JP7738261B2 - Transfer mold and method for manufacturing transfer mold - Google Patents

Description

The present invention relates to a transfer mold and a method for manufacturing a transfer mold.

A variety of medical devices and materials have been developed using biocompatible materials, including cell culture substrates, scaffolding materials for regenerative medicine (e.g., materials for regenerating cartilage, bone, spine, nucleus pulposus, ligament, corneal stroma, skin, oral mucosa, gums, periodontal ligament, blood vessels, nerves, and liver tissue), transplant materials, wound dressing materials, bone filling agents, hemostatic materials, adhesion prevention materials, and drug delivery carriers.

In order to induce or enhance interactions between cells or tissues and biocompatible materials, microstructures such as irregularities, holes, and micropatterns may be formed on the surface of the biocompatible material. Surface structures are formed by contacting the surface of the biocompatible material with a mold having a structure complementary to the structure to be imparted to the biocompatible material, and then transferring the complementary structure of the mold to the surface of the biocompatible material. The application of inkjet technology and lithography technology to the formation of the complementary structure of the mold is being considered (see, for example, JP 2016-533244 A).

JP 2016-533244 A

The above technology makes it possible to form simple (semi)spherical or linear micropatterns on the surface of biocompatible materials by transferring the complementary structure of a mold. However, human tissues and organs are not composed of simple spheres or straight lines, but rather of complex combinations of curved and curved surfaces. For example, while the surface of biocompatible materials used in regenerative medical products that comes into contact with cells is naturally curved, the interface with the epithelial cell layer in scaffolds such as cultured skin is often flat or has linear irregularities. In such cases, the cultured tissue may not adhere to the scaffold, resulting in floating or peeling, making it difficult to obtain cultured tissue suitable for practical use.

Moreover, molds used to transfer surface structures to biocompatible materials require low toxicity, ease of processing, strength, durability, and other characteristics from their constituent materials. However, developing low-toxicity materials poses many challenges. Furthermore, attempts to increase strength and durability result in a decrease in processability. Conventional materials have not yet achieved a balanced and sufficient level of these properties.

The object of the present invention is to provide a transfer mold that can transfer a surface structure formed by a biomimetic curved surface onto the surface of a biocompatible material, and that is stable and safe in terms of the material, as well as a method for manufacturing such a transfer mold that can be efficiently produced.

The inventors conducted extensive research to solve the above problems and discovered that the above objectives could be achieved by adopting the following configuration, leading to the completion of the present invention.

In one embodiment, the present invention provides
A transfer mold for transferring a surface structure to a transfer target,
the transfer mold has a stainless steel substrate and a transfer surface structure formed on a surface of the stainless steel substrate;
the surface structure for transfer has, when viewed along a normal direction to the surface of the stainless steel substrate, a concave curved surface that is concave in an inward direction toward the inside of the stainless steel substrate, a convex curved surface that is convex in an outward direction opposite to the inward direction, or a combination thereof;
The transfer mold has a shoulder portion formed on at least a part of the concave curved surface or at least a part of the convex curved surface, where the curvature of the curved surface changes.

In another embodiment, the present invention provides
The method for manufacturing the transfer mold includes:
The present invention relates to a method for manufacturing a transfer mold, which includes a step of irradiating a laser onto the surface of a stainless steel substrate to form a transfer surface structure.

In this transfer mold, the transfer surface structure formed on the surface of the stainless steel substrate has a concave curved surface that is concave inward, a convex curved surface that is convex outward, or a combination of these. Furthermore, in this transfer mold, not only are these simple curved surfaces formed, but at least a portion of the concave curved surface or at least a portion of the convex curved surface has a shoulder portion where the curvature of the curved surface changes. Therefore, simply by bringing the transfer surface structure of the transfer mold into contact with the surface of the object to be transferred, a complex biomimetic curved surface can be imparted to the object to be transferred. Furthermore, because stainless steel is used as the constituent material of the transfer mold, it exhibits high levels of low toxicity, ease of processing, strength, durability, etc.

This method for manufacturing a transfer mold allows for the formation of a transfer surface structure simply by irradiating a laser onto the surface of a stainless steel substrate, without any complicated processes. Therefore, it is possible to efficiently manufacture transfer molds with complex, biomimetic transfer surface structures that could not be achieved using conventional inkjet or lithography technologies.

FIG. 1 is a perspective view schematically illustrating a transfer mold according to one embodiment of the present invention. FIG. 2 is a partially enlarged cross-sectional view taken along line AA of FIG. 1. FIG. 2 is a partially enlarged cross-sectional view schematically illustrating an example of a concave curved surface of the transfer surface structure. FIG. 2 is a partially enlarged cross-sectional view schematically illustrating an example of a convex curved surface of a transfer surface structure. 1 shows an enlarged view of a portion of a micropattern designed for forming a surface structure for transfer. 4B shows a wide-angle view of the design pattern of FIG. 4A. 4B shows a cross-sectional view of the design pattern of FIG. 4A taken along line A1-B1. 1 shows a planar SEM photograph (100x magnification) of the transfer surface structure of the transfer mold of Example 1. 1 shows an SEM photograph (300x magnification) of a part of the transfer surface structure of the transfer mold of Example 1. 1 shows a shape profile when the fine protrusions in the transfer surface structure of the transfer mold of Example 1 are scanned obliquely. 1 shows an SEM photograph (100x magnification) of a transfer mold having fine protrusions in the shape of a square pillar in Comparative Example 1. 1 shows SEM photographs (narrow field of view (left) 300x magnification; wide field of view (right) 100x magnification) of the surface structure of a fibrous collagen gel obtained using the transfer mold of Example 1. 1 shows SEM photographs (narrow field of view (left) 300x magnification; wide field of view (right) 30x magnification) of the surface structure of a fibrous collagen gel obtained using the transfer mold of Comparative Example 1. 1 is a tissue image showing a cross section of a cultured tissue stained with HE staining, obtained from a fibrous collagen gel that has been transferred using the transfer mold of Example 1. 1 is a tissue image showing a cross section of a cultured tissue stained with HE staining, obtained from a fibrous collagen gel that had been transferred using a transfer mold in Comparative Example 1.

A transfer mold and a method for manufacturing a transfer mold according to one embodiment of the present invention will be described below with reference to the drawings. The present invention is not limited to these embodiments. In some or all of the figures, parts not necessary for the explanation have been omitted, and some parts have been enlarged or reduced in size to facilitate explanation. Terms indicating positional relationships, such as up and down, referred to with reference to the figures are used solely to facilitate explanation and are in no way intended to limit the configuration of the present invention.

<Transfer mold>
Fig. 1 is a perspective view schematically showing a transfer mold according to one embodiment of the present invention. Fig. 2 is a partially enlarged cross-sectional view taken along line A-A in Fig. 1. Fig. 3A is a partially enlarged cross-sectional view schematically showing an example of a concave curved surface of the transfer surface structure, and Fig. 3B is a partially enlarged cross-sectional view schematically showing an example of a convex curved surface of the transfer surface structure.

The transfer mold 1 is a transfer mold for transferring a surface structure to a transfer target object. The transfer method will be described later. The transfer mold 1 has a stainless steel substrate 2 and a transfer surface structure 4 formed on the surface 3 of the stainless steel substrate 2. In the transfer mold 1 shown in Figure 1, the transfer surface structure 4 is formed on a portion of the surface 3 of the stainless steel substrate 2 (a portion of the upper surface of the stainless steel substrate 2 in Figure 1), but this is not limited thereto, and the transfer surface structure 4 may also be formed on the entire surface 3 of the stainless steel substrate 2.

The stainless steel substrate 2 is a component that constitutes the main body of the transfer mold 1 and is made of stainless steel. The shape of the stainless steel substrate 2 is typically plate-like as shown in Figure 1, but is not limited to this. As described below, forming the transfer surface structure 4 on the surface 3 of the stainless steel substrate 2 simply requires irradiating the surface 3 of the stainless steel substrate 2 with a laser, so laser processing can be performed regardless of the shape of the stainless steel substrate 2. Other shapes of the stainless steel substrate 2 can be any shape, such as a cube, rectangular parallelepiped, linear, rod-like, spherical, or cylindrical, as long as they can be transferred to the object to be transferred. An appropriate shape can be adopted depending on the method for transferring the surface structure to the object to be transferred.

The stainless steel is not particularly limited, and may be martensitic stainless steel, ferritic stainless steel, austenitic stainless steel, austenitic-ferritic stainless steel, precipitation hardening stainless steel, or a combination thereof.

As shown in Figure 2, when viewed along the normal direction z of the surface 3 of the stainless steel substrate 2, the transfer surface structure 4 has a concave curved surface 41 that is concave in the inward direction D _in toward the inside of the stainless steel substrate 2, a convex curved surface 42 that is convex in the outward direction D _out opposite the inward direction D _in , or a combination thereof. In the embodiment of Figure 2, the transfer surface structure 4 has a combination of a concave curved surface 41 and a convex curved surface 42, but is not limited to this. It may have only a concave curved surface 51 as shown in Figure 3A, or a convex curved surface 52 as shown in Figure 3B. Furthermore, a portion of the transfer surface structure 4 formed on the surface 3 of the stainless steel substrate 2 may have only the concave curved surfaces 41, 51, and the remaining region of the transfer surface structure 4 may have only the convex curved surfaces 42, 52.

In the transfer surface structure 4, the concave curved surfaces 41, 51 are preferably part of a plurality of micropores having a predetermined depth formed on the surface 3 of the stainless steel substrate 2. The convex curved surfaces 42, 52 are preferably part of a plurality of microprotrusions having a predetermined height formed on the surface 3 of the stainless steel substrate 2. In particular, the convex curved surfaces are preferably at least part of the surface of dome-shaped protrusions that are convex in the outward direction D _out .

In the transfer surface structure 4, the micropores and/or microprotrusions are preferably formed in the form of triangular lattice points or square lattice points. The depth and formation pitch of the micropores, and the height and formation pitch of the microprotrusions can be set appropriately depending on the target surface structure (micropattern) to be transferred to the transfer target object. The depth and formation pitch of the micropores may be the same or different from each other, and the height and formation pitch of the microprotrusions may also be the same or different from each other. The lower limit of the formation pitch of the micropores and/or microprotrusions (the distance between the lattice points) is preferably 50 μm, more preferably 100 μm, even more preferably 150 μm, and particularly preferably 200 μm. The upper limit of the formation pitch is preferably 800 μm, more preferably 500 μm, even more preferably 300 μm, and particularly preferably 250 μm.

In this embodiment, as shown in Figures 2, 3A, and 3B, not only are the concave surfaces 41, 51 and the convex surfaces 42, 52 formed, but also shoulder portions 41s, 42s, 51s, and 52s are formed on at least a portion of the concave surfaces 41, 51 or at least a portion of the convex surfaces 42, 52, where the curvature of the curved surfaces changes. By providing the transfer surface structure 4 with such shoulder portions 41s, 42s, 51s, and 52s, it is possible to impart to the transfer surface structure 4 a complex biomimetic shape that cannot be obtained with a simple spherical, flat, or curved surface.

The shoulders 41s, 42s, 51s, and 52s are portions of the curved surface where they are formed that change in curvature, and are portions that protrude from the outer surface of the curved surface (including both concave and convex curved surfaces) toward the outward direction _Dout . Specifically, for example, when focusing on the convex curved surface 42 shown in Fig. 2, the shoulders 42s are protrusions that result from the displacement of the convex curved surface 42 toward the outward direction Dout relative to a plane (a plane including a dashed line connecting both ends of the base of the shoulder 42s in Fig. 2) that would result if the convex curved surface 42 were continuously and monotonically connected from one end (e.g., the top) to the other end (e.g., the _bottom ) in the normal direction z to the surface of the stainless steel substrate 2. Similarly, with regard to the concave curved surface 41, the shoulder 41 s is a protrusion that is generated by displacement of the concave curved surface 41 toward the outward direction D _out relative to a surface (a surface including a dashed line connecting both ends of the base of the shoulder 41 s in FIG. 2 ) that is assumed to be continuously and monotonously connected from one end to the other end in the normal direction z to the surface of the stainless steel substrate 2.

As shown in Figure 3A, even when the transfer surface structure 4 has only a concave curved surface 51, the concave curved surface 51 has a shoulder 51s. The shoulder 51s is formed by the concave curved surface 51 being displaced in the outward direction _Dout relative to a surface that would be assumed to be continuously and monotonically connected. As shown in Figure 3B, even when the transfer surface structure 4 has only a convex curved surface 52, the convex curved surface 52 also has a shoulder 52s. The shoulder 52s is formed by the convex curved surface 52 being displaced in the outward direction _Dout relative to a surface that would be assumed to be continuously and monotonically connected.

In the transfer mold 1, it is sufficient that shoulders 41s, 42s, where the curvature of the curved surface changes, are formed on at least a portion of the concave curved surface 41 or at least a portion of the convex curved surface 42; shoulders do not have to be formed on all concave or convex curved surfaces. Furthermore, when shoulders are formed on a curved surface, the number of shoulders on one curved surface is not limited to one, as shown in Figures 2, 3A, and 3B, and may be two, three, or more.

The shoulder portion does not necessarily have to be convex in the outward direction _Dout and have an extreme value in the cross section shown in Figure 2. As long as the protrusions are formed by deviating from a curved surface (hereinafter also referred to as a "virtual curved surface") that would be formed if the protrusions were continuously and monotonically connected, those protrusions are included in the shoulder portion. For example, the lower limit of the ratio of the height of the shoulder portion to the height of the micro-protrusions (the maximum amount of displacement from the virtual curved surface that gives rise to the protrusion) is preferably 1%, more preferably 3%, and even more preferably 5%. The upper limit of the ratio is preferably 30%, more preferably 25%, and even more preferably 20%.

When the stainless steel substrate 2 is viewed in plan view (when the stainless steel substrate 2 shown in Figure 1 is viewed from above), the shoulder portion 42s may be formed around the entire outer surface (convex curved surface 42) of the micro-protrusion, or may be formed on only a portion of the outer surface.

While there are no particular limitations on the maximum height Rz of the transfer surface structure 4, the lower limit is preferably 10 μm, more preferably 20 μm, even more preferably 30 μm, and particularly preferably 50 μm. The upper limit of the maximum height Rz is preferably 500 μm, more preferably 300 μm, even more preferably 200 μm, and particularly preferably 150 μm. By keeping the maximum height Rz of the transfer surface structure 4 within the above range, the surface structure can be efficiently transferred to the transfer target object. The maximum height Rz can be measured in accordance with JIS B 0601-2001.

The surface 3 of the transfer mold 1 may be subjected to appropriate surface treatments such as hydrophilic treatment, hydrophobic treatment, hardening treatment, and release treatment.

<<Manufacturing method of transfer mold>>
The method for manufacturing a transfer mold according to this embodiment includes a step of irradiating a surface of a stainless steel substrate with a laser to form a transfer surface structure. This embodiment employs laser irradiation of the surface of the stainless steel substrate to form the transfer surface structure, thereby enabling efficient and simple manufacture of a transfer mold having a transfer surface structure with a shoulder formed on the curved surface. Conventional inkjet and lithography techniques can form concave or convex curved surfaces in a transfer surface structure by integral processing such as droplet injection or etching after micropattern formation, but shoulders on curved surfaces are difficult to form even with these techniques. The formation of shoulders on the curved surface of a transfer surface structure can be achieved by appropriately controlling the type of laser, laser irradiation conditions (output, pulse width, repetition frequency, scanning speed), the hardness and crystallinity of the stainless steel substrate, the temperature of the stainless steel substrate during laser irradiation, processing time, etc.

Specifically, data for the desired transfer surface structure is input into a laser irradiation device, and the surface 3 of the stainless steel substrate 2 is irradiated with a laser while scanning it. The stainless steel substrate 2 may be placed on an xyz stage, and the stainless steel substrate 2 side may be moved three-dimensionally. For example, when forming micro-protrusions that give a convex curved surface in a square lattice pattern, multiple grooves may be formed in a square lattice pattern by irradiating with a laser. A transfer surface structure of the desired shape can be obtained by repeating laser irradiation as necessary.

The processing using laser irradiation can be either photothermal processing or ablation processing, but ablation processing is preferred from the standpoint of minimizing damage to the stainless steel substrate surface and suppressing debris generation.

The laser to be irradiated is not particularly limited as long as it can process stainless steel substrates, but a picosecond laser is preferred from the standpoints of processing speed, shape controllability, processing accuracy, and the ability to perform ablation processing.

The irradiation conditions for the picosecond laser should be set to conditions suitable for forming a shoulder on the curved surface of the transfer surface structure. The lower limit of the output is preferably 10 mW, more preferably 50 mW, and even more preferably 90 mW. The upper limit of the output is preferably 150 mW, but may be 130 W or 110 mW. The lower limit of the pulse width is preferably 1 ps, more preferably 3 ps, even more preferably 5 ps, and particularly preferably 10 ps. The upper limit of the pulse width is preferably 30 ps, more preferably 25 ps, and even more preferably 20 ps. The lower limit of the repetition frequency is preferably 1 kHz, more preferably 10 kHz, and even more preferably 20 kHz. The upper limit of the repetition frequency is preferably 100 kHz, more preferably 80 kHz, and even more preferably 50 kHz.

After laser irradiation, various processes such as cleaning, drying, and surface treatment may be performed. By simply performing laser irradiation and any optional post-processing, it is possible to efficiently and easily manufacture transfer molds with biomimetic, complex surface structures for transfer.

<Transfer Method>
A method for transferring a surface structure to a transfer target using a transfer mold includes at least a step of contacting the transfer target surface structure of the transfer mold with the transfer target. As a result, a surface structure complementary to the transfer target surface structure of the transfer mold is transferred to the transfer target. The manner of contact can be selected appropriately depending on the material and properties of the transfer target. When the transfer target is solid, transfer can be achieved by contacting the transfer mold and the transfer target, preferably under pressure. If necessary, contact or pressing can be performed while heated to a temperature above the softening point of the transfer target. When the transfer target is liquid or semi-solid and can be solidified (including gelation), transfer can be achieved by performing a solidification treatment (e.g., heating, radiation exposure, etc.) while the transfer mold and the transfer target are in contact.

The transfer target material is preferably a scaffold material containing one or more selected from the group consisting of collagen, gelatin, hyaluronic acid, fibrin, fibrinogen, alginate, agarose, chitosan, chitin, cellulose, pectin, starch, laminin, gluten, casein, albumin, vitronectin, tenascin, entactin (night gene), heparan sulfate proteoglycan (perlecan), poly(acrylic acid) and its derivatives, poly(ethylene oxide) and its copolymers, poly(vinyl alcohol), polyphosphazene, and Matrigel. The microenvironment (niche) in which stem cells reside is three-dimensional, and an environment with a mechanical gradient (unevenness) is considered physiologically similar to human tissues/organs. For example, if the uneven (papilla-like) micropattern at the human epithelium/connective tissue interface could be reproduced on a scaffold material, physiological cellular responses more similar to those in vivo could be obtained, potentially facilitating applications and development in regenerative medicine. The transfer mold has a biomimetic, complex surface structure for transfer, making it possible to reproduce a microstructure similar to that of human tissue as described above on the scaffold material to be transferred, which could contribute to applications in regenerative medicine, etc.

This section describes a method for transferring a surface structure when using collagen gel as a scaffold material. One suitable form of this method involves contacting a gel-forming collagen aqueous solution with a transfer mold, degassing any remaining air in the gaps as necessary, and then maintaining this contact state for a predetermined period of time to produce a fibrous collagen gel with the desired surface structure.

While there are no particular limitations on the type of collagen used as the raw material for the fibrous collagen gel, type I collagen, which is abundant in the body, is preferred, and atelocollagen, from which the antigenic determinant telopeptides have been removed, is even more preferred. Collagen derived from biological sources such as mammals, seafood, birds, and reptiles can usually be used, but collagen derived from seafood, which does not share viruses with humans, is preferred. Collagen derived from fish is particularly preferred, and examples of fish-derived collagen include scales and skin. Scales have the advantage of being low in impurities such as lipids that cause a fishy odor, allowing for the production of highly pure collagen.

In this specification, "collagen" refers to collagen molecules with a triple helix structure and aggregates or assemblies of these collagen molecules, and does not include thermally denatured collagen (gelatin) or collagen peptides in which the triple helix structure has been broken.

Known methods for solubilizing collagen contained in biological tissue to obtain a solubilized collagen aqueous solution include solubilization treatment with enzymes, extraction treatment with dilute acid, and solubilization treatment with alkali. In this specification, unless otherwise specified, the term "solubilized collagen aqueous solution" refers to an aqueous collagen solution solubilized by any processing method.

When a fibrous agent such as a buffer solution is added to a solubilized collagen aqueous solution to adjust the solubilized collagen aqueous solution to an appropriate ionic strength and pH, the collagen molecules orient and assume a structure similar to collagen fibers found in vivo, resulting in a collagen gel with a specific shape. This collagen gel is called a "fibrous collagen gel." Furthermore, in this specification, a solubilized collagen aqueous solution with an ionic strength and pH suitable for forming a fibrous collagen gel is called a "gel-forming collagen aqueous solution."

A gel-forming collagen aqueous solution is obtained by providing a solubilized collagen aqueous solution with an ionic strength and pH suitable for forming a fibrous collagen gel. Specifically, the ionic strength and pH are adjusted by adding a fibrosing agent to the solubilized collagen aqueous solution. Suitable examples of fibrosing agents include saline, buffer solutions, buffered saline, acidic salt solutions, neutral salt solutions, and alkaline salt solutions. The pH of the aqueous solution is preferably set appropriately within a range of, for example, pH 3 to 10 depending on the type of collagen (acid-solubilized collagen, enzyme-solubilized collagen, alkali-solubilized collagen, etc.). For enzyme-solubilized collagen, for example, a buffer solution, buffered saline solution, or neutral salt solution with a pH range of 6 to 8 is preferably used. Specific examples of buffer solutions and buffered saline solutions include phosphate buffer, Tris buffer, HEPES buffer, acetate buffer, carbonate buffer, citrate buffer, phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (D-PBS), Tris-buffered saline, and HEPES-buffered saline.

As a method for bringing the gel-forming collagen aqueous solution into contact with the transfer mold, for example, the following two methods can be mentioned.
(Contact method 1): A method in which a transfer mold is placed with the area having the transfer surface structure facing upward, and an aqueous collagen solution for gel formation is poured into the area.
(Contact method 2): A method in which a transfer mold is placed on top of a gel-forming collagen aqueous solution contained in a specified container, with the area having the transfer surface structure facing downward.
In the above contact method 1, when the transfer mold alone is not able to hold the gel-forming collagen aqueous solution, it is preferable to use a predetermined frame or container that surrounds the transfer mold.

If unintended residual gaps form during contact between the gel-forming collagen aqueous solution and the transfer mold, it is preferable to remove the air present in the residual gaps by degassing. Degassing is an effective method when the gel-forming collagen aqueous solution is difficult to penetrate into the recesses of the transfer mold due to its viscosity, or when it is difficult to uniformly pour the gel-forming collagen aqueous solution into the recesses of the transfer mold. There are no particular limitations on the degassing method, and known methods using an aspirator, vacuum pump, vacuum dryer, etc. can be used. When degassing, it is desirable to take appropriate measures such as fixing the transfer mold or applying pressure to the gel-forming collagen aqueous solution.

The degree to which air present in the remaining gaps is removed can be appropriately determined so that the desired surface structure is imparted to the fibrous collagen gel, and it is not necessary to completely remove the air. In other words, it is not necessary for the surface structure complementary to the transfer surface structure of the transfer mold to be completely transferred to the fibrous collagen gel.

After the solubilized collagen aqueous solution is given an ionic strength and pH suitable for forming a fibrous collagen gel, it is preferable to hold the solution at a temperature of, for example, 15 to 30°C (but below the collagen denaturation temperature) for a certain period of time until the fibrous collagen gel is formed. The holding time is, for example, 6 to 24 hours.

In parallel with or after the gelation reaction, γ-ray irradiation, electron beam irradiation, U irradiation, etc. may be performed as necessary.
Crosslinking may be performed by irradiation with radiation such as V irradiation or plasma irradiation, or by ionizing radiation irradiation. Alternatively, chemical crosslinking may be performed using a carbodiimide crosslinking agent (for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride).

When observing a fibrous collagen gel with a scanning electron microscope at, for example, 10,000x magnification, the presence of numerous fibrous structures can be confirmed. Furthermore, although it is generally not easy to confirm that fibrous collagen has a D period using a scanning electron microscope, if a D period is confirmed in even a portion of the fibrous collagen, it is generally safe to conclude that the entire fibrous collagen has a D period.

The present invention will be described in detail below using examples, but the present invention is not limited to the following examples as long as it does not depart from the gist of the invention.

<<Manufacturing of transfer molds>>
Example 1
A stainless steel plate (SUS316L-H) measuring 40 mm long, 50 mm wide, and 0.4 mm thick was prepared as the stainless steel substrate. A 17 mm x 17 mm area near the center of one side of the stainless steel plate was irradiated with laser light to form a surface structure for transfer. The surface structure for transfer was designed to have 7,225 microprotrusions (85 x 85) approximately 130 μm high formed in a square lattice pattern with a formation pitch of 0.2 mm. Figure 4A shows an enlarged view of a portion of the designed micropattern, Figure 4B shows a wide-angle view of Figure 4A, and Figure 4C shows a cross-sectional view of the design pattern taken along line A1-B1. Laser irradiation was performed according to this design pattern. The laser was a picosecond pulse laser with a wavelength of 532 nm, and the irradiation conditions were an output of 100 mW, a pulse width of 15 ps, a repetition frequency of 20 kHz, and a scanning speed of 1 mm/s. Figure 5A shows a planar SEM photograph (100x magnification) of the resulting transfer surface structure, and Figure 5B shows a partially enlarged SEM photograph (300x magnification) of the transfer surface structure. Figure 5C shows the shape profile of the micro-protrusions in the transfer surface structure when scanned obliquely. As is clear from Figures 5A, 5B, and 5C, the resulting transfer surface structure has dome-shaped protrusions that are convex outward as the micro-protrusions, and shoulders are formed on the curved surfaces corresponding to the side surfaces of the dome-shaped protrusions.

<Comparative Example 1>
The same stainless steel plate as in Example 1 was prepared, and square pillar-shaped micro-projections (top surface dimensions: 100 μm × 100 μm) were formed in a square lattice pattern using lithography. Figure 6 shows an SEM photograph (100x magnification) of the resulting transfer mold having square pillar-shaped micro-projections. It can be seen that no shoulders were formed in the transfer mold of Comparative Example 1.

<Transfer of surface structure to collagen gel>
"Cellcampus FD-08G" (lyophilized product) manufactured by Taki Chemical Industry Co., Ltd., which was produced from tilapia scales, was dissolved in an HCl solution of pH 3, and the collagen concentration was then adjusted to 1.1% and the pH to 3, yielding a colorless, transparent aqueous solution of solubilized collagen. A silicon plate (2.5 mm thick) with a hole in its center was placed on top of the transfer mold of Example 1, which had been placed so that the area having the surface structure for transfer was facing upward. This hole was circular and had a diameter of 17 mm, and the silicon plate was placed so that the area of the transfer mold having the surface structure for transfer was located in the center of the hole.

Next, 0.78 mL of a mixture of 9 parts by volume of the solubilized collagen aqueous solution and 1 part by volume of 10x Dulbecco's phosphate-buffered saline (D-PBS) was poured into the holes in the silicon plate. The mixture easily filled the recesses in the transfer surface structure of the transfer mold. It was then held at 25°C for 12 hours to obtain a fibrous collagen gel (0.78 g, collagen concentration 1% by mass). The fibrous collagen gel was easily removed from the transfer mold, and no residue remained on the transfer mold.

SEM photographs of the surface of the fibrous collagen gel that had been in contact with the area of the transfer mold that had the surface structure for transfer were confirmed to contain areas with complex surface structures that were complementary to the surface structure for transfer. Figure 7 shows SEM photographs (narrow field of view (left) 300x; wide field of view (right) 100x) of the surface structure of the fibrous collagen gel obtained using the transfer mold of Example 1.

The surface structure was similarly transferred to the surface of a fibrous collagen gel using the transfer mold of Comparative Example 1 instead of the transfer mold of Example 1. Figure 8 shows SEM photographs (narrow field of view (left) 300x; wide field of view (right) 30x) of the surface structure of a fibrous collagen gel obtained using the transfer mold of Comparative Example 1.

Cell culture test
Using the transfer molds of Example 1 and Comparative Example 1, circular fibrous collagen gels (17 mm in diameter in plan view) were used as cell culture substrates (scaffolding materials) to conduct cell culture tests according to the following procedure. Each cell culture substrate had a thickness of approximately 2.5 mm. Primary cultured cells derived from the oral mucosal epithelium of patients who visited the Department of Oral Surgery at Niigata University Medical and Dental Hospital were used. This experiment was approved by the Ethics Committee of the Niigata University School of Dentistry.

(Day 0)
Each cell culture substrate was placed in a 12-well plate. For each fibrous collagen gel, the surface with the surface structure was placed on top. Each well was coated with a mixture of 25 μL of 1 μg/μL type IV collagen aqueous solution and 500 μL of phosphate buffer, and then left to stand overnight at 4°C.
(Day 1)
A cell suspension was prepared by mixing the oral mucosal epithelium-derived primary cultured cells with Medium A (EpiLife® Thermo Fisher Scientific) supplemented with EDGS (EpiLife Defined Growth Supplements) and 1.2 mM Ca++ (high calcium). 1 mL of the cell suspension was seeded onto the surface of the fibrous collagen gel at 1 x ¹⁰ cells/well, and 3.8 mL of Medium A was then poured into the well, bringing the total medium volume to 4.8 mL. The cells were then cultured in liquid phase (Submerged Culture), with the medium replaced daily until Day 4 of culture.
(Day 4)
The cells were then transferred to air-liquid interface culture, and the medium was changed every other day. The culture was continued until day 11 of culture.
(Day 11)
Each cell culture substrate was removed and immersed in 4% paraformaldehyde overnight (4°C) to fix the resulting cultured tissue.
Thereafter, the paraffin-embedded specimens were stained with hematoxylin and eosin (HE) by the usual method and subjected to morphological observation under an optical microscope.

(result)
Figure 9 is a histological image showing a cross section of HE-stained cultured tissue obtained from the fibrous collagen gel transferred using the transfer mold of Example 1. Figure 10 is a histological image showing a cross section of HE-stained cultured tissue obtained from the fibrous collagen gel transferred using the transfer mold of Comparative Example 1. From Figure 9, the formation of a continuous epithelial layer over the entire surface of the cell culture substrate (collagen gel scaffold material) was observed. Furthermore, epithelial leg-like cells proliferated in the recesses of the cell culture substrate, which could be said to have a structure similar to the oral mucosal epithelial tissue of a living body having a papilla-like structure. It should be noted that Example 1 was more similar to the oral mucosal epithelial tissue of a living body. On the other hand, from Figure 10, in the image of the cultured tissue of the fibrous collagen gel made using the transfer mold of Comparative Example 1, the applied square pillar-shaped microprotrusions were deformed, crushed, and flattened, dissimilar to the oral mucosal epithelial tissue of a living body. Furthermore, the formed cultured tissue was easily detached from the substrate (scaffold material).

REFERENCE SIGNS LIST 1 Transfer mold 2 Stainless steel substrate 3 Surface of stainless steel substrate 4 Transfer surface structure 41, 51 Concave curved surface 41s, 51s Shoulder portion formed on concave curved surface 42, 52 Convex curved surface 42s, 52s Shoulder portion formed on convex curved surface D _in: Inward direction toward the inside of the stainless steel substrate D _out : Outward direction toward the outside of the stainless steel substrate z: Normal direction to the surface of the stainless steel substrate

Claims (6)

A transfer mold for transferring a surface structure to a transfer target,
the transfer mold has a stainless steel substrate and a transfer surface structure formed on a surface of the stainless steel substrate;
the surface structure for transfer has a convex curved surface that is convex in an outward direction opposite to an inward direction toward the inside of the stainless steel substrate when viewed along a normal direction to the surface of the stainless steel substrate,
a shoulder portion is formed on at least a portion of the convex curved surface, the curvature of the curved surface changing ;
A transfer mold in which the maximum height Rz of the transfer surface structure is 10 μm or more and 500 μm or less . The transfer mold according to claim 1 , wherein the convex curved surface is a surface of at least a part of the dome-shaped protrusion that is convex in the outward direction. 3. The transfer mold according to claim 1 or 2, wherein the transfer target is a scaffold material comprising one or more selected from the group consisting of collagen, gelatin, fibrin, fibrinogen, alginic acid, agarose, chitosan, chitin, pectin, laminin, gluten, casein, albumin, vitronectin, tenascin, entactin (night gene), heparan sulfate proteoglycan (perlecan), poly(acrylic acid) and derivatives thereof, poly(ethylene oxide) and copolymers thereof, polyphosphazene , and Matrigel . A method for manufacturing a transfer mold according to claim 1,
A method for manufacturing a transfer mold, comprising the step of irradiating a surface of a stainless steel substrate with a laser to form a transfer surface structure. The method for manufacturing a transfer mold according to claim 4 , wherein the laser is a picosecond laser. 6. The method for manufacturing a transfer mold according to claim 5 , wherein the picosecond laser irradiation conditions are an output of 10 mW to 150 mW, a pulse width of 1 ps to 30 ps, and a repetition frequency of 1 kHz to 100 kHz.