Microfiber for loading pancreas islet, preparation method and application

Abstract

The invention provides a microfiber for loading pancreas islet, a preparation method and application, the preparation method comprises the following steps: S1, preparing a micro-fluidic chip and building a pneumatic control micro-fluidic spinning device, the micro-fluidic chip is provided with channels for conveying a core flow, a sample flow and a sheath flow, the micro-fluidic chip further comprises a PDMS chamber, and the PDMS chamber is provided with a PDMS cavity; a channel for periodically pressurizing to expand so as to squeeze and close the core flow; s2, adding collagen into the core flow, then adding beta-TC6 cells to enable the cell density of the core flow to be 4 * 10 < 6 > mL <-1 > to 8 * 10 < 6 > mL <-1 >, respectively controlling the flow rates of the core flow, the sample flow and the sheath flow to be 0.8-1.2 [mu] L / min, 18-22 [mu] L / min and 80-120 [mu] L / min, and carrying out preparation under the frequency of 0.8-1.2 Hz; according to the method, a pneumatic control droplet generation method is combined with microfluidics, so that the high-flux islet-like can be prepared more simply and quickly, and the difference between the islet-like and an in-vivo complex microenvironment is small; the prepared microfiber is continuous and complete, and a large number of water-phase liquid drops containing beta-TC6 cells are uniformly distributed in the microfiber.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a microfiber for loading pancreatic islets, a preparation method and an application thereof. Background Art

[0002] Diabetes is a serious chronic disease, predicted by the World Health Organization to become the seventh leading cause of death by 2030. Currently, basic diabetes research, drug screening, and evaluation of new treatments typically require the use of animal models. However, this approach is limited by species-specific differences. For example, GK rats exhibit pancreatic islet cell damage during fetal and early childhood, which is inconsistent with the pathogenesis of type 2 diabetes mellitus (T2DM) in humans.

[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, providing new disease models for diabetes and its complications and new methods for evaluating therapeutic drugs.

[0004] Microfibers are important biomaterials for constructing microphysiological systems that are closer to the in vivo environment. They can not only provide a three-dimensional culture environment for cells, but can also be used to simulate tissues such as blood vessels, muscles and nerves, which is conducive to studying cell-cell interactions and tissue functions. High-level assembly through stacking, weaving, 3D printing and other methods will expand their application in the biomedical field and tissue and organ engineering.

[0005] To this end, Chinese patent application number 202311217116.6 discloses a hydrogel microfiber loaded with pancreatic α- and β-cells, its preparation method, and its application. The preparation method comprises dissolving a photoinitiator in a cross-linked water-soluble polymer solution, filtering and sterilizing it, and then adding pancreatic α- and β-cells to obtain a hydrogel cell prepolymer solution. This hydrogel cell prepolymer solution is passed into an inner phase tube, and an ionically cross-linked aqueous phase solution is passed into an outer phase tube. The flow rates of the solutions in the inner and outer phase tubes are adjusted to achieve a mutually immiscible laminar flow state. In a microfluidic device, upon contact between the inner phase solution and the outer phase solution, the ionically cross-linked components in the inner phase solution undergo ionically cross-linking with the outer phase solution, initially forming hydrogel microfibers. Subsequently, secondary cross-linking is performed by irradiation with 405nm blue light, ultimately forming hydrogel microfibers loaded with pancreatic α- and β-cells. This scheme simulates the cell type ratio, facilitating the study of cell-cell interactions, but does not form a functional islet structure. This represents "co-encapsulation" rather than "self-organization into islets," significantly different from the complex microenvironment in the body.

[0006] 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 microfibers loaded with pancreatic islets have the defects of low loading rate, poor uniformity, unsatisfactory cell survival rate, and the like.

[0008] To solve the above problems, the present invention provides a method for preparing microfibers for loading pancreatic islets, comprising: S1, preparing a microfluidic chip and building a pneumatically controlled microfluidic spinning device, wherein the microfluidic chip is provided with channels for conveying a core flow, a sample flow, and a sheath flow, respectively, and the microfluidic chip further comprises a PDMS chamber for periodically pressurizing to expand and squeeze the channel of the core flow and close it; S2, adding collagen to the core flow, and then adding β-TC6 cells to make the cell density of the core flow be 4×10 6 mL -1 -8×10 6 mL -1 , the flow rates of the core flow, sample flow and sheath flow were controlled to be 0.8-1.2 μL / min, 18-22 μL / min and 80-120 μL / min, respectively, and the preparation was performed at a frequency of 0.8-1.2 Hz.

[0009] Preferably, step S2 is to add collagen to the core flow, and then add β-TC6 cells to make the cell density of the core flow be 6×10 6 mL -1 The flow rates of the core flow, sample flow and sheath flow were controlled to be 1.0 μL / min, 20 μL / min and 100 μL / min, respectively, and the preparation was performed at a frequency of 1.0 Hz.

[0010] Preferably, the microfluidic chip in step S1 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 the 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 the second position to form a sixth channel for flowing out the formed microfibers.

[0011] Preferably, the gas control device includes a gas storage tank connected to the third channel for storing nitrogen, a control valve is provided between the gas storage tank and the third channel for controlling the opening and closing frequency of the core flow, and the control valve is electrically connected to the control module.

[0012] Preferably, the width of the first channel is 0.8-1.2 mm and the height is 1000-1200 μm, the width of the second channel is 430-460 μm and the height is 650-750 μm, the width of the third channel and the fourth channel is 180-220 μm and the height is 400-600 μm, the length of the PDMS chamber is 0.8-1.2 mm, the width is 180-220 μm, the height is 400-600 μm and the spacing with the fourth channel is 130-180 μm.

[0013] Preferably, the width of the first channel 1 is 1 mm and the height is 1100 μm, the width of the second channel 2 is 450 μm and the height is 700 μm, the width of the third channel 3 is 200 μm and the height is 500 μm, the width of the fourth channel 4 is 70 μm and the height is 500 μm, the length of the PDMS chamber is 1 mm, the width is 200 μm, the height is 500 μm and the spacing between the PDMS chamber and the fourth channel 4 is 150 μm.

[0014] Preferably, the core flow is a 13-16% w / w Dextran solution, the sample flow is a mixed solution of 0.8-1.2% w / w NaA + 15-18% w / w PEG, and the sheath flow is a mixed solution of 2.5-4.5% w / w CaCl2 + 16-18% w / w PEG, which are injected separately through micro-syringe pumps.

[0015] Preferably, the core flow is a 15% w / w Dextran solution, the sample flow is a mixed solution of 1% w / w NaA + 17% w / w PEG, and the sheath flow is a mixed solution of 4% w / w CaCl2 + 17% w / w PEG, which are injected into the microfluidic chip through a microinjection pump.

[0016] 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.

[0017] 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 10:1 is poured on the resin template, and after vacuum debubbling, it is cured at 80°C for 1.0h, and the PDMS layer with the microstructure is peeled off to obtain a PDMS microchip.

[0018] Preferably, the specific operation of adding collagen to the core flow in step S2 is: mixing equal volumes of 26-32% w / w dextran solution and 3-4 mg / mL rat tail type I collagen, and then adjusting the pH to 7.35-7.45 with 0.5 M NaOH.

[0019] Preferably, the specific operation of adding collagen to the core flow in step S2 is: mixing equal volumes of 30% (w / w) dextran solution and 3.34 mg / mL rat tail type I collagen, and adding a small amount of 0.5 M NaOH to adjust the pH to 7.4.

[0020] The present invention provides a pancreatic islet-loaded microfiber prepared by the above method.

[0021] The present invention also discloses the use of the pancreatic islet-loaded microfibers in preparing drugs for treating diabetes or preparing pancreatic islet pathology models.

[0022] Compared with the existing technology, the preparation method of microfibers loaded with pancreatic islets described in the present invention has the following beneficial effects: 1) By combining the pneumatically controlled droplet generation method with microfluidics, high-throughput pancreatic islets can be prepared more simply and quickly, with little difference from the complex microenvironment in the body; 2) After optimization, the aqueous phase droplets are larger in size, have high flux, and good dispersion, which is conducive to loading more cells and forming independent three-dimensional culture microunits with good dispersion. The microfibers finally prepared are continuous and complete, and a large number of aqueous phase droplets containing β-TC6 cells are evenly distributed in the microfibers; 3) Compared with traditional microfluidic droplet three-dimensional cell culture, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 8 This 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 These are the expression results of INS-1, INS-2, and PDX-1 in β-TC6 cells in different culture modes in Example 3 of the present invention. DETAILED DESCRIPTION

[0024] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following describes in detail the specific embodiments of the present invention in conjunction with the accompanying drawings. The features of the embodiments of the present invention may be combined with each other without conflict.

[0025] 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.

[0026] Microphysiological systems can more realistically simulate the response of human organs, such as pancreatic islets, to drugs, and can be used to quickly screen the effectiveness and toxicity of drugs, greatly shortening the drug development cycle and reducing R&D costs. Among them, microfiber materials prepared based on microfluidic technology have shown great potential in constructing microphysiological systems. Existing microfibers are still unable to load islets. If only a limited number of islets are supported through a microporous array, the cell loading flux is low, resulting in low accuracy in the final drug response prediction. 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 1-3As 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 within a certain range, the core flow rate and droplet size are positively correlated. Larger droplets can be obtained by increasing the core flow rate. Furthermore, the core flow rate has little effect on the fiber width.

[0036] 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 within a certain range, the sample flow rate, droplet spacing and fiber width are positively correlated. 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.

[0037] 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.

[0038] 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 The prepared microfibers are continuous and intact, with numerous aqueous droplets containing β-TC6 cells uniformly distributed within them. Blue fluorescent polystyrene microspheres (5µm, excitation / emission: 400 / 450nm) and green polystyrene microspheres (5µm, excitation / emission: 488 / 518nm) were mixed at a concentration of 0.1mg / ml in the sample and core stream solutions, respectively. Three-dimensional images obtained using confocal microscopy revealed the spatial independence of the two colored microspheres, with a clear distinction between the droplets and the fibers, indicating that the core and sample streams do not diffuse into each other. Replacing the PS microspheres within the droplets with cells of similar size provides a space for three-dimensional cell culture, and the aqueous components within the droplets also provide biochemical conditions for cell survival. This demonstrates that the prepared aqueous droplet-loaded microfibers can serve as carriers for a variety of chemical substances or for three-dimensional cell culture.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 life and death conditions on day 0, day 1, and day 7 showed that the cells still showed good activity after multiple days of culture.

[0043] The proliferation of β-TC6 cells in microfibers on day 0 and day 3 was detected by MTT assay. Figure 7 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.

[0044] 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 .

[0045] 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 9It 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.

[0046] This application combines a pneumatically controlled droplet generation method with microfluidics to prepare high-throughput islets more simply and quickly. 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. Although the present invention is disclosed as above, the present invention is not limited to this. 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 preparing microfibers for loading pancreatic islets, characterized in that: include: S1. Prepare a microfluidic chip and build a pneumatically controlled microfluidic spinning device, wherein the microfluidic chip is provided with channels for conveying a core flow, a sample flow, and a 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; S2. Add collagen to the core flow, and then add β-TC6 cells to make the cell density of the core flow 4×10 6 mL -1 -8×10 6 mL -1 The flow rates of the core flow, sample flow and sheath flow were controlled to be 0.8-1.2 μL / min, 18-22 μL / min and 80-120 μL / min, respectively, and the preparation was performed at a frequency of 0.8-1.2 Hz.

2. The method for preparing microfibers for loading pancreatic islets according to claim 1, characterized in that: The microfluidic chip in step S1 includes 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 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, the PDMS chamber has two channels and is respectively located on both sides of the fourth channel (4), and is used to expand by pressurization and squeeze the fourth channel (4) from both sides to close it; the microfluidic chip also includes 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 the formed microfibers.

3. The method for preparing microfibers for loading pancreatic islets according to claim 2, characterized in that: The gas control device comprises 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) for controlling the opening and closing frequency of the core flow, and the control valve is electrically connected to the control module.

4. The method for preparing microfibers for loading pancreatic islets according to claim 3, characterized in that: The width of the first channel (1) is 0.8-1.2 mm, and the height is 1000-1200 μm, the width of the second channel (2) is 430-460 μm, and the height is 650-750 μm, the width of the third channel (3) and the fourth channel (4) are 180-220 μm, and the height is 400-600 μm, the length of the PDMS chamber is 0.8-1.2 mm, the width is 180-220 μm, and the height is 400-600 μm, and the spacing between the PDMS chamber and the fourth channel (4) is 130-180 μm.

5. The method for preparing microfibers for loading pancreatic islets according to claim 1, characterized in that: The core flow is a 13-16% w / w Dextran solution, the sample flow is a mixed solution of 0.8-1.2% w / w NaA+15-18% w / w PEG, and the sheath flow is a mixed solution of 2.5-4.5% w / w CaCl2+16-18% w / w PEG, which are injected separately through micro-syringe pumps.

6. The method for preparing microfibers for loading pancreatic islets according to claim 1, 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.

7. The method for preparing microfibers for loading pancreatic islets according to claim 1, characterized in that: The specific operation of adding collagen to the core flow in step S2 is as follows: 26-32% w / w dextran solution and 3-4 mg / mL rat tail type I collagen are mixed in equal volumes, and then the pH is adjusted to 7.35-7.45 with 0.5 M NaOH.

8. A pancreatic islet-loaded microfiber prepared by the method according to any one of claims 1 to 7.

9. Use of the pancreatic islet-loaded microfiber according to claim 7 in preparing a drug for treating diabetes or preparing a pancreatic islet pathology model.

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