A high-throughput anaerobic intestinal chip system and its application in evaluating the function of probiotics in alleviating enteritis

Abstract

The present application relates to a kind of high flux anaerobic intestinal chip system and the application in the function of evaluating probiotic bacteria to alleviate enteritis, high flux anaerobic intestinal chip system includes microfluidic chip, fluid perfusion device, oxygen monitoring chip and micro anaerobic box, fluid perfusion device can inject culture medium into microfluidic chip, oxygen monitoring chip is arranged in microfluidic chip upstream, microfluidic chip is arranged in micro anaerobic box, microfluidic chip is equipped with intestinal cell, high flux anaerobic intestinal chip can simulate intestinal environment, by different kinds of probiotic bacteria are added to intestinal chip, can be obtained by detecting culture product, or detecting intestinal cell after culture, to evaluate probiotic bacteria function;Further can combine high flux anaerobic intestinal chip with machine learning, utilize machine learning technology to analyze and process the multidimensional data characteristics (gene, protein, cell level) of enteritis model based on intestinal chip, to carry out global, overall comprehensive evaluation;Realize high flux, intelligent screening of better efficacy probiotic bacteria strain finally.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidic chip, and particularly relates to a high-throughput anaerobic intestinal chip system and application thereof in evaluation of functions of probiotics in relieving enteritis. BACKGROUND

[0002] Nowadays, probiotics and their related products have become a continuously growing industry and are one of the most commonly used food supplements in the world. As probiotics are beneficial to human health, they have been widely recognized by the academic community, and more and more people have begun to pay attention to how to replace drugs with biological therapy to regulate chronic diseases. In recent years, the use of probiotics has also been widely supported by the medical community, especially in the field of gastroenterology. Related studies have shown that probiotics can achieve the purpose of preventing / treating intestinal diseases through the bacteria themselves or their metabolites or by regulating the intestinal microecology. However, there are many types of probiotics that can be used to prevent and treat intestinal diseases, and the strains of probiotics are different, the metabolites produced are different, and the treatment effects and mechanisms are also different. Therefore, in-depth excavation and analysis of the efficacy and health mechanism of probiotics, screening of specific functional probiotics, and realization of more precise, effective and safe support of probiotics to the health of the human body have become a global research hotspot and the main direction of industrial innovation and development.

[0003] Currently, for the development of probiotic products, the screening of probiotic functions is generally carried out through in vitro screening and efficacy evaluation of probiotics before clinical experimental research. The in vitro screening and efficacy evaluation of probiotics mainly use the characteristics of acid production, gas production and growth metabolism of probiotics for selective culture, and then evaluate the efficacy of the probiotics such as gastric acid tolerance, bile salt tolerance, inhibition of pathogenic bacteria and reduction of cholesterol. The interaction between probiotics and specific cells is studied based on cell models, and then the efficacy of mucosal adhesion, antioxidant and immune regulation is evaluated. The traditional screening method has many drawbacks, such as complicated operation, long detection period, poor identification ability, low throughput and the like. In order to simplify the operation steps, improve the screening throughput and reduce the time and economic cost of screening, researchers propose functional gene screening based on molecular biology technology and study the influence of probiotics and prebiotics on the composition and metabolic activity of intestinal flora based on in vitro fermentation model. Although the above methods have greatly improved the current situation of probiotic screening and overall improved the screening level, they can only screen probiotics and cannot further analyze and verify the functions of the screened probiotics, and cannot determine the mechanism of probiotics to play physiological functions. Animal experiments and human experiments are of great significance to the study of the probiotic effects of probiotics. This research can determine the health benefits of probiotic intake and the correlation with disease relief. However, the existing animal model screening of probiotics has problems such as long cycle, high cost and low screening efficiency. In addition, due to ethical and economic reasons, the use of mouse or pig animal experiment model is not always feasible, and the research results using these models may be inconsistent. At the same time, the sample size of most clinical studies is relatively low, which limits the reliability of the research results. Therefore, it is an urgent need to develop a convenient, fast, accurate and high-throughput probiotic function evaluation technology for probiotic screening.

[0004] In recent years, the development of intestinal chip technology has made it possible to quickly, accurately and high-throughput screen probiotics with specific functions. Intestinal chip is a simulation of human intestinal chip system based on microfluidic technology. Although intestinal chip can realize high-throughput culture of functional probiotics, it is still a challenge to accurately evaluate the efficacy of multiple probiotic strains with the same function and screen the best probiotic. The traditional efficacy evaluation standard relies on the simple comparison of detection indexes such as intestinal barrier function and cytokine levels between different experimental groups, which can only evaluate whether the probiotics have probiotic functions, and cannot accurately determine the optimal efficacy of a strain. Therefore, most of the current probiotic screening technologies can only screen a certain type of strain with the same function, and cannot further screen the best strain based on multi-dimensional efficacy evaluation detection indexes. SUMMARY

[0005] To solve the above technical problems, the application provides a high-throughput anaerobic intestinal chip system and application thereof in evaluation of probiotic bacteria in relieving intestinal inflammation.

[0006] The technical scheme adopted by the application is as follows: a high-throughput anaerobic intestinal chip system comprises,

[0007] A microfluidic chip is internally provided with intestinal cells and a culture medium;

[0008] A fluid perfusion device comprises a syringe pump, an input pipeline and a recovery pipeline, the syringe pump is connected to the microfluidic chip through the input pipeline, and the recovery pipeline is connected to the microfluidic chip downstream;

[0009] An oxygen monitoring chip is connected to the microfluidic chip upstream;

[0010] A micro anaerobic box comprises a box body, anaerobic bags and a heating device, one or more anaerobic bags are arranged in the box body, the heating device can be used for heating the box body, and the microfluidic chip is arranged in the box body.

[0011] Preferably, the microfluidic chip comprises an upper chip, a middle chip and a lower chip, the upper chip is provided with an upper rhombic chamber and an upper S-shaped chamber, the middle chip is provided with a middle rhombic chamber and a middle S-shaped chamber, the middle chip is arranged between the upper chip and the lower chip, the upper S-shaped chamber and the middle S-shaped chamber are in position correspondence and are separated by a microporous membrane, and the upper rhombic chamber and the middle rhombic chamber are not in communication.

[0012] Preferably, the oxygen monitoring chip comprises an upper monitoring chip and a lower monitoring chip, the lower monitoring chip is provided with a culture medium flow channel and a fluorescent sensing patch capable of reacting with oxygen in the culture medium, and the upper monitoring chip is provided with a light collection device capable of collecting the change in fluorescent intensity on the fluorescent sensing patch.

[0013] Preferably, the upper chip and the middle chip are both connected with the oxygen monitoring chip upstream.

[0014] Preferably, intestinal cells and a culture medium are injected into the channel of the upper chip, and the culture medium is injected into the channel of the lower chip.

[0015] Preferably, the intestinal cells are Caco-2 and HT29 MTX mixed and co-cultured at a ratio of 9:1.

[0016] The application of the high-throughput anaerobic intestinal chip system in evaluation of probiotic bacteria in relieving intestinal inflammation.

[0017] Preferably, the application comprises the following steps:

[0018] Step one: build a high-throughput anaerobic intestinal chip system, inject cells into the upper layer chip, and then continuously perfuse cell culture medium into the upper layer chip and the middle layer chip in the microfluidic chip;

[0019] Step two: inject probiotics into the upper layer chip, perfuse anaerobic culture medium into the microfluidic chip, and build a probiotic intervention intestinal cell scenario; after culture, the evaluation indexes are detected;

[0020] Step three: perform steps 1 and 2 on multiple strains to obtain multiple sets of evaluation index data, train the evaluation analysis model through machine learning, and build a comprehensive efficacy score standard based on principal component weights;

[0021] Step four: sort multiple strains according to comprehensive efficacy.

[0022] Preferably, the culture medium composition includes 77% DMEM, 20% FBS, 1% double antibody and 1% glutamine; the anaerobic culture medium composition includes 77% DMEM, 20% FBS, 1% double antibody, 1% glutamine and 1% L-cysteine.

[0023] Preferably, in step three, a normal intestinal model group and a colonitis group are also built for comparison with the probiotic intervention group.

[0024] Preferably, the evaluation indexes include cytokine detection, barrier protein immunofluorescence detection, and inflammation-related gene expression detection.

[0025] Preferably, the cytokine detection object is the culture medium collected downstream in the microfluidic chip, and the inflammatory factor IL-1β is detected.

[0026] Preferably, the barrier protein immunofluorescence detection object is the Caco-2 / HT29MTX cells loaded on the porous membrane.

[0027] Preferably, the inflammation-related gene expression detection object is the Caco-2 / HT29MTX cells loaded on the porous membrane.

[0028] The present application has the advantages and positive effects that: based on high-throughput intestinal chip and machine learning technology, a functional probiotic screening system with high efficiency, speed, precision and intelligence is built, which provides a reliable evaluation tool for probiotic screening, and solves the problems of time-consuming, high cost and inability to provide comprehensive and reliable data information for precise evaluation of probiotic function in the current probiotic high-throughput screening;

[0029] Based on the above intestinal chip evaluation system, a high-throughput anaerobic intestinal chip is constructed to provide a platform for high-throughput culture of probiotics; machine learning technology is used to analyze and process the multi-dimensional data features (genes, proteins, and cell levels) of the intestinal inflammation model based on the intestinal chip for global and overall comprehensive evaluation; and finally, high-throughput and intelligent screening of probiotic strains with better efficacy is realized. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Model of anaerobic intestinal chip;

[0031] Figure 2 Oxygen content in the high-throughput anaerobic intestinal chip system;

[0032] Figure 3 Intestinal chip cell live and dead staining detection results;

[0033] Figure 4 Co-culture of intestinal cells and probiotics in the intestinal chip;

[0034] Figure 5 Correlation coefficient matrix of 23 intestinal chip colonitis detection indicators;

[0035] Figure 6 Variance contribution rate and cumulative contribution rate of each principal component;

[0036] Figure 7 Probiotic comprehensive efficacy score. DETAILED DESCRIPTION

[0037] The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0038] The present application relates to a high-throughput anaerobic intestinal chip system and its application in evaluating the function of probiotics in relieving intestinal inflammation, which comprises a miniature anaerobic box that can provide an anaerobic environment, a high-throughput intestinal chip for co-culturing probiotics and intestinal cells, an oxygen real-time monitoring sensor and related chips, a heating table that provides a constant temperature environment, and other accessories such as injection pumps and catheters for fluid perfusion.

[0039] Co-culture of intestinal cells with probiotics is the basis for evaluating the function of probiotics in relieving colitis. Since 90% of intestinal probiotics are anaerobic or facultative anaerobic bacteria, a low-oxygen environment is necessary for co-culture of probiotics and intestinal cells. Currently, a cell model is difficult to construct a low-oxygen environment for more than 24 hours by external conditions, thereby limiting its research in the evaluation of specific functions of probiotics. The microfluidic intestinal chip anaerobic culture system provides an effective tool for co-culture of intestinal cells and probiotics. The high-throughput anaerobic intestinal chip system of the present invention provides a low-oxygen environment for the intestinal chip by loading a special anaerobic box with anaerobic bags. At the same time, the flow injection pump continuously perfuses the chip channel with anaerobic medium at a certain speed through the pipeline to ensure the low-oxygen environment inside the intestinal chip. In addition, the oxygen sensor is connected to the pipeline of the perfusion medium, which can realize real-time monitoring of the low-oxygen environment in the microfluidic channel. Through the control of the low-oxygen concentration in the environment and the chip and the real-time monitoring of the sensor, the intestinal chip anaerobic system can ensure the stable co-culture of probiotics and intestinal cells for up to 96h, providing a favorable tool for evaluating the function of probiotics in relieving colitis.

[0040] A high-throughput anaerobic intestinal chip system, comprising a microfluidic chip, a fluid perfusion device, an oxygen detection chip and a micro anaerobic box. The microfluidic chip is provided with intestinal cells and culture medium; the fluid perfusion device comprises a syringe pump, an input pipeline and a recovery pipeline, the syringe pump is connected to the microfluidic chip through the input pipeline, and the recovery pipeline is connected to the downstream of the microfluidic chip, and the fluid perfusion device can continuously perfuse the culture medium into the microfluidic chip; the oxygen monitoring chip is connected to the upstream of the microfluidic chip and can detect the oxygen content in the microfluidic chip; an anaerobic environment is provided by the micro anaerobic box, the micro anaerobic box comprises a box body, anaerobic bags and a heating device, one or more anaerobic bags are arranged in the box body, the heating device can be used for heating the box body, and the microfluidic chip is arranged in the box body.

[0041] The microfluidic chip comprises an upper chip, a middle chip and a lower chip, the upper chip is provided with an upper rhombic chamber and an upper S-shaped chamber, the upper rhombic chamber is used for injecting probiotics; the middle chip is provided with a middle rhombic chamber and a middle S-shaped chamber, the middle rhombic chamber is used for storing the culture medium after culture, the middle chip is arranged between the upper chip and the lower chip, the upper S-shaped chamber and the middle S-shaped chamber are in position correspondence, and the intestinal cells are arranged in the upper S-shaped chamber through a microporous membrane.

[0042] In some embodiments of the present application, a high-throughput anaerobic intestinal chip for screening probiotics is provided, the microfluidic chip mainly comprises an upper chip, a porous filter membrane, a middle layer chip and a lower chip, as shown in Figure 1The upper layer chip is an intestinal cell and probiotic perfusion layer, containing 16 S-shaped cell culture chambers and communicating with 16 rhombic probiotic addition chambers, the 16 channels are connected by single-inlet and single-outlet shunt channels for cell shunting and waste liquid collection. The probiotics are injected into the upper layer rhombic chamber by a microneedle injector. The middle layer chip is a culture medium perfusion layer, both sides contain channels, the channels contacting the upper layer chip are S-shaped cavities, and the channels contacting the bottom layer chip contain rhombic chambers and single-inlet and single-outlet shunt channels, the rhombic chambers are used to collect the culture medium after the intestinal cells and probiotics interact, and the shunt channels are used for shunting and waste liquid collection of the culture medium. The 16 S-shaped cavities of the upper layer and the middle layer chip are separated by a microporous membrane of polycarbonate. The microporous membrane is used for co-culture of cells and probiotics; the lower layer chip serves as a channel-free substrate.

[0043] The rapid proliferation and differentiation of intestinal cells benefit from the continuous supply and constant update of the culture medium, therefore, the upper layer chip and the middle layer chip need to be perfused with culture medium at the same time. The fluid flow direction of the upper layer chip is opposite to that of the lower layer chip. The fluid of the upper layer chip flows through the inlet, shunt channel, rhombic chamber, S-shaped chamber, shunt channel, and outlet, and then the waste liquid is collected by a syringe. The fluid of the middle layer chip channel flows through the inlet, shunt channel, S-shaped chamber, rhombic chamber, shunt channel, and outlet, and then the waste liquid is collected by a syringe.

[0044] In order to ensure the low-oxygen environment for co-culture of probiotics and intestinal cells, the oxygen content in the fluid flowing into the intestinal chip needs to be monitored in real time. Some embodiments of the present application involve an oxygen real-time monitoring chip, as shown in Figure 1 for real-time monitoring of the oxygen content in the culture medium flowing into the upper and middle layer channels of the intestinal chip. The chip includes an upper monitoring chip and a lower monitoring chip, and the lower monitoring chip is provided with inlet and outlet connecting conduits, and the conduits of the inlet and outlet are connected with a syringe and an intestinal chip, respectively. The interface of the upper monitoring chip contacting the outside is provided with a 2cm×2cm square groove for fixing an optical fiber adapter. The center of the groove contains a 5mm diameter micropore, and the interface of the micropore contacting the outside is connected with an optical fiber, and the interface contacting the lower monitoring chip is used for bonding a fluorescence sensing patch. The fluorescence sensing patch is pasted in the channel of the lower monitoring chip, and the culture medium can flow in the channel, and the fluorescence substance contained in the patch can react with the oxygen in the culture medium, thereby affecting the fluorescence intensity, and the signal can be captured in real time by the optical fiber of the upper monitoring chip and the matching detection equipment. In order to ensure the sealing, the optical fiber adapter is bonded with the chip contact interface by sealing glue. Two oxygen sensing chips are respectively arranged, and are connected with the inlet of the upper layer chip and the inlet of the middle layer chip of the intestinal chip through PVDF conduits, so as to realize monitoring of the oxygen content in the culture medium flowing through the upper layer chip and the middle layer chip of the intestinal chip.

[0045] In some embodiments of the present application, Caco-2 and HT29 MTX are selected and co-cultured according to a cell quantity ratio of 9:1 to simulate the human intestinal environment, and a certain amount of LPS is used to stimulate the cells to simulate the colitis microenvironment. The mixed culture of Caco-2 and HT29 MTX is used to construct the intestinal chip, which can more simulate the intestinal microenvironment. The human colon adenocarcinoma cell line Caco-2 is derived from human rectal and colon cancer cells, and has similarities with small intestinal epithelial cells in terms of marker enzyme, morphological function expression, and permeability. Caco-2 cells have become one of the most classic in vitro models for studying drug absorption and metabolism, and are currently the best intestinal transport model and epithelial transport model. However, Caco-2 cells lack the function of secreting mucus and cannot perfectly simulate the barrier function of the small intestine. Compared with Caco-2, HT29-MTX is a goblet cell differentiated from HT29 cells, which can secrete mucus, thereby more realistically simulating the mucus layer absorption barrier function of intestinal cells.

[0046] Caco-2 and HT-29MTX cells are separately cultured and passaged for 2 times, and then mixed according to a total cell amount of 10 6 cell / mL and a cell quantity ratio of 9:1, and then introduced into an intestinal chip special anaerobic box for flow culture. The intestinal model is evaluated by intestinal barrier function related indexes such as transmembrane resistance, permeability, and mucin secretion amount, so as to construct the intestinal model. After the intestinal model is successfully constructed, LPS is introduced to induce the formation of an intestinal inflammation model. LPS is one of the most widely used inflammatory agents for replicating colitis models in vitro. When the LPS signal is transmitted into the cells, it can occur through intracellular signal transmission and cascade reaction, so as to change the gene expression and induce the synthesis and release of various pro-inflammatory factors (such as TNF-α, IL-1, IL-6, etc.). The colitis model is constructed by evaluating the intestinal barrier function related indexes such as transmembrane resistance, mucin secretion amount, and inflammatory factors. After the colitis model is successfully constructed, different strains of probiotics are injected into the rhombic chamber on the upper layer of the chip through a microsyringe, and interact with the intestinal cells in the S-shaped chamber through the flowing culture medium. The colitis-related data of the probiotics and intestinal cells after the interaction are collected as the probiotic intervention group.

[0047] The intestinal chip culture system is combined with machine learning to construct a probiotic function evaluation model. First, the intestinal chip normal group, the colitis group, and the probiotic intervention group are constructed, and the colitis-related genes and protein indexes of the three groups are determined. Then, based on the machine learning algorithm, the colitis-related detection indexes of the three groups are screened and analyzed, and based on the principal component weighting method, a probiotic function evaluation model for relieving colitis is constructed to obtain a comprehensive efficacy score, so as to sort the efficacy of various functionally similar probiotics and screen the probiotic with the optimal efficacy.

[0048] Firstly, the colon inflammation related index data of the three groups of intestinal chips, such as colon inflammation related genes, barrier proteins, inflammatory factors, etc., are collected to construct a multi-dimensional feature data matrix. Then, based on machine learning technology, high-throughput data obtained from the intestinal chip is analyzed. The high-throughput anaerobic intestinal chip system can be used to evaluate the function of probiotics in relieving intestinal inflammation. The specific steps are as follows:

[0049] Step one: Construct a high-throughput anaerobic intestinal chip system, and continuously infuse cell culture medium into the upper chip and the middle chip in the microfluidic chip, wherein the infusion direction of the medium in the upper chip and the middle chip is opposite;

[0050] Step two: inject probiotics into the upper chip, and infuse anaerobic culture medium into the microfluidic chip to construct an intestinal cell scenario intervened by probiotics; after culture, the evaluation indexes are detected;

[0051] Step three: perform steps 1 and 2 on multiple strains to obtain multiple sets of evaluation index data, train an evaluation analysis model by machine learning, and construct a comprehensive efficacy score standard based on principal component weights; specifically including (1) feature correlation analysis, (2) unsupervised principal component analysis, and (3) construction of comprehensive efficacy score based on principal component weights;

[0052] Feature correlation analysis: collect colon inflammation related detection index data of the above groups of intestinal chips (such as colon inflammation related genes, barrier proteins, inflammatory factors, etc.), and construct a multi-dimensional feature data matrix; use the Spearman rank correlation method to analyze the correlation between each feature and obtain a correlation coefficient matrix; then perform Kaiser-Meyer-Olkin (KMO) test and Bartlett's sphericity test on the correlation coefficient matrix of the original data;

[0053] Unsupervised principal component analysis: considering that the intestinal inflammation related detection index data has different dimensions or orders of magnitude, the multi-dimensional detection index is standardized and the correlation coefficient matrix is calculated. Based on the correlation coefficient matrix, the unsupervised machine learning algorithm principal component analysis (PCA) is used to solve the eigenvalues of the correlation coefficient matrix and the corresponding variance contribution rate, cumulative variance contribution rate, and unit orthogonal eigenvector. The cumulative variance contribution rate and Scree plot are used to determine the appropriate number of principal components to realize data dimension reduction;

[0054] Construction of comprehensive efficacy score based on principal component weight: the linear combination of the above selected first m principal components Z1, Z2, …, Zm is used, and the variance contribution rate of each principal component Zm is used as the weight to construct a comprehensive evaluation index, i.e. comprehensive efficacy score Y. m i <000012

[0055] Y = a1Z1 + a2Z2 +... + a m Z m ;

[0056] Step four: compare the differences in efficacy of different probiotics, sort multiple strains according to the comprehensive efficacy, calculate the comprehensive efficacy score (Y value) of each experiment observation, further use variance analysis (ANOVA) and Dunnett's t Tests multiple comparisons to test the differences between different probiotic groups, and sort the comprehensive efficacy score of probiotics, so as to screen out probiotic strains with better efficacy.

[0057] The combination of high-throughput anaerobic enteric chip system and machine learning can grade the efficacy of probiotics in relieving colitis, and can more accurately distinguish the efficacy of functionally similar probiotics, providing a new evaluation platform for high-throughput and accurate evaluation of probiotics, and the evaluation results are more accurate and clear.

[0058] The present application will be described below in conjunction with the drawings, wherein the experimental methods not specifically described in the operation steps are performed according to the corresponding product instructions. The instruments, reagents and consumables used in the examples can be purchased from commercial companies unless otherwise specified.

[0059] Example 1: Construction of high-throughput anaerobic enteric chip system

[0060] 1.1 Chip preparation and assembly

[0061] The enteric chip includes an upper chip, a porous filter membrane, an intermediate layer chip and a lower chip. The upper, middle and lower three-layer chips are prepared by using PDMS based on the flip-mold method. The S channels of the upper and middle layers are separated by a polycarbonate microporous membrane. The multi-layer chip and the polycarbonate microporous membrane are sealed by using a plasma processor. The oxygen monitoring chip is composed of an upper chip, an oxygen sensing fluorescent patch and a lower chip. For example Figure 1The upper chip is the intestinal cell and probiotic perfusion layer, containing 16 S-shaped cell culture chambers connected to 16 diamond-shaped probiotic addition chambers. Each of these 16 channels is connected by a single-inlet, single-outlet shunt channel, which is used for cell diversion and waste collection, respectively. The shunt channel, diamond-shaped chamber, and S-shaped chamber of the upper chip are all located on the same side facing the middle chip. Fluid in the upper chip flows through the inlet, shunt channel, diamond-shaped chamber, S-shaped chamber, shunt channel, and outlet, and waste fluid is then collected by a syringe. The middle chip is the culture medium perfusion layer and consists of interconnected S-shaped and diamond-shaped chambers. The S-shaped chamber is located on the side facing the upper chip, and the diamond-shaped chamber is located on the side facing the lower chip. Both the S-shaped and diamond-shaped chambers have shunt channels at both ends, and the shunt channels at both ends are located on the side facing the lower chip. Fluid in the middle chip's channels flows through an inlet, a shunt channel, an S-shaped chamber, a diamond-shaped chamber, a shunt channel, and an outlet, with waste fluid collected by a syringe. The 16 S-shaped chambers of the upper and middle chips are separated by a polycarbonate microporous membrane. The lower chip serves as a channel-free substrate.

[0062] The upper, middle and lower layer chips were all prepared using PDMS based on the molding method: the curing glue was mixed in a mass ratio of 10:1, centrifuged at 5000rpm for 3 minutes to remove bubbles, poured into a mold, and cured in an 80℃ forced air drying oven for 30 minutes, followed by demolding and punching; the polycarbonate microporous membrane used to load cells was cut into 1mm×0.5mm strips; the upper chip, porous filter membrane, middle chip and lower chip were oxygen activated by a plasma treatment machine for 120s, and then placed in an 80℃ forced air drying oven for 30 minutes before bonding the three chips; the bonded intestinal chip was placed in a 121℃ high pressure sterilizer for 30 minutes, taken out, and dried in a 60℃ drying oven for use.

[0063] The intestinal chip, oxygen monitoring chip and injection pump are connected through a steel needle and rubber hose.

[0064] 1.2 Preparation of anaerobic culture medium

[0065] The anaerobic culture medium includes the following components by volume: 77% DMEM, 20% FBS, 1% double antibody (penicillin-streptomycin), 1% L-glutamine and 1% L-cysteine. After preparation, nitrogen blowing is performed for 15 minutes to deoxygenate.

[0066] 1.3 Construction of Intestine Chip Anaerobic System

[0067] The anaerobic intestinal chip was placed in a miniature anaerobic box with a constant temperature heating platform; the blue-capped bottle storing the upper culture medium was connected to the oxygen sensor chip channel inlet via a 1.5mm rubber hose, the oxygen sensor chip channel outlet was connected to the upper channel inlet of the intestinal chip via a 1.5mm rubber hose, and the upper chip channel outlet was connected to a 20mL syringe via a 1.5mm rubber hose; the blue-capped bottle storing the lower culture medium was connected to the sensor chip channel inlet via a 1.5mm rubber hose, the sensor chip channel outlet was connected to the lower channel inlet of the intestinal chip via a 1.5mm rubber hose, and the lower channel outlet was connected to a 20mL syringe via a 1.5mm rubber hose; the optical fiber of the oxygen sensor was connected to the oxygen sensor host and computer through the opening of the upper cover.

[0068] The anaerobic bags used were Mitsubishi anaerobic gas-producing bags from Japan. The dedicated anaerobic chamber was constructed from thick acrylic board, with four holes on each side for the culture medium pipeline and two small holes on the top for the anaerobic sensor fiber. The oxygen sensor monitors the anaerobic environment of the chip in real time. After connecting the culture medium, oxygen sensor, intestinal chip, and computer, three 25L anaerobic gas-producing bags were placed in the anaerobic chamber and the lid was sealed.

[0069] 1.4 Validation of the High-Throughput Anaerobic Intestine Chip System

[0070] The oxygen sensor is placed in the constructed high-throughput anaerobic intestinal chip system to detect the oxygen content in the chip in real time. The test results are as follows: Figure 2 As shown in the figure, after stabilization for 2 hours, the oxygen content in the chip can be stably lower than 1%, ensuring a good anaerobic environment.

[0071] Example 2: Verification of Intestinal Chip Cell Viability

[0072] 2.1 Seeding and culture of intestinal chip cells

[0073] Preparation of cell culture medium: The cell culture medium was prepared by volume with 77% DMEM, 20% FBS, 1% double antibody and 1% L-glutamine.

[0074] Preparation of gel matrix culture medium: basal collagen concentration is 8.5 mg / mL, collagen I (rat tail collagen type I, Beijing Solebeau Technology Co., Ltd., C8062) original concentration is 2 mg / mL, thaw on ice and dispense into small tubes; aspirate 353 μL basal gel and 250 μL collagen I, and fill up to 10 mL with cell culture medium.

[0075] Preparation of cell suspension: Intestinal cells Caco-2 and HT29MTX frozen in liquid nitrogen were respectively thawed and revived at 37℃; after the Caco2 and HT29MTX cells were subcultured twice and trypsinized, the cells were counted, mixed uniformly in a ratio of 9:1, and adjusted to a cell suspension of 1-5x10 6 cells / mL with gel matrix medium.

[0076] The intestinal chip constructed in Example 1 was taken, and the cell suspension was placed in a 5 mL sterile syringe, and added to the S-shaped channel of the upper chip of the intestinal chip at a speed of 5 μL / s, and the chip with cells was placed in a 37℃ constant temperature anaerobic incubator for static culture for 2 h; then the cell culture medium was continuously flowed in the upper and lower layers.

[0077] 2.2 Cell detection of intestinal chip

[0078] According to the Calcein / PI cell activity and cytotoxicity detection kit instructions (Shanghai Biyun Tian Biotechnology Co., Ltd., C2015M), 1 μL of Calcein AM reagent (staining live cells, green) and 1 μL of Propidium Iodide (staining dead cells, red) were added to 1 mL of detection buffer in the kit, and the staining detection solution was mixed uniformly.

[0079] The cells of the intestinal chip cultured for 1-5 days were taken for live and dead staining detection. The cells in the intestinal chip were washed with PBS, and 1 mL of the prepared staining detection solution was injected into the chip at a speed of 5 μL / s, and incubated in the dark for 30 min, then washed with PBS, and then photographed and analyzed under a upright fluorescence microscope (ECLIPSE Si). The detection results are shown in Figure 3 Fig. 2, the results of live and dead staining of cells for 5 consecutive days showed that the cells maintained high activity within 5 days, and after 5 days of culture, the cells were highly differentiated, and the TEER value and permeability results showed that after 5 days of culture, the intestinal cells had differentiated to form a dense intestinal barrier, which could be used for the construction of a colonitis model.

[0080] Example 3: Interaction of intestinal chip cells with probiotics

[0081] 3.1 Co-culture of intestinal chip cells with probiotics

[0082] 1 μL of CMTPX (red, 10 mM) was taken and added to 1 mL of DMEM to prepare a cell tracer.

[0083] The probiotics frozen in liquid nitrogen were thawed, revived at 37℃, and subcultured twice in an anaerobic workstation for 24 h before use. After the subculture of probiotics 3-4 / 3-12 / 3-13 / 3-14 / LT, they were washed twice with PBS, and stained with a cell tracer (CMTPX, red) for 30 min in the dark, and washed three times with PBS to remove excess dye.

[0084] The concentration of the stained probiotics was adjusted to OD≈0.25 with cell culture medium, and different strains were injected into the upper diamond-shaped chamber with a 1 mL syringe. After 2 h of static culture, anaerobic culture medium (prepared by mixing DMEM, FBS, double-antibiotic, L-glutamine, and L-cysteine at a volume ratio of 77:20:1:1:1, and deoxygenated by nitrogen blowing for 15 min) and cell culture medium (prepared by mixing DMEM and FBS at a volume ratio of 78:20, and adding double-antibiotic and L-glutamine) were introduced at a flow rate of 625 μL / h for 24 h of flow culture.

[0085] After the culture, the non-adherent bacteria were washed away with PBS at a rate of 5 μL / s using a 5 mL syringe. The cell viability was tested by introducing a 0.1% Calcein AM reagent (staining live cells, green) into the cells, followed by 30 min of incubation in the dark, PBS washing, and inverted fluorescence microscopy for image analysis.

[0086] The results are shown in Figure 4 The fluorescence results showed that the red-labeled probiotics could adhere to the cell surface, and most of the cells were green, indicating that the intestinal chip system could be used for co-culture of intestinal cells and probiotics.

[0087] 3.2 Construction of intestinal model

[0088] Intestinal model chips were constructed for the normal group, the colitis group, and the probiotic intervention group. After the assembly of the intestinal chip culture system, the micro-porous membrane was pretreated with the gel matrix culture medium. Then, the intestinal cells Caco-2 and HT29MTX were mixed uniformly and added to the upper chip S-shaped channel at a certain speed. After 2 h of static culture, the upper and lower layers were connected to the flowing culture medium for continuous flow culture. After 5 days, the cells were fully differentiated to form a dense intestinal barrier, serving as the normal group. After 5 days of culture, the cells of the normal group were fully differentiated to form a dense intestinal barrier, the culture medium was removed, and a cell culture medium containing LPS was added for 24 h of flow culture, serving as the colitis group. After 5 days of culture, the intestinal cells of the colitis group were washed twice with PBS, and the probiotics were resuspended in a cell culture medium containing LPS. Different strains were injected into the diamond-shaped chamber of the upper chip of the intestinal chip using a microsyringe, and after 2 h of static culture, anaerobic bacterial culture medium and cell culture medium were introduced for flow culture, serving as the probiotic intervention group.

[0089] Wherein, the probiotic group respectively uses the strain 3-4, 3-12, 3-13, 3-14 and LT to intervene, and different intestinal chips are obtained.

[0090] 3.3 Influence of probiotics on inflammatory factors, barrier proteins and transmembrane resistance of colitis cells

[0091] After culturing the normal group, the colitis group and the probiotic intervention group respectively, the 24h cell culture medium was collected, and the inflammatory factor IL-1β was detected according to the ELISA kit instruction, so as to detect the influence of probiotics on inflammatory factors of colitis cells, and the test results are shown in Tables 1-2.

[0092] After culturing the normal group, the colitis group and the probiotic intervention group respectively, the chips of each group were gently disassembled with scissors, and the porous membranes were taken out and placed in PBS for detecting TEER value. Three porous membranes were collected from each group, and the micro-porous membrane resistance in three different directions was detected by Millicell ERS resistance meter, and the average value was multiplied by the total area of the micro-porous membrane to calculate the intestinal chip cell resistance, so as to detect the influence of probiotics on the resistance of colitis cells, and the results are shown in Tables 1-2.

[0093] After culturing the normal group, the colitis group and the probiotic intervention group respectively, the chips of each group were gently disassembled with scissors, and the porous membranes (3 from each group) were taken out and placed in 4% paraformaldehyde for 10min, and washed with PBS for 3 times; 0.2% Triton-X100 (2μL Triton-X 100 was dissolved in 1mL PBS) was used to break the membrane for 10min, and washed with PBS for 3 times; 5% bovine serum albumin (500mg was dissolved in 10mL PBS) was used for blocking for 1h; the primary antibody (10μL Occludin was dissolved in 5mL 5% bovine serum albumin) was incubated at 4℃ overnight, and washed with PBS for 3 times; the secondary antibody (10μL secondary antibody was dissolved in 10mL PBS) was incubated at room temperature for 1h; the mounting medium containing DAPI was used for mounting, and after drying, image acquisition was performed; Image J was used for image result statistics, and the results are shown in Tables 1-2. The influence of probiotics on barrier proteins of colitis cells was detected. Among them, the C group is the normal group sample, 23 repeated samples are detected, and the average value of each type of protein is shown in Table 1 and Table 2 as C group; the L group is the colitis group sample, 21 repeated samples are detected, and the average value of each type of protein is shown in Table 1 and Table 2 as L group; LT, 3-4, 3-12, 3-13 and 3-14 are different types of probiotic intervention groups, 5 repeated samples are detected for each type, and the average value of each type of protein is shown in Table 1 and Table 2 as specific data.

[0094] Table 1

[0095]

[0096] Table 2

[0097]

[0098] 3.4 Effect of probiotics on the expression of inflammation-related genes in colitis cells

[0099] After the intervention of each group, the chips were gently disassembled with scissors, and the multi-well membranes (3 per group) were placed in 1 mL of TRIZOL for lysis for 10 min; 0.2 mL of chloroform was added, and the mixture was thoroughly shaken and mixed for 30 s on a vortex shaker, and then allowed to stand at room temperature for 3 min; after centrifugation at 12,000 x g at 4°C for 10 min, the upper aqueous phase containing total RNA was transferred to a new centrifuge tube, 0.5 mL of isopropanol was added to the aqueous phase, and the tube was inverted and allowed to stand at room temperature for 10 min; after centrifugation at 12,000 x g at 4°C for 10 min, the liquid was discarded, and a white RNA precipitate was obtained at the bottom of the tube, 1 mL of 75% ethanol was added, and the precipitate was washed by inverting the tube; after centrifugation at 12,000 x g at 4°C for 2 min, the liquid was discarded, the residual ethanol at the bottom of the tube was aspirated, and then the centrifuge tube was left open in a fume hood for 5-10 min. An appropriate amount of sterile DEPC-treated water was added to dissolve the RNA precipitate, and the RNA concentration was determined using a Nanodrop instrument.

[0100] Using cDNA was prepared using a RevertAid reverse transcription kit. The reaction system for reverse transcription was 20 μL:

[0101] Oligo(dT)18 Primer 1 μL

[0102] 5 x Reaction Buffer 4 μL

[0103] RiboLock RNase Inhibitor (20 U / μL) 1 μL

[0104] 10 mM dNTP Mix 2 μL

[0105] RevertAid RT (200 U / μL) 1 μL

[0106] Total RNA 1000 ng

[0107] H2O, nuclease-free 11 μL

[0108] After vortex mixing, the reverse transcription was performed on a PCR instrument, and the reverse transcription program was as follows: 42°C for 60 min, and 70°C for 5 min.

[0109] The cDNA after reverse transcription was diluted 2-fold with sterile DEPC-treated water, mixed by shaking, and stored at -20°C.

[0110] The cDNA obtained by reverse transcription in the above step was used as a template for qRT-PCR. ChamQUniversal SYBR qPCR Master Mix reagent was used and the sample was added according to the following reaction system:

[0111] 5×ChamQ Universal SYBR qPCR Master Mix 4μL

[0112] Upstream primer (10 μM) 0.4 μL

[0113] Downstream primer (10 μM) 0.4 μL

[0114] cDNA 2 μL

[0115] H2O, nuclease-free 13.2μL

[0116] The reaction conditions for qRT-PCR were as follows: pre-denaturation at 95°C for 30 seconds; cycling at 95°C for 10 seconds, 60°C for 30 seconds, 40-45 cycles; and 65-95°C for 5 seconds. The Ct values ​​obtained were used to calculate the transcription level of the target gene. The calculation formula was based on 2 -ΔΔt conduct.

[0117] The results of inflammatory factors, barrier proteins, transmembrane electrical resistance, and inflammation-related genes in the normal group, colitis group, and intervention group are shown in Appendix 1 , which were used as a multidimensional data set for machine learning analysis and scoring model construction.

[0118] Example 4: Probiotic screening method based on machine learning technology

[0119] 4.1 Feature Correlation Analysis Results

[0120] The correlation coefficient matrix of 23 intestinal chip colitis detection indicators is as follows Figure 5 As shown, many of the test indicators are highly correlated, with some overlap in information. A Kaiser-Meyer-Olkin (KMO) test on the correlation coefficient matrix of the raw data showed KMO = 0.743 > 0.7, and Bartlett's test of sphericity showed P < 0.05, indicating that further principal component analysis is suitable.

[0121] 4.2 Unsupervised principal component analysis results

[0122] As shown in Table 3, after PCA analysis, the eigenvectors corresponding to each principal component are obtained. Then the variance contribution rate and cumulative variance contribution rate of each principal component are obtained. The cumulative variance contribution rate of the first 6 principal components reaches 77.15% (>70%), as shown in Table 4 and Figure 6The variance contribution rate and cumulative contribution rate of each principal component are shown, and in combination with the Scree plot result, the first 6 principal components are sufficient to describe the intervention effect of probiotics on colonitis cells.

[0123] Table 3 Characteristic vector corresponding to each principal component

[0124]

[0125]

[0126] Table 4 Eigenvalue, variance contribution rate and cumulative contribution rate of correlation matrix

[0127]

[0128] 4.3 Construction of comprehensive efficacy score result based on principal component weight

[0129] The linear combination of the first 6 principal components Z1, Z2, …, Z6 is used, and the variance contribution rate of each principal component is used as the weight to construct the comprehensive evaluation index Y, that is:

[0130] Y = 0.4332 * Z1 + 0.1064 * Z2 + 0.0792 * Z3 + 0.058 * Z4 + 0.0499 * Z5 + 0.0447 * Z6 4.4 Comparison of efficacy differences of different probiotics

[0131] As shown in Table 4, the variance analysis (ANOVA) and Dunnett's t Tests multiple comparison method are used to test the differences between different probiotic groups and to sort the comprehensive efficacy scores of probiotics. By comparison, among the 5 probiotic strains used, the efficacy of probiotic strains from high to low is in the order of Figure 7

[0132] 3-12 > 3-14 > LT > 3-13 > 3-4. This method can effectively score and sort different probiotic strains.

[0133] The embodiments of the present application are described in detail above, but the content described is only the preferred embodiments of the present application and cannot be considered as limiting the scope of the present application. Any equivalent changes and improvements made in the scope of the present application should still be within the scope of the present application.​​

Claims

1. Application of a high-throughput anaerobic intestinal chip system to evaluate the ability of probiotics to alleviate enteritis, characterized by: The high-throughput anaerobic Intestine Chip system includes: A microfluidic chip having intestinal cells and culture medium therein; the microfluidic chip comprising an upper chip, a middle chip, and a lower chip; the upper chip having an upper rhombus-shaped chamber and an upper S-shaped chamber; the middle chip having a middle rhombus-shaped chamber and a middle S-shaped chamber; the middle chip being disposed between the upper chip and the lower chip; the upper S-shaped chamber and the middle S-shaped chamber corresponding in position and separated by a microporous membrane; the upper rhombus-shaped chamber and the middle rhombus-shaped chamber not communicating with each other; A fluid perfusion device, comprising a syringe pump, an input line, and a recovery line, wherein the syringe pump is connected to the microfluidic chip through the input line, and the recovery line is connected to the downstream of the microfluidic chip; an oxygen monitoring chip connected to the upstream of the microfluidic chip; A micro anaerobic box comprises a box body, an anaerobic bag and a heating device, wherein one or more anaerobic bags are arranged in the box body, the heating device can be used to heat the box body, and the microfluidic chip is arranged in the box body; The steps include: Step 1: Construct a high-throughput anaerobic intestine chip system, inject cells into the upper chip, and then continuously perfuse cell culture medium into the upper and middle chips of the microfluidic chip; Step 2: Inject probiotics into the upper chip and perfuse anaerobic culture medium into the microfluidic chip to construct an intestinal cell scenario with probiotic intervention; after culture, test the evaluation indicators; Step 3: Perform steps 1 and 2 on multiple strains to obtain multiple sets of evaluation index data, train the evaluation and analysis model through machine learning, and construct a comprehensive efficacy scoring standard based on the principal component weights; It includes feature correlation analysis, unsupervised principal component analysis, and construction of a comprehensive efficacy score based on principal component weights; Among them, feature correlation analysis: Based on the evaluation indicators, a multidimensional feature data matrix is ​​constructed; the Spearman rank correlation method is used to perform correlation analysis between each feature and obtain a correlation coefficient matrix; the Kaiser-Meyer-Olkin test and Bartlett sphericity test are then performed on the correlation coefficient matrix of the original data; the evaluation indicators include cytokine detection, barrier protein immunofluorescence detection, and inflammation-related gene expression detection; Unsupervised principal component analysis: Data is standardized for multidimensional detection indicators and a correlation coefficient matrix is ​​calculated. Based on the correlation coefficient matrix, principal component analysis is performed using an unsupervised machine learning algorithm to determine the characteristic roots of the correlation coefficient matrix and the corresponding variance contribution rate, cumulative variance contribution rate, and unit orthogonal eigenvector. The appropriate number of principal components is determined based on the cumulative variance contribution rate and Screeplot to achieve data dimensionality reduction. Step 4: Sort multiple strains according to their comprehensive efficacy.

2. The use according to claim 1, characterized in that: The oxygen monitoring chip includes an upper monitoring chip and a lower monitoring chip. The lower monitoring chip is provided with a culture medium circulation channel and a fluorescent sensor patch that can react with oxygen in the culture medium. The upper monitoring chip is provided with a light collection device that can collect changes in fluorescence intensity on the fluorescent sensor patch. The upper chip and the middle chip are both connected upstream with the oxygen monitoring chip.

3. The use according to claim 1, characterized in that: Intestinal cells and culture medium are injected into the upper chip channel, and culture medium is injected into the middle chip channel.

4. The use according to any one of claims 1 to 3, characterized in that: The intestinal cells are Caco-2 and HT29 MTX mixed and co-cultured in a ratio of 9:

1.

5. The use according to claim 1, characterized in that: The culture medium components include 78% DMEM, 20% FBS, 1% double antibody and 1% glutamine; the anaerobic culture medium components include 77% DMEM, 20% FBS, 1% double antibody, 1% glutamine and 1% L-cysteine.

6. The use according to claim 1, characterized in that: In step three, a normal intestinal model group and a colitis group were also constructed for comparison with the probiotic intervention group.

7. The use according to claim 1, characterized in that: The cytokine assay targets the culture medium collected from the downstream of the microfluidic chip, detecting the inflammatory factor IL-1β; the barrier protein immunofluorescence assay targets Caco-2 / HT29 MTX cells loaded on the porous membrane; Inflammation-related gene expression detection The detection objects were Caco-2 / HT29MTX cells loaded on the porous membrane.

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