Pancreatic islet-blood vessel co-culture system based on micro-fluidic chip as well as establishment method and application of pancreatic islet-blood vessel co-culture system

Abstract

The invention provides a pancreas islet-blood vessel co-culture system based on a micro-fluidic chip, and an establishment method and application thereof. The establishment method of the pancreas islet-blood vessel co-culture system comprises the following steps: S1, preparing pancreas islet-loaded microfibers; s2, an assembled chip is prepared, the assembled chip comprises a containing cavity, a first micro-channel network and a second micro-channel network are symmetrically arranged on the two sides of the containing cavity, and the ends, away from the containing cavity, of the first micro-channel network and the second micro-channel network communicate with a liquid inlet and a liquid outlet correspondingly; and S3, controlling the flow of a liquid inlet to be 6-8 [mu] L / min, and carrying out dynamic culture. According to the method, key structures and characteristics of pancreas islet tissues can be simulated, in-vitro evaluation can be carried out on diabetes treatment drugs, gaps between in-vitro and in-vivo models are expected to be bridged, and new tools and methods are provided for the fields of drug development, disease model construction, personalized medical treatment and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a pancreatic islet-blood vessel co-culture system based on a microfluidic chip, an establishment method and an application thereof. Background Art

[0002] Currently, basic research on diabetes, screening of therapeutic drugs, and evaluation of new treatments usually require the construction of disease models. Animal models have species-specific differences, and two-dimensional models have difficulty simulating islet-vascular interactions and lack a dynamic fluid environment, resulting in low accuracy in drug response predictions.

[0003] Microphysiological systems (MPS) are complex organ physiological microsystems constructed in vitro using microfluidic 3D cell culture devices. They integrate multiple cell structures, 3D cell structures, cell components, fluid flow, dynamic pressure or stretching, and interactions between multiple organs. This makes it possible to simulate the characteristics of human tissues and organs and the interactions between tissues and organs in vitro, and shows significant application prospects in fields such as life sciences and new drug research and development.

[0004] To this end, Chinese patent application number 201810977419.0 discloses a method for establishing an organ-on-a-chip (OOC) model of pancreatic islet pathology, comprising the following steps: chip preparation and modification, spheroidization of pancreatic β-TC6 cells, streptozotocin-induced 3D spheroid injury, and functional testing. This approach enables the preparation of controllable and uniform pancreatic β-TC6 spheroids and utilizes streptozotocin to induce a 3D spheroid-based pancreatic islet injury model, providing a novel approach for establishing a type 1 diabetes model.

[0005] Furthermore, Chinese patent application number 202211113469.7 discloses a method for establishing an in vitro model of type 1 diabetes based on a microfluidic chip and its application. The microfluidic chip comprises a pancreatic islet functional area and a sugar uptake functional area, connected by a communication channel. The microfluidic chip is surface-modified with collagen. The pancreatic islet functional area is used to seed pancreatic islet cells with insulin secretion function, while the sugar uptake functional area is used to seed cells with sugar uptake function. This approach can simulate type 1 diabetes with insufficient insulin secretion levels while also possessing the two key characteristics of sugar metabolism: insulin secretion and sugar uptake, reflecting the interactive characteristics of sugar regulation in the body.

[0006] The vascularization strategy in the above scheme is limited to perfusion simulation and does not integrate endothelial cell co-culture. The model construction is highly complex and difficult to standardize for drug screening. In view of this, the present invention is proposed. Summary of the Invention

[0007] The problem solved by the present invention is that the existing in vitro diabetes model established based on microfluidic chips is limited to perfusion of simulated blood vessels, and is unable to integrate pancreatic islets with vascular endothelium and achieve dynamic interaction, resulting in a large difference from the in vivo microenvironment.

[0008] To solve the above problems, the present invention provides a method for establishing an islet-blood vessel co-culture system based on a microfluidic chip, comprising: S1, preparing microfibers loaded with islets; S2, preparing an assembly chip, wherein the assembly 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, and the first microchannel network and the second microchannel network are respectively connected to a liquid inlet and a liquid outlet at one 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, Matrigel is laid on the bottom of the accommodating cavity in step S2, and a vascular ring structure is formed on the Matrigel. The vascular ring structure is formed by inoculating and culturing HUVECs. A plurality of loaded microfibers that have been fully cultured and developed into a cluster are placed on the vascular ring structure. Matrigel is laid again on the loaded microfibers, and then a PDMS layer is used as a cover to clamp and seal the accommodating cavity.

[0010] Preferably, the specific preparation method of step S2 is as follows: add 100 μL of Matrigel to the bottom of the holding chamber, place it in a 37°C incubator for 10 min to form a gel; digest HUVECs, resuspend them in culture medium, and add 2×10 5 mL -1 The cells were seeded at a density of 100 μL in the holding cavity and left to stand in the incubator for 12 hours to spontaneously form a vascular ring structure. The supernatant of the vascular culture layer with the formed vascular ring structure in the holding cavity was aspirated, and the microfibers loaded with β cells were cultured in vitro for three days to fully develop into clusters. The microfibers 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 the incubator to gel, thereby achieving high-order assembly of the microfibers. The PDMS layer was used as a cover, and the exposed vascular culture layer chip was clamped and closed with a customized clamp.

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

[0012] Preferably, the islet-loaded microfibers in step S1 are prepared using a microfluidic chip, and the microfluidic chip is provided with channels for conveying core flow, sample flow and sheath flow respectively. The microfluidic chip also includes a PDMS chamber for periodically pressurizing to expand and squeeze the core flow channel and close it.

[0013] Preferably, the microfluidic chip includes a second channel and a fourth channel, which are respectively used to transport sample flow and core flow, and there are two second channels and they are respectively located on both sides of the fourth channel. The second channel and the fourth channel are merged at a first position to form a first convergence channel. The chip also includes a third channel, one end of the third channel is connected to the gas control device, and the other end is provided with a connected PDMS chamber, and there are two PDMS chambers and they are respectively located on both sides of the fourth channel, which are used to expand by pressurization and squeeze the fourth channel from both sides to close it; the microfluidic chip also includes a first channel for transporting sheath flow, and there are two first channels and they are located on both sides of the first convergence channel. The first channel and the first convergence channel are merged at a second position to form a sixth channel for flowing out the formed microfibers.

[0014] Preferably, the preparation process parameters of the islet-loaded microfibers are as follows: collagen is added to the core flow, and β-TC6 cells are added to make the cell density of the core flow 4×10 6 mL -1 -8×10 6 mL -1 , the flow rates of core flow, sample flow and sheath flow were controlled to be 0.8-1.2μL / min, 18-22μL / min, 80-120μL / min, and the frequency was 0.8-1.2Hz, 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 a curing agent with a weight ratio of 8-12:1 is poured on the resin template, and after vacuum debubbling, it is cured at 75-85°C for 0.8-1.1h, and the PDMS layer with the microstructure is peeled off to obtain a PDMS microchip; the PDMS layers of two PDMS microchips with the same structure are subjected to oxygen plasma surface treatment, and the two PDMS microchips are aligned and sealed to obtain the result.

[0015] The present invention also provides a pancreatic islet-blood vessel co-culture system based on a microfluidic chip, which is obtained by the above-mentioned establishment method.

[0016] The present invention also discloses the application of a pancreatic islet-blood vessel co-culture system based on a microfluidic chip in evaluating drugs for treating diabetes or preparing a pancreatic islet model.

[0017] Compared with the prior art, the method for establishing and applying the pancreatic islet-vascular co-culture system based on a microfluidic chip of the present invention has the following beneficial effects: 1) Design of a pneumatically controlled microvalve chip, utilizing periodic nitrogen pressure regulation to precisely cut off the core flow, and combined with microfluidic spinning technology to generate monodisperse aqueous droplet microfibers, achieving efficient encapsulation of pancreatic beta cells, overcoming the shortcomings of traditional passive shear force droplet technology, such as poor uniformity and low cell viability; 2) Dynamic vascularization: RGD peptide modification of the calcium alginate microfiber surface promotes endothelial cell adhesion and directional growth. Combined with a microfluidic dynamic perfusion system to simulate capillary blood flow, this enables physiological-level interaction in islet-vascular co-culture. 3) Bionic microenvironment integration: By stacking microfibers and assembling chip chambers, a multilayer structure containing a large number of pancreatic islet clusters and a three-dimensional vascular network is formed. Biochemical factors and biophysical stimuli are coordinated to simulate a physiological microenvironment closer to the body through the synergistic effect of biophysical and biochemical factors, significantly improving the model's prediction accuracy for diabetes drug responses.

[0018] 4) This invention combines pneumatic control technology with dynamic microfiber co-culture for the first time, simultaneously addressing the two major technical bottlenecks of "high-throughput islet loading" and "functional vascularization," providing an in vitro evaluation platform for diabetes research that is closer to human physiology. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic structural diagram of a chip in the pneumatically controlled microfluidic spinning device according to Example 1 of the present invention; Figure 2 This is a physical picture of the template of the chip in the pneumatically controlled microfluidic spinning device described in Example 1 of the present invention; Figure 3 This is a schematic diagram of the pneumatically controlled microfluidic spinning device described in Example 1 of the present invention; Figure 4 : is a size histogram of the droplet spacing l and droplet length Δl under different conditions in Example 2 of the present invention (n=10); Figure 5 These are the bright field image and fluorescence image of the aqueous droplet microfiber loaded with β-TC6 cells in Example 2 of the present invention; Figure 6 These are bright field images and fluorescence images of β-TC6 cells cultured in microfiber droplets on days 0, 1, and 7 in Example 3 of the present invention; Figure 7 The proliferation of β-TC6 cells in microfibers in Example 3 of the present invention on day 0 and day 3, n = 3; Figure 8This is an immunofluorescence staining image of β-TC6 cells cultured in microfibers on the third day in Example 3 of the present invention; Figure 9 The expression results of INS-1, INS-2 and PDX-1 in β-TC6 cells under different culture modes in Example 3 of the present invention are shown; Figure 10 Schematic diagram and actual image of the chip of the pancreatic islet-vascular co-culture system based on the microfluidic chip in Example 4 of the present invention; Figure 11 The cell survival and growth of β-TC6 cells (n=6) and HUVECs (n=4) were detected by MTT assay in Example 5 of the present invention; Figure 12 Insulin secretion was detected by ELISA in Example 5 of the present invention (n=6); Figure 13 The NO release was detected by chemical method in Example 5 of the present invention, including a control group (n=3) and an experimental group (n=10); Figure 14 This is a diagram showing the results of fluorescence staining of angiogenesis-related cells in Example 5 of the present invention; Figure 15 The expression results of mRNA related to HUVECs angiogenesis in Example 5 of the present invention; Figure 16 This is a flow chart for preparing the pancreatic islet-vascular co-culture system based on a microfluidic chip according to an embodiment of the present invention.

[0020] Description of reference numerals: 1-first channel; 2-second channel; 3-third channel; 4-fourth channel; 5-second position; 6-sixth channel; 7-first position. DETAILED DESCRIPTION

[0021] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following detailed description of the specific embodiments of the present invention is made in conjunction with the accompanying drawings. The technical features of the embodiments of the present invention can be combined with each other without conflict.

[0022] Unless otherwise stated, the chemical reagents and biological materials used in this invention were obtained from commercial sources and processed and used according to standard operating procedures. All experimental steps were performed under conventional laboratory conditions to ensure the reproducibility and reliability of the technical solutions. The specific structural units, connection relationships, and coordination relationships are described in detail in the following sections.

[0023] The microphysiological system can simulate the response of human organs to drugs more realistically, can quickly screen the effectiveness and toxicity of drugs on the chip, greatly shorten the drug development cycle, reduce research and development costs, and show great application prospects in the fields of life sciences and new drug development; among them, the pancreatic islet microphysiological system can be used to construct a disease model of diabetes and its complications. Since the capillary network in the pancreatic islets can not only provide blood supply and assist the pancreatic islets in sensing blood glucose concentration, but also provide structural support through its basement membrane and endothelial cells to maintain the morphology and function of the pancreatic islets, the role of blood vessels should be considered as an important part of constructing the pancreatic islet microphysiological system. The existing technology for establishing an in vitro model of diabetes based on microfluidic chips often uses a perfusable microfluidic system to simulate capillary blood flow. How to integrate the pancreatic islets with the vascular endothelium and achieve dynamic interaction still faces major challenges. To this end, the applicant proposes the following technical solutions: Example 1 Pneumatically controlled microfluidic spinning device for preparing microfibers For the convenience of explanation, the pneumatically controlled microfluidic spinning device for preparing microfibers is first described as follows: like Figure 1-3 As shown, a pneumatically controlled microfluidic spinning device includes a microfluidic chip, the microfluidic chip including a second channel 2 and a fourth channel 4, respectively used to transport a sample flow and a core flow, the second channel 2 having two channels and located on either side of the fourth channel 4, the second channel 2 and the fourth channel 4 merge at a first position 7 to form a first confluence channel, the chip also including 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, the two PDMS chambers being located on either side of the fourth channel 4, for expanding by pressurization and squeezing the fourth channel 4 from both sides to close it; The microfluidic chip also includes a first channel 1 for transporting 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 merge at the second position 5 to form a sixth channel 6 for flowing out the formed microfibers.

[0024] When the gas control device is turned on, a PDMS chamber is formed. Due to the elasticity of PDMS itself, the PDMS chamber continues to expand and squeeze 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 opened or closed, thereby periodically cutting off the core flow, and precise control can be achieved by adjusting the frequency and flow rate of the core flow, which can be used for controllable encapsulation and even has the potential to carry out independent organoid culture.

[0025] 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. 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.

[0026] 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, and a height of 500 μm, and is spaced 150 μm apart from the fourth channel 4. It should be noted that the dimensions of the channels and resin template of the microfluidic chip were measured using an inverted fluorescence microscope (IX73, Olympus), and the height was measured using a vernier caliper.

[0027] The core flow was a 15% w / w Dextran solution, the sample flow was a mixed solution of 1% w / w NaA and 17% w / w PEG, and the sheath flow was a mixed solution of 4% w / w CaCl2 and 17% w / w PEG. These solutions were injected into the corresponding channels of the microfluidic chip via a microsyringe pump (Harvard), and deionized water was used to collect the microfibers.

[0028] This setup utilizes the Ca in NaA and CaCl2 solutions. 2+ An instantaneous ionic crosslinking reaction occurs to form microfibers. At the same time, due to the intermolecular interaction and physicochemical property differences between PEG and DEX in aqueous solution, a two-phase aqueous system can be formed. Therefore, at the first position 7, the core flow controlled by air pressure can be sheared to form droplets and stored in the microfibers. At the second position 5, NaA and CaCl2 in the solution are mixed. 2+ An instantaneous ionic cross-linking reaction occurs to form microfibers, which then flow out and are collected through the sixth channel 6. It should be noted that the above solutions are all prepared with deionized water.

[0029] 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 a curing agent with a weight ratio of 10:1 is poured on the resin template, and after vacuum removal of bubbles, the PDMS is cured at 80°C for 1 hour, and the PDMS layer with the microstructure is peeled off to obtain a PDMS microchip; the PDMS layers of two PDMS microchips with the same structure are subjected to oxygen plasma surface treatment, and the PDMS layers are aligned and sealed to generate the final microfluidic chip, which is sterilized and set aside.

[0030] Example 2 Optimization of process parameters for preparing microfibers To visualize the microfibers, green fluorescent polystyrene microspheres (5 µm, excitation / emission: 488 / 518 nm) were mixed in the core flow solution (0.1 mg / ml). The flow rates of the core flow, sample flow, and sheath flow were in the range of 1-3 μL / min, 5-40 μL / min, and 50-200 μL / min, respectively. The frequency range of the pump valve switching was 0.625-1 Hz. An inverted fluorescence microscope was used to photograph the microfibers and perform size statistics on the droplet length Δl, the head-to-head distance l of the droplets, and the microfiber width.

[0031] 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 adjusted in the range of 0.625-2.5 Hz. The results are shown in Figure 2. Figure 4 -a; the flow rates of the sample flow and sheath flow were set to 20 and 100 μL / min respectively and the frequency was 1 Hz, and the flow rate of the core flow was adjusted to vary in the range of 0.5-2.0 μL / min. The results are shown in Figure 4 -b; Set the core flow and sheath flow rates to 1 and 100 μL / min and the frequency to 1 Hz, and adjust the sample flow rate to vary in 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 to vary in the range of 50-200 μL / min. The results are shown in Figure 4 -d.

[0032] Depend on Figure 4 -a shows that as the frequency slows down, Δl increases from 494.7±60.14μm to 1111.99±113.73μm, l increases from 854.55±51.74μm to 2587.49±398.21μm, while the width of the microfiber does not change significantly. This shows that higher frequencies will produce denser droplets, which can effectively increase the flux of droplets in the microfiber, but it also means that the droplets are more difficult to distinguish. Figure 4 As shown in Figure 2, when the core flow rate 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 rate reaches 2.0 μL / min, even with the assistance of the valve, dispersed droplets cannot form, and the core flow becomes turbulent. This indicates that the core flow rate and droplet size are positively correlated within a certain range. Larger droplets can be obtained by increasing the core flow rate. Furthermore, the core flow rate has little effect on the fiber width.

[0033] Depend on Figure 4-c shows that as the sample flow rate increases, the head-to-head distance l of the droplets increases from 1303.544±69.13μm to 2772.82±188.23μm, and the fiber width increases from 388.95±4.47μm to 643.24±10.77μm, while Δl does not change significantly. When the sample flow rate is 5μL / min, the core flow is severely stacked and droplets cannot be formed, indicating that the sample flow rate, droplet spacing and fiber width are positively correlated within a certain range. Figure 4 -d shows that as the sheath flow increases, the effect on l and Δl is 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, and the higher the flow velocity, the thinner the fiber.

[0034] In summary, droplet size is primarily determined by the core flow and valve frequency, droplet spacing is primarily determined by the sample flow, and fiber width is primarily determined by the sheath flow. In this study, to achieve 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, sample, 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 were larger, with high flux and good dispersion, which facilitated the loading of more cells and the formation of independent three-dimensional culture microunits, resulting in more uniform cell microspheres or microtissues.

[0035] Microfibers were prepared under optimal conditions and the morphology of the microfibers was characterized using laser scanning confocal microscopy. Figure 5 .Depend on Figure 5 It can be seen that the prepared microfibers are continuous and complete, and a large number of aqueous phase droplets containing β-TC6 cells are evenly distributed in the microfibers.

[0036] Blue fluorescent polystyrene microspheres (PS microspheres, 5µm, excitation / emission: 400 / 450nm) and green polystyrene microspheres (5µm, excitation / emission: 488 / 518nm) were mixed in the sample flow and core flow solutions at a concentration of 0.1mg / ml, respectively. The three-dimensional images obtained by confocal microscopy showed that the two colors of microspheres existed independently within the spatial range, and there was a clear distinction between the droplets and the fibers, indicating that the core flow and the sample flow did not diffuse into each other. When the PS microspheres in the droplets were replaced with cells of the same size, a space for three-dimensional culture of the cells could be provided, and the aqueous phase components in the droplets could also provide biochemical conditions for cell survival, indicating that the prepared aqueous droplet-loaded microfibers can be used as carriers for loading a variety of chemical substances or for three-dimensional cell culture.

[0037] Example 3 Preparation and Characterization of Islet-Loaded Microfibers Based on the above preparation of aqueous droplet-loaded microfibers, PS microspheres were replaced with mouse insulinoma cells β-TC6, and type I collagen, an extracellular matrix component, was added to the core flow 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.

[0038] In order to encapsulate cells, the spinning device involved was sterilized with ultraviolet light for 30 minutes before operation, the NaA / PEG mixed solution and Dextran solution were filtered with a 0.22 μm sterile membrane, and the CaCl2 solution was sterilized by high pressure.

[0039] When preparing islet-loaded fibers, type I collagen was mixed into the core flow. The specific operation was as follows: 30% (w / w) dextran solution was mixed with 3.34 mg / mL rat tail type I collagen in equal volumes, and a small amount of 0.5M NaOH was added to adjust the pH to approximately 7.4. β-TC6 cells (purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences and cultured in DMEM supplemented with penicillin-streptomycin (1% v / v) and FBS (15% v / v)) were digested with 0.25% trypsin-EDTA and resuspended in the core flow solution to obtain a density of 6×10 6 mL -1 The cell suspension was used for microfluidic experiments.

[0040] The collected microfibers were transferred to a culture dish containing DMEM high glucose medium and cultured for 1-7 days, with the medium changed every day. To track the cells and their location in the fibers, Cell Tracker was used. TM Green CMFDA was used to label β-TC6 cells, and PI was used to stain dead cells. The specific labeling method was based on existing techniques. During the culture period, the prepared microfibers were cut and fluorescent images of the cells were obtained using an inverted fluorescence microscope, such as Figure 6 The cell viability on day 0, day 1, and day 7 indicated that the cells still showed good activity after multiple days of culture. The proliferation of β-TC6 cells in microfibers on day 0 and day 3 was detected by MTT assay. Figure 7 .

[0041] 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 good cell proliferation.

[0042] In order to further explore the growth of β-TC6 cells in microfibers and the formation of pancreatic islets, the islets were cultured in vitro for three days, and the secretion of cytoskeleton protein F-actin and insulin was characterized using laser confocal microscopy. Figure 8 In addition, RT-qPCR was used to measure the expression levels of INS-1, INS-2, and PDX-1 mRNA in microfibers (three-dimensional culture) and culture plates (two-dimensional culture). The results are shown in Figure 9 .

[0043] Depend on Figure 8 It can be seen that the staining results of cytoskeleton F-actin showed that pancreatic islet cells grew in clusters in a three-dimensional culture state and formed multiple islet-like clusters. At the same time, the staining results of insulin proved that the islet-like clusters had normal endocrine function. Figure 9 It can be seen that compared with the two-dimensional culture on the plane, the expression of INS-1 of the β-TC6 cells in the microfibers described in the present application increased by about 340.69 times (*p<0.05), the expression of INS-2 increased by about 1.74 times (**p<0.01), and the expression of PDX-1 increased by about 1026.64 times (*p<0.05). This shows that the aqueous phase droplets in the microfibers provide cells with a three-dimensional space that promotes the growth of cell clusters. It can not only exchange substances with the outside world through the thinner hydrogel medium layer of the microfibers, but also ensure that the cells can be protected from external interference (such as bacteria, various studies have reported that calcium alginate materials have antibacterial properties) inside the aqueous phase droplets. In addition, due to the addition of extracellular matrix components (collagen), biochemical factors are provided to the cells, which further promotes the formation and functional remodeling of islets.

[0044] This application combines pneumatically controlled droplet generation with microfluidics to more simply and quickly prepare high-throughput pancreatic islets. Compared with traditional three-dimensional cell culture in microfluidic droplets, it is more conducive to the recovery and biological analysis of cell-loaded droplets, and can avoid accidental loss or damage of droplets during transfer, cell staining, etc.

[0045] Example 4 Method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip In the pancreatic islet physiological microenvironment, in addition to cellular and biochemical factors, 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. Research has shown that because pancreatic islet cell clusters grow in close proximity to capillaries, they can constantly sense the fluid flow from these capillaries, and fluid shear force can significantly improve the development and function of pancreatic islet cells. Dynamic culture methods, as the main 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.

[0046] For ease of explanation, the following description is given of the assembled chip used to establish the islet-vascular co-culture system: like Figure 10As shown, an assembly chip includes a receiving chamber 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 chamber. The first microchannel network and the second microchannel network are respectively connected to a liquid inlet and a liquid outlet at one end away from the receiving chamber. A syringe pump is provided at the liquid inlet for perfusing the receiving chamber with fluid at a flow rate of 5-8 μl / min.

[0047] The assembled chip was fabricated using the following method: a microfluidic chip resin template was prepared using a 3D printer (nanoArch® SI40, BMF Material Technology Inc.). This resin template was then used as a foundation for the PDMS chip through molding. The cavity of one PDMS layer was hollowed out with a scalpel and then aligned and sealed with the other PDMS layer to form the final assembled chip. The chip was then sterilized and ready for use. In one example, the liquid inlet and outlet were 0.15 mm high, and the accommodating cavity was 8 mm wide and 1 mm high.

[0048] To simulate capillary blood flow and match physiological flow rates, a fluid dynamics simulation was performed. The model was constructed in a three-dimensional configuration, with dimensions consistent with the experimental setup. By employing laminar single-phase flow, changes in shear stress were effectively monitored. in, and are the density and dynamic viscosity of the fluid respectively; is the fluid velocity vector; For pressure.

[0049] In this model, a flow rate of 7 μL / min was set at the inlet, with laminar flow conditions. The fluid density and dynamic viscosity were set to 1 × 10³ kg / cm³ and 1 × 10⁻³ Pa·s, respectively. Normally, the physiological flow rate in capillaries is 0.3-0.7 mm / s. By adjusting the inlet flow rate, the simulated flow rate in the microchannel was matched to this, ultimately confirming a system perfusion flow rate of 7 μL / min.

[0050] Matrigel is laid on the bottom of the accommodating cavity, and a vascular ring structure is formed on the Matrigel. The vascular ring structure is formed by inoculating and culturing HUVECs. Multiple sections of loaded microfibers that have been fully cultured and developed into clusters are placed on the top of the vascular ring structure. Matrigel is laid on the loaded microfibers again, and then a PDMS layer is used as a cover to clamp and seal the accommodating cavity.

[0051] Matrigel, a soluble basement membrane matrix, mimics the in vivo extracellular matrix environment and provides HUVECs with the necessary support and signals to form vascular structures, promoting cell attachment and migration. By modifying the bottom wall of the containment chamber of the assembled chip with Matrigel, HUVEC growth and vascularization were promoted. After 24 hours of culture, HUVECs within the containment chamber formed abundant networks of vascular rings on the Matrigel and expressed the endothelial marker CD31. This is primarily attributed to the endothelial cells' ability to form tubes, enabling them to migrate, proliferate, and form tubular structures on an appropriate matrix. Microfibers are inherently tubular structures, and the surface of CaA microfibers can be modified to facilitate HUVEC adhesion. Therefore, using microfibers as cell carriers can encourage the formation of complex vascular-like structures on the microfiber surface. Matrigel was then applied to the surface of unloaded microfibers, cut into small pieces, and placed in the assembled chip with formed vascular rings. Continuous perfusion with a syringe pump applied shear force.

[0052] The specific operation is as follows: sterilize the assembled chip with UV light for 30 minutes in advance, add 100 μL of Matrigel to the bottom of the cavity, and place it in a 37°C incubator for 10 minutes to make it gel; digest HUVECs, resuspend them in culture medium, and add 2×10 5 mL -1 The cells were seeded at a density of 100 μL in the holding cavity and placed in an incubator for 12 hours to allow them to spontaneously form a vascular ring structure. The supernatant of the vascular culture layer with the formed vascular ring structure in the holding cavity was aspirated, and the microfibers loaded with β cells were cultured in vitro for three days to fully develop into clusters. The microfibers were cut into small pieces 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 and allowed to gel to form, thereby achieving high-order assembly of the microfibers. The PDMS layer was used as a cover, and the exposed vascular culture layer chip was clamped and closed with a customized clamp, such as Figure 16 shown.

[0053] In order 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. When the flow rate of the liquid inlet was 6-8 μL / min, it matched the physiological fluid flow rate in the capillaries (0.3-0.7 mm / s).

[0054] Example 5 Drug Evaluation Method and Vascular Function Verification 5.1 GLP-1 Drug Evaluation GLP-1 (glucagon-like peptide-1) is a type of incretin hormone that enhances insulin secretion by activating the GLP-1 receptor on the surface of pancreatic β-cells, effectively managing blood glucose 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 endothelial cells, thereby inhibiting platelet aggregation, reducing vascular plaque, and exerting cardiovascular protective effects. Effective blood glucose management, improving pancreatic function, protecting blood vessels, and reducing diabetic vascular complications are all key to the clinical treatment of diabetes. To evaluate the physiological function of β-TC6 in the assembly system and to evaluate the hypoglycemic drug GLP-1, the assembly system was continuously perfused for three days using medium containing 100 nM GLP-1. A control group containing medium without GLP-1 was used.

[0055] On the 0th and 3rd day of culture, the above two cell types were taken and the cell activity was determined using the MTT method. Specifically, the supernatant was carefully aspirated, 90 μL of fresh culture medium and 10 μL of MTT solution were added, and the culture was continued for 4 hours. Then the supernatant was aspirated, and 110 μL of Formazan 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 full-wavelength microplate reader (Multiskan SkyHigh, Thermo Scientific™) to evaluate the proliferation of β-TC6 cells in the microfibers and HUVECs in the vascular culture layer chip. The experimental setting was zeroed (culture medium, MTT, Formazan solution), and the results are shown in the table. Figure 11 ,in Figure 11 a, b represent the activity of β-TC6 cells (n=6) and HUVECs (n=4), respectively.

[0056] On the third day of dynamic culture, the concentration of insulin was determined using a mouse insulin ELISA kit. The results are shown in Figure 12 Specifically: The microfibers were removed and dissolved with citric acid, and the cells were repeatedly frozen and thawed. The supernatant was centrifuged and tested. The insulin concentration was expressed in pg / mL. The specific operation process was carried out according to the instruction manual of the ELISA kit. On the third day, 100 μl of the supernatant was taken and measured using the nitric oxide detection kit. The results are shown in Figure 13 The specific operation process is carried out according to the instructions of the kit.

[0057] As shown in the figure, for β-TC6 cells, compared to the control group without GLP-1 (7-37), the proliferation activity of β-TC6 cells on day 3 was significantly increased (*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-fold (**p<0.01). This result is consistent with the conclusions of previous reports, demonstrating that GLP-1 (7-37) can stimulate pancreatic β-cell proliferation and regeneration, exert anti-apoptotic effects through multiple signaling pathways, and enhance insulin synthesis and secretion. This result also demonstrates that the microphysiological system constructed using islet-like microfibers in this invention can be used to evaluate pancreatic islet function and has the potential to screen diabetes drugs.

[0058] In the pancreatic islet-vascular co-culture system, HUVEC cell proliferation activity was significantly enhanced compared to the control group without GLP-1 (7-37) (**p < 0.01). 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 previous reports and suggest that GLP-1 (7-37) promotes endothelial cell proliferation, reduces injury and apoptosis, improves endothelial cell function, and maintains vascular integrity.

[0059] 5.2 Vascular Function Verification Complete ECM culture medium was injected from the liquid inlet at a flow rate of 7 μL / min using a syringe pump for three days of dynamic co-culture. Alexa Fluor® 488 phalloidin was used to characterize the cytoskeletal actin (F-actin) of β-TC6 cells in the microfibers and HUVECs in the vascular culture layer chip, respectively, according to the manufacturer's instructions. Platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31) expression in HUVECs was detected by immunofluorescence staining. The results are shown in Table 1. Figure 14 Specifically, the cells in the microchip were fixed in 4% w / w paraformaldehyde at room temperature for 60 minutes, immersed in blocking reagent for 1 hour, and then the corresponding primary antibody was added at a dilution ratio of 1:100 and incubated at 4°C overnight. Finally, the corresponding secondary antibody AlexaFluor TM 568 (dilution 1:100) for 1 h, cell nuclei were stained with DAPI for 10 min, and fluorescence images of the cells were obtained using an inverted fluorescence microscope and a laser confocal microscope.

[0060] To evaluate the expression levels of vascularization genes in HUVECs on the chip, after 3 days of dynamic co-culture, cells were extracted and total RNA was isolated according to the kit's protocol. Subsequently, cDNA synthesis and PCR reactions were performed on the total RNA according to the protocol of the one-step RT-qPCR kit (dye method), and detection was performed using a Lightcycle® 96 (Roche). The results are shown in Figure 15 .

[0061] The expression level of each gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). HUVECs-specific genes include CD31, VCAM-1, and KLF-2. The primer sequences are shown in Table 1.

[0062] Table 1 Primer sequences for RT-qPCR gene expression analysis Figure 14 Figure B1 is a panoramic image obtained by stitching together multiple confocal images. F-actin (green) shows that HUVECs are distributed on the surface of the microfibers and between the microfibers, and it can be seen that the fibers have a certain outline after cell attachment. A laser confocal microscope was used to scan one area in the Z-axis direction with a layer thickness of 5μm. Due to the thickness of the chip and the working distance of the microscope, only 37 layers (about 185 microns) of scanning results were obtained. However, from Figure 14 -B 2a (21 / 37) and Figure 14 -B 2b (29 / 37) The images of different slices clearly show that HUVECs attached to the surface of the two microfibers, proliferating and migrating from the bottom to the top. 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 layer show that HUVECs can express CD31, indicating that HUVECs can be vascularized on microfibers. In addition, HUVECs are arranged in a single layer on the microfibers, and the arched shape formed by their cross-section is similar to the vascular structure, reflecting the morphological adaptability of HUVECs in a specific microenvironment and can better simulate the vascular structure in the physiological microenvironment of pancreatic islets.

[0063] Depend on Figure 15It can be seen that in the pancreatic islet-vascular co-culture system based on the microfluidic chip described in the present application, the expression of HUVECs CD31 increased by about 4.25 times (*p<0.05) compared with the two-dimensional culture, the expression of VCAM-1 increased by about 14.88 times (***p<0.005), and the expression of KLF-2 increased by about 38.88 times (***p<0.005), which indicates that angiogenesis is active and cell-to-cell interactions (such as cell-to-cell adhesion and signal transduction) are enhanced.

[0064] Specifically, CD31 is a specific marker for endothelial cells, and its increased expression indicates enhanced endothelial cell differentiation and angiogenesis. Increased expression of VCAM-1 is associated with inflammatory responses and leukocyte adhesion, which plays an important role in the angiogenesis process. Increased expression of KLF-2 is associated with the functional quiescence state and vascular protection function of endothelial cells, and its high expression helps maintain vascular stability and functionality. These results all indicate that in the assembly system, the prepared modified microfibers can achieve vascularization through the attachment, proliferation, and migration of HUVECs on their surface.

[0065] 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 pancreatic islet and endothelial function, and demonstrate its important role in treating diabetes and delaying diabetic vascular complications. In addition, it has been demonstrated that the constructed pancreatic islet microphysiological system can simulate the key structures and characteristics of pancreatic islet tissue and can be used for in vitro evaluation of diabetes treatment drugs. It is expected to bridge the gap between in vitro and in vivo models and provide new tools and methods for drug development, disease model construction, and personalized medicine.

[0066] This study successfully constructed a pancreatic islet microphysiological system based on microfiber assembly and conducted a comprehensive evaluation of the diabetes drug GLP-1 (7-37). The system consists of two main components: first, microfibers loaded with large numbers 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 surfaces. This construction method simulates a physiological microenvironment closer to that in vivo through the synergistic effects of biophysical and biochemical factors. Using this constructed microphysiological system, GLP-1 (7-37) was evaluated in vitro, combined with cell viability and functional assays. 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 biomimetic, drug evaluation, and food safety, promoting the development of related research and applications.

[0067] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by 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 assembly chip, wherein the assembly 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, and the first microchannel network and the second microchannel network are respectively connected to a liquid inlet and a liquid outlet at one end away from the receiving cavity; S3. Control the flow rate of the liquid inlet to 6-8 μL / min for dynamic culture.

2. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 1, characterized in that: 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 sections of loaded microfibers that have been fully cultured and developed into clusters are placed on the top of the vascular ring structure. Matrigel is laid again on the loaded microfibers, and then a PDMS layer is used as a cover to clamp and seal the receiving cavity.

3. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 2, characterized in that: The specific preparation method of step S2 is as follows: add 100 μL of Matrigel to the bottom of the holding chamber and place it in a 37°C incubator for 10 min to form a gel; digest HUVECs, resuspend them in culture medium, and add 2×10 5 mL -1 The cells were seeded at a density of 100 μL in the holding cavity and left to stand in the incubator for 12 hours to spontaneously form a vascular ring structure. The supernatant of the vascular culture layer with the formed vascular ring structure in the holding cavity was aspirated, and the microfibers loaded with β cells were cultured in vitro for three days to fully develop into clusters. The microfibers 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 the incubator to gel, thereby achieving high-order assembly of the microfibers. The PDMS layer was used as a cover, and the exposed vascular culture layer chip was clamped and closed with a customized clamp.

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

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 islet-loaded microfibers in step S1 are prepared using a microfluidic chip, which is provided with channels for conveying core flow, sample flow, and sheath flow, respectively. The microfluidic chip also includes a PDMS chamber for periodically pressurizing to expand and squeeze the core flow channel and close it.

6. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 5, characterized in that: The microfluidic chip comprises a second channel (2) and a fourth channel (4), which are respectively used to transport a sample flow and a core flow. The second channel (2) has two channels and is respectively 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 comprises 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. The PDMS chamber has two channels and is respectively located on both sides of the fourth channel (4), and is used to expand by pressurizing and squeezing the fourth channel (4) from both sides to close it. The microfluidic chip also comprises a first channel (1) for transporting a sheath flow. The first channel (1) has two channels and is 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 flowing out microfibers that form islet-loaded microfibers.

7. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 6, characterized in that: The preparation process parameters of the pancreatic islet-loaded microfibers are as follows: collagen is added to the core flow, and β-TC6 cells are added to make the cell density of the core flow 4×10 6 mL -1 -8×10 6 mL -1 , the flow rates of core flow, sample flow and sheath flow were controlled to be 0.8-1.2 μL / min, 18-22 μL / min, 80-120 μL / min, respectively, and the frequency was 0.8-1.2 Hz.

8. The method for establishing a pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 5, characterized in that: The microfluidic chip is prepared by the following method: using a 3D printing device to prepare a microfluidic chip resin template, pouring a mixture of PDMS and a curing agent with a weight ratio of 8-12:1 on the resin template, vacuum de-bubbling and curing at 75-85°C for 0.8-1.1 hours, peeling off the PDMS layer with the microstructure to obtain a PDMS microchip; performing oxygen plasma surface treatment on the PDMS layers of two PDMS microchips with the same structure, aligning and sealing them to obtain the result.

9. A pancreatic islet-vascular co-culture system based on a microfluidic chip, characterized in that: Obtained by the establishment method according to any one of claims 1 to 8.

10. Use of the pancreatic islet-vascular co-culture system based on a microfluidic chip according to claim 9 in evaluating drugs for treating diabetes or preparing pancreatic islet models.

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