In-vitro bionic intestinal barrier injury micro-physiological system

Abstract

The invention discloses an in-vitro bionic intestinal barrier injury micro-physiological system. The device comprises a stomach digestion simulation system, a small intestine digestion simulation system, a small intestine interaction simulation system, a large intestine glycolysis simulation system and a large intestine interaction simulation system. According to the invention, the physicochemical environment for digestion of patients with inflammatory bowel diseases (IBD) is simulated by adjusting parameters such as pH, and the physiological system of the patients with IBD is simulated through human-derived organs and microorganisms. The in-vitro substitution model not only simulates gastrointestinal digestion, absorption and glycolysis processes of a patient with intestinal barrier injury in vitro, but also simulates interaction between food and metabolites thereof and intestinal tracts through introduction of organoids. The model can be used for exploring the metabolic process or toxic effect of food in the body of a patient suffering from intestinal barrier injury, evaluating the bioaccessibility, metabolic fate, interaction with microorganisms and influence on intestinal physiology of the food, and realizing precise risk assessment.

Description

Technical Field

[0001] The invention relates to an in vitro bionic intestinal barrier damage microphysiological system, belonging to the technical field of food. Background Art

[0002] Inflammatory bowel disease (IBD) is a disease that causes chronic inflammation of the intestine. The intestinal barrier function of patients is impaired, manifested as inflammation and ulcers of the intestinal mucosa. This damage not only affects the absorption of nutrients, but also leads to an imbalance of intestinal flora and the invasion of pathogenic microorganisms, further aggravating the inflammatory response. Studies have shown that the integrity of the intestinal barrier plays a key role in the onset and progression of IBD, and protecting and repairing the intestinal barrier function is of great significance for the effective treatment of IBD. The development of an in vitro bionic intestinal barrier damage microphysiological system containing intestinal tissue and microorganisms will help to better study the pathological mechanism of IBD and screen potential therapeutic drugs.

[0003] The human digestive organs are usually divided into digestive glands and digestive tracts including the mouth, esophagus, stomach, small intestine and large intestine, which together constitute the human digestive system. Digestive conditions and digestive environments vary from one digestive organ to another. Since in vivo digestion experiments are limited by high experimental costs, long cycles, poor reproducibility of results, and ethical condemnation, in vitro simulations of the composition of digestive fluids and the digestive environment in the human body are now often used to study the changes of various substances during the digestive process.

[0004] At present, most of the in vitro digestive simulation systems focus on simulating healthy people. There is no in vitro simulation model designed for the physical and chemical environment of gastrointestinal digestion in IBD patients. At the same time, important parameters such as changes in gastrointestinal fluid pH, gastrointestinal fluid secretion rate, and gastrointestinal emptying rate in the model are inconsistent with the actual situation in IBD patients. In addition, most in vitro digestive models related to the simulation of human gastrointestinal digestion and intestinal microecology have failed to simulate the interaction between food and the digestive tract. Summary of the invention

[0005] To solve the above problems, the present invention provides a human in vitro bionic intestinal barrier injury microphysiological system, which simulates the gastrointestinal digestion, absorption, and fermentation processes of patients with intestinal barrier injury in vitro through microfluidic chip technology, and simulates the interaction between food and its metabolites and the intestine through the introduction of organoids. This model can be used to explore the metabolic process or toxic effects of food in patients with intestinal barrier injury, evaluate the bioaccessibility, metabolic fate, interaction with microorganisms, and effects on intestinal physiology of food, and achieve precise risk assessment.

[0006] The first object of the present invention is to provide a digestive system for simulating inflammatory bowel disease damage in vitro, the digestive system comprising: an inflammatory bowel disease organoid chip and an inflammatory bowel disease colon digestion device;

[0007] The method for preparing the inflammatory bowel disease organoid chip is as follows:

[0008] (1) mixing the organoid suspension with the chitosan solution to obtain an organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution;

[0009] (2) mixing the organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-0.1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain the organoid matrix gel;

[0010] (3) implanting the organoid matrix gel into a chip with a chamber to obtain an organoid chip;

[0011] (4) Treating the organoid chip with lipopolysaccharide to obtain an inflammatory bowel disease organoid chip;

[0012] Among them, organoids include small intestinal organoids and colonic organoids;

[0013] The inflammatory bowel disease colon digestion device contains mucosal spheres, and the preparation method of the mucosal spheres is as follows:

[0014] Agar is added into water to dissolve, and mucin is added to obtain a mucosal gel solution; the mucosal gel solution is solidified to obtain mucosal spheres.

[0015] In one embodiment, the concentration of chitosan in the small intestinal organoid-chitosan mixture of step (1) is 60 mg / mL to 100 mg / mL;

[0016] In the sodium alginate-fibronectin solution of step (1), the concentration of sodium alginate is 70 mg / mL to 90 mg / mL, and the concentration of fibronectin is 80 μg / mL to 120 μg / mL.

[0017] In one embodiment, in step (2), the organoid-chitosan mixture and the sodium alginate-fibronectin solution are mixed at a ratio of 0.8 to 1.2: 1 to 1.4;

[0018] In the small intestinal organoid chip of step (3), the organoid concentration is 5 / μL to 20 / μL.

[0019] In one embodiment, in step (4), 200 mg / L lipopolysaccharide (culture medium) is induced for 24 hours to obtain an inflammatory bowel disease organoid chip.

[0020] In one embodiment, the digestate and culture medium can be connected to the organoid chip at a flow rate of 5 μL / min to 15 μL / min and interact for 3 to 24 hours.

[0021] In one embodiment, the small intestinal organoids are intestinal crypt cells of human or murine origin.

[0022] In one embodiment, the chip (and the microwell array of the chip) can be designed by CAD software. The microwell depth of the microwell array of the organoid chip is 1.0 mm, the microwell diameter is 2.0 mm, the middle chamber length is 4.5 mm, the chamber diameter is 1.5 mm, the chamber depth is 0.5-1.5 mm, and the connecting channel size is 100 μm×100 μm×100 μm. Within this size range, the normal growth of organoids can be guaranteed while facilitating fluid replacement operations.

[0023] Optionally, the chip has an elliptical cylindrical chamber with a chamber length of 3 to 4.5 mm, a chamber diameter of 1.5 to 2.5 mm, and a chamber depth (height) of 0.5 to 1.5 mm.

[0024] In one embodiment, the flow rate of digestate and culture medium into the small intestinal organoid chip is 5 μL / min to 15 μL / min.

[0025] In one embodiment, the inflammatory bowel disease colon digestion device comprises: an interconnected large intestine fermentation simulation system, a central intelligent control and display system, an exhaust gas detection system, and an online detection system for bacterial count and short-chain fatty acids;

[0026] Among them, the large intestine fermentation simulation system includes: culture medium feeding bottle, pH control feeding bottle, ascending colon fermentation tank, transverse colon fermentation tank, descending colon fermentation tank, and anaerobic cylinder;

[0027] The culture medium feeding bottle includes a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottle includes a first pH control feeding bottle, a second pH control feeding bottle and a third pH control feeding bottle

[0028] The central intelligent control and display system includes the overall control and display screen, pH control system, temperature control system, stirring rate control system, gas circuit system, and flow rate control system;

[0029] The tail gas detection system includes a tail gas detector and a product collection bottle;

[0030] The online detection system for bacterial count and short-chain fatty acids includes a bacterial count and short-chain fatty acid detector; the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector, a second bacterial count and short-chain fatty acid detector and a third bacterial count and short-chain fatty acid detector.

[0031] In one embodiment, in the large intestine fermentation simulation system, the first culture medium feeding bottle, the large intestine first pH control feeding bottle and the ascending colon fermentation tank are connected by a silicone tube; the second culture medium feeding bottle, the second pH control feeding bottle and the transverse colon fermentation tank are connected by a silicone tube; the third culture medium feeding bottle, the third pH control feeding bottle and the descending colon fermentation tank are connected by a silicone tube;

[0032] The first culture medium feeding bottle, the second culture medium feeding bottle and the third culture medium feeding bottle are connected to the general control and display screen through a communication line, and the first pH control feeding bottle, the second pH control feeding bottle and the third pH control feeding bottle are connected to the general control and display screen through a communication line;

[0033] The anaerobic cylinder is connected to the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank through a silicone tube; the anaerobic cylinder is connected to the main control and display screen through a communication line;

[0034] The tail gas detector is connected to the descending colon fermentation tank and the product collection bottle through a silicone tube; the tail gas detector is connected to the main control and display screen through a communication line;

[0035] The bacteria quantity and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the bacteria quantity and short-chain fatty acid detector is located at the bottom of the transverse colon fermentation tank, and the bacteria quantity and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank; the bacteria quantity and short-chain fatty acid detector is connected to the main control and display screen through a communication line.

[0036] In one embodiment, the usage ratio of agar, mucin and water is 1-3 g: 0.3-0.6 g: 50 mL.

[0037] In one embodiment, the intestinal mucosal spheres have a diameter of 0.3 to 0.7 cm.

[0038] In one embodiment, the intestinal mucosal spheres are located in the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank of the colon digestion simulation device, and the number is 3 to 7.

[0039] In one embodiment, the pH values ​​of the ascending colon fermentation zone, the transverse colon fermentation zone, and the descending colon fermentation zone are 4.6-5.0, 5.0-5.4, and 5.4-5.9, respectively.

[0040] The present invention also provides an in vitro method for simulating the digestion of the small intestine and colon of patients with inflammatory bowel disease damage, wherein the material to be digested (which may be a simulated digest of the small intestine or a simulated digest of the colon) is interacted with an inflammatory bowel disease organoid chip (including an inflammatory bowel disease small intestine organoid chip and an inflammatory bowel disease colon organoid chip), respectively, and reacted through an inflammatory bowel disease colon digestion device; after the reaction, the growth status of the organoid is detected, and the fermentation products of the inflammatory bowel disease colon digestion device are subjected to flora analysis, short-chain fatty acid analysis, etc.

[0041] In one embodiment, 100-200 mL is transferred from the ascending colon fermenter, the transverse colon fermenter and the descending colon fermenter to the next fermenter at a rate of 10-20 mL / min every 6-8 hours, and the colon digestate is fermented for 3-24 hours to obtain the colon digestate.

[0042] In one embodiment, the simulated digest of the small intestine or the simulated digest of the colon enters the pipe connected to the chip hole 1 of the small intestine organoid chip of IBD patients (or the colon organoid chip of IBD patients) through a pressure-driven pump, and the flow rate is 5-15 μL / min; the pipe connected to the chip hole 2 is inserted into the differentiation culture medium, and the flow rate is 5-15 μL / min, that is, the introduction ratio of digestive fluid and culture medium is 1:0.8-1.2; the interaction takes 3-24 hours.

[0043] In one embodiment, simulated gastric digestive fluid is added to a suspension of acetylated starch sample (or ordinary starch) to react to obtain gastric fluid simulated digestion products; the gastric fluid simulated digestion products are added to simulated small intestinal digestive fluid to react to obtain small intestinal simulated digestion products.

[0044] The second object of the present invention is to provide the use of any of the above-mentioned digestive systems in simulating in vitro digestion of patients with inflammatory bowel disease injuries or in screening drugs.

[0045] Beneficial Effects of the Invention

[0046] The present invention provides a human in vitro bionic intestinal barrier injury microphysiological system, which can simulate the gastrointestinal digestion, absorption, and fermentation processes of patients with intestinal barrier injury in vitro, and at the same time simulate the interaction between food and its metabolites and the intestine through the introduction of organoids.

[0047] Specifically:

[0048] (1) The simulation effect of the present invention is consistent with the unique digestive and absorption physiological environment of patients with intestinal barrier damage. By using temperature control and heating systems, stirring, peristaltic pumps, pH meters and other devices, dynamic simulations such as the dynamic secretion of digestive juices and the dynamic changes in gastrointestinal emptying rate during digestion are achieved, and the pH value of the digestive environment can be monitored in real time;

[0049] (2) The present invention uses microfluidic chip technology to accurately control fluid dynamics, oxygen concentration and biofluid flow, simulate the physiological environment of the small intestine and colon, and improve the accuracy of in vitro simulation of food digestion and absorption in patients with intestinal barrier damage;

[0050] (3) The present invention simulates the real dynamic digestion and absorption physiological process in vitro by introducing organoids into the interactive device. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1Schematic diagram of colon simulation fermentation and multi-dimensional online analysis system;

[0052] Figure 2 Schematic diagram of the small intestine organoid chip;

[0053] Figure 3 This is the growth diagram of small intestinal organoids in Example 1 and Comparative Example 1;

[0054] Figure 4 1 is the ZO-1 fluorescence staining image and ZO-1 / DAPI ratio of small intestinal organoids in Example 1 and Comparative Example 1;

[0055] Figure 5 These are muc2 fluorescence staining images and muc2 / DAPI ratios of small intestinal organoids of Example 1 and Comparative Example 1;

[0056] Figure 6 It is a growth diagram of colon organoids of Example 1 and Comparative Example 1;

[0057] Figure 7 It is the ZO-1 fluorescence staining image and ZO-1 / DAPI ratio of colon organoids in Example 1 and Comparative Example 1;

[0058] Figure 8 The muc2 fluorescence staining images and muc2 / DAPI ratios of colon organoids in Example 1 and Comparative Example 1;

[0059] Fig. 9 It is a graph showing the distribution results of short-chain fatty acids in the fermentation liquid of Example 1 and Comparative Example 1 and in mouse feces. DETAILED DESCRIPTION

[0060] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0061] Device:

[0062] Intestinal barrier damage digestive system Figure 1 As shown, it includes interconnected large intestine fermentation simulation system, central intelligent control and display system (pH control system, temperature control system, stirring rate control system, gas path system, flow rate control system), tail gas detection system, bacterial quantity and short-chain fatty acid online detection system;

[0063] The large intestine fermentation simulation system includes culture medium feeding bottles 201-203, pH control feeding bottles 301-303, ascending colon fermentation tank 101, transverse colon fermentation tank 102, descending colon fermentation tank 103, and anaerobic cylinder 601;

[0064] Among them, the feeding bottles include a first culture medium feeding bottle 201, a second culture medium feeding bottle 202 and a third culture medium feeding bottle 203, and the pH control feeding bottles include a first pH control feeding bottle 302, a second pH control feeding bottle 303 and a third pH control feeding bottle 304;

[0065] The central intelligent control and display system includes a general control and display screen 703, a pH control system, a temperature control system, a stirring rate control system, a gas path system, and a flow rate control system.

[0066] The tail gas detection system includes a tail gas detector 501 and a product collection bottle 401 .

[0067] The bacterial count and short-chain fatty acid online detection system includes bacterial count and short-chain fatty acid detectors 801-803;

[0068] Among them, the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector 801, a second bacterial count and short-chain fatty acid detector 802 and a third bacterial count and short-chain fatty acid detector 803.

[0069] In the large intestine fermentation simulation system, the first culture medium feeding bottle 201, the large intestine first pH control feeding bottle 301 and the ascending colon fermentation tank 101 are connected by a silicone tube; the second culture medium feeding bottle 202, the second pH control feeding bottle 302 and the transverse colon fermentation tank 102 are connected by a silicone tube; the third culture medium feeding bottle 203, the third pH control feeding bottle 303 and the descending colon fermentation tank 103 are connected by a silicone tube;

[0070] The first culture medium feeding bottle 201, the second culture medium feeding bottle 202 and the third culture medium feeding bottle 203 are connected to the main control and display screen 703 via a communication line, and the first pH control feeding bottle 301, the second pH control feeding bottle 302 and the third pH control feeding bottle 303 are connected to the main control and display screen 703 via a communication line;

[0071] The anaerobic cylinder 601 is connected to the ascending colon fermentation tank 101, the transverse colon fermentation tank 102, and the descending colon fermentation tank 103 through a silicone tube; the anaerobic cylinder 601 is connected to the general control and display screen 703 through a communication line.

[0072] The tail gas detector 501 is connected to the descending colon fermentation tank 103 and the product collection bottle 401 through a silicone tube; the tail gas detector 501 is connected to the main control and display screen 703 through a communication line.

[0073] The bacterial count and short-chain fatty acid detector 801 is located at the bottom of the ascending colon fermentation tank 101, the bacterial count and short-chain fatty acid detector 802 is located at the bottom of the transverse colon fermentation tank 102, and the bacterial count and short-chain fatty acid detector 803 is located at the bottom of the descending colon fermentation tank 103; the bacterial count and short-chain fatty acid detectors 801-803 are connected to the main control and display screen 703 via a communication line.

[0074] Source of raw materials:

[0075] Corn starch was purchased from Hangzhou Prostar Starch Co., Ltd.;

[0076] The preparation process of acetylated starch is as follows: a certain amount of corn starch is dispersed in deionized water to prepare a 30% (w / w) starch suspension, which is placed on a heating plate and maintained at a temperature of 40°C. The pH of the suspension is adjusted to 8-9 with a 1 mol / L NaOH solution, and then 15% (v / w) acetic anhydride is added dropwise within 0.5 h, and the reaction is continued for 2 h. Finally, the pH of the system is adjusted to 6.5 with a 1 mol / L HCl solution to terminate the reaction. The obtained acylated starch is washed twice with deionized water and ethanol, then dried in an oven at 40°C, ground and sieved through a 100-mesh sieve to obtain a sample;

[0077] Fibronectin was purchased from Thermo Fisher Scientific;

[0078] α-Amylase was purchased from Sigma-Aldrich with an enzyme activity of 50 U / mg;

[0079] Pancreatic enzyme was purchased from Sigma-Aldrich Company with an enzyme activity of 1000 U / mg;

[0080] Glycoamylase was purchased from Sigma-Aldrich with an enzyme activity of 260 U / mL;

[0081] Pepsin was purchased from Merrill, with an enzyme activity of 3000 U / mg;

[0082] Trypsin was purchased from Adamas Life Company with an enzyme activity of 2500 U / mg.

[0083] SHIME medium: 1.0 g / L arabinogalactan, 2.0 g / L pectin, 3.0 g / L starch, 1.0 g / L xylan, 0.5 g / L cysteine, 0.4 g / L glucose, 3.0 g / L yeast extract, 4.0 g / L mucin and 1.0 g / L peptone.

[0084] Experimental methods:

[0085] 1. Small Intestinal Organoid Culture:

[0086] (1) Isolation of intestinal crypts

[0087] Fresh small intestine was obtained from the donor, cold PBS was mixed with human small intestine tissue in a conical tube and washed several times until the supernatant was clear, and the PBS solution needed to be sucked out after each wash; 10 mL of 2 mM EDTA / DPBS buffer was added to a 15 mL conical tube, and digested at 4°C for 20 min, with slight shaking every 5 minutes; after digestion, it was naturally precipitated, EDTA was sucked out, the pipette tip was rinsed with 0.1% BSA / DPBS (w / v), and the colon segment was gently washed three times with 0.1% BSA / DPBS, and the supernatant needed to be gently sucked out each time; the pipette tip was rinsed, the front section was cut off, and the colon segment was rinsed with 0.1% BSA / DPBS to release the crypts; the supernatant was filtered through a 70 μm filter after rinsing; the above steps were repeated three times, the supernatants were combined, centrifuged at 600 r / min for 3 min, and the supernatant was removed to obtain intestinal crypt cells.

[0088] (2) Organoid culture, passaging and differentiation:

[0089] Organoid culture:

[0090] After the matrix gel melts at 4°C, the matrix gel is used to dissolve the crypt precipitate (operated on ice); the number of crypts cultured is 100-150 / 50μL matrix gel, and 55μL matrix gel containing crypts is added to a 24-well plate (55μL matrix gel / well). The 24-well plate is inverted and placed in a 37°C incubator for 10-20 minutes to allow the matrix gel to solidify. After the matrix gel polymerizes, 500μL of growth medium is added to each well.

[0091] Organoid passaging:

[0092] After 5 to 7 days of culture, when the density reaches 15 to 25 organoids per well, organoids are passaged at a ratio of 1:2 to avoid overgrowth of organoids and accumulation of excessive debris in the cavity; rinse the pipette tip with 0.1% BSA / DPBS, place the tissue culture plate on ice to thaw the matrix gel, and scrape the bottom of the well with the pipette tip to remove the matrix gel droplets; transfer the organoids, matrix gel and culture medium to a 1.5mL centrifuge tube, place it on a pre-cooled module, gently pipette up and down the mixture, centrifuge at 300×g at 4°C for 3 minutes, aspirate the upper layer of culture medium and matrix gel, leaving the bottom layer of organoid sediment, resuspend the organoids in the matrix gel and allow the matrix gel to polymerize; after plating, add 500μL of fresh growth medium to each well and continue passage growth.

[0093] Organoid Differentiation:

[0094] In order to carry out the next experiment, the growth medium of some organoids was replaced with differentiation medium after 5 to 7 days of culture. The organoids began to differentiate cells. The medium was replaced every two days and the cells were cultured until maturity to obtain small intestinal organoids.

[0095] Following the same method, fresh colon tissue was obtained from donors to prepare colon organoids.

[0096] The basal culture medium used is one or more of DMEM / F12 or Advanced DMEM / F12 supplemented with 10 mM HEPEs, 2 mM Glutamax, and 100 units / mL Penicillin-streptomycin.

[0097] The growth medium was basal medium supplemented with 0.5nM Wnt3a, B27, N2, 500μM N-acetyl cysteine, 500nM A83-01, 10nM Human-Gastrin, 5nM Prostaglandin E2, 10μM SB 202190, 10mM Nicotinamide, 10μM Y027632 dihydrochloride, 50ng / mL EGF, 500ng / mL R-spondin1, and 100ng / mL Noggin.

[0098] The differentiation medium is a basal medium supplemented with 500 ng / mL R-spondin1, 100 ng / mL Noggin, B27, N2, 500 μM N-acetyl cysteine, 500 μM N-acetyl cysteine, 500 nM A83-01, 50 ng / mL EGF, and 10 nM Human-Gastrin.

[0099] 2. Construction of IBD mouse model

[0100] SPF male C57BL / 6J mice drank 3% (w / w) DSS water for DSS modeling for 7 days. The mice were monitored every day, their weight was recorded, the feces of the mice were observed, and the rectal bleeding was measured using a fecal blood concealment kit to confirm the typical symptoms of colitis such as weight loss, diarrhea, and blood in the stool. After the modeling was successful, the mice were fasted but not watered for 20 hours to digest or empty the contents of the gastrointestinal tract as much as possible. The starch sample to be tested was prepared into a suspension of a certain concentration (2.5g / mL), mixed, and the desired dose (0.4mL) was drawn through a syringe for gavage on the mice. Fresh feces of the mice were collected for the detection of short-chain fatty acids.

[0101] 3. Preparation of digestive juice for IBD patients

[0102] Preparation of simulated gastric digestive fluid (SGF): SGF was prepared with water as solvent, containing 2000 U / L of pepsin, 6.9 mM potassium chloride, 0.9 mM potassium dihydrogen phosphate, 12.5 mM sodium bicarbonate, 11.8 mM sodium chloride, 0.4 mM magnesium chloride, 0.5 mM ammonium carbonate, and 0.005 mM calcium chloride, and the pH value was adjusted to 3.0 with hydrochloric acid;

[0103] Preparation of simulated intestinal digestive fluid (SIF): SIF was prepared with water as solvent, containing 4000U / L trypsin, 1200U / L saccharifying enzyme, 6.8mM potassium chloride, 0.8mM potassium dihydrogen phosphate, 42.5mM sodium bicarbonate, 1.1mM magnesium chloride, 9.6mM sodium chloride, 0.044mM calcium chloride, and the pH was adjusted to 5.0-5.5 with hydrochloric acid (the pH within this range had no effect on the experimental results);

[0104] Preparation of simulated gastric digest: 60 mL of simulated gastric digestive fluid (SGF, pH 3.0) was added to 200 mL of acetylated starch sample suspension (10%, m / v). The food liquid was digested at 37°C and 30 rpm. After 1 hour of reaction, simulated gastric digest was obtained.

[0105] Preparation of simulated small intestinal digest: 20 mL of gastric juice simulated digestion product was added to 18 mL of simulated small intestinal digestive fluid (SIF, pH 5.0-5.5), and digested at 37°C for 2 h at a rotation speed of 30 rpm to obtain 38 mL of simulated small intestinal digest.

[0106] Test method:

[0107] 1. Immunofluorescence experiment

[0108] Aspirate the medium from the wells and wash the Matrigel with 1 mL PBS without disrupting the 3D matrix. Add 1 mL of cold cell recovery solution to each well and incubate at 4°C for 30 min. After incubation, the Matrigel is dissolved and dispersed. Use a pipette tip rinsed with 0.1% BSA in cold PBS to handle subsequent organoids to avoid organoids adsorbing to the pipette tip. Wash the organoids twice with 0.1% BSA in cold PBS and then resuspend in 4% paraformaldehyde for 30 min. Wash twice more with organoid wash buffer (1 mL Triton X-100 and 2 g BSA in 1 L PBS) and incubate at 4°C for 30 min for the third time to achieve permeabilization. Aspirate the organoid wash buffer and add 200 μL of organoid wash buffer containing primary antibody to each well and incubate overnight at 4°C with gentle shaking. The ZO-1 primary antibody dilution ratio (v / v) is 1:50. Wash the organoids 5 times with organoid wash buffer for 5 min each. Add 200 μL of fluorescently labeled secondary antibody and incubate at room temperature for 2 h. Subsequently, wash the organoids 5 times with organoid wash buffer for 5 min each time. Counterstain the nuclei and live cell nuclei with DAPI and PI, scan layer by layer with a laser confocal microscope, and stitch and reconstruct the images.

[0109] 2. Determination of short-chain fatty acids (SCFAs)

[0110] Take 25 mg of the sample to be tested in a 2 mL EP tube, add pure water and vortex evenly, centrifuge at 4 ° C for 20 min (5000 r / min). Take 0.8 mL of the supernatant in a 2 mL EP tube, add 0.1 mL of H2SO4 solution (50%) and 0.8 mL of the extract, centrifuge for 15 min (4 ° C, 10000 r / min) and let stand at -20 ° C for 30 min. Finally, take the supernatant in an injection bottle and detect it by gas chromatography-mass spectrometry (GC-MS). Chromatographic conditions: chromatographic column: Agilent HP-FFAP capillary column (30m×250μm×0.25μm); temperature program: 80 ° C for 1 min, increase to 200 ° C at 10 ° C / min, maintain for 5 min, increase to 240 ° C at 40 ° C / min, maintain for 1 min; carrier gas (He) flow rate 3 mL / min, injection volume 1 μL; split ratio: 5:1. Mass spectrometry conditions: ionization voltage -70 eV; transfer line temperature 240 °C; ion source temperature 200 °C; quadrupole temperature 150 °C; mass scan range m / z 33-150.

[0111] Example 1: In vitro intestinal barrier damage model

[0112] 1. Preparation of polydimethylsiloxane (PDMS) chip

[0113] The microwell array of the chip was designed using CAD software. The microwell depth of the microwell array of the organoid chip is 1.0 mm, the microwell diameter is 2.0 mm, the middle chamber length is 4.5 mm, the chamber diameter is 1.5 mm, the chamber depth is 1 mm, and the connecting channel size is 100 μm × 100 μm × 100 μm. Within this size range, the normal growth of the organoid can be ensured while facilitating the liquid replacement operation. Figure 2 shown.

[0114] 2. Organoid colonization

[0115] The steps for preparing artificial matrix gel and implanting organoids by double-layer cross-linking method are as follows:

[0116] (1) Take the organoids of mature small intestine and resuspend them in DPBS solution (PBS solution without calcium ions) to obtain 20 / μL organoid suspension; mix the chitosan solution and the intestinal organoid suspension at a ratio of 1:1 (v / v), the final concentration of the chitosan solution is 8% (w / v), and the concentration of the intestinal organoids is 10 / μL, so that the organoids are evenly distributed in the solution to obtain an intestinal organoid-chitosan mixture; prepare an 8% (w / v) sodium alginate solution, add fibronectin to 100 μg / mL, fully dissolve and mix, and obtain a sodium alginate-fibronectin solution;

[0117] (2) Mix the sodium alginate-fibronectin solution and the intestinal organoid-chitosan mixture in a ratio of 1:1 (V / V); add an excess of 0.1M calcium chloride solution (just enough to cover) for 10 minutes of preliminary cross-linking to form a soft gel in the inner layer, and absorb the calcium chloride solution; then add an excess of 0.2M calcium chloride solution for 5 minutes of secondary cross-linking to further enhance the hardness of the outer layer; wash the matrix gel three times with sterile water, and implant it into the culture cavity of the chip, implant 20 organoids / chip, continue to culture for 5 days, replace the growth medium with differentiation medium, and obtain a small intestinal organoid chip. After 5 days of differentiation culture, a complete small intestinal organoid is formed;

[0118] (3) The small intestinal organoid chip was induced with 200 ng / mL lipopolysaccharide solution (flow rate 10 μL / min) and organoid culture medium (flow rate 10 μL / min) for 24 h to simulate the physiological environment of the small intestine of patients with intestinal barrier damage, thus constructing the small intestinal organoid chip of IBD patients;

[0119] The same method was used to prepare the colon organoid chip of IBD patients.

[0120] 3. Collection and inoculation of bacterial flora in IBD patients

[0121] Feces were collected from 3 to 5 IBD patients who had not taken antibiotics for 6 months. 50 g of feces were sampled and mixed, and a phosphate buffer solution with pH 7.0 was added at a ratio of 1:10 (g:mL), and 1 g of sodium thioacetate was added as a reducing agent. The sample was homogenized using a homogenizer;

[0122] After homogenization, the sample was centrifuged at 3000 rpm for 5 minutes. After centrifugation, 50 mL, 80 mL and 50 mL of intestinal microbial culture were inoculated into the ascending colon fermentation tank (101), transverse colon fermentation tank (102) and descending colon fermentation tank (103) in the large intestine fermentation simulation system, respectively. Then, 5 mucosal spheres were added to the three reactors, and then 500 mL, 800 mL and 500 mL of SHIME culture medium were added respectively. The mixture was stirred at 30 rpm for 24 hours at 37°C, and anaerobic gas (5% hydrogen, 10% carbon dioxide, 85% nitrogen) was passed for 10 minutes every 8 hours. After stabilization, the pH values ​​of the three reactors were controlled within the ranges of 4.6-5.0, 5.0-5.4 and 5.4-5.9, respectively (different pH values ​​within the ranges will not affect the results). The magnetic stirring device was turned on at a speed of 30 rpm to simulate fermentation at 37°C.

[0123] After the first inoculation and cultivation for 24 hours, in order to maintain the normal growth of microorganisms, nutrients were added and 300 mL of culture medium was discharged daily to maintain the constant volume, and the mucosal pellets in the tank were replaced with 3 new mucosal pellets to simulate mucosal regeneration. After the inoculation of fecal intestinal flora, continuous fermentation for 7 days tended to be stable, and part of the fermentation liquid was stored in a -80℃ refrigerator for flora determination.

[0124] The preparation method of the above-mentioned mucosal beads is as follows: weigh 2g of agar and heat it to 100°C in 50mL of distilled water to dissolve it. After the solution becomes clear and transparent, cool it to 60°C and add 0.5g of mucin. After it is fully dissolved, adjust the pH of the solution to 8 to obtain a mucosal gel solution; add the mucosal gel solution into a mold to solidify it into a small ball with a diameter of 0.5cm, and sterilize it with ultraviolet light in a clean bench for 20 minutes.

[0125] 4. Simulate in vitro digestion and fermentation in patients with intestinal barrier damage

[0126] 15 mL of simulated small intestinal digest (pH 5.0-5.5, pH within the range does not affect the experimental results) was taken into the pipe connected to the chip hole 1 of the small intestinal organoid chip of the small intestinal IBD patient through a pressure-driven pump, with a flow rate of 10 μL / min; the pipe connected to the chip hole 2 was inserted into the differentiation medium, with a flow rate of 10 μL / min, that is, the ratio of digestive fluid to culture medium was 1:1; the chip hole 3 discharged the waste liquid at a flow rate of 20 μL / min, and the organoid chip was placed in the environmental culture chamber in the cell culture instrument (carbon dioxide concentration was 5%, temperature was 37°C), and the small intestinal organoid chip of the small intestinal IBD patient after 24 hours of interaction was obtained;

[0127] The remaining small intestine simulated digest (23 mL) was passed into the ascending colon fermenter of the inflammatory bowel disease colon digestion device. 100 mL was transferred from the ascending colon fermenter, transverse colon fermenter, and descending colon fermenter to the next fermenter at a rate of 20 mL / min every 8 hours. The colon digest was fermented for 24 hours to obtain the colon digest;

[0128] 15 mL of colon digesta was taken and entered into the pipe connected to chip hole 1 of the colon organoid chip of the small intestine IBD patient through a pressure-driven pump, with a flow rate of 10 μL / min; the pipe connected to chip hole 2 was inserted into the differentiation medium, with a flow rate of 10 μL / min, that is, the ratio of digestive fluid and culture medium was 1:1; chip hole 3 discharged the waste liquid with a flow rate of 20 μL / min, and the organoid chip was placed in the environmental culture chamber in the cell culture instrument (carbon dioxide concentration was 5%, temperature was 37°C), and the small intestine organoid chip of the colon IBD patient after 24 hours of interaction was obtained.

[0129] Comparative Example 1: Simulating healthy digestion in people

[0130] On the basis of Example 1, the lipopolysaccharide-induced IBD model in step 2 was omitted; the stool source in step 3 was changed to healthy adults who had not taken antibiotics for 6 months; the pH values ​​of the ascending colon, transverse colon, and descending colon fermentation zones in step 3 were changed to 5.6-6.0, 6.0-6.4, and 6.4-6.9, respectively (different pH values ​​within the range would not affect the results); the pH value of the simulated small intestinal digestive fluid in step 4 was changed to 6.8; the remaining steps were consistent with the examples, and small intestinal organoid chips treated with digestive fluid from healthy people and colon organoid chips treated with digestive fluid from healthy people were obtained, respectively.

[0131] The colon digesta obtained in Example 1 and Comparative Example 1, the small intestinal organoid chip of the small intestine IBD patient after interaction, and the colon organoid chip of the small intestinal IBD patient after interaction were used for subsequent testing.

[0132] (1) Intestinal damage

[0133] The organoid chips obtained in Example 1, which simulate the digestion of small intestine damage in patients with IBD in vitro, and the organoid chips obtained in Example 1, which simulate the digestion of colon damage in patients with IBD in vitro, and the small intestine organoid chips treated with digestive fluid from healthy people and the colon organoid chips treated with digestive fluid from healthy people, were used to detect the growth of the organoids in the chips. Figure 3 As shown, the growth of colon organoids Figure 6 The results showed that the organoids of the example were significantly damaged, and more proliferating cells were observed in the organoids of the control group, which were not observed in the example.

[0134] The above organoid chips and mouse intestines were fluorescently stained for tight junction protein ZO-1 and mucin Muc-2. The results of small intestinal organoid fluorescent staining are shown in Figure 2. Figure 4 , Figure 5 The results of fluorescence staining of tight junction protein ZO-1 and mucin Muc-2 in colon organoids are shown in Figure 7 , Figure 8 shown.

[0135] The average fluorescence intensity ratio of ZO-1 and DAPI can reflect the tight junction of cells, and the average fluorescence intensity ratio of Muc-2 and DAPI can reflect the stability of the overall structure of the organoid. The higher the ratio, the more stable the overall structure of the organoid. The results show that the tight junction and structural stability of the organoid chip in Example 1 are poor, which is closer to intestinal barrier damage in vivo.

[0136] (2) Short-chain fatty acids

[0137] The short-chain fatty acid content in Example 1, Comparative Example 1 and mouse intestinal digesta was analyzed by a short-chain fatty acid online detection system. The results are as follows: Fig. 9 shown.

[0138] The results show that the effect of simulating intestinal damage in vitro in Example 1 is close to that of IBD mice, and the data are very different. It can be seen that the effect of the organoid chip in Example 1 in simulating intestinal damage in vitro is closer to the actual situation.

[0139] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An in vitro digestive system simulating inflammatory bowel disease, characterized in that: The digestive system includes: an inflammatory bowel disease organoid chip and an inflammatory bowel disease colon digestion device; The method for preparing the inflammatory bowel disease organoid chip is as follows: (1) mixing the organoid suspension with the chitosan solution to obtain an organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution; (2) mixing the organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-0.1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain the organoid matrix gel; (3) implanting the organoid matrix gel into a chip with a chamber to obtain an organoid chip; (4) Treating the organoid chip with lipopolysaccharide to obtain an inflammatory bowel disease organoid chip; Among them, organoids include small intestinal organoids and colonic organoids; The inflammatory bowel disease colon digestion device contains mucosal spheres, and the preparation method of the mucosal spheres is as follows: Agar is added into water to dissolve, and mucin is added to obtain a mucosal gel solution; the mucosal gel solution is solidified to obtain mucosal spheres.

2. The digestive system according to claim 1, characterized in that The concentration of chitosan in the small intestinal organoid-chitosan mixture of step (1) is 60 mg / mL to 100 mg / mL; In the sodium alginate-fibronectin solution of step (1), the concentration of sodium alginate is 70 mg / mL to 90 mg / mL, and the concentration of fibronectin is 80 μg / mL to 120 μg / mL.

3. The digestive system according to claim 1, characterized in that In step (2), the organoid-chitosan mixture and the sodium alginate-fibronectin solution are mixed in a ratio of 0.8 to 1.2: 1 to 1.

4.

4. The digestive system according to claim 1, characterized in that In the organoid chip of step (3), the concentration of organoids is 5 / μL to 20 / μL.

5. The digestive system according to claim 1, characterized in that The inflammatory bowel disease colon digestion device includes: an interconnected large intestine fermentation simulation system, a central intelligent control and display system, an exhaust gas detection system, and an online detection system for bacterial count and short-chain fatty acids; Among them, the large intestine fermentation simulation system includes: culture medium feeding bottle, pH control feeding bottle, ascending colon fermentation tank, transverse colon fermentation tank, descending colon fermentation tank, and anaerobic cylinder; The culture medium feeding bottle includes a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottle includes a first pH control feeding bottle, a second pH control feeding bottle and a third pH control feeding bottle; The central intelligent control and display system includes the overall control and display screen, pH control system, temperature control system, stirring rate control system, gas circuit system, and flow rate control system; The tail gas detection system includes a tail gas detector and a product collection bottle; The online detection system for bacterial count and short-chain fatty acids includes a bacterial count and short-chain fatty acid detector; the bacterial count and short-chain fatty acid detector includes a first bacterial count and short-chain fatty acid detector, a second bacterial count and short-chain fatty acid detector and a third bacterial count and short-chain fatty acid detector.

6. The digestive system according to claim 2, characterized in that In the large intestine fermentation simulation system, the first culture medium feeding bottle, the large intestine first pH control feeding bottle and the ascending colon fermentation tank are connected by a silicone tube; the second culture medium feeding bottle, the second pH control feeding bottle and the transverse colon fermentation tank are connected by a silicone tube; the third culture medium feeding bottle, the third pH control feeding bottle and the descending colon fermentation tank are connected by a silicone tube; The first culture medium feeding bottle, the second culture medium feeding bottle and the third culture medium feeding bottle are connected to the general control and display screen through a communication line, and the first pH control feeding bottle, the second pH control feeding bottle and the third pH control feeding bottle are connected to the general control and display screen through a communication line; The anaerobic cylinder is connected to the ascending colon fermentation tank, the transverse colon fermentation tank, and the descending colon fermentation tank through a silicone tube; the anaerobic cylinder is connected to the main control and display screen through a communication line; The tail gas detector is connected to the descending colon fermentation tank and the product collection bottle through a silicone tube; the tail gas detector is connected to the main control and display screen through a communication line; The bacteria quantity and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the bacteria quantity and short-chain fatty acid detector is located at the bottom of the transverse colon fermentation tank, and the bacteria quantity and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank; the bacteria quantity and short-chain fatty acid detector is connected to the main control and display screen through a communication line.

7. The digestive system according to claim 1, characterized in that The dosage ratio of agar, mucin and water is 1-3 g: 0.3-0.6 g: 50 mL; the diameter of the intestinal mucosal sphere is 0.3-0.7 cm.

8. The digestive system according to claim 1, characterized in that The intestinal mucosal spheres are located in the ascending colon fermentation tank, the transverse colon fermentation tank and the descending colon fermentation tank of the colon digestion simulation device, and the number of the spheres is 3 to 7.

9. The digestive system according to claim 1, characterized in that The pH values ​​of the ascending colon, transverse colon, and descending colon fermentation zones were 4.6-5.0, 5.0-5.4, and 5.4-5.9, respectively.

10. Use of the digestive system according to any one of claims 1 to 9 in simulating in vitro digestion of patients with inflammatory bowel disease injury or in screening drugs.

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