A bionic liver lobule structure multi-component microsphere and its preparation method and application

Abstract

The invention discloses a bionic liver lobule structure multi-component microsphere and its preparation method and application, using capillary glass tubes of various sizes, designing and constructing a multi-channel microfluidic chip, adding sodium alginate to deionized water, and configuring to obtain pre-gel solution A; adding calcium chloride, methacrylic anhydride gelatin and 2-hydroxy-2-methyl-1-phenyl-1-acetone to deionized water, and configuring to obtain methacrylic anhydride gelatin pre-gel solution. Hepatocytes, mesenchymal stem cells and endothelial cells are mixed with methacrylic anhydride gelatin pre-gel solution respectively, and pre-gel solution B, pre-gel solution C and pre-gel solution D are configured; the prepared pre-gel solution A, pre-gel solution B, pre-gel solution C and pre-gel solution D are passed into the prepared multi-channel microchannel chip, silicone oil is passed into the collecting tube, and the collecting tube is continuously irradiated with ultraviolet light to obtain the bionic liver lobule structure multi-component microsphere. The method of the present invention has the advantages of low cost, simple operation, and controllable size.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a bionic liver lobule structure multi-component microsphere and a preparation method and application thereof. Background Art

[0002] Liver diseases often cause liver failure or partial liver resection. As an effective solution strategy, liver tissue engineering has broad application prospects in the treatment and prognosis of liver diseases. The liver lobule is the basic component and functional unit of the liver, composed of hepatocytes, fibroblasts, Kupffer cells, endothelial cells, etc. At present, there are many technologies used to make in vitro liver models that simulate the microstructure and composition of the liver lobule. For example, two-dimensional micropatterning technology can achieve specific positioning and co-culture of multiple cells. Although this technology realizes the interaction between cells and promotes the improvement of liver bionic function, it still has limitations in forming three-dimensional liver microstructure. In recent years, three-dimensional bioprinting technology has shown great advantages in constructing ideal three-dimensional tissues and organs. A large number of three-dimensional printed artificial livers have shown good application prospects in clinical transformation. However, bioprinting has certain limitations in achieving mass production of independent liver lobule units. Therefore, new strategies for large-scale construction of bionic liver lobules with morphology and function similar to the liver are worth looking forward to.

[0003] Microfluidics has significant advantages in preparing microcarriers with fine structures and special components. Compared with other tissue engineering methods, microfluidics can encapsulate a variety of cells in a gentle and highly operable manner. In addition, the hand-made microfluidic chip based on glass capillaries makes the micron-scale core structure of the prepared microcarriers highly customizable, with low manufacturing process cost and high preparation efficiency. At present, various studies have prepared bionic microcarriers with various core-shell structures, anisotropic structures, and multi-component structures through microfluidics. However, in order to ensure the material exchange and nutrient supply between cells in the microcarriers and the outside world, the size and cell content of these microcarriers are strictly limited. In addition, these microcarriers cannot well mimic the complex structure of the liver lobule, resulting in insufficient cell-cell and cell-extracellular matrix interactions, which limits their liver bionic functions. Therefore, new bionic microcarriers with effective nutrient exchange pathways prepared based on microfluidics are expected to be applied to liver tissue engineering and clinical transformation applications. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing multi-component microspheres with a bionic liver lobule structure in view of the deficiencies in the prior art, so as to obtain multi-component microspheres with precisely controllable structure and size, thereby highly simulating the complex liver lobule structure.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing multi-component microspheres with a bionic liver lobule structure comprises the following steps:

[0007] (1) seven capillary glass tubes are connected in parallel and fixed together, wherein the six outer capillary glass tubes are enclosed in a circle, and the seventh capillary glass tube is enclosed in the center; an eighth capillary glass tube is coaxially placed in the seventh capillary glass tube; eight input pipes are respectively connected to the inlets of the eight capillary glass tubes, and a collection tube is coaxially sleeved at the outlets of the eight capillary glass tubes, thereby preparing a multi-channel microfluidic chip;

[0008] (2) Adding sodium alginate into deionized water to prepare pre-gel solution A;

[0009] (3) adding calcium chloride, methacrylic anhydride gelatin and 2-hydroxy-2-methyl-1-phenyl-1-propanone into deionized water to prepare a methacrylic anhydride gelatin pregel solution;

[0010] Subsequently, the hepatocytes were added into the methacrylic anhydride gelatin pre-gel solution to prepare the pre-gel solution B;

[0011] adding mesenchymal stem cells to methacrylic anhydride gelatin pre-gel solution to obtain pre-gel solution C;

[0012] Adding endothelial cells to methacrylic anhydride gelatin pre-gel solution to prepare pre-gel solution D;

[0013] (4) Silicone oil is introduced into the collecting tubes at the outlets of the eight capillary glass tubes, and ultraviolet light is continuously used to irradiate the collecting tubes;

[0014] (5) At the same time, the pregel liquid A is introduced into the eighth capillary glass tube through the input pipe, the pregel liquid D is introduced into the seventh capillary glass tube through the input pipe, and the pregel liquid B and the pregel liquid C are introduced into the six outer capillary glass tubes through the input pipe at intervals; and the pregel liquids are collected and solidified in the collection tube to obtain the obtained product.

[0015] Furthermore, in step (1), the inner diameters of the eight capillary glass tubes are consistent, all of which are 300-1000 μm. The outlet ends of the seven capillary glass tubes assembled in parallel are heated and thinned to a total diameter of 600-800 μm; the eighth capillary glass tube is heated and thinned and then coaxially placed into the seventh capillary glass tube in the middle.

[0016] Furthermore, in step (1), the collecting tube is a capillary glass tube with an inner diameter of 1.0-1.2 mm, and the inlet of the collecting tube is fixed by a flat needle.

[0017] Furthermore, in step (1), the input channel is composed of eight capillary glass tubes that are bent after heating, which are respectively connected to the inlets of the corresponding eight capillary glass tubes and fixed by flat-mouth needles, so as to facilitate the subsequent inflow of pre-gel liquid.

[0018] Furthermore, in step (2), the viscosity of the sodium alginate is low, and the viscosity value is preferably 300-700 mPa·s; and the mass concentration of the sodium alginate in the pre-gel solution A is 0.5-1.5%.

[0019] Furthermore, in step (3), in the methacrylic anhydride gelatin pre-gel solution, the mass concentration of calcium chloride is 0.5-1.5%, the mass concentration of methacrylic anhydride gelatin is 5-10%, and the mass concentration of 2-hydroxy-2-methyl-1-phenyl-1-propanone is 0.05-0.2%.

[0020] Furthermore, in step (3), the cell density of the hepatocytes in the pre-gel solution B is 2 - 3 × 10 5 cells / ml; the cell density of mesenchymal stem cells in pre-gel solution C is 2 - 3 × 10 5 cells / mL; the cell density of endothelial cells in pre-gel solution D is 2 - 3 × 10 5 cells / mL.

[0021] Furthermore, in step (5), the flow rate of the pregel liquid A is 0.05-0.2 mL / h; the flow rate of the pregel liquid D is 0.05-0.2 mL / h; the flow rate of the pregel liquid B into each capillary glass tube is 0.1-0.5 mL / h, and the flow rate of the pregel liquid C into each capillary glass tube is 0.1-0.5 mL / h; the flow rate of the silicone oil into the collection tube is 1-10 mL / h.

[0022] Furthermore, the present invention also claims to protect the bionic liver lobule structure multi-component microspheres prepared by the above preparation method.

[0023] Furthermore, the use of the above-mentioned bionic liver lobule structure multi-component microspheres in the preparation of devices for treating acute liver failure is also within the protection scope of the present invention. Beneficial Effects

[0024] (1) The present invention uses capillary glass tubes of various sizes to design and construct a multi-channel microfluidic chip, providing a method for preparing bionic multi-component microspheres, which has the advantages of low cost, simple operation, and controllable size. Combined with microfluidic technology, a large number of bionic liver lobule structure multi-component microspheres can be efficiently prepared for in situ implantation in failing livers, promoting liver tissue regeneration and inhibiting hepatocyte apoptosis, thereby promoting the repair of damaged livers.

[0025] (2) The present invention is based on alginate and methacrylic anhydride gelatin, and four mixed hydrogel solutions are prepared for the preparation of multi-component microspheres. Pregel solution A is prepared from alginate; methacrylic anhydride gelatin pregel solution is prepared from calcium chloride, methacrylic anhydride gelatin, and 2-hydroxy-2-methyl-1-phenyl-1-propanone; pregel solution B is prepared from methacrylic anhydride gelatin pregel solution and liver cells; pregel solution C is prepared from methacrylic anhydride gelatin pregel solution and mesenchymal stem cells; pregel solution D is prepared from methacrylic anhydride gelatin pregel solution and endothelial cells. The materials used, alginate and methacrylic anhydride gelatin, have certain biological safety and stability, and microfluidic technology can be used to obtain multi-component microspheres with precisely controllable structure and size.

[0026] (3) The bionic liver lobule multi-component microspheres prepared by the present invention, the rapid ionic cross-linking and curing of alginate and calcium ions enables the in-situ generation of calcium alginate fibers in the collecting tube, the peripheral methacrylic anhydride gelatin mixed solution forms emulsified particles to wrap the calcium alginate fibers under the shear force of silicone oil, and the methacrylic anhydride gelatin cross-links and cures under the action of ultraviolet light to form multi-component microspheres. After the microspheres are separated from the fibers, multi-component microspheres with coaxial through-holes are obtained, which simulate the cord-like structure and spatial anisotropic structure of the liver lobule, are conducive to the exchange of nutrients, and highly simulate the liver lobule structure. Compared with existing materials, the bionic liver lobule multi-component microspheres prepared by the present invention can promote liver tissue regeneration and inhibit hepatocyte apoptosis after being transplanted into the damaged liver, thereby promoting the repair of the damaged liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0028] Figure 1 This is a schematic diagram of building a multi-channel microfluidic chip using capillary glass tubes in the present invention;

[0029] Figure 2 A real shot of the multi-channel microfluidic chip constructed by the present invention (a), a stereoscopic microscope image of a bent capillary glass tube implanted with a seven-link capillary tube as an inflow channel (b), and a stereoscopic microscope image of the multi-channel outlet in a collection tube (c);

[0030] Figure 3 The present invention uses the prepared multi-channel microfluidic chip to collect high-speed camera images of calcium alginate fibers and methacrylic anhydride gelatin emulsion droplets in the tube at different flow rates (a) and statistics of fiber and droplet size changes at different flow rates (b);

[0031] Figure 4A schematic diagram of a multi-channel microfluidic chip prepared for use in the invention, wherein eight inflow channels have different inputs and gel solutions;

[0032] Figure 5 Stereoscopic microscope images of multi-component methacrylic anhydride gelatin microspheres prepared by the present invention wrapped on calcium alginate fibers (a), stereoscopic microscope images of multi-component methacrylic anhydride gelatin microspheres sliding freely on calcium alginate fibers (b), and stereoscopic microscope images of multi-component methacrylic anhydride gelatin microspheres after falling off from calcium alginate fibers (c);

[0033] Figure 6 The electron scanning microscope image (a) of the bionic liver lobule structure multi-component microsphere as a whole and the electron scanning microscope image (b) after longitudinal sectioning;

[0034] Figure 7 This is a laser confocal scanning microscope fluorescence image of each component of the bionic liver lobule structure multi-component microsphere prepared by the present invention carrying fluorescent nanoparticles;

[0035] Figure 8 Fluorescence images of different cells carried in each component of the multi-component microspheres of the bionic liver lobule structure prepared by the present invention.

[0036] Fig. 9 The liver function test (a) and the liver cell damage and repair function test (b) of the multi-component microspheres with multi-cell bionic liver lobule structure prepared by the present invention;

[0037] Fig.10 Schematic diagram of the experimental process of using the multi-component microspheres with multi-cell bionic liver lobule structure prepared by the present invention to treat acute liver failure in mice (a), changes in blood alanine aminotransferase in each group of mice during the treatment process (b), changes in aspartate aminotransferase (c), and statistics of mouse survival rate (d);

[0038] Fig.11 Hematoxylin-eosin staining, Ki67 staining, and TUNEL fluorescence staining images of liver tissues of mice in each group after seven days of using the multi-cell bionic liver lobule structure multi-component microspheres prepared by the present invention to treat acute liver failure in mice (a), and statistics on Ki67 cell positive rate (b) and nuclear TUNEL cell positive rate (c);

[0039] Fig.12 The CD68, NRF2, and HO-1 fluorescence co-staining images (a) and quantitative statistics of CD68 (b), NRF2 (c), and HO-1 (d) fluorescence intensities of the liver tissues of each group of mice were shown seven days after the multi-component microspheres with multi-cell-carrying bionic liver lobule structure prepared by the present invention were used to treat acute liver failure in mice. DETAILED DESCRIPTION

[0040] The present invention can be better understood with reference to the following examples.

[0041] Example 1: Preparation of a multi-channel microfluidic chip:

[0042] Since hexagonal close packing can maximize space utilization and improve structural stability, we assembled seven capillary glass tubes with an inner diameter of 580 μm in a hexagonal close packing manner and fixed them with light-curing glue. The tail ends were heated with an alcohol lamp and then stretched. Figure 1 After the seven-linked glass tubes are fixed on a glass slide using light-curing glue, a thin capillary glass tube is coaxially placed in the middle capillary tube, a collection tube with an inner diameter of 1 mm is coaxially placed at the outlet of the seven-linked glass tube, and the inlets of all capillaries are sealed and fixed using flat needles, and finally a multi-channel microfluidic chip is prepared, as shown in FIG. Figure 2 As shown. Among them, Figure 2 a is a real picture of the multi-channel microfluidic chip constructed by the present invention. Figure 2 b is a stereomicroscope picture of a bent glass capillary tube implanted into a seven-unit capillary as an inflow channel. Figure 2 c is a stereomicroscope image of the multichannel outlet in the collection tube.

[0043] Example 2: Preparation of multi-component microspheres with bionic liver lobule structure:

[0044] (1) Using deionized water as solvent, 1% sodium alginate (ALG, viscosity 300-700 mPa·s) was prepared into mixed pregel solution A; using deionized water as solvent, 1% calcium chloride, 10% gelatin methacrylate anhydride (GelMA), and 0.1% 2-hydroxy-2-methyl-1-phenyl-1-propanone were prepared into GelMA pregel solution; first, the appropriate flow rate of each channel was explored, pregel solution A was passed into the eighth channel, GelMA pregel solution was passed into the other seven channels, and silicone oil was passed into the collection tube. A high-speed camera was used to record the fiber formation and droplet emulsification at different flow rates, and their relationship was statistically analyzed, such as Figure 3 As shown. Among them, Figure 3 a is a high-speed camera image of calcium alginate fibers and methacrylic anhydride gelatin emulsion droplets collected in the tube at different flow rates using the prepared multi-channel microfluidic chip of the present invention. By accurately controlling the fluid flow rate within a certain range (the flow rate range of the pregel liquid A is 0.02-0.08 mL / h, the flow rate of the GelMA pregel liquid in the seven channels is kept consistent, ranging from 0.001-0.5 mL / h; the silicone oil flow rate range is 0.5-1.5 mL / h), a multi-component microsphere fiber assembly with different droplet transverse diameters, droplet long diameters and fiber diameters can be obtained. Figure 3b is the statistical change of fiber and droplet size when the flow rates of pregel solution A and silicone oil remain unchanged and the flow rate of GelMA pregel solution is adjusted (0.001-0.5 mL / h). It can be seen that with the increase of the flow rate of GelMA pregel solution, the transverse diameter and long diameter of the droplet increase, while the fiber diameter does not show significant change.

[0045] (2) Using GelMA pregel solution as solvent, 0.01% green fluorescent nanoparticles are prepared into green fluorescent GelMA pregel solution; using GelMA pregel solution as solvent, 0.01% red fluorescent nanoparticles are prepared into red fluorescent GelMA pregel solution; using GelMA pregel solution as solvent, 0.01% blue fluorescent nanoparticles are prepared into blue fluorescent GelMA pregel solution. The mixed pregel solution A is passed into the eighth channel, the green fluorescent GelMA pregel solution is passed into the middle channel of the seven capillaries assembled in parallel, the red fluorescent GelMA pregel solution and the blue fluorescent GelMA pregel solution are passed into the outer six channels of the seven capillaries assembled in parallel at intervals, silicone oil is passed into the collection tube, and ultraviolet light is continuously irradiated to the collection tube, such as Figure 4 Finally, an ALG fiber with several GelMA microspheres was prepared. Figure 5 .like Figure 5 (a) shows that GelMA microspheres can slide freely on ALG fibers. Figure 5 (b) As shown; GelMA microspheres with hollow through-channel structures were obtained by peeling the GelMA microspheres from the ALG fibers. Figure 5 (c) As shown. The hollow structure of the GelMA microspheres can be seen by electron scanning microscopy. Figure 6 As shown; among them, Figure 6 a is an electron scanning microscope image of the bionic liver lobule structure multi-component microspheres prepared by the present invention, Figure 6 b is an electron scanning microscope image of the multi-component microspheres with bionic liver lobule structure after longitudinal section. The multi-component structure of the GelMA microspheres can be seen by laser confocal scanning microscopy, as shown in Figure 2. Figure 7 shown.

[0046] Example 3: Preparation and functional verification of bionic liver lobule structure multi-component microspheres carrying multiple cells:

[0047] Using deionized water as solvent, 1% ALG was prepared into pre-gel solution A. Using deionized water as solvent, 1% calcium chloride, 10% GelMA, and 0.1% 2-hydroxy-2-methyl-1-phenyl-1-propanone were prepared into GelMA pre-gel solution. Using GelMA pre-gel solution as solvent, hepatocytes were added to prepare a solution containing 2.5 × 10 5Hepatocyte pregel solution B: GelMA pregel solution was used as solvent, and mesenchymal stem cells were added to prepare a solution containing 2.5 × 10 5 Mesenchymal stem cell pregel solution C: GelMA pregel solution was used as solvent, and endothelial cells were added to prepare a solution containing 2.5 × 10 5 Pregel solution D for endothelial cells. The mixed pregel solution A is passed into the eighth channel, the gel solution D is passed into the middle channel of seven capillaries assembled in parallel, the pregel solution B and the pregel solution C are passed into the six outer channels of the seven capillaries assembled in parallel at intervals, the silicone oil is passed into the collecting tube, and the collecting tube is continuously irradiated with ultraviolet light. Hepatocytes, mesenchymal stem cells, and endothelial cells are stained with green fluorescence, red fluorescence, and blue fluorescence, respectively, before being suspended in the pregel solution. The multi-component structure of the multi-component microspheres of the bionic liver lobule structure carrying multiple cells can be seen by fluorescence microscopy, as shown in FIG. Figure 8 shown.

[0048] By constructing an in vitro hepatocyte injury model, we further verified the liver-like function of multi-component microspheres with bionic liver lobule structures carrying multiple cells. First, we added lipopolysaccharide to the culture medium of normal hepatocytes to construct an in vitro hepatocyte injury model. The hepatocytes treated with lipopolysaccharide were divided into four groups: hepatocyte group (Control), hepatocyte spheroids group (Hepatocytes spheroids), multi-cell spheroids group (Multi-cell spheroids), and hepatocyte lobule bionic microsphere group (BMMPs). After co-culture with damaged hepatocytes, it was found that the hepatocyte albumin secretion and urea synthesis in the BMMPs group were significantly enhanced, and the levels of alanine aminotransferase and aspartate aminotransferase in the supernatant were significantly reduced, such as Fig. 9 As shown. Among them, Fig. 9 a is a liver function test of multi-component microspheres with multi-cell bionic liver lobule structure prepared by the present invention, Fig. 9 b is the detection of the multi-component microspheres with multi-cell bionic liver lobule structure prepared by the present invention to promote the repair function of liver cell damage. The above results show that our liver lobule bionic microspheres can effectively play a liver-like function and promote the recovery of damaged liver cells.

[0049] Example 4: Evaluation of the in vivo liver repair efficacy of multi-component microspheres with bionic liver lobule structure:

[0050] We established an acute liver injury rat model by intraperitoneal injection of D-galactosamine and treated it with liver lobule biomimetic microspheres. The results are shown in Fig.10 .like Fig.10As shown in (a). Healthy rats were set as the control group (Control), and rats with acute liver injury were divided into three groups: phosphate buffer treatment group (ALF), hepatocyte treatment group (Cell), and liver lobule biomimetic microsphere treatment group (BMMPs). Rats in the ALF group, Cell group, and BMMPs group were injected with phosphate buffer, hepatocyte suspension, and liver lobule biomimetic microspheres, respectively. During the seven-day treatment period, by detecting the levels of alanine aminotransferase and aspartate aminotransferase on the 1st, 3rd, and 7th days, we found that the levels of alanine aminotransferase and aspartate aminotransferase in the blood of rats with acute liver failure treated with liver lobule biomimetic microspheres were significantly decreased, as shown in Figure 2. Fig.10 (b, c) At the same time, we also found that rats with acute liver failure treated with liver lobule biomimetic microspheres had a higher survival rate, as shown in Fig.10 (d) shown.

[0051] In addition, the liver tissues of rats in each group were stained with hematoxylin-eosin. Fig.11 It can be observed that the cavitation of liver tissue in rats with acute liver failure treated with liver lobule bionic microspheres was significantly reduced, the inflammatory area was significantly reduced, and liver damage was significantly repaired. Fig.11 (a) As shown. After Ki67 immunostaining and Ki67 positive rate determination of the liver tissues of rats in each group, we found that the proliferation activity of hepatocytes was significantly increased after treatment with liver lobule biomimetic microspheres, as shown in Fig.11 We further evaluated the apoptosis of hepatocytes in the liver tissues of each group by terminal deoxynucleotidyl transferase nick end labeling. The results showed that the apoptosis rate of hepatocytes in the liver tissues of rats with acute liver failure treated with liver lobule biomimetic microspheres was significantly reduced. Fig.11 (c) The above results show that liver lobule bionic microspheres can effectively improve liver damage and promote liver repair.

[0052] When acute liver injury occurs, there is inevitably inflammatory damage and oxidative stress in the damaged area of ​​the liver, and the resulting cytokine storm further aggravates liver injury, forming a vicious cycle. To further explore the role of liver lobule biomimetic microspheres in promoting the repair of damaged liver, we performed immunofluorescence staining of CD68, nuclear factor E2-related factor (Nrf2), and heme oxygenase-1 (HO-1) on liver samples from different groups of rats and performed relative quantitative statistics. The results are shown in Fig.12 Compared with the control group and ALF group, the expression of CD68 in the BMMPs group was increased, indicating that macrophages were aggregated and the anti-inflammatory ability was enhanced ( Fig.12a, b). It has been reported that activation of the Nrf2 / HO-1 antioxidant pathway can promote liver recovery. The expression of Nrf2 and HO-1 genes in the BMMPs group increased significantly, indicating that the antioxidant capacity of liver tissue in rats with acute liver injury treated with BMMPs was enhanced ( Fig.12 a, c, d). These results indicate that liver lobule biomimetic microspheres transplantation can alleviate liver damage through anti-inflammatory and antioxidant effects. The above results indicate that liver lobule biomimetic microspheres can effectively improve liver damage and promote liver repair.

[0053] The present invention provides a bionic liver lobule structure multi-component microsphere and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing multi-component microspheres with a bionic liver lobule structure, characterized in that: The steps include: (1) seven capillary glass tubes are connected in parallel and fixed together, wherein the six outer capillary glass tubes are enclosed in a circle, and the seventh capillary glass tube is enclosed in the center; an eighth capillary glass tube is coaxially placed in the seventh capillary glass tube; eight input pipes are respectively connected to the inlets of the eight capillary glass tubes, and a collection tube is coaxially sleeved at the outlets of the eight capillary glass tubes, thereby preparing a multi-channel microfluidic chip; (2) Adding sodium alginate into deionized water to prepare pre-gel solution A; (3) adding calcium chloride, methacrylic anhydride gelatin and 2-hydroxy-2-methyl-1-phenyl-1-propanone into deionized water to prepare a methacrylic anhydride gelatin pregel solution; Subsequently, the hepatocytes were added into the methacrylic anhydride gelatin pre-gel solution to prepare the pre-gel solution B; adding mesenchymal stem cells to methacrylic anhydride gelatin pre-gel solution to obtain pre-gel solution C; Adding endothelial cells to methacrylic anhydride gelatin pre-gel solution to prepare pre-gel solution D; (4) Silicone oil is introduced into the collecting tubes at the outlets of the eight capillary glass tubes, and ultraviolet light is continuously used to irradiate the collecting tubes; (5) At the same time, the pregel solution A is introduced into the eighth capillary glass tube through the input pipe, the pregel solution D is introduced into the seventh capillary glass tube through the input pipe, and the pregel solution B and the pregel solution C are introduced into the six outer capillary glass tubes through the input pipe at intervals; after being collected and solidified in the collection tube, the methacrylic anhydride gelatin microspheres are peeled off from the sodium alginate fibers to obtain GelMA microspheres with a hollow through-channel structure, that is, In step (5), the flow rate of the pregel solution A is in the range of 0.02-0.08 mL / h, the flow rate of the methacrylic anhydride gelatin pregel solution in the seven channels is kept consistent, in the range of 0.001-0.5 mL / h; and the flow rate of the silicone oil is in the range of 0.5-1.5 mL / h.

2. The method for preparing the bionic liver lobule structure multi-component microspheres according to claim 1, characterized in that: In step (1), the inner diameters of the eight capillary glass tubes are consistent, all of which are 300-1000 μm, and the outlet ends of the seven capillary glass tubes assembled in parallel are heated and drawn to a total diameter of 600-800 μm; The eighth capillary glass tube is heated, drawn, and then coaxially placed into the seventh capillary glass tube in the middle.

3. The method for preparing the bionic liver lobule structure multi-component microspheres according to claim 1, characterized in that: In step (1), the collection tube is a capillary glass tube with an inner diameter of 1.0-1.2 mm, and the inlet of the collection tube is fixed by a flat needle.

4. The method for preparing the bionic liver lobule structure multi-component microspheres according to claim 1, characterized in that: In step (1), the input channel is composed of eight capillary glass tubes that are bent after heating, and are respectively connected to the inlets of the corresponding eight capillary glass tubes and fixed by flat-mouth needles, which facilitates the subsequent inflow of pre-gel liquid.

5. The method for preparing the bionic liver lobule structure multi-component microspheres according to claim 1, characterized in that: In step (2), the viscosity of the sodium alginate is 300-700 mPa·s; and the mass concentration of the sodium alginate in the pregel solution A is 0.5-1.5%.

6. The method for preparing the bionic liver lobule structure multi-component microspheres according to claim 1, characterized in that: In step (3), in the methacrylic anhydride gelatin pregel solution, the mass concentration of calcium chloride is 0.5-1.5%, the mass concentration of methacrylic anhydride gelatin is 5-15%, and the mass concentration of 2-hydroxy-2-methyl-1-phenyl-1-propanone is 0.05%-0.2%.

7. The method for preparing the bionic liver lobule structure multi-component microspheres according to claim 1, characterized in that: In step (3), the cell density of hepatocytes in pre-gel solution B is 2 - 3 × 10 5 cells / ml; the cell density of mesenchymal stem cells in pre-gel solution C is 2 - 3 × 10 5 cells / ml; the cell density of endothelial cells in pre-gel solution D is 2 - 3 × 10 5 cells / mL.

8. The bionic liver lobule structure multi-component microspheres prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the bionic liver lobule structure multi-component microspheres according to claim 8 in the preparation of a device for treating acute liver failure.

Images