Patterned cell culture substrate for cell behavior regulation and control as well as preparation and use methods of patterned cell culture substrate

Abstract

The invention discloses a patterned cell culture substrate for cell behavior regulation and a preparation and use method thereof. The preparation method comprises the following steps: preparing the patterned cell culture substrate on the inner surface of a conventional cell culture substrate through a patterning technology; the patterned cell culture substrate has a three-dimensional microgroove structure. The three-dimensional microgroove structure can provide various three-dimensional microenvironments for cell growth, so that the contact guide effect of cells is triggered, the cell behaviors are regulated and controlled, and a good cell behavior regulation and control effect is achieved. By adopting the laser patterning technology, the photoetching technology and the electron beam photoetching technology, the rapid manufacturing of the patterned cell culture base material and the manufacturing of the cell culture base material with high-precision and high-complexity micro-nano scale patterns can be realized. Meanwhile, the problem that the pattern stability is insufficient when the patterned cell culture substrate is prepared by adopting a chemical modification method is avoided.

Description

Technical Field

[0001] This invention relates to the field of cell culture media, and more particularly to a patterned cell culture medium for cell behavior regulation and its preparation and use methods. Background Technology

[0002] Cell patterning generally refers to the use of microfabrication technology to create microscale cell-related physicochemical parameters on a substrate, such as morphology, hardness, roughness, temperature, extracellular matrix proteins, and cell growth factors, to produce cell-addressable patterned substrates. This induces cells to respond through their own corresponding mechanisms, thereby controlling cell growth, proliferation, differentiation, migration, and related gene expression.

[0003] Surface chemical modification and surface topological morphology preparation are two main methods for obtaining patterned substrates. Surface chemical modification involves using chemical substances to create patterns on the substrate; these substances regulate cell adhesion through properties such as hydrophobicity, thereby achieving cell patterning. Surface topological morphology preparation generally refers to the fabrication of micrometer, nanometer, or micro-nano scale structures such as grooves, channels, pits, pillars, and islands on the surface of culture media. These topological morphologies primarily regulate cell behavior by triggering "contact guidance," adjusting the cell's adhesion arrangement.

[0004] Currently, common methods for preparing patterned culture media for cell behavior regulation include surface chemical modification, photolithography, and casting. However, these methods generally suffer from complex processes. Surface chemical modification, in particular, suffers from short-lived patterning time, difficulty in preservation, and potential adverse effects on cell viability. Furthermore, processing pre-defined patterns on transparent polymer-based culture media often results in low processing precision, leading to poor pattern quality and directly impacting the regulation of cell orientation, thus failing to meet the experimental requirements for high-precision cell behavior regulation. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a patterned cell culture medium for cell behavior regulation, as well as its preparation and use methods.

[0006] To achieve one of the above objectives, the present invention provides a method for preparing a patterned cell culture medium for cell behavior regulation, the method comprising: preparing a patterned cell culture medium on the inner surface of a conventional cell culture medium by patterning technology; the patterned cell culture medium having a three-dimensional microgroove structure; the patterning technology including one or more of laser patterning technology, photolithography technology, and electron beam lithography.

[0007] In this invention, a three-dimensional microgroove structure is fabricated on the inner surface of conventional cell culture media using patterning technology. The pattern type, precision, density, and size of the three-dimensional microgroove structure can be flexibly controlled. This structure provides diverse three-dimensional microenvironments for cell growth, triggering cell contact guidance and resulting in different adhesion arrangements, thereby regulating cell behavior and achieving good cell behavior regulation. The laser patterning, photolithography, and electron beam lithography technologies employed in this invention not only enable the rapid preparation of patterned cell culture media but also allow for the precise manufacturing of complex patterned cell culture media. Simultaneously, it avoids the problem of insufficient pattern stability encountered when using chemical modification methods to prepare patterned cell culture media.

[0008] Furthermore, the laser patterning technique method includes: The conventional cell culture medium is pretreated and then fixed on the processing table; the target pattern is drawn and the pattern information is imported into the laser processing system; the laser processing parameters are set, and the laser spot is focused on the inner surface of the conventional cell culture medium to ablate the target pattern on the inner surface of the conventional cell culture medium; a patterned cell culture medium is obtained.

[0009] In this invention, conventional cell culture media are first pretreated and then fixed on a laser processing stage. This pretreatment optimizes the substrate condition for better laser ablation results. A target pattern is drawn on drafting software and imported into the laser processing system. Next, matching laser parameters are set on the laser processing system, and the laser spot is focused on the inner surface of the conventional cell culture media, directly ablating the target pattern onto its inner surface. Finally, impurities generated during processing are cleaned to obtain a patterned cell culture media. This direct laser ablation method can produce microgroove structures with a depth of 40μm-130μm and a width of 120μm-270μm.

[0010] Preferably, the pretreatment of conventional cell culture medium includes: coating the surface of the conventional cell culture medium with a photosensitive coating.

[0011] Coating the surface of conventional cell culture media with a photosensitive coating can improve the substrate's absorption rate of laser wavelengths and precisely control the accuracy of the processed pattern, achieving simplified, efficient, and high-quality patterned fabrication. The specific steps are as follows: A photosensitive coating is uniformly coated onto the surface of the conventional cell culture media, covering the target patterned processing area. The coating thickness can be set according to the pattern requirements. The conventional cell culture media is fixed on a laser processing stage. The target pattern is drawn on drafting software and imported into the laser processing system. Next, matching laser parameters are set on the laser processing system, and the laser spot is focused onto the photosensitive coating on the inner surface of the conventional cell culture media, ablating the target pattern onto the inner surface. Finally, the laser-ablated cell culture media is placed in a 75% ethanol solution and ultrasonically cleaned for 15 minutes, repeated 2-3 times to thoroughly clean the photosensitive coating and impurities generated during processing, obtaining the patterned cell culture media. This photosensitive coating-assisted ablation processing method can obtain microgrooves with a depth of 0.6μm-40μm and a width of 45μm-85μm. Preferably, the photosensitive coating is black ink.

[0012] Preferably, the laser used in the laser patterning technology includes one or more of millisecond pulse lasers, microsecond pulse lasers, nanosecond pulse lasers, picosecond pulse lasers, and femtosecond pulse lasers.

[0013] The laser patterning technology described in this invention is compatible with lasers of different pulse widths to adapt to the differentiated requirements for pattern accuracy, density, and size in cell behavior regulation; preferably, the lasers used include, but are not limited to, infrared lasers, visible lasers, and ultraviolet lasers.

[0014] Furthermore, the material of the conventional cell culture medium includes one or more of the following: polystyrene, polytetrafluoroethylene, polypropylene, polycarbonate, polymethyl methacrylate, glass, siliconized glass, and biodegradable materials.

[0015] The preparation method of this invention involves fabricating a three-dimensional microgroove structure on the inner surface of a conventional cell culture medium, which includes cell culture dishes. The conventional cell culture medium is made of various materials, including but not limited to polystyrene, polytetrafluoroethylene, polypropylene, polycarbonate, polymethyl methacrylate, glass, siliconized glass, and biodegradable materials. In the preparation of patterned cell culture media, culture dishes of different materials can be selected according to the cell culture requirements.

[0016] Furthermore, the pattern type includes one or more of the following: grid pattern, beaded pattern, horizontal line pattern, cross pattern, dot matrix pattern, rhombus pattern, and micro-groove array pattern.

[0017] The patterned cell culture medium prepared by this invention has a three-dimensional microgroove structure. The pattern type, precision, density, and size of the three-dimensional microgroove structure can be flexibly adjusted. Different pattern types and densities of three-dimensional microgroove structures have different effects on cell behavior regulation. For example, when the pattern is set to a high-density microgroove array pattern, cell orientation can be regulated; when the pattern type is set to a horizontal line pattern, the secretion of Fibronectin protein in cells can be regulated.

[0018] To achieve the second objective mentioned above, a patterned cell culture medium for regulating cell behavior is provided, wherein the patterned cell culture medium is prepared according to the above-described method for preparing a patterned cell culture medium for regulating cell behavior.

[0019] The patterned cell culture medium prepared in this invention uses conventional cell culture medium as raw material. Through patterning technology, three-dimensional microgroove structures with various pattern types are processed on the inner surface of the conventional cell culture medium. The three-dimensional microgroove structures on the patterned cell culture medium affect cell arrangement, which in turn affects the secretion of extracellular matrix. The extracellular matrix then reacts in turn to cell growth, forming an interactive regulatory effect. That is, the extracellular matrix and the three-dimensional microgroove structures on the culture medium together constitute the three-dimensional microenvironment for cell growth, influencing cell behavior.

[0020] Furthermore, the regulation of cell behavior includes at least one of cell orientation regulation and extracellular matrix protein expression regulation.

[0021] The patterned culture medium prepared by this invention can form a three-dimensional microgroove structure, providing different three-dimensional microenvironments for cell growth, thereby triggering cell contact guidance and achieving good regulation of cell orientation and extracellular matrix-related protein expression, providing an experimental platform and technical support for the preparation of target proteins.

[0022] To achieve the third objective mentioned above, a method for using patterned cell culture media for cell behavior regulation is provided, comprising the following steps: S1: Sterilize and disinfect the patterned cell culture medium, and rinse it with phosphate buffer solution; S2: Cells are routinely cultured and passaged, and a cell suspension is prepared using a standard culture medium for later use; the cell suspension is seeded into a patterned area within a patterned cell culture medium and placed in a 37°C, 5% CO2 incubator to allow the cells to adhere and grow on the patterned culture medium; after the cells have completed adhesion, behavioral regulation cells are obtained. S3: After cell culture is completed, trypsin is added to the patterned cell culture medium for cell digestion. After washing, sterilization and drying, the patterned cell culture medium can be reused.

[0023] The patterned cell culture medium of the present invention can regulate cell behavior. The specific method of use is as follows: First, the patterned cell culture medium is sterilized. The sterilization methods include, but are not limited to, autoclaving, chemical disinfectants, dry heat sterilization, ultraviolet (UV) irradiation, gamma-ray irradiation, and ethylene oxide (EO) sterilization. After sterilization, the patterned cell culture medium is immersed in 75% ethanol for 15 minutes. The immersed patterned cell culture medium is then removed and placed under a UV lamp for 15 minutes for sterilization. The patterned cell culture medium is rinsed 2-3 times with phosphate-buffered saline (PBS) to prepare for cell culture. The cells are then cultured and passaged using conventional culture media, including DMEM low-glucose medium, to prepare a cell suspension for later use. The cell suspension is then seeded into the patterned areas within the patterned cell culture medium. Preferably, the number of cells seeded is 600,000 to 1,000,000. Patterned cell culture medium was placed in a 37°C, 5% CO2 incubator for 2-3 days to allow cells to adhere and grow on the medium. Once adhesion was complete, behavioral regulatory cells were obtained; these cells included fibroblasts, 3T3 cells, and other adherent cells. After cell culture, a suitable amount of trypsin (enough to completely cover the medium) was added to the patterned cell culture medium, and digestion was performed for at least 5 minutes. The medium was then rinsed 2-3 times with phosphate-buffered saline (PBS), and finally, ultrasonically cleaned with 75% ethanol solution for 15 minutes, repeated 2-3 times. After air drying, the medium was stored in a clean environment and could be reused. The patterned cell culture medium prepared in this invention can be reused after processing, saving on culture medium costs for the preparation of target proteins and three-dimensional structured cell sheets. Furthermore, step S2 also includes: after the cells have completed adhesion, the conventional culture medium is replaced with a membrane-promoting culture medium, and the cells are cultured in a 37°C, 5% CO2 incubator for 5 days, with the culture medium being replaced every two days, to obtain behavioral regulation cell membranes.

[0024] The patterned cell culture medium of the present invention can also be used to prepare cell sheets. The specific method is as follows: First, the patterned cell culture medium is sterilized. The sterilization methods include, but are not limited to, autoclaving, chemical disinfection, dry heat sterilization, ultraviolet (UV) irradiation, gamma-ray irradiation, and ethylene oxide (EO) sterilization. After sterilization, the patterned cell culture medium is immersed in 75% ethanol for 15 minutes. The immersed patterned cell culture medium is then removed and placed under a UV lamp for 15 minutes for sterilization. The patterned cell culture medium is rinsed 2-3 times with phosphate-buffered saline (PBS) to prepare for cell culture. The cells are then cultured and passaged using conventional culture media, including DMEM low-glucose medium, and prepared into a suspension for later use. The cell suspension is then seeded into the patterned area within the patterned cell culture medium. Preferably, the number of cells seeded is 600,000 to 1,000,000. Patterned cell culture media were cultured at 37°C in a 5% CO2 incubator for 2-3 days to allow cells to adhere and grow on the patterned media. After cell adhesion was complete, the conventional culture medium was replaced with a membrane-promoting medium, including DMEM high-glucose medium, and the cells were cultured at 37°C in a 5% CO2 incubator for 5-6 days, with the medium being changed every two days, to obtain behavior-regulated cell sheets. The patterned cell culture media prepared by this invention can produce three-dimensional structured cell sheets with adjustable protein content, which can promote the application of cell sheets in regenerative medicine, personalized wound repair treatment, and the development of functional organoid tissues by multilayer stacking of cell sheets.

[0025] The beneficial effects of this invention, a patterned cell culture medium for cell behavior regulation, and its preparation and application methods, are as follows: In this invention, a three-dimensional microgroove structure is fabricated on the inner surface of conventional cell culture media using patterning technology. The pattern type, precision, density, and size of the three-dimensional microgroove structure can be flexibly controlled, resulting in good cell behavior regulation. The laser patterning, photolithography, and electron beam lithography technologies employed in this invention not only enable the rapid preparation of patterned cell culture media but also allow for the precise manufacturing of complex patterned cell culture media. Simultaneously, it avoids the problem of insufficient pattern stability encountered when using chemical modification methods to prepare patterned cell culture media.

[0026] The patterned cell culture medium prepared in this invention uses conventional cell culture medium as raw material. Through patterning technology, three-dimensional microgroove structures with various pattern types are processed on the inner surface of the cell culture medium, providing different three-dimensional microenvironments for cells. This triggers cell contact guidance, enabling good regulation of cell orientation and extracellular matrix-related protein expression, and providing an experimental platform and technical support for the preparation of target proteins.

[0027] The patterned cell culture medium prepared using this invention can also be used to prepare three-dimensional structured cell sheets with adjustable protein content, which can promote the application of cell sheets in regenerative medicine and personalized wound repair treatment, as well as promote the development of functional organoid tissues by stacking multiple cell sheets. Attached Figure Description

[0028] Figure 1 The images show the effect of microgroove array patterns with spacing of 20μm, 50μm, 100μm, 200μm, 300μm, 500μm, and 1000μm. Figure 2 Pattern renderings with grid densities of 99%, 92%, 85%, 80%, and 73%; Figure 3 Examples of beaded patterns, horizontal lines, crosses, dot matrix patterns, and rhombus patterns; Figure 4 Histograms showing cell morphology under optical microscope, cell morphology under fluorescence microscope, and the distribution of cell angles. Figure 5 This is a graph showing the relationship between the microgroove spacing and the orientation and standard deviation of cells. Figure 6 The process by which patterned polystyrene culture dishes regulate cell behavior; Figure 7 The expression levels of extracellular matrix-related protein genes are regulated. Note: P < 0.05 is indicated by *; P < 0.01 is indicated by **; P < 0.001 is indicated by ***. Detailed Implementation

[0029] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0030] Example 1: Preparation of microgroove array patterned polystyrene culture dishes This embodiment provides a method for preparing a microgroove array patterned polystyrene culture dish, specifically including the following steps: Step 1: Evenly coat a BIOFIL® 35mm polystyrene petri dish with black ink (the coating area should be 8.5cm², the same as the culture area). 2 50 μl of black ink was dropped into a petri dish, and the petri dish was placed on a shaker to allow the ink to be evenly coated on the surface. Then it was placed on a horizontal test bench to air dry naturally. Based on volume conversion, the coating thickness was approximately 58.82 μm. Step 2: Use CAD software on the computer to draw a rectangle with a length of 15mm and a width of 25μm. Fill the rectangle with a single line using the center line of the rectangle's width, and array them with spacing of 45μm, 75μm, 125μm, 225μm, 325μm, 525μm, and 1025μm to form a microgroove array pattern. Step 3: Fix a 35mm polystyrene plate coated with black ink (BIOFIL®) onto a 1064nm picosecond pulsed laser machine. Focus the laser using the machine's probe. The laser is set with a pulse overlap rate of 99.90%, a single pulse energy of 3μJ, and a scanning overlap rate of 75% (pulse overlap rate of 2MHz, scanning speed of 100mm / s, and average power of 6W). A microgroove array pattern with spacings of 20μm, 50μm, 100μm, 200μm, 300μm, 500μm, and 1000μm is ablated onto the polystyrene culture dish. Each microgroove is 15mm long and 50μm wide. The height from the highest point to the lowest point in the cross-sectional view of the microgroove is 10-20μm. Step 4: Immerse the processed polystyrene culture dish in an ethanol solution and clean it with an ultrasonic cleaner for 15 minutes. Repeat 2-3 times to obtain a microgroove array patterned polystyrene culture dish. The microgroove array pattern effect of the prepared microgroove array patterned polystyrene culture dish is as follows. Figure 1 As shown.

[0031] Figure 1 middle, Figure 1 (a) to Figure 1 (f) are effect diagrams of microgroove array patterns with spacings of 20μm, 50μm, 100μm, 200μm, 300μm, and 1000μm, respectively. Figure 1 (h) shows the effect without a pattern. As can be seen from the comparison, the number of strip grooves in the array decreases as the spacing between the microgrooves gradually increases.

[0032] Example 2: Preparation of polystyrene petri dishes with grid patterns. This embodiment provides the preparation of a polystyrene petri dish with a grid pattern, specifically including the following steps: Step 1: Use CAD software on your computer to draw grid patterns of different densities; Step 2: Fix the BIOFIL® 60mm polystyrene culture dish on a 1064nm picosecond pulsed laser machine tool, and use the machine tool's probe to focus the laser; using processing parameters of 99.60% pulse overlap rate and 44μJ single pulse energy (with a pulse overlap rate of 500KHz, a scanning speed of 100mm / s, and an average power of 22W), ablate the polystyrene culture dish to create grid patterns of different densities drawn in CAD software; Step 3: Immerse the processed polystyrene culture dishes in an ethanol solution and clean them using an ultrasonic cleaner for 15 minutes. Repeat this process 2-3 times to obtain polystyrene culture dishes with a grid pattern. The grid pattern effect of the prepared grid-patterned polystyrene culture dishes is as follows: Figure 2 As shown.

[0033] Figure 2 middle, Figure 2 (a) to Figure 2 (e) Graph patterns with spacing of 99%, 92%, 85%, 80%, and 73% are shown. Figure 1 (f) shows the effect without a pattern. The comparison shows that as the grid density gradually decreases, the number of grids decreases.

[0034] Example 3: Preparation of polystyrene petri dishes with other patterns This embodiment provides the preparation of polystyrene petri dishes with other patterns, specifically including the following steps: Step 1: Use CAD software on your computer to draw 20mm×20mm array bead patterns, horizontal line patterns, cross patterns, dot matrix patterns, and diamond patterns; Step 2: Fix the BIOFIL® 60mm polystyrene culture dish on a 1064nm picosecond pulsed laser machine tool, and use the machine tool's probe to focus the laser; using processing parameters of 99.60% pulse overlap rate and 44μJ single pulse energy (with a pulse overlap rate of 500KHz, a scanning speed of 100mm / s, and an average power of 22W), ablate the polystyrene culture dish to create beaded patterns, horizontal lines, cross patterns, dot matrix patterns, and diamond patterns drawn in CAD software; Step 3: Immerse the processed polystyrene petri dishes in an ethanol solution and clean them using an ultrasonic cleaner for 15 minutes. Repeat this process 2-3 times to obtain polystyrene petri dishes with beaded patterns, horizontal lines, cross patterns, dot matrix patterns, and diamond patterns, respectively. The patterned polystyrene petri dishes are shown in the image below. Figure 3 As shown.

[0035] Figure 3 middle, Figure 3 (a) to Figure 3 (e) are renderings of the beaded pattern, horizontal line pattern, cross pattern, dot matrix pattern, and diamond pattern, respectively. Figure 3 (f) is a comparison image without a pattern.

[0036] Example 4: Method for regulating NHDF cell orientation using microgroove array patterns This embodiment provides a method for regulating the orientation of NHDF cells using microgroove array patterns, specifically including the following steps: S1: Place the patterned polystyrene culture dish with a microgroove array of 35mm series spacing prepared in Example 1 after cleaning on a clean bench, sterilize it by UV irradiation for 15 minutes, and then clean the patterned polystyrene culture dish with 2-3ml PBS buffer 2-3 times, and let it air dry for later use. S3: NHDF cells were routinely cultured, and then a cell suspension was prepared using ordinary low-glucose complete culture medium for later use; cells were then cultured in patterned polystyrene culture dishes and unpatterned polystyrene culture dishes at a rate of 10,000 cells / cm². 2 NHDF cells were seeded at a specific cell density and cultured in a constant temperature incubator at 37°C and 5% CO2 concentration for 3 days to obtain behavioral regulation cells.

[0037] Images 4 (a1, b1, c1, d1, e1, f1, g1, h1) showing cell orientation under optical and fluorescence microscopy are shown. The angles between the long axis of the cell nucleus and the long side of the pattern were obtained by ImageJ software analysis. The histogram of the angle distribution is shown below. Figure 4 As shown in (a2, b2, c2, d2, e2, f2, g2, h2), the pattern of different groove spacings affects cell orientation. The angle with the highest relative frequency is taken as the main orientation angle. Using the orientation degree calculation formula, P=2(cos2θ)-1 (P is the orientation degree, θ is the angle between the long axis of the cell nucleus and the long side of the groove), the NHDF cell orientation degree of polystyrene culture dishes with different groove spacings is calculated and plotted as follows. Figure 5 (a) shows the relationship between groove spacing and orientation; the standard deviation of NHDF cell orientation angle was obtained by analyzing the data using ImageJ software, and plotted as shown in Figure 1. Figure 5(b) shows the relationship between groove spacing and standard deviation. Comparing the effects of patterned and unpatterned culture dishes on cell orientation regulation, it demonstrates that this striped groove patterned polystyrene culture dish has a regulatory effect on the orientation of NHDF cells. When the spacing is 20-200 μm, the cell orientation degree of NHDF is very close to 1, indicating a significant cell orientation regulation ability. When the spacing is greater than 300 μm, the cell orientation regulation ability begins to decrease significantly. Patent JP2021-129501A discloses a continuous laser-based polystyrene culture dish for directional cell growth, with a standard deviation angle range of 30°-60° for cell orientation. In contrast, the striped groove patterned polystyrene culture dish prepared by this invention using picosecond laser coating with black ink has a standard deviation angle range of 4°-23° for cell orientation, indicating that the culture dish prepared by this invention has a better cell orientation regulation effect.

[0038] S4: After cell culture, add an appropriate amount of trypsin to the patterned cell culture medium (enough to completely cover it), and digest with trypsin for at least 5 minutes. Then rinse the patterned polystyrene culture dish 2-3 times with phosphate-buffered saline (PBS). Finally, use 75% ethanol solution and ultrasonic cleaning for 15 minutes, 2-3 times. After air drying, store in a clean environment. This patterned cell culture medium can be reused.

[0039] Example 5: Method for regulating extracellular matrix-related proteins using patterned polystyrene culture dishes This invention provides patterned polystyrene culture dishes for regulating proteins related to the extracellular matrix, such as... Figure 6 As shown, the specific steps include: S1: Patterned polystyrene culture dishes with various pattern types and a diameter of 60 mm were prepared according to the steps of Examples 2 and 3, and then cleaned. In this example, the patterned area is set in the middle area of ​​the inner surface of the patterned polystyrene culture dish, and the outer contour of the patterned area is square.

[0040] A silicone separator ring with a square hollow inner ring was prepared and immersed in 75% ethanol for 15 minutes. In this embodiment, the shape and size of the inner ring of the silicone separator ring are adapted to the outer contour shape and size of the patterned area of ​​the patterned polystyrene culture dish. The silicone separator ring can be prepared by referring to the method of Example 1 of patent CN202211280988.2.

[0041] Remove the soaked silicone spacer ring, rinse the patterned polystyrene culture dish 2-3 times with phosphate-buffered saline (PBS), place it under a UV lamp for 15 minutes to sterilize, and let it air dry for later use. S2: Place the cleaned patterned polystyrene culture dishes and unpatterned polystyrene culture dishes on a clean bench and sterilize them by UV irradiation for 15 minutes. Then clean the patterned polystyrene culture dishes with 2-3 ml PBS buffer, repeating the cleaning process 2-3 times. Allow them to air dry for later use. Attach the silicone spacer ring to the patterned polystyrene culture dish, ensuring that the inner ring of the silicone spacer ring aligns with the outer contour edge of the patterned area. S3: Culture NHDF cells using standard methods, and then prepare a cell suspension using ordinary low-glucose complete culture medium for later use; 600,000 cells were seeded into a silica gel separator ring. After the NHDF cells had adhered to the culture vessel, the low-glucose medium was replaced with a cell membrane-forming medium, and the cells were cultured for 5 days to obtain cell membrane sheets. In this embodiment, the cell membrane-forming medium was prepared according to Example 1 of patent CN202210710094.6.

[0042] Total RNA was extracted from cells using the Trizol method, reverse transcribed, and then the expression levels of related protein genes were detected using PCR. The regulatory effects of extracellular matrix-related protein gene expression levels were as follows: Figure 7 As shown. Figure 7 In (a), the expression level of the Col-1 gene was significantly increased compared with the blank control group when the grid density was 99% and 92%. Figure 7 In (b), the expression level of the Col-6 gene was slightly higher than that of the blank control group when the grid density was 99% and 85%. Figure 7 In (c), the expression level of the Fibronectin gene was slightly increased compared with the blank control group when the grid density was 92% and 85%. Figure 7 In (d), when the pattern type was beaded, horizontal line, or cross pattern, the expression level of the Col-1 gene was lower than that of the blank control group. Figure 7 In (e), when the pattern type is a horizontal line pattern, the expression level of the Col-6 gene is higher than that of the blank control group. Figure 7 In (f), when the pattern type is a horizontal line pattern, the expression level of the Fibronectin gene is higher than that of the blank control group.

[0043] Therefore, it can be concluded that the genes for type I alpha1 collagen, type VI alpha1 collagen, and Fibronectin protein in NHDF cell sheets can be regulated by different types of patterned culture dishes.

[0044] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. A method for preparing patterned cell culture materials for cell behavior regulation, characterized in that, The method includes: preparing a patterned cell culture medium on the inner surface of a conventional cell culture medium using a patterning technique; the patterned cell culture medium has a three-dimensional microgroove structure; the patterning technique includes one or more of laser patterning, photolithography, and electron beam lithography.

2. The method for preparing a patterned cell culture medium for cell behavior regulation according to claim 1, characterized in that, The laser patterning technique includes: The conventional cell culture medium is pretreated and then fixed on the processing table; the target pattern is drawn and the pattern information is imported into the laser processing system; the laser processing parameters are set, and the laser spot is focused on the inner surface of the conventional cell culture medium to ablate the target pattern on the inner surface of the conventional cell culture medium; a patterned cell culture medium is obtained.

3. The method for preparing a patterned cell culture medium for cell behavior regulation according to claim 2, characterized in that, The pretreatment of conventional cell culture media includes coating the surface of the conventional cell culture media with a photosensitive coating.

4. The method for preparing a patterned cell culture medium for cell behavior regulation according to claim 1, characterized in that, The lasers used in the laser patterning technology include one or more of the following: millisecond pulse lasers, microsecond pulse lasers, nanosecond pulse lasers, picosecond pulse lasers, and femtosecond pulse lasers.

5. The method for preparing a patterned cell culture medium for cell behavior regulation according to claim 1, characterized in that, The materials used in conventional cell culture media include one or more of the following: polystyrene, polytetrafluoroethylene, polypropylene, polycarbonate, polymethyl methacrylate, glass, siliconized glass, and biodegradable materials.

6. The method for preparing a patterned cell culture medium for cell behavior regulation according to claim 1, characterized in that, The types of patterns include one or more of the following: grid pattern, beaded pattern, horizontal line pattern, cross pattern, dot matrix pattern, rhombus pattern, and micro-groove array pattern.

7. A patterned cell culture medium for regulating cell behavior, characterized in that, The patterned cell culture medium for cell behavior regulation is prepared according to any one of claims 1-6.

8. A patterned cell culture medium for regulating cell behavior according to claim 7, characterized in that, The regulation of cell behavior includes at least one of cell orientation regulation and extracellular matrix protein secretion regulation.

9. A method of using the patterned cell culture medium for cell behavior regulation as described in claim 7 or 8, characterized in that, Includes the following steps: S1: Sterilize and disinfect the patterned cell culture medium, and rinse it with phosphate buffer solution; S2: Cells are routinely cultured and passaged, and a cell suspension is prepared using a standard culture medium for later use; the cell suspension is seeded into a patterned area within a patterned cell culture medium and placed in a 37°C, 5% CO2 incubator to allow the cells to adhere and grow on the patterned culture medium; after the cells have completed adhesion, behavioral regulation cells are obtained. S3: After cell culture is completed, trypsin is added to the patterned cell culture medium for cell digestion. After washing, sterilization and drying, the patterned cell culture medium can be reused.

10. The method of using the patterned cell culture medium for cell behavior regulation according to claim 9, characterized in that, Step S2 also includes: after the cells have completed adhesion, the regular culture medium is replaced with a membrane-promoting culture medium and cultured in a 37°C, 5% CO2 incubator for 5 days, with the culture medium being replaced every two days to obtain behavioral regulation cell membranes.

Images