Islet-vascular co-culture system based on microfluidic chip, establishment method and application

Abstract

The application provides an islet-vascular co-culture system based on a microfluidic chip, a building method and application, and the building method of the islet-vascular co-culture system comprises the following steps: S1, preparing microfibers loaded with islets; S2, preparing an assembled chip, the assembled chip comprises a containing cavity, first microchannel networks and second microchannel networks are symmetrically arranged on two sides of the containing cavity, and the first microchannel networks and the second microchannel networks are respectively communicated with a liquid inlet and a liquid outlet at an end away from the containing cavity; S3, controlling the flow of the liquid inlet to be 6-8 muL / min to carry out dynamic culture. The application can simulate the key structure and characteristics of islet tissue, can evaluate a diabetes treatment drug in vitro, can bridge the gap between in vitro and in vivo models, and can provide new tools and methods for the fields of drug development, disease model construction and personalized medical treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chip, in particular to an islet-vascular co-culture system based on a microfluidic chip, a method for establishing the same and an application. BACKGROUND

[0002] Currently, the basic research on diabetes, the screening of therapeutic drugs and the evaluation of new treatment methods usually need to construct disease models, among which animal models have species-specific differences, and two-dimensional models are difficult to simulate the interaction between islets and blood vessels, and lack of dynamic fluid environment, resulting in low prediction accuracy of drug response.

[0003] Microphysiological systems (MPS) is a kind of complex organ physiological microsystem constructed in vitro by microfluidic 3D cell culture device, which integrates various cell structures, 3D cell structures, cell components, fluid flow, dynamic pressure or stretching and the interaction between multiple organs, providing the possibility of simulating the characteristics of human tissues and organs and the interaction between tissues and organs in vitro, and showing great application prospects in the fields of life science and new drug research and development.

[0004] Therefore, the patent with the application number 201810977419.0 discloses a method for establishing an islet pathological model based on an organ chip, which includes the following steps: chip preparation and modification, islet beta-TC6 cell ball formation, streptozotocin-induced 3D cell ball injury, and function detection. This scheme can realize the preparation of controllable and uniform islet beta-TC6 cell balls, and use streptozotocin to induce 3D cell-based islet injury model based on islet beta-TC6 cell balls, providing a new method for the construction of type 1 diabetes model.

[0005] In addition, the patent with the application number 202211113469.7 discloses a method for establishing an I-type diabetes in vitro model based on a microfluidic chip and its application. The microfluidic chip has an islet function area and a sugar uptake function area, which are connected through a communication channel. The microfluidic chip is surface modified with collagen. The islet function area is used for inoculating islet cells with insulin secretion function, and the sugar uptake function area is used for inoculating cells with sugar uptake function. This scheme can simulate type I diabetes with insufficient insulin secretion, and also has two key characteristics of sugar metabolism, insulin secretion and sugar uptake, which can reflect the interaction characteristics of in vivo sugar regulation.

[0006] The vascularization strategy in the above scheme is limited to perfusion simulation and does not integrate endothelial cell co-culture. The complexity of model construction is high, and it is difficult to standardize the application in drug screening. In view of this, the present application is proposed. SUMMARY

[0007] The problem addressed by this invention is that existing in vitro diabetes models based on microfluidic chips are limited to perfusion simulation of blood vessels, and cannot integrate pancreatic islets with vascular endothelium and achieve dynamic interaction, resulting in significant differences from the in vivo microenvironment.

[0008] To address the aforementioned problems, this invention provides a method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip, comprising: S1, preparing microfibers loaded with pancreatic islets; S2, preparing an assembled chip, wherein the assembled chip includes a receiving cavity, and a first microchannel network and a second microchannel network are symmetrically arranged on both sides of the receiving cavity, the first microchannel network and the second microchannel network being connected to a liquid inlet and a liquid outlet respectively at the end away from the receiving cavity; S3, controlling the flow rate of the liquid inlet to 6-8 μL / min for dynamic culture.

[0009] Preferably, in step S2, a Matrigel is laid above the bottom of the receiving cavity, and a vascular ring structure is formed on the Matrigel. The vascular ring structure is formed by inoculating and culturing HUVECs. Multiple segments of fully cultured and developed into clusters of load microfibers are placed on the upper part of the vascular ring structure. Matrigel is then laid on the load microfibers again, and then a PDMS layer is used as a cover to clamp and seal the receiving cavity.

[0010] Preferably, the specific preparation method for step S2 is as follows: 100 μL of Matrigel is added to the bottom of the container, and the container is placed in a 37°C incubator for 10 min to allow it to gel; HUVECs are digested, resuspended in the culture medium, and incubated at 2 × 10⁻⁶ ppm. 5 mL -1 The microfibers were seeded at a density in the containment chamber and placed in an incubator for 12 hours to allow them to spontaneously form vascular ring structures. The supernatant of the vascular culture layer with the formed vascular ring structure was aspirated from the containment chamber. The microfibers loaded with β cells were cultured in vitro for three days to allow them to fully develop into clusters. They were cut into small segments and placed on the vascular ring structure. 100 μL of Matrigel was added to completely cover the microfibers. The microfibers were placed in an incubator to allow them to gel, thereby achieving high-order assembly of the microfibers. A PDMS layer was used as a cap, and the exposed vascular culture layer chip was clamped and sealed using a custom clamp.

[0011] Preferably, the assembled chip is prepared by the following method: a microfluidic chip resin template is prepared using a 3D printing device, and a PDMS chip is made based on the resin template by molding. The cavity of one PDMS layer is hollowed out and aligned and sealed with another PDMS layer to obtain the chip.

[0012] Preferably, the microfibers loaded with pancreatic islets in step S1 are fabricated using a microfluidic chip. The microfluidic chip is provided with channels for conveying the core flow, sample flow, and sheath flow, respectively. The microfluidic chip also includes a PDMS chamber for periodically pressurizing to expand and thereby compress and close the channel of the core flow.

[0013] Preferably, the microfluidic chip includes a second channel and a fourth channel for conveying a sample flow and a core flow, respectively. The second channel has two sections, each located on either side of the fourth channel. The second and fourth channels merge at a first position to form a first confluence channel. The chip also includes a third channel, one end of which is connected to a gas control device, and the other end is connected to a PDMS chamber. There are two PDMS chambers located on either side of the fourth channel, used to close the fourth channel by pressurizing and expanding it from both sides. The microfluidic chip also includes a first channel for conveying a sheath flow. The first channel has two sections, each located on either side of the first confluence channel. The first channel and the first confluence channel merge at a second position to form a sixth channel for the outflow of formed microfibers.

[0014] Preferably, the preparation process parameters for the islet-loaded microfibers are as follows: collagen is added to the core stream, followed by the addition of β-TC6 cells to achieve a core stream cell density of 4 × 10⁻⁶. 6 mL -1 -8×10 6 mL -1 The flow rates of the core flow, sample flow, and sheath flow were controlled at 0.8–1.2 μL / min, 18–22 μL / min, and 80–120 μL / min, respectively, with frequencies of 0.8–1.2 Hz.

[0015] Preferably, the microfluidic chip is prepared by the following method: a microfluidic chip resin template is prepared using a 3D printing device; a mixture of PDMS and curing agent with a weight ratio of 8-12:1 is poured onto the resin template; after vacuum degassing, it is cured at 75-85℃ for 0.8-1.1h; the PDMS layer with microstructure is peeled off to obtain the PDMS microchip; the PDMS layers of two PDMS microchips with the same structure are subjected to oxygen plasma surface treatment, aligned and sealed to obtain the final product.

[0016] The present invention also provides a pancreatic islet-blood vessel co-culture system based on a microfluidic chip, which is obtained by the above-described establishment method.

[0017] This invention also discloses the application of a microfluidic chip-based pancreatic islet-vascular co-culture system in the evaluation of drugs for the treatment of diabetes or in the preparation of pancreatic islet models.

[0018] Compared with existing technologies, the method for establishing and applying the pancreatic islet-vascular co-culture system based on microfluidic chips described in this invention has the following beneficial effects:

[0019] 1) Design a pneumatically controlled microvalve chip, utilize periodic nitrogen pressure regulation to precisely cut off the core flow, and combine it with microfluidic spinning technology to generate monodisperse aqueous droplet microfibers to achieve efficient encapsulation of pancreatic β cells, overcoming the defects of poor uniformity and low cell survival rate of traditional passive shear force droplet technology;

[0020] 2) Dynamic vascularization construction: RGD peptide modification is performed on the surface of calcium alginate microfibers to promote the adhesion and directional growth of vascular endothelial cells. Combined with a microfluidic dynamic perfusion system to simulate capillary blood flow, physiological-level interaction of pancreatic islet-vascular co-culture is achieved.

[0021] 3) Biomimetic microenvironment integration: By stacking microfibers and assembling chip chambers, a multi-layered structure containing a large number of pancreatic islet clusters and a three-dimensional vascular network is formed. Through the synergistic effect of biochemical factors and biophysical stimulation, the physiological microenvironment closer to that in vivo is simulated, significantly improving the accuracy of the model's response prediction to diabetes drugs.

[0022] 4) This invention is the first to combine pneumatic control technology with dynamic co-culture of microfibers, and simultaneously solves the two major technical bottlenecks of "high-throughput pancreatic islet loading" and "functional vascularization", providing an in vitro evaluation platform for diabetes research that is closer to human physiology. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the chip structure in the pneumatically controlled microfluidic spinning device described in Embodiment 1 of the present invention;

[0024] Figure 2 This is a physical image of the chip template in the pneumatically controlled microfluidic spinning device described in Embodiment 1 of the present invention;

[0025] Figure 3 This is a schematic diagram of the pneumatically controlled microfluidic spinning device described in Embodiment 1 of the present invention;

[0026] Figure 4 This is a histogram of the droplet spacing l and droplet length Δl under different conditions in Embodiment 2 of the present invention (n=10).

[0027] Figure 5 Bright-field and fluorescence images of aqueous droplet microfibers loaded with β-TC6 cells in Example 2 of this invention;

[0028] Figure 6 These are bright-field and fluorescence images of β-TC6 cells cultured in microfiber droplets on days 0, 1, and 7 in Example 3 of this invention.

[0029] Figure 7 The proliferation of β-TC6 cells in microfibrils in Example 3 of this invention on day 0 and day 3, n=3;

[0030] Figure 8 This is an immunofluorescence staining image of β-TC6 cells cultured in microfibrils on day 3 in Example 3 of the present invention;

[0031] Figure 9 The results of INS-1, INS-2, and PDX-1 expression in β-TC6 cells under different culture modes in Example 3 of this invention;

[0032] Figure 10 The diagram shows the chip schematic and physical image of the pancreatic islet-blood vessel co-culture system based on microfluidic chip in Embodiment 4 of the present invention.

[0033] Figure 11 The MTT assay was used in Example 5 of this invention to detect cell survival and growth of β-TC6 cells (n=6) and HUVECs (n=4);

[0034] Figure 12 The ELISA method used in Example 5 of this invention to detect insulin secretion (n=6);

[0035] Figure 13 This invention provides an example of a chemical method for detecting NO release, comprising a control group (n=3) and an experimental group (n=10).

[0036] Figure 14 This is a diagram showing the fluorescence staining results of angiogenesis-related cells in Example 5 of the present invention;

[0037] Figure 15 The expression results of angiogenesis-related mRNAs in HUVECs in Example 5 of this invention;

[0038] Figure 16 This is a flowchart illustrating the fabrication process of the pancreatic islet-blood vessel co-culture system based on a microfluidic chip, as described in an embodiment of the present invention.

[0039] Explanation of reference numerals in the attached figures:

[0040] 1-First channel; 2-Second channel; 3-Third channel; 4-Fourth channel; 5-Second position; 6-Sixth channel; 7-First position. Detailed Implementation

[0041] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Without conflict, the technical features of the embodiments of the present invention can be combined with each other.

[0042] Unless otherwise stated, all chemical reagents and biological materials used in this invention were commercially obtained and processed and applied in accordance with standard operating procedures. All experimental steps were performed under normal laboratory conditions, ensuring the reproducibility and reliability of the technical solution. Specific structural units, connection relationships, and mating relationships will be described in detail in subsequent sections.

[0043] Microphysiological systems can more realistically simulate the human body's organ response to drugs, enabling rapid screening of drug efficacy and toxicity on-chip, significantly shortening drug development cycles and reducing development costs, showing great application potential in life sciences and new drug development. Among them, the pancreatic islet microphysiological system can be used to construct disease models of diabetes and its complications. Since the capillary network within the islets not only provides blood supply and assists the islets in sensing blood glucose concentration, but also provides structural support through its basement membrane and endothelial cells, maintaining the morphology and function of the islets, the role of blood vessels should be considered as an important part of constructing the pancreatic islet microphysiological system. Existing technologies for establishing in vitro diabetes models based on microfluidic chips often use perfusionable microfluidic systems to simulate capillary blood flow. However, integrating the islets with the vascular endothelium and achieving dynamic interaction remains a significant challenge. Therefore, the applicant proposes the following technical solution:

[0044] Example 1: Pneumatically Controlled Microfluidic Spinning Device for Microfiber Preparation

[0045] For ease of explanation, the pneumatically controlled microfluidic spinning device used to prepare microfibers will be described first as follows:

[0046] like Figures 1-3 As shown, a pneumatically controlled microfluidic spinning device includes a microfluidic chip. The microfluidic chip includes a second channel 2 and a fourth channel 4, which are used to transport sample flow and core flow, respectively. There are two second channels 2, which are located on both sides of the fourth channel 4. The second channel 2 and the fourth channel 4 are merged at a first position 7 to form a first confluence channel. The chip also includes a third channel 3. One end of the third channel 3 is connected to a gas control device, and the other end is provided with a connected PDMS chamber. There are two PDMS chambers, which are located on both sides of the fourth channel 4, and are used to close the fourth channel 4 by pressurizing expansion and squeezing it from both sides.

[0047] The microfluidic chip also includes a first channel 1 for conveying sheath flow. There are two first channels 1 located on both sides of the first confluence channel. The first channel 1 and the first confluence channel are merged at the second position 5 to form a sixth channel 6 for the outflow of the formed microfibers.

[0048] When the gas control device is turned on, the PDMS chamber is formed. Due to the elasticity of PDMS itself, the PDMS chamber continues to expand and compress the fourth channel 4 as the gas pressure increases. When the gas pressure reaches 50 kPa, the fourth channel 4 is completely closed. If the gas control device is turned on periodically at a certain frequency, the fourth channel 4 will be periodically turned on or off, thereby periodically cutting off the core flow. Precise control can be achieved by adjusting the frequency and the flow rate of the core flow. It can be used for controllable encapsulation and even has the potential to carry out independent organoid culture.

[0049] As an example of the present invention, the gas control device includes a gas storage tank connected to the third channel 3 for storing nitrogen gas, and a control valve is provided between the gas storage tank and the third channel 3, and the control valve is electrically connected to the control module.

[0050] As an example of the present invention, the first channel 1 has a width of 1 mm and a height of 1100 μm, the second channel 2 has a width of 450 μm and a height of 700 μm, the third channel 3 has a width of 200 μm and a height of 500 μm, the fourth channel 4 has a width of 70 μm and a height of 500 μm, and the PDMS chamber has a length of 1 mm, a width of 200 μm, a height of 500 μm, and a distance of 150 μm between it and the fourth channel 4. It should be noted that the dimensions of the channels of the microfluidic chip and the resin template were measured using an inverted fluorescence microscope (IX73, Olympus), and the height was measured using vernier calipers.

[0051] The core stream is a 15% w / w Dextran solution, the sample stream is a mixed solution of 1% w / w NaA and 17% w / w PEG, and the sheath stream is a mixed solution of 4% w / w CaCl2 and 17% w / w PEG. These solutions are injected into the corresponding channels of the microfluidic chip via a micro-injection pump (Harvard), and the microfibers are collected using deionized water.

[0052] This setup utilizes the Ca in the NaA and CaCl2 solution. 2+ A transient ionic cross-linking reaction occurs to form microfibers, while the differences in intermolecular interactions and physicochemical properties of PEG and DEX in aqueous solution enable the formation of a two-phase aqueous system. Therefore, at the first position 7, the pressure-controlled core flow can be sheared to form droplets and stored within the microfibers, while at the second position 5, NaA and CaCl2 solution... 2+ A transient ionic cross-linking reaction occurs to form microfibers, which flow out through channel 6 and are collected. It should be noted that all the above solutions were prepared with deionized water.

[0053] As an example of the present invention, the microfluidic chip is prepared by the following method: a microfluidic chip resin template is prepared using a 3D printing device (nanoArch®SI40, BMF Material Technology Inc). A mixture of PDMS and curing agent with a weight ratio of 10:1 is poured onto the resin template. After vacuum degassing, the PDMS is cured at 80°C for 1 hour. The PDMS microchip is obtained by peeling off the PDMS layer with microstructure. The PDMS layers of two PDMS microchips with the same structure are subjected to oxygen plasma surface treatment, aligned and sealed to generate the final microfluidic chip, which is then sterilized for later use.

[0054] Example 2: Optimization of process parameters for preparing microfibers

[0055] To visualize the microfibers, green fluorescent polystyrene microspheres (5µm, excitation / emission: 488 / 518nm) were mixed in a core flow solution (0.1mg / ml). The flow rates of the core flow, sample flow, and sheath flow ranged from 1-3μL / min, 5-40μL / min, and 50-200μL / min, respectively. The frequency range of the pump valve switching was 0.625-1Hz. The microfibers were characterized by photographs using an inverted fluorescence microscope, and the droplet length Δl, the droplet head-to-head spacing l, and the microfiber width were statistically analyzed.

[0056] Specifically, the flow rates of the core flow, sample flow, and sheath flow were set to 1, 20, and 100 μL / min, respectively, and the frequency was varied within the range of 0.625–2.5 Hz. The results are as follows: Figure 4 As shown in -a; the flow rates of the sample flow and sheath flow were set to 20 and 100 μL / min, respectively, with a frequency of 1 Hz. The flow rate of the core flow was varied within the range of 0.5-2.0 μL / min. The results are shown in [Figure 1]. Figure 4 -b; Set the core flow and sheath flow rates to 1 and 100 μL / min and the frequency to 1 Hz, respectively. Adjust the sample flow rate within the range of 5-40 μL / min. The results are shown in […]. Figure 4 -c; Set the core flow and sample flow rates to 1 and 20 μL / min respectively, the valve frequency to 1 Hz, and adjust the sheath flow rate within the range of 50-200 μL / min. See the results below. Figure 4 -d.

[0057] Depend on Figure 4 As shown in -a, as the frequency decreases, Δl increases from 494.7±60.14μm to 1111.99±113.73μm, and l increases from 854.55±51.74μm to 2587.49±398.21μm, while the width of the microfiber does not change significantly. This indicates that higher frequencies produce denser droplets, which can effectively increase the droplet flux within the microfiber, but also means that the droplets are more difficult to distinguish.Figure 4 As shown in Figure -b, when the core flow velocity increases from 0.5 to 1.0 μL / min, Δl increases from 325.97±50.16 μm to 786.17±58.81 μm. However, when the core flow velocity reaches 2.0 μL / min, even with the assistance of the valve, it is no longer possible to form dispersed droplets, and the core flow exhibits a turbulent morphology. This indicates that the core flow velocity and droplet size are positively correlated within a certain range. Larger droplets can be obtained by increasing the core flow velocity; furthermore, the core flow velocity has almost no effect on the fiber width.

[0058] Depend on Figure 4 As shown in -c, with the increase of sample flow rate, the droplet head-to-head distance *l* increased from 1303.544±69.13 μm to 2772.82±188.23 μm, and the fiber width increased from 388.95±4.47 μm to 643.24±10.77 μm, while Δl did not change significantly. Specifically, when the sample flow rate was 5 μL / min, the core flow exhibited severe stacking, preventing droplet formation, indicating a positive correlation between sample flow rate, droplet distance, and fiber width within a certain range. Figure 4 As shown in the diagram, with the increase of sheath flow velocity, the effects on l and Δl are not significant, while the fiber width decreases from 573.79±7.89μm to 476.23±7.57μm. This indicates that the sheath flow velocity is negatively correlated with the fiber width; the higher the flow velocity, the finer the fiber.

[0059] In summary, droplet size mainly depends on the core flow and valve frequency, droplet spacing mainly depends on the sample flow, and fiber width mainly depends on the sheath flow. In this study, to obtain aqueous droplets with moderate spacing and high flux, the optimal spinning conditions were determined by comparing the size parameters of microfibers and droplets under different conditions. The optimal spinning conditions were: core flow, sample flow, and sheath flow rates of 1, 20, and 100 μL / min, respectively, and a valve frequency of 1 Hz. Under these optimal conditions, the aqueous droplets are larger, have higher flux, and better dispersion, which is beneficial for loading more cells and forming independent three-dimensional culture microunits to obtain more homogeneous cell microspheres or microtissues.

[0060] Microfibers were prepared under optimal conditions, and their morphology was characterized using laser scanning confocal microscopy. The results are shown in [Figure number missing]. Figure 5 .Depend on Figure 5 It can be seen that the prepared microfibers are continuous and intact, with a large number of aqueous droplets containing β-TC6 cells evenly distributed in the microfibers.

[0061] Blue fluorescent polystyrene microspheres (PS microspheres, 5µm, excitation / emission: 400 / 450nm) and green polystyrene microspheres (5µm, excitation / emission: 488 / 518nm) were mixed at a concentration of 0.1 mg / ml in the sample stream and core stream solutions, respectively. Three-dimensional images obtained using confocal microscopy showed that the two colors of microspheres existed independently within the spatial range, with a clear distinction between the droplets and fibers. This indicates that there was no interdiffusion between the core stream and the sample stream. When the PS microspheres in the droplets were replaced with cells of the same size, a three-dimensional culture space could be provided for the cells. Furthermore, the aqueous phase within the droplets could provide biochemical conditions for cell survival. This suggests that the prepared aqueous droplet-loaded microfibers can serve as a carrier for loading various chemical substances or for three-dimensional cell culture.

[0062] Example 3: Preparation and characterization of pancreatic islet-loaded microfibers

[0063] Based on the above preparation of aqueous droplet-loaded microfibers, PS microspheres were replaced with mouse islet tumor cells β-TC6, and type I collagen, an extracellular matrix component, was added to the core stream to provide cells with a better three-dimensional growth environment and biochemical factors, thereby realizing the preparation of islet-loaded microfibers and in vitro three-dimensional culture of islets.

[0064] To encapsulate the cells, the spinning apparatus involved was pre-sterilized with ultraviolet light for 30 minutes before the operation. The NaA / PEG mixed solution and Dextran solution were filtered through a 0.22 μm sterile membrane, and the CaCl2 solution was autoclaved.

[0065] In preparing islet-loaded fibers, type I collagen was mixed into the core stream. Specifically, an equal volume of 30% (w / w) dextran solution and 3.34 mg / mL rat tail type I collagen was mixed, and a small amount of 0.5 M NaOH was added to adjust the pH to approximately 7.4. β-TC6 cells (purchased from the Chinese Academy of Sciences Cell Bank / Stem Cell Bank, cultured in DMEM medium supplemented with penicillin-streptomycin (1% v / v) and FBS (15% v / v)) were digested with 0.25% trypsin-EDTA and resuspended in the core stream solution to obtain a density of 6 × 10⁻⁶ cells. 6 mL -1 Cell suspensions are used in microfluidic experiments.

[0066] The collected microfibrils were transferred to culture dishes containing DMEM high-glucose medium and cultured for 1–7 days, with the medium changed daily. Cell Tracker was used to track cells and their location within the fibrils. TMGreen CMFDA was used to label β-TC6 cells, and PI staining was performed on dead cells using existing techniques. Microfibrils were harvested during culture, and fluorescence images of the cells were obtained using an inverted fluorescence microscope. Figure 6 The cell viability on days 0, 1, and 7 indicates that the cells maintained good activity even after multiple days of culture. The MTT assay was used to detect the proliferation of β-TC6 cells in microfilaments on days 0 and 3; the results are shown below. Figure 7 .

[0067] The A570 value (i.e., absorbance at 570 nm) increased from 0.116 on day 0 to 0.787 on day 3, an increase of approximately 6.78 times (***p<0.001), indicating that the cells were proliferating well.

[0068] To further investigate the aggregation and growth of β-TC6 cells and the formation of islets within microfibrils, islet-loaded microfibrils were cultured in vitro for three days. The secretion of the cytoskeletal protein F-actin and insulin was then characterized using laser confocal microscopy. The results are shown in [Figure number missing]. Figure 8 As shown in the figure. Furthermore, RT-qPCR was used to determine the expression levels of INS-1, INS-2, and PDX-1 mRNAs in microfibrils (three-dimensional culture) and culture plates (two-dimensional culture), respectively. The results are shown in the figure. Figure 9 .

[0069] Depend on Figure 8 It was found that F-actin staining of the cytoskeleton indicated that pancreatic islet cells grew in clusters and formed multiple islet-like clusters in three-dimensional culture. Simultaneously, insulin staining confirmed that these islet-like clusters possess normal endocrine function. Figure 9 It is known that, compared with two-dimensional culture in a planar environment, the expression of INS-1 in β-TC6 cells in the microfibrils described in this application increased by approximately 340.69 times (*p<0.05), the expression of INS-2 increased by approximately 1.74 times (**p<0.01), and the expression of PDX-1 increased by approximately 1026.64 times (*p<0.05). This indicates that the aqueous droplets in the microfibrils provide a three-dimensional space for cells to promote cell cluster growth. The microfibrils can exchange substances with the outside world through a thin hydrogel medium layer, while ensuring that the cells are protected from external interference (such as bacteria, and various studies have reported that calcium alginate materials have antibacterial properties) inside the aqueous droplets. Furthermore, the addition of extracellular matrix components (collagen) provides cells with biochemical factors, which further promotes the formation and functional remodeling of islets.

[0070] This application combines a pneumatically controlled droplet generation method with microfluidics, which can more easily and quickly prepare high-throughput islets. Compared with traditional three-dimensional cell culture within microfluidic droplets, it is more conducive to the recovery of cell-loaded droplets and biological analysis, and can avoid accidental loss or damage of droplets during transfer, cell staining and other processes.

[0071] Example 4: Method for establishing a pancreatic islet-vascular co-culture system based on microfluidic chip

[0072] In the physiological microenvironment of the pancreatic islets, in addition to cellular and biochemical factors, the fluid shear force generated by blood flow and intercellular fluid flow, as a key biophysical factor, is an indispensable part of the in vitro biomimetic construction of the microenvironment. Studies have shown that because pancreatic islet cell clusters grow close to capillaries, they can constantly sense fluid flow from the capillaries, and fluid shear force can significantly improve the development and function of islet cells. Dynamic culture methods, as a major advantage of microfluidic chips, make it feasible to simulate biophysical factors from a fluid dynamics perspective to construct organ-on-a-chip or physiological microsystems.

[0073] For ease of explanation, the assembled chip used to establish the islet-vessel co-culture system will be described first as follows:

[0074] like Figure 10 As shown, an assembled chip includes a receiving cavity for three-dimensional culture of vascular endothelial cells and assembly of pancreatic islet-loaded microfibers. A first microchannel network and a second microchannel network are symmetrically arranged on both sides of the receiving cavity. The first microchannel network and the second microchannel network are respectively connected to a liquid inlet and a liquid outlet at the end away from the receiving cavity. The liquid inlet is equipped with an injection pump for fluid perfusion of the receiving cavity at a flow rate of 5-8 μl / min.

[0075] The assembled chip is prepared using the following method: a microfluidic chip resin template is prepared using a 3D printing device (nanoArch® SI40, BMF Material Technology Inc.). Based on this resin template, a PDMS chip is further fabricated using a molding method. The cavity of one PDMS layer is hollowed out with a scalpel and aligned with another PDMS layer to form the final assembled chip, which is then sterilized for later use. As an example of the present invention, the height of the liquid inlet and liquid outlet is 0.15 mm, and the width of the receiving cavity is 8 mm and the height is 1 mm.

[0076] To simulate capillary blood flow and match physiological flow velocities, a fluid dynamics simulation was performed. The model was built in three dimensions, consistent with experimental settings. By employing laminar single-phase flow, changes in shear stress could be effectively monitored.

[0077] in, and These are the fluid's density and dynamic viscosity, respectively. For fluid velocity vector; This refers to pressure.

[0078] In this model, a flow rate of 7 μL / min was set at the inlet, under laminar flow conditions. The fluid density and dynamic viscosity were set to 1 × 10³ kg / cm³ and 1 × 10⁻³ Pa·s, respectively. The physiological fluid velocity in capillaries is typically 0.3–0.7 mm / s. By adjusting the inlet flow rate, the simulated flow velocity in the microchannel was matched to this velocity, and the system perfusion flow rate of 7 μL / min was ultimately confirmed.

[0079] Matrigel is laid on the bottom of the receiving cavity, and a vascular ring structure is formed on the Matrigel. The vascular ring structure is formed by inoculating and culturing HUVECs. Multiple segments of fully cultured and developed into clusters of load microfibers are placed on the upper part of the vascular ring structure. Matrigel is laid on the load microfibers again, and then a PDMS layer is used as a cover to clamp and seal the receiving cavity.

[0080] Matrigel, a soluble basement membrane matrix, can mimic the extracellular matrix environment in vivo and provide the necessary support and signals for HUVECs to form vascular structures, promoting cell attachment and migration. By modifying the bottom wall of the assembly chip's containment cavity with Matrigel, the growth and vascularization of HUVECs were promoted. After 24 hours of culture, HUVECs within the containment cavity formed abundant network-like vascular rings on Matrigel and expressed the endothelial-specific marker CD31. This is mainly attributed to the tubular ability of endothelial cells, enabling them to migrate, proliferate, and form tubular structures on a suitable matrix. Microfibrils themselves are tubular structures, and the surface of CaA microfibrils can be modified to allow HUVECs to adhere. Therefore, using microfibrils as cell carriers can promote the formation of complex, blood vessel-like structures on the microfibril surface. Matrigel was modified on the surface of unloaded microfibrils, cut into small segments, and placed in the assembly chip with pre-formed vascular rings. Fluid shear force was applied through continuous perfusion using an injection pump.

[0081] The specific procedure is as follows: The assembled chip is sterilized for 30 minutes using UV light beforehand; 100 μL of Matrigel is added to the bottom of the housing cavity, and the mixture is incubated at 37°C for 10 minutes to allow it to gel; the HUVECs are digested and resuspended in the culture medium at a concentration of 2 × 10⁻⁶. 5 mL -1The microfibers were seeded at a density within the containment chamber and incubated for 12 hours to allow spontaneous formation of vascular ring structures. The supernatant of the vascular culture layer with the formed vascular ring structures was aspirated. Microfibers loaded with β cells were cultured in vitro for three days to allow them to fully develop into clusters. These clusters were then cut into small segments and placed on top of the vascular ring structures. 100 μL of Matrigel was added to completely cover the microfibers, and the mixture was placed in an incubator to allow gelation, thus achieving high-order assembly of the microfibers. A PDMS layer was used as a cap, and a custom-designed clamp was used to secure and seal the exposed vascular culture chip. Figure 16 As shown.

[0082] To match the flow rate of the liquid in the chip with the physiological flow rate of the capillaries, fluid simulation was performed using software. The flow rate of the liquid inlet was 6-8 μL / min, which matched the physiological fluid flow rate in the capillaries (0.3-0.7 mm / s).

[0083] Example 5: Drug Evaluation Methods and Vascular Function Verification

[0084] 5.1 Evaluation of GLP-1 drugs

[0085] GLP-1 (glucagon-like peptide-1) is an incretin that enhances insulin secretion by activating the GLP-1 receptor on the surface of pancreatic β-cells, thus achieving effective glycemic management in diabetic patients. It also inhibits β-cell apoptosis and promotes β-cell proliferation and regeneration. Furthermore, studies have shown that GLP-1 (7-37) can activate endothelial nitric oxide synthase (eNOS), increasing nitric oxide (NO) production in vascular endothelial cells, thereby inhibiting platelet aggregation, reducing vascular plaque, and exerting a cardiovascular protective effect. Effective glycemic management, improved pancreatic function, vascular protection, and reduction of diabetic vascular-related complications are all crucial for the clinical treatment of diabetes. To evaluate the physiological function of β-TC6 in the assembled system and to evaluate the hypoglycemic drug GLP-1, the assembled system was continuously perfused for three days using a medium containing 100 nM GLP-1, with a control group not containing GLP-1 medium.

[0086] On days 0 and 3 of culture, both cell types were harvested, and cell viability was determined using the MTT assay. Specifically: the supernatant was carefully aspirated, and 90 μL of fresh culture medium and 10 μL of MTT solution were added. Culture was continued for 4 hours. Then, the supernatant was aspirated, and 110 μL of Formazan dissolving solution was added to each well to fully dissolve the crystals. The supernatant was transferred to a 96-well plate, and the absorbance of each well was measured at 570 nm using a multi-wavelength microplate reader (Multiskan SkyHigh, Thermo Scientific™) to assess the proliferation of β-TC6 cells within microfibrils and HUVECs in the vascular culture chip. Zero wells were set up (culture medium, MTT, Formazan dissolving solution). Results are shown in [Figure number missing]. Figure 11 ,in Figure 11 a and b represent the activity of β-TC6 cells (n=6) and HUVECs (n=4), respectively.

[0087] Insulin concentration was measured using a mouse insulin ELISA kit on day 3 of dynamic culture. Results are shown below. Figure 12 Specifically: Microfibrils were extracted and dissolved in citric acid, and cells were repeatedly frozen and thawed to disrupt them. The supernatant was collected by centrifugation for testing. Insulin concentration is expressed in pg / mL. The specific procedure was performed according to the ELISA kit instructions. On day 3, 100 μl of the supernatant was taken and measured using a nitric oxide assay kit. The results are shown in the table below. Figure 13 The specific operating procedure should be performed according to the kit's instruction manual.

[0088] As shown in the figure, compared with the control group without GLP-1 (7-37), the proliferation activity of β-TC6 cells was significantly increased on day 3 (*p<0.05), and insulin secretion increased from 39.18±3.91 pg / mL / chip to 53.31±7.24 pg / mL / chip, an increase of approximately 1.36 times (**p<0.01). This result is consistent with previously reported conclusions, demonstrating that GLP-1 (7-37) can stimulate the proliferation and regeneration of pancreatic β-cells, exert anti-apoptotic effects through multiple signaling pathways, and enhance insulin synthesis and secretion. This result also indicates that the microphysiological system constructed using islet-like loaded microfibers in this invention can be used to evaluate pancreatic islet function and has the potential to screen diabetes drugs.

[0089] For vascularized HUVECs in the islet-vascular co-culture system, the cell proliferation activity was significantly increased compared to the control group without GLP-1 (7-37) (**p<0.01). The results showed an increase in mean NO concentration, from 0.068±0.010 μmol / mL / chip to 0.085±0.012 μmol / mL / chip. These results are consistent with previously reported conclusions, reflecting that GLP-1 (7-37) helps promote endothelial cell proliferation, reduce damage and apoptosis, improve endothelial cell function, and maintain vascular integrity.

[0090] 5.2 Vascular function verification

[0091] ECM complete culture medium was injected into the liquid inlet using a syringe pump at a flow rate of 7 μL / min for three days of dynamic co-culture. Following the manufacturer's instructions, Alexa Fluor® 488 phalloidin was used to characterize the cytoskeletal actin (F-actin) of β-TC6 cells in microfibrils and HUVECs in the vascular culture chip, respectively. Immunofluorescence staining was used to detect the platelet-endothelial cell adhesion molecule CD31 (PECAM-1 / CD31) of HUVECs. Results are shown in [Figure number missing]. Figure 14 Specifically, cells within the microchip were fixed in 4% w / w paraformaldehyde at room temperature for 60 min, then immersed in blocking reagent for 1 h, followed by the addition of the corresponding primary antibody at a dilution of 1:100, incubation at 4°C overnight, and finally the addition of the corresponding secondary antibody Alexa Fluor at room temperature. TM 568 (diluted 1:100) for 1 h, cell nuclei stained with DAPI for 10 min, and fluorescence images of cells obtained using an inverted fluorescence microscope and a laser confocal microscope.

[0092] To assess the expression level of vascularization genes in HUVECs on a microarray, cells were extracted after 3 days of dynamic co-culture, and total RNA was isolated according to the kit's instructions. Subsequently, cDNA synthesis and PCR were performed on the total RNA using a one-step RT-qPCR kit (dye method), and the results were detected using a Lightcycle® 96 (Roche) PCR instrument. The results are shown below. Figure 15 .

[0093] The expression levels of each gene were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). HUVECs-specific genes include CD31, VCAM-1, and KLF-2, and their primer sequences are shown in Table 1.

[0094] Table 1 Primer sequences used for RT-qPCR gene expression detection analysis

[0095] Figure 14 Image B1 is a panoramic view obtained by stitching together multiple confocal images. F-actin (green) shows that HUVECs are distributed both on and between microfibrils, and the fibers show a certain outline after cell attachment. A section of the region was scanned along the Z-axis using laser confocal microscopy at a layer thickness of 5 μm. Due to limitations in chip thickness and microscope working distance, only 37 layers (approximately 185 micrometers) of scanning results were obtained. However, from... Figure 14 -B 2a (21 / 37) and Figure 14 -B 2b (29 / 37) Images of different layers clearly show that HUVECs are attached to the surface of two microfibers, proliferating and migrating from the bottom up. Figure 14 -B 2c (29 / 37) and 14-B 2d (29 / 37) is the same as 14-B 2b (29 / 37) The staining results of cell nuclei (blue) and CD31 (red) in the same sheet show that HUVECs can express CD31, indicating that HUVECs can be vascularized on microfibrils. Moreover, HUVECs are arranged in a monolayer on microfibrils, and the arch-shaped cross-section formed by them is similar to the vascular structure, reflecting the morphological adaptation of HUVECs in a specific microenvironment, which can better simulate the vascular structure in the physiological microenvironment of pancreatic islets.

[0096] Depend on Figure 15 It is evident that in the microfluidic chip-based pancreatic islet-vascular co-culture system described in this application, the expression of CD31 in HUVECs increased by approximately 4.25 times (*p<0.05), the expression of VCAM-1 increased by approximately 14.88 times (***p<0.005), and the expression of KLF-2 increased by approximately 38.88 times (***p<0.005) compared to two-dimensional culture. This indicates that angiogenesis is active and intercellular interactions (such as intercellular adhesion and signal transduction) are enhanced.

[0097] Specifically, increased expression of CD31, a specific marker for endothelial cells, indicates enhanced endothelial cell differentiation and angiogenesis. Increased expression of VCAM-1 is associated with inflammatory responses and leukocyte adhesion, which play an important role in angiogenesis. Increased expression of KLF-2 is related to the functional resting state of endothelial cells and vascular protection; its high expression helps maintain vascular stability and function. All these results indicate that in the assembled system, the prepared modified microfibers can achieve vascularization through the attachment, proliferation, and migration of HUVECs on their surface.

[0098] In summary, it can be seen that in the constructed pancreatic islet microphysiological system, GLP-1 (7-37), as a diabetes treatment drug, can regulate insulin secretion and NO release, improve the function of the islets and endothelium, demonstrating its important role in treating diabetes and delaying diabetic vascular-related complications. Furthermore, it has been demonstrated that the constructed pancreatic islet microphysiological system can simulate the key structures and characteristics of islet tissue and can perform in vitro evaluation of diabetes treatment drugs, potentially bridging the gap between in vitro and in vivo models and providing new tools and methods for drug development, disease model construction, and personalized medicine.

[0099] This invention successfully constructed a pancreatic islet microphysiological system based on microfiber assembly and conducted a comprehensive evaluation of the diabetes treatment drug GLP-1 (7-37). The system consists of two main parts: first, microfibers loaded with a large number of β-TC6 cells were prepared using a combination of microfluidic spinning and droplet microfluidics; subsequently, these microfibers were assembled to promote the growth of vascular endothelial cells on their surface. This construction method simulates a physiological microenvironment closer to that in vivo through the synergistic effect of biophysiological and biochemical factors. Using this constructed microphysiological system, combined with cell viability and functional analysis, GLP-1 (7-37) was evaluated in vitro. The results showed that GLP-1 (7-37) increased the cell viability of β-TC6 and HUVECs by approximately 1.13-fold and 1.43-fold, respectively, and increased insulin secretion by approximately 1.36-fold. This novel pancreatic islet microphysiological system is expected to provide innovative technologies and methods for fields such as tissue biomimicry, drug evaluation, and food safety, promoting the development of related research and applications.

[0100] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip, characterized in that, include: S1. Prepare microfibers loaded with pancreatic islets; S2. Prepare an assembled chip, the assembled chip including a receiving cavity, a first microchannel network and a second microchannel network symmetrically arranged on both sides of the receiving cavity, the first microchannel network and the second microchannel network being connected to a liquid inlet and a liquid outlet respectively at the end away from the receiving cavity; S3. Control the flow rate of the liquid inlet to 6-8 μL / min for dynamic culture; The islet-loaded microfibers in step S1 are prepared using a microfluidic chip. The microfluidic chip includes a second channel (2) and a fourth channel (4), which are used to transport sample flow and core flow, respectively. There are two second channels (2) and they are located on both sides of the fourth channel (4). The second channel (2) and the fourth channel (4) are merged at a first position (7) to form a first confluence channel. The microfluidic chip also includes a PDMS chamber for periodically pressurizing to expand and squeeze the channel of the core flow and close it. The microfluidic chip also includes a first channel (1) for transporting sheath flow. There are two first channels (1) and they are located on both sides of the first confluence channel. The first channel (1) and the first confluence channel are merged at a second position (5) to form a sixth channel (6) for the outflow of the islet-loaded microfibers. Collagen was added to the core stream, followed by the addition of β-TC6 cells to achieve a cell density of 4 × 10⁻⁶ cells in the core stream. 6 mL -1 -8×10 6 mL -1 The flow rates of the core flow, sample flow, and sheath flow were controlled at 0.8-1.2 μL / min, 18-22 μL / min, and 80-120 μL / min, respectively, with a frequency of 0.8-1.2 Hz. In step S2, Matrigel is laid on the bottom of the receiving cavity, and a vascular ring structure is formed on the Matrigel. The vascular ring structure is formed by inoculating and culturing HUVECs. Multiple segments of fully cultured and developed load microfibers are placed on the upper part of the vascular ring structure. Matrigel is laid on the load microfibers again, and then a PDMS layer is used as a cover to clamp and seal the receiving cavity.

2. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 1, characterized in that, The specific preparation method for step S2 is as follows: Add 100 μL of Matrigel to the bottom of the container and incubate at 37°C for 10 min to allow it to gel; digest the HUVECs, resuspend them in the culture medium, and incubate at 2 × 10⁻⁶. 5 mL -1 The microfibers were seeded at a density in the containment chamber and placed in an incubator for 12 hours to allow them to spontaneously form vascular ring structures. The supernatant of the vascular culture layer with the formed vascular ring structure was aspirated from the containment chamber. The microfibers loaded with β cells were cultured in vitro for three days to allow them to fully develop into clusters. They were cut into small segments and placed on the vascular ring structure. 100 μL of Matrigel was added to completely cover the microfibers. The microfibers were placed in an incubator to allow them to gel, thereby achieving high-order assembly of the microfibers. A PDMS layer was used as a cap, and the exposed vascular culture layer chip was clamped and sealed using a custom clamp.

3. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 1, characterized in that, The assembled chip is prepared by the following method: a microfluidic chip resin template is prepared using 3D printing equipment, and a PDMS chip is made based on the resin template by molding. The cavity of one PDMS layer is hollowed out and aligned and sealed with another PDMS layer to obtain the chip.

4. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 1, characterized in that, The microfluidic chip also includes a third channel (3), one end of which is connected to a gas control device, and the other end is provided with a connected PDMS chamber. There are two PDMS chambers located on both sides of the fourth channel (4), which are used to close the fourth channel (4) by pressurizing and expanding and squeezing it from both sides.

5. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 1, characterized in that, The microfluidic chip is prepared using the following method: a resin template for the microfluidic chip is prepared using 3D printing equipment; a mixture of PDMS and curing agent with a weight ratio of 8-12:1 is poured onto the resin template; after vacuum degassing, it is cured at 75-85℃ for 0.8-1.1 hours; the PDMS layer with microstructure is peeled off to obtain the PDMS microchip; the PDMS layers of two PDMS microchips with the same structure are subjected to oxygen plasma surface treatment, aligned and sealed to obtain the final product.

6. A pancreatic islet-vascular co-culture system based on a microfluidic chip, characterized in that, Obtained by the method described in any one of claims 1-5.

7. The application of the microfluidic chip-based pancreatic islet-vascular co-culture system as described in claim 6 in the evaluation of drugs for the treatment of diabetes or in the preparation of pancreatic islet models.

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