Bionic intestinal chip for magnetically controlling intestinal villus movement and preparation method thereof

Abstract

The invention discloses a bionic intestinal chip for magnetic control intestinal villus movement and a preparation method thereof, and relates to the field of organ chip preparation technology and culture.The bionic intestinal chip comprises an upper chip body and a lower chip body which are tightly attached, a magnetic bionic porous membrane is clamped between the upper chip body and the lower chip body, and swinging of bionic intestinal villus can be controlled by an external magnetic field. The magnetic bionic porous membrane with the micro-column structure is prepared through template recarving; assembling the porous membrane, an upper-layer chip and a lower layer, and respectively culturing an intestinal epithelial cell line and an endothelial cell line in an upper-layer chamber and a lower-layer chamber; and connecting the chip after cell culture with a circulating peristaltic pump, and placing the chip in an external circulating magnetic field to control the swinging of the bionic intestinal villus so as to realize dynamic cell culture. The prepared bionic intestinal tract chip can highly simulate intestinal tract villus and crypt structures, dynamic culture of the intestinal tract organ chip is achieved, related biological mechanism research and high-throughput drug screening can be carried out, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of organ chip preparation and cultivation, and specifically relates to a bionic intestinal chip for magnetically controlling intestinal villus movement and a preparation method thereof. Background Art

[0002] The intestinal mucosal barrier is the body's first line of defense against the invasion of external pathogens and is crucial to maintaining the stability of the body's internal environment. When the intestinal mucosal barrier is damaged, harmful substances may enter the body and cause infection or inflammatory reactions. In severe cases, it may cause symptoms such as diarrhea, abdominal pain, fever, and even complications such as dehydration and electrolyte disorders. Therefore, restoring the intestinal mucosal barrier structure and maintaining intestinal functional homeostasis are of great significance in first aid. The intestinal chip is a new technology that combines microfluidics and tissue engineering. It can highly mimic the human intestinal microenvironment and reproduce the intestinal barrier function, absorption function and immune function. Compared with traditional in vitro models, the intestinal chip has many advantages such as high degree of bionics, good stability and low cost.

[0003] Current intestinal chips usually use static porous membranes, which only establish physical isolation between the intestinal cavity and the vascular cavity. They cannot simulate the unique physiological microstructures (such as villi and crypts) and movement characteristics (such as peristalsis and villus swing) of the intestine, resulting in limitations in simulating intestinal functions and disease pathological mechanisms. In addition, traditional materials mostly use rigid porous membranes, which have poor biocompatibility and flexibility. Summary of the invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a bionic intestinal chip for magnetically controlled intestinal villus movement and a preparation method thereof, so as to solve the above-mentioned technical problems existing in the prior art.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A bionic intestinal chip for magnetically controlling intestinal villus movement, comprising an upper chip, a lower chip, and a magnetic bionic porous membrane;

[0007] An upper chamber opened downward is provided at the bottom of the upper chip, an upper microfluidic channel is penetrated in the upper chip, one end of the upper microfluidic channel is connected to the upper chamber, and the other end extends to the upper surface of the upper chip and outward to form an upper flow channel inlet and an upper flow channel outlet, an intestinal epithelial cell line is cultured in the upper chamber, and the intestinal epithelial cell line in the upper chamber is circulated between the upper flow channel inlet and the upper flow channel outlet;

[0008] A lower chamber is provided on the top of the lower chip, which is open upward. A lower microchannel is passed through the lower chip, so that one end of the lower microchannel is connected to the upper chamber, and the other end extends to the lower surface of the lower chip and outward to form a lower channel inlet and a lower channel outlet. An endothelial cell line is cultured in the lower chamber, and the intestinal epithelial cell line in the lower chamber is circulated between the lower channel inlet and the lower channel outlet.

[0009] The magnetic bionic porous membrane is located between the bonding surfaces of the upper chip and the lower chip, and simultaneously realizes the separation and independence of the upper chamber and the lower chamber. The magnetic bionic porous membrane adopts a flexible Ecoflex matrix and is doped with magnetic particles.

[0010] The swing of magnetic particles located on the magnetic bionic porous membrane is controlled by an external cyclic magnetic field.

[0011] Furthermore, the magnetic bionic porous membrane is bonded between the bonding surfaces of the upper chip and the lower chip by plasma treatment, and the conditions of the plasma treatment are 200W, 30s.

[0012] Furthermore, the magnetic particles are one or more of elemental iron, magnetite, carbonyl iron, and cobalt ferrite, and the particle size is 50 to 150 nanometers.

[0013] Furthermore, the diameter D of the upper chamber where the upper chip is located is 上腔 =4mm, height H 上腔 =2mm;

[0014] The lower chamber diameter D of the lower chip 下腔 =4mm, height H 下腔 =1mm.

[0015] Furthermore, the frequency of the external circulating magnetic field is 0.1 Hz to 0.3 Hz, and the magnetic field strength is 0.5 to 4 KGs.

[0016] The method for preparing the bionic intestinal chip with magnetically controlled intestinal villus movement comprises the following steps:

[0017] S1, preparing an upper chip and a lower chip;

[0018] S2, prepare the magnetic bionic porous membrane, using CO 2 The through-hole positive mold is prepared by laser cutting, and the porous membrane magnetic raw material solution is filled into the through-hole structure of the positive membrane, and the excess porous membrane magnetic raw material solution is scraped off; the porous membrane raw material solution is then evenly coated on one side of the positive membrane, and a magnetic bionic membrane is obtained after curing; the magnetic porous membrane after curing is separated from the through-hole positive mold, and then a magnetic bionic porous membrane is obtained;

[0019] S3, placing the magnetic bionic porous membrane on the bonding surface of the upper chip and the lower chip for hydrophilization treatment, and then placing it in an oven at 80°C for bonding for 1 hour;

[0020] S4. Then, intestinal epithelial cell lines and endothelial cell lines are implanted in the upper chamber of the upper chip and the lower chamber of the lower chip, respectively.

[0021] Further, in S2, the through hole male mold is one or more of polyethylene terephthalate, polycarbonate, polymethyl methacrylate, polypropylene, and polystyrene;

[0022] The through hole male mold thickness H 膜 =1.0mm, through hole diameter Φ 孔 =0.5mm, through hole spacing D 孔 =1.0mm.

[0023] The porous membrane magnetic raw material solution comprises Ecoflex, pore-forming agent and magnetic particles, which are mixed in a mass ratio of 2:1:1;

[0024] The pore-forming agent is one of sodium chloride, ammonium chloride, sucrose and sodium sulfate.

[0025] Furthermore, in S2, the curing temperature is 75° C. and the time is 2 hours.

[0026] Furthermore, the S1 specifically includes the following steps:

[0027] S101, firstly, printing out an upper chip positive mold and a lower chip positive mold respectively through a 3D printing solution;

[0028] S102, using PDMS prepolymer to cast the upper chip positive mold and the lower chip positive mold respectively, to obtain the upper chip primary mold and the lower chip primary mold having a channel pattern;

[0029] S103, drilling an upper channel inlet, an upper channel outlet, an upper microchannel and an upper chamber on the upper chip mold to form an upper chip; drilling a lower channel inlet, a lower channel outlet, a lower microchannel and a lower chamber on the lower chip mold to form a lower chip.

[0030] Furthermore, the S4 specifically includes the following steps:

[0031] S401, encapsulating the inner surface of the upper chamber of the upper chip and the lower chamber of the lower chip with rat tail collagen I, and incubating at 37° C. for 2 h;

[0032] S402, absorb 10 6 / mL of intestinal epithelial cells were introduced into the upper chamber of the upper chip and incubated in an incubator at 37°C for 2 hours to allow the cells to adhere to the upper surface of the magnetic bionic porous membrane;

[0033] S403, absorb 10 6 / mL of endothelial cells were introduced into the lower chamber of the lower chip and incubated in an incubator at 37°C for 2 hours to allow the cells to adhere to the lower surface of the magnetic bionic porous membrane;

[0034] S404, respectively, performing microfluidic circulation in the upper chamber and the lower chamber, controlling the flow rate to be set to 30-45 μL / hr, and continuously culturing for 7 days.

[0035] Beneficial effects of the present invention:

[0036] 1. The magnetically controlled intestinal villus motion bionic intestinal chip of the present invention has successfully simulated the intestinal microenvironment through precise design and material selection, and can provide strong support for biological research and drug screening. Through optimized structure and dynamic control mechanism, the chip has a wide range of application potential.

[0037] 2. The present invention prepares a bionic organ chip that can simulate the movement of intestinal villi through positive mold replication and external magnetic field manipulation, which can more accurately reproduce the mechanical, structural and functional characteristics of the intestine. This bionic design enables the chip to simulate complex processes such as intestinal barrier function, absorption of nutrients, and drug transport, providing a more realistic experimental platform for related research.

[0038] 3. The present invention improves the repeatability of experimental results: Since the experimental conditions in the chip can be precisely controlled, the experimental results have high repeatability, which helps to reduce experimental errors and improve the reliability of research conclusions. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0040] Figure 1 Schematic diagram of the exploded structure of the bionic intestinal chip according to an embodiment of the present invention;

[0041] Figure 2 is a schematic diagram of the upper chip structure of an embodiment of the present invention;

[0042] Figure 3 is a schematic diagram of the structure of the lower chip according to an embodiment of the present invention;

[0043] Figure 4 This is an immunofluorescence image of the proliferation and fatty acid absorption of the surface cells of the intestinal chip according to an embodiment of the present invention;

[0044] Figure 5 is a light microscope image of the intestinal villus structure swinging under different magnetic fields according to an embodiment of the present invention;

[0045] Figure 6 This is an immunofluorescence image of the intestinal barrier tight junction protein ZO-1 according to an embodiment of the present invention;

[0046] Figure 7 It is a schematic flow chart of a method for preparing a bionic intestinal chip for magnetically controlling intestinal villus movement according to an embodiment of the present invention;

[0047] Figure 8 It is a schematic diagram of the process of preparing an upper chip and a lower chip according to an embodiment of the present invention;

[0048] Fig. 9 is a schematic diagram of the process of implanting intestinal epithelial cell lines and endothelial cell lines according to an embodiment of the present invention; DETAILED DESCRIPTION

[0049] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] like Figure 1-Figure 3 As shown, an embodiment of the present invention provides a bionic intestinal chip for magnetically controlling the movement of intestinal villi, including an upper chip 1, a lower chip 2, and a magnetic bionic porous membrane 3.

[0051] An open upper chamber 11 is provided at the lower part of the upper chip 1, and an upper microfluidic channel 12 runs through the upper chip 1, so that one end of the upper microfluidic channel 12 is connected to the upper chamber 11, and the other end extends to the upper surface of the upper chip 1 and outward to form an upper channel inlet 101 and an upper channel outlet 102. An intestinal epithelial cell line is cultured in the upper chamber 11, and the intestinal epithelial cell line in the upper chamber 11 is circulated between the upper channel inlet 101 and the upper channel outlet 102; the circulation of the cell culture fluid is supported to simulate the cell environment in the intestine.

[0052] An open lower chamber 21 is provided at the upper part of the lower chip 2, and a lower microfluidic channel 22 runs through the lower chip 2, so that one end of the lower microfluidic channel 22 is connected to the upper chamber 11, and the other end extends to the lower surface of the lower chip 2 and outward to form a lower channel inlet 201 and a lower channel outlet 202. An endothelial cell line is cultured in the lower chamber 21, and the intestinal epithelial cell line in the lower chamber 21 is circulated between the lower channel inlet 201 and the lower channel outlet 202; the circulation of the cell culture fluid is supported to simulate the cell environment in the intestine.

[0053] The magnetic bionic porous membrane 3 is located between the bonding surfaces of the upper chip 1 and the lower chip 2. The magnetic bionic porous membrane 3 is bonded by plasma treatment, which enhances the bonding strength between the membrane and the chip and ensures that no separation occurs during use. The treatment conditions are 200W and 30s, which optimizes the surface properties of the membrane and improves adhesion.

[0054] And simultaneously realize the separation and independence of the upper chamber 11 and the lower chamber 21 (i.e., forming a double-chamber structure. The upper chamber 11 is used to culture intestinal epithelial cells, and the lower chamber 21 is used to culture endothelial cells). The magnetic bionic porous membrane 3 adopts a flexible Ecoflex matrix and incorporates magnetic particles; adopts a flexible Ecoflex matrix and incorporates magnetic particles. The porous membrane not only achieves the isolation of the upper and lower chambers, but also has the ability to regulate the movement of intestinal villi. The swing of the magnetic particles is controlled by an external circulating magnetic field to simulate the movement of intestinal villi. This movement can promote the absorption of nutrients and the interaction between cells, enhancing the physiological relevance of the chip.

[0055] The bonding surfaces of the chips 2 are bonded by plasma treatment, and the plasma treatment conditions are 200 W for 30 seconds.

[0056] The magnetic particles are one or more of elemental iron, magnetite, carbonyl iron, and cobalt ferrite, and the particle size is 50 to 150 nanometers. Particles in this size range can effectively respond to external magnetic fields and produce appropriate movements.

[0057] The diameter D of the upper chamber 11 where the upper chip 1 is located 上腔 =4mm, height H 上腔 =2mm;

[0058] The diameter D of the lower chamber 21 of the lower chip 2 下腔 =4mm, height H 下腔 =1mm. This design can provide sufficient space for cell growth while maintaining the effectiveness of fluid flow.

[0059] The frequency of the external circulating magnetic field is 0.1Hz~0.3Hz, and the magnetic field strength is 0.5~4KGs. The external magnetic field adopts a circulating constant magnetic field (1KGs) as needed, the swing frequency of the bionic villi is 0.15Hz, and the swing amplitude is about 8%. Therefore, the swing amplitude under the condition of 1KGs is closest to the physiological range. These parameters can adjust the swing frequency and amplitude of the intestinal villi and simulate the movement of the intestine under physiological conditions. For details, refer to Figure 5 shown.

[0060] Under the magnetic field of 0.5KGs, the oscillation frequency of the intestinal villi is smaller, showing smaller oscillations. According to the images, the movement amplitude of the intestinal villi is smaller at this intensity.

[0061] Under a magnetic field of 1 kg, the intestinal villi swing at a frequency of 0.15 Hz, showing a relatively obvious swing. According to the image, the movement amplitude of the intestinal villi at this intensity is relatively large, which can effectively simulate the swing amplitude of about 10% under physiological conditions.

[0062] When the magnetic field strength increases to 2KGs and 4KGs, the swing amplitude of the intestinal villi will increase, but it will exceed the swing amplitude under physiological conditions by about 10%.

[0063] Under a magnetic field of 0 KGs, the oscillation frequency of the intestinal villi is 0, so the intestinal villi will not oscillate under this intensity.

[0064] The frequency and intensity of the external circulating magnetic field have a significant effect on the movement of intestinal villi. Appropriate magnetic field intensity (such as 1KGs) helps to better simulate the movement of the intestine under physiological conditions.

[0065] By simulating the intestinal microenvironment and the movement of intestinal villi, the chip can more realistically reflect the behavior of cells under physiological conditions, providing a more reliable platform for drug screening and biological research. It can also study the interaction between intestinal epithelial cells and endothelial cells, revealing their mechanism of action in drug metabolism and absorption.

[0066] like Figure 7 As shown, the method for preparing a bionic intestinal chip for magnetically controlling intestinal villus movement comprises the following steps:

[0067] S1, preparing an upper chip 1 and a lower chip 2;

[0068] The specific steps include:

[0069] like Figure 8 As shown, S101, firstly, a 3D printing scheme with a resolution of 50 microns is used for printing (the 3D printing scheme can be customized through a program, can efficiently and accurately manufacture complex structures and patterns, is suitable for the design of microfluidic chips, and can customize the shape and size of the chip according to specific needs to meet the special requirements of experiments or applications), and the upper chip positive mold and the lower chip positive mold are printed respectively;

[0070] S102, using PDMS prepolymer (which has good biocompatibility and chemical stability and is very suitable for microfluidic applications) to cast the upper chip positive mold and the lower chip positive mold respectively, after printing, clean with isopropyl alcohol and UV-cured for 60 minutes, to obtain the upper chip primary mold and the lower chip primary mold with channel patterns;

[0071] S103, drill the upper channel inlet 101, the upper channel outlet 102, the upper microchannel 12 and the upper chamber 11 on the upper chip mold to form the upper chip 1; drill the lower channel inlet 201, the lower channel outlet 202, the lower microchannel 22 and the lower chamber 21 on the lower chip mold to form the lower chip 2.

[0072] S2, prepare magnetic bionic porous membrane 3, cutting power is 150W, scanning rate is 40mm / s CO 2 Laser cutting technology is used to prepare a through-hole positive mold on polyethylene terephthalate (PET) material. The positive mold thickness is H 膜 =1.0mm, through hole diameter Φ 孔 =0.2mm, through hole spacing D 孔 =0.5mm. Appropriate thickness can ensure that the male mold has sufficient mechanical strength during the manufacturing process to avoid deformation or breakage, and the thickness affects the flow characteristics of the fluid in the through hole. Appropriate thickness can optimize fluid flow and reaction time. At the same time, the size of the pore size directly affects the flux and flow rate of the fluid. The appropriate pore size can ensure the effective transmission of the fluid in the microfluidic system. The pore spacing affects the overall structural stability of the male mold. Appropriate spacing can prevent mutual interference between the pores.

[0073] Prepare a porous membrane raw material solution, which consists of Ecoflex and sodium chloride pore formers in a mass ratio of 2:1. At the same time, prepare a porous membrane magnetic raw material solution, which consists of Ecoflex, sodium chloride pore formers and carbonyl iron magnetic particles in a mass ratio of 2:1:1. After mixing, store at -20°C; Ecoflex is an elastomer that provides membrane flexibility and is suitable for maintaining structural integrity in microfluidic applications. The pore former is one of sodium chloride, ammonium chloride, sucrose, and sodium sulfate. The addition of the pore former can form pores in the membrane, affecting the permeability of the membrane and the fluid flow characteristics.

[0074] The porous membrane raw material solution is evenly dispersed under low-speed stirring (200 rpm), and after degassing for 15 minutes, the porous membrane magnetic raw material solution is filled into the through-hole structure of the positive membrane, and the excess porous membrane magnetic raw material solution is scraped off; the porous membrane raw material solution is evenly coated on one side of the positive membrane, and a magnetic bionic membrane is obtained after curing; the magnetic porous membrane after curing is separated from the through-hole positive mold (the curing molding temperature is 75°C and the time is 2h), placed in ultrapure water, and the sodium chloride pore-forming agent is removed through multiple heating cycles, and then a magnetic bionic porous membrane 3 is obtained.

[0075] At this time, the through-hole positive mold is one or more of polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), polypropylene (PP) and polystyrene (PS). PET has good optical transparency and is suitable for microfluidic applications that require optical observation. It has good tolerance to a variety of chemicals and is suitable for biological experiments. PC has high impact resistance and is suitable for applications that require mechanical strength. It also has good transparency and is suitable for optical detection. PMMA has very high transparency and is suitable for optical experiments. Complex structures can be made by laser cutting, thermoforming and other methods. Polypropylene has a low density and is suitable for lightweight microfluidic devices. It has good tolerance to many chemicals and is suitable for biological experiments. Polystyrene can be easily formed by injection molding and other methods, which is suitable for large-scale production. While having a certain degree of transparency, its cost is relatively low.

[0076] S3. Place the magnetic bionic porous membrane 3 on the bonding surface where the upper chip 1 and the lower chip 2 are located, and perform hydrophilic treatment through a plasma instrument. After the hydrophilic treatment, the surface is more likely to form good contact with the adhesive or other materials, thereby enhancing the bonding strength between different layers. This plasma treatment can increase the surface energy of the material, making it more hydrophilic. This helps to improve the wettability of the material and promote the subsequent bonding or coating process. At the same time, the plasma treatment can remove organic pollutants and oxides on the surface to ensure the surface is clean, thereby improving the bonding effect. Then place it in an 80°C oven for bonding for 1 hour; bonding in an 80°C oven for 1 hour can promote the curing or cross-linking of the adhesive, and also help to eliminate the stress generated during the treatment process and ensure the stability between the layers.

[0077] S4, then implanting intestinal epithelial cell lines and endothelial cell lines in the upper chamber 11 of the upper chip 1 and the lower chamber 21 of the lower chip 2, respectively. Fig. 9 As shown, the specific steps include:

[0078] S401. Use rat tail collagen I to encapsulate the inner surface of the upper chamber 11 of the upper chip 1 and the lower chamber 21 of the lower chip 2, and incubate at 37°C for 2 hours. After encapsulation, cells are more likely to adhere to the surface in subsequent steps, thereby improving the growth environment of cells.

[0079] S402, absorb 10 6 / mL of intestinal epithelial cells are passed into the upper chamber 11 of the upper chip 1 and incubated in an incubator at 37°C for 2 hours to allow the cells to adhere to the upper surface of the magnetic bionic porous membrane 3; by injecting intestinal epithelial cells into the upper chamber 11, the physiological environment in the intestine can be simulated, providing a corresponding model for research. In a culture environment of 37°C, the cells can adapt to and adhere to the surface encapsulated by rat tail collagen I to form a single-layer cell structure.

[0080] S403, absorb 10 6 / mL of endothelial cells are introduced into the lower chamber 21 of the lower chip 2 and incubated in an incubator at a temperature of 37°C for 2 hours to allow the cells to adhere to the lower surface of the magnetic bionic porous membrane 3; by injecting endothelial cells into the lower chamber 21, the physiological conditions in the blood vessels can be simulated, providing a model for studying blood vessel-related biological processes. Similar to the upper chamber, endothelial cells can adhere to and adapt to the surface of rat tail collagen I in a 37°C culture environment to form a functional endothelial cell layer.

[0081] S404, respectively, the upper chamber 11 and the lower chamber 21 are subjected to microfluidic circulation, the flow rate is controlled to be set to 30-45 μL / hr, and the chip is placed in an external circulating magnetic field (3 KGs), the swing frequency of the bionic hair is 0.1 Hz, and the culture is continued for 7 days. The cell growth status is shown in Figure 4 Circulating fluid can simulate the blood flow in the body and the liquid flow in the intestinal cavity, providing cells with a culture environment that is closer to physiological conditions. Fluid circulation helps to provide cells with continuous nutrients and oxygen, while taking away metabolic waste. Long-term fluid culture can promote cell growth, differentiation and function, and is suitable for long-term biological experiments and drug screening.

[0082] The magnetically controlled movement ability of the bionic intestinal chip prepared above was tested.

[0083] The test method is: place the assembled bionic intestinal chip in a left-right circulating magnetic field (3KGs) above the chip, and control the swing of the bionic intestinal villi through the movement of the magnetic field. The swing frequency is 0.1Hz, thereby realizing the controlled swing of the magnetic villi under the control of the external magnetic field, simulating the movement of the intestinal villi in the real intestine.

[0084] Test results see Figure 5 : Magnetically controlled bionic villi can be controlled by an external magnetic field and swing left and right in sequence along the direction of the magnetic flux lines. Therefore, this swinging mode is conducive to strengthening the exchange of fluid between intestinal villi and external fluid, reducing radial mass transfer resistance, and thus highly simulating the intestinal barrier function and absorption function in vitro.

[0085] The intestinal barrier function of the bionic intestinal chip prepared above was tested.

[0086] The test method is: pass ZO-1 antibody and VE-Cadherin antibody into the upper and lower channels of the intestinal chip, stain Caco-2 cells and HUVEC cells respectively, and observe the immunofluorescence staining results under a laser confocal microscope. Specifically: pass the paraformaldehyde solution into the upper and lower channels to fix the cells for 10 minutes, and then block the cells with immunostaining blocking solution to enhance the signal-to-noise ratio. Then, pass ZO-1 antibody and VE-Cadherin antibody from the intestinal cavity and the vascular end respectively, and incubate at 4°C for 12 hours. Finally, pass the fluorescent secondary antibody into the upper and lower ends for incubation for 1 hour, and use a laser confocal microscope to observe the expression of ZO-1 and VE-Cadherin.

[0087] Test results see Figure 6 : Compared with the planar culture group and the magnetic villus static culture group, the intestinal barrier function in the magnetically controlled dynamic culture group was more complete, and the expression of ZO-1 and VE-Cadherin was significantly increased, proving that the bionic intestinal chip prepared by the present invention can accurately reproduce the intestinal barrier function.

[0088] The present invention prepares a bionic intestinal chip that simulates the movement of intestinal villi. A magnetic bionic porous membrane is obtained by a template replication method. The porous membrane is then combined with a two-layer PDMS chip. Endothelial cell lines and intestinal epithelial cell lines are cultured on both sides of the porous membrane to simulate the intestinal barrier and endothelial barrier functions. The chip that forms the barrier function is connected to a microfluidic pump to simulate blood flow and intestinal fluid flow, thereby achieving dynamic cell culture. Under the control of an external magnetic field, the magnetic villi can swing back and forth repeatedly, simulating the physiological swing of intestinal villi, and having more bionic value. This simulation not only truly simulates the tissue structure of the intestine, but also reproduces the physiological function of the swing of intestinal villi. It can be used for related biological mechanism research, disease modeling and drug screening, and has certain application prospects.

[0089] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A bionic intestinal chip for magnetically controlling the movement of intestinal villi, comprising an upper chip (1), a lower chip (2), and a magnetic bionic porous membrane (3), characterized in that: An upper chamber (11) is provided at the bottom of the upper chip (1) and is open downwards. An upper microfluidic channel (12) runs through the upper chip (1), so that one end of the upper microfluidic channel (12) is connected to the upper chamber (11), and the other end extends to the upper surface of the upper chip (1) and outwards to form an upper flow channel inlet (101) and an upper flow channel outlet (102). An intestinal epithelial cell line is cultured in the upper chamber (11), and the intestinal epithelial cell line in the upper chamber (11) is circulated between the upper flow channel inlet (101) and the upper flow channel outlet (102); A lower chamber (21) open to the top of the lower chip (2) is provided, and a lower microchannel (22) is passed through the lower chip (2), so that one end of the lower microchannel (22) is connected to the upper chamber (11), and the other end extends to the lower surface of the lower chip (2) and outwards to form a lower channel inlet (201) and a lower channel outlet (202). An endothelial cell line is cultured in the lower chamber (21), and the intestinal epithelial cell line in the lower chamber (21) is circulated between the lower channel inlet (201) and the lower channel outlet (202); The magnetic bionic porous membrane (3) is located between the bonding surfaces of the upper chip (1) and the lower chip (2), and simultaneously realizes the separation and independence of the upper chamber (11) and the lower chamber (21); the magnetic bionic porous membrane (3) adopts a flexible Ecoflex matrix and is doped with magnetic particles; The swing of magnetic particles located on the magnetic bionic porous membrane (3) is controlled by an external circulating magnetic field.

2. The bionic intestinal chip for magnetically controlled intestinal villus movement and the preparation method thereof according to claim 1, characterized in that: The magnetic bionic porous membrane (3) is bonded between the bonding surfaces of the upper chip (1) and the lower chip (2) by plasma treatment, and the conditions of the plasma treatment are 200W for 30s.

3. The bionic intestinal chip for magnetically controlled intestinal villus movement according to claim 1, characterized in that: The magnetic particles are one or more of elemental iron, magnetite, carbonyl iron, and cobalt ferrite, and the particle size is 50 to 150 nanometers.

4. The bionic intestinal chip for magnetically controlled intestinal villus movement according to claim 1, characterized in that: The diameter D of the upper chamber (11) where the upper chip (1) is located is 上腔 =4mm, height H 上腔 =2mm; The diameter D of the lower chamber (21) of the lower chip (2) 下腔 =4mm, height H 下腔 =1mm.

5. The bionic intestinal chip for magnetically controlled intestinal villus movement according to claim 1, characterized in that: The frequency of the external circulating magnetic field is 0.1 Hz to 0.3 Hz, and the magnetic field strength is 0.5 to 4 KGs.

6. The method for preparing the bionic intestinal chip for magnetically controlled intestinal villus movement according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, preparing an upper chip (1) and a lower chip (2); S2, preparing a magnetic bionic porous membrane (3), using CO2 laser cutting to prepare a through-hole positive mold, filling the porous membrane magnetic raw material solution into the through-hole structure of the positive membrane, scraping off excess porous membrane magnetic raw material solution; then evenly coating the porous membrane raw material solution on one side of the positive membrane; separating the magnetic porous membrane from the through-hole positive mold after curing, and then obtaining the magnetic bionic porous membrane (3); S3, placing the magnetic bionic porous membrane (3) on the bonding surface of the upper chip (1) and the lower chip (2) for hydrophilization treatment, and then placing it in an oven at 80° C. for bonding for 1 hour; S4. Then, intestinal epithelial cell lines and endothelial cell lines are implanted in the upper chamber (11) of the upper chip (1) and the lower chamber (21) of the lower chip (2), respectively.

7. The method for preparing the bionic intestinal chip for magnetically controlled intestinal villus movement according to claim 6, characterized in that: In S2, the through hole male mold is one or more of polyethylene terephthalate, polycarbonate, polymethyl methacrylate, polypropylene, and polystyrene; The through hole male mold thickness H 膜 =1.0mm, through hole diameter Φ 孔 =0.5mm, through hole spacing D 孔 =1.0mm. The porous membrane magnetic raw material solution comprises Ecoflex, pore-forming agent and magnetic particles, which are mixed in a mass ratio of 2:1:1; The pore-forming agent is one of sodium chloride, ammonium chloride, sucrose and sodium sulfate.

8. The method for preparing the bionic intestinal chip for magnetically controlled intestinal villus movement according to claim 6, characterized in that: In S2, the curing temperature is 75° C. and the curing time is 2 hours.

9. The method for preparing the bionic intestinal chip for magnetically controlled intestinal villus movement according to claim 6, characterized in that: The S1 specifically includes the following steps: S101, firstly, printing out an upper chip positive mold and a lower chip positive mold respectively through a 3D printing solution; S102, using PDMS prepolymer to cast the upper chip positive mold and the lower chip positive mold respectively, to obtain the upper chip primary mold and the lower chip primary mold having a channel pattern; S103, drilling an upper channel inlet (101), an upper channel outlet (102), an upper microchannel (12) and an upper chamber (11) on the upper chip primary mold to form an upper chip (1); drilling a lower channel inlet (201), a lower channel outlet (202), a lower microchannel (22) and a lower chamber (21) on the lower chip primary mold to form a lower chip (2).

10. The method for preparing the bionic intestinal chip for magnetically controlled intestinal villus movement according to claim 6, characterized in that: The S4 specifically includes the following steps: S401, using rat tail collagen I to encapsulate the inner surface of the upper chamber (11) of the upper chip (1) and the lower chamber (21) of the lower chip (2), and incubate at 37° C. for 2 h; S402, absorb 10 6 / mL of intestinal epithelial cells are introduced into the upper chamber (11) of the upper chip (1), and incubated in an incubator at 37°C for 2 hours to allow the cells to adhere to the upper surface of the magnetic bionic porous membrane (3); S403, absorb 10 6 / mL of endothelial cells are introduced into the lower chamber (21) of the lower chip (2), and incubated in an incubator at 37°C for 2 hours to allow the cells to adhere to the lower surface of the magnetic bionic porous membrane (3); S404, respectively, performing microchannel fluid circulation in the upper chamber (11) and the lower chamber (21), controlling the flow rate to be set to 30-45 μL / hr, and continuously culturing for 7 days.

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