System and method for assessing hypoglycemic effects of product

Abstract

The invention provides a system and method for evaluating the hypoglycemic effect of a product, the system comprises a multi-organ chip and a driving device, the multi-organ chip comprises a plurality of chambers arranged in an array, the chambers comprise a second chamber, a third chamber and a fourth chamber which are independently used for culturing liver organs, pancreas islet organs and muscle organs respectively, and the driving device is connected with the second chamber, the third chamber and the fourth chamber; the second chamber, the third chamber and the fourth chamber are connected in series through a fluid micropipe; the driving device is connected with the multiple organ chips and used for driving the multiple organ chips to move. By creating a relatively independent culture environment, the system can be used for constructing an in-vitro platform for evaluating the hypoglycemic curative effect and metabolic pathway of a product, and can also be combined with other sugar regulating organs to further clarify metabolic crosstalk with a sugar regulating organ system, so that a unique and reliable research platform is provided for development of hypoglycemic drugs in the future. Besides, the system can provide a plurality of fluid channels to realize high-throughput detection, so that the time and the economic cost of drug research and development are remarkably reduced, and the application value is high.

Description

Technical Field

[0001] This application relates to the field of biology. Specifically, this application relates to systems and methods for evaluating the blood glucose lowering effect of products. Background Art

[0002] Organ-on-a-chip uses microfluidic technology to control fluid flow, combines cell-cell interactions, matrix properties, and biochemical and biomechanical properties to construct a three-dimensional physiological micro-system of the human organ on the chip. In addition, precise fluid flow control allows for appropriate exchange of nutrients, oxygen, drugs, and waste between the culture medium and cells. Compared with single-organ-on-a-chip models, multi-organ-on-a-chip provides an effective technical means for studying the interactions between different organs. However, current organ-on-chip models face technical challenges such as complex manufacturing processes and single culture modes. Therefore, how to design a multi-organ-on-a-chip system that can switch between "static isolation" and "dynamic interaction" culture modes to improve the above problems has become an important technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0003] It should be noted that this application is based on the inventor's discovery and recognition of the following facts and problems:

[0004] The core pathological mechanism of type 2 diabetes mellitus (T2DM) involves multi-organ interactions, including insulin resistance, pancreatic β-cell dysfunction, and abnormal hepatic gluconeogenesis. Traditional in vitro models (such as monolayer cell culture) cannot simulate the dynamic interactions between organs. In contrast, organ-on-a-chip technology integrates multiple cell types through a microfluidic system, can replicate metabolic coupling and signal transduction between organs, and provides a new platform for T2DM mechanism research and drug development. Organ-on-a-chip technology uses photolithography or printing technology to construct microtissues and provides a suitable microenvironment for their growth and habitation, so as to summarize the specific functions of single or multiple tissues. The core of the multi-organ-on-a-chip lies in connecting multiple organ models through microfluidic technology to simulate their physiological and pathological interactions. It realizes the flow between chambers through fluid strategies dominated by pump drive, gravity drive, or diffusion, and relies on passive diffusion, convective diffusion, or vascularized networks to complete mass transfer. Although significant progress has been made in multi-organ-on-a-chip technology, its manufacturing and application still face challenges. Multi-organ-on-a-chip requires precise microfabrication technology and highly biomimetic cell culture conditions, such as constructing multiple organs and precisely controlling the fluid flow between organs. In addition, the operation and data analysis of the chip require professional knowledge and skills, which limit its application in the clinical and industrial fields.

[0005] The multi-organ chip has shown great potential in type 2 diabetes research, especially in simulating the interactions among the islets of Langerhans, the liver, and the muscles, as well as in drug screening. By simulating the interactions among the islets of Langerhans (insulin secretion), the liver (glucose metabolism), and the muscles (glucose uptake), the multi-organ chip can more accurately study the disease mechanism and the efficacy of drugs. Previous studies have shown that the co-culture of islets of Langerhans and hepatocytes not only successfully simulated the interaction between the function of islet β cells and hepatic glycolipid metabolism but also replicated the hyperglycemic state in pre-diabetes. These findings provide a new in vitro model of T2DM for drug screening or pathological mechanism research. Although the design of microphysiological systems can be used to simulate the interactions between tissues, existing models mainly utilize a single type of islet / hepatocyte and lack the participation of other metabolism-related organs (such as muscles, the intestine, and adipose tissue, etc.). This limitation hinders the comprehensive simulation of the regulation of the in vivo multi-organ network.

[0006] In this study, we developed a new multi-organ interaction platform that enables the dual-mode switching between "static isolation" and "dynamic interaction" through micropore tension regulation and gravity-driven operation. In the static mode, hydrophobic micropore isolation chambers ensure the independent growth of hepatocytes, islet cells, and myocytes. When gravity-driven flow is initiated, the fluid infiltrates into the microtubes to disrupt the surface tension and trigger the interaction mode. Under these conditions, the convective diffusion mechanism achieves multi-organ mass balance within 2 - 3 hours. This platform eliminates the manufacturing dependence on lithography and allows modular assembly within minutes. The validation of the liver-islet-muscle glucose metabolism model shows that the multi-organ system dynamically reproduces physiological glucose fluctuations (2 - 3 hours response time, mimicking postprandial regulation), overcoming the long-standing challenges of independent culture and functional coupling in traditional models. This platform provides a bionic tool for studying diabetes mechanisms, drug evaluation, and the development of personalized therapies, demonstrating significant translational potential.

[0007] To this end, in one aspect of the present application, the present application proposes a system for evaluating the hypoglycemic effect of a product. According to an embodiment of the present application, the system includes: a multi-organ chip, including: a plurality of chambers arranged in an array, each of the chambers including: a first chamber, a second chamber, a third chamber, a fourth chamber, and a fifth chamber, the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber being connected in series through fluid microtubes; the second chamber is used for culturing liver organs; the third chamber is used for culturing islet organs; the fourth chamber is used for culturing muscle organs; the inlet of the fluid microtube is located in the first chamber, and the outlet of the fluid microtube is located in the fifth chamber; the inner and outer wall surfaces of the fluid microtube have hydrophobicity; along the diameter direction of the fluid microtube, a plurality of micropores are distributed on the side wall of the fluid microtube; a driving device, the driving device is connected to the multi-organ chip and is used for driving the multi-organ chip to move.

[0008] The multi-organ chip adopts a physically separated chamber design. Before the interaction of multiple organs, it ensures that each organ can be independently cultured in its respective chamber, so that cell growth is not interfered by cells in other chambers, thus solving the problems of cell viability and state caused by the mixing of culture media in the multi-organ model. The liquid inlet of the fluid microtube can be in contact with the culture solution containing the blood glucose-lowering product, and the liquid inlet and outlet and the inside of the microtube are kept dry under static conditions. Due to the hydrophobicity of the microtube (such as a stainless steel microtube), the culture solution in the second to fourth chambers cannot flow into the fluid microtube, thereby providing an independent space for organ growth. When the organs cultured under static conditions all reach the optimal growth state, the culture medium at the liquid inlet is driven by a gravity drive device to flow into the microtube under the action of gravity, and flows through the micropores on the fluid microtube to flow through the multi-organ chamber, breaking the surface tension above the micropores to promote the contact between the liquid in the microtube and the liquid outside the microtube, thereby realizing the material exchange between multiple organs. Specifically, after the blood glucose-lowering product contacts the islets, it can stimulate insulin secretion. The secreted insulin flows into the second and fourth chambers through the micropores and binds to the insulin receptor, starting the sugar metabolism process, thereby playing a blood glucose-lowering role. By detecting the sugar content and / or metabolites in the cell supernatant, the blood glucose-lowering effect of the product can be effectively evaluated.

[0009] Thus, the system of the present application can be used to construct an in vitro platform for evaluating the blood glucose-lowering efficacy and metabolic pathway of products by creating a relatively independent culture environment. It can be combined with other sugar-regulating organs, and screen blood glucose-lowering drugs by reflecting the metabolic network of the sugar-regulating system, further clarifying the metabolic crosstalk between the sugar-regulating organ systems, providing a unique and reliable research platform for future blood glucose-lowering drug development. In addition, the system of the present application can provide high throughput for simultaneous detection of multiple fluid channels, significantly reducing the time and economic costs of drug research and development.

[0010] According to an embodiment of the present application, the above system for evaluating the blood glucose-lowering effect of a product may further have the following additional technical features:

[0011] According to an embodiment of the present application, the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber are connected in series through a fluid microtube in sequence.

[0012] According to an embodiment of the present application, the lengths of the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber are 5-10 mm respectively, and the widths are 5-10 mm respectively.

[0013] According to an embodiment of the present application, the pore diameter of the micropore is 15-25 μm; the interval between the multiple micropores is 5-10 μm; the tube diameter of the fluid microtube is 2-0.8 mm.

[0014] According to an embodiment of the present application, the fluid microtube is made of stainless steel.

[0015] According to an embodiment of the present application, the system further includes: an extraction instrument for extracting the cell supernatant in the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber; a detection device for detecting glucose, biomarkers, and / or metabolites in the cell supernatant.

[0016] In another aspect of the present application, the present application provides a method for evaluating the hypoglycemic effect of a product using the system for evaluating the hypoglycemic effect of a product described above. According to an embodiment of the present application, the method includes: placing liver cells and a liver culture medium in the second chamber for culture, placing pancreatic islet cells and a pancreatic islet culture medium in the third chamber for culture, placing muscle cells and a muscle culture medium in the fourth chamber for culture; driving the multi-organ chip to move through the driving device so that the culture solution containing the test sample in the first chamber flows into the fluid microtube through the liquid inlet, and flows through the second chamber, the third chamber, and the fourth chamber through the micropores, and the excess liquid flows out through the liquid outlet; collecting and detecting the cell supernatant in the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber.

[0017] According to an embodiment of the present application, the multi-organ chip is placed on a gravity-driven platform, and the gravity-driven platform is driven to tilt up and down by the driving device, with an inclination angle of 5° to 15° and an inclination interval of 45 s to 90 s.

[0018] According to an embodiment of the present application, the culture solution containing the test product continuously flows into the fluid microtube for 2 h to 4.5 h.

[0019] According to an embodiment of the present application, the glucose concentration in the culture solution containing the test product is 20 to 30 mmol / l.

[0020] According to an embodiment of the present application, the culture solution containing the test product is selected from DMEM medium containing the test sample.

[0021] According to an embodiment of the present application, evaluating the hypoglycemic effect of the product includes screening products with hypoglycemic effects.

[0022] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, wherein:

[0024] Figure 1 Shows a schematic diagram of a multi-organ chip structure according to an embodiment of the present application;

[0025] Figure 2 Shows a physical diagram of a multi-organ chip according to an embodiment of the present application;

[0026] Figure 3 Shows a schematic diagram of the multi-organ interaction platform of Example 1; (A) Independent culture conditions for each organ chamber before interaction: no liquid at the inlet and outlet, the microtubes are kept dry, placed horizontally for 24 h to ensure independent cell growth (day 0). (B) Organ chamber interaction conditions (day 1); the drug is added to the inlet, and as the fluid flows into the organ chamber, the substances secreted by the organ chamber after drug stimulation interact with adjacent chambers through micropores. (C) Schematic working principle of the embeddable multi-organ interaction platform;

[0027] Figure 4 Shows the effect of perfusing 70 kDa FITC-dextran fluorescent molecules at the inlet on the microchannel permeability in Example 1; A(i) and A(ii) are for 0 h of perfusion; B(i) and B(ii) are for 1 h of perfusion; C(ii) and C(iii) are for 2 h of perfusion; the data are expressed as mean ± standard deviation, n = 10. One-way ANOVA was used for statistical analysis, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0028] Figure 5 Shows the effect of perfusing 70 kDa FITC-dextran fluorescent molecules in the middle chamber on the microchannel permeability in Example 1; A(i) and A(ii) are for 0 h of perfusion; B(i) and B(ii) are for 1 h of perfusion; C(i) and C(ii) are for 2 h of perfusion; D(i) and D(ii) are for 3 h of perfusion; the data are expressed as mean ± standard deviation, n = 10. One-way ANOVA was used for statistical analysis, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0029] Figure 6 Shows the comparison of dynamic glucose concentrations between the single-organ group and the normal interaction group (NI) in Example 3; the data are expressed as mean ± standard deviation, n = 8 - 10; one-way ANOVA was used for statistical analysis, *P < 0.05, **P < 0.01, ***P < 0.001;

[0030] Figure 7Shows the comparison of dynamic glucose concentrations in different groups in the multi-organ interaction system of Example 3; data are expressed as mean ± standard deviation, n = 8 - 10; one-way ANOVA was used for statistical analysis, *P < 0.05, **P < 0.01, ***P < 0.001;

[0031] Figure 8 Shows the untargeted metabolomics analysis of the combined administration of metformin and repaglinide on the multi-organ interaction platform in Example 3; A: OPLS-DA analysis of the metabolic profiles of the model group, metformin and repaglinide group; B: Volcano plot of differential metabolites between the model group and the metformin and repaglinide group; C: Heat map of differential metabolites between the model group and the metformin and repaglinide group; D - F: Graphs showing the changes in metabolite contents involved in metabolic pathways after treatment with the metformin and repaglinide group; G: Metabolic pathway enrichment after 1 day of treatment with metformin and repaglinide.

[0032] Reference numerals:

[0033] 1000: Multi-organ chip; 100: First chamber; 200: Second chamber; 300: Third chamber; 400: Fourth chamber; 500: Fifth chamber; 600: Fluid microtube. Detailed implementation mode

[0034] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention and should not be construed as a limitation of the present invention.

[0035] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, "a plurality" means two or more.

[0036] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0037] In this article, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0038] The present application provides a system for evaluating the hypoglycemic effect of a product and a method for evaluating the hypoglycemic effect of a product using the same, which will be described in detail below respectively.

[0039] System for evaluating the hypoglycemic effect of a product

[0040] In one aspect of the present application, the present application provides a system for evaluating the hypoglycemic effect of a product (hereinafter, also referred to as "multi-organ interaction platform" in this document). According to an embodiment of the present application, the system includes a multi-organ chip and a driving device, which will be described in detail below respectively.

[0041] According to an embodiment of the present application, referring to Figure 1 and Figure 2 , the multi-organ chip 1000 includes: a plurality of chambers arranged in an array, and each chamber includes: a first chamber 100, a second chamber 200, a third chamber 300, a fourth chamber 400, and a fifth chamber 500. The second chamber 200 is used for culturing liver organs; the third chamber 300 is used for culturing islet organs; the fourth chamber 400 is used for culturing muscle organs. The multi-organ chip adopts a physically separated chamber design to ensure that each organ can grow to the best state in its respective chamber before multi-organ interaction. Exemplarily, the multi-organ chip includes a bottom plate, and a plurality of chambers arranged in an array are formed on the bottom plate.

[0042] According to an embodiment of the present application, referring to Figure 1 and Figure 2 , the first chamber 100, the second chamber 200, and the third chamber 300, the fourth chamber 400, and the fifth chamber 500 are connected in series through a fluid microtube 600. The liquid inlet of the fluid microtube 600 is located in the first chamber 100, and the liquid outlet of the fluid microtube 600 is located in the fifth chamber 500. The inner wall and outer wall surfaces of the fluid microtube 600 have hydrophobicity; along the diameter direction of the fluid microtube 600, a plurality of micropores are distributed on the side wall of the fluid microtube 600.

[0043] When at rest, the liquid inlet and outlet of the fluid microtube remain dry without liquid. Due to the hydrophobicity of the inner wall and outer wall surfaces of the fluid microtube, surface tension will form a stable liquid surface, preventing liquid from entering the hydrophobic pipeline, thereby ensuring the stable growth of cells. When the multi-organ chip is in the interaction mode, the liquid inlet of the fluid microtube can contact the culture solution containing the hypoglycemic product accommodated in the first chamber. The external force of the gravity driving device (such as vibration or tilting) will break the equilibrium state of the liquid surface, overcome the resistance of the surface tension, and the cohesion between liquid molecules will weaken, enabling the liquid to overcome the resistance of the hydrophobic surface and enter the pipeline. Moreover, the micropores on the side wall of the fluid microtube further promote the flow of the liquid. When the liquid is driven into the pipeline during movement, the micropores allow the liquid to pass through and flow through the multi-organ chambers to contact the organs.

[0044] It should be noted that the number of the first chamber, the second chamber, the third chamber, the fourth chamber and the fifth chamber in the multi-organ chip is each one or more, and among them, the second chamber, the third chamber and the fourth chamber are each multiple. Thus, it is beneficial to high-throughput analysis and screening of blood glucose-lowering products. For the number of the first chamber and the fifth chamber being each one or more, the first / fifth chamber can correspond to different fluid microtubes or the same fluid microtube. For example, the first chamber can accommodate the inlets of two or more fluid flow channels, and the fifth chamber can accommodate the outlets of two or more fluid flow channels.

[0045] According to an embodiment of the present application, the product can include food or medicine.

[0046] According to an embodiment of the present application, the first chamber 100, the second chamber 200, the third chamber 300, the fourth chamber 400 and the fifth chamber 500 are connected in series through a fluid microtube 600 in sequence. Since after the blood glucose-lowering preparation contacts the islets of Langerhans, it can stimulate insulin secretion, and the secreted insulin flows into the second chamber and the fourth chamber through the micropores, binds to the insulin receptor, and initiates the sugar metabolism process, thereby playing a blood glucose-lowering role. Therefore, arranging the third chamber accommodating the islet organ between the second chamber accommodating the liver organ and the fourth chamber accommodating the muscle organ is beneficial for the secreted islets to quickly transfer into the second chamber and the fourth chamber, initiate the sugar metabolism process, and play a blood glucose-lowering role.

[0047] According to an embodiment of the present application, the lengths of the first chamber 100, the second chamber 200, the third chamber 300, the fourth chamber 400 and the fifth chamber 500 are respectively 5 - 10 mm, and the widths are respectively 5 - 10 mm. Thus, it provides enough space to accommodate cells and culture medium, while maintaining the compactness of the system, facilitating high-throughput experiments, contributing to the uniform distribution of nutrients, oxygen and metabolic wastes, ensuring the stability of the cell growth environment, accelerating the transport of substances (such as insulin), and reducing the diffusion time.

[0048] According to an embodiment of the present application, the pore diameter of the micropores is 15 - 25 μm. Thus, it allows the efficient diffusion of small molecules (insulin, glucose), prevents cell migration; the surface tension maintains static independence, and the gravity drive realizes dynamic interaction; the periodic flow matches the metabolic rhythm, accelerating the insulin-receptor binding and the blood glucose-lowering process; the structure is stable, and the high-density micropore design balances the flux and mechanical strength, supporting long-term culture.

[0049] According to an embodiment of the present application, the interval between multiple micropores is 5-10 μm. Thus, the maximum limit achievable by laser drilling under the condition of the largest number of holes is ensured; otherwise, overlapping between holes will occur, affecting the size. It allows efficient diffusion of small molecules (insulin, glucose), prevents cell migration; the surface tension maintains static independence, and the gravity drive realizes dynamic interaction; the periodic flow matches the metabolic rhythm, accelerating insulin-receptor binding and the blood glucose lowering process; the structure is stable, and the high-density micropore design balances the flux and mechanical strength, supporting long-term culture.

[0050] According to an embodiment of the present application, the diameter of the fluid microtube is 0.2-0.8 mm. Thus, the fluid microtube has a lower flow resistance, enabling the liquid to pass through more smoothly; it has a higher flow rate, which can improve efficiency, reduce the time for the liquid to pass through, and facilitate rapid liquid delivery; it reduces the risk of the microtube being blocked by particulate matter or air bubbles; it is more suitable for high-viscosity liquids, such as culture media containing cells or proteins, reducing the flow resistance and making it easier for the liquid to pass through.

[0051] According to an embodiment of the present application, the fluid microtube is made of stainless steel. Thus, it provides a hydrophobic surface.

[0052] According to an embodiment of the present application, the driving device of the present application can be a commonly used gravity-driven platform in the art, which can achieve swinging up and down and / or left and right.

[0053] According to an embodiment of the present application, the system further includes: an extraction instrument, which is used to extract the cell supernatant in the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber. Exemplarily, the extraction instrument can be a pipette, a pipette tube, etc. The chambers of the multi-organ chip can be of an open design or can have a detachable cover. When detection is required, the cover can be opened or removed to facilitate the extraction of the cell supernatant.

[0054] According to an embodiment of the present application, the system further includes: a detection device, which is used to detect glucose, biomarkers, and / or metabolites in the cell supernatant. Exemplarily, the detection device can include a liquid chromatograph, a gas chromatograph, a centrifuge, an electrophoresis instrument, a PCR instrument, etc.

[0055] In another aspect of the present application, the present application proposes a method for evaluating the hypoglycemic effect of a product using the method described above. According to an embodiment of the present application, the method includes: placing liver cells and liver culture medium in the second chamber for culture, placing pancreatic islet cells and pancreatic islet culture medium in the third chamber for culture, and placing muscle cells and muscle culture medium in the fourth chamber for culture; driving the multi-organ chip to move by the driving device so that the culture solution containing the product to be tested in the first chamber flows into the fluid microtube through the liquid inlet, and flows through the second chamber, the third chamber and the fourth chamber through the micropores, and the excess liquid flows out from the liquid outlet; collecting the cell supernatants in the first chamber, the second chamber, the third chamber, the fourth chamber and the fifth chamber and performing detection.

[0056] Thus, an evaluation platform closer to the in vivo metabolic regulation network is constructed using the method of the present application, which is beneficial to accurately and efficiently evaluating the hypoglycemic effect of a product, and helps to identify key biomarkers and metabolic pathways (such as amino acid metabolism and glucose metabolism) closely related to disease progression. This multi-organ interaction system overcomes the limitations of traditional models and provides a unique and reliable research platform for the future development of type 2 diabetes drugs. In addition, the multi-organ chip in the present invention can provide high-throughput simultaneous detection of multiple fluid channels, significantly reducing the time and economic costs of drug research and development.

[0057] It should be noted that the placement implementation order of "placing liver cells and liver culture medium in the second chamber", "placing pancreatic islet cells and pancreatic islet culture medium in the third chamber" and "placing muscle cells and muscle culture medium in the fourth chamber" is not strictly limited, and can be added to the chamber according to the growth status of these three organs as appropriate, as long as it is ensured that the best growth state can be achieved at the same time or at a close time (for example, within 1, 2, 3, 4, 5 or 6 hours), so that when the sample to be tested acts on the three organs, the hypoglycemic effect can be fully exerted.

[0058] According to an embodiment of the present application, the glucose concentration in the culture solution containing the product to be tested is 20-30 mmol / l.

[0059] According to an embodiment of the present application, the culture solution containing the product to be tested is selected from DMEM medium containing the sample to be tested.

[0060] According to an embodiment of the present application, the multi-organ chip is placed on a gravity-driven platform, and the gravity-driven platform is driven to tilt up and down by the driving device, and the tilt angle is 5°-15°, and the tilt interval is 45 s-90 s. Thus, the fluid can flow in laminar flow under the above conditions, accelerating mass exchange and efficient mass transfer.

[0061] According to an embodiment of the present application, the culture medium containing the sample to be tested continuously flows into the fluid microtube for 2 h to 4.5 h, such as 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h. Thus, on the one hand, the product to be tested can fully act on the islet organ to stimulate its insulin secretion, and on the other hand, insulin can be fully dispersed into the muscle organ and the liver organ to bind to insulin receptors, thereby giving full play to the blood glucose lowering effect. Therefore, the blood glucose lowering effect of the product can be accurately and efficiently evaluated. If the time for the sample to be tested to continuously flow into the fluid microtube is too long, problems such as decreased cell activity, liquid evaporation, increased risk of blockage, depletion of nutrients, accumulation of metabolic wastes, temperature changes, liquid stratification or precipitation may occur.

[0062] According to an embodiment of the present application, the culture medium containing the product to be tested contains glucose, and the glucose concentration is 20 to 30 mmol / l. Thus, it is avoided that the cell fluid in the first to fifth chambers is diluted by adding a culture medium without glucose, resulting in the inability to determine whether the decrease in the sugar content in the cell fluid is due to the blood glucose lowering effect of the cells themselves or dilution, and thus the blood glucose lowering effect of the product to be tested cannot be accurately evaluated. In some embodiments, the liver culture medium, the islet culture medium, and the muscle culture medium each independently contain glucose with a concentration of 20 to 30 mmol / l.

[0063] According to an embodiment of the present application, evaluating the blood glucose lowering effect of a product includes screening products with blood glucose lowering effect.

[0064] It should be noted that the features and advantages described above for the system for evaluating the blood glucose lowering effect of a product also apply to this method and will not be repeated here.

[0065] The solution of the present invention will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0066] Example 1

[0067] 1. Provide a system for evaluating the blood glucose lowering effect of a product:

[0068] As Figures 1-3As shown, the multi-organ chip consists of an array of 96-well rectangular chambers, each with a size of 7.2 mm × 7.2 mm. The liver chamber, islet chamber, and muscle chamber are connected in series by fluid microtubes in sequence. The fluid microtube (stainless steel tube) has a diameter of 0.5 mm, and micropores are distributed along the diameter direction on the side wall. The pore diameter of the micropores is 20 μm, and the interval between multiple micropores is 6 μm. A gravity rocker platform is used as the driving device.

[0069] 2. Cell culture and drug action

[0070] C2C12 muscle cells: C2C12 cells were inoculated into the muscle chamber of the 96-well multi-organ interaction platform at 2×10 4 and cultured with 10% FBS and DMEM medium (containing 25 mol / L glucose).

[0071] HepG2 cells: HepG2 cells were inoculated into the liver chamber of the 96-well multi-organ interaction platform at 1×10 5 and continuously cultured with 10% FBS and MEM medium (containing 25 mol / L glucose).

[0072] Beta-tc-6 cells: Beta-tc-6 cells were inoculated into the islet chamber of the 96-well multi-organ interaction platform at 1×10 5 After culturing for 24 h with 10% FBS and DMEM medium (containing 25 mol / L glucose), a 0.3 mM palmitic acid solution was added and cultured for two days to form a type 2 diabetes model with insufficient insulin secretion.

[0073] After culturing until completely adherent, the test solution was applied, and the gravity rocker platform was tilted up and down with an inclination angle of 10° and an inclination interval of 60 s. The drug molecules penetrated into the organ chamber through the micropores. The supernatant samples were aspirated from the chamber with a pipette, and according to the manufacturer's instructions of the glucose kit, the absorbance of each group was measured using an F-02 multimode microplate detection system to obtain the dynamic glucose level and glucose consumption. The cell supernatant after running for 24 h was collected and analyzed by LC-MS.

[0074] Example 2: Testing the permeation performance of the multi-organ high-throughput circulation device

[0075] To verify that substances such as drug molecules and protein molecules in the chamber can effectively exchange substances through the micropores, a quantitative analysis of the permeation of the open microtubes in the multi-organ chip of Example 1 was carried out. The specific experimental steps are as follows:

[0076] At the inlet ( Figure 4 the left 1 chamber of A(i)) or the middle chamber ( Figure 5The middle chamber of A(i) was perfused with DMEM medium containing 10 μm of 70 kDa FITC-dextran fluorescent molecules. The gravity rocker platform was driven to tilt up and down (the operation was the same as step 2 of Example 1). The liquid flowing out from the outlet ( Figure 4 A(i) / Figure 5 the right 1 chamber of A(i)) was circulated and perfused to the inlet. Prism software was used to perform one-way ANOVA on the measurement data of the fluorescence molecules in each microporous medium over time.

[0077] Fluorescent molecules were perfused into the inlet to simulate the penetration of drug molecules. The results were as Figure 4 shown. Compared with 0 h, the fluorescence intensity of the medium in the middle 3 micropores gradually increased over time after 2 h, and there was no significant difference among the micropores. It indicated that on the way from the inlet to the outlet through the hollow microtubes, the fluorescent molecules at the inlet transported substances into each chamber through the micropores, and finally formed a multi-organ interaction platform for "vascular" administration with a circulatory function.

[0078] Analysis of perfusing fluorescent molecules into the middle chamber to simulate the penetration of protein molecules. The results were as Figure 5 shown. Compared with 0 h, the fluorescence intensity among the micropores gradually increased over time after 3 h, and there was no significant difference among the micropores. It indicated that during the gravity flow of the fluorescent molecules in the middle chamber, substances were transported into the adjacent 2 chambers through the hollow channels of the open microtubes, and finally formed a multi-organ interaction platform for supporting substance exchange.

[0079] Example 3 Detection of dynamic glucose concentration in the multi-organ interaction platform

[0080] To further evaluate the effect of the multi-organ interaction system under different conditions on glucose regulation, in step 2 of Example 1, the experimental designs of different groups were as follows:

[0081] Multi-organ normal interaction group (Normal interaction group, abbreviated as NI group): C2C12 muscle cells, HepG2 cells, and Beta-tc-6 cells were co-inoculated and cultured. After complete adherence, DMEM medium containing 25 mmol / l glucose was applied at the inlet position, and a multi-organ interaction platform was formed through fluid driving.

[0082] Multi-organ interaction model group (Model group): Beta-tc-6 cells induced by palmitic acid injury were first inoculated into the islet chamber of the 96-well multi-organ interaction platform at a density of 1×10 5 . C2C12 muscle cells and HepG2 cells were co-inoculated at the same time. After all cells were completely adherent, DMEM medium containing 25 mmol / l glucose was applied at the inlet position, and a type 2 diabetes model with insufficient insulin secretion was formed through fluid driving.

[0083] Multi-organ interaction drug administration group: On the basis of the above model group, DMEM medium containing 25 mmol / l glucose and drugs were applied at the inlet position. The drugs were respectively: 1) 100 μM metformin + 10 μM glibenclamide (Met+G); 2) 100 μM metformin + 100 nM liraglutide (Met+L); 3) 100 μM metformin + 5 nM repaglinide (Met+R); 4) 100 μM metformin (Met).

[0084] Single-organ culture group (S-group): C2C12 muscle cells (S-muscle), HepG2 liver cells (S-liver) and Beta-tc-6 islet cells (S-islet) were respectively inoculated in the well plate. After complete adherence, DMEM medium containing 25 mmol / l glucose was added to the well plate respectively.

[0085] The changes in glucose concentration in each chamber at different time points were analyzed in detail, and the experimental results are shown as Figure 6 and Figure 7 shown.

[0086] Compare the changes in glucose concentration and consumption in single-organ and multi-organ systems, and monitor the supernatant of each group at different time points. Compared with the glucose concentration and consumption in single culture at 3 h, 9 h and 24 h, the glucose concentration in the NI group decreased significantly, and the glucose consumption increased significantly ( Figure 6 ). This indicates that, compared with a single organ, the interaction between multiple organs leads to a decrease in glucose concentration and an increase in glucose utilization rate.

[0087] After 3 h, 9 h and 24 h in the NI group, the glucose concentration decreased significantly to 12 - 14 mmol / l, indicating that glucose metabolism is higher in the multi-organ interaction system ( Figure 7 A). In contrast, the glucose concentration in the model group decreased to 17 - 19 mmol / l at the same time points. Although there was also a significant decrease, the consumed glucose concentration was lower (6 - 7 mmol / l), suggesting that impaired insulin may affect the cooperation of multiple organs, resulting in a decrease in glucose metabolism efficiency ( Figure 7 B).

[0088] In the combined drug intervention group, after metformin and glibenclamide were used in combination, the glucose concentration decreased to 12 - 14 mmol / l after 3 h, 9 h and 24 h, indicating that the combined use of these two drugs can effectively promote glucose metabolism ( Figure 7 C). Similarly, after metformin and liraglutide were used in combination, the glucose concentration decreased to about 13 mmol / l after 3 h, 9 h and 24 h ( Figure 7D), further confirmed the synergistic effect of these two drugs in promoting glucose metabolism.

[0089] In addition, after metformin and repaglinide were used in combination, the glucose concentration decreased to 15 - 16 mmol / l at 3 h, and further decreased to about 13 mmol / l after 9 h and 24 h ( Figure 7 E). This result indicates that the promoting effect of repaglinide on glucose metabolism is relatively significant, and its effect gradually stabilizes over time.

[0090] Finally, when metformin was used alone, the glucose concentration decreased to 15 - 17 mmol / l after 3 h, 9 h, and 24 h ( Figure 7 F). Compared with the combined medication, the effect of metformin used alone was slightly weaker, suggesting that metformin can exert a stronger hypoglycemic effect when used in combination with other drugs.

[0091] To further evaluate the differences between groups, the glucose concentrations at different time points were compared in detail. After 24 h, compared with the Model group, the glucose concentrations in the normal group and each drug administration group in the multi-organ interaction system were significantly decreased, indicating that these groups showed better effects in glucose metabolism regulation. It is worth noting that compared with the single drug administration groups, the glucose concentrations in each combined drug administration group in the multi-organ interaction system gradually approached the glucose levels in the normal interaction group over time, as shown in Figure 7 G. These results not only confirmed the ability of the liver-pancreas-muscle multi-organ interaction system to effectively regulate glucose concentration in vitro, but also suggested the potential advantages of the combined drug administration strategy in improving glucose metabolism.

[0092] In summary, the multi-organ interaction system plays an important role in glucose regulation, and there are significant differences in the effects of different drug combinations on glucose metabolism. The combined medication shows a stronger effect in promoting glucose metabolism, especially the combination of metformin with glibenclamide, liraglutide, or repaglinide, which can significantly improve the glucose consumption efficiency.

[0093] Example 4 Metabolomic Characteristics of the Multi-Organ Interaction Platform

[0094] Untargeted LC-MS metabolomic analysis was performed on the supernatant extracted from the multi-organ interaction platform to detect changes in metabolic pathways. Among them, the design of each group is referred to Example 3. The results are as shown in Figure 8 shown.

[0095] Untargeted LC-MS metabolomic analysis was performed on the supernatant extracted from the multi-organ interaction platform to detect metabolic pathways. As shown in Figure 8As shown in the OPLS-DA score plot of A, there was a distinct separation between the metabolic profiles of the model group and the metformin and repaglinide group, indicating successful establishment of the model. The volcano plot of differential metabolites showed that 73 metabolites were upregulated and 55 metabolites were downregulated after being mediated by metformin and repaglinide. The clustering heat map and the metabolite content change map showed the change trends of metabolites in the two groups. These metabolites were mainly related to glucose metabolism, amino acid metabolism, lipid metabolism, and fatty acid metabolism. Key metabolic pathways in the multi-organ interaction platform such as Figure 8 as shown in G.

[0096] In specific metabolic pathways, the metabolite FumaricAcid involved in Arginine biosynthesis, Citrate cycle (TCA cycle), Pyruvate metabolism, Alanine, aspartate and glutamate metabolism was significantly downregulated after administration. Fumaric acid metabolism is closely related to diabetic metabolic disorders, and the level of fumaric acid in T2DM rats is increased. Fumaric acid is a dicarboxylic acid involved in the TCA cycle. Previous studies have shown that metformin treatment significantly affects the inhibition of TCA cycle metabolism and the concentration level of fumaric acid decreases. In the multi-organ in vitro platform, after administration of metformin and repaglinide, the level of fumaric acid decreased significantly, which was consistent with previous reports.

[0097] The metabolite sorbitol involved in Fructose and mannose metabolism and Galactose metabolism pathways was significantly downregulated after administration. Sorbitol levels increase under hyperglycemic conditions, leading to activation of the polyol pathway (sorbitol-aldehyde reductase pathway), which in turn affects glucose homeostasis and causes metabolic disorders such as diabetes, obesity, and cardiovascular diseases. In a hyperglycemic environment, the depletion of the intracellular inositol pool is related to the increased synthesis of sorbitol, which in turn affects the activity of Na+ / K+ ATPase and the synthesis of phosphatidylinositol. Previous studies have shown that repaglinide improves hyperglycemia by regulating inositol consumption and reducing sorbitol levels. In the multi-organ in vitro platform, after administration of metformin and repaglinide, the level of sorbitol decreased significantly.

[0098] The metabolite docosahexaenoic acid (DHA), which is involved in the biosynthesis of unsaturated fatty acids, is a long-chain polyunsaturated fatty acid that plays an important role in regulating glucose tolerance and insulin sensitivity. Previous studies have shown that a dihydroxylated product of DHA, protectin DX (PDX), exerts glucose-regulatory activity through interleukin (IL)-6 released from skeletal muscle. These results indicate that the liver-pancreas-muscle multi-organ interaction platform after 1 day of combined drug treatment increases glucose utilization by regulating metabolic pathways such as Arginine biosynthesis, thereby reducing the overall glucose concentration of the multi-organ interaction platform.

[0099] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A system for evaluating the blood sugar lowering effect of a product, characterized in that, Comprising: A multi-organ chip, comprising: a plurality of chambers arranged in an array, each of the chambers comprising: a first chamber, a second chamber, a third chamber, a fourth chamber, and a fifth chamber, the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber being connected in series through fluid microtubes; The second chamber is used for culturing liver organs; The third chamber is used for culturing pancreatic islet organs; The fourth chamber is used for culturing muscle organs; The inlet of the fluid microtube is located in the first chamber, and the outlet of the fluid microtube is located in the fifth chamber; the inner and outer wall surfaces of the fluid microtube are hydrophobic; along the diameter direction of the fluid microtube, a plurality of micropores are distributed on the side wall of the fluid microtube; A driving device, the driving device is connected to the multi-organ chip and is used for driving the multi-organ chip to move.

2. The system according to claim 1, wherein The first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber are connected in series through fluid microtubes in sequence.

3. The system according to claim 1, wherein The lengths of the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber are 5-10 mm respectively, and the widths are 5-10 mm respectively.

4. The system according to claim 1, characterized in that, The aperture of the micropores is 15-25 μm; The interval between the plurality of micropores is 5-10 μm; The diameter of the fluid microtube is 0.2-0.8 mm.

5. The system according to any one of claims 1-4, characterized in that, The fluid microtube is made of stainless steel; Optionally, the system further comprises: An extraction instrument, the extraction instrument is used for extracting the cell supernatant in the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber; A detection device, the detection device is used for detecting glucose, biomarkers, and / or metabolites in the cell supernatant.

6. A method for evaluating the hypoglycemic effect of a product using the system for evaluating the hypoglycemic effect of a product according to any one of claims 1-5, characterized in that, Comprising: Placing liver cells and a liver culture medium in the second chamber for culturing, placing pancreatic islet cells and a pancreatic islet culture medium in the third chamber for culturing, and placing muscle cells and a muscle culture medium in the fourth chamber for culturing; Driving the multi-organ chip to move through the driving device, so that the culture solution containing the sample to be tested in the first chamber flows into the fluid microtube through the inlet, and flows through the second chamber, the third chamber, and the fourth chamber through the micropores, and the excess liquid flows out from the outlet; Collecting and detecting the cell supernatant in the first chamber, the second chamber, the third chamber, the fourth chamber, and the fifth chamber.

7. The method according to claim 6, wherein The multi-organ chip is placed on a gravity-driven platform, and the gravity-driven platform is driven to tilt up and down through the driving device, the tilt angle is 5°-15°, and the tilt interval is 45 s-90 s.

8. The method according to claim 6, characterized in that, The culture solution containing the sample to be tested continuously flows into the fluid microtube for 2 h-4.5 h.

9. The method according to claim 6, wherein The glucose concentration in the culture solution containing the product to be tested is 20-30 mmol / l; Optionally, the culture solution containing the product to be tested is selected from a DMEM culture medium containing the sample to be tested.

10. The method according to claim 6, wherein Evaluating the hypoglycemic effect of the product includes screening products with hypoglycemic effects.

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