A method for producing curved protein micropatterns

Abstract

This invention relates to a method for fabricating curved protein micropatterns, a field of materials and tissue engineering, involving the complex patterning of curved surfaces and the inner surfaces of tubular lumens, and the subsequent controlled cell arrangement. The method comprises: fabricating and surface treating a PDMS curved surface or tubular lumen substrate; tightly fitting a Parylene C porous membrane to the tubular lumen; incubating FN protein on the curved surface to create a complex protein pattern through infiltration; and seeding a cell sample at a predetermined density to achieve controlled cell arrangement and growth. This experiment can closely simulate the living environment of cells on complex curved surfaces and organs in the body, is simple to perform, and is inexpensive, providing new insights for studying cells on the surfaces of specialized tissues and organs.

Description

Technical Field

[0001] The present invention belongs to the field of materials and tissue engineering, and involves the production of microprotein patterns on curved surfaces and the capture and arrangement of cells. It can simulate the living environment of cells in typical human tissues with complex curved surfaces, tubular and spiral structures, and can be used in tissue regeneration medicine. Background Art

[0002] The natural functionality of the human body is demonstrated through the adaptation of tissue and organ structures to their functions. The continuous growth and regeneration of these structures is a process of continuous cell proliferation and interaction. The cellular microenvironment is the environment in which cells survive, maintaining their normal proliferation, differentiation, and metabolic functions. This microenvironment primarily consists of neighboring cells, the extracellular matrix, and the extracellular matrix of other cells. Numerous adhesion factors facilitate interaction between cells and the extracellular matrix, so regulating cell behavior can be achieved by modulating cell adhesion.

[0003] Microcontact printing is a common method for preparing surface micropatterns for cell attachment. It was initially established and applied in microelectronics (Singhvi et al., 1994). With the recent development of microelectronics in the biomedical field, microcontact printing has been widely used in cell biology, providing cells with a suitable substrate for adhesion and growth.

[0004] Microcontact printing technology primarily utilizes the incubation of active proteins on a patterned PDMS surface, allowing the proteins to settle. The protein pattern is then transferred to the substrate via contact printing to create hydrophilic and hydrophobic regions for selective cell adhesion. This technique can be used to control cell shape and activity (Chen et al., 1997) and dictate cell extension and cytoskeletal organization (Wang et al., 2014). In stem cell research, the shape of the micropattern influences the direction of cell differentiation (McBeath et al., 2004). Furthermore, patterning can help cells generate functional tissues (Rachelle et al., 2014).

[0005] The advantages of microcontact printing are clear: simple operation, easy observation of results, no need for a rigorous laboratory environment, low-cost substrates and materials, and the ability to achieve precise surface patterning, making it highly convenient and feasible for studying cell behavior and cell-cell interactions. However, a disadvantage is that the flatness of solid stamps makes it difficult to pattern proteins on non-planar structures.

[0006] Currently, most cell research is based on flat or sharply angled surfaces, whereas complex curved surfaces and tubular structures account for a significant proportion of tissues and organs in the body. Many current studies are striving to closely mimic these structures, recreating the real-world environments in which cells live. For example, micropatterning on curved surfaces using fiber-assisted molding (Vahid Hosseini et al., 2014) or plasma etching (Mathur et al., 2012) has successfully guided cell alignment. However, these methods are unable to create fine and complex micropatterns, making it difficult to control the cytoskeletal organization of specific cells. Furthermore, the micropatterns formed on curved surfaces by these techniques are not flat but rather three-dimensional with topological structures, making it impossible to culture cells on flat surfaces. 3D printing (Polzin et al., 2013) can create complex patterns, but it struggles to achieve accuracy of tens of micrometers at the cellular scale, making it difficult to create micropatterns within the lumen of tubes several millimeters in diameter.

[0007] Research references related to the present invention:

[0008] Singhvi R,Kumar A,Lopez GP,et al.Engineering cell shape and function[J].Science,1994,264(5159):696-8.

[0009] Chen CS,Mrksich M,Huang S,et al.Geometric control of cell life and death[J].Science,1997,276(5317):1425-8.

[0010] Wang D, Zheng W, Xie Y, et al. Tissue-specific mechanical and geometrical control of cell viability and actin cytoskeleton alignment[J]. Scientific Reports, 2014, 4(6160).

[0011] McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and Rho A regulate stem cell lineage commitment. Dev Cell, 2004, 6: 483–495.

[0012] Rachelle N. Palchesko, Kira L. Lathrop, James L. Funderburgh. In Vitro Expansion of Corneal Endothelial Cells on Biomimetic Substrates. Scientific Repores, 2014;5, 7955 - 7964.

[0013] Vahid Hosseini, Philip Kollmannsberger, Samad Ahadian. Fiber - Assisted Molding (FAM) of Surfaces with Tunable Curvature to Guide Cell Alignment and Complex Tissue Architecture. Small 2014, 10, 4851–4857.

[0014] A. Mathur, S. W. Moore, M. P. Sheetz, J. Hone. Role of feature curvature in contact guidance. Acta Biomater. 2012, 8, 2597.

[0015] C. Polzin, S. Spath, H. Seitz. Characterization and evaluation of a PMMA - based 3D printing process. Rapid Prototyping Journal 2013, 19, 37. Summary of the Invention

[0016] The technology of this invention solves the problem of limited research on cell behavior on curved surfaces, complex operations, and insufficient precision. In the field of materials and tissue engineering, it provides high-precision cell capture and arrangement in large arrays of multiple curved surfaces. It also provides a method for producing curved protein micropatterns, achieving the effect of producing micropatterns on curved surfaces and curved surfaces within lumens. By seeding cells, it simulates the growth and adhesion of cells on curved structures in vivo to study cell behavior. This technology provides high-precision cell capture and arrangement in large arrays of multiple curved surfaces in the field of materials and tissue engineering for the study of cell behavior.

[0017] The present invention provides a method for producing curved protein micropatterns, comprising:

[0018] pouring a polymer material medium into a removable mold;

[0019] Inserting smooth rods of different diameters into the polymer material in the mold, and peeling the smooth rods from the polymer material and the mold to obtain empty tubes of polymer material with different pore diameters;

[0020] Fabricate porous membranes containing micropores of varying shapes, sizes, and defined pattern distributions;

[0021] The porous membrane is in close contact with the inner curved surface of the tube cavity of the polymer material hollow tube;

[0022] adding a reagent containing an adhesive protein onto the porous membrane, causing the adhesive protein to penetrate the micropores of the porous membrane onto the inner curved surface of the tubular cavity below the porous membrane, peeling off the porous membrane from the inner curved surface of the tubular cavity, and distributing the adhesive protein on the inner curved surface of the tubular cavity according to the set micropattern of the micropores on the porous membrane;

[0023] The micropore pattern on the porous membrane is consistent with the micropattern on the inner curved surface of the lumen, and the micropattern is a specific pattern set according to different cell adhesion requirements;

[0024] Before inoculating cells, the cells were treated with 1 mg / ml of Pluronic 127 for 5 minutes to enhance the hydrophobicity of the inner surface of the protein-free lumen.

[0025] Cells are seeded on the inner curved surface of the tube cavity, and the cells adhere and grow on the inner curved surface of the tube cavity according to the set micro-pattern of the adhesion protein.

[0026] The polymer material is PDMS polydimethylsiloxane (PDMS for short).

[0027] The smooth rods refer to metal rods or glass rods of different diameters that have been polished and have a smooth and flawless surface.

[0028] The diameter of the smooth rod is 1 mm to 6 mm, which is consistent with the diameter range of small-caliber blood vessels.

[0029] The method further includes vacuuming the polymer material to remove air bubbles, heating the polymer material at 65° C. for 4 hours, and placing the polymer material in acetone after solidification to allow the polymer material to swell, thereby causing the smooth rod to peel off naturally.

[0030] The porous membrane material is parylene (PAC for short).

[0031] The porous membrane is made from a pre-designed micro pattern through photolithography technology.

[0032] The method also includes heating and softening the porous membrane and then immersing it in alcohol until the holes on the porous membrane are filled with alcohol, and at the same time performing a hydrophilic treatment on the inner curved surface of the tube cavity, and then placing the porous membrane on the inner curved surface of the tube cavity, and drying it so that the porous membrane is tightly attached to the inner curved surface of the tube cavity.

[0033] The adhesion proteins are extracellular matrix proteins, including fibronectin, laminin, collagen, and the like.

[0034] The micropore pattern on the porous membrane matches the micropore pattern on the inner curved surface of the tube lumen. The micropore pattern is customized to meet the needs of different cell adhesion requirements: endothelial cells have a hexagonal pattern with a side length of 17.5 μm, while cardiac, skeletal, and smooth muscle cells have a rectangular pattern with a width of 20 μm and a length of 40 μm. The size, shape, and position of the micropores are customized to meet the specific cell culture requirements.

[0035] Before inoculating cells, the cells were treated with 1 mg / ml of Pluronic 127 for 5 minutes to enhance the hydrophobicity of the inner surface of the protein-free lumen, facilitating cell adhesion to the adhesion protein micropattern.

[0036] The cells in the method can be any adherent cells including mammalian cells and protozoan cells.

[0037] The advantages of the present invention compared with the prior art are:

[0038] (1) The present invention can realize the preparation of arrayed protein micropatterns on PDMS curved surfaces with good chemical stability and biocompatibility, so that cells can be arranged, adhered and grown on the curved surfaces as expected, which can make up for the gap in the current research methods for curved surface structure cells, thereby providing possibilities for in vitro curved surface tissue cell research. The current curved surface micropatterning methods generally use topological surfaces as templates to prepare curved surface topological micropatterns. The prepared micropatterns are not on the same plane and cannot meet the needs of subsequent cell biology research. The micropatterns prepared on the curved surface of the present invention are on the same plane, providing a good platform for subsequent cell adhesion, spreading and connection research.

[0039] (2) The adhesive protein micropattern of the present invention conforms to the shape, size, and position of the micropores on the porous membrane. By designing the shape, size, and position of the micropores on the porous membrane, an adhesive protein micropattern can be created on a curved surface. The size of the micropores can be arbitrarily varied from 2 μm to several tens of microns. Because the porous membrane can conform well to the curved surface, the diameter of the curved surface can be varied within a range of 1-6 mm. Therefore, the method of the present invention has the advantage of being able to adapt different curved surfaces and different micropatterns according to experimental needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is an empty PDMS tube;

[0041] Figure 2 This is a schematic diagram of a porous membrane. Its internal pattern can be designed based on the shape and arrangement of cells in different tissues, with an accuracy of at least 2μm and an error of less than 1μm.

[0042] Figure 3 Schematic diagram of the lumen after protein micropatterning, where 1 is a schematic diagram of the lumen cross section, 2 is a schematic diagram of the protein micropattern, and 3 is a schematic diagram of cell adhesion;

[0043] Figure 4 Schematic diagram of the method for fabricating curved protein micropatterns, including 4-molds with different diameters, 5-diagram of the PDMS lumen, 6-porous membrane, 7-adhesion protein solution, 8-protein micropattern, and 9-cell solution;

[0044] Figure 5 Fluorescence image of a 1mm diameter curved protein micropattern (left), and endothelial cells grown on the protein micropattern (right);

[0045] Figure 6 Fluorescence image of a 6mm diameter curved protein micropattern (left), and endothelial cells growing on the protein micropattern (right)

[0046] Figure 7 Cell adhesion and cytoskeleton analysis images of 5 mm diameter curved micropatterns distributed at angles of 0 (left), 45 (middle), and 90° (right) to the surface axis. DETAILED DESCRIPTION

[0047] The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0048] Example 1: Culturing HUVECs on 1mm and 6mm diameter curved surfaces

[0049] (1) Preparation of PDMS hollow tubes: Sylgard 184 prepolymer and cross-linker were mixed at a mass ratio of 10:1 and poured into a mold. Polished glass rods with diameters of 1 mm and 6 mm were inserted into the center of the mold. After vacuuming to remove bubbles, the mold was heated at 65 °C for 4 hours. After solidification, the mold was placed in acetone to allow PDMS to swell and the glass rod to peel off naturally, thereby obtaining tubes with different inner diameters to simulate curved surface structures with different diameters in the body (such as Figure 1 , Figure 3 1 in Figure 4 5). The diameter of the polished glass rod can vary from 1 to 6 mm. Figure 4 4, this embodiment uses 1mm and 6mm.

[0050] (2) Design patterns according to the arrangement and shape of different tissue cells, and use photolithography technology to produce porous membranes of different shapes and sizes. In this embodiment, the micropores are 20 μm wide and 40 μm long. The micropores are arranged head to tail at 5 μm intervals in the vertical direction, with an overall length of 3 mm. The micropores are arranged horizontally at 50 μm intervals, with an overall length of 3 mm (such as Figure 2 Heat it to 90℃ to soften it and then immerse it in alcohol. When the hole is filled with alcohol, use the guiding effect of the liquid to place the membrane in the cavity and blow it dry to make it completely tight. Figure 4 6). Prepare 50 μg / mL adhesion protein fibronectin solution and drop 200 μL on the membrane ( Figure 4 7), after the protein permeates for 20 minutes, the porous membrane is peeled off to form a protein micropattern ( Figure 3 2 of them, Figure 4 8). The cells were washed three times with PBS, treated with 1 mg / ml Pluronic 127 for 5 minutes to make the protein-free area more hydrophobic, and then washed three times with PBS. The adhesion proteins are extracellular matrix proteins, including fibronectin, laminin, collagen, etc. Fibronectin was used in the present embodiment.

[0051] (3) The concentration is 2x10 5 pieces / cm 2 Endothelial cells were mixed and seeded on the above curved surface ( Figure 4 9), after culturing at 37° for 2 hours, cells can be observed to grow in the designed pattern ( Figure 3 3) Long-term culture can be used to observe the skeleton arrangement and direction to study the growth and arrangement of cells in the body. Figure 5 The left side is the immunofluorescence staining of fibronectin in an empty tube with a diameter of 1 mm, and the right side is the cell adhesion diagram. Figure 6 The left side shows immunofluorescence staining of fibronectin in an empty tube with a diameter of 6 mm, and the right side shows cell adhesion, demonstrating that the present invention is applicable to micropatterning of curved surfaces with a diameter of 1-6 mm and can also allow cell adhesion.

[0052] Example 2: Culturing HUVECs at different angles on a 5 mm diameter curved surface

[0053] (1) Preparation of PDMS hollow tubes: Sylgard 184 prepolymer and cross-linker were mixed at a mass ratio of 10:1 and poured into a mold. A 5 mm diameter polished glass rod was inserted into the center of the mold. After vacuuming to remove bubbles, the mold was heated at 65 °C for 4 hours. After solidification, the mold was placed in acetone to allow PDMS to swell and the glass rod to peel off naturally, thereby obtaining tubes with different inner diameters to simulate curved surface structures with different diameters in the body (such as Figure 1 , Figure 3 1 in Figure 4 5). The diameter of the polished glass rod can vary from 1 to 6 mm. Figure 4 4 in the figure, this embodiment uses 5mm.

[0054] (2) Design patterns according to the arrangement and shape of different tissue cells, and use photolithography technology to produce porous membranes of different shapes and sizes. In this embodiment, the micropores are 20 μm wide and 40 μm long. The micropores are arranged head to tail at 5 μm intervals in the vertical direction, with an overall length of 3 mm. The micropores are arranged horizontally at 50 μm intervals, with an overall length of 3 mm (such as Figure 2 The angle between the micropore arrangement angle and the axis of the empty tube is 0° ( Figure 7 Left image in the figure), 45°( Figure 7 in the middle figure) and 90°( Figure 7 Heat it to 90℃ to soften it and then immerse it in alcohol. Once the pores are filled with alcohol, use the liquid's guiding effect to place the membrane in the cavity and blow dry it to make it completely adhered. Figure 4 6). Prepare 50 μg / mL adhesion protein fibronectin solution and drop 200 μL on the membrane ( Figure 4 7), after the protein permeates for 20 minutes, the porous membrane is peeled off to form a protein micropattern ( Figure 3 2 of them, Figure 4 8). The cells were washed three times with PBS, treated with 1 mg / ml Pluronic 127 for 5 minutes to make the protein-free area more hydrophobic, and then washed three times with PBS. The adhesion proteins are extracellular matrix proteins, including fibronectin, laminin, collagen, etc. Fibronectin was used in this example.

[0055] (3) The concentration is 2x10 5 pieces / cm 2 Endothelial cells were mixed and seeded on the above curved surface ( Figure 4 9), culture at 37o for 12 hours to observe the skeleton arrangement. Figure 7The results show that the present invention can prepare micropatterns with different angles to the axis of the curved surface on a curved surface with a diameter of 5 mm, and allows cell adhesion. Cytoskeleton analysis shows that the arrangement direction of the cells is consistent with the micropatterning method.

[0056] According to the technical solution provided by the present invention, curved surface micropatterning technology involves creating a biocompatible curved surface, applying a hydrophilic treatment, and then covering it with a porous membrane and drying it to ensure a tight fit. Through the permeation action of the porous membrane, a finely structured, well-defined micropattern is formed on the curved surface, enabling cell adhesion and subsequent growth. By designing different micropatterns, the cytoskeletal spread and arrangement of cells can be controlled.

[0057] In summary, the diameter of the curved surface in the present invention ranges from 1 mm to 6 mm, the design accuracy of the pattern can reach up to 2 μm, and the error does not exceed 1 μm, so that cells can be accurately positioned on the patterned curved surface.

[0058] The above embodiments are provided for the purpose of describing the present invention only and are not intended to limit the scope of the present invention. The scope of the present invention is defined by the appended claims. Various equivalent substitutions and modifications made without departing from the spirit and principles of the present invention are intended to be within the scope of the present invention.

Claims

1. A method for culturing cells on a curved surface, characterized in that include: pouring a polymer material medium into a removable mold; Inserting smooth rods of different diameters into the polymer material in the mold, and peeling the smooth rods from the polymer material and the mold to obtain empty tubes of polymer material with different pore diameters; Fabricate porous membranes containing micropores of varying shapes, sizes, and defined pattern distributions; The prepared porous membrane is closely attached to the inner curved surface of the polymer material hollow tube; that is, the porous membrane is heated and softened, and then immersed in alcohol until the pores on the porous membrane are filled with alcohol, and the inner curved surface of the tube cavity is hydrophilized, and then the porous membrane is placed on the inner curved surface of the tube cavity, and dried to make the porous membrane closely attached to the inner curved surface of the tube cavity; adding a reagent containing an adhesive protein onto the porous membrane, causing the adhesive protein to penetrate the micropores of the porous membrane onto the inner curved surface of the tubular cavity below the porous membrane, peeling off the porous membrane from the inner curved surface of the tubular cavity, and distributing the adhesive protein on the inner curved surface of the tubular cavity according to the set micropattern of the micropores on the porous membrane; The porous membrane is fabricated using photolithography using a pre-designed micropattern. The micropore pattern on the porous membrane matches the shape, size, and position of the micropattern on the curved surface within the lumen. The micropattern is a specific pattern set according to different cell adhesion requirements. The scale of the micropores can be freely varied between 2 μm and several tens of microns, with an error of less than 1 μm. The specific pattern includes a hexagonal micropattern with a side length of 17.5 μm for endothelial cells and a rectangular micropattern with a width of 20 μm and a length of 40 μm for cardiac muscle, skeletal muscle, and smooth muscle cells. Because the porous membrane adheres well to the curved surface, the diameter of the curved surface varies within a range of 1-6 mm. Before inoculating the cells, the membrane is treated with a surfactant to enhance the hydrophobicity of the protein-free lumen surface. Inoculating cells on the inner curved surface of the tube cavity, the cells will adhere and grow on the inner curved surface of the tube cavity according to the set micropattern of the adhesion protein; The polymer material is PDMS; The porous membrane material is polyparaxylene.

2. The method according to claim 1, wherein: The smooth rods refer to metal rods or glass rods of different diameters that have been polished and have a smooth and flawless surface.

3. The method according to claim 1, wherein: The diameter of the smooth rod is 1 mm to 6 mm.

4. The method according to claim 1, wherein: The method further includes vacuuming the polymer material to remove air bubbles, heating the polymer material at 65° C. for 4 hours, and placing the polymer material in acetone after solidification to allow the polymer material to swell, thereby causing the smooth rod to peel naturally.

5. The method according to claim 1, wherein: The adhesion protein is an extracellular matrix protein, including fibronectin, laminin or collagen.

6. The method according to claim 1, wherein: Before inoculating cells, the surface active agent Pluronic 127 (1 mg / ml) was used to treat the microstructure for 5 minutes to enhance the hydrophobicity of the inner surface of the protein-free lumen and facilitate cell adhesion to the adhesion protein micropattern.

7. The method according to claim 1, wherein: The cells include mammalian cells or protozoan cells.

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