Double-layer cross-linked artificial matrigel and application thereof in small intestine micro-physiological system

Abstract

The invention discloses double-layer cross-linked artificial matrigel and an application method of the double-layer cross-linked artificial matrigel in a small intestine micro-physiological system. Low-concentration calcium ions are adopted for primary crosslinking to form a soft inner layer structure, and then high-concentration calcium ions are used for secondary crosslinking to improve the overall rigidity. The matrigel is used for dynamic culture of small intestine organoid and is combined with a micro-fluidic chip to form a small intestine micro-physiological system. In the system, the digestive absorption process of the small intestine is dynamically simulated by controlling the flow ratio and rate of digestive fluid and culture fluid. Compared with the traditional natural matrigel, the matrigel disclosed by the invention has more appropriate mechanical strength and structural stability, supports long-term growth of the organoids, and improves the multiplication capacity, tight connection and structural integrity of the organoids in an in-vitro environment. The method can be used for researching intestinal diseases, food digestion and drug absorption, and is an in-vitro digestion simulation platform with high biocompatibility and stability.

Description

Technical Field

[0001] The invention relates to a double-layer cross-linked artificial matrix glue and application thereof in a small intestine microphysiological system, belonging to the technical field of food digestion and tissue engineering. Background Art

[0002] Organoid technology plays an important role in simulating the three-dimensional structure and function of human organs. It is an effective tool for studying organ physiology and pathology and is widely used in drug screening and disease model construction. Small intestinal organoids can simulate the digestion and absorption process of the small intestine in vitro and are an important model for studying intestinal function. In the human body, the main digestion and absorption process of food occurs in the small intestine. After initial digestion in the stomach, food enters the small intestine, where it is further broken down into small molecules and absorbed into the blood through small intestinal epithelial cells. Therefore, small intestinal organoids have broad application prospects in food digestion and drug absorption research.

[0003] At present, the culture matrix of small intestinal organoids mainly uses naturally extracted matrix gel, such as matrix gel taken from mouse sarcoma. This type of matrix gel has complex components and significant differences between batches, resulting in poor stability of organoids during culture and showing great heterogeneity. In addition, the mechanical properties of natural matrix gel are weak and it is easy to disintegrate during in vitro digestion simulation, which affects the repeatability of the experiment and the accuracy of the results, limiting its application in dynamic simulation environments.

[0004] In contrast, artificial matrix gel can be customized through synthetic materials and cross-linking technology to more precisely control material composition and mechanical properties, thereby improving batch consistency and biocompatibility. However, although artificial matrix gel has solved some of the problems of natural matrix gel to a certain extent, there are still some limitations. Most current artificial matrix gels lack mechanical strength and stability, and are easily disintegrated or dissolved in dynamic simulation environments such as microfluidic systems, making it difficult to meet the long-term culture requirements of small intestinal organoids under dynamic digestion conditions. This limits its application in small intestinal microphysiological systems.

[0005] Therefore, there is an urgent need for an improved artificial matrix gel material that can maintain high strength and stability in the microfluidic system and support the long-term growth of organoids under dynamic digestion conditions. This method should be able to be closer to the real small intestinal digestion and absorption process in the human body, simulate the dynamic physiological environment, and provide a reliable and efficient research platform. Summary of the invention

[0006] To solve the above problems, the present invention provides a sodium alginate-fibronectin-chitosan artificial matrix glue prepared by a double-layer cross-linking method, and uses it in a small intestinal microphysiological system to achieve a combination of dynamic digestion simulation and organoid culture. Specifically, it includes the following contents:

[0007] The first object of the present invention is to provide a method for simulating in vitro small intestinal digestion, by inserting digesta and culture medium into a small intestinal organoid chip to simulate in vitro small intestinal digestion;

[0008] The method for constructing the small intestine organoid chip is as follows:

[0009] (1) mixing a small intestinal organoid suspension with a chitosan solution to obtain a small intestinal organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution;

[0010] (2) mixing the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-0.1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain the small intestinal organoid matrix gel;

[0011] (3) The small intestinal organoid matrix gel is implanted into a chip with a chamber and cultured to obtain a small intestinal organoid chip.

[0012] 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;

[0013] 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.

[0014] In one embodiment, in step (2), the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution are mixed in a volume ratio of 0.8-1.2:1-1.4;

[0015] In the small intestinal organoid chip of step (3), the number of small intestinal organoids is 5 to 20 per chip.

[0016] In one embodiment, the small intestinal organoid suspension is obtained by resuspending the organoid pellet in culture medium.

[0017] In one embodiment, the flow rate of digestate and culture medium into the small intestinal organoid chip is 5-15 μL / min.

[0018] In one embodiment, the small intestinal organoids are intestinal crypt cells of human or murine origin.

[0019] In one embodiment, the chip (and the microwell array of the chip) can be designed by CAD software. The microwell depth of the microwell array of the organoid chip is 1.0 mm, the microwell diameter is 2.0 mm, the middle chamber length is 4.5 mm, the chamber diameter is 1.5 mm, the chamber depth is 0.5-1.5 mm, and the connecting channel size is 100 μm×100 μm×100 μm. Within this size range, the normal growth of organoids can be guaranteed while facilitating fluid replacement operations.

[0020] In one embodiment, after the initial cross-linking with 0.05-0.1 M calcium chloride, the supernatant needs to be discarded, and then 0.1-0.3 M calcium chloride solution is added for secondary cross-linking.

[0021] A second object of the present invention is to provide a method for preparing a double-layer cross-linked artificial matrix glue, the method comprising:

[0022] (1) mixing the organoid suspension with the chitosan solution to obtain an organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution;

[0023] (2) mixing the organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain a double-layer cross-linked artificial matrix glue;

[0024] Optionally, the concentration of chitosan in the organoid-chitosan mixture of step (1) is 60 mg / mL to 100 mg / mL;

[0025] Optionally, 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;

[0026] Optionally, the number of organoids in the double-layer cross-linked artificial matrix gel in step (2) is 5 to 20 per sheet.

[0027] The third object of the present invention is to provide a double-layer cross-linked artificial matrix glue prepared by the above method.

[0028] The fourth object of the present invention is to provide a method for improving the growth state of organoids in an organoid chip, using the above-mentioned double-layer cross-linked artificial matrix gel to wrap the organoids and implant them in the organoid chip.

[0029] A fifth object of the present invention is to provide a method for constructing a small intestinal organoid chip, the method comprising:

[0030] (1) mixing a small intestinal organoid suspension with a chitosan solution to obtain a small intestinal organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution;

[0031] (2) mixing the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-0.1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain the small intestinal organoid matrix gel;

[0032] (3) implanting the small intestinal organoid matrix gel into a chip with a chamber to obtain a small intestinal organoid chip;

[0033] Optionally, the concentration of chitosan in the small intestinal organoid-chitosan mixture of step (1) is 60 mg / mL to 100 mg / mL;

[0034] Optionally, 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;

[0035] Optionally, in step (2), the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution are mixed in a ratio of 0.8 to 1.2: 1 to 1.4;

[0036] Optionally, in the small intestinal organoid chip of step (3), the number of small intestinal organoids is 5 to 20 per chip;

[0037] Optionally, the intestinal organoids are derived from intestinal crypt cells of human or mouse origin.

[0038] Optionally, in a chip with an elliptical cylindrical chamber, the chamber length is 3 to 4.5 mm, the chamber diameter is 1.5 to 2.5 mm, and the chamber depth (height) is 0.5 to 1.5 mm.

[0039] The sixth objective of the present invention is to provide a small intestinal organoid chip prepared by the above method.

[0040] Beneficial effects of the present invention

[0041] The present invention provides a sodium alginate-fibronectin-chitosan artificial matrix glue prepared based on a double-layer cross-linking method, and uses it in a small intestinal microphysiological system to achieve a combination of dynamic digestion simulation and organoid culture;

[0042] Specifically:

[0043] (1) The composition of the artificial matrix gel of the present invention is controllable and highly reproducible, and the composition can be precisely controlled and optimized to avoid the presence of unnecessary growth factors or immunogenic substances, thereby providing an environment more conducive to the stable growth of organoids;

[0044] (2) The double-layer cross-linked matrix gel of the present invention has high mechanical strength, which can improve the structural stability of small intestinal organoids and help maintain the integrity of organoids in a dynamic microfluidic system.

[0045] (3) The organoid chip of the present invention has a long-lasting and stable effect in simulating long-term digestion and absorption in the small intestine, thereby improving the overall dissolution efficiency.

[0046] (4) The simulation results of the present invention are highly similar to the digestion results in mice and have high authenticity. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the small intestine organoid chip;

[0048] Figure 2 is the Matrigel strength value;

[0049] Figure 3 Comparison diagram of organoids before and after differentiation under high mechanical strength matrix gel (Comparative Example 7 (2), Comparative Example 8 (2));

[0050] Figure 4 are the results of fluorescence staining; A is the Edu fluorescence staining image, and B is the Edu / DAPI ratio;

[0051] Figure 5 are the results of fluorescence staining; A is the ZO-1 fluorescence staining image, and B is the ZO-1 / DAPI ratio;

[0052] Figure 6 Are the results of fluorescence staining; A is the Muc-2 fluorescence staining image, and B is the Muc-2 / DAPI ratio.

[0053] Figure 7 It is the result of starch hydrolysis rate.

[0054] Figure 8 Chain length distribution of starch digestion products, compared with the data in mice. DETAILED DESCRIPTION

[0055] 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.

[0056] raw material:

[0057] Corn starch was purchased from Prostar Starch Co., Ltd.;

[0058] Fibronectin was purchased from Thermo Fisher Scientific;

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

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

[0061] The basal culture medium used was DMEM / F12 or Advanced DMEM / F12 supplemented with 10 mM HEPEs, 2 mM Glutamax, and 100 units / mL Penicillin-streptomycin.

[0062] 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.

[0063] 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.

[0064] Experimental methods:

[0065] 1. Crypt Isolation and Extraction

[0066] Crypts were extracted from the colon of 4-6 week old C57BL / 6 mice as follows:

[0067] Mice were anesthetized and killed by cervical vertebrae dissection. The middle section of the colon (3-4 cm) was removed and stored in pre-cooled phosphate buffered saline (DPBS). The contents were removed, the colon was cut open, the villi and adipose tissue were scraped off, and the colon was washed 10 times with pre-cooled DPBS. The colon was cut into 2 mm segments and gently washed 10 times with DPBS until the supernatant was clear. The DPBS was aspirated after each wash. 10 mL of 2 mM Digest with EDTA / DPBS solution at 4°C for 20 min, with gentle shaking at intervals of 5 min; after digestion, allow to precipitate naturally, absorb EDTA, rinse the pipette tip with 0.1% BSA / DPBS, and then gently wash the colon segment three times with 0.1% BSA / DPBS, and gently absorb the supernatant each time; rinse the pipette tip (absorb and beat in BSA / DPBS several times), cut off the front section, and then blow the colon segment with 0.1% BSA / DPBS for 30-40 times, and the supernatant can be seen to be turbid; filter the supernatant through a 70μm filter after rinsing, repeat three times, combine the supernatants, centrifuge at 600r / min for 3min, remove the supernatant, and obtain intestinal crypts.

[0068] 2. Organoid Passaging and Culture

[0069] After 7 to 10 days in culture, when the density of intestinal crypts reaches 150 organoids per well, the organoids are passaged at a 1:2 ratio to avoid overgrowth of organoids and accumulation of excess debris in the cavity.

[0070] Rinse the pipette tip with cold DPBS containing 0.1% BSA, 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, gently pipet up and down, centrifuge at 300×g at 4°C for 3 minutes, discard the upper layer of culture medium and matrix gel to obtain the organoid precipitate.

[0071] Detection method:

[0072] 1. Proliferation:

[0073] 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.

[0074] 2. Immunofluorescence experiment:

[0075] Aspirate the medium from the wells and wash the Matrigel with 1 mL PBS without disrupting the 3D matrix. Add 1 mL of cold cell recovery solution to each well and incubate at 4°C for 30 min. After incubation, the Matrigel is dissolved and dispersed. Use a pipette tip rinsed with 0.1% BSA in cold PBS to handle subsequent organoids to avoid 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 Muc2 primary antibody is diluted at a ratio of 1:500 and the ZO-1 primary antibody is diluted at a ratio of 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 with shaking. Subsequently, wash the organoids 5 times with organoid wash buffer for 5 minutes each time. Counterstain the nuclei and live cell nuclei with DAPI and PI, scan layer by layer with a laser confocal microscope, and stitch and reconstruct the images.

[0076] 3. Matrigel stiffness test

[0077] 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.

[0078] Example 1: Preparation of small intestinal organoid chips

[0079] 1. Preparation of polydimethylsiloxane (PDMS) chip

[0080] (1) The microwell array of the chip was designed using CAD software. The microwell depth of the microwell array of the organoid chip was 1.0 mm, the microwell diameter was 2.0 mm, the middle chamber length was 4.5 mm, the chamber diameter was 1.5 mm, the chamber depth was 1 mm, and the connecting channel size was 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 1 shown.

[0081] 2. Organoid colonization

[0082] The steps for preparing artificial matrix gel and implanting organoids by double-layer cross-linking method are as follows:

[0083] (1) Take the intestinal organoid precipitate and resuspend it with DPBS solution (PBS solution without calcium ions) to obtain 20 / μL organoid suspension; mix the chitosan solution and the intestinal organoid suspension in a volume ratio of 1:1 (v / v), the final concentration of the chitosan solution is 8% (w / v, g / mL), and the intestinal organoid concentration is 10 / μL, so that the organoids are evenly distributed in the solution to obtain an intestinal organoid-chitosan mixture; prepare an 8% (w / v, g / mL) sodium alginate solution, add fibronectin to 100 μg / mL, fully dissolve and mix, and obtain a sodium alginate-fibronectin solution;

[0084] (2) Sodium alginate-fibronectin solution and intestinal organoid-chitosan mixture were mixed in a ratio of 1:1 (v / v); an excess of 0.1 M 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; an excess of 0.2 M 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 implanted per chip, and the culture was continued 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.

[0085] Comparative Example 1: Low-concentration single-layer cross-linked matrix glue

[0086] On the basis of Example 1, in step 2 "organoid colonization", only 0.1 M calcium chloride was used for primary cross-linking for 10 minutes, and no secondary cross-linking was performed. The remaining steps were consistent with Example 1.

[0087] Comparative Example 2: High-concentration single-layer cross-linked matrix glue

[0088] On the basis of Example 1, in step 2 "organoid colonization", only 0.2 M calcium chloride was used for primary cross-linking for 10 minutes, and no secondary cross-linking was performed. The remaining steps were consistent with Example 1.

[0089] Comparative Example 3: Changing the addition ratio of sodium alginate

[0090] On the basis of Example 1, in step 2 "organoid colonization", the addition ratio of sodium alginate was changed to 6% w / v, and the remaining steps were consistent with Example 1 to prepare a small intestinal organoid chip.

[0091] Comparative Example 4: Changing the addition ratio of sodium alginate

[0092] On the basis of Example 1, in step 2 "organoid colonization", the addition ratio of sodium alginate was changed to 10% w / v, and the remaining steps were consistent with Example 1 to prepare a small intestinal organoid chip.

[0093] Comparative Example 5: Changing the Source of Matrigel

[0094] On the basis of Example 1, steps 1 and 2 were changed, and the artificial matrix gel was replaced with mouse sarcoma soluble basement membrane extract (i.e., natural matrix gel) at a concentration of 5 mg / mL. The operation was performed on ice, and the intestinal organoids were mixed with Matrigel matrix gel (10 cells / μL) and directly placed in the culture chamber of the chip. 20 organoids / well were colonized and cultured for 5 days. The growth medium was replaced with differentiation medium to obtain a small intestinal organoid chip. After 5 days of differentiation culture, complete small intestinal organoids were formed.

[0095] The remaining steps were the same as in Example 1 to prepare a small intestinal organoid chip.

[0096] Comparative Example 6: Changing the Source of Matrigel

[0097] On the basis of Comparative Example 5, the concentration of mouse sarcoma soluble basement membrane extract was changed to 10 mg / mL, and the remaining steps were consistent with the embodiment and Comparative Example 5 to prepare a small intestinal organoid chip.

[0098] Comparative Example 7: Changing the concentration of calcium chloride

[0099] (1) On the basis of Example 1, in step 2 "organoid colonization", the calcium chloride concentration during the initial cross-linking was changed to 0.05 M. The remaining steps were consistent with Example 1 to prepare a small intestinal organoid chip.

[0100] (2) On the basis of Example 1, in step 2 "organoid colonization", the concentration of calcium chloride during the initial cross-linking was changed to 0.5 M, and the remaining steps were consistent with Example 1 to prepare a small intestinal organoid chip.

[0101] Comparative Example 8: Changing the concentration of calcium chloride

[0102] (1) On the basis of Example 1, in step 2 "organoid colonization", the concentration of calcium chloride during secondary cross-linking was changed to 0.05 Mxxx, and the remaining steps were consistent with Example 1 to prepare a small intestinal organoid chip.

[0103] (2) On the basis of Example 1, in step 2 "organoid colonization", the concentration of calcium chloride during secondary cross-linking was changed to 0.5 M, and the remaining steps were consistent with Example 1 to prepare a small intestinal organoid chip.

[0104] Example 2: Detecting organoid chip performance

[0105] The matrix glue prepared in Example 1 and Comparative Examples 1 to 8 was tested for stiffness;

[0106] (1) Matrigel stiffness test

[0107] Matrigel stiffness Figure 2 As shown, the results show that the G' (storage modulus) values ​​of the matrix glue prepared in Example 1 and Comparative Examples 1 to 8 are compared. It can be seen that Example 1 has suitable mechanical strength, and the stiffness is not significantly increased compared with the naturally extracted matrix glue (such as Comparative Examples 5 and 6). As shown in Comparative Examples 7 and 8, the calcium chloride concentration during the primary and secondary cross-linking is too low, which will cause the mechanical strength of the artificial matrix glue to be significantly reduced, but increasing the calcium chloride concentration will also significantly increase the mechanical strength of the artificial matrix glue. The results show that the double-layer cross-linking method can effectively improve the stiffness of the matrix glue and increase the stability of the matrix glue, which is consistent with the natural matrix glue, making it more suitable for use in the dynamic environment of the microfluidic small intestine organoid chip.

[0108] Natural matrix gel may contain residual biological components, undesirable trace substances and immunogenic substances, which can adversely affect the growth and differentiation of organoids. In addition, due to the batch variability of natural components, the concentration and composition of growth factors and cytokines in natural matrix gel will vary, resulting in unstable organoid state. In contrast, artificial matrix gel can precisely control the composition and optimize the design due to its controllable and highly reproducible composition, avoiding the presence of unnecessary growth factors or immunogenic substances, and providing an environment for organoids that is more conducive to stable growth. Therefore, although natural and artificial matrix gels are similar in stiffness, the high consistency and composition advantages of artificial matrix gel ensure a more stable state and good growth effect of organoids.

[0109] Example 3: A method for simulating small intestinal digestion based on small intestinal organoid chips

[0110] 1. In vitro digestion simulation of the intestine

[0111] Preparation of mixed enzyme: 4.5 g of pancreatic enzyme was dissolved in 40 mL of deionized water, vortexed for 10 min to make it evenly dispersed, centrifuged at 5000×g for 10 min, and 27 mL of supernatant was mixed with 27 mg of amyloglucosidase to obtain mixed coal.

[0112] Weigh 200 mg of starch sample in a centrifuge tube, add 4 mL of deionized water, 4 mL of sodium acetate buffer (pH 5.2) and 10 glass balls in sequence, mix well, and place in a 37°C water bath shaker (170 r / min) for 30 min; take 2 mL of mixed enzyme and add it to the mixed system to obtain digestion solution, and keep it at a low temperature of 4°C to avoid increasing enzyme activity. The digestion solution can be mixed with the organoid culture medium in the organoid chip through a pressure-driven pump to achieve simulated digestion.

[0113] 2. Simulate small intestinal digestion

[0114] Take the small intestinal organoid chip prepared in Example 1, and the digestive fluid enters the pipe connected to the chip hole 1 through a pressure-driven pump at a flow rate of 10 μL / min; the pipe connected to the chip hole 2 is inserted into the differentiation medium at a flow rate of 10 μL / min, that is, the introduction ratio of the digestive fluid and the culture medium is 1:1; the chip hole 3 discharges the waste liquid at a flow rate of 20 μL / min, and the organoid chip is placed in the environmental culture chamber in the cell culture instrument (carbon dioxide concentration is 5%, temperature is 37°C) and simulated for 3 days.

[0115] The simulation effects of the small intestinal organoid chips prepared in Comparative Examples 1 to 8 were tested in the same manner, and the results were as follows:

[0116] (1) Organoid growth assay

[0117] according to Figure 3 The results show that high concentrations of calcium chloride have a significant effect on the growth of the matrix gel in Comparative Example 7 (2) and Comparative Example 8 (2). High concentrations of calcium chloride increase the hardness of the matrix gel during the cross-linking process, inhibit the expansion and growth of organoids, and cause the morphology of the differentiated organoids to be incomplete or significantly reduced. At the same time, low concentrations of calcium chloride in Comparative Example 7 (1) and Comparative Example 8 (1) failed to form a sufficient cross-linking network, and the matrix gel was difficult to form and no further culture was performed. Therefore, high concentrations of calcium chloride have an adverse effect on the normal growth of organoids during the cross-linking process of the matrix gel, while too low a concentration leads to insufficient matrix formation, ultimately affecting the development and morphological stability of the organoids.

[0118] The EdU staining results of the small intestinal organoids in the small intestinal organoid chip after 3 days of culture are shown in Figure 2. Figure 4 As shown in A, the fluorescence intensity ratio of EdU / DAPI is Figure 4 B. The results show that the ratio of Example 1 is significantly higher than that of Comparative Examples 1 to 6 (p<0.0001), indicating that the small intestinal organoids cultured in a double-layer cross-linked matrix gel have higher proliferation activity. In contrast, in Comparative Examples 3 and 4 (high concentration of sodium alginate), the proliferation activity of the small intestinal organoids decreased significantly (p<0.05), showing the negative effect of the amount of sodium alginate added on the growth of the organoids. The results show that the matrix gel configuration of Example 1 is more conducive to the proliferation of organoids.

[0119] (2) Organoid tight junction detection

[0120] The results of ZO-1 staining after 3 days of culture of small intestinal organoids in the small intestinal organoid chip are shown in Figure 5 As shown in A, the fluorescence intensity ratio of ZO-1 / DAPI is Figure 5B. The results showed that the expression of ZO-1 in Example 1 was significantly higher than that in all comparative samples (p<0.0001), especially the high-concentration single-layer cross-linked sample (Comparative Example 2) showed a lower tight junction (p<0.001). This shows that the double-layer cross-linked matrix gel can maintain the tight junction between cells of the organoid while improving the mechanical strength, which helps to maintain the intact structure of the organoid.

[0121] (3) Organoid stability testing

[0122] The results of Muc-2 staining after 3 days of culture of small intestinal organoids in the small intestinal organoid chip are shown in Figure 6 As shown in A, the fluorescence intensity ratio of Muc-2 / DAPI is Figure 6 B. The results show that the expression of Muc-2 in Example 1 is significantly higher than that in the comparative sample (p<0.05), showing better structural stability. In contrast, in Comparative Examples 5 and 6 (naturally extracted matrix gel), the expression of Muc-2 in the organoids is lower, indicating that they are more easily disintegrated in a dynamic digestive environment. The results show that the double-layer cross-linked matrix gel significantly improves the structural stability of the small intestinal organoids and helps to maintain the integrity of the organoids in the dynamic microfluidic system.

[0123] Comparative Example 9: Comparison with dynamic digestion without small intestinal organoids

[0124] On the basis of Example 3, the small intestinal organoid chip was not inoculated with the organoid, and only the chip and the artificial matrix gel carrier were retained. On the third day of culture, the glucose content in the waste liquid was detected every 3 hours.

[0125] The glucose oxidase-peroxidase method was used to determine the glucose content released at each time point. The operating conditions were as follows: collect the waste liquid at each time point, add 50 μL of glucose determination reagent, put the ELISA plate into the ELISA reader, and determine the absorbance of the supernatant at 520 nm. The ELISA test conditions were as follows: oscillation for 10 seconds, constant temperature at 37°C, and insulation for 10 minutes.

[0126] Hydrolysis rate = mass of hydrolyzed starch / mass of total starch × 100%.

[0127] In the small intestinal digestion and absorption experiment, the digestion conditions of Example 3 (with organoids added) and Comparative Example 9 (without organoids added) at different time points (3h, 6h, 9h, 12h, 15h, 18h, 21h, 24h) were compared and analyzed. The experimental results showed that the digestion efficiency (dissolution rate) in Example 3 was significantly higher than that in Comparative Example 9 (see Table 1). Figure 7). Digestion efficiency is significantly improved: During the entire 24-hour digestion process, the dissolution rate of Example 3 at each time point was significantly higher than that of Comparative Example 9, and the difference was statistically significant (p<0.05). This shows that the addition of organoids significantly improves the digestion efficiency. The dissolution rate advantage is obvious: Especially in the initial stage of digestion (3h, 6h, 9h), the dissolution rate of Example 3 is significantly ahead of Comparative Example 9, indicating that the addition of organoids helps to simulate a more realistic intestinal environment, making the digestion and absorption process of nutrients in the small intestine more efficient. Sustained and stable effect: Even at subsequent time points (15h, 18h, 21h, 24h), the dissolution rate of Example 3 still remains at a high level, indicating that the role of organoids in simulating long-term digestion and absorption in the small intestine is long-lasting and stable, thereby improving the overall dissolution efficiency.

[0128] Comparative Example 10: Products after starch digestion in the small intestine of mice

[0129] Healthy male 8-week-old C57BL / 6J mice were selected as in vivo controls. Before the experiment, the mice were fasted but not watered for 12 hours to allow the original contents in the gastrointestinal tract to be digested or emptied as much as possible. The starch samples were prepared into a 2.5g / mL suspension with deionized water. After mixing, the mice were gavaged at a dose (2mg / g body weight) and then anesthetized with isoflurane. 30 minutes after gavage, the mice were killed by cervical dislocation, and then the intestinal contents were collected by dissection, and immediately frozen in liquid nitrogen, and the samples were stored at -80°C for later use.

[0130] The chain length distribution of the starch digestion products of Example 3 and Comparative Example 10 was determined using a high pressure ion chromatography system. A mixture of the sample (10 mg) and sodium acetate buffer (5 mL, pH 4.5) was heated at 100°C for 30 min with continuous stirring. Subsequently, 2 U of isoamylase (E-ISAMYHP, 500 U / mL, Novozymes) was added and stirred continuously at 40°C for 12 h. After the reaction was complete, the enzyme was inactivated by heating at 100°C for 10 min. After the solution was centrifuged, the supernatant was filtered through a 0.45 μm filter for testing.

[0131] Figure 8Show the chain length distribution of starch digestion products, and compare the DP (Degree of Polymerization) of Example 3 (organoid-based small intestine digestive system) and Comparative Example 10 (digestion products in mice) respectively. There are significant differences in the product chain length distribution between Example 3 and the original starch sample before digestion, but there is no significant difference from Comparative Example 10. Example 3 significantly increases the proportion of short-chain products with DP≤5, and the content of medium-chain products (such as 6<DP≤12 and 12<DP≤24) is less, indicating that starch is decomposed in the small intestine digestive system with added organoids. It can be seen that Example 3 is close to the digestion conditions in the mouse body in terms of simulating the product characteristics of starch digestion in the small intestine and can effectively restore the digestion process in the mouse small intestine environment.

[0132] Therefore, the present invention provides an in vitro method that effectively simulates the in vivo digestion process through the small intestine organoid digestive system, has a high similarity with the in vivo digestion results in mice, and at the same time realizes the effective regulation of the starch chain length distribution.

[0133] The above results show that the double-layer cross-linked artificial matrix glue of Example 1 is significantly superior to other comparative example samples. This matrix glue not only provides good mechanical support, but also promotes the healthy growth and function maintenance of organoids, and is suitable for dynamic digestion simulation in the small intestine microphysiological system.

[0134] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for simulating small intestinal digestion in vitro, characterized in that: By inserting digesta and culture medium into the small intestinal organoid chip, in vitro small intestinal digestion was simulated; The method for constructing the small intestine organoid chip is as follows: (1) mixing a small intestinal organoid suspension with a chitosan solution to obtain a small intestinal organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution; (2) mixing the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-0.1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain the small intestinal organoid matrix gel; (3) The small intestinal organoid matrix gel is implanted into a chip with a chamber and cultured to obtain a small intestinal organoid chip.

2. The method according to claim 1, characterized in that In the small intestinal organoid-chitosan mixture of step (1), the concentration of chitosan 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 method according to claim 1, characterized in that In step (2), the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution are mixed in a volume ratio of 0.8-1.2:1-1.4; In the small intestinal organoid chip of step (3), the number of small intestinal organoids is 5 to 20 per chip.

4. The method according to claim 1, characterized in that: The flow rate of digestate and culture medium into the small intestinal organoid chip is 5μL / min~15μL / min.

5. The method according to claim 1, characterized in that Small intestinal organoids are intestinal crypt cells of human or mouse origin.

6. A method for preparing a double-layer cross-linked artificial matrix glue, characterized in that: The method comprises: (1) mixing the organoid suspension with the chitosan solution to obtain an organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution; (2) mixing the organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain a double-layer cross-linked artificial matrix glue; Optionally, the concentration of chitosan in the organoid-chitosan mixture of step (1) is 60 mg / mL to 100 mg / mL; Optionally, 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; Optionally, the number of organoids in the double-layer cross-linked artificial matrix gel in step (2) is 5 to 20 per sheet.

7. The double-layer cross-linked artificial matrix glue prepared by the method according to claim 6.

8. A method for improving the growth state of organoids in an organoid chip, characterized in that: The organoids are encapsulated with the double-layer cross-linked artificial matrix gel as claimed in claim 7 and implanted in the organoid chip.

9. A method for constructing a small intestinal organoid chip, characterized in that: The method comprises: (1) mixing a small intestinal organoid suspension with a chitosan solution to obtain a small intestinal organoid-chitosan mixture; dissolving sodium alginate and fibronectin in water, and mixing to obtain a sodium alginate-fibronectin solution; (2) mixing the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution, adding 0.05-0.1 M calcium chloride solution for preliminary cross-linking for 5-15 min, then adding 0.1-0.3 M calcium chloride solution for secondary cross-linking for 5-10 min, and washing to obtain the small intestinal organoid matrix gel; (3) implanting the small intestinal organoid matrix gel into a chip with a chamber to obtain a small intestinal organoid chip; Optionally, the concentration of chitosan in the small intestinal organoid-chitosan mixture of step (1) is 70 mg / mL to 90 mg / mL; Optionally, in the sodium alginate-fibronectin solution of step (1), the concentration of sodium alginate is 60 mg / mL to 100 mg / mL, and the concentration of fibronectin is 80 μg / mL to 120 μg / mL; Optionally, in step (2), the small intestinal organoid-chitosan mixture and the sodium alginate-fibronectin solution are mixed in a ratio of 0.8 to 1.2: 1 to 1.4; Optionally, in the small intestinal organoid chip of step (3), the number of small intestinal organoids is 5 to 20 per chip; Optionally, the small intestinal organoids are intestinal crypt cells of human or mouse origin; Optionally, in the chip with the elliptical cylindrical chamber, the chamber length is 3 to 4.5 mm, the chamber diameter is 1.5 to 2.5 mm, and the chamber depth is 0.5 to 1.5 mm.

10. The small intestinal organoid chip prepared by the method according to claim 9.

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