Currently, liver and kidney organ chips derived from primary cells, cell lines, and induced pluripotent stem cells have been successfully constructed in the literature. The advantages and disadvantages of using cells from different sources to construct organ chips may vary depending on the design of the organ chip, which places requirements on the design capabilities of the organ chip.

[0005] Secondly, high-throughput screening based on organ chips has not yet been realized. At present, most organ chip applications are usually combined with offline detection, and the variety and quantity of detected substances are limited. The online detection methods currently combined with organ chips mainly include two aspects: ① Through electrochemical monitoring of the chip microenvironment, some biological functions of the chip model are determined, including the integrity of the cell layer, the function of mitochondria, etc.; ② Optical detection, electrochemical sensing and discontinuous mass spectrometry analysis (see below) and other analytical methods are integrated with organ chip devices to analyze target molecules; but these so-called "online detection methods" have practical problems to varying degrees, such as the organ chip's failure to fully simulate the microenvironment in the body and the incomplete online analysis of the system.

[0006] There are certain technical bottlenecks in the transition from basic research to application. In the analysis and detection of biological samples, omics technology can observe the changes of hundreds or thousands of metabolites in one analysis, analyze the structure and function of the test object at different levels, including the molecular gene level ("genomics"), protein level ("proteomics") and metabolite level ("metabolomics"), and achieve a comprehensive analysis of the target sample information at different molecular levels. Omics analysis has become a mainstream research method in many aspects such as disease diagnosis, biomarker discovery, drug safety evaluation and pharmacology research. Specifically, proteomics studies the existence and activity of proteins at the overall level under specific conditions; metabolomics studies the metabolites of living organisms, mainly small molecules with a molecular weight of less than 1000; genomics studies the structure, function, evolution, location, editing, etc. of the genome, as well as their effects on organisms; when analyzing biological samples, information at several omics levels can be integrated to dig deep into candidate key factors; by integrating information at different levels such as genes, mRNA, regulatory factors, proteins, and metabolism, a metabolic regulatory network can be constructed to deeply understand the regulation and causal relationship between various omics, and to more deeply understand the molecular mechanism and genetic basis of complex traits in biological processes and disease processes, namely "multi-omics integrated analysis". When conducting omics research, there are many substances contained in biological samples, and it is extremely challenging to achieve a panoramic analysis of them.

[0007] With the continuous development of detection technology, high-resolution mass spectrometry (HRMS), which has the advantages of high sensitivity, high mass resolution, high mass accuracy, and wide dynamic range, has been increasingly widely used in complex sample detection and omics research. It can measure the mass of compounds to four decimal places with high precision, thereby accurately inferring the exact structure of small molecules. In addition, HRMS does not need to use standard samples to optimize the analytical conditions one by one when qualitatively detecting complex samples containing multiple substances, which reduces the tediousness of pre-analysis processing and has low requirements for chromatography. When targeted quantitative research is to be carried out, internal standards can be used to develop standard curves, and commercial detection of absolute quantification of 1,000 target substances in one test has been achieved. At present, non-targeted metabolomics methods based on ultra-high liquid chromatography high-resolution mass spectrometry can obtain tens of thousands of mass spectral characteristic peaks (i.e., liquid-mass system) at one time. It is believed that with the continuous improvement and expansion of mass spectrometry databases, more and more spectral information will be annotated, and more and more accurate material information will be expected to be obtained when conducting metabolomics, lipidomics, and proteomics tests.

[0008] Combining organ chips with multi-omics integrated analysis technology based on high-resolution mass spectrometry, and even expanding it to online monitoring methods, to instantly, quickly and efficiently evaluate drug cardio-liver-kidney toxicity, will be a major development prospect of organ chips. In summary, the existing research field of drug safety evaluation organ chips is not yet fully developed and there are still many problems, such as limited variety and quantity of test substances; lack of universal and standardized products both internationally and domestically; long detection time and cumbersome manual processing; the so-called "online detection means" have organ chips that fail to fully simulate the microenvironment in the body, and incomplete online analysis of the system; it is urgent to establish a standard toxicological safety evaluation operation mechanism model for drug safety evaluation organ chip systems, simulate the true internal environment of organ cells and tissues, comprehensively analyze damage markers, simplify processes and shorten detection time (avoid loss of biological information), develop internationally universal standardized products, realize true online analysis, build a quality control system, and achieve technical breakthroughs in the coordinated control of drug testing applications based on multi-omics integrated analysis. Summary of the invention

[0009] In order to solve the above technical problems existing in the background technology, the present invention provides an organ microfluidic chip and a drug safety evaluation system which can directly and conveniently evaluate the safety or effectiveness of a drug, have a short detection time and simplified processing.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] An organ microfluidic chip, characterized in that: the organ microfluidic chip comprises a chip substrate, a cell culture matrix network and a chamber for accommodating the network, a cell injection loading port, an organ chip perfusion fluid output port and a drug perfusion port; the chip substrate is provided with a groove; the cell culture matrix network and the chamber for accommodating the network are embedded in the groove on the chip substrate; the cell culture matrix network is provided with holes; the cell injection loading port is arranged on the top of the cell culture matrix network and is connected to the cell culture matrix network; the cell injection loading port injects cells required for the organ chip into the cell culture matrix network, and the cells are finally gathered in the holes; the drug perfusion port is connected with the organ chip perfusion fluid output port through the cell culture matrix network.

[0012] Preferably, the cell culture matrix network adopted in the present invention has a 3D structure as a whole, which is a bionic loofah sac structure, and the matrix network is densely covered with holes of different diameters, and the holes are arranged on the 3D structure; the cell culture matrix network is obtained by 3D printing or MOF material synthesis mode (the pore size is achieved by 3D printing equipment, or precursor, concentration, temperature and pressure control technical parameters when synthesizing MOF materials); there are multiple holes, and the pore sizes of the holes are not equal. Some holes have pore sizes larger than or much larger than the diameter of the cells to be loaded, which are used for the flow, immersion and cell penetration of the organ chip perfusion fluid, and some holes have pore sizes smaller than the diameter of the cells to be loaded, which are used for incarceration or hanging of the cells to be loaded. Finally, after the small holes are filled with cells, a simulated organ effect of bionic hanging grapes is formed.

[0013] Preferably, the cells used in the present invention are derived from commercially available human or animal hepatic epithelial cells, cardiomyocytes, mesangial cells, other organ cells, primary cells, cell lines, induced pluripotent stem cells, tumor cells or disease pathological cells; for example, when the cells are hepatic epithelial cells, cardiomyocytes, mesangial cells, the hepatic epithelial cells are human cell line THLE-2; the cardiomyocytes are human cell line AC16; the mesangial cells are human cell line HRMC, etc.

[0014] Preferably, the organ microfluidic chip used in the present invention also includes a constant temperature and humidity living cell culture device, in which the chip substrate is arranged; the cell injection loading port, the organ chip perfusion fluid output port and the drug perfusion port respectively penetrate the constant temperature and humidity living cell culture device and then communicate with the cell culture matrix network; the constant temperature and humidity living cell culture device is a 36-37°C constant temperature and humidity environment device, and the constant temperature and humidity living cell culture device is filled with clean air containing 5% concentration of carbon dioxide; the constant temperature and humidity living cell culture device is provided with a first electron microscope, and its lens is used to directly observe the cell morphology on the matrix network.

[0015] A drug safety evaluation system formed based on the organ microfluidic chip as described above, characterized in that: the drug safety evaluation system includes an organ microfluidic chip, an automated processing device for polar metabolites, an automated processing device for fat-soluble substances, an automated processing device for protein and polypeptide hydrolysis, a waste liquid storage tank and a multi-omics integrated analysis system; the organ microfluidic chip is respectively connected to the automated processing device for polar metabolites, the automated processing device for fat-soluble substances, and the automated processing device for protein extraction and polypeptide hydrolysis; the automated processing device for polar metabolites, the automated processing device for fat-soluble substances, and the automated processing device for protein extraction and polypeptide hydrolysis are respectively connected to the waste liquid storage tank through the multi-omics integrated analysis system.

[0016] Preferably, the polar metabolite automated processing equipment used in the present invention comprises a first vortex mixer, a first high-speed centrifuge, a first automated clamping track, a first injection bottle liner, a first high-resolution liquid phase mass spectrometry system and a first constant temperature box; the first injection bottle liner is placed on the first automated clamping track and moves synchronously with the first automated clamping track; the perfusion fluid in the organ microfluidic chip flows into the first vortex mixer through the organ chip perfusion fluid outlet; the first vortex mixer vortex-mixes the perfusion fluid and then injects it into the EP centrifuge tube of the first high-speed centrifuge; the first After a high-speed centrifuge centrifuge centrifuges the EP centrifuge tube, the centrifuged perfusion fluid is injected into the inner liner tube of the first sample injection bottle; the first automated clamping track transports the inner liner tube of the first sample injection bottle to the first high-resolution liquid phase mass spectrometry system for detection; the first high-resolution liquid phase mass spectrometry system is connected to the waste liquid storage tank through a multi-omics integrated analysis system; the first vortex mixer, the first high-speed centrifuge, and the first automated clamping track are all arranged in a first constant temperature box; preferably, the temperature of the first constant temperature box is a constant temperature of 4°C; the first vortex mixer is a vortex mixer with a self-cleaning function.

[0017] Preferably, the automated processing equipment for fat-soluble substances used in the present invention comprises a ball mill, a second vortex mixer, a second high-speed centrifuge, a second automated clamping track, a second injection bottle liner tube, a second high-resolution liquid phase mass spectrometry system and a second constant temperature box; the perfusion fluid in the organ microfluidic chip flows into the ball mill through the organ chip perfusion fluid outlet; the ball mill injects the crushed perfusion fluid into the second vortex mixer; the second vortex mixer vortex-mixes the crushed perfusion fluid and then injects it into the EP centrifuge tube of the second high-speed centrifuge; the second high-speed centrifuge centrifuges the EP centrifuge tube and then centrifuges it. , inject the perfusion fluid after centrifugation into the inner liner of the second sampling bottle; the second automated clamping track transports the inner liner of the second sampling bottle to the second high-resolution liquid phase mass spectrometry system for detection; the second high-resolution liquid phase mass spectrometry system is connected to the waste liquid storage tank through a multi-omics integrated analysis system; the ball mill, the second vortex mixer, the second high-speed centrifuge, and the second automated clamping track are all arranged in a second constant temperature box; preferably, the temperature of the second constant temperature box is 4°C; the second vortex mixer is a vortex mixer with self-cleaning and ultrasonic functions; the ball mill is a ball mill with self-cleaning function.

[0018] Preferably, the protein extraction and polypeptide hydrolysis automated processing equipment used in the present invention comprises a third vortex mixer, a third high-speed centrifuge, a darkroom, a protein concentration detector, a second electron microscope, an SDS-PAGE detector, a third automated clamping track, a third sample bottle liner, a third high-resolution liquid phase mass spectrometry system and a third constant temperature box; the perfusion fluid in the organ microfluidic chip flows into the third vortex mixer through the organ chip perfusion fluid outlet; the third vortex mixer vortex-mixes the perfusion fluid and then injects it into the EP centrifuge tube of the third high-speed centrifuge; the third high-speed centrifuge centrifuge centrifuges the EP centrifuge tube and then injects the centrifuged perfusion fluid into the darkroom for alkylation reaction; the perfusion fluid after the alkylation reaction is injected into the protein concentration detector and SDS-PAGE detector, the perfusion fluid after measurement is injected into the inner liner of the third sampling bottle; the third automated clamping track transports the inner liner of the third sampling bottle to the third high-resolution liquid phase mass spectrometry system for detection; the third high-resolution liquid phase mass spectrometry system is connected to the waste liquid storage tank through a multi-omics integrated analysis system; the third vortex mixer, the third high-speed centrifuge, the darkroom, the protein concentration detector, the second electron microscope, the SDS-PAGE detector, and the third automated clamping track are all arranged in a third constant temperature box; preferably, the temperature of the third constant temperature box is a constant temperature of 37°C; the third vortex mixer is a vortex mixer with a self-cleaning function; preferably, the protein concentration detector is provided with a second electron microscope.

[0019] Preferably, the waste liquid storage tanks used in the present invention include a first waste liquid storage tank, a second waste liquid storage tank and a third waste liquid storage tank; the polar metabolite automated processing equipment is connected to the first waste liquid storage tank through a multi-omics integrated analysis system; the fat-soluble substance automated processing equipment is connected to the second waste liquid storage tank through a multi-omics integrated analysis system; the protein and polypeptide hydrolysis automated processing equipment is connected to the third waste liquid storage tank through a multi-omics integrated analysis system.

[0020] A drug safety evaluation method based on the drug safety evaluation system as described above, characterized in that the drug safety evaluation method comprises the following steps:

[0021] 1) preparing an organ microfluidic chip and obtaining the perfusion fluid after passing through the organ microfluidic chip;

[0022] 2) dividing the perfusate into three portions, respectively labeled as a first perfusate, a second perfusate, and a third perfusate, wherein the first perfusate, the second perfusate, and the third perfusate have exactly the same composition;

[0023] 3) injecting the first portion of the perfusate into the polar metabolite automated processing equipment to detect polar metabolites and obtain polar metabolite detection results; injecting the second portion of the perfusate into the fat-soluble substance automated processing equipment to detect fat-soluble substances and obtain fat-soluble substance detection results; injecting the third portion of the perfusate into the protein and polypeptide hydrolysis automated processing equipment to detect protein and polypeptide hydrolysis, and obtaining protein and polypeptide hydrolysis detection results by reverse calculation;

[0024] 4) The polar metabolite detection results, fat-soluble substance detection results, and protein and peptide hydrolysis detection results are injected into the multi-omics integrated analysis system, and after being processed in a human-computer combination mode, a comprehensive analysis result is obtained;

[0025] 5) Conduct a comprehensive evaluation of drug safety based on the comprehensive analysis results;

[0026] Preferably, in step 3), the specific implementation method of injecting the first perfusion fluid into the polar metabolite automated processing equipment for polar metabolite detection is:

[0027] a.1) 50 μl of the first perfusion solution was drawn and injected into the first vortex mixer, and 150 μl of pre-cooled ice methanol was injected into the first vortex mixer, and the mixture was vortexed automatically for 3 minutes; the ice methanol contained 1 μg / ml of 2-chlorophenylalanine;

[0028] a.2) After the vortexing is completed, the perfusate of the first vortex mixer is injected into the EP centrifuge tube of the first high-speed centrifuge, and the first centrifugation is performed at 12000r / min for 10min. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube, and then the second centrifugation is performed at 12000r / min for 5min. After the centrifugation is completed, the supernatant is injected into the liner tube of the first injection bottle;

[0029] a.3) The inner liner of the first injection bottle is transported to the first high-resolution liquid phase mass spectrometry system through the first automated clamping track to perform polar metabolite detection to obtain polar metabolite detection results; the first high-resolution liquid phase mass spectrometry system is an ultra-high performance liquid chromatography or a tandem mass spectrometry system;

[0030] The steps a.1) and a.2) are both carried out at a constant temperature of 4°C;

[0031] Preferably, in step 3), the specific implementation method of injecting the second perfusion fluid into the fat-soluble substance automated processing equipment for fat-soluble substance detection is:

[0032] b.1) taking microliters of the second perfusion solution and injecting it into the ball mill, and injecting 1 ml of lipid extract into the ball mill at the same time, breaking the second perfusion solution to obtain a homogenate;

[0033] b.2) injecting the homogenate into a second vortex mixer, injecting 200 μl of water into the second vortex mixer, and vortexing automatically for 3 minutes;

[0034] b.3) After the vortexing is completed, the perfusate of the second vortex mixer is injected into the EP centrifuge tube of the second high-speed centrifuge and centrifuged at 12000r / min for 10min. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube for concentration to obtain a concentrated product;

[0035] b.4) injecting lipid reconstitution solution into the concentrated product for reconstitution, and injecting the reconstituted product into the inner liner of the second injection bottle;

[0036] b.5) ​​The inner liner of the second injection bottle is transported to a second high-resolution liquid phase mass spectrometry system through a second automated clamping track to perform fat-soluble substance detection to obtain fat-soluble substance detection results; the second high-resolution liquid phase mass spectrometry system is an ultra-high performance liquid chromatography or a tandem mass spectrometry system;

[0037] The steps b.1), b.2), b.3) and b.4) are all carried out at a constant temperature of 4°C;

[0038] Preferably, the specific implementation method of injecting the third perfusion fluid into the protein extraction and polypeptide hydrolysis automated processing equipment for protein extraction and polypeptide hydrolysis detection is:

[0039] c.1) aspirating 50 μl of the third perfusion solution and injecting it into a third vortex mixer, and injecting an appropriate amount of lysis solution into the third vortex mixer, and vortexing to mix thoroughly; the lysis solution is 1.5% SDS / 100 mM Tris-Cl;

[0040] c.2) After the vortexing is completed, the perfusate of the third vortex mixer is injected into the EP centrifuge tube of the third high-speed centrifuge, and the first centrifugation is performed at 12000 r / min for 10 minutes. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube, and an appropriate amount of acetone is injected into the EP centrifuge tube; after the second centrifugation is performed at 12000 r / min for 3 minutes, the precipitate is obtained after the centrifugation is completed;

[0041] c.3) After injecting a reconstitution solution into the precipitate for reconstitution, dithiothreitol was added and incubated at 37°C for 1 hour; then, iodoacetamide was added and an alkylation reaction was performed at room temperature in the dark to block the sulfhydryl group; the reconstitution solution was 8M Urea / 100mM Tris-Cl;

[0042] c.4) Determine the protein concentration of the product obtained in step c.3) by a protein concentration detector, perform SDS-PAGE detection by an SDS-PAGE detector, add 100mM Tris-HCl solution to the result, dilute the Urea concentration to less than 2M, add trypsin at a mass ratio of 1:50 between enzyme and protein, incubate at 37°C with shaking overnight for enzyme cleavage; add TFA after 12 hours to terminate the enzyme cleavage; desalt the supernatant of the enzyme cleavage product; and drain to form a peptide sample;

[0043] c.5) Dissolve the peptide sample in the loading buffer and then inject it into the inner liner of the third injection bottle;

[0044] c.6) The inner liner of the third injection bottle is transported to the third high-resolution liquid phase mass spectrometry system through the third automated clamping track to perform protein and polypeptide hydrolysis detection to obtain protein and polypeptide hydrolysis detection results; the third high-resolution liquid phase mass spectrometry system is an ultra-high performance liquid chromatography or a tandem mass spectrometry system;

[0045] The steps c.1), c.2), c.3), c.4) and c.5) are all carried out at a constant temperature of 37°C.

[0046] As described above, the application of organ microfluidic chips in constructing in vitro tumor organ models.

[0047] As mentioned above, the organ microfluidic chip is used to construct a full replacement model for animal experiments or a semi-replacement model for animal experiments.

[0048] The advantages of the present invention are:

[0049] 1) Three automated sample processing devices were designed, which are used to extract polar metabolites in the perfusion fluid, peptides obtained by protein extraction and subsequent hydrolysis, and detection fluids of fat-soluble metabolites online, and enter high-resolution mass spectrometry corresponding to the detection conditions of the three types of substances for qualitative and quantitative analysis. Its advantage is that the perfusion fluid can be processed by classification, and the information of substances of the same category can be analyzed by classification.

[0050] 2) It replaces and changes the cumbersome process and instability of the original manual processing of perfusion fluid samples. Its advantage is that it controls the standardization and uniformity of sample preparation.

[0051] 3) The time for the material information contained in the perfusion fluid to enter the high-resolution mass spectrometer is greatly shortened. The advantage is that it avoids the loss of biological information contained in the sample due to long sample processing time.

[0052] 4) Created a unique "drug safety evaluation organ chip spatiotemporal tracking technology" to achieve real-time online mass spectrometry detection of the substance information contained in the perfusion fluid, superimposed with a high-power electron microscope mounted on a 3D multi-aperture cell stereostructure matrix network, and dynamically observed the changes in cell morphology under the perfusion of toxic substances. Compared with the traditional organ chip coupled biochemical detection machine technology, which measures a limited number of substances (classic cell deep damage markers), its advantage is that it can discover and determine the currently unknown cell shallow damage markers, and clarify the relationship between the appearance and time of these markers, and merge deep damage markers. So far, there is no high-resolution organ chip based on wireless or offline mass spectrometry detection and multi-omics integrated analysis function, nor is there an organ chip and system that can fully analyze all metabolites theoretically contained in the perfusion fluid.

[0053] 5) By loading different types of human cells (heart, liver, kidney or other human organ cells), human simulated organs are formed, which can be used to simulate human organs and used for pre-clinical research on drug safety evaluation. It provides an effective tool for estimating and judging the risks that may occur when Phase I clinical volunteers use new drugs for the first time, and realizes innovation in pre-clinical research.

[0054] 6) The existing so-called "online detection methods" have, to varying degrees, practical problems such as "the organ chip fails to fully simulate the in vivo microenvironment" and "the system is not fully online analyzed". The advantage of this system is that the unique structure of the "cell-containing three-dimensional matrix network with a variety of different pore sizes contained in the chip chamber" simulates the microenvironment state in the organism to the greatest extent and makes online perfusion possible, so as to achieve complete online analysis of the organ chip.

[0055] 7) The chip system can also be loaded with tumor cells or pathological and physiological cells, and expanded to become an organ chip for drug effectiveness evaluation, or an in vitro organ model, becoming a universal, general-purpose, and functionally extended organ chip system. It is an important innovation in the field of life sciences with multidisciplinary characteristics, and can develop standardized commercial products that meet specific functional requirements.

[0056] 8) The chip system can also be loaded with different types of animal cells, which can replace or semi-replace animal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 It is a schematic diagram of the structure of the drug safety evaluation system provided by the present invention;

[0058] Figure 2 It is a schematic diagram of the structure of the organ microfluidic chip provided by the present invention;

[0059] Figure 3It is a schematic diagram of the structure of the polar metabolite automated processing equipment used in the present invention;

[0060] Figure 4 It is a schematic structural diagram of the automated processing equipment for fat-soluble substances used in the present invention;

[0061] Figure 5 It is a schematic diagram of the structure of the protein polypeptide hydrolysis automated processing equipment used in the present invention;

[0062] Figure 6 Schematic diagram of the structure of the vortex mixer with self-cleaning function adopted by the present invention;

[0063] Figure 7 It is a schematic structural diagram of a vortex mixer with self-cleaning and ultrasonic functions adopted in the present invention;

[0064] Figure 8 It is a structural schematic diagram of a ball mill with a self-cleaning function adopted in the present invention;

[0065] Fig. 9 It is a structural schematic diagram of the matrix network of the bionic loofah capsule structure provided by the present invention;

[0066] in:

[0067] 1-organ microfluidic chip; 11-constant temperature and humidity living cell culture device; 12-cell injection loading port; 13-first electron microscope; 14-drug infusion port; 15-organ chip perfusion fluid output port; 2-polar metabolite automated processing equipment; 21-first vortex mixer; 22-first high-speed centrifuge; 23-first automated clamping track; 24-first injection bottle liner; 25-first high-resolution liquid phase mass spectrometry system; 26-first constant temperature box; 3-lipid-soluble substance automated processing equipment; 31-ball mill; 32-second vortex mixer; 33-second high-speed centrifuge; 34-second automated clamping track; 35 - the second injection bottle liner; 36 - the second high-resolution liquid phase mass spectrometry system; 37 - the second constant temperature box; 4 - protein and polypeptide hydrolysis automated processing equipment; 41 - the third vortex mixer; 42 - the third high-speed centrifuge; 43 - the darkroom; 44 - the protein concentration detector; 45 - the second electron microscope; 46 - the SDS-PAGE detector; 47 - the third automated clamping track; 48 - the third injection bottle liner; 49 - the third high-resolution liquid phase mass spectrometry system; 410 - the third constant temperature box; 5 - the waste liquid storage tank; 51 - the first waste liquid storage tank; 52 - the second waste liquid storage tank; 53 - the third waste liquid storage tank; 6 - the multi-omics integrated analysis system. DETAILED DESCRIPTION

[0068] See also Figure 2The present invention provides an organ microfluidic chip, which includes a chip substrate (a mature commercial product), a cell-accommodating chamber, a cell culture matrix network placed in the cell-accommodating chamber, a cell injection loading port 12, an organ chip perfusion fluid output port 15, and a drug perfusion port 14; a groove is provided on the chip substrate; the cell-accommodating chamber and the cell culture matrix network contained therein are embedded in the groove on the chip substrate; holes are provided on the cell culture matrix network; the cell injection loading port 12 is provided on the top of the cell-accommodating chamber and is connected to the cell culture matrix network; the cell injection loading port 12 injects cells required by the organ chip into the cell culture matrix network, and the cells are finally gathered in the holes; the drug perfusion port 14 is connected to the organ chip perfusion fluid output port 15 through the cell culture matrix network of the cell-accommodating chamber. Among them, the cell culture matrix network is an overall 3D structure, and the holes are set on the 3D structure; the cell culture matrix network is prepared by 3D printing photocurable resin or MOF material; there are multiple holes, the pore diameters of the holes are not equal, and the holes with multiple pore diameters are set on the 3D structure to form a 3D structure similar to a loofah. The size of the hole diameter is designed and made according to two situations: ① According to the size of the diameter of the cells to be loaded. One part of the diameter is larger than the diameter of the cells to be loaded, and its purpose is to facilitate the passage of cells, facilitate the perfusion of perfusion fluid, and form a perfusion fluid circulation immersion system with other wide diameter holes; the diameter of one part is smaller than the diameter of the cells to be loaded, and its purpose is to intercept and entrap the target perfusion cells, so that the cells are evenly distributed on the matrix network, simulating the distribution state of the internal environment of the organ cells to the greatest extent; ② The control of the hole diameter preparation is achieved by adjusting the technical parameters of the 3D printing equipment or the technical parameters of the precursor, concentration, temperature and pressure control when synthesizing MOF materials. The cells are mature commercial human or animal cells, including but not limited to hepatic epithelial cells, cardiomyocytes, mesangial cells, primary cells, cell lines, induced pluripotent stem cells, tumor cells or disease pathological cells; when the cells are hepatic epithelial cells, cardiomyocytes, and mesangial cells, the hepatic epithelial cells are human cell lines THLE-2; the cardiomyocytes are human cell lines AC16; and the mesangial cells are human cell lines HRMC. When loading cells, only one of the above cells is loaded at a time, forming a single organ chip; when it is necessary to prepare a multi-organ chip, it is only necessary to connect multiple single organ chip chambers loaded with different cells in series, forming a multi-organ chip modular assembly mode. The specific correspondence between the pore size design of the cell-containing matrix network material and the loaded cells is shown in Table 1.

[0069] Table 1 Correspondence between the pore size design of cell-containing matrix network materials and loaded cells

[0070]

[0071]

[0072] Specifically, there is a channel for inputting cells on the chip matrix (a mature microfluidic chip material in the market), through which multiple cells can be loaded and injected. These cells will be distributed in a chamber on the chip (an area of ​​the size of the chamber is cut out on the chip, and then the prepared chamber is embedded in the corresponding area. The microfluidic channel and the interface between the chip and the chamber are printed in advance before embedding. The chamber is made of the same material as the chip, and its design and production can meet the functional requirements). This chamber is densely covered with 3D structures (3D printing method or MOF material synthesis mode (pore size is obtained through 3D printing equipment, or precursor, concentration, temperature and pressure control technology when synthesizing MOF materials). Parameter realization), compared with the cell diameter, pores larger than the cell diameter are passed through by the cells and play a role in allowing the perfusion fluid to pass through, so that the entire chamber can be soaked in the perfusion fluid; pores smaller than the cells are used for interception and incarceration. The overall design makes the organ chip as close to the internal environment of human organs as possible. Because of the existence of pores larger than the cell diameter, the entire matrix network is immersed in the cell perfusion fluid. After the cells are injected, they will be evenly distributed on the matrix network to form a simulated organ cell composition, and together with the microenvironment formed by the perfusion fluid system, they form an overall simulated organ, that is, an organ chip. Finally, a matrix network with a bionic loofah sac structure and a bionic grape hanging effect that simulates blood perfusion is formed. Its structural schematic diagram is shown below. Fig. 9 As shown. When the target drug is perfused into the organ chip, the perfusion fluid at the back end of the chip contains the metabolites of the organ chip (including polar metabolites, lipid metabolites, and proteomes). A high-power microscope, such as the NIKON Ti2-E fully electric microscope, is mounted above the cell matrix network on the organ chip device to observe the morphological changes of cells before and after drug perfusion.

[0073] The organ microfluidic chip also includes a constant temperature and humidity living cell culture device 11, in which the chip substrate is arranged; the cell injection loading port 12, the organ chip perfusion fluid output port 15 and the drug perfusion port 14 respectively penetrate the constant temperature and humidity living cell culture device 11 and are connected with the cell culture matrix network; the constant temperature and humidity living cell culture device 11 is a 36-37°C constant temperature and humidity environment device, and the constant temperature and humidity living cell culture device 11 is filled with gas containing 5% concentration of carbon dioxide. The constant temperature and humidity living cell culture device 11 simulates the micro-growth environment in which organ cells grow in the body, and the gas in the device is filtered by a clean filter device to avoid bacterial contamination; a first electron microscope 13 is arranged on the constant temperature and humidity living cell culture device 11.

[0074] See also Figure 1While providing the organ microfluidic chip as described above, the present invention also provides a drug safety evaluation system formed based on the organ microfluidic chip, the drug safety evaluation system comprising an organ microfluidic chip 1, an automated polar metabolite processing device 2, an automated fat-soluble substance processing device 3, an automated protein and polypeptide hydrolysis processing device 4, a waste liquid storage tank 5 and a multi-omics integrated analysis system 6; the organ microfluidic chip 1 is respectively connected to the automated polar metabolite processing device 2, the automated fat-soluble substance processing device 3, and the automated protein and polypeptide hydrolysis processing device 4; the automated polar metabolite processing device 2, the automated fat-soluble substance processing device 3, and the automated protein and polypeptide hydrolysis processing device 4 are respectively connected to the waste liquid storage tank 5 through the multi-omics integrated analysis system 6.

[0075] See also Figure 3 The polar metabolite automated processing device 2 used in the present invention comprises a first vortex mixer 21, a first high-speed centrifuge 22, a first automated clamping track 23, a first sample bottle liner 24, a first high-resolution liquid phase mass spectrometry system 25 and a first constant temperature box 26; the first sample bottle liner 24 is placed on the first automated clamping track 23 and moves synchronously with the first automated clamping track 23; the perfusion liquid in the organ microfluidic chip 1 flows into the first vortex mixer 21 through the organ chip perfusion liquid outlet 15; the first vortex mixer 21 vortex-mixes the perfusion liquid and then injects it into the EP centrifuge tube of the first high-speed centrifuge 22; the first high-speed centrifuge 22 mixes the E After the P centrifuge tube is centrifuged, the centrifuged perfusion fluid is injected into the first sample bottle liner 24; the first automated clamping track 23 transports the first sample bottle liner 24 to the first high-resolution liquid mass spectrometry system 25 for detection; the first high-resolution liquid mass spectrometry system 25 is connected to the waste liquid storage tank 5 through the multi-omics integrated analysis system 6; the first vortex mixer 21, the first high-speed centrifuge 22, and the first automated clamping track 23 are all arranged in the first constant temperature box 26; preferably, the temperature of the first constant temperature box 26 is 4°C; the first vortex mixer 21 is a vortex mixer with a self-cleaning function, and its purpose is to realize the required automatic processing function of the perfusion fluid. The structure of the vortex mixer with a self-cleaning function is as follows Figure 6 As shown, it is equipped with a high-speed jet outlet, which is connected to a high-pressure water delivery device. When the self-cleaning function is needed, it will drive the clean water flow to wash the inside of the device at high speed. All devices with self-cleaning function are also equipped with negative pressure drain outlets to discharge waste water from the device during and after the self-cleaning function is implemented to complete self-cleaning.

[0076] See also Figure 4The automated processing equipment 3 for fat-soluble substances used in the present invention comprises a ball mill 31, a second vortex mixer 32, a second high-speed centrifuge 33, a second automated clamping track 34, a second sample bottle liner 35, a second high-resolution liquid phase mass spectrometry system 36 and a second constant temperature box 37; the perfusion liquid in the organ microfluidic chip 1 flows into the ball mill 31 through the organ chip perfusion liquid outlet 15; the ball mill 31 injects the crushed perfusion liquid into the second vortex mixer 32; the second vortex mixer 32 vortex-mixes the crushed perfusion liquid and injects it into the EP centrifuge tube of the second high-speed centrifuge 33; the second high-speed centrifuge 33 vortex-mixes the crushed perfusion liquid After the centrifuge tube is centrifuged, the centrifuged perfusion fluid is injected into the second sample bottle liner 35; the second automated clamping track 34 transports the second sample bottle liner 35 to the second high-resolution liquid mass spectrometry system 36 for detection; the second high-resolution liquid mass spectrometry system 36 is connected to the waste liquid storage tank 5 through the multi-omics integrated analysis system 6; the ball mill 31, the second vortex mixer 32, the second high-speed centrifuge 33, and the second automated clamping track 34 are all arranged in the second constant temperature box 37; preferably, the temperature of the second constant temperature box 37 is 4°C; the second vortex mixer 32 is a vortex mixer with self-cleaning and ultrasonic functions, and its structure is as follows Figure 7 As shown, the structure is in Figure 6 On the basis of, an ultrasonic generator device is added, the purpose of which is to maximize the possibility of fat-soluble substances being incorporated into fat-soluble solvents through ultrasonic vibration while vortex oscillation; ball mill 31 is a ball mill with self-cleaning function, and its structure is as follows Figure 8 As shown, an ultrasonic generator device is added to the existing ball mill. Its purpose is to maximize the possibility of fat-soluble substances being integrated into the fat-soluble solvent through ultrasonic vibration while the ball mill is working, so as to realize the required automatic processing function of the perfusion fluid. It is equipped with a self-cleaning high-pressure scattered flow water spray pipe inlet and an outlet for the self-cleaning post-guiding cleaning fluid; automatic cleaning can be achieved after homogenization.

[0077] See also Figure 5The protein extraction and polypeptide hydrolysis automated processing equipment 4 used in the present invention comprises a third vortex mixer 41, a third high-speed centrifuge 42, a darkroom 43, a protein concentration detector 44, a second electron microscope 45, an SDS-PAGE detector 46, a third automated clamping track 47, a third sample bottle liner 48, a third high-resolution liquid phase mass spectrometry system 49 and a third constant temperature box 410; the perfusate in the organ microfluidic chip 1 flows into the third vortex mixer 41 through the organ chip perfusate outlet 15; the third vortex mixer 41 vortex-mixes the perfusate and then injects it into the EP centrifuge tube of the third high-speed centrifuge 42; the third high-speed centrifuge 42 centrifuges the EP centrifuge tube and then injects the centrifuged perfusate into the darkroom 43 for alkylation reaction; the perfusate after the alkylation reaction is sequentially injected into the protein concentration detector The third automatic clamping track 47 transports the third injection bottle liner 48 to the third high-resolution liquid phase mass spectrometry system 49 for detection; the third high-resolution liquid phase mass spectrometry system 49 is connected to the waste liquid storage tank 5 through the multi-omics integrated analysis system 6; the third vortex mixer 41, the third high-speed centrifuge 42, the darkroom 43, the protein concentration detector 44, the second electron microscope 45, the SDS-PAGE detector 46, and the third automatic clamping track 47 are all arranged in the third constant temperature box 410; preferably, the temperature of the third constant temperature box 410 is 37°C; the third vortex mixer 41 is a vortex mixer with a self-cleaning function; preferably, the protein concentration detector 44 is provided with a second electron microscope 45. The purpose of the second electron microscope 45 is to realize the automatic processing function of the perfusate.

[0078] See also Figure 1 , the waste liquid storage tank 5 used in the present invention includes a first waste liquid storage tank 51, a second waste liquid storage tank 52 and a third waste liquid storage tank 53; the polar metabolite automated processing equipment 2 is connected to the first waste liquid storage tank 51 through a multi-omics integrated analysis system 6; the fat-soluble substance automated processing equipment 3 is connected to the second waste liquid storage tank 52 through a multi-omics integrated analysis system 6; the protein extraction and polypeptide hydrolysis automated processing equipment 4 is connected to the third waste liquid storage tank 53 through a multi-omics integrated analysis system 6. The multi-omics integrated analysis system 6 is a cutting-edge and mature human-machine combined data analysis operation mode, not a hardware in the traditional sense. The purpose of the diagram is to express the existence of such a human-machine fusion operation mode, not hardware. Because there is a continuous or discontinuous flow of perfusion fluid under control, each type of perfusion fluid is collected into a waste liquid pool for storage and post-processing after being extracted and the test fluid is tested.

[0079] In addition, based on the drug safety evaluation system as described above, the present invention also provides a drug safety evaluation method, the theoretical basis of which is derived from "Microfluidic Platform with In-Chip Electrophoresis Coupled to Mass Spectrometry for Monitoring Neurochemical Release from Nerve Cells" (Xiangtang Li, Hankun Hu, Shulin Zhao, Yi-Ming Liu. Anal. Chem. 2016, 88: 5338-5344; 2), which specifically includes the following steps:

[0080] 1) preparing an organ microfluidic chip and obtaining the perfusion fluid after passing through the organ microfluidic chip;

[0081] 2) dividing the perfusate into three portions, respectively labeled as the first perfusate, the second perfusate, and the third perfusate, wherein the first perfusate, the second perfusate, and the third perfusate have exactly the same composition;

[0082] 3) Injecting the first portion of the perfusion fluid into the polar metabolite automated processing device 2, extracting and processing to obtain a detection fluid, selecting a high-resolution mass spectrometer adapted to the detection conditions of polar substances to perform polar metabolite detection, and obtaining polar metabolite detection results; injecting the second portion of the perfusion fluid into the fat-soluble substance automated processing device 3, extracting and processing to obtain a detection fluid, selecting a high-resolution mass spectrometer adapted to the detection conditions of fat-soluble substances to perform fat-soluble substance detection, and obtaining fat-soluble substance detection results; injecting the third portion of the perfusion fluid into the protein extraction and polypeptide hydrolysis automated processing device 4 to perform protein extraction and polypeptide hydrolysis detection to obtain a polypeptide detection fluid, and the polypeptide detection fluid is subjected to a high-resolution mass spectrometer adapted to the detection conditions of polypeptide substances, and the protein extraction and polypeptide hydrolysis detection results are obtained by reverse calculation;

[0083] in:

[0084] In step 3), the specific implementation method of injecting the first perfusion fluid into the polar metabolite automatic processing device 2 for polar metabolite detection is:

[0085] a.1) 50 μl of the first perfusion solution is drawn and injected into the first vortex mixer 21, and 150 μl of pre-cooled ice methanol is injected into the first vortex mixer 21, and the mixture is vortexed automatically for 3 minutes; the ice methanol contains 1 μg / ml of 2-chlorophenylalanine;

[0086] a.2) After the vortexing is completed, the perfusion fluid of the first vortex mixer 21 is injected into the EP centrifuge tube of the first high-speed centrifuge 22, and the first centrifugation is performed at 12000r / min for 10min. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube, and then the second centrifugation is performed at 12000r / min for 5min. After the centrifugation is completed, the supernatant is injected into the liner tube 24 of the first injection bottle;

[0087] a.3) The inner liner tube 24 of the first injection bottle is transported to the first high-resolution liquid phase mass spectrometry system 25 through the first automated clamping track 23 to detect polar metabolites and obtain polar metabolite detection results; the first high-resolution liquid phase mass spectrometry system 25 is an ultra-high liquid chromatography or tandem mass spectrometry; the detection conditions, self-built library and public library of the first high-resolution liquid phase mass spectrometry system 25 are directly called as needed (existing technology, the present invention will not repeat it). The first high-resolution liquid phase mass spectrometry system 25 can be an ultra-high liquid chromatography (www.shimadzu.com.cn) or a tandem mass spectrometry (www.appliedbiosystems.com.cn). According to the material information obtained by mass spectrometry, the self-built library and the public library are matched to obtain the qualitative and quantitative information of the polar metabolites contained in the test solution. This is a mature market technology; its results can be analyzed separately or integrated according to the lipid group and protein group. The evaluation method is a mature market evaluation method: qualitative (presence or absence of substances), quantitative (absolute quantification or relative quantification). Generally, it is to compare the difference changes in different time periods or different concentration gradients.

[0088] Step a.1) and step a.2) are both carried out at a constant temperature of 4°C;

[0089] Preferably, in step 3), the specific implementation method of injecting the second perfusion fluid into the fat-soluble substance automated processing device 3 for fat-soluble substance detection is:

[0090] b.1) 20 μl of the second perfusion solution is taken and injected into the ball mill 31 , and 1 ml of lipid extract is injected into the ball mill 31 to crush the second perfusion solution to obtain a homogenate;

[0091] b.2) injecting the homogenate into the second vortex mixer 32, injecting 200 μl of water into the second vortex mixer 32, and vortexing automatically for 3 minutes;

[0092] b.3) After the vortexing is completed, the perfusion fluid of the second vortex mixer 32 is injected into the EP centrifuge tube of the second high-speed centrifuge 33 and centrifuged at 12000r / min for 10min. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube for concentration to obtain a concentrated product;

[0093] b.4) injecting a lipid reconstitution solution into the concentrated product for reconstitution, and injecting the reconstituted product into the liner tube 35 of the second injection bottle;

[0094] b.5) ​​The inner liner tube 35 of the second injection bottle is transported to the second high-resolution liquid mass spectrometry system 36 through the second automated clamping track 34 to detect fat-soluble substances and obtain fat-soluble substance detection results; the detection conditions, self-built library and public library of the second high-resolution liquid mass spectrometry system 36 are directly called as needed (existing technology, the present invention will not repeat them). The second high-resolution liquid mass spectrometry system 36 can be an ultra-high liquid chromatography (www.shimadzu.com.cn) or a tandem mass spectrometry (www.appliedbiosystems.com.cn).

[0095] Step b.1), step b.2), step b.3) and step b.4) are all carried out at a constant temperature of 4°C;

[0096] Preferably, the specific implementation method of injecting the third perfusion fluid into the protein and polypeptide hydrolysis automatic processing equipment 4 for protein and polypeptide hydrolysis detection is:

[0097] c.1) 50 μl of the third perfusion solution is drawn and injected into the third vortex mixer 41, and at the same time, an appropriate amount of lysis solution is injected into the third vortex mixer 41, and vortexed to mix thoroughly; the lysis solution is 1.5% SDS / 100 mM Tris-Cl;

[0098] c.2) After the vortexing is completed, the perfusion fluid of the third vortex mixer 41 is injected into the EP centrifuge tube of the third high-speed centrifuge 42, and the first centrifugation is performed at 12000r / min for 10min. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube, and an appropriate amount of acetone is injected into the EP centrifuge tube; the second centrifugation is performed at 12000r / min for 3min. After the centrifugation is completed, the precipitate is obtained;

[0099] c.3) After injecting the reconstitution solution into the precipitate for reconstitution, dithiothreitol was added and incubated at 37°C for 1 hour; then, iodoacetamide was added and an alkylation reaction was performed at room temperature in the dark to block the sulfhydryl group; the reconstitution solution was 8M Urea / 100mM Tris-Cl;

[0100] c.4) The protein concentration of the product obtained in step c.3) is determined by a protein concentration detector 44, and SDS-PAGE detection is performed by an SDS-PAGE detector 46. 100 mM Tris-HCl solution is added to the result, the Urea concentration is diluted to less than 2 M, trypsin is added at a mass ratio of 1:50 between enzyme and protein, and the mixture is incubated at 37°C with shaking overnight for enzyme cleavage; TFA is added after 12 hours to terminate the enzyme cleavage; the supernatant of the enzyme cleavage product is desalted; the peptide sample is formed after being drained; 50 micrograms of sample is grabbed after protein quantification, and SDS-PAGE detection is performed by polyacrylamide gel electrophoresis, and protein bands are observed after Coomassie brilliant blue staining through the microscope window set on the machine.

[0101] c.5) injecting the peptide sample into the loading buffer to dissolve it, and then injecting it into the inner liner tube 48 of the third injection bottle;

[0102] c.6) The inner liner tube 48 of the third injection bottle is transported to the third high-resolution liquid phase mass spectrometry system 49 through the third automated clamping track 47 to perform protein and polypeptide hydrolysis detection to obtain protein and polypeptide hydrolysis detection results; the detection conditions, self-built library and public library of the third high-resolution liquid phase mass spectrometry system 49 are directly called as needed (existing technology, the present invention will not repeat them). The third high-resolution liquid phase mass spectrometry system 49 can be an ultra-high liquid chromatography (www.shimadzu.com.cn) or a tandem mass spectrometry (www.appliedbiosystems.com.cn).

[0103] Step c.1), step c.2), step c.3), step c.4) and step c.5) are all carried out at a constant temperature of 37°C.

[0104] 4) The polar metabolite detection results, fat-soluble substance detection results, and protein and peptide hydrolysis detection results are injected into the multi-omics integrated analysis system 6 for human-machine combined mode processing to obtain comprehensive analysis results;

[0105] 5) Conduct a comprehensive evaluation of drug safety based on the comprehensive analysis results.

[0106] The present invention provides an organ model for evaluating drug effectiveness and a new method for applying the model. Different types of tumor cells are loaded to form an in vitro tumor organ model that simulates the growth state of tumors on organs in vivo. The organ chip transfected with tumor cells is perfused with a candidate drug perfusion solution, and the perfusion solution is classified and processed online and multi-omics integrated analysis is performed, which can be expanded into a drug effectiveness evaluation model. In addition, the present invention provides a new method for replacing or semi-replacing organ chips in animal experiments. Loading animal cells to form an animal organ chip is also a method for evaluating the perfusion solution of the animal organ chip using mass spectrometry online multi-omics, which can achieve animal experiment replacement or semi-replacement and become a potential iterative technology to subvert the animal experiment industry. In order to achieve the "3R principle of animal experiment reduction, substitution, and optimization" advocated and pursued by the global academic and environmental protection circles to protect animal welfare ethics. Moreover, the present invention can also develop standardized products that can be used for scientific research experiments with specific functional purposes, and has a broad market space.

Claims

1. A drug safety evaluation system based on an organ microfluidic chip, characterized in that: The drug safety evaluation system comprises an organ microfluidic chip (1), an automated polar metabolite processing device (2), an automated fat-soluble substance processing device (3), an automated protein extraction and polypeptide hydrolysis processing device (4), a waste liquid storage tank (5), and a multi-omics integrated analysis system (6); the organ microfluidic chip (1) is respectively connected to the automated polar metabolite processing device (2), the automated fat-soluble substance processing device (3), and the automated protein extraction and polypeptide hydrolysis processing device (4); the automated polar metabolite processing device (2), the automated fat-soluble substance processing device (3), and the automated protein extraction and polypeptide hydrolysis processing device (4) are respectively connected to the waste liquid storage tank (5) via the multi-omics integrated analysis system (6); The organ microfluidic chip (1) comprises a chip substrate, a cell culture matrix network, a cell injection loading port (12), a constant temperature and humidity living cell culture device (11), an organ chip perfusion fluid output port (15) and a drug perfusion port (14); the chip substrate is provided with a groove; the cell culture matrix network is embedded in the groove on the chip substrate; the cell culture matrix network is provided with holes; the cell injection loading port (12) is arranged on the top of the cell culture matrix network and is connected to the cell culture matrix network; the cell injection loading port (12) injects cells required by the organ chip into the cell culture matrix network, and the cells are finally gathered in the holes; the drug perfusion port (14) is connected to the organ chip perfusion fluid output port (15) through the cell culture matrix network; The cell culture matrix network is an overall 3D structure, and the holes are arranged on the 3D structure; the cell culture matrix network is prepared by 3D printing or bubble filling mode; there are multiple holes, and the pore diameters of the holes are non-equivalent; the 3D structure is similar to a loofah-like structure; the size of the hole diameter is designed according to the size of the diameter of the cells to be loaded, and the preparation of the hole diameter is achieved by adjusting the technical parameters of the 3D printing equipment or the precursor, concentration, temperature and pressure control technical parameters when synthesizing MOF materials; The chip substrate is arranged in a constant temperature and humidity living cell culture device (11); the cell injection loading port (12), the organ chip perfusion fluid output port (15) and the drug perfusion port (14) respectively penetrate the constant temperature and humidity living cell culture device (11) and are connected to the cell culture matrix network; the constant temperature and humidity living cell culture device (11) is a 36-37°C constant temperature and humidity anaerobic environment device, and the constant temperature and humidity living cell culture device (11) is filled with carbon dioxide; a first electron microscope (13) is arranged on the constant temperature and humidity living cell culture device (11); The polar metabolite automated processing equipment (2) comprises a first vortex mixer (21), a first high-speed centrifuge (22), a first automated clamping track (23), a first injection bottle liner tube (24), a first high-resolution liquid phase mass spectrometry system (25) and a first constant temperature box (26); the first injection bottle liner tube (24) is placed on the first automated clamping track (23) and moves synchronously with the first automated clamping track (23); the perfusion fluid in the organ microfluidic chip (1) flows into the first vortex mixer (21) through the organ chip perfusion fluid outlet (15); the first vortex mixer (21) vortex-mixes the perfusion fluid and then injects it into the EP centrifuge tube of the first high-speed centrifuge (22); After the first high-speed centrifuge (22) centrifuges the EP centrifuge tube, the centrifuged perfusion fluid is injected into the first injection bottle liner tube (24); the first automated clamping track (23) transports the first injection bottle liner tube (24) to the first high-resolution liquid phase mass spectrometry system (25) for detection; the first high-resolution liquid phase mass spectrometry system (25) is connected to the waste liquid storage tank (5) through the multi-omics integrated analysis system (6); the first vortex mixer (21), the first high-speed centrifuge (22), and the first automated clamping track (23) are all arranged in a first constant temperature box (26); The automated processing equipment (3) for fat-soluble substances comprises a ball mill (31), a second vortex mixer (32), a second high-speed centrifuge (33), a second automated clamping track (34), a second sample bottle liner (35), a second high-resolution liquid phase mass spectrometry system (36) and a second constant temperature box (37); the perfusion fluid in the organ microfluidic chip (1) flows into the ball mill (31) through the organ chip perfusion fluid outlet (15); the ball mill (31) injects the crushed perfusion fluid into the second vortex mixer (32); the second vortex mixer (32) vortex-mixes the crushed perfusion fluid and then injects it into the EP centrifuge tube of the second high-speed centrifuge (33); After the second high-speed centrifuge (33) centrifuges the EP centrifuge tube, the centrifuged perfusion fluid is injected into the second injection bottle inner liner (35); the second automated clamping track (34) transports the second injection bottle inner liner (35) to the second high-resolution liquid phase mass spectrometry system (36) for detection; the second high-resolution liquid phase mass spectrometry system (36) is connected to the waste liquid storage tank (5) through the multi-omics integrated analysis system (6); the ball mill (31), the second vortex mixer (32), the second high-speed centrifuge (33), and the second automated clamping track (34) are all arranged in a second constant temperature box (37); The protein extraction and polypeptide hydrolysis automated processing equipment (4) comprises a third vortex mixer (41), a third high-speed centrifuge (42), a darkroom (43), a protein concentration detector (44), a second electron microscope (45), an SDS-PAGE detector (46), a third automated clamping track (47), a third injection bottle liner tube (48), a third high-resolution liquid phase mass spectrometry system (49) and a third constant temperature box (410); the perfusion fluid in the organ microfluidic chip (1) flows into the third vortex mixer (41) through the organ chip perfusion fluid outlet (15); the third vortex mixer (41) vortex-mixes the perfusion fluid and then injects it into the EP centrifuge tube of the third high-speed centrifuge (42); the third high-speed centrifuge (42) centrifuges the EP centrifuge tube and then injects the centrifuged perfusion fluid into the EP centrifuge tube. The alkylation reaction is carried out in a darkroom (43); the perfusion fluid after the alkylation reaction is injected into a protein concentration detector (44) and an SDS-PAGE detector (46); the perfusion fluid after the measurement is injected into a third injection bottle liner tube (48); the third automated clamping track (47) transports the third injection bottle liner tube (48) to a third high-resolution liquid phase mass spectrometry system (49) for detection; the third high-resolution liquid phase mass spectrometry system (49) is connected to a waste liquid storage tank (5) via a multi-omics integrated analysis system (6); the third vortex mixer (41), the third high-speed centrifuge (42), the darkroom (43), the protein concentration detector (44), the second electron microscope (45), the SDS-PAGE detector (46), and the third automated clamping track (47) are all arranged in a third constant temperature box (410).

2. The drug safety evaluation system according to claim 1, characterized in that: The temperature of the first constant temperature box (26) is a constant temperature of 4°C; and the first vortex mixer (21) is a vortex mixer with a self-cleaning function.

3. The drug safety evaluation system according to claim 2, characterized in that: The temperature of the second constant temperature box (37) is a constant temperature of 4°C; the second vortex mixer (32) is a vortex mixer with self-cleaning and ultrasonic functions; and the ball mill (31) is a ball mill with self-cleaning functions.

4. The drug safety evaluation system according to claim 3, characterized in that: The temperature of the third constant temperature box (410) is a constant temperature of 37°C; and the third vortex mixer (41) is a vortex mixer with a self-cleaning function.

5. The drug safety evaluation system according to claim 4, characterized in that: The protein concentration detector (44) is provided with a second electron microscope (45).

6. The drug safety evaluation system according to claim 2, 3, 4 or 5, characterized in that: The waste liquid storage tank (5) includes a first waste liquid storage tank (51), a second waste liquid storage tank (52) and a third waste liquid storage tank (53); the polar metabolite automated processing equipment (2) is connected to the first waste liquid storage tank (51) via a multi-omics integrated analysis system (6); the fat-soluble substance automated processing equipment (3) is connected to the second waste liquid storage tank (52) via a multi-omics integrated analysis system (6); and the protein extraction and polypeptide hydrolysis automated processing equipment (4) is connected to the third waste liquid storage tank (53) via a multi-omics integrated analysis system (6).

7. A drug safety evaluation method based on the drug safety evaluation system according to any one of claims 2 to 6, characterized in that: The drug safety evaluation method comprises the following steps: 1) Prepare organ microfluidic chip and obtain perfusion fluid after passing through the organ microfluidic chip; 2) dividing the perfusate into three portions, respectively labeled as the first perfusate, the second perfusate, and the third perfusate, wherein the first perfusate, the second perfusate, and the third perfusate have exactly the same composition; 3) Injecting the first portion of the perfusion fluid into the polar metabolite automated processing device (2) to perform polar metabolite detection and obtain polar metabolite detection results; injecting the second portion of the perfusion fluid into the fat-soluble substance automated processing device (3) to perform fat-soluble substance detection and obtain fat-soluble substance detection results; injecting the third portion of the perfusion fluid into the protein extraction and polypeptide hydrolysis automated processing device (4) to perform protein and polypeptide hydrolysis detection and obtain protein and polypeptide hydrolysis detection results through reverse calculation; 4) The polar metabolite detection results, fat-soluble substance detection results, and protein and peptide hydrolysis detection results are injected into the multi-omics integrated analysis system (6), and after being processed in a human-computer combination mode, a comprehensive analysis result is obtained; 5) Conduct a comprehensive evaluation of drug safety based on the comprehensive analysis results.

8. The method according to claim 7, characterized in that: In step 3), the specific implementation method of injecting the first perfusion fluid into the polar metabolite automatic processing equipment (2) for polar metabolite detection is: a.1) 50 μl of the first perfusion solution is drawn and injected into the first vortex mixer (21), and 150 μl of pre-cooled ice methanol is injected into the first vortex mixer (21), and the mixture is vortexed automatically for 3 minutes; the ice methanol contains 1 μg / ml of 2-chlorophenylalanine; a.2) After the vortexing is completed, the perfusion fluid of the first vortex mixer (21) is injected into the EP centrifuge tube of the first high-speed centrifuge (22), and the first centrifugation is performed at 12000 r / min for 10 minutes. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube, and the second centrifugation is performed at 12000 r / min for 5 minutes. After the centrifugation is completed, the supernatant is injected into the liner tube (24) of the first injection bottle; a.3) The inner liner tube (24) of the first injection bottle is transported to the first high-resolution liquid phase mass spectrometry system (25) through the first automated clamping track (23) to perform polar metabolite detection to obtain polar metabolite detection results; the first high-resolution liquid phase mass spectrometry system (25) is an ultra-high performance liquid chromatography or a tandem mass spectrometry; The steps a.1) and a.2) are both carried out at a constant temperature of 4°C.

9. The method according to claim 8, characterized in that: In the step 3), the specific implementation method of injecting the second perfusion fluid into the fat-soluble substance automated processing device (3) for fat-soluble substance detection is: b.1) 20 μl of the second perfusion solution is drawn and injected into the ball mill (31), and 1 ml of lipid extract is injected into the ball mill (31) to crush the second perfusion solution to obtain a homogenate; b.2) injecting the homogenate into the second vortex mixer (32), injecting 200 μl of water into the second vortex mixer (32), and vortexing automatically for 3 minutes; b.3) After the vortexing is completed, the perfusion fluid of the second vortex mixer (32) is injected into the EP centrifuge tube of the second high-speed centrifuge (33) and centrifuged at 12000r / min for 10 minutes. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube for concentration to obtain a concentrated product; b.4) injecting the lipid reconstitution solution into the concentrated product for reconstitution, and injecting the reconstituted product into the inner liner tube (35) of the second injection bottle; b.5) ​​The inner liner tube (35) of the second injection bottle is transported to the second high-resolution liquid phase mass spectrometry system (36) through the second automated clamping track (34) to perform fat-soluble substance detection to obtain fat-soluble substance detection results; the second high-resolution liquid phase mass spectrometry system (36) is an ultra-high performance liquid chromatography or a tandem mass spectrometry; The steps b.1), b.2), b.3) and b.4) are all carried out at a constant temperature of 4°C.

10. The method according to claim 9, characterized in that: In step 3), the specific implementation method of injecting the third portion of the perfusion fluid into the protein extraction and polypeptide hydrolysis automated processing equipment (4) for protein extraction and polypeptide hydrolysis detection is: c.1) aspirate 50 μl of the third perfusion solution and inject it into the third vortex mixer (41), and inject an appropriate amount of lysis solution into the third vortex mixer (41), and vortex to mix thoroughly; the lysis solution is 1.5% SDS / 100 mM Tris-Cl; c.2) After the vortexing is completed, the perfusion fluid of the third vortex mixer (41) is injected into the EP centrifuge tube of the third high-speed centrifuge (42), and the first centrifugation is performed at 12000r / min for 10 minutes. After the centrifugation is completed, the supernatant is sucked and injected into another EP centrifuge tube, and an appropriate amount of acetone is injected into the EP centrifuge tube; the second centrifugation is performed at 12000r / min for 3 minutes. After the centrifugation is completed, the precipitate is obtained; c.3) After injecting the reconstitution solution into the precipitate for reconstitution, dithiothreitol was added and incubated at 37°C for 1 hour; then, iodoacetamide was added and an alkylation reaction was performed at room temperature in the dark to block the thiol group; the reconstitution solution was 8M Urea / 100 mM Tris-Cl; c.4) The protein concentration of the product obtained in step c.3) is determined by a protein concentration detector (44), and SDS-PAGE detection is performed by an SDS-PAGE detector (46). 100 mM Tris-HCl solution is added to the result to dilute the Urea concentration to less than 2 M, and trypsin is added at a mass ratio of 1:50 between enzyme and protein, and the mixture is incubated at 37°C with shaking overnight for enzyme cleavage; TFA is added after 12 hours to terminate the enzyme cleavage; the supernatant of the enzyme cleavage product is desalted; and the peptide sample is formed after being drained; c.5) injecting the peptide sample into the loading buffer to dissolve it, and then injecting it into the inner liner tube (48) of the third injection bottle; c.6) The inner liner tube (48) of the third injection bottle is transported to the third high-resolution liquid phase mass spectrometry system (49) through the third automated clamping track (47) to perform protein and polypeptide hydrolysis detection to obtain protein and polypeptide hydrolysis detection results; the third high-resolution liquid phase mass spectrometry system (49) is an ultra-high performance liquid chromatography or tandem mass spectrometry; The steps c.1), c.2), c.3), c.4) and c.5) are all carried out at a constant temperature of 37°C.

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