Gastrointestinal tract dynamic digestion and small intestine organoid short-term interaction system

Abstract

The invention discloses a gastrointestinal tract dynamic digestion and small intestine organoid short-term interaction system, and belongs to the technical field of digestion simulation and food. According to the invention, methacrylic acid starch is selected to prepare matrigel, and starch gel can form a firmer three-dimensional network structure due to the existence of functional groups; on the basis, a photocuring agent polymer is introduced into the starch to be cross-linked with a photocuring functional group, and compared with non-cross-linked starch, the starch shows more excellent mechanical properties. The in-vitro simulation device for dynamic digestion of the small intestine organoids and the gastrointestinal tract is integrated, digestion and absorption in the human body can be better simulated, recycling can be achieved after interaction, and the utilization rate of the organoids is further increased.

Description

Technical Field

[0001] The present invention relates to a gastrointestinal dynamic digestion and small intestinal organoid short-term interaction system, belonging to the field of digestion simulation and food technology. Background Art

[0002] Organoid technology can simulate the 3D environment and physiological functions of human organs in vitro, and is derived from primary tissues or human stem cells. Intestinal organoids can simulate the physiological environment and physiological functions of the small intestine. The gastrointestinal tract is the main place for the human body to digest food. When food reaches the small intestine after a series of digestive processes, it is further digested into small molecules in the small intestine and absorbed into the blood through intestinal epithelial cells. It will also have a certain impact on the intestinal barrier. However, currently, intestinal organoids are mostly used in disease research, drug testing and other fields, and there is a lack of applications that integrate small intestinal organoids with in vitro digestion simulation devices.

[0003] The gastrointestinal dynamic digestion device can simulate the digestive enzyme environment in the gastrointestinal tract, while the small intestinal organoid chip can simulate the microscopic physiological environment of the small intestinal organ. The integration of the two allows the digestive fluid in the gastrointestinal dynamic digestion device to fully contact the small intestinal organoids placed in the built-in tank, which is more in line with the real gastrointestinal digestion and absorption process in the human body.

[0004] At present, organoid culture is mainly based on matrix gel culture, but the organoids obtained by matrix gel culture show great heterogeneity in shape, size and cell composition, and the matrix gel is fragile and easily damaged in liquid flow. At the same time, the organoids are severely damaged after in vitro interaction, and most of them are used once and cannot be recycled for other experiments or tests; the organoid culture cycle is long and the cost is high, and it will also be affected by the cell state during the experiment.

[0005] Starch is a natural polymer that is abundant in resources, low in cost, safe, non-toxic, renewable and biodegradable. When the temperature rises, it can absorb water and swell, and the hydrogen bonds between molecules are broken. This process is called starch gelatinization. After that, as the temperature decreases, the starch molecules rearrange and condense to form an orderly structure, and build a three-dimensional gel grid structure. This process is called starch retrogradation. Starch has good biocompatibility and biodegradability. It can provide physical support while avoiding adverse immune responses, and can be degraded and digested by biological enzymes.

[0006] Starch is an excellent precursor for preparing hydrogels. If it is modified and groups that can form a cross-linked network are introduced into it, its mechanical properties can be improved while maintaining good biocompatibility. In order to cross-link starch molecules, the introduction of photocuring technology can be considered. This technology is green and efficient, and can use photocuring agents to make the functional groups in starch form a three-dimensional cross-linked network.

[0007] Therefore, when using organoids to simulate gastrointestinal digestion in vitro, finding a method that can reuse organoids without affecting the experimental results of organoids can improve the utilization efficiency of organoids and reduce experimental costs, which has extremely high practical and economic value. Summary of the invention

[0008] To solve the above problems, the present invention uses methacrylated starch to prepare matrix glue. The presence of functional groups can form a more solid three-dimensional network structure of starch gel; on this basis, a photocuring agent is introduced into the starch to cross-link with the photocuring functional groups, which shows better mechanical properties than non-cross-linked starch. The present invention integrates small intestinal organoids with an in vitro simulation device for dynamic digestion of the gastrointestinal tract, which can better simulate digestion and absorption in the human body.

[0009] The first object of the present invention is to provide a method for simulating gastrointestinal digestion based on short-term organoid interactions, wherein the composite gel with small intestinal organoids is transferred to a gastrointestinal digestion device for interaction; wherein,

[0010] The preparation method of the composite gel is as follows:

[0011] Mixing methacrylated starch, phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite and water to obtain a starch mixed solution; mixing the starch mixed solution and matrix glue at a volume ratio of 1-1.5:10-15 to obtain a composite gel, wherein the concentration of methacrylated starch is 10%-12%, and the concentration of phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite is 1%-1.5% (w / w);

[0012] The gastrointestinal digestion device includes: a gastric fluid simulation digestion tank, a pump and an intestinal fluid simulation digestion tank; the intestinal fluid simulation digestion tank is provided with a built-in groove for carrying the composite gel with small intestinal organoids;

[0013] The gastric fluid simulation digestion tank, the pump and the intestinal fluid simulation digestion tank are connected through a silicone tube.

[0014] In one embodiment, the content of small intestinal organoids in the composite gel is 15-20 / 55 μL.

[0015] In one embodiment, the sprouting rate of small intestinal organoids in the composite gel is 0% to 10%.

[0016] In one embodiment, the interaction time is 10 to 16 hours.

[0017] In one embodiment, the discharge flow rate of the digestive fluid from the gastric fluid simulation digestion tank to the intestinal fluid simulation digestion tank is 1.5 mL / min to 2.5 mL / min.

[0018] The second object of the present invention is to provide a gastrointestinal digestion simulation system with recyclable organoids, comprising a gastrointestinal digestion device and a composite gel with small intestinal organoids; wherein:

[0019] The gastrointestinal digestive system includes:

[0020] A gastric fluid simulation digestion tank, a pump, and an intestinal fluid simulation digestion tank; the intestinal fluid simulation digestion tank has a built-in tank for carrying a composite gel with small intestinal organoids;

[0021] The gastric fluid simulation digestion tank, the pump and the intestinal fluid simulation digestion tank are connected through a silicone tube;

[0022] The preparation method of the composite gel is as follows:

[0023] Methacrylated starch, phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite and water are mixed to obtain a starch mixed solution; the starch mixed solution and matrix glue are mixed at a volume ratio of 1-1.5:10-15 to obtain a composite gel, in which the concentration of methacrylated starch is 10%-12% (w / v), and the concentration of phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite is 1%-1.5% (w / w).

[0024] In one embodiment, the content of small intestinal organoids in the composite gel is 15 to 20 / 55 μL; and the budding rate of small intestinal organoids in the composite gel is 0% to 10%.

[0025] The parameters of the gastric fluid simulation digestion tank are as follows: height 9-11 cm, outer diameter 6.5-7.5 cm, inner diameter 6.0-6.5 cm, and capacity 250-275 mL; the parameters of the intestinal fluid simulation digestion tank are as follows: height 9-11 cm, outer diameter 6.5-7.5 cm, inner diameter 6.0-6.5 cm, and capacity 250-275 mL; the parameters of the built-in tank are as follows: inner diameter 1.8-2.2 cm, height 2.2-2.8 cm, metal mesh aperture 0.4-0.5 mm, and the metal mesh is the bottom of the built-in tank for carrying matrix glue.

[0026] In one embodiment, the parameters of the gastric fluid simulation digestion tank are a height of 9 to 11 cm, an outer diameter of 6.5 to 7.5 cm, an inner diameter of 6.5 to 7.5 cm, and a capacity of 250 to 275 mL; the parameters of the intestinal fluid simulation digestion tank are a height of 9 to 11 cm, an outer diameter of 6.5 to 7.5 cm, an inner diameter of 6.0 to 6.5 cm, and a capacity of 250 to 275 mL; the parameters of the built-in tank are an inner diameter of 1.8 to 2.2 cm, a height of 2.2 to 2.8 cm, and a metal mesh aperture of 0.4 to 0.5 mm.

[0027] In one embodiment, the parameters of the gastric fluid simulation digestion tank and the intestinal fluid simulation digestion tank are: height 10 cm, outer diameter 7 cm, inner diameter 6.2 cm, and capacity 250 mL.

[0028] In one embodiment, the inner diameter of the built-in tank in the intestinal fluid simulation digestion tank is 2 cm, the height is 2.5 cm, and the pore size of the metal mesh is 0.5 mm.

[0029] In one embodiment, 40-55 μL of the composite gel is transferred into the inner tank.

[0030] In one embodiment, the digestive juice is prepared by:

[0031] (1) preparing a corn starch suspension, adding SSF electrolyte stock solution to the suspension, vortexing and mixing, and then adding SSF electrolyte stock solution containing α-amylase, CaCl2 and ultrapure water, and mixing thoroughly; finally, adding hydrochloric acid solution to inactivate α-amylase to obtain an oral digestion product;

[0032] (2) adding the original electrolyte solution of SGF to the oral digestion product, and then adding the electrolyte reserve solution of SGF without adding pepsin, CaCl2, and ultrapure water, adjusting the pH of the solution to 3.0 with hydrochloric acid solution, and reacting to obtain gastric digestion product;

[0033] (3) First, add SIF electrolytic reserve solution, pancreatic enzyme, and saccharifying enzyme into an intestinal digestion tank, and then continue to add CaCl2, sodium hydroxide solution, and ultrapure water. After mixing, the pH of the mixed solution reaches 7.0, and the gastric digestate is transferred to the intestinal digestion tank for reaction to obtain intestinal digestate.

[0034] The third object of the present invention is to provide a composite gel, the preparation method of which comprises:

[0035] Methacrylated starch, phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite and water are mixed to obtain a starch mixed solution; the starch mixed solution and matrix glue are mixed according to a volume ratio of 1-1.5:10-15 to obtain a composite gel, in which the concentration of methacrylated starch is 10%-12% (w / v, g / mL), and the concentration of phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite is 1%-1.5% (w / w).

[0036] The fourth object of the present invention is to provide the use of the above-mentioned composite gel in organoids to simulate gastrointestinal digestion.

[0037] In one embodiment, the composite gel is used to encapsulate organoids; the composite gel with organoids is placed in a digestive ring to simulate gastrointestinal digestion.

[0038] Beneficial effects of the present invention

[0039] The present invention provides a short-term interaction system between human gastrointestinal dynamic digestion and small intestinal organoids. The small intestinal organoids can be introduced into an in vitro small intestinal simulated digestive system to achieve the interaction between the in vitro small intestinal simulated digestive system and the organoids, and can be used to study the effects of small molecules of digestive products on the organoids. Specifically:

[0040] (1) The composite gel prepared by the present invention has good mechanical strength (above 1000 Pa) and a stable structure, which is more suitable for organoid digestion simulation;

[0041] (2) After 12 hours of interaction with the simulated digestive fluid, the organoids of the present invention were in good condition and still had a strong proliferation ability, with functions close to those of the non-interacting organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a dynamic digestion device for the gastrointestinal tract;

[0043] Figure 2 It is the growth diagram of small intestinal organoids of Example 1 and Comparative Example 3;

[0044] Figure 3 The Edu fluorescence staining images (A) and Edu / DAPI ratios (B) of small intestinal organoids in Example 1 and some comparative examples;

[0045] Figure 4 The ZO-1 fluorescence staining images (A) and ZO-1 / DAPI ratios (B) of small intestinal organoids of Example 1 and some comparative examples;

[0046] Figure 5 The Muc-2 fluorescence staining images (A) and Muc-2 / DAPI ratios (B) of small intestinal organoids of Example 1 and some comparative examples;

[0047] Figure 6 The mechanical properties of the small intestinal organoid matrix gel of Examples, Comparative Examples 1-2 and Comparative Examples 6-7;

[0048] Figure 7 The mechanical properties of the matrix gel of small intestinal organoids in Example and Comparative Examples 4 to 5. DETAILED DESCRIPTION

[0049] 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.

[0050] Device:

[0051] Gastrointestinal dynamic digestive apparatus Figure 1 As shown, it includes a gastric fluid simulation digestion tank, a pump and an intestinal fluid simulation digestion tank; the intestinal fluid simulation digestion tank has a built-in groove for carrying a composite gel with small intestinal organoids;

[0052] The gastric fluid simulation digestion tank, the pump and the intestinal fluid simulation digestion tank are connected through a silicone tube.

[0053] Optionally, the parameters of the gastric fluid simulation digestion tank are 9-11 cm in height, 6.5-7.5 cm in outer diameter, 6.5-7.5 cm in inner diameter, and 250-275 mL in capacity; the parameters of the intestinal fluid simulation digestion tank are 9-11 cm in height, 6.5-7.5 cm in outer diameter, 6.0-6.5 cm in inner diameter, and 250-275 mL in capacity; the parameters of the built-in tank are 1.8-2.2 cm in inner diameter, 2.2-2.8 cm in height, and 0.4-0.5 mm in metal mesh aperture.

[0054] In the present invention, the parameters of the gastric fluid simulation digestion tank and the intestinal fluid simulation digestion tank are: height 10 cm, outer diameter 7 cm, inner diameter 6.2 cm, and capacity 250 mL.

[0055] In the present invention, the inner diameter of the built-in tank in the intestinal fluid simulation digestion tank is 2 cm, the height is 2.5 cm, and the aperture of the metal mesh is 0.5 mm.

[0056] raw material:

[0057] Corn starch was purchased from Yuanye Technology Biological Co., Ltd.;

[0058] Wheat starch, dimethyl sulfoxide, methacrylic anhydride, triethylamine, and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0059] α-Amylase was purchased from Sigma-Aldrich with an enzyme activity of 50 U / mg;

[0060] Trypsin was purchased from Sigma-Aldrich with an enzyme activity of 1000 U / mg;

[0061] Glycoamylase was purchased from Sigma-Aldrich Company with an enzyme activity of 260 U / mL.

[0062] Preparation method of methacrylated starch:

[0063] Weigh 6g corn starch and 200mL dimethyl sulfoxide (DMSO) in a round-bottom flask, stir and heat to 70°C, heat for 30 minutes until completely gelatinized; after cooling to room temperature (25°C), slowly drop 12mL methacrylic anhydride and 0.24mL triethylamine, wherein triethylamine is used as a nucleophilic substituent. After the reactants are stirred at room temperature for 18h, the product is precipitated in anhydrous ethanol, the precipitated product is dissolved in deionized water, and then precipitated in anhydrous ethanol, repeated 3 times, and finally freeze-dried to obtain methacrylated starch.

[0064] Experimental methods:

[0065] 1. Small Intestinal Organoid Culture:

[0066] (1) Isolation of intestinal crypts

[0067] In a conical tube, cold PBS and human small intestinal tissue were mixed and washed several times until the supernatant was clear, and the PBS solution needed to be sucked off 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 off, the gun 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 off each time; the gun 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.

[0068] (2) Organoid culture, passaging and differentiation:

[0069] Organoid culture:

[0070] 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.

[0071] Organoid passaging:

[0072] 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.

[0073] Organoid Differentiation:

[0074] 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 and the medium was replaced every two days.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 2. Gastrointestinal digestion in vitro:

[0079] Starch needs to be digested by the mouth, stomach and small intestine in the human body. Therefore, in the present invention, in vitro digestion simulation of the mouth and stomach is required before the interaction between the small intestinal digestive juice and the organoids.

[0080] (1) Preparation of simulated digestion electrolyte solution

[0081] Simulated salivary fluid (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) are composed of corresponding electrolyte stock solutions, enzymes, CaCl2 and water. Specifically:

[0082] Electrolyte concentration: KCl solution is 37.3g / L, KH2PO4 solution is 68g / L, NaHCO3 solution is 84g / L, NaCl solution is 117g / L, MgCl2(H2O)6 solution is 30.5g / L, (NH4)2CO3 solution is 48g / L, NaOH solution is 1mol / L, HCl solution is 6mol / L, and the solvents are all water.

[0083] 15.1 mL of KCl solution, 3.7 mL of KH2PO4 solution, 6.8 mL of NaHCO3 solution, 0.5 mL of MgCl2(H2O)6 solution, and 0.06 mL of (NH4)2CO3 solution were added to the SSF electrolytic stock solution, and 0.09 mL of HCl solution was added to adjust the pH to 7.0, and the mixed solution was fixed to 500 mL with deionized water.

[0084] 6.9 mL of KCl solution, 0.9 mL of KH2PO4 solution, 12.5 mL of NaHCO3 solution, 0.4 mL of MgCl2(H2O)6 solution, and 0.5 mL of (NH4)2CO3 solution were added to the SGF electrolytic stock solution, and 1.3 mL of HCl solution was added to adjust the pH to 3.0, and the mixed solution was fixed to 500 mL with deionized water.

[0085] To the SIF electrolytic stock solution were added 6.8 mL of KCl solution, 0.8 mL of KH2PO4 solution, 42.5 mL of NaHCO3 solution, 9.6 mL of MgCl2(H2O)6 solution, and 1.1 mL of (NH4)2CO3 solution, and 0.7 mL of HCl solution was added to a pH of 7.0, and the mixed solution was fixed to 500 mL with deionized water.

[0086] (2) Oral simulated digestion

[0087] Prepare 20mL corn starch suspension (10%, g / mL), add 14mL SSF electrolytic stock solution to the suspension, vortex oscillate to mix, continue to add 2.0mL SSF electrolytic stock solution containing 1500U / mL α-amylase, then add 100μL of 0.3mol / LCaCl2 and 3900μL ultrapure water, and mix thoroughly. All the above solutions need to be heated to 37°C before adding, and then the mixed solution is reacted in a 37°C water bath for 2min, wherein the stirring speed is 100r / min, and finally 0.6mL of 1.0mol / L hydrochloric acid solution is added to inactivate the α-amylase to obtain oral digestive material.

[0088] (3) Gastric juice simulated digestion

[0089] Add 30 mL of SGF electrolytic stock solution to 40 mL of oral digestion product, then add 6.4 mL of SGF electrolytic stock solution without pepsin added, and continue to add 20 μL of 0.3 mol / L CaCl2, 2780 μL of ultrapure water and 0.8 mL of 1.0 mol / L hydrochloric acid solution to adjust the solution pH to 3.0. Then, react at 37°C and a rotation speed of 30 r / min for 60 min to obtain gastric digestate. Transfer the gastric simulated digestion fluid to the intestinal simulated digestion tank at a flow rate of 2 mL / min until all the digestion fluid is transferred.

[0090] (4) Intestinal fluid simulated digestion

[0091] First, add 44 mL of SIF electrolyte reserve solution, 20 mL of pancreatic enzyme (weigh 2 g in 20 mL of SIF electrolyte solution), 0.4 mL of saccharifying enzyme (add 0.2 mL of saccharifying enzyme for every gram of dry starch) into the intestinal digestion tank, and then add 160 μL of 0.3 mol / LCaCl2, 0.60 mL of 1.0 mol / L sodium hydroxide solution and 5.24 mL of ultrapure water. After mixing, the pH of the mixed solution reaches 7.0. Then, the gastric digestate is transferred to the intestinal digestion tank and reacted at 37°C and 30 r / min for 2 h to obtain the intestinal digestate.

[0092] Test method:

[0093] 1. Immunofluorescence experiment of small intestinal organoids

[0094] (1) Proliferation:

[0095] The proliferation of intestinal organoids was detected using the BeyoClickTM EdU cell proliferation kit. Intestinal organoids were cultured in a 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. DPAI counterstained the cell nucleus, and the layer-by-layer scanning was performed using a laser confocal microscope, and the 3D image was reconstructed.

[0096] (2) Immunofluorescence experiment:

[0097] ZO-1 can reflect the expression of tight junction proteins in the intestine, while Muc-2 can reflect the expression of mucin in the intestine. The secretion of these two proteins is beneficial to the intestinal barrier function.

[0098] Aspirate the culture medium from the wells of the culture plate 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 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 organoid adsorption 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 antibodies to each well and incubate overnight at 4°C with gentle shaking. The dilution ratio of Muc2 primary antibody is 1:500 and the dilution ratio of ZO-1 primary antibody is 1:50. Wash the organoids 5 times with organoid wash buffer for 5 minutes each time. Add 200 μL of fluorescently labeled secondary antibody and incubate at room temperature for 2 hours. Subsequently, wash the organoids 5 times with organoid wash buffer for 5 minutes each time. Counterstain the nuclei and live cell nuclei with DAPI, scan layer by layer with a laser confocal microscope, and stitch and reconstruct the images.

[0099] 2. Photorheology experiment

[0100] When the composite gel is formed, the photorheology of the composite gel is performed using a MARS 60 instrument (Thermo Fisher Scientific) equipped with an accessory light module. Before the photorheological test, the starch and matrix gel mixture was subjected to a 30s non-irradiated rheological test to reach a steady state before irradiation. The UV light intensity was set to 28mW / cm2, and irradiation was performed from the bottom of the sample. A time scanning experiment was performed in the linear viscoelastic region with a constant strain amplitude of 0.5% and a constant frequency of 6rad / s to track the change of the elastic storage modulus G' over time to monitor the formation of in situ gel. The storage modulus G' measures how much elastic deformation modulus a material can store when it is denatured, reflecting the elastic characteristics of the material. In the present invention, starch gel is added to improve the mechanical properties of the organoid matrix gel, so that the mechanical strength of the composite hydrogel is maximized while the organoid can grow normally. Since the organoid needs to grow in the three-dimensional network of the hydrogel, the mechanical strength is too high, or too much starch is added, which affects the growth space of the organoid, and then affects its growth.

[0101] 3. Rheological test

[0102] For the formed gel, rheological tests can be performed directly. The rheological properties of the gel were measured by a DHR-3 rheometer at 25°C, 40 mm, and 1 mm gap on a plate. Dynamic oscillation frequency analysis was performed within the linear viscoelastic range, and the dynamic oscillation frequency range was 0.1 to 100 rad / s.

[0103] Example 1: A method for reusable organoids and simulating short-term interaction between digestive fluid and organoids

[0104] A method for reusable organoids and simulating short-term interaction between digestive fluid and organoids, comprising the following steps:

[0105] (1) Preparation of composite gel

[0106] Preparation of starch mixed solution: methacrylated starch and water are mixed and dissolved, and a photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite) is added in an amount of 1% by weight of the methacrylated starch, and mixed to prepare a starch mixed solution;

[0107] Preparation of mixed matrix gel: 500 μL matrix gel and 50 μL starch mixed solution (the starch mixed solution contains 55 mg methacrylated starch and 0.55 mg LAP) were used to prepare a composite gel, i.e., methacrylated starch accounted for 10% (w / v, g / mL) of the composite gel;

[0108] (2) Preparation of small intestinal organoids

[0109] Take the small intestinal organoid precipitate, dissolve it with 5 μL growth medium, add it to 55 μL composite gel and mix well; after mixing, add 55 μL / well of composite gel into a 24-well plate (8-10 organoids per well) and incubate at a light intensity of 115 mW / cm 2 Irradiate under UV-DIMAX UV lamp for 120s to construct starch gel network structure; place the 24-well plate in a 37°C incubator for 10-20min (the duration has no effect on the gel structure), and after the composite gel is polymerized, add 500μL growth medium to each well, and replace the growth medium every two days;

[0110] (3) Small intestinal organoid interactions

[0111] When the number of organoids in the 24-well plate reaches 15 to 20 per well, the growth medium is replaced with differentiation medium, i.e., on the first day of differentiation, the organoid budding rate in each well is cultured until it reaches 10%; the composite gel containing the differentiated organoids (55 μL; 15 to 20) is transferred to the built-in glass tank of the intestinal fluid simulation digestion tank (containing intestinal digesta) of the small intestinal digestion simulation system for interaction for 12 hours;

[0112] The germination rate calculation formula is as follows:

[0113]

[0114] In the intestinal fluid simulation digestion tank, the interaction conditions were: 37°C, stirring speed 30r / min, and interaction 12h.

[0115] After the interaction is completed, the organoids are transferred back to the cell culture plate and fresh differentiation medium is added for buffering. After 12 hours, the organoids can differentiate normally.

[0116] Comparative Example 1: Changing the amount of methacrylated starch added in the composite gel

[0117] On the basis of Example 1, the proportion of methacrylated starch in the composite gel in step (1) was changed to 8% (g / mL), the amount of photoinitiator LAP added was still 1% of the mass of starch, and the remaining steps were consistent with Example 1.

[0118] Comparative Example 2: Changing the amount of methacrylated starch added in the composite gel

[0119] On the basis of Example 1, the proportion of methacrylated starch in the composite gel in step (1) was changed to 15% (g / mL), the amount of photoinitiator LAP added was still 1% of the mass of starch, and the remaining steps were consistent with Example 1.

[0120] Comparative Example 3: Changing the state of organoids during interaction

[0121] On the basis of Example 1, when the organoid budding rate in step (3) exceeds 50%, the composite gel containing the differentiated organoids is transferred to the built-in glass tank of the digestion tank of the small intestine digestion simulation system for interaction, and the remaining steps are consistent with Example 1.

[0122] Comparative Example 4: No addition of methacrylated starch

[0123] On the basis of Example 1, in step (1), methacrylated starch and photoinitiator LAP are not added, only matrix glue is used, and the remaining steps are consistent with Example 1.

[0124] Comparative Example 5: No LAP added

[0125] On the basis of Example 1, no photoinitiator LAP is added to the methacrylated starch in step (1), and the remaining steps are consistent with Example 1.

[0126] Comparative Example 6: Changing the type of starch

[0127] On the basis of Example 1, in step (1), methacrylated starch was replaced with corn starch, the added amount was 10% of the mixture of matrix gelatin and starch solution, the added amount of LAP was 1% of the starch, and the remaining steps were consistent with Example 1.

[0128] Comparative Example 7: Changing the Type of Starch

[0129] On the basis of Example 1, in step (1), methacrylated starch was replaced with wheat starch, the added amount was 10% of the mixture of matrix gel and starch solution, the added amount of LAP was 1% of the starch, and the remaining steps were consistent with Example 1.

[0130] The organoids after the interaction of Example 1 and Comparative Examples 1 to 7 were transferred back to the wells of the cell culture plate for recovery, and the steps were as follows:

[0131] Place the cell culture plate on ice to thaw the matrix gel, rinse the pipette tip with cold DPBS containing 0.1% BSA, and gently scrape the bottom of the well with the pipette tip to remove the composite gel droplets, transfer the culture medium and composite gel to a centrifuge tube, centrifuge at 300×g for 3 min at 4°C, aspirate the upper culture medium and gel, calculate 50 μL of matrix gel per well, dilute the organoid precipitate with culture medium that is less than 10% (v / v) of matrix gel, and then dissolve it in the matrix gel, mix well, add 55 μL / well of matrix gel to the culture plate, place the culture plate in a 37°C incubator for 10 to 20 min, after the matrix gel is polymerized, add 500 μL of fresh differentiation culture medium to each well, and place the 24-well culture plate in a 37°C 5% CO2 incubator.

[0132] After the organoids were recovered and cultured for 12 hours, the organoids of Example 1, Comparative Examples 1-2 and Comparative Examples 4-5 were fixed and immunostained with Muc-2 and ZO-1, and the 3D images of the organoids were reconstructed using a laser confocal microscope; the proliferation of the organoids of Comparative Example 3 was detected by the Edu method; when the composite gel was formed, the photorheological test was performed on Example 1 and Comparative Examples 6-7; three holes were selected in Example 1 and Comparative Examples 4-5 to perform rheological tests on the composite gel of the organoids after interaction. The results are as follows Figures 2 to 7 shown.

[0133] The morphology of the organoids before and after the interaction in Example 1 and Comparative Example 3 is as follows Figure 2 As shown, it can be seen that the organoid state of Example 1 is significantly better than that of Comparative Example 3.

[0134] As the ultraviolet irradiation progresses, the G' of the composite gel in Comparative Example 1 is lower than that in Example 1, indicating that the mechanical strength of the composite gel is weaker than that of the composite gel in the example ( Figure 6), the G' of the composite gel of Comparative Example 2 and Example 1 is not much different, but the immunostaining fluorescence intensity of Muc-2 and ZO-1 proteins in the organoid of Comparative Example 2 after interaction is lower than that in Example 1 ( Figure 4 , Figure 5 ), which shows that the growth condition of the small intestinal organoids in Comparative Example 2 is relatively poor;

[0135] After the interaction, the organoids in Comparative Example 3 had incomplete morphology, poor proliferation in the last 12 h of culture, and the fluorescence intensity of Edu staining was significantly lower than that in Example 1 ( Figure 3 );

[0136] In Comparative Examples 4 and 5, no starch gel compounding or light curing was performed. Although the starch was also polymerized due to retrogradation, the network structure formed was relatively fragile, and the mechanical strength of the gel was much lower than that of Example 1 (above 1000 Pa). Figure 7 );

[0137] The G' of the composite gels of Comparative Examples 6 and 7 does not increase with the progress of UV irradiation, which indicates that corn starch and wheat starch cannot be photocured. Although they can be coagulated by starch retrogradation, their mechanical strength is much lower than that of the composite gel of Example 1 ( Figure 6 ).

[0138] Example 2: Recovering organoids for other experiments or testing

[0139] The organoids after the interaction in Example 1 were collected and cultured for 12 hours, and the proliferation of small intestinal organoids was detected using the BeyoClickTM EdU cell proliferation kit. The organoids that did not participate in the interaction were used as controls. The results are as follows: Figure 3 shown.

[0140] The results showed that the proliferation ability of the organoids after interaction was close to that of the non-interacting organoids. It can be seen that the organoids after interaction still have good functions and structures and can be suitable for subsequent experiments.

[0141] 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. A method for simulating gastrointestinal digestion based on short-term organoid interactions, characterized in that: The composite gel with small intestinal organoids was transferred to the gastrointestinal digestive apparatus for interaction; wherein, The preparation method of the composite gel is as follows: Mixing methacrylated starch, phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite and water to obtain a starch mixed solution; mixing the starch mixed solution and matrix gel at a volume ratio of 1-1.5:10-15 to obtain a composite gel, wherein the concentration of methacrylated starch is 10%-11% g / mL, and the concentration of phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite is 1%-1.5% w / w; The gastrointestinal digestion device includes: a gastric fluid simulation digestion tank, a pump and an intestinal fluid simulation digestion tank; the intestinal fluid simulation digestion tank is provided with a built-in groove for carrying the composite gel with small intestinal organoids; The gastric fluid simulation digestion tank, the pump and the intestinal fluid simulation digestion tank are connected through a silicone tube.

2. The method according to claim 1, characterized in that: The content of small intestinal organoids in the composite gel was 15 to 20 / 55 μL.

3. The method according to claim 1, characterized in that The budding rate of small intestinal organoids in the composite gel was 0% to 10%.

4. The method according to claim 1, characterized in that: The interaction time is 10 to 16 hours.

5. The method according to claim 1, characterized in that The flow rate of the digestive fluid discharged from the gastric fluid simulation digestion tank to the intestinal fluid simulation digestion tank is 1.5 to 2.5 mL / min.

6. A gastrointestinal digestion simulation system with recyclable organoids, characterized in that: It includes a gastrointestinal digestion device and a composite gel with small intestinal organoids; wherein, The gastrointestinal digestive system includes: A gastric fluid simulation digestion tank, a pump, and an intestinal fluid simulation digestion tank; the intestinal fluid simulation digestion tank has a built-in tank for carrying a composite gel with small intestinal organoids; The gastric fluid simulation digestion tank, the pump and the intestinal fluid simulation digestion tank are connected through a silicone tube; The preparation method of the composite gel is as follows: Methacrylated starch, phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite and water are mixed to obtain a starch mixed solution; the starch mixed solution and matrix glue are mixed at a volume ratio of 1-1.5:10-15 to obtain a composite gel, in which the concentration of methacrylated starch is 10%-12% g / mL, and the concentration of phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite is 1%-1.5% w / w.

7. The system according to claim 6, characterized in that The content of small intestinal organoids in the composite gel is 15 to 20 / μL; the budding rate of small intestinal organoids in the composite gel is 0% to 10%.

8. The system according to claim 6, characterized in that The parameters of the gastric fluid simulation digestion tank are as follows: height 9-11 cm, outer diameter 6.5-7.5 cm, inner diameter 6.0-6.5 cm, and capacity 250-275 mL; the parameters of the intestinal fluid simulation digestion tank are as follows: height 9-11 cm, outer diameter 6.5-7.5 cm, inner diameter 6.0-6.5 cm, and capacity 250-275 mL; the parameters of the built-in tank are as follows: inner diameter 1.8-2.2 cm, height 2.2-2.8 cm, and metal mesh aperture 0.4-0.5 mm.

9. A composite gel, characterized in that: The preparation method comprises: Methacrylated starch, phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite and water are mixed to obtain a starch mixed solution; the starch mixed solution and matrix glue are mixed at a volume ratio of 1-1.5:10-15 to obtain a composite gel, in which the concentration of methacrylated starch is 10%-12% g / mL, and the concentration of phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite is 1%-1.5% w / w.

10. Use of the composite gel according to claim 9 in organoids simulating gastrointestinal digestion.

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