Method for producing membrane, membrane, water collecting membrane, cell scaffold material, and method for producing cell scaffold material

Abstract

To provide a membrane having a novel pattern.SOLUTION: A method for producing a membrane comprises: a first step S1 of fixing a first position and a second position of a base while pulling the base in a first direction; a second step S2 of forming a membrane on a surface of a region including at least a portion between the first position and the second position of the base; and a third step S3 of contracting the base in the first direction connecting the first position and the second position. In the second step S2, a first uneven pattern is formed in the membrane by thermal stress in the first direction in the base; and in the third step S3, the base is returned from a stretched state to its original state, whereby the base contracts in the first direction, forming a second uneven pattern on the membrane.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present disclosure relates to a method for producing a membrane, a membrane, a water collecting membrane, a cell scaffolding material, and a method for producing a cell scaffolding material. [Background technology]

[0002] When an in-plane compressive stress acts on a hard film on a soft substrate, bifurcation buckling occurs, and various patterns are formed on the surface of the hard film. In recent years, the application of hard films with patterns on their surfaces to various fields has been studied (for example, see Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] S. Nagashima, SM Yoon, DH Kim, A. Nakatani, MWMoon: Wrinkle-assisted capillary bridging for the directed assembly of single-level DNA nanowire arrays, Advanced Materials Interfaces, 9 (2022), 2102243 Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have developed a technique for producing novel patterned films.

[0005] The present disclosure has been made in view of these problems, and an object of the disclosure is to provide a novel patterned film. [Means for solving the problem]

[0006] In order to solve the above problems, a method for producing a film according to an embodiment of the present disclosure includes a first step of fixing a first position and a second position of the substrate while the substrate is stretched in a first direction, a second step of forming a film on a surface of an area including at least a part between the first position and the second position of the substrate, and a third step of returning the substrate from the stretched state to its original state, thereby causing the substrate to shrink in a first direction connecting the first position and the second position. In the second step, a first uneven pattern is formed in the film by thermal stress in the substrate in the first direction, and in the third step, a second uneven pattern is formed in the film by shrinkage of the substrate in the first direction.

[0007] Another aspect of the present disclosure is a film having a hierarchical concave-convex pattern, which has a first concave-convex pattern repeatedly arranged in one direction and a second concave-convex pattern repeatedly arranged in the same direction as the first concave-convex pattern, and in each of the multiple second concave-convex patterns, a first concave-convex pattern smaller than the second concave-convex pattern is formed.

[0008] Yet another aspect of the present disclosure is a water collecting membrane for collecting moisture in the air, the water collecting membrane comprising the membrane described above.

[0009] Yet another aspect of the present disclosure is a cell scaffold material, which is provided with the above-mentioned membrane and allows cells to adhere and grow on the cell scaffold material.

[0010] Yet another aspect of the present disclosure is a method for producing a cell scaffold material, comprising the step of transferring a hierarchical relief pattern to a raw material using the above-mentioned film as a template. Effect of the Invention

[0011] According to the present disclosure, novel patterned films can be provided. [Brief description of the drawings]

[0012] [Figure 1] 1 is an electron microscope photograph showing the structure of the membrane of the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram illustrating a method for producing a membrane according to the present disclosure. [Diagram 3] FIG. 2 is a schematic diagram showing a method for producing a cell scaffold material according to the present disclosure. [Figure 4] FIG. 1 shows the cell morphology evaluation of human mesenchymal stem cells after culture on a cell scaffold material of an example. [Diagram 5] FIG. 1 is a diagram showing the definitions of area, aspect ratio, and angle. [Figure 6] FIG. 1 shows mRNA expression normalized to Gapdh in human mesenchymal stem cells passaged for 3 days on a flat substrate and on an example cell scaffold material. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 1 is an electron microscope photograph showing the structure of the film of the present disclosure. The film 10 of the present disclosure has wrinkles 11, which are a first uneven pattern repeatedly arranged in one direction, and ridges 12, which are a second uneven pattern repeatedly arranged in the same direction as the first uneven pattern.

[0014] The wrinkles 11 and ridges 12 are a type of pattern formed by bifurcation buckling that occurs when an in-plane compressive stress acts on a hard film adhered to a soft substrate. The wrinkles 11 have a waveform similar to a sine wave. The ridges 12 have a shape like a hanging ridge with a linear crease as a peak. The film 10 has a hierarchical uneven pattern in which the ridges 12 are formed as large uneven patterns, and the wrinkles 11 are formed as small uneven patterns on each of the multiple ridges 12.

[0015] In the film 10, the depth (amplitude) of the unevenness of the ridge 12 is larger than the amplitude of the wrinkles 11. The amplitude of the ridge 12 may be, for example, 25 μm or more. The amplitude of the ridge 12 may be 1 μm or more, 2 μm or more, 3 μm or more, 4 μm or more, 5 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, 25 μm or more, 30 μm or more, 35 μm or more, 40 μm or more, 45 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, or 100 μm or more. The amplitude of the wrinkles 11 may be, for example, less than 100 nm. The amplitude of the wrinkles 11 may be less than 1 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, or less than 100 nm.

[0016] In the film 10, the distance (wavelength) between the peaks of the ridges 12 is longer than the wavelength of the wrinkles 11. The wavelength of the ridges 12 may be, for example, 65 μm or more. The wavelength of the ridges 12 may be 5 μm or more, 10 μm or more, 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, or 100 μm or more. The amplitude of the wrinkles 11 may be, for example, less than 3 μm. The amplitude of the wrinkles 11 may be less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, or less than 1 μm.

[0017] In the film 10, the aspect ratio (wavelength / amplitude) of the ridges 12 is higher than the aspect ratio of the wrinkles 11. The aspect ratio of the ridges 12 may be, for example, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, or 1 or more. The aspect ratio of the wrinkles 11 may be, for example, less than 0.3.

[0018] The film 10 of the present disclosure can be used as a water collection film for condensing and collecting moisture in the air. In this case, the wrinkles 11 mainly have the function of condensing moisture in the air. The ridges 12 mainly function as flow paths for the water condensed in the wrinkles 11. In the film 10 of the present disclosure, the wrinkles 11 and the ridges 12 are arranged in the same direction, so that the water condensed in the wrinkles 11 can be efficiently collected by the ridges 12.

[0019] The membrane 10 of the present disclosure can be used as a cell scaffold material for adhering and growing cells. In this case, the ridges 12 mainly function to adhere cells, and the wrinkles 11 mainly function to adhere specific proteins contained in the cells. In the membrane 10 of the present disclosure, the wrinkles 11 and the ridges 12 are aligned in the same direction, so that the positions of the proteins contained in the cells can be well controlled while the cells are stretched and grown.

[0020] The membrane 10 may be made of a material such as a metal. The membrane 10 may be made of gold, platinum, aluminum, titanium, copper, stainless steel, carbon materials, etc. When the membrane 10 is used as a water collection means, the membrane 10 is preferably made of copper, stainless steel, aluminum, etc. When the membrane 10 is used as a cell scaffold material, the membrane 10 is preferably made of gold, platinum, titanium, stainless steel, carbon materials, etc.

[0021] FIG. 2 illustrates a schematic diagram of a method for producing a membrane according to the present disclosure.

[0022] In the first step S1, the flexible substrate 20 is pulled in one direction (the left-right direction in FIG. 2) to apply pre-strain (pre-stretch), and the first position (the left end in FIG. 2) and the second position (the right end in FIG. 2) are fixed in that state. The other directions (the front-back direction perpendicular to the paper in FIG. 2) are left free and unconstrained.

[0023] In a second step S2, a film 10 is formed on the surface of an area including at least a part between the first position and the second position on the substrate 20. For example, a metal or the like is evaporated onto the surface of the substrate 20 by a sputtering device. At this time, the heated and ejected metal adheres to the surface of the substrate 20, causing the substrate 20 to thermally expand. However, since both ends of the substrate 20 in the left-right direction are fixed, wrinkles 11 extending in the front-rear direction perpendicular to the left-right direction (direction perpendicular to the paper surface) are formed in the film 10 so as to be arranged in the left-right direction.

[0024] In a third step S3, the substrate 20 is returned from the stretched state to its original state, thereby contracting the substrate 20 in a first direction connecting the first position and the second position (the left-right direction in FIG. 2). At this time, the compressive strain applied to the film 10 causes ridges 12 extending in the front-back direction to be arranged in the left-right direction in the film 10 on which the wrinkles 11 have been formed, similar to the wrinkles 11. The substrate 20 is softer than the metal film 10, and therefore deforms in accordance with the deformation of the film 10.

[0025] The substrate 20 is made of a material having a smaller Young's modulus than the film 10. The Young's modulus of the material constituting the substrate 20 may be, for example, less than 1 MPa. The Young's modulus of the material constituting the substrate 20 may be less than 100 MPa, less than 50 MPa, less than 10 MPa, less than 5 MPa, less than 4 MPa, less than 3 MPa, less than 2 MPa, less than 1 MPa, less than 0.5 MPa, or less than 0.1 MPa. The Young's modulus of the material constituting the film 10 may be, for example, 70 GPa or more. The Young's modulus of the material constituting the film 10 may be 1 GPa or more, 5 GPa or more, 10 GPa or more, 20 GPa or more, 30 GPa or more, 40 GPa or more, 50 GPa or more, 60 GPa or more, 70 GPa or more, 80 GPa or more, 90 GPa or more, or 100 GPa or more.

[0026] The substrate 20 may be made of resin, rubber, elastomer, etc. The base for forming the film 10 may be a plate-shaped substrate 20, or may have any shape.

[0027] The membrane 10 may be composed of a metal such as gold.

[0028] In the first step S1, the substrate 20 may be fixed in a stretched state such that its length is 1.4 times or more its original length. The greater the pre-strain, the higher the aspect ratio of the ridge 12 formed in the third step S3. The substrate 20 may be fixed in a stretched state such that its length is 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more its original length. The amplitude and wavelength of the ridge 12 vary depending on the thickness of the film 10.

[0029] In the third step S3, the substrate 20 may be compressed so that the length of the substrate 20 becomes shorter than the original length. In this case, in the first step S1, the substrate 20 may be fixed without being stretched.

[0030] The cell scaffold material of the present disclosure includes the membrane 10 of the present disclosure. The cell scaffold material may be made of a raw material having bioabsorbability or biocompatibility. The cell scaffold material may be made of a polymeric material such as polydimethylsiloxane (PDMS), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), copolymer of lactic acid and glycolic acid (PLGA), polycaprolactone (PCL), collagen, gelatin, glycosaminoglycan, chitin, chitosan, hyaluronic acid, and polypeptide.

[0031] 3 is a schematic diagram showing a method for producing a cell scaffold material according to the present disclosure. The cell scaffold material 30 according to the present disclosure can be produced by transferring a hierarchical concavo-convex pattern to the above-mentioned raw material using the membrane 10 according to the present disclosure as a template (plate).

[0032] (Example) Cells were cultured using a cell scaffold material 30 having a hierarchical concave-convex pattern produced by the method according to the embodiment.

[0033] (PDMS pattern preparation) The hierarchical concave-convex pattern was transferred to polydimethylsiloxane (PDMS). The fabricated PDMS was washed several times with pure water and sterilized by autoclaving at 120 °C for 15 min. After autoclaving, the PDMS surface was treated with oxygen plasma for 2 min to make it hydrophilic. To promote cell attachment, fibronectin coating, diluted 20-fold in PBS, was performed at 37 °C for 30 min immediately after plasma treatment.

[0034] (cell culture) Human bone marrow-derived mesenchymal stem cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture at 37°C in a humidified incubator with 5% CO2. Cells were passaged every 3-4 days until they reached 80-90% confluence. After setting the PDMS pattern in each well of a 24-well culture plate, 20,000 cells / cm2 were cultured in each well. 2 were seeded and cultured for 3 days.

[0035] (Area, aspect ratio, angle of attached cells) Figure 4 shows cell morphology evaluation of human mesenchymal stem cells after culture on hierarchical wrinkle patterns for 3 days. Phase contrast images (a, b) and fluorescence images of actin filaments and nuclei (c, d) are shown for flat and wrinkled substrates, respectively. White bars indicate 200 μm. Cell area (e), aspect ratio (f), and distribution (g, h) were evaluated for cells passaged on flat substrates (n = 307) and wrinkled substrates (n = 328), respectively. Graphs show the mean ± standard deviation obtained from four independent experiments.

[0036] Human mesenchymal stem cells cultured for 3 days on the hierarchical concave-convex pattern were harvested and stained. As shown in the fluorescent staining images in Figure 4 (cell nuclei: Hoechst 33342; cytoskeleton: Alexa Flour 546 Phalloidin), the cells were oriented along the large concave-convex pattern. Quantitative evaluation of the cell area, aspect ratio, and angle from the stained images of actin filaments revealed that the cells passaged on the flat PDMS (7949 ± 6837 μm 2), cells passaged on hierarchically patterned PDMS had a smaller area (4343 ± 3618 μm 2 ) The aspect ratio of cells on the hierarchical patterned PDMS was larger (5.75 ± 3.97) than that of cells on flat PDMS (2.62 ± 1.45). As shown in the angle histogram, cells on the hierarchical pattern were oriented along the large concave-convex pattern (90.7 ± 12.1 degrees), whereas cells on the flat surface were randomly oriented (88.3 ± 54.0 degrees). The definitions of area, aspect ratio, and angle are as shown in Figure 5.

[0037] (Effect of hierarchical concave-convex patterns on cell differentiation potential) To investigate the effect of the geometry of the hierarchical concave-convex patterns on the differentiation capacity of mesenchymal stem cells, we performed real-time qPCR on cells passaged for 3 days on hierarchically patterned PDMS in general cell maintenance medium without chemically induced differentiation medium.

[0038] Figure 6 shows mRNA expression normalized to Gapdh in human mesenchymal stem cells passaged for 3 days on flat and hierarchical wrinkle substrates. Osteoblast markers (Runx2, Alp, Opn), chondrocyte markers (Sox9), and tenocyte markers (Mkx, Col1) were evaluated. Graphs show the mean ± standard deviation from six independent experiments. p values ​​<0.05 were considered significant ( * p<0.05). p values ​​were calculated using paired t-test.

[0039] As shown in Figure 6, the hierarchical concave-convex pattern reduced the osteoblast differentiation markers Runx2 (0.89-fold change; p=0.69), Alp (0.57-fold change; p<0.05), and Opn (0.57-fold change; p<0.05), as well as the chondrocyte differentiation marker Sox9 (0.50-fold change; p<0.05), while significantly increasing the tendon differentiation markers Mkx (1.48-fold change; p<0.05) and Col1 (1.60-fold change; p<0.05). These results suggest that the use of hierarchical concave-convex patterns makes it possible to induce differentiation of mesenchymal stem cells into tendon cells.

[0040] The present disclosure has been described above based on examples. These examples are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible in the combination of each component and each treatment process, and that such modifications are also within the scope of the present disclosure. [Explanation of symbols]

[0041] 10 membranes, 11 wrinkles, 12 ridges, 20 substrates, 30 cell scaffold materials.

Claims

1. A first step of fixing a first position and a second position of the substrate while pulling the substrate in a first direction; a second step of depositing a film on a surface of an area including at least a portion between the first position and the second position of the substrate; a third step of contracting the substrate in a first direction connecting the first position and the second position by returning the substrate from the tensioned state to its original state; Equipped with In the second step, a first uneven pattern is formed in the film by the thermal stress in the first direction in the substrate; In the third step, a second uneven pattern is formed on the film by shrinking the substrate in the first direction. Method for producing the membrane.

2. The film has a hierarchical concave-convex pattern in which a first concave-convex pattern smaller than the second concave-convex pattern is formed in each of a plurality of second concave-convex patterns. The method of claim 1 .

3. The aspect ratio of the second concave-convex pattern is higher than that of the first concave-convex pattern. The method according to claim 1 or 2.

4. The first concave-convex pattern and the second concave-convex pattern are arranged in the same direction. The method according to claim 1 or 2.

5. The first uneven pattern and the second uneven pattern extending in a second direction perpendicular to the first direction are formed to be aligned in the first direction. The method according to claim 4.

6. The wavelength of the second uneven pattern is longer than that of the first uneven pattern. The method according to claim 1 or 2.

7. The amplitude of the second concave-convex pattern is greater than that of the first concave-convex pattern. The method according to claim 1 or 2.

8. The Young's modulus of the substrate is smaller than that of the film. The method according to claim 1 or 2.

9. The film is made of a metal. The method according to claim 1 or 2.

10. The first uneven pattern is a wrinkle pattern, and the second uneven pattern is a ridge pattern. The method according to claim 1 or 2.

11. A first concave-convex pattern repeatedly arranged in one direction; a second concave-convex pattern repeatedly arranged in the same direction as the first concave-convex pattern; having A hierarchical concave-convex pattern is formed in each of a plurality of second concave-convex patterns, the first concave-convex pattern being smaller than the second concave-convex pattern. film.

12. The aspect ratio of the second concave-convex pattern is higher than that of the first concave-convex pattern. The membrane of claim 11.

13. The aspect ratio of the second concave-convex pattern is 0.3 or more. The membrane of claim 12.

14. The wavelength of the second uneven pattern is longer than that of the first uneven pattern.

13. A membrane according to claim 11 or 12.

15. The amplitude of the second concave-convex pattern is greater than that of the first concave-convex pattern.

13. A membrane according to claim 11 or 12.

16. The film is made of a metal.

13. A membrane according to claim 11 or 12.

17. The first uneven pattern is a wrinkle pattern, and the second uneven pattern is a ridge pattern.

13. A membrane according to claim 11 or 12.

18. A water collecting membrane for collecting moisture in the air, comprising a membrane according to claim 11 or 12.

19. A cell scaffold material for adhering and growing cells, comprising the membrane according to claim 11 or 12.

20. Made from bioabsorbable or biocompatible raw materials The cell scaffold material according to claim 19.

21. The method further comprises a step of transferring the hierarchical concavo-convex pattern to a raw material using the film according to claim 11 or 12 as a template. A method for producing a cell scaffold material.

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