JP2020184894A - Flat plate having fine holes and method of manufacturing the same, and cell culture substrate and method of manufacturing the same - Google Patents

Abstract

To obtain a flat plate having fine holes, the flat plate being configured to be able to reduce the influence on cells in contact with inner surfaces of the fine holes as compared with the prior art.SOLUTION: Provided is a flat plate 2 having fine holes 3 formed at a predetermined depth. On an inner surface 4 of the fine holes 3 in the flat plate 2 are formed a plurality of vertical grooves 5 which extend in a streak shape along the depth direction of the fine holes 3 and are arranged at intervals in the circumferential direction. The inner surface 4 includes: a bottom side region 4A from the bottom of the fine hole to the side of the opening of the fine hole up to 1/3 of the predetermined depth; an opening side region 4C from the opening of the fine hole to the bottom side up to 1/3 of the predetermined depth; and a middle region 4B between the opening side region and the bottom side region. When the surface roughness Ry of the bottom side region is RyA, the surface roughness Ry of the middle region is RyB, and the surface roughness Ry of the opening side region is RyC, a relationship of RyB/RyA<0.5 and a relationship of 0.8<RyC/RyB<1.2 hold.SELECTED DRAWING: Figure 3

Description

The present invention relates to a flat plate having micropores and a method for producing the same, and a cell culture substrate and a method for producing the same.

Three-dimensional cell culture technology has been developed for culturing cells in an environment closer to the living body by imitating the pericellular environment and morphology in the living body. In recent years, as a culture container for three-dimensional cell culture, a container provided with a plate having a plurality of micro-order micropores has been proposed.

In order to form such fine holes, cutting is often used because of the ease of processing and the low cost. When fine holes are formed on a resin flat plate by cutting, tool marks are likely to occur due to the influence of various external factors such as mechanical vibration. The tool mark is a tool mark formed on a surface machined by a cutting tool such as an end mill (see Patent Document 1).

The micropores formed in the cell culture vessel may have a deep concave shape, and the cutting tool with a long protrusion length used to form such micropores is prone to swing during machining and also. Since the rigidity tends to decrease, the tool mark tends to be noticeable.

The presence of toolmarks can interfere with cell culture and optical analysis and evaluation of cultured cells. Such tool marks can be reduced by polishing, but it is not easy to polish microholes with various micro-ordered shapes.

Japanese Unexamined Patent Publication No. 2007-283437

The cells seeded with the medium in the micropores pass through the center or the vicinity of the micropores and move from the opening side (entrance side) to the bottom side, or from the opening side of the micropores along the inner surface of the micropores. It moves toward the bottom side. It is considered that the influence on cells and the influence on optical analysis can be reduced if the tool mark on the inner surface of the microhole is not as much as possible.

The present invention has been created in view of the above circumstances, and has a structure capable of reducing the influence of tool marks formed on the inner surface of the fine holes as compared with the conventional case. It is an object of the present invention to provide a flat plate having a hole and a method for producing the flat plate.

The flat plate having fine holes according to the present invention is a flat plate having fine holes formed at a predetermined depth, and the inner surface of the fine holes in the flat plate is along the depth direction of the fine holes. A plurality of vertical grooves are formed that extend in a streak pattern and are arranged at intervals in the circumferential direction, and the inner surface thereof has the predetermined depth from the bottom of the microhole toward the opening side of the microhole. The bottom side region up to 1/3 of the roughness, the opening side region up to 1/3 of the predetermined depth from the opening of the fine hole toward the bottom side, the opening side region and the above. The surface roughness Ry of the bottom side region is RyA, the surface roughness Ry of the middle region is RyB, and the surface roughness Ry of the opening side region is RyC, including the middle region between the bottom side region and the bottom side region. If, the relationship of RyB / RyA <0.5 is provided, and the relationship of 0.8 <RyC / RyB <1.2 is provided.

The method for producing a flat plate having fine holes based on the present invention includes a step of forming fine holes in a resin flat plate by cutting and a step of irradiating the surface of the flat plate on which the fine holes are formed with vacuum ultraviolet rays. The integrated irradiation amount of the vacuum ultraviolet rays in the irradiation step is larger than 0 and 2600 mJ / cm ² or less.

The method for producing a cell culture substrate based on the present invention includes a step of forming fine holes in a resin flat plate by cutting, and a step of irradiating the surface of the flat plate on which the fine holes are formed with vacuum ultraviolet rays. The integrated irradiation amount of the vacuum ultraviolet rays in the irradiation step includes the step of forming the wall portion surrounding the fine holes of the flat plate irradiated with the vacuum ultraviolet rays, which is larger than 0 and 2600 mJ / cm ² or less.

Other aspects of the method for producing a cell culture substrate based on the present invention include a step of forming fine holes in a resin flat plate by cutting, and vacuum ultraviolet rays on the surface of the flat plate on which the fine holes are formed. A cell culture medium having a hole to which the fine holes of the master have been transferred on the bottom surface using a mold prepared by using a flat plate having fine holes irradiated with vacuum ultraviolet rays as an electrocast master. The integrated irradiation amount of the vacuum ultraviolet rays in the irradiation step including the step of forming the material is larger than 0 and 2600 mJ / cm ² or less.

According to the above configuration, it is possible to reduce the influence of the tool mark formed on the inner surface of the fine hole as compared with the conventional case.

(A) is a perspective view showing the cell culture base material 1, and (B) is a perspective view showing the plate portion 2 of the cell culture base material 1. FIG. 5 is an enlarged plan view showing a plurality of microholes 3 formed in a region 7 on the bottom surface (plate portion 2) of the cell culture substrate 1. It is a cross-sectional view taken along the line III-III in FIG. It is a photograph which shows an example of the vertical groove 5 formed in the inner surface 4 of the fine hole 3 formed in a plate part 2. It is a figure which shows typically the state of irradiating vacuum ultraviolet rays using an excimer lamp 8. It is a table which shows the relationship between the irradiation distance L and the radiation intensity I. It is an image which shows the cross section of the fine hole used in Example 1, and is the figure for demonstrating the measurement condition concerning Example 1. FIG. FIG. 5 is a graph showing the relationship between the integrated irradiation amount (irradiation time) and the surface roughness RyA, RyB, and RyC with respect to the first embodiment. It is a table which shows the relationship between the irradiation time and RyB / RyA, and the relationship between the irradiation time and RyC / RyB with respect to Example 1. It is an image which shows the cross section of the fine hole used in Example 2, and is the figure for demonstrating the measurement condition concerning Example 2. FIG. 5 is a graph showing the relationship between the integrated irradiation amount obtained when the irradiation distance is set to 0.2 mm and the surface roughness Ra, Ry, and Rz with respect to the second embodiment. FIG. 5 is a graph showing the relationship between the integrated irradiation amount obtained when the irradiation distance is set to 0.5 mm and the surface roughness Ra, Ry, and Rz with respect to the second embodiment. FIG. 5 is a graph showing the relationship between the integrated irradiation amount obtained when the irradiation distance is set to 2.5 mm and the surface roughness Ra, Ry, and Rz with respect to the second embodiment. It is a graph which shows the relationship between the integrated irradiation amount and surface roughness Ry obtained when the irradiation distance was set to 0.2mm, 0.5mm, 2.5mm with respect to Example 2. FIG. It is an image which shows the cross section of the microhole used in Example 3, and is the figure for demonstrating the measurement condition concerning Example 3. FIG. 5 is a graph showing the relationship between the integrated irradiation amount (irradiation time) and the surface roughness Ra, Ry, and Rz with respect to the third embodiment.

Hereinafter, embodiments for carrying out the invention will be described with reference to the drawings. In the following description, the same parts and equivalent parts may be given the same reference numbers, and duplicate explanations may not be repeated. FIG. 1 (A) is a perspective view showing the cell culture base material 1, and FIG. 1 (B) is a perspective view showing the plate portion 2 of the cell culture base material 1. FIG. 2 is an enlarged plan view showing a plurality of microholes 3 formed in the region 7 of the bottom surface (plate portion 2) of the cell culture substrate 1. FIG. 3 is a cross-sectional view taken along the line III-III in FIG.

(Cell culture substrate 1)
As shown in FIG. 1 (A), the cell culture base material 1 includes a resin plate portion 2 and a wall portion 6. As shown in FIG. 1 (B), a plurality of fine holes 3 having a predetermined depth H (FIG. 3) are formed in the region 7 on the surface 2S of the plate portion 2. In the cell culture substrate 1, the region 7 is the bottom surface, and each microhole 3 is a portion for culturing cells. The plate portion 2 is, for example, an acrylic flat plate. The microhole 3 has, for example, a substantially conical shape, the diameter of the opening 3H is 500 μm, the diameter D of the bottom 3B is 10 μm, the opening angle θ is 75 °, and the depth H is 320 μm. .. The distance between the central axes of the two adjacent openings 3H is, for example, 459 μm to 530 μm. The size of each part of the fine hole 3 is not limited, but it is preferably a size that cannot be polished. For example, the diameter of the opening 3H of the fine hole 3 is less than 1 mm, preferably 10 to 900 μm, and more preferably 10 to 500 μm. Further, the depth H of the fine hole 3 is less than 1 mm, preferably 10 to 900 μm, and more preferably 10 to 500 μm. The wall portion 6 has, for example, an acrylic flat plate formed with a plurality of through holes including the region 7 or having a size substantially equal to that of the region 7. The plate portion 2 and the wall portion 6 are joined to each other by an appropriate method.

FIG. 4 is an image showing the inside state of the fine holes 3 formed in the acrylic flat plate (plate portion) 2 with a diamond tool. As shown in this example, the inner surface (surface) 4 of the plate portion 2 on which the microholes 3 are formed extends in a streak shape along the depth direction of the microholes 3 and is spaced in the circumferential direction. A plurality of vertical grooves 5 are formed so as to be arranged with a space between them. As shown in FIG. 3, the inner peripheral surface (inner surface) 4 in which the fine holes 3 of the plate portion 2 are formed includes the bottom side region 4A, the middle ventral region 4B, and the opening side region 4C.

The bottom side region 4A is a region from the bottom 3B of the fine hole 3 toward the side of the opening 3H of the fine hole 3 to 1/3 of a predetermined depth H. The opening side region 4C is a region from the opening 3H of the fine hole 3 toward the bottom 3B side to 1/3 of the predetermined depth H. The middle abdominal region 4B is a region between the opening side region 4C and the bottom side region 4A.

In the plate portion 2, the surface roughness Ry of the bottom side region 4A is defined as RyA, the surface roughness Ry of the middle region 4B is defined as RyB, and the surface roughness Ry of the opening side region 4C is defined as RyC. In the case, it has a relationship of RyB / RyA <0.5 and a relationship of 0.8 <RyC / RyB <1.2.

The microhole 3 having the above configuration has an excimer on the machined surface of the plate part 2 as shown in FIG. 5 after the plate part 2 is machined using a cutting tool such as a diamond tool or a cemented carbide tool. It can be formed by irradiating vacuum ultraviolet rays with the lamp 8. The wavelength of the vacuum ultraviolet light is around 10 nm to 200 nm, for example, 172 nm.

Here, the radiation intensity I ₀ is 10 [mW / cm ² ], the absorption coefficient α is 16 [atm ^-1 · cm ^-1 ], and the oxygen partial pressure p in the atmosphere is 0.21 [atm]. When the irradiation distance is L [cm], the relationship between the radiation intensity I [mW / cm ² ] of the vacuum ultraviolet rays and the irradiation distance L [mm] can be expressed by the following equation.
I = I ₀ × exp (−pαL)
FIG. 6 shows the relationship between the irradiation distance L and the radiation intensity I created based on the above formula. The integrated irradiation dose [mJ / cm ² ] can be calculated by integrating the radiation intensity I [mW / cm ² ] over time. By changing the integrated irradiation amount, each value of RyA, RyB and RyC can be changed.

The presence or absence of the tool mark and its degree can be evaluated by the surface roughness which is one of the indexes of the surface texture. The relative relationship between the surface roughness RyA, RyB, and RyC was examined. Specifically, an acrylic flat plate (thickness 1 mm) was prepared, and a conical fine hole was formed on the surface of the flat plate (thickness 1 mm) by cutting with a diamond tool. The diameter of the conical opening was 500 μm, the depth was 320 μm, the opening angle was 75 °, the processing conditions were a rotation speed of 50,000 rpm, a feed rate of 3 mm / min, and a dwell of 10 μsec. Then, vacuum ultraviolet rays were irradiated toward the fine holes. The irradiation conditions were a wavelength of 172 nm, an irradiation distance of 0.5 mm, and an irradiation time of 1 to 10 min.

The surface roughness Ry at each irradiation time was calculated by observing with a laser microscope. Specifically, an acrylic plate having fine holes after being irradiated with vacuum ultraviolet rays for a predetermined time is cut with an acrylic cutter so that the fine holes are cut along the lines III-III of FIG. 2, and the cut surface thereof is cut. The three-dimensional shape is measured with a laser microscope VK-9710 (light source wavelength: 408 nm) manufactured by KEYENCE, and the surface roughness is determined from the three-dimensional shape using the attached surface roughness calculation software (VK-Analyzer VK-H1A1). Calculated. As shown in FIG. 7, the surface roughness is on a straight line extending vertically from the center of the bottom 3B of the microhole toward the surface on which the opening 3H is formed (in FIG. 3, the surface 2S of the plate portion 2). At a certain measurement position 1 43.5 μm away from the bottom 3B, a measurement position 2 87 μm away from the measurement position 1, and a measurement position 3 87 μm away from the measurement position 2, the circumferential direction (that is, the bottom) around each measurement position. A line segment having a length of 3 μm was measured in a direction (at an equal distance from the center of 3B) (measurement points 1, measurement points 2, and measurement points 3 in FIG. 7). The surface roughness Ry measured at the measurement point 1, the measurement point 2, and the measurement point 3 was designated as RyA, RyB, and RyC, respectively.

FIG. 8 is a graph showing the relationship between the integrated irradiation amount (irradiation time) and the surface roughness RyA, RyB, and RyC. FIG. 9 is a table showing the relationship between the irradiation time and RyB / RyA, and the relationship between the irradiation time and RyC / RyB. As shown in FIGS. 8 and 9, in the state after cutting and before irradiating with vacuum ultraviolet rays (untreated state), the relationship of RyB / RyA <0.5 is not established, and the bottom side. It can be seen that the value (0.512) of RyB / RyA, which is a relative value of Ry (RyB) of the blunt region 4B with respect to Ry (RyA) of the region 4A, is higher than that after the treatment.

Further, in the untreated state, the relationship of 0.8 <RyC / RyB <1.2 is not established, and the value of RyC / RyB which is a relative value of RyC of the opening side region 4C with respect to RyB of the middle region 4B It can be seen that (0.540) is lower than that after the treatment. On the other hand, the states after irradiation with vacuum ultraviolet rays all have a relationship of RyB / RyA <0.5 and a relationship of 0.8 <RyC / RyB <1.2. It is considered that by lowering the surface roughness of the opening side region 4C with respect to the bottom side region 4A in this way, the tool mark can be reduced and the influence on the cells in contact with the plate portion 2 can be reduced.

According to the results of Example 1, it was confirmed that the surface roughness increases when the integrated irradiation amount exceeds 2600 [mJ / cm ² ]. Therefore, when the integrated irradiation amount is 2600 [mJ / cm ² ] or less, the relationship of RyC / RyB ≦ 1.0 is established, and it can be seen that a lower surface roughness can be obtained.

The relationship between the integrated irradiation amount and the surface roughness Ra, Ry, and Rz was examined. Specifically, a cone-shaped fine hole was formed on an acrylic flat plate having a thickness of 1 mm by cutting with a diamond tool (see FIG. 3). The diameter of the conical opening was 500 μm, the depth was 320 μm, the opening angle was 75 °, the processing conditions were a rotation speed of 50,000 rpm, a feed rate of 3 mm / min, and a dwell of 10 μsec. Then, vacuum ultraviolet rays were irradiated toward the fine holes.

Regarding the irradiation conditions, the irradiation distance L was 0.2 mm, 0.5 mm, and 2.5 mm, respectively, and the irradiation time was 1 to 10 min. The surface roughness is measured by measuring the three-dimensional shape of the cross section of the fine hole with a laser microscope VK-9710 (light source wavelength: 408 nm) and calculating the attached surface roughness from the three-dimensional shape as in Example 1. Calculated using software (VK-Analyzer VK-H1A1). Specifically, as shown in FIG. 10, from the bottom 3B on a straight line extending vertically from the center of the bottom 3B of the microhole toward the surface (surface 2S of the plate 2) where the opening 3H is formed. The surface roughness Ra, Ry, and Rz of the measurement point 4, which is a line segment from a position 50 μm away to a position further 200 μm away, were calculated.

11 to 13 are graphs showing the relationship between the integrated irradiation amount obtained when the irradiation distances are set to 0.2 mm, 0.5 mm, and 2.5 mm and the surface roughness Ra, Ry, and Rz, respectively. is there. FIG. 14 is a graph showing the relationship between the integrated irradiation amount obtained when the irradiation distances are set to 0.2 mm, 0.5 mm, and 2.5 mm and the surface roughness Ry. As shown in FIGS. 11 to 14, the surface roughness Ry is high when the integrated irradiation amount is 2600 [mJ / cm ² ] or less at any of the irradiation distances of 0.2 mm, 0.5 mm, and 2.5 mm. It was confirmed that the surface roughness Ry increased when it decreased and exceeded 2600 [mJ / cm ² ]. In the micropores of the cell culture substrate, the surface roughness Ry has a dominant influence on the culture and optical analysis and evaluation of cell aggregates as compared with Ra and Rz. Therefore, it can be seen that the integrated irradiation dose is preferably 2600 [mJ / cm ² ] or less.

According to the results shown in FIGS. 11 to 14, it can be seen that the effect of improving the surface roughness Ry can be obtained immediately after the irradiation with the vacuum ultraviolet rays. Considering the results of the irradiation distances of 0.2 mm and 0.5 mm, it can be seen that if the integrated irradiation amount is 500 [mJ / cm ² ] or more, a sufficient effect of improving the surface roughness Ry can be obtained. In view of the result of the irradiation distance of 2.5 mm, it can be seen that if the integrated irradiation amount is 250 [mJ / cm ² ] or more, a sufficient effect of improving the surface roughness Ry can be obtained.

The cutting tool was changed to a carbide tool, and the relationship between the integrated irradiation amount and the surface roughness Ra, Ry, and Rz was examined. A conical fine hole was formed on an acrylic flat plate having a thickness of 1 μm by cutting with a cemented carbide tool. The diameter of the conical opening was 500 μm, the depth was 320 μm, the opening angle was 75 °, the processing conditions were a rotation speed of 50,000 rpm, a feed rate of 3 mm / min, and a dwell of 10 μsec. Then, vacuum ultraviolet rays were irradiated toward the fine holes.

Regarding the irradiation conditions, the irradiation distance L was 0.5 mm, and the irradiation time was 1 to 10 min. The surface roughness is measured by measuring the three-dimensional shape of the cross section of the fine hole with a laser microscope VK-9710 (light source wavelength: 408 nm) and calculating the three-dimensional shape by the attached surface roughness calculation in the same manner as in Example 1. Calculated using software (VK-Analyzer VK-H1A1). Specifically, as shown in FIG. 15, on a straight line extending vertically from the center of the bottom portion 3B of the microhole toward the surface on which the opening 3H is formed (in FIG. 3, the surface 2S of the plate portion 2). The surface roughness Ra, Ry, and Rz of the measurement point 5, which is a line segment from a position 50 μm away from the bottom 3B to a position further 200 μm away, were calculated.

FIG. 16 is a graph showing the relationship between the integrated irradiation amount and the surface roughness Ra, Ry, and Rz. According to the results shown in FIG. 16, it can be seen that the effect of improving the surface roughness Ry can be obtained immediately after the irradiation with the vacuum ultraviolet rays. It was confirmed that the surface roughness Ry decreased when the integrated irradiation amount was 2600 [mJ / cm ² ] or less, and the surface roughness Ry increased when the integrated irradiation amount exceeded 2600 [mJ / cm ² ].

Although the embodiments and examples have been described above, the above disclosure contents are examples in all respects and are not restrictive. The technical scope of the present invention is indicated by the scope of claims, and is intended to include all modifications within the meaning and scope equivalent to the scope of claims.

For example, in the above embodiment, an acrylic flat plate is used for the plate portion and the wall portion, but for example, an appropriate resin flat plate such as polystyrene resin can be used. Further, in the above embodiment, a plurality of microholes are formed and a flat plate irradiated with ultraviolet rays is used as the plate portion 2 of the cell culture substrate 1. However, a flat plate after forming a plurality of fine holes and irradiating with ultraviolet rays. It is also possible to form a mold by electroplating using the above as a master, and to form a flat plate having a plurality of fine holes from this mold by an appropriate molding technique (for example, resin molding). Since the flat plate formed from this mold is formed by transferring the master with reduced tool marks, it can be said that it has the same configuration as that of the above embodiment. Further, this mold may further include a mold for the wall portion. In this case, the step of joining the wall portion 6 to the plate portion 2 becomes unnecessary, and the plate portion and the wall portion can be integrally molded to prepare a cell culture base material.

1 Cell culture substrate, 2 Plate (flat plate), 2S surface, 3 Microholes, 3B bottom, 3H opening, 4 inner surface, 4A bottom side area, 4B middle area, 4C opening side area, 5 flutes, 6 Walls, 7 areas where microholes are formed, 8 excimer lamps.

Claims (9)

A flat plate having fine holes formed at a predetermined depth.
On the inner surface of the microholes in the flat plate, a plurality of vertical grooves extending in a streak shape along the depth direction of the microholes and arranging at intervals in the circumferential direction are formed.
The inner surface faces the bottom side region up to 1/3 of the predetermined depth from the bottom of the microhole toward the opening side of the microhole, and from the opening of the microhole toward the bottom side. Includes an opening-side region up to 1/3 of the predetermined depth and a middle region between the opening-side region and the bottom-side region.
When the surface roughness Ry of the bottom side region is RyA, the surface roughness Ry of the middle region is RyB, and the surface roughness Ry of the opening side region is RyC,
It has a relationship of RyB / RyA <0.5,
A flat plate having fine holes having a relationship of 0.8 <RyC / RyB <1.2. The flat plate having a fine hole according to claim 1, which has a relationship of RyC / RyB ≦ 1.0. The flat plate having fine holes according to claim 1 or 2, which is made of acrylic. The flat plate having the fine holes according to any one of claims 1 to 3, wherein the diameter of the opening of the fine holes is 10 to 900 μm. A cell culture substrate comprising a flat plate having the micropores according to any one of claims 1 to 4. The process of forming fine holes in a resin flat plate by cutting,
The step of irradiating the surface of the flat plate on which the fine holes are formed with vacuum ultraviolet rays is included.
A method for producing a flat plate having fine holes, wherein the integrated irradiation amount of the vacuum ultraviolet rays in the irradiation step is larger than 0 and 2600 mJ / cm ² or less. The method for producing a flat plate having fine holes according to claim 6, wherein the integrated irradiation amount of the vacuum ultraviolet rays in the irradiation step is 250 mJ / cm ² or more. The process of forming fine holes in a resin flat plate by cutting,
A step of irradiating the surface of the flat plate on which the fine holes are formed with vacuum ultraviolet rays,
Including a step of forming a wall portion surrounding the fine holes of the flat plate irradiated with vacuum ultraviolet rays.
A method for producing a cell culture substrate, wherein the integrated irradiation amount of the vacuum ultraviolet rays in the irradiation step is greater than 0 and 2600 mJ / cm ² or less. The process of forming fine holes in a resin flat plate by cutting,
A step of irradiating the surface of the flat plate on which the fine holes are formed with vacuum ultraviolet rays,
A step of forming a cell culture substrate having holes transferred to the fine holes of the master on the bottom surface by using a mold prepared by using a flat plate having fine holes irradiated with vacuum ultraviolet rays as an electrocast master. Including
A method for producing a cell culture substrate, wherein the integrated irradiation amount of the vacuum ultraviolet rays in the irradiation step is greater than 0 and 2600 mJ / cm ² or less.