In-vitro bionic intestinal barrier injury microphysiological system

Abstract

The application discloses an in-vitro bionic intestinal barrier damage microphysiological system, which comprises a stomach digestion simulation system, a small intestinal digestion simulation system, a small intestinal interaction simulation system, a large intestinal fermentation simulation system and a large intestinal interaction simulation system. The application simulates the physicochemical environment of the digestion of an inflammatory bowel disease (IBD) patient by adjusting parameters such as pH, and simulates the physiological system of the IBD patient by using human organoids and microorganisms. The in-vitro substitute model not only simulates the digestion, absorption and fermentation process of the gastrointestinal tract of an intestinal barrier damage patient, but also simulates the interaction between food and its metabolites and the intestinal tract by introducing organoids. The model can be used to explore the metabolism or toxic effect of food in the body of an intestinal barrier damage patient, evaluate the bioaccessibility, metabolic fate, interaction with microorganisms and influence on the intestinal tract physiology of food, and realize 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. Patients suffer from impaired intestinal barrier function, which manifests as inflammation and ulcers of the intestinal mucosa. This damage not only affects the absorption of nutrients, but also leads to an imbalance in the 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. Protecting and repairing intestinal barrier function is of great significance for the effective treatment of IBD. The development of an in vitro biomimetic intestinal barrier damage microphysiological system containing intestinal tissue and microorganisms will help to better study the pathological mechanisms of IBD and screen potential therapeutic drugs.

[0003] The human digestive system is generally divided into the digestive glands and the digestive tract, which includes the mouth, esophagus, stomach, small intestine, and large intestine. Digestive conditions and environments vary depending on the digestive organ. Because in vivo digestion experiments are limited by high costs, long cycles, poor reproducibility, and ethical concerns, in vitro methods are now often used to simulate the composition of human digestive fluids and the digestive environment to study the changes in various substances during digestion.

[0004] Current in vitro digestive simulation systems mostly 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. Moreover, most in vitro digestion 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 address these issues, the present invention provides an in vitro biomimetic microphysiological system for intestinal barrier damage. This system uses microfluidic chip technology to simulate the digestion, absorption, and fermentation processes of the gastrointestinal tract in patients with intestinal barrier damage. Furthermore, through the introduction of organoids, it simulates the interaction between food and its metabolites and the intestine. This model can be used to investigate the metabolic processes and toxic effects of foods in patients with intestinal barrier damage, assess their bioaccessibility, metabolic fate, interactions with microorganisms, and impact on intestinal physiology, and enable 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 organ-on-chip is as follows:

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

[0009] (2) The organoid-chitosan mixture and the sodium alginate-fibronectin solution were mixed, and 0.05-0.1 M calcium chloride solution was added for preliminary cross-linking for 5-15 min, and then 0.1-0.3 M calcium chloride solution was added for secondary cross-linking for 5-10 min, and then washed 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 and dissolved, and mucin is added to obtain a mucosal gel solution; the mucosal gel solution is solidified to obtain mucosal pellets.

[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 0.8-1.2:1-1.4;

[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 organ-on-a-chip.

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

[0021] In one embodiment, the small intestinal organoids are derived from 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 the organoid can be ensured while facilitating the fluid exchange operation.

[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 the 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 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 main control and display screen via 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 main control and display screen via 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 bacterial count and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the bacterial count and short-chain fatty acid detector is located at the bottom of the transverse colon fermentation tank, and the bacterial count and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank; the bacterial count 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 pellet has a diameter of 0.3 to 0.7 cm.

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

[0039] In one embodiment, the pH values ​​of the ascending colon, transverse colon, and descending colon glycolysis zones 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 digested material (which can be a simulated small intestine digestive material or a simulated colon digestive material) is interacted with an inflammatory bowel disease organ-on-chip (including an inflammatory bowel disease small intestine organ-on-chip and an inflammatory bowel disease colon organ-on-chip), 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 bacterial flora analysis, short-chain fatty acid analysis, etc.

[0041] In one embodiment, 100-200 mL of liquid 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.

[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, with a flow rate of 5 to 15 μL / min; the pipe connected to the chip hole 2 is inserted into the differentiation culture medium, with a flow rate of 5 to 15 μL / min, that is, the ratio of the digestive fluid and the culture medium is 1:0.8 to 1.2; the interaction lasts for 3 to 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 a 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 present invention

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

[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 devices such as a temperature control heating system, stirring, a peristaltic pump, and a pH meter, the dynamics of the secretion of digestive fluid and the dynamic changes in gastrointestinal emptying rate during digestion are simulated, and the pH value of the digestive environment can be monitored in real time.

[0049] (2) The present invention uses microfluidic chip technology to precisely control fluid dynamics, oxygen concentration, and biofluid flow, simulating the physiological environment of the small intestine and colon, and improving 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 the colon simulated fermentation and multi-dimensional online analysis system;

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

[0053] Figure 3 This is a diagram of the growth 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 the muc2 fluorescence staining images and muc2 / DAPI ratios of the small intestinal organoids of Example 1 and Comparative Example 1;

[0056] Figure 6 1 is a graph showing the growth of colon organoids in Example 1 and Comparative Example 1;

[0057] Figure 7 The ZO-1 fluorescence staining images and ZO-1 / DAPI ratios of colon organoids in Example 1 and Comparative Example 1 are shown;

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

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

[0060] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended 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 system, flow rate control system), tail gas detection system, bacterial count 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; 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 via silicone tubes; the anaerobic cylinder 601 is connected to the main control and display screen 703 via 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 1mol / L NaOH solution, and then 15% (v / w) acetic anhydride is added dropwise within 0.5h, and the reaction is continued for 2h. Finally, the pH of the system is adjusted to 6.5 with a 1mol / L HCl solution to terminate the reaction. The obtained acylated starch is washed twice with deionized water and ethanol, and then dried in a 40°C oven. The sample is ground and passed through a 100-mesh sieve;

[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 with an enzyme activity of 1000 U / mg;

[0080] Glucoamylase 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, 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 a donor. Cold PBS was mixed with human small intestinal tissue in a conical tube and washed several times until the supernatant was clear. The PBS solution needed to be aspirated 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 minutes, with gentle shaking every 5 minutes. After digestion, the tube was allowed to precipitate naturally, the EDTA was aspirated, 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, with the supernatant gently aspirated 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 rpm for 3 minutes, and the supernatant was removed to obtain intestinal crypt cells.

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

[0089] Organoid culture:

[0090] After Matrigel has melted at 4°C, dissolve the crypt pellet in Matrigel (perform on ice). Crypts are cultured at a concentration of 100–150 per 50 μL of Matrigel. Add 55 μL of Matrigel containing crypts to a 24-well plate (55 μL / well). Invert the plate and place it in a 37°C incubator for 10–20 minutes to allow the Matrigel to solidify. After the Matrigel has polymerized, add 500 μL of growth medium 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 organoid pellet, resuspend the organoids in 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 undergo cell differentiation. 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 0.5 nM Wnt3a, B27, N2, 500 μM N-acetyl cysteine, 500 nM A83-01, 10 nM Human-Gastrin, 5 nM Prostaglandin E2, 10 μM SB 202190, 10 mM Nicotinamide, 10 μM Y027632 dihydrochloride, 50 ng / mL EGF, 500 ng / mL R-spondin 1, and 100 ng / mL Noggin.

[0098] Differentiation medium is a basal medium supplemented with 500 ng / mL R-spondin 1, 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 were fed 3% (w / w) DSS water for 7 days to establish a DSS model. The mice were monitored daily, their weights were recorded, their feces were observed, and rectal bleeding was measured using a fecal blood occultation kit to confirm typical symptoms of colitis, such as weight loss, diarrhea, and bloody stools. After successful modeling, the mice were fasted for 20 hours but not water to allow the original contents of the gastrointestinal tract to be digested or emptied as much as possible. The starch sample to be tested was prepared into a suspension of a certain concentration (2.5g / mL). After mixing, the desired dose (0.4mL) was drawn through a syringe and gavage was performed 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 using water as the solvent, containing 2000 U / L 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. The pH was adjusted to 3.0 with hydrochloric acid.

[0103] Preparation of simulated intestinal fluid (SIF): SIF was prepared using water as the solvent and contained 4000 U / L trypsin, 1200 U / L saccharifying enzyme, 6.8 mM potassium chloride, 0.8 mM potassium dihydrogen phosphate, 42.5 mM sodium bicarbonate, 1.1 mM magnesium chloride, 9.6 mM sodium chloride, and 0.044 mM calcium chloride. 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 for 1 hour to obtain simulated gastric digest.

[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 of PBS without disrupting the 3D matrix. Add 1 mL of cold cell recovery solution to each well and incubate at 4°C for 30 minutes. After incubation, the Matrigel dissolves and disperses. Use pipette tips rinsed with cold 0.1% BSA in PBS to handle subsequent organoids to prevent them from adhering to the pipette tips. Wash the organoids twice with cold 0.1% BSA in PBS and then resuspend in 4% paraformaldehyde for 30 minutes. 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 minutes a third time to permeabilize. Aspirate the organoid wash buffer and add 200 μL of primary antibody in organoid wash buffer to each well. Incubate overnight at 4°C with gentle shaking. Use a 1:50 dilution (v / v) of ZO-1 primary antibody. Wash the organoids five times with organoid wash buffer for 5 minutes each. Add 200 μL of fluorescently labeled secondary antibody and incubate with shaking at room temperature for 2 hours. Subsequently, wash the organoids five times with organoid wash buffer for 5 minutes each. Counterstain nuclei and live cell nuclei with DAPI and PI. Scan each layer with a laser confocal microscope and reconstruct the images.

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

[0110] 25 mg of the sample to be tested was placed in a 2 mL EP tube, purified water was added, and the mixture was vortexed and centrifuged at 4°C for 20 min (5000 rpm). 0.8 mL of the supernatant was then transferred to a 2 mL EP tube, 0.1 mL of 50% H₂SO₄ solution and 0.8 mL of the extract were added. The tube was centrifuged for 15 min (4°C, 10,000 rpm), and then allowed to stand at -20°C for 30 min. Finally, the supernatant was transferred to an injection vial and analyzed by gas chromatography-mass spectrometry (GC-MS). Chromatographic conditions: Column: Agilent HP-FFAP capillary column (30 m × 250 μm × 0.25 μm); Temperature program: 80°C for 1 min, then increase at 10°C / min to 200°C, hold for 5 min, then increase at 40°C / min to 240°C, hold 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 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 colonizing organoids using the double-layer cross-linking method are as follows:

[0116] (1) Organoids of mature small intestine were taken and resuspended in DPBS solution (PBS solution without calcium ions) to obtain 20 / μL organoid suspension; chitosan solution and intestinal organoid suspension were mixed at a ratio of 1:1 (v / v), with the final concentration of chitosan solution being 8% (w / v) and the concentration of intestinal organoids being 10 / μL, so that the organoids were evenly distributed in the solution to obtain an intestinal organoid-chitosan mixture; 8% (w / v) sodium alginate solution was prepared, and fibronectin was added to 100 μg / mL, fully dissolved and mixed to obtain a sodium alginate-fibronectin solution;

[0117] (2) Sodium alginate-fibronectin solution and intestinal organoid-chitosan mixture were mixed in a ratio of 1:1 (V / V); excess 0.1M calcium chloride solution (just enough to cover) was added for 10 minutes of preliminary cross-linking to form a soft gel in the inner layer, and the calcium chloride solution was absorbed; excess 0.2M calcium chloride solution was then added for 5 minutes of secondary cross-linking to further enhance the hardness of the outer layer; the matrix gel was washed three times with sterile water and implanted into the culture cavity of the chip, with 20 organoids / chip implanted, and cultured for 5 days. The growth medium was replaced with differentiation medium to obtain a small intestinal organoid chip, and complete small intestinal organoids were formed after 5 days of differentiation culture;

[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 IBD patient small intestinal organoid chip;

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

[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 fecal samples were mixed and added to pH 7.0 phosphate buffer solution at a ratio of 1:10 (g:mL). 1 g of sodium thioacetate was added as a reducing agent, and 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 liquid were inoculated into the ascending colon fermenter (101), transverse colon fermenter (102), and descending colon fermenter (103) in the large intestine fermentation simulation system, respectively. Then, 5 mucosal pellets 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 range 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] 24 hours after the initial inoculation, to maintain normal microbial growth, nutrients were added daily and 300 mL of culture medium was removed to maintain a constant volume. The mucosal pellets in the tank were replaced with three new ones to simulate mucosal regeneration. After the fecal intestinal flora was inoculated, fermentation continued for 7 days until it stabilized. A portion of the fermentation broth was stored at -80°C for bacterial flora analysis.

[0124] The mucosal spheres were prepared as follows: 2 g of agar was weighed and dissolved in 50 mL of distilled water, heated to 100°C, and after the solution became clear and transparent, it was cooled to 60°C and 0.5 g of mucin was added. After complete dissolution, the pH of the solution was adjusted to 8 to obtain a mucosal gel solution. The mucosal gel solution was added to a mold to solidify into small spheres with a diameter of 0.5 cm, and then sterilized by ultraviolet light in a clean bench for 20 minutes.

[0125] 4. Simulating 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 this range does not affect the experimental results) was taken and introduced 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 at a flow rate of 10 μL / min; the pipe connected to the chip hole 2 was inserted into the differentiation medium at 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 of the cell culture instrument (carbon dioxide concentration of 5%, temperature of 37°C) to obtain the small intestinal organoid chip of the small intestinal IBD patient after 24 hours of interaction;

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

[0128] 15 mL of colon digest was taken and entered into the pipe connected to the chip hole 1 of the colon organoid chip of the small intestinal IBD patient through a pressure-driven pump at a flow rate of 10 μL / min; the pipe connected to the chip hole 2 was inserted into the differentiation culture medium at a flow rate of 10 μL / min, that is, the ratio of digestive fluid and 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 colon IBD patient after 24 hours of interaction was obtained.

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

[0130] Based on Example 1, the lipopolysaccharide-induced IBD model in step 2 was omitted; the feces source in step 3 was changed to healthy adults who had not taken antibiotics for 6 months; the pH values ​​of the ascending colon glycolysis zone, transverse colon glycolysis zone, and descending colon glycolysis zone 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 will 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 example, 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 intestinal 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 growth of the organoids in the chips was tested using the in vitro simulated digestion of small intestine damage in IBD patients and the in vitro simulated digestion of colon damage in IBD patients obtained in Example 1, and the in vitro simulated digestion of small intestine damage in healthy people and the in vitro simulated digestion of colon damage in healthy people obtained in Comparative Example 1. Figure 3 As shown, the growth of colon organoids is as follows 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 group.

[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 fluorescence staining are as follows: 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 mean fluorescence intensity ratio of ZO-1 and DAPI reflects the tight junctions of cells, while the mean fluorescence intensity ratio of Muc-2 and DAPI reflects the stability of the overall organoid structure. A higher ratio indicates a more stable organoid structure. The results show that the tight junctions and structural stability of the organoid chip in Example 1 were both poor, 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: Figure 9 shown.

[0138] The results showed that the effect of simulating intestinal damage in Example 1 in vitro was close to that of IBD mice, with little difference in the data. It can be seen that the effect of the organoid chip in Example 1 in simulating intestinal damage in vitro is closer to the real situation.

[0139] Although the present invention has been disclosed above in terms of preferred embodiments, 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 preparation method of the inflammatory bowel disease organ 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 them to obtain a sodium alginate-fibronectin solution; (2) The organoid-chitosan mixture and the sodium alginate-fibronectin solution were mixed, and 0.05-0.1 M calcium chloride solution was added for preliminary cross-linking for 5-15 min, and then 0.1-0.3 M calcium chloride solution was added for secondary cross-linking for 5-10 min, and then washed to obtain the organoid matrix gel; (3) The organoid matrix gel is implanted in 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 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; 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 and dissolved, and mucin is added to obtain a mucosal gel solution; the mucosal gel solution is solidified to obtain mucosal pellets.

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

4.

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

5. The digestive system according to claim 1, wherein The large intestine fermentation simulation system includes: a culture medium feeding bottle, a pH control feeding bottle, an ascending colon fermentation tank, a transverse colon fermentation tank, a descending colon fermentation tank, and an anaerobic cylinder; The culture medium feeding bottles include a first culture medium feeding bottle, a second culture medium feeding bottle and a third culture medium feeding bottle; the pH control feeding bottles include 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 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 5, 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 through a silicone tube; the second culture medium feeding bottle, the second pH control feeding bottle and the transverse colon fermentation tank are connected through a silicone tube; the third culture medium feeding bottle, the third pH control feeding bottle and the descending colon fermentation tank are connected through 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 main control and display screen via 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 main control and display screen via 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 bacterial count and short-chain fatty acid detector is located at the bottom of the ascending colon fermentation tank, the first bacterial count and short-chain fatty acid detector is located at the bottom of the transverse colon fermentation tank, the second bacterial count and short-chain fatty acid detector is located at the bottom of the descending colon fermentation tank, and the third bacterial count 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, wherein The usage ratio of agar, mucin and water is 1-3 g: 0.3-0.6 g: 50 mL; the diameter of the mucosal ball is 0.3-0.7 cm.

8. The digestive system according to claim 5, characterized in that The mucosal balls 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 mucosal balls is 3 to 7.

9. The digestive system according to claim 5, 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 in patients with inflammatory bowel disease injury or in screening drugs.

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