A cardiomyocyte-driven photonic crystal micropillar array and its preparation method and application

Abstract

The present invention discloses a cardiomyocyte-driven photonic crystal micropillar array and its preparation method and application. The method comprises preparing a micropillar array of micrometer-sized micropillars, replicating the micropillar array to obtain a micropit template; dripping a first hydrogel precursor solution dispersed with charged colloidal particles and a conductive component into the micropit template and solidifying it; then dripping a second hydrogel precursor solution free of charged colloidal particles into the micropit template and solidifying it, and stripping the template to obtain a photonic crystal micropillar array; and modifying the photonic crystal micropillar array with polydopamine to obtain a cardiomyocyte-driven photonic crystal micropillar array for cardiomyocyte culture and mechanical sensing. The photonic crystal micropillar array of the present invention has conductive properties and can simulate the extracellular matrix environment in the body to promote electrical conduction and synchronous contraction between cardiomyocytes, thereby realizing mechanical sensing of cardiomyocytes at the single-cell level.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and in particular to a myocardial cell-driven photonic crystal microcolumn array, a preparation method thereof, and applications thereof. Background Art

[0002] Cell mechanics plays a crucial role in biological processes at the cellular and tissue levels. Due to its importance in maintaining normal physiological function, dysregulation of cell mechanics is often considered to be associated with the development and progression of specific diseases. For example, reduced mechanical strength in muscle cells is associated with diseases such as heart failure and muscular dystrophy. Therefore, developing technologies that can effectively sense cell mechanics is crucial for uncovering potential links between cell mechanics and disease and for developing therapeutics.

[0003] Currently, many mechanical measurement technologies, including microcantilevers, atomic force microscopy, traction force microscopy, optical tweezers, and magnetic tweezers, have been widely used to measure cellular forces. However, most of these measurement methods require complex instrumentation and specialized software analysis and processing. The high cost and time-consuming professional operation limit the further promotion and use of these instruments. In addition, these technologies may cause irreversible damage to cells during the measurement process, making it impossible to achieve long-term continuous measurement. Therefore, a simple and biosafe cellular mechanical sensing technology remains to be developed. Summary of the Invention

[0004] In order to study the mechanical changes of myocardial cells under pathological conditions, a visual and non-invasive sensing strategy is provided to solve the problems of traditional mechanical sensing technology, such as high cost, long time consumption and irreversible damage to cells during the measurement process. The present invention provides a myocardial cell-driven photonic crystal microcolumn array and its preparation method and application.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for preparing a cardiomyocyte-driven photonic crystal microcolumn array comprises the following steps:

[0007] preparing a micropillar array of micrometer-sized micropillars, and replicating the micropillar array to obtain a micropit template;

[0008] A first hydrogel precursor solution containing charged colloidal particles and a conductive component is dripped into the micro-pit template and solidified; a second hydrogel precursor solution containing no charged colloidal particles is then dripped into the micro-pit template and solidified, and the template is peeled off to obtain a photonic crystal microcolumn array;

[0009] Photonic crystal microcolumn arrays were modified with polydopamine to obtain cardiomyocyte-driven photonic crystal microcolumn arrays for cardiomyocyte culture and mechanical sensing.

[0010] To optimize the above technical solutions, specific measures / limitations adopted also include:

[0011] The micro-pillar array is prepared by 3D printing technology; polydimethylsiloxane is used to replicate the micro-pillar array to obtain a micro-pit template.

[0012] The charged colloidal particles are charged sulfonated silicon dioxide nanoparticles; the first hydrogel precursor polymer solution contains 10%-25% w / v of the charged colloidal particles.

[0013] The conductive component is selected from at least one of carbon nanotubes, MXene, and graphene; the first hydrogel prepolymer solution and the second hydrogel prepolymer solution contain 0.05%-0.2% w / v of the conductive component.

[0014] Furthermore, after the micro-pit template is hydrophilically treated, it is filled with the first hydrogel precursor solution in a vacuum manner.

[0015] Furthermore, the hydrogel in the first hydrogel prepolymer solution and the second hydrogel prepolymer solution is selected from at least one of polyethylene glycol diacrylate and polyacrylamide.

[0016] Furthermore, a photoinitiator is added to the first hydrogel prepolymer solution and the second hydrogel prepolymer solution, and the solutions are cured by irradiation with ultraviolet light.

[0017] Preferably, the micropillars in the micropillar array have a diameter of 1-10 μm, a height of 10-50 μm, and a spacing between micropillars of 5-15 μm.

[0018] The present invention also protects a myocardial cell-driven photonic crystal microcolumn array prepared by the method, the structure of which includes an upper conductive structural color microcolumn sensing layer composed of a microcolumn array and a lower hydrogel support layer.

[0019] The present invention also protects the application of the photonic crystal microcolumn array in myocardial cell culture and mechanical sensing, which is used for the inoculation and culture of myocardial cells, in which the myocardial cells can restore their autonomous beating performance and drive the microcolumns in the conductive structural color microcolumn sensing layer to produce displacement and visual structural color changes.

[0020] Furthermore, the contractile force generated by the myocardial cells is monitored in real time based on the color changes produced by the conductive structure color micro-column sensing layer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The photonic crystal microcolumn array of the present invention has conductive properties and can simulate the extracellular matrix environment in the body to promote electrical conduction and synchronous contraction between myocardial cells.

[0023] 2) The present invention utilizes the deformation of the photonic crystal microcolumn array during the contraction of myocardial cells, which does not damage the activity of myocardial cells and can be used for long-term cell mechanical sensing.

[0024] 3) The sensing strategy adopted by the present invention is to observe the visual structural color changes produced by the photonic crystal microcolumn array driven by myocardial cells. The sensing strategy is simple and easy to promote.

[0025] 4) The scale of the photonic crystal micropillar array of the present invention is at the level of several microns. Thanks to this fine micro-nanostructure, the structural color micropillar can produce sensitive bending and structural color changes in response to weaker cellular forces (from the μN level to the nN level), thereby improving the sensitivity of mechanical sensing; the size of the micropillar matches the size of myocardial cells, and the micropillar array of the present invention can realize mechanical sensing of myocardial cells at the single-cell level.

[0026] 5) The micropillars in the present invention are independent of each other, and can simultaneously achieve high-throughput mechanical sensing of hundreds or even thousands of myocardial cells in different regions.

[0027] 6) The present invention utilizes the sensitive structural color changes of the photonic crystal micropillar array to reveal the changing trend of weak contractility of myocardial cells in the development process of diseases such as myocardial hypertrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the preparation process of the photonic crystal microcolumn array of the present invention.

[0029] Figure 2 This is a diagram showing the process of displacement and structural color changes caused by the contraction of cardiomyocytes driving the photonic crystal micropillars after cardiomyocytes are cultured on a photonic crystal micropillar array.

[0030] Figure 3 The structural color changes produced by the photonic crystal microcolumn array during the induction of myocardial hypertrophy: a is the structural color change process diagram at 0 hours of induction; b is the structural color change process diagram at 6 hours of induction; c is the structural color change process diagram at 48 hours of induction. DETAILED DESCRIPTION

[0031] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0033] Other descriptions of the present invention are as follows:

[0034] Before each solidification, the excess first or second hydrogel precursor solution must be removed.

[0035] Modifying a photonic crystal micropillar array with a polydopamine layer to promote cell adhesion: The photonic crystal micropillar array was immersed in an alkaline dopamine solution for 12 hours to form a biocompatible polydopamine layer on its surface. After thorough washing, the array was then used for cardiomyocyte inoculation and culture. After two days of culture, the cardiomyocytes regained their spontaneous beating ability, driving the displacement of the structural color micropillars and visualizing structural color changes.

[0036] The micropillars in the micropillar array of the present invention are micron-sized, with a diameter of 1-10 μm, a height of 10-50 μm, and an interval between the micropillars of 5-15 μm.

[0037] The cardiomyocytes cultured in the present invention can be primary extracted cardiomyocytes, stem cell-induced cardiomyocytes or cardiac organoids.

[0038] The present invention is further described in detail below with reference to specific embodiments:

[0039] Example 1 Preparation of a Cardiomyocyte-Driven Photonic Crystal Micropillar Array

[0040] (1) Preparation steps of photonic crystal micropillar array

[0041] A micropillar array template was fabricated using a 3D printer. PDMS and a curing agent were mixed in a 9:1 ratio and replicated to create a PDMS micropit template. An acrylamide solution (15%, w / v) containing highly charged sulfonated silica nanoparticles (15%, w / v), carbon nanotubes (0.05%, w / v), and 1% (v / v) photoinitiator (2-hydroxy-2-methylpropiophenone) was added. The hydrogel precursor solution was vacuum-treated for 5 minutes to fill the micropits. After removing excess solution, the solution in the micropits was irradiated with UV light for 30 seconds to cure the micropillars. Then, pure acrylamide solution (30%, w / v) was added and UV-cured for a second time for 1 minute. The PDMS template was removed, and the photonic crystal micropillar array was immersed in a dopamine solution (pH = 8.5) for 12 hours for modification. The resulting structure was then rinsed thoroughly with deionized water.

[0042] (2) Cardiomyocyte culture based on polyacrylamide photonic crystal micropillar arrays

[0043] The photonic crystal micropillar array prepared in the previous step was sterilized with UV light. Cardiomyocytes from newborn rats were then seeded on one side of the micropillars. After 48 hours of incubation at 37°C and 5% CO₂, spontaneous beating resumed. Under halogen light, the color changes of the photonic crystal micropillar array driven by the contraction of the cardiomyocytes were observed directly under a microscope.

[0044] Example 2 Preparation and Application of a Cardiomyocyte-Driven Photonic Crystal Micropillar Array

[0045] (1) Preparation steps of photonic crystal micropillar array

[0046] A micropillar array template was fabricated using a 3D printer. PDMS and a curing agent were mixed in a 9:1 ratio and replicated to create a PDMS micropit template. A 10% w / v solution of polyethylene glycol diacrylate containing highly charged sulfonated silica nanoparticles (12.5%, w / v), carbon nanotubes (0.05%, w / v), and 1% (v / v) photoinitiator (2-hydroxy-2-methylpropiophenone) was added. The hydrogel precursor solution was vacuum-treated for 5 minutes to fill the micropits. After removing excess solution, the solution in the micropits was irradiated with UV light for 20 seconds to cure the micropillars. A 20% w / v solution of polyethylene glycol diacrylate containing carbon nanotubes (0.05%, w / v) was then added and UV-cured for a second time for 1 minute. Finally, the PDMS template was removed, and the photonic crystal micropillar array was immersed in a dopamine solution (pH = 8.5) for 12 hours for modification and then rinsed thoroughly with deionized water.

[0047] (2) Cardiomyocyte culture based on polyethylene glycol diacrylate photonic crystal micropillar arrays

[0048] The photonic crystal micropillar array prepared in the previous step was sterilized by ultraviolet light. Cardiomyocytes from newborn rats were extracted and inoculated on one side of the micropillars. After 48 hours of culture at 37°C and 5% CO2, spontaneous beating was restored. Under the illumination of a halogen light source, the color change of the photonic crystal micropillar array caused by the contraction of the cardiomyocytes was observed directly under a microscope. Figure 2 shown.

[0049] (3) Research on myocardial hypertrophy based on cardiomyocyte-driven photonic crystal micropillar arrays

[0050] The myocardial hypertrophy model was induced by treating the cardiomyocyte-driven photonic crystal microcolumn array with angiotensin II for 48 hours. During this period, the contractile force of the myocardial cells was monitored using the structural color changes produced by the photonic crystal microcolumns to study the changes in the mechanical behavior of the myocardial cells: ordinary myocardial cells can basically maintain a stable contractile force level within 48 hours, and this contractile performance can last for more than a week, but will gradually weaken as the culture time increases; in the early stage of induction (6 hours) of myocardial hypertrophy disease, the contractile force of the myocardial cells will be enhanced, but as the induction time increases (48 hours), the contractile force of the myocardial cells will be significantly weakened, such as Figure 3 shown.

[0051] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any simple modification, equivalent replacement and improvement made by any technician familiar with the profession to the above embodiment without departing from the scope of the technical solution of the present invention and based on the technical essence of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a cardiomyocyte-driven photonic crystal micropillar array, characterized in that: The following steps are involved: preparing a micropillar array of micrometer-sized micropillars, and replicating the micropillar array to obtain a micropit template; A first hydrogel precursor solution containing charged colloidal particles and a conductive component is dripped into the micro-pit template and solidified; a second hydrogel precursor solution containing no charged colloidal particles is then dripped into the micro-pit template and solidified, and the template is peeled off to obtain a photonic crystal microcolumn array; Polydopamine was used to modify a photonic crystal micropillar array to obtain a cardiomyocyte-driven photonic crystal micropillar array for cardiomyocyte culture and mechanical sensing. The structure includes an upper conductive structural color micropillar sensing layer composed of a micropillar array and a lower hydrogel scaffold layer. The charged colloidal particles are charged sulfonated silica nanoparticles; the first hydrogel precursor solution contains 10%-25% w / v of charged colloidal particles; The conductive component is selected from at least one of carbon nanotubes, MXene, and graphene; the first hydrogel prepolymer solution and the second hydrogel prepolymer solution contain 0.05%-0.2% w / v of the conductive component.

2. The method for preparing a cardiomyocyte-driven photonic crystal micropillar array according to claim 1, characterized in that: The micro-pillar array is prepared by 3D printing technology; polydimethylsiloxane is used to replicate the micro-pillar array to obtain a micro-pit template.

3. The method for preparing a cardiomyocyte-driven photonic crystal micropillar array according to claim 1, characterized in that: After the micro-pit template is hydrophilic treated, it is filled with the first hydrogel precursor solution in a vacuum manner.

4. The method for preparing a cardiomyocyte-driven photonic crystal micropillar array according to claim 1, characterized in that: The hydrogel in the first hydrogel prepolymer solution and the second hydrogel prepolymer solution is selected from at least one of polyethylene glycol diacrylate and polyacrylamide.

5. The method for preparing a cardiomyocyte-driven photonic crystal micropillar array according to claim 1, characterized in that: A photoinitiator is added to the first hydrogel prepolymer solution and the second hydrogel prepolymer solution, and is cured by irradiation with an ultraviolet lamp.

6. A cardiomyocyte-driven photonic crystal micropillar array prepared by the method according to any one of claims 1 to 5, characterized in that: Its structure includes an upper conductive structural color micro-column sensing layer composed of a micro-column array and a lower hydrogel scaffold layer.

Images