An alveolus biomimetic microfluidic device for simulating alveolar three-dimensional structure and a preparation method thereof

Abstract

This invention provides a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli. The fabrication method includes: preparing an inverse opal membrane; bonding the inverse opal membrane to the bottom of a transwell chamber structure (with the bottom filter membrane removed); bonding the top of the transwell chamber to a perforated plate cover; inserting one end of a gas delivery tube through the perforation in the perforated plate cover into the transwell chamber; and connecting the other end to an injection pump; culturing alveolar epithelial cells and vascular endothelial cells on the front and back sides of the inverse opal membrane, respectively, to obtain the biomimetic microfluidic device. The biomimetic microfluidic device provided by this invention can achieve three-dimensional stretching under the action of a microfluidic injection pump, simulating the periodic stretch-recovery process of human lung respiratory movement in vitro. The fabrication method is simple and reliable.

Description

Technical fields:

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli containing an inverse opal structure film, and its preparation method. Background technology:

[0002] The human lungs are respiratory organs characterized by a unique mechanical microenvironment, including periodic respiratory movements and a blood-air barrier. Due to their complex structure and function, establishing reliable in vitro models remains challenging and in high demand for basic biological research, disease treatment, and targeted drug testing. Increasing evidence suggests that the mechanical microenvironment influences cellular function, including mechanical forces, extracellular matrix, and intracellular factors. However, to date, effective alveolar microphysiological systems that truly mimic the three-dimensional periodic stretching of human alveoli are extremely rare.

[0003] Inverse opal refers to a type of porous structure with orderly arranged uniform pores and interconnected windows, which is beneficial for cell distribution, differentiation, and diffusion of biomolecules. As a biomimetic scaffold in biomedicine, its highly tunable and precisely controllable characteristics will lead to a paradigm shift in tissue engineering and regenerative medicine research. Furthermore, it can respond to changes in external environmental signals, generating intuitive and visual response signals to complex environmental signals, and has enormous application potential in fields such as biomedical sensing and tissue medicine.

[0004] Therefore, in this invention, we start by constructing an inverse opal structure membrane and design a novel alveolar biomimetic microfluidic device based on microfluidic technology. This novel alveolar biomimetic microfluidic device can be used to study the effect of mechanical stretching on the proliferation and differentiation of alveolar stem cells by periodically stretching the inverse opal structure membrane inside it. Summary of the Invention:

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli and its preparation method. By bonding an inverse opal polyurethane membrane to a transwell chamber device to form a sealed structure, continuous infusion / extraction by an injection pump can cause the membrane to undergo a certain degree of stretching. This degree and manner of stretching is similar to that of physiological conditions, which can simulate alveolar respiration in vitro and allow for further research.

[0006] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0007] (I) This invention provides a method for preparing a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli, comprising the following steps:

[0008] S1. Prepare an inverse opal structure membrane and bond the inverse opal structure membrane to the bottom of the transwell chamber structure after removing the bottom filter membrane.

[0009] S2. Culture alveolar epithelial cells and vascular endothelial cells on the front and back sides of the inverted opal membrane, respectively.

[0010] S3. Attach the top of the transwell chamber to the perforated plate cover with through holes, pass one end of the gas delivery tube through the through hole of the perforated plate cover into the transwell chamber, and connect the other end to a syringe and a microfluidic injection pump to obtain the alveolar biomimetic microfluidic device.

[0011] Furthermore, the preparation method of the inverse opal structure film is as follows: first, a positive structure template is prepared, a polyurethane solution is added to the positive structure template, and a polyurethane film with a certain thickness is obtained by spin coating with a spin coater. Then, silica particles are etched to obtain the inverse opal structure polyurethane film.

[0012] Furthermore, the preparation method of the positive structure template is as follows: silica particles are mixed with anhydrous ethanol and ultrasonically vibrated to make the silica particles uniformly distributed in anhydrous ethanol to obtain a silica suspension. The silica suspension is uniformly coated on a glass slide and allowed to stand to allow the anhydrous ethanol to evaporate, leaving a uniformly distributed silica particle film as the positive structure template.

[0013] Furthermore, the slides are cleaned before use. The cleaning process involves washing the slides twice in an ultrasonic vibrator with a surfactant solution, ultrapure water, and anhydrous ethanol, and then drying them to ensure that the slides meet the required cleanliness standards.

[0014] Furthermore, the diameter of the silica particles is 270 nm; and the weight ratio of silica particles to anhydrous ethanol in the silica suspension is 20:100.

[0015] Furthermore, the polyurethane solution is prepared by dissolving polyurethane in N,N-dimethylformamide solvent and heating at 80°C for more than 2 hours to ensure uniform dissolution of the polyurethane, thereby obtaining a polyurethane solution; the mass concentration of the polyurethane is 20% (w / w).

[0016] Furthermore, during the spin coating process, the spin coater speed was set to 300 rpm for 30 seconds, followed by 400 rpm for 30 seconds; then the spin-coated glass slide carrying a thin layer of polyurethane solution was placed on a hot plate with a temperature of 80°C, and dried after 2 hours to obtain a polyurethane film with a thickness of 5 micrometers.

[0017] Furthermore, the etching method for the silica particles is as follows: a glass slide carrying silica particles and a polyurethane film is immersed in 4% hydrofluoric acid overnight. After the silica particle layer is etched by hydrofluoric acid, a polyurethane film floating on the liquid surface is obtained. The film is then retrieved with a clean glass slide and washed 5 times with ultrapure water to remove the hydrofluoric acid adhering to the film, thus obtaining an inverse opal structure polyurethane film.

[0018] Furthermore, the transwell chamber is side-sealed, forming a closed space structure with the inverse opal structure membrane and the orifice plate top cover; the bottom diameter of the transwell chamber is 7mm; and the gas delivery pipe is a Teflon tube with a pore size of 1mm.

[0019] (II) This invention also provides a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli, prepared by the method described above. It includes an inverted opal membrane, a transwell chamber with a bottom filter removed, a perforated plate cap, and an air delivery tube. The bottom of the transwell chamber with the bottom filter removed is bonded to the inverted opal membrane, and the top is bonded to the perforated plate cap. The perforated plate cap has a through-hole, through which one end of the air delivery tube extends into the transwell chamber, and the air delivery tube is sealed to the perforated plate cap. The other end of the air delivery tube is connected to an injection pump. Alveolar epithelial cells and vascular endothelial cells are cultured on the front and back sides of the inverted opal membrane, respectively. This device can achieve periodic stretching of the inverted opal membrane under the action of a microfluidic injection pump, thereby simulating the three-dimensional respiration of alveoli.

[0020] (III) The present invention also provides the application of the alveolar biomimetic microfluidic device prepared by the above preparation method in the in vitro simulation of the three-dimensional structure of alveoli.

[0021] The beneficial effects of this invention are:

[0022] 1) The present invention provides a method for preparing a biomimetic microfluidic device for simulating alveolar structure using an inverse opal structure membrane. The inverse opal structure membrane is prepared by preparing an inverse opal structure. The inverse opal structure membrane is combined with a transwell chamber and placed in a well plate of the same size to form a sealed structure. It is then connected to an air delivery tube and a syringe. Under the action of a microfluidic injection pump, the inverse opal structure membrane is stretched in three dimensions. The preparation method is simple and reliable.

[0023] 2) The alveolar biomimetic microfluidic device provided by the present invention contains an inverse opal structure membrane. Under the action of the microfluidic injection pump, the inverse opal structure membrane can achieve three-dimensional stretching, which better simulates the stretching-recovery process of lung respiratory movement in the human body in vitro.

[0024] 3) The alveolar biomimetic microfluidic device provided by the present invention can culture alveolar epithelial cells and vascular endothelial cells in the upper and lower layers of the inverse opal membrane, respectively. In conjunction with the periodic stretching of the inverse opal membrane, the microfluidic injection pump parameters are adjusted to 0.25Hz so that the periodic stretching frequency of the inverse opal membrane is the same as the respiratory frequency under physiological conditions, thus better simulating the respiratory process of human alveoli in vitro. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the stretching process of the inverse opal structure polyurethane membrane used in this invention to simulate the alveolar structure.

[0026] Figure 2 This is a flowchart illustrating the preparation process of the inverse opal structure polyurethane membrane of this invention.

[0027] Figure 3 This is a scanning electron microscope image of the inverse opal structure polyurethane film prepared in Example 2 of the present invention.

[0028] Figure 4 The graph shows the relationship between the stretching degree of the inverse opal structure membrane in the alveolar biomimetic microfluidic device prepared in Example 2 of the present invention and the corresponding statistical graph.

[0029] Figure 5 Immunofluorescence staining image of human alveolar epithelial cells-vascular endothelial cell barrier cultured using the alveolar biomimetic microfluidic device prepared in Example 2 of this invention. Detailed implementation method:

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Implementation conditions not specified in the embodiments are generally conditions in conventional experiments.

[0031] Example 1

[0032] This invention provides a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli, comprising an inverted opal membrane, a transwell chamber with a bottom filter removed, a perforated plate cap, and an air delivery tube. The bottom of the transwell chamber is bonded to the inverted opal membrane, and the top is bonded to the perforated plate cap. The perforated plate cap has a through-hole, through which one end of the air delivery tube extends into the transwell chamber, and the air delivery tube is sealed to the perforated plate cap. The other end of the air delivery tube is connected to a syringe, which is fixed to a microfluidic injection pump. Alveolar epithelial cells and vascular endothelial cells are cultured on the front and back sides of the inverted opal membrane, respectively. This device can achieve periodic stretching of the inverted opal membrane under the action of the microfluidic injection pump, thereby simulating the three-dimensional respiration of alveoli.

[0033] Example 2

[0034] This invention provides a method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli, comprising the following steps:

[0035] (I) Preparation of inverse opal structured membranes

[0036] 1) Prepare clean glass slides: Before use, glass slides are cleaned. The cleaning process is as follows: the glass slides are cleaned twice in an ultrasonic oscillator with a solution containing surfactant, ultrapure water and anhydrous ethanol respectively, then dried and plasma treated for 5 minutes to make the glass slides reach the required cleanliness standard.

[0037] 2) Preparation of polyurethane solution: Thermosetting polyurethane is dissolved in N,N-dimethylformamide solvent (DMF) at a ratio of 20% (w / w), and heated at 80°C for more than 2 hours to make the polyurethane uniformly dissolved, so as to obtain a polyurethane solution, which is used as the raw material for inverse opal structure membrane.

[0038] 3) Preparation of positive structure template:

[0039] Silica particles with a particle size of 270 nm were mixed with anhydrous ethanol at a weight ratio of 20:100. After mixing, the mixture was ultrasonically vibrated to make the silica particles uniformly distributed in the anhydrous ethanol, thus obtaining a silica suspension.

[0040] A 40 μl droplet of silica suspension is pipetted onto a clean glass slide. The droplet is then spread evenly on another glass slide and allowed to air dry at room temperature until the anhydrous ethanol in the droplet evaporates. A thin film of uniformly distributed silica particles adheres to the glass slide, serving as a positive-structure silica template.

[0041] 4) Preparation of inverse opal structured polyurethane films:

[0042] The polyurethane solution was added to the positive structural template and then spin-coated using a spin coater. The spin coater speed was set to 300 rpm for 30 seconds and then 400 rpm for 30 seconds. The spin-coated glass slide containing a thin layer of polyurethane solution was then placed on a heating stage with a temperature of 80°C. After 2 hours, the slide was dried to allow the DMF to evaporate, resulting in a cured polyurethane layer adhering to the positive structural silica template.

[0043] An aqueous solution containing 4% hydrofluoric acid by volume was prepared as an etching solution for etching the positive structure silica template and for peeling the inverse opal structure film off the glass slide.

[0044] A glass slide containing silica particles and a polyurethane film was placed in a plastic box containing a 4% hydrofluoric acid aqueous solution. The hydrofluoric acid in the solution reacted chemically with the silica particles, etching the positive structure template on the glass slide. The polyurethane film attached to the positive structure silica particles floated on the liquid surface after the silica particles were etched away. The film was retrieved with a clean glass slide and washed five times with ultrapure water to remove the hydrofluoric acid attached to the film, thus obtaining an inverse opal structure polyurethane film.

[0045] (II) Preparation of alveolar biomimetic microfluidic device

[0046] 1) Take a transwell chamber with a bottom diameter of 7mm and closed sides, separate and remove the original PET film at the bottom, and bond the inverse opal polyurethane film to the bottom of the transwell chamber.

[0047] 2) Take the top cover of the 24-hole plate and drill holes in the top cover;

[0048] 3) Attach the top of the transwell chamber to the perforated plate cover with through holes. At this time, the transwell device, the inverse opal polyurethane membrane, and the perforated plate cover form a closed space structure. Insert one end of a 1mm diameter Teflon tube through the through hole in the perforated plate cover into the transwell chamber, and connect the other end to a syringe. The syringe is fixed on a microfluidic injection pump to obtain an alveolar biomimetic microfluidic device. By setting the parameters of the microfluidic injection pump, the amount and frequency of gas entering the device can be adjusted, which can achieve the effect of periodic stretching of the inverse opal membrane, thereby simulating the three-dimensional respiration of alveoli.

[0049] Experimental Example 1 – Stretch Test of Alveolar Bionic Microfluidic Device

[0050] The alveolar biomimetic microfluidic device prepared in Example 2 was subjected to a stretching test.

[0051] A perfusion value is set on the microfluidic injection pump, and the microfluidic injection pump is started. Gas is injected into the alveolar biomimetic microfluidic device through a Teflon tube driven by the injection pump. The inverse opal polyurethane membrane undergoes elastic deformation and tension change due to the change in gas volume. By setting different perfusion values, different volumes of gas are injected into the alveolar biomimetic microfluidic device, causing the inverse opal polyurethane membrane to undergo different degrees of tension deformation.

[0052] Figure 4 This is a statistical graph showing the change in the stretching degree of the inverse opal membrane in the alveolar biomimetic microfluidic device with the gas perfusion volume and the relationship between the two. Figure 4 (a) Real-time generated images of different stretching degrees of the inverse opal structure film caused by different gas injection volumes observed by a high-speed camera. Figure 4 (b) A statistical graph showing the relationship between changes in volumetric infusion volume and the degree of stretching of the inverse opal structure membrane. Figure 4 (a) and Figure 4 (b) It can be seen that the more gas is injected into the alveolar biomimetic microfluidic device, the greater the stretching of the inverse opal structure membrane.

[0053] The perfusion volume was controlled from 0 mL and gradually increased to 0.7 mL, while other factors remained constant. Figure 4 (a) It is evident that as the gas injection volume increases within this range, the surface area of ​​the inverse opal membrane gradually increases; the stretching degree of the inverse opal membrane is obtained by comparing the increased surface area with the static surface area, and a statistical graph showing the relationship between the two and the gas injection volume is plotted. Figure 4 (b) It can be seen that, within the range of 0–0.7 mL, the stretching degree of the inverse opal membrane increases with the increase of gas perfusion volume. These results indicate that the stretching degree of the inverse opal membrane can be adjusted by the gas perfusion volume, thereby simulating alveoli under different stretching states due to physiological or pathological conditions.

[0054] Experimental Example 2 – Bionic microfluidic device for alveoli used to construct epithelial cell-endothelial cell interface

[0055] The alveolar biomimetic microfluidic device prepared in Example 2 was used to construct the epithelial cell-endothelial cell interface.

[0056] 1) After cleaning and drying the anti-opal polyurethane membrane prepared in Example 2, it was placed in the reaction chamber of a vacuum plasma cleaner and plasma-treated for 30 seconds to make its hydrophobic surface hydrophilic, which facilitates the subsequent cell adhesion and reproduction.

[0057] 2) Dilute the matrix gel with serum-free culture medium at a ratio of 1:3 and drop it onto the inverted opal polyurethane membrane at the bottom of the transwell device. Place it at 37°C for 2 hours to allow the matrix gel dilution to solidify and adhere uniformly to the membrane surface. Then, in the same way, attach a thin layer of matrix gel to the other side of the inverted opal polyurethane membrane.

[0058] 3) Human alveolar epithelial cells were seeded onto the surface (front) of the inverted opal polyurethane membrane at the bottom of the transwell device, and vascular endothelial cells were seeded onto the other side (back) of the inverted opal polyurethane membrane. Human alveolar epithelial cells and vascular endothelial cells were cultured in a 1:1 mixture of DMEM and ECM culture medium and cultured at 37°C until the cell density was >80%.

[0059] To facilitate observation of the epithelial-endothelial cell interface, the two types of cells in step 3) were fixed with 4% paraformaldehyde solution for 30 minutes, and the residual paraformaldehyde solution was washed off with PBS.

[0060] Human alveolar epithelial cells were stained with E-Cadherin, and the E-Cadherin primary antibody solution was prepared by diluting it with PBS to a ratio of 1:500. One-tenth of the cell culture medium volume of the E-Cadherin solution was added to the human alveolar epithelial cell layer.

[0061] Vascular endothelial cells were stained with VE-Cadherin, and the VE-Cadherin primary antibody solution was diluted with PBS to prepare a 1:500 solution. 1 / 10 volume of the VE-Cadherin solution was added to the endothelial cell layer.

[0062] The two groups were incubated overnight at 4°C. The residual solution was then washed off with PBS. A secondary antibody solution corresponding to E-Cadherin diluted in PBS at a concentration of 1:1000 was added to the human alveolar epithelial cell layer, and a secondary antibody solution corresponding to VE-Cadherin diluted in PBS at a concentration of 1:1000 was added to the vascular endothelial cells. The fluorescence images of the two cell layers were then observed using a laser scanning confocal microscope.

[0063] like Figure 5 As shown in Figure ac, E-Cadherin-stained human alveolar epithelial cells grow on the surface of the inverse opal polyurethane membrane and emit bright red fluorescence, while VE-Cadherin-stained vascular endothelial cells grow on the membrane surface opposite to the epithelial cells and emit bright green fluorescence. Human alveolar epithelial cells and vascular endothelial cells grow densely on both sides of the inverse opal polyurethane membrane, forming a cell-cell interface similar to that in human alveoli.

[0064] This invention discloses a biomimetic microfluidic device for simulating alveolar structure, comprising an inverse opal membrane and its preparation method. The method involves pouring adhesive onto a layer of positively structured silica particles and etching the silica particles to obtain an inverse opal membrane with a vibrant structural color. This inverse opal membrane is then combined with a transwell chamber, placed in a well plate of matching dimensions, and connected to a syringe. Under the action of a microfluidic injection pump, the inverse opal membrane is stretched in three dimensions, thereby simulating the expansion-contraction function of alveoli. The biomimetic microfluidic device prepared by this invention can culture alveolar epithelial cells and vascular endothelial cells in two separate layers. Under the action of the microfluidic pump, the inverse opal membrane and the cells on the membrane are stretched within a physiological range. Simultaneously, the good elasticity of the inverse opal membrane allows it to perfectly return to its original position, ensuring that the cells on the membrane undergo a stretch-recovery cycle. Furthermore, the inverse opal structural color of the membrane changes visibly with the degree of membrane stretching, serving as an indicator of the stretching degree. The device of this invention can more realistically simulate the physiological and pathological conditions of alveoli, and can be used for related basic research, disease modeling and drug screening. It has advantages that traditional in vitro culture and animal experiments cannot provide, and has broad application prospects.

[0065] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions that fall within the scope of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principle of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli, characterized in that, include: S1. Prepare an inverse opal structure membrane and bond the inverse opal structure membrane to the bottom of the transwell chamber structure after removing the bottom filter membrane. S2. Culture alveolar epithelial cells and vascular endothelial cells on the front and back sides of the inverted opal membrane, respectively. S3. Attach the top of the transwell chamber to the perforated plate cover with through holes, pass one end of the gas delivery tube through the through hole in the perforated plate cover into the transwell chamber, and connect the other end to a syringe and a microfluidic injection pump to obtain an alveolar biomimetic microfluidic device; The preparation method of the inverse opal structure film is as follows: first, a normal structure template is prepared, a polyurethane solution is added to the normal structure template, and a polyurethane film with a certain thickness is obtained by spin coating with a spin coater. Then, silica particles are etched to obtain the inverse opal structure polyurethane film.

2. The method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli according to claim 1, characterized in that, The method for preparing the positive structure template is as follows: Silica particles were mixed with anhydrous ethanol and ultrasonically vibrated to obtain a silica suspension. The silica suspension was then uniformly coated onto a glass slide and allowed to stand to allow the anhydrous ethanol to evaporate, leaving a uniformly distributed silica particle film as a positive structure template.

3. The method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli according to claim 2, characterized in that, The diameter of the silicon dioxide particles is 270 nm; In the silica suspension, the weight ratio of silica particles to anhydrous ethanol is 20:

100.

4. The method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli according to claim 1, characterized in that, The polyurethane solution is prepared by: Polyurethane was dissolved in N,N-dimethylformamide solvent and heated at 80°C for more than 2 hours to obtain a polyurethane solution. The mass concentration of the polyurethane is 20%.

5. The method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli according to claim 1, characterized in that, During the spin coating process, the spin coater speed was set to 300 rpm for 30 seconds, then 400 rpm for 30 seconds. The spin-coated glass slide containing a thin layer of polyurethane solution was then placed on a hot plate with a temperature of 80°C. After 2 hours, it was dried to obtain a polyurethane film with a thickness of 5 micrometers.

6. The method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli according to claim 1, characterized in that, The etching method for the silicon dioxide particles is as follows: A glass slide containing silica particles and a polyurethane film was immersed in 4% hydrofluoric acid overnight. The silica particle layer was corroded by the hydrofluoric acid to obtain an inverse opal structure polyurethane film.

7. The method for fabricating a biomimetic microfluidic device for simulating the three-dimensional structure of alveoli according to claim 1, characterized in that, The transwell chamber is side-closed, forming a sealed spatial structure with the inverse opal structure membrane and the perforated plate top cover; The bottom diameter of the transwell chamber is 7 mm; The gas transmission pipe is a Teflon tube with a pore size of 1 mm.

8. A biomimetic microfluidic device for simulating the three-dimensional structure of alveoli, characterized in that, Prepared by the preparation method according to any one of claims 1 to 7, This includes an inverted opal structure membrane, a transwell chamber with the bottom filter membrane removed, a perforated plate top cover, and a gas delivery pipe; The bottom of the transwell chamber with the bottom filter membrane removed is bonded and fixed to the inverse opal structure membrane, and the top is bonded and fixed to the top cover of the perforated plate. The orifice plate top cover has a through hole, one end of the gas delivery pipe extends into the transwell chamber through the through hole, and the gas delivery pipe is sealed to the orifice plate top cover; the other end of the gas delivery pipe is connected to the injection pump. Alveolar epithelial cells and vascular endothelial cells were cultured on the front and back sides of the inverted opal membrane, respectively.

9. The application of the alveolar biomimetic microfluidic device prepared by the preparation method according to any one of claims 1 to 7 in the in vitro simulation of the three-dimensional structure of alveoli.

Images