Colon micro-ecology dynamic interaction system

Abstract

The invention discloses a dynamic interaction system for colon micro-ecology, and belongs to the field of digestive system simulation. By combining the colon-like organ with the colon dynamic glycolysis in-vitro simulation device, the fermentation process of undigested food molecules in a human body and the influence of the undigested food molecules on the colon can be better simulated, and many defects existing in traditional static culture are overcome; for example, the cell tissue size is not uniform, the dynamic regulation capability is lacked, and stable culture conditions cannot be maintained for a long time, so that the problem of non-uniform distribution of nutrition and oxygen is solved.

Description

Technical Field

[0001] The invention relates to a colon microecological dynamic interaction system, and belongs to the field of digestive system simulation. Background Art

[0002] The interaction between colon organoids and colonic flora is crucial for simulating the microecological environment of the human colon. However, existing in vitro models generally lack a long-term dynamic interaction system and cannot accurately simulate the complex material metabolism and microecological environment in the colon. This limitation greatly affects the authenticity and reliability of in vitro models, especially in the study of cell metabolism and microbial interactions. At the same time, the growth of organoids requires a specific oxygen concentration, and the existing technology lacks a system that can accurately regulate the oxygen concentration, making it difficult for organoid culture to stably maintain an ideal physiological environment. Therefore, there is an urgent need for a new in vitro dynamic interaction system to improve the long-term co-culture model of colon organoids and intestinal flora, thereby improving the authenticity and reliability of in vitro simulations.

[0003] Organoid technology can simulate the three-dimensional structure and physiological function of human organs in vitro, and is usually derived from human stem cells or primary tissues. As an in vitro model of colon organs, colon organoids can simulate the real colon physiological environment. After the food ingested by the human body is digested and decomposed in the gastrointestinal tract, some undigested small molecules enter the colon and are fermented by microorganisms in the intestine. Current in vitro colon fermentation experiments usually only focus on the fermentation process, but do not fully consider the interaction between fermentation products and colon organoids. This defect greatly limits the simulation of the complex functions of the colon microecology. Summary of the invention

[0004] To solve the above problems, the present invention combines colon organoids with an in vitro simulation device for dynamic colon fermentation, which can better simulate the fermentation process of undigested food molecules in the human body and its impact on the colon, overcome many defects existing in traditional static culture, such as uneven cell tissue size, lack of dynamic adjustment ability, and inability to maintain stable culture conditions for a long time, thereby solving the problem of uneven distribution of nutrients and oxygen.

[0005] The first object of the present invention is to provide a method for simulating colon digestion, wherein organoid culture medium and colon fermentation solution are passed into a microfluidic chip containing colon organoids to simulate colon digestion;

[0006] The preparation method of the colon fermentation liquid comprises: inoculating artificial intestinal flora into an intestinal culture medium containing small intestinal digesta, and fermenting to obtain;

[0007] The artificial intestinal flora consists of Prevotella copri ATCC-33547, Faecalibacterium prausnitzii ATCC-27768, Roseburia faecis Mingzhoubio-B84313, Bifidobacterium longum ATCC-15707, Ruminococcus bromii ATCC-27255, Bifidobacterium adolescentis ATCC-15707, Bacteroides uniformis ATCC-25285, Collinsella aerofaciens ATCC-25986, Ruminococcus gnavus ATCC-29149 and Akkermansia muciniphila ATCC-baa-835 according to the number of viable bacteria (10 8 CFU / mL) ratio 0.10~0.30:0.10~0.30:0.05~0.20:0.05~0.20:0.05~0.20:0.005~0.1:0.005~0.1:0.01~0.03:0.01~0.03:0.01~0.03;

[0008] The above strains can all be purchased commercially.

[0009] In one embodiment, colon organoids can be obtained by stem cell culture and differentiation; or by tissue isolation and culture.

[0010] In one embodiment, colon organoids are cultured to differentiation and maturity and then implanted into a microfluidic chip.

[0011] In one embodiment, the microfluidic chip is the BeonChip electrochemical-3D cell culture microfluidic chip BE-GRADIENT.

[0012] In one embodiment, the number of viable bacteria in the colon fermentation solution reaches 1 to 10×10 9 CFU / mL.

[0013] In one embodiment, the number of organoids in each microfluidic chip is 20 to 50.

[0014] In one embodiment, the flow rate of the organoid culture medium into the microfluidic chip is 0.5-1 μL / min; the flow rate of the colon lysate into the microfluidic chip is 0.5-1 μL / min.

[0015] In one embodiment, air is aerated into the organoid culture medium and anaerobic gas is aerated into the colon lysate.

[0016] In one embodiment, the composition of the air aerated in the organoid culture medium (v / v) is 5% carbon dioxide, 15% oxygen, and 80% nitrogen.

[0017] In one embodiment, the anaerobic gas composition (v / v) of 80% nitrogen, 10% carbon dioxide, and 10% hydrogen is introduced into the colonic fermentation solution.

[0018] In one embodiment, the simulated colonic digestion is carried out at 37°C for 5 to 10 days.

[0019] In one embodiment, the method for preparing an intestinal culture medium containing small intestinal digesta is:

[0020] The sample is mixed with simulated oral fluid, and CaCl2 solution and water are added, and the mixture is mixed to obtain oral digesta; the oral digesta are mixed with simulated gastric fluid, and CaCl2 solution, gastric lipase, pepsin and water are added, and the mixture is stirred to obtain gastric digesta; the gastric digesta are mixed with simulated small intestinal fluid, and CaCl2 solution, trypsin, pancreatic lipase, bile salt and water are added, and the mixture is stirred to obtain small intestinal digesta; the small intestinal digesta are centrifuged to obtain undigested solid residue and small intestinal digestion fluid, and 8-12% v / v of the small intestinal digestion fluid and the undigested solid residue are transferred to an intestinal culture medium to obtain an intestinal culture medium containing the small intestinal digesta;

[0021] Among them, samples included starch, whey protein, and fatty acids;

[0022] The simulated oral fluid was prepared from KCl, NaHCO3, KH2PO4, MgCl2·6H2O, (NH4)2CO3 and water;

[0023] Simulated gastric fluid was prepared from NaCl, KCl, NaHCO3, KH2PO4, MgCl2·6H2O, (NH4)2CO3 and water;

[0024] The simulated small intestinal fluid was prepared from NaCl, KCl, NaHCO3, KH2PO4, MgCl2·6H2O and water.

[0025] The second object of the present invention is to provide the use of any of the above methods in detecting digestion of polysaccharide-based foods, drug screening or constructing disease models.

[0026] In one embodiment, the polysaccharide-based food includes cereal foods, starch, and resistant starch; optionally, the starch includes but is not limited to corn starch, acetylated starch, propionylated starch, and butyrylated starch.

[0027] In one embodiment, drug screening involves intestinal absorption of drugs.

[0028] In one embodiment, the disease model is an intestinal disease model, including but not limited to inflammatory bowel disease, intestinal infectious diseases, colorectal cancer, intestinal fibrosis model, and diarrhea model.

[0029] The third object of the present invention is to provide a colon microecological interaction system, which comprises an organoid culture medium bottle, a culture medium ventilation bottle, an artificial intestinal flora fermentation tank, an anaerobic bottle and a microfluidic chip;

[0030] Among them, the organoid culture medium bottle and the microfluidic chip are connected by a silicone tube; the artificial intestinal flora fermentation tank and the microfluidic chip are connected by a silicone tube; the culture medium ventilation bottle and the organoid culture medium bottle are connected by a silicone tube; the artificial intestinal flora fermentation tank and the anaerobic bottle are connected by a silicone tube;

[0031] The artificial intestinal flora fermentation tank is filled with colonic fermentation liquid, and the preparation method of the colonic fermentation liquid comprises: inoculating the artificial intestinal flora into an intestinal culture medium containing small intestinal digesta, and fermenting to obtain;

[0032] The artificial intestinal flora consists of Prevotella copri ATCC-33547, Faecalibacterium prausnitzii ATCC-27768, Roseburia faecis Mingzhoubio-B84313, Bifidobacterium longum ATCC-15707, Ruminococcus bromii ATCC-27255, Bifidobacterium adolescentis ATCC-15707, Bacteroides uniformis ATCC-25285, Collinsella aerofaciens ATCC-25986, Ruminococcus gnavus ATCC-29149 and Akkermansia muciniphila ATCC-baa-835 according to the number of viable bacteria (10 8 CFU / mL) ratio 0.10~0.30:0.10~0.30:0.05~0.20:0.05~0.20:0.05~0.20:0.005~0.1:0.005~0.1:0.01~0.03:0.01~0.03:0.01~0.03;

[0033] The microfluidic chip is populated with colon organoids.

[0034] In one embodiment, the number of viable bacteria in the colon fermentation solution reaches 1 to 10×10 9 CFU / mL; the number of organoids in each microfluidic chip is 20 to 50.

[0035] The beneficial effects of the present invention are:

[0036] (1) The present invention constructs a dynamic interaction system between colon organoids and flora by introducing colon fermentation liquid with a specific flora ratio, thereby improving the authenticity and reliability of the cell state of the organoids in in vitro simulation and the in vitro model in simulating the microecological environment of the human colon;

[0037] (2) The system of the present invention can accurately simulate the metabolism and microbiome interaction process of the colon in the human body, and the simulation effect is closer to the real human body, which significantly enhances the physiological relevance of the model;

[0038] (3) The present invention provides a new colon organoid interaction system, which helps to more deeply understand the role of colon microbiota in human health; at the same time, the method of the present invention can also be used for drug screening and the establishment of related disease models, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a system concept flow chart of the present invention, showing the dynamic interaction process between organoids and colon fermentation fluid;

[0040] Figure 2 The PCA analysis graphs and flora abundance heat maps of intestinal bacteria of Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 show the flora changes under different culture conditions; A is the PCA analysis graph; B is the flora abundance heat map;

[0041] Figure 3 is a graph showing the relative abundance changes of intestinal bacteria in Example 1, Comparative Example 2 and Comparative Example 3, showing the abundance differences of the flora;

[0042] Figure 4 It is a graph of the relative abundance changes of Bifidobacterium longum, Bifidobacterium adolescentis, and Akkermansia muciniphila intestinal bacteria of the initial flora, Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, showing the abundance differences of the flora;

[0043] Figure 5 It is a comparison chart of the organoid growth and the number of buds of Example 1, Comparative Example 1, Comparative Example 3 and Comparative Example 4; A is a photo; B is the number of buds;

[0044] Figure 6Comparison of Edu staining images and average fluorescence intensity of organoids on the seventh day in Example 1, Comparative Example 1, Comparative Example 3 and Comparative Example 4; A is the staining image; B is the fluorescence intensity;

[0045] Figure 7 Comparison of PI staining images and average fluorescence intensity of dead cells in organoids on the seventh day of Example 1, Comparative Example 1, Comparative Example 3 and Comparative Example 4; A is the staining image; B is the fluorescence intensity;

[0046] Figure 8 The intestinal flora changes on the seventh day of Example 2 and the human body real environment comparison; A is the abundance of organoid flora; B is the abundance of human flora;

[0047] Fig. 9 Comparison chart of short-chain fatty acid content on the seventh day between Example 2 and the real human environment comparison example; A is the short-chain fatty acid content; B is the digestion data result. DETAILED DESCRIPTION

[0048] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0049] Device:

[0050] The colon microecological interaction system of the present invention is shown in the schematic diagram Figure 1 As shown, the colon microecological interaction system includes an organoid culture medium bottle, a culture medium aeration bottle, an artificial intestinal flora fermentation tank, an anaerobic bottle, and a microfluidic chip;

[0051] Among them, the organoid culture medium bottle and the microfluidic chip are connected by a silicone tube; the artificial intestinal flora fermentation tank and the microfluidic chip are connected by a silicone tube; the culture medium ventilation bottle and the organoid culture medium bottle are connected by a silicone tube; and the artificial intestinal flora fermentation tank and the anaerobic bottle are connected by a silicone tube.

[0052] raw material:

[0053] Intestinal culture medium preparation: 8g corn starch, 4.50g yeast extract, 3g tryptone, 0.43g bile salt No. 3, 4.50g NaCl, 2.50g KCl, 0.40g KH2PO4, 4.50g MgCl2·6H2O, 0.20g CaCl2·2H2O, 1.0mL Tween 80 and 1L deionized water. After autoclaving at 121℃ for 15 minutes, 0.5g mucin, 0.8g L-cysteine ​​hydrochloride, 0.05g heme and 2mL trace element stock solution were added;

[0054] Preparation of trace element stock solution: 3.0 g MgSO4·7H2O, 0.10 g FeSO4·7H2O, 0.10 g CaCl2·2H2O, 0.32 g MnCl2·4H2O, 0.18 g CoSO4·7H2O, 0.01 g CuSO4·5H2O, 0.18 g ZnSO4·7H2O, 0.092 g NiSO4·6H2O and 1 L deionized water.

[0055] BeonChip electrochemical-3D cell culture microfluidic chip was purchased from Micro-Nano Cube Technology (Beijing) Co., Ltd., and the product model is BE-GRADIENT.

[0056] High amylose corn starch was purchased from Hangzhou Prostar Starch Co., Ltd.

[0057] Gastric lipase, pepsin, trypsin, and pancreatic lipase were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0058] Experimental methods:

[0059] 1. Construction of colon organoids

[0060] (1) Culture and proliferation of pluripotent stem cells:

[0061] Human induced pluripotent stem cells (hiPSCs) were cultured using a standard feeder-free culture system, mTeSR1 medium. hiPSCs were cultured in a culture dish coated with Matrigel at 37°C and 5% CO2, and the culture medium was changed every day. When the confluence of hiPSCs reached 70-80%, the cells were dissociated with EDTA solution, gently pipetted to form a single cell suspension, and re-seeded in a culture dish newly coated with Matrigel and continued to be cultured.

[0062] (2) Directed differentiation into mesoderm:

[0063] When the confluence of hiPSCs cells reaches about 70%, directed differentiation is started; RPMI culture medium containing 10 μM CHIR99021 (Wnt signal agonist) is used to induce cells to differentiate into mesoderm. After culturing for 2 to 3 days, cell morphological changes are observed every day, and the cells will gradually develop mesoderm characteristics.

[0064] (3) Formation of colon-specific precursor cells:

[0065] Continue to culture the mesoderm cells in the basal medium to promote their differentiation into intestinal precursor cells for 3 to 4 days. During this period, replace the medium to ensure that the cells maintain growth.

[0066] (4) Formation of three-dimensional colon organoids:

[0067] After the cells differentiated into colonic progenitor cells, the cells were resuspended in ice-cold Matrigel matrix at an appropriate density (1000-5000 cells / 10 μL) and seeded in the center of a 24-well plate, 55 μL per well; the 24-well plate was inverted and placed in a 37°C incubator for 20 minutes to allow Matrigel to solidify. Use growth medium and continue to culture at 37°C, 5% CO2. Change the medium every 3 days and continue to observe organoid formation until visible structures are formed.

[0068] After visible structures are formed, the growth medium is replaced with differentiation medium and culture is continued for 5 to 7 days (the time is determined according to the state of the organoids) to promote the differentiation of organoids into mature colon cells and obtain colon organoids with mature three-dimensional structures.

[0069] 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.

[0070] 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 Y027632dihydrochloride, 50ng / mL EGF, 500ng / mL R-spondin1, and 100ng / mL Noggin.

[0071] 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.

[0072] Detection method:

[0073] 1. Detection of intestinal flora abundance

[0074] DNA of intestinal flora was extracted using a DNA purification kit, and the V3-V4 region of the bacterial 16SrRNA gene was PCR amplified using primers 341F and 806R. After detection and purification by 2% agarose gel electrophoresis, the PCR products were sequenced using the Novasek 6000 PE250 platform, providing at least 50,000 raw data for each sample. The sequencing data were quality controlled using the DADA2 plug-in in QIIME 2.0, including denoising, splicing, and chimera removal, to form amplicon sequencing variants (ASVs). Subsequently, the ASVs were annotated with species using the RDP classifier (http: / / rdp.cme.msu.edu / ), and the Greengenes2 database was used for reference comparison, accurate to species-level bacterial classification, and the composition and diversity of intestinal flora were analyzed.

[0075] 2. Organoid proliferation detection

[0076] Using BeyoClick TM The EdU cell proliferation kit (Biyuntian Biotech, Nantong, China) was used to detect the proliferation of colon organoids. Intestinal organoids were incubated with medium containing 10 μmol / L EdU for 12 h, and then the organoids were dissolved and fixed in 4% paraformaldehyde for 30 min. After permeabilization (0.5% Triton X-100 / PBS) for 10 min, the cells were reacted with the reagents in the kit for 30 min. The nuclei were counterstained with DAPI, and the cells were scanned layer by layer using a laser confocal microscope, and the 3D images were reconstructed.

[0077] 3. Organoid dead cell detection

[0078] PI (propidium iodide) staining is used to detect dead cells in colon organoids. First, the organoids after culture are gently washed with PBS to remove the residual culture medium. The organoids are then fixed in 4% paraformaldehyde for 30 minutes. After fixation, 0.5% Triton X-100 / PBS solution is used for cell permeabilization for 10 minutes. Next, the organoids are reacted with 50μg / mL of PI dye for 30 minutes. PI can enter the damaged cell membrane and bind to DNA, thereby marking dead cells. After staining, the sample is washed again with PBS and observed by laser confocal microscopy. Cells positive for PI staining show red fluorescence, representing dead cells or cells with damaged cell membranes.

[0079] 4. Determination of short-chain fatty acids

[0080] Take 100 mg of the sample to be tested (metabolism solution) and place it in a 2 mL EP tube, add an appropriate amount of pure water and vortex mix. Centrifuge at 5000 r / min for 20 minutes at 4 ° C, take 0.8 mL of supernatant in another 2 mL EP tube, add 0.1 mL 50% H2SO4 solution and 0.8 mL extract, and centrifuge again at 10000 r / min for 15 minutes at 4 ° C. After centrifugation, let the sample stand at -20 ° C for 30 minutes. Finally, take the supernatant and transfer it to the injection bottle, and analyze it by gas chromatography-mass spectrometry (GC-MS). The chromatographic conditions were as follows: using an Agilent HP-FFAP capillary column (30m×250μm×0.25μm), the temperature program was 80℃ for 1 minute, then increased to 200℃ at 10℃ / min and maintained for 5 minutes, then increased to 240℃ at 40℃ / min and maintained for 1 minute; the carrier gas was helium (He), the flow rate was 3mL / min, the injection volume was 1μL, and the split ratio was 5:1. The mass spectrometry conditions were as follows: ionization voltage -70eV, transmission line temperature 240℃, ion source temperature 200℃, quadrupole temperature 150℃, and mass scanning range m / z 33-150. This GC-MS method can accurately detect the content of short-chain fatty acids in samples. Prepare a series of short-chain fatty acid standard solutions with known concentrations (e.g., 0.01, 0.05, 0.1, 0.5, 1 mM and other concentration gradients). Use GC-MS to detect each standard solution and record the peak area at each concentration. Based on the linear relationship between concentration and peak area, draw a standard curve. By comparing the peak area in the sample with the standard curve, calculate the actual concentration of short-chain fatty acids in the sample.

[0081] 5. Data processing

[0082] In all experiments, data were repeated at least 3 times and expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism 7.0 (GraphPad Prism Software Inc., San Diego, California) software. The comparison between two groups was performed using the bilateral Student's t-test method. For more than two groups, the p value was calculated using the Dunnett's post-hoc testing method in One-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, all compared with the examples.

[0083] Example 1: Dynamic interaction system of colon organoids and colon lysate

[0084] 1. Preparation of intestinal flora

[0085] The strains were cultured and mixed according to the proportions shown in Table 1. The mixing method was as follows: culture the strains separately until the logarithmic growth phase, collect the cells, resuspend them in sterile PBS, and adjust the number of each cell to 10. 8 CFU / mL, and the intestinal flora was obtained by compounding.

[0086] Table 1 Artificial intestinal flora

[0087]

[0088]

[0089] 2. Preparation of Colonic Digestives

[0090] (1) Oral digestion stage:

[0091] Preparation of simulated oral fluid: 1.408 g KCl, 1.428 g NaHCO3, 0.629 g KH2PO4, 0.038 g MgCl2·6H2O, 0.007 g (NH4)2CO3 and 1 L deionized water.

[0092] Sample preparation: Mix 25 mL of sample (0.2 g / mL corn starch solution) with 20 mL of simulated oral fluid, add 125 μL of 0.3 mol / L CaCl2 solution and 4.875 mL of deionized water, and mix in a 37°C water bath for 2 minutes to simulate the mechanical digestion process in the mouth.

[0093] (2) Stomach digestion stage:

[0094] Gastric buffer preparation: 2.808 g NaCl, 0.643 g KCl, 2.625 g NaHCO3, 0.153 g KH2PO4, 0.031 g MgCl2·6H2O, 0.06 g (NH4)2CO3 and 1 L deionized water.

[0095] Gastric digestion reaction: 40 mL of the sample solution after oral digestion was mixed with 40 mL of simulated gastric buffer, and 20 μL of 0.3 mol / L CaCl2, 20 mg of gastric lipase (final concentration 60 U / mL), 2000 mg of pepsin (final concentration 2000 U / mL) and 9.475 mL of deionized water were added and mixed.

[0096] The samples were placed in an in vitro digestion system, stirred at 150 r / min, the pH was controlled at 3.0, and digestion was continued at a constant temperature of 37°C for 1 hour.

[0097] (3) Small Intestinal Digestion Stage

[0098] Preparation of simulated small intestinal fluid: 3.452 g NaCl, 0.634 g KCl, 8.952 g NaHCO3, 0.136 g KH2PO4, 0.084 g MgCl2·6H2O and 1 L deionized water.

[0099] Small intestinal digestion reaction: 40 mL of the sample solution after gastric digestion was mixed with 40 mL of simulated small intestinal buffer, and 200 μL of 0.3 mol / L CaCl2, 90 mg of trypsin (final concentration 100 U / mL), 13.35 mg of pancreatic lipase (final concentration 2000 U / mL), 1361.90 mg of bile salt (final concentration 10 mmol / L) and 56.3 mL of deionized water were added and mixed.

[0100] The sample was placed in an in vitro digestion system, stirred at 150 r / min, the pH was controlled at 7.0, and digested continuously for 2 hours at a constant temperature of 37°C to obtain small intestinal digesta.

[0101] (4) Colonic metabolism simulation

[0102] About 10% of the chyme in the small intestinal digesta is not absorbed. 10% (v / v) of the small intestinal soluble part and the undigested solid residue are used as fermentation substrates. The small intestinal digesta are centrifuged at 4000×g for 10 minutes at 4°C to collect the small intestinal undigested solid residue and 50 mL of the small intestinal digestive fluid (supernatant). 5 mL of the small intestinal digestive fluid and the small intestinal undigested solid residue are added to 300 mL of intestinal culture medium, inoculated with artificial intestinal flora (inoculation amount is 10% v / v), and fermented until the colony forming unit reaches 10 9 CFU / mL.

[0103] The reaction temperature in the fermentation system was maintained at 37°C, and the pH was adjusted to 6.8 using an automatic pH control system, and 1 mol / L NaOH and 0.5 mol / L HCl were used for automatic adjustment. Before fermentation, anaerobic gases (nitrogen, carbon dioxide, and hydrogen) were introduced to exhaust the air to ensure a strict anaerobic environment, and the bacterial solution was circulated. At the same time, after 24 hours of fermentation every day, 100 mL of waste liquid was discharged and 100 mL of new intestinal flora culture medium was introduced to maintain normal growth of the flora, and a usable colon fermentation solution (10 9 CFU / mL).

[0104] 3. Colon Organoids and Colon Fermentation Fluid

[0105] Based on the BeonChip electrochemical-3D cell culture microfluidic chip BE-GRADIENT, the colon organoids and colon lysate were interacted as follows:

[0106] The colon organoids with mature three-dimensional structures were transferred to the microfluidic chip, with 30 organoids per chip. The microfluidic chip was made of PDMS material, with a channel width of 1μm, a length of 300μm, a depth of 50μm, and a culture chamber volume of 14μL. The fluid flow rate was controlled at 0.5-1μL / min by an external pump to ensure the continuous dynamic interaction between the organoids and the colon lysate, and the culture was continued at 37°C for 7 days;

[0107] The gas control system introduces air (5% carbon dioxide, 15% oxygen, 80% nitrogen) into the organoid differentiation medium; while anaerobic gas (80% nitrogen + 10% carbon dioxide + 10% hydrogen) is introduced into the colon fermenter.

[0108] Comparative Example 1: No colonic fermentation solution added (no intestinal flora)

[0109] On the basis of Example 1, the colon lysate was not introduced into the microfluidic chip in "3. Colon organoids and colon lysate", and the remaining steps were consistent with Example 1.

[0110] Comparative Example 2: Fermentation of colonic fermentation liquid alone (without organoids and their culture medium)

[0111] On the basis of Example 1, a colon fermentation solution was prepared and fermented for 7 days. The remaining steps were the same as those in Example 1.

[0112] Comparative Example 3: Anaerobic culture of organoids

[0113] On the basis of Example 1, the microfluidic chip in "3. Colon organoids and colon lysate" was changed to also introduce anaerobic gas, and the remaining steps were consistent with Example 1.

[0114] Comparative Example 4: Changing the Type and Ratio of Intestinal Bacteria

[0115] On the basis of Example 1, the types of intestinal flora in the colon fermentation solution were changed, and only the three most commonly used probiotics were selected, as shown in Table 2. The remaining steps were consistent with Example 1.

[0116] Table 2 Artificial intestinal flora

[0117]

[0118] The bacterial community structure Figure 2 The bacterial abundance results are shown in Figure 3As shown, the results show that the flora in Example 1 is different from those in Comparative Examples 2, 3, and 4, indicating that the dynamic interaction between the organoid and the fermentation solution is different from that of a single variable, and can effectively simulate the colon microecological environment in vivo; the abundance of beneficial bacteria such as Prevotella copri and Faecalibacterium prausnitzii in Example 1 is significantly higher than that in the comparative example, further supporting the effect that the organoid and the flora jointly maintain ecological stability and proliferate beneficial bacteria.

[0119] Figure 4 The results showed that the relative abundance of Bifidobacterium longum, Bifidobacteriumadolescentis and Akkermansia muciniphila in Example 1 was significantly higher than that in Comparative Examples 2, 3 and 4. In particular, the abundance of Bifidobacterium longum and Akkermansia muciniphila in Example 1 was significantly increased, reflecting the significant effect of Example 1 on the optimization of intestinal flora. In addition, Example 1 showed statistically significant differences in the abundance of different strains (p<0.01 or p<0.001), which further confirmed the effectiveness of the artificial intestinal flora of Example 1 in organoid interaction.

[0120] The number and growth of organoid buds on the seventh day Figure 5 As shown, the results show that the number of buds in Example 1 is significantly higher than that in Comparative Example 1, Comparative Example 3 and Comparative Example 4, showing a significant statistical difference (p<0.05, p<0.01). Compared with Comparative Example 4, the bacterial community composed of only 3 probiotics, the organoids in Example 1 grew well, and the number of buds in the differentiation stage increased significantly, which shows that in the microfluidic chip, Example 1 is more conducive to promoting the healthy growth and development of organoids, reflecting its positive effect on the regeneration ability of intestinal tissue.

[0121] The results of Edu staining of organoids on day 7 of culture are as follows Figure 6 As shown, the results show that Example 1 has outstanding performance in cell proliferation activity, and its average fluorescence intensity is significantly higher than that of Comparative Example 1, Comparative Example 3 and Comparative Example 4. This shows that Example 1 can effectively promote the proliferation activity of cells in organoids. The results of PI staining of organoids on the 7th day of culture are shown in Figure 7 As shown, the results show that the average fluorescence intensity of Example 1 is significantly lower than that of each comparative example group, indicating that under the conditions of Example 1, the mortality rate of organoids is lower and the stability is higher, which has obvious advantages.

[0122] In summary, the results show that the conditions of Example 1 are more conducive to the colonization of organoids in the microfluidic chip, and the growth state of the organoids is significantly better than that of Comparative Examples 1 to 4.

[0123] Example 2: Using high amylose corn starch

[0124] 1. Organoid Interaction

[0125] On the basis of Example 1, corn starch was replaced with high-amylose corn starch, and the remaining steps were consistent with Example 1. The state of the organoids and the changes in the flora in the colon lysate were detected.

[0126] The results of organoid-simulated bacterial abundance are as follows Figure 8 As shown in A, the results showed that the relative abundance of strains Prevotella copri, Faecalibacterium prausnitzii, and Roseburia faecis increased significantly with the addition of high-amylose corn starch.

[0127] Short chain fatty acids Fig. 9 As shown in A, the results showed that the relative abundance of these strains increased significantly with the addition of high-amylose corn starch. Butyrate and propionate are not only energy sources for intestinal epithelial cells, but also participate in regulating intestinal barrier function and anti-inflammatory response. This result proves that high-amylose corn starch has a significant promoting effect on intestinal flora metabolism and organoid function. The results showed that the addition of high-amylose corn starch also promoted expression in organoids, showing that it not only improved the metabolic capacity of intestinal flora, but also promoted cell proliferation and intestinal barrier function of organoids, further restoring the authenticity of intestinal epithelial cells.

[0128] 2. Human digestion data

[0129] Three volunteers consumed high-amylose corn starch every day for one week (0.5g / Kg). Fecal samples showed an increase in short-chain fatty acids and significant changes in the intestinal flora structure.

[0130] Figure 8 Bacterial abundance analysis showed that the abundance of key bacterial groups such as Prevotella copri and Faecalibacterium prausnitzii in the volunteers increased, which was consistent with the results of the in vitro experiment (Example 2) ( Figure 8 A), further supporting the positive effect of high amylose corn starch on promoting intestinal health and flora stability. Fig. 9 As shown in Figure B, the results showed that the concentrations of acetic acid, propionic acid, and butyric acid increased significantly in the fecal samples of volunteers, indicating that high-amylose corn starch also promoted the production of short-chain fatty acids when metabolized in the body. This shows that the application of high-amylose corn starch in vivo and in vitro can effectively promote intestinal flora metabolism and improve intestinal function.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simulating colon digestion, characterized in that: The organoid culture medium and colon fermentation solution were passed into the microfluidic chip containing colon organoids to simulate colon digestion; The preparation method of the colon fermentation liquid comprises: inoculating artificial intestinal flora into an intestinal culture medium containing small intestinal digesta, and fermenting to obtain; The artificial intestinal flora consists of Prevotella copri ATCC-33547, Faecalibacterium prausnitzii ATCC-27768, Roseburia faecis Mingzhoubio-B84313, Bifidobacterium longum ATCC-15707, Ruminococcus bromii ATCC-27255, Bifidobacterium adolescentis ATCC-15707, Bacteroides uniformis ATCC-25285, Collinsella aerofaciens ATCC-25986, Ruminococcus gnavus ATCC-29149 and Akkermansia muciniphila ATCC-baa-835 is composed of 0.10~0.30:0.10~0.30:0.05~0.20:0.05~0.20:0.05~0.20:0.005~0.1:0.005~0.1:0.01~0.03:0.01~0.03:0.01~0.

03.

2. The method according to claim 1, characterized in that The number of viable bacteria in the colonic fermentation fluid reached 1 to 10×10 9 CFU / mL.

3. The method according to claim 1, characterized in that: The number of organoids in each microfluidic chip ranges from 20 to 50.

4. The method according to claim 1, characterized in that: The flow rate of the organoid culture medium into the microfluidic chip is 0.5-1 μL / min; the flow rate of the colon lysate into the microfluidic chip is 0.5-1 μL / min.

5. The method according to claim 1, characterized in that Air was aerated into the organoid culture medium; anaerobic gas was aerated into the colonic fermentation fluid.

6. The method according to claim 1, characterized in that Simulated colonic digestion was performed at 37°C for 5 to 10 days.

7. The method according to claim 1, characterized in that The preparation method of intestinal culture medium containing small intestinal digesta is as follows: The sample is mixed with simulated oral fluid, and CaCl2 solution and water are added, and the mixture is mixed to obtain oral digesta; the oral digesta are mixed with simulated gastric fluid, and CaCl2 solution, gastric lipase, pepsin and water are added, and the mixture is stirred to obtain gastric digesta; the gastric digesta are mixed with simulated small intestinal fluid, and CaCl2 solution, trypsin, pancreatic lipase, bile salt and water are added, and the mixture is stirred to obtain small intestinal digesta; the small intestinal digesta are centrifuged to obtain undigested solid residue and small intestinal digestion fluid, and 8-12% v / v of the small intestinal digestion fluid and the undigested solid residue are transferred to an intestinal culture medium to obtain an intestinal culture medium containing the small intestinal digesta; Among them, samples included starch, whey protein, and fatty acids; The simulated oral fluid was prepared from KCl, NaHCO3, KH2PO4, MgCl2·6H2O, (NH4)2CO3 and water; Simulated gastric fluid was prepared from NaCl, KCl, NaHCO3, KH2PO4, MgCl2·6H2O, (NH4)2CO3 and water; The simulated small intestinal fluid was prepared from NaCl, KCl, NaHCO3, KH2PO4, MgCl2·6H2O and water.

8. Use of the method according to any one of claims 1 to 7 in detecting digestion of polysaccharide-based foods, drug screening or constructing disease models.

9. A colon microecological interaction system, characterized in that: The colon microecological interaction system comprises an organoid culture medium bottle, a culture medium ventilation bottle, an artificial intestinal flora fermentation tank, an anaerobic bottle and a microfluidic chip; Among them, the organoid culture medium bottle and the microfluidic chip are connected by a silicone tube; the artificial intestinal flora fermentation tank and the microfluidic chip are connected by a silicone tube; the culture medium ventilation bottle and the organoid culture medium bottle are connected by a silicone tube; the artificial intestinal flora fermentation tank and the anaerobic bottle are connected by a silicone tube; The artificial intestinal flora fermentation tank is filled with colonic fermentation liquid, and the preparation method of the colonic fermentation liquid comprises: inoculating the artificial intestinal flora into an intestinal culture medium containing small intestinal digesta, and fermenting to obtain; The artificial intestinal flora consists of Prevotella copri ATCC-33547, Faecalibacterium prausnitzii ATCC-27768, Roseburia faecis Mingzhoubio-B84313, Bifidobacterium longum ATCC-15707, Ruminococcus bromii ATCC-27255, Bifidobacterium adolescentis ATCC-15707, Bacteroides uniformis ATCC-25285, Collinsella aerofaciens ATCC-25986, Ruminococcus gnavus ATCC-29149 and Akkermansia muciniphila ATCC-baa-835 is composed of 0.10-0.30:0.10-0.30:0.05-0.20:0.05-0.20:0.05-0.20:0.005-0.1:0.005-0.1:0.01-0.03:0.01-0.03:0.01-0.03; The microfluidic chip is populated with colon organoids.

10. The colon microecological interaction system according to claim 9, characterized in that: The number of viable bacteria in the colonic fermentation fluid reached 1 to 10×10 9 CFU / mL; the number of organoids in each microfluidic chip is 20 to 50.

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