Container manufacturing method, container, and mask member

Abstract

To provide a container manufacturing method capable of providing a container with a simple structure suitable for imaging technology.SOLUTION: A container manufacturing method manufactures a container 2000 including at least one container body 2030 in which a sample 15 is placed, by performing a hydrophilic treatment on a container 200 to be treated including at least one container body 203 to be treated. The container manufacturing method includes a step S1 of covering a first region 221 of a bottom surface 209 of the container body 203 to be treated with a mask member 100, and a step S2 of performing a hydrophilic treatment on a second region 222 of the bottom surface 209 after the first region 221 is covered with the mask member 100. The hydrophilic treatment refers to a treatment that makes the water contact angle of the second region 222 smaller than the water contact angle of the second region 222 before the hydrophilic treatment is performed. The first region 221 refers to a region adjacent to the second region 222 from the outside of the second region 222.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to a container manufacturing method, a container, and a mask member.

Background Art

[0002] Patent Document 1 describes live cell imaging technology using state-of-the-art microscopes such as imaging cytometers, laser confocal microscopes, two-photon excitation laser microscopes, and ultrasonic microscopes. In live cell imaging, when observing cells attached to the bottom surface of a cell culture container as they are, an inverted microscope that can observe from the bottom surface side of the container is preferably used.

[0003] The cell culture container described in Patent Document 1 has a bottom portion provided with a through hole, a wall portion rising from the bottom portion, and a thin film portion welded to the bottom portion so as to cover the through hole. Since the bottom portion and the thin film portion contain the same type of synthetic resin, welding can be suitably performed even when the thickness of the thin film portion is very thin. As a result, a cell culture container suitable for cell observation with an inverted microscope and free from the influence of an adhesive on cultured cells can be obtained, and thus an image with high resolution can be acquired by the inverted microscope.

[0004] In particular, the surface on the cell culture compartment side of the thin film portion is subjected to a surface modification treatment. By the modification treatment, cell adhesiveness is optimized. The water contact angle of the surface after the modification treatment is 60° to 100°.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, the cell culture container described in Patent Document 1 has a thin film portion welded to the bottom so as to cover the through holes in the bottom, and the structure of the cell culture container is complicated.

[0007] The present invention has been made in view of the above problems, and an object thereof is to provide a container manufacturing method, a container, and a mask member capable of providing a container having a simple structure suitable for imaging technology.

Means for Solving the Problems

[0008] According to one aspect of the present invention, a container manufacturing method manufactures a container including at least one container body on which a sample is placed by performing a hydrophilic treatment on a processing target container including at least one processing target container body. The container manufacturing method includes a step of covering a first region of the bottom surface of the processing target container body with a mask member, and a step of performing the hydrophilic treatment on a second region of the bottom surface after the first region is covered with the mask member. The hydrophilic treatment indicates a treatment that makes the water contact angle of the second region smaller than the water contact angle of the second region before the hydrophilic treatment is performed. The first region indicates a region adjacent to the second region from the outside of the second region.

[0009] In one aspect of the present invention, it is preferable that the mask member has elasticity.

[0010] In one aspect of the present invention, it is preferable that the heat-resistant temperature of the mask member is 120° C. or higher.

[0011] In one aspect of the present invention, in the step of performing the hydrophilic treatment, it is preferable to perform the hydrophilic treatment so that the water contact angle of the second region is 10° or more and less than 60°.

[0012] In one aspect of the present invention, in the step of performing the hydrophilic treatment, it is preferable to perform the hydrophilic treatment with a non-liquid.

[0013] In one aspect of the present invention, it is preferable that the first region surrounds the second region.

[0014] In one aspect of the present invention, it is preferable that the container to be processed includes a plurality of the container bodies to be processed and a container base portion. It is preferable that the plurality of container bodies to be processed are arranged on the container base portion. It is preferable that the mask member includes a plurality of mask portions and a mask base portion. It is preferable that the plurality of mask portions are provided corresponding to the plurality of container bodies to be processed respectively. It is preferable that the plurality of mask portions are arranged on the mask base portion. In the step of covering the first region with the mask member, each of the plurality of mask portions preferably covers the first region of the corresponding container body to be processed.

[0015] In one aspect of the present invention, it is preferable that each of the plurality of mask portions protrudes downward from the mask base portion. It is preferable that each of the plurality of mask portions includes a mask bottom portion and a mask wall portion. The mask bottom portion preferably covers the first region of the corresponding container body to be processed. The mask wall portion preferably extends from the mask bottom portion to the mask base portion.

[0016] In one aspect of the present invention, it is preferable that the surface of the mask bottom portion facing the first region has a shape along the first region. It is preferable that the outer wall surface of the mask wall portion has a shape along the inner wall surface extending from the bottom surface of the container body to be processed.

[0017] In one aspect of the present invention, it is preferable that the mask bottom portion has an annular shape.

[0018] According to another aspect of the present invention, the container includes at least one container body on which a sample is disposed. The container body has a bottom surface and an inner wall surface extending from the bottom surface. The bottom surface has a first region and a second region where the sample is disposed. The first region indicates a region adjacent to the second region from the outside of the second region. The water contact angle of the second region is smaller than the water contact angle of the first region.

[0019] In one embodiment of the present invention, it is preferable that the first region surrounds the second region.

[0020] In one embodiment of the present invention, it is preferable that the container further includes a container base portion on which a plurality of the container bodies are disposed.

[0021] In one embodiment of the present invention, the water contact angle of the second region is preferably 10 degrees or more and less than 60 degrees.

[0022] According to still another aspect of the present invention, the mask member covers a part of the processing target container including a plurality of processing target container bodies. The mask member includes a plurality of mask portions and a mask base portion. The plurality of mask portions are provided corresponding to the plurality of processing target container bodies respectively. The plurality of mask portions are disposed on the mask base portion. Each of the plurality of mask portions prohibits hydrophilization treatment of the first region by covering the first region of the bottom surface of the corresponding processing target container body. The first region indicates a region adjacent to the second region where a sample can be disposed on the bottom surface from the outside of the second region. The hydrophilization treatment indicates a treatment that makes the water contact angle of the second region smaller than the water contact angle of the second region before the execution of the hydrophilization treatment. The mask portion does not prohibit the hydrophilization treatment of the second region by exposing the second region.

Advantages of the Invention

[0023] According to the present invention, it is possible to provide a container with a simple structure suitable for imaging technology.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Mode for Carrying Out the Invention

[0025] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, the same or corresponding parts are denoted by the same reference numerals and the description will not be repeated. In the present specification, for ease of understanding of the invention, the X-axis, Y-axis, and Z-axis orthogonal to each other may be described. Typically, the X-axis and Y-axis are parallel to the horizontal direction, and the Z-axis is parallel to the vertical direction.

[0026] With reference to FIGS. 1 to 9, a method for manufacturing a container according to an embodiment of the present invention will be described. In the method for manufacturing a container according to the present embodiment, a container 2000 (FIG. 8(a)) including at least one container body 2030 (FIG. 8(a)) in which a sample is disposed is manufactured by performing a hydrophilic treatment on a processing target container 200 (FIG. 1) including at least one processing target container body 203 (FIG. 1). The container 2000 is, for example, a well plate or a dish. The well plate has, for example, a size according to the ANSI / SBS standard.

[0027] FIG. 1 is a perspective view showing a mask member 100 and a processing target container 200. As shown in FIG. 1, when the container manufacturing method is executed, the mask member 100 is fitted into the processing target container 200. As a result, the mask member 100 covers a part of the processing target container 200.

[0028] The processing target container 200 includes a container base portion 201. The container base portion 201 has, for example, a substantially flat plate shape. In the example of FIG. 1, the container base portion 201 has a substantially flat plate shape that is substantially quadrangular.

[0029] The processing target container 200 further includes at least one processing target container body 203. In the example of FIG. 1, the processing target container 200 includes a plurality of processing target container bodies 203. The number of the processing target container bodies 203 is not particularly limited. The number of the processing target container bodies 203 is, for example, 6, 12, 24, or 96. The plurality of processing target container bodies 203 are arranged on the container base portion 201.

[0030] The processing target container body 203 is recessed with respect to the container base portion 201. That is, the processing target container body 203 protrudes downward from the container base portion 201. The processing target container body 203 is hollow and has a space 213. Further, the processing target container body 203 has an opening 215. The processing target container body 203 has, for example, a substantially bottomed cylindrical shape. In the example of FIG. 1, the processing target container body 203 has a substantially bottomed cylindrical shape.

[0031] Specifically, the container body 203 to be processed includes a bottom portion 205 and a wall portion 207. The bottom portion 205 has a substantially flat plate shape. In the example of FIG. 1, the bottom portion 205 has a substantially disc shape. The wall portion 207 extends from the bottom portion 205 to the container base portion 201. Specifically, the wall portion 207 extends from the outer edge of the bottom portion 205 to the container base portion 201. The wall portion 207 has a substantially cylindrical shape. In the example of FIG. 1, the wall portion 207 has a substantially cylindrical shape.

[0032] The material of the container 200 to be processed is, for example, a synthetic resin or glass. In this case, the synthetic resin is, for example, polystyrene, polyethylene, or polyethylene terephthalate. From the viewpoints of ease of processing and cost, preferably, the material of the container 200 to be processed is polystyrene.

[0033] Note that the container 200 to be processed is, for example, an integrally molded product. Also, for example, the container base portion 201 and the container body 203 to be processed may be separate parts, and the container body 203 to be processed may be joined to the container base portion 201. Further, for example, the container 200 to be processed may have the same heat resistance temperature as the mask member 100 described later. Further, for example, the container 200 to be processed may have the same alcohol resistance as the mask member 100 described later.

[0034] The mask member 100 includes a mask base portion 101. The mask base portion 101 has, for example, a substantially flat plate shape. In the example of FIG. 1, the mask base portion 101 has a substantially flat plate shape of a substantially quadrangle.

[0035] The mask member 100 further includes at least one mask portion 103. In the example of FIG. 1, the mask member 100 includes a plurality of mask portions 103. The number of mask portions 103 is not particularly limited. The number of mask portions 103 is, for example, 6, 12, 24, or 96. In the example of FIG. 1, the number of mask portions 103 is the same as the number of container bodies 203 to be processed. The plurality of mask portions 103 are provided corresponding to the plurality of container bodies 203 to be processed respectively. The plurality of mask portions 103 are arranged on the mask base portion 101.

[0036] The mask portion 103 is recessed with respect to the mask base portion 101. That is, the mask portion 103 protrudes downward from the mask base portion 101. Therefore, according to the present embodiment, the mask portion 103 can be easily fitted into the processing target container body 203. The mask portion 103 is hollow and has a space 113. Further, the mask portion 103 has a first opening 115 and a second opening 117.

[0037] Specifically, the mask portion 103 includes a mask bottom portion 105 and a mask wall portion 107. The mask bottom portion 105 has a substantially annular shape. In the example of FIG. 1, the mask bottom portion 105 has a substantially circular ring shape.

[0038] The mask wall portion 107 extends from the mask bottom portion 105 to the mask base portion 101. Specifically, the mask wall portion 107 extends from the outer edge of the mask bottom portion 105 to the mask base portion 101. The mask wall portion 107 has a substantially cylindrical shape. In the example of FIG. 1, the mask wall portion 107 has a substantially cylindrical shape.

[0039] The mask member 100 preferably has elasticity. This is because when the mask member 100 is fitted into the processing target container 200, it is possible to suppress the formation of a gap between the mask member 100 and the processing target container 200. For example, the elastic modulus of the material of the mask member 100 is smaller than the elastic modulus of the material of the processing target container 200. In the present embodiment, elasticity refers to the property of a substance that can undergo a reversible deformation in which deformation occurs due to an external force and returns to its original shape when the external force is removed. Elasticity can also be described as rubber elasticity.

[0040] The material of the mask member 100 is, for example, resin. In this case, the resin is preferably, for example, rubber. This is because rubber has elasticity. In this case, the rubber is, for example, natural rubber, nitrile rubber, or silicone rubber. The material of the mask member 100 is, for example, polydimethylsiloxane (PDMS).

[0041] The heat-resistant temperature of the mask member 100 (mask base portion 101 and mask portion 103) is preferably, for example, 120°C or higher. This is because deformation and deterioration of the mask member 100 can be suppressed even when sterilization, disinfection, or decontamination is performed in an environment at a temperature higher than room temperature. For example, if the heat-resistant temperature of the mask member 100 is 120°C or higher, deformation and deterioration of the mask member 100 can be prevented even when autoclave treatment is performed on the mask member 100. More preferably, the heat-resistant temperature of the mask member 100 is 150°C or higher. The heat-resistant temperature refers to the lowest temperature at which a substance undergoes alteration, deformation, or a change in state compared to before heating, and is a temperature higher than room temperature.

[0042] The mask member 100 preferably has alcohol resistance. This is because deformation and alteration of the mask member 100 can be prevented even when the mask member 100 is cleaned with alcohol such as ethanol. Alcohol resistance refers to the property of a substance that does not deform or alter when alcohol adheres to it.

[0043] The material of the mask member 100 preferably has resistance to hydrophilic treatment. Resistance to hydrophilic treatment refers to the property of a substance that does not undergo alteration, deformation, or a change in state in response to hydrophilic treatment. For example, carbon black (fine particles of carbon) may be included in the mask member 100.

[0044] Note that the mask member 100 is, for example, an integrally molded product. In this case, as a method for manufacturing the mask member 100, general molding methods can be used, for example, compression molding, extrusion molding, injection molding, or die molding can be used.

[0045] Also, for example, the mask base portion 101 and the mask portion 103 may be separate parts, and the mask portion 103 may be joined to the mask base portion 101.

[0046] FIG. 2(a) is a plan view showing the mask member 100. FIG. 2(b) is a plan view showing the processing target container 200. As shown in FIG. 2(b), the processing target container body 203 has a bottom surface 209. The bottom surface 209 is the upper surface of the bottom portion 205. The bottom surface 209 is substantially circular. The bottom surface 209 is a plane. The bottom surface 209 has a first region 221 and a second region 222. In FIG. 2(b), for ease of understanding, the boundary between the first region 221 and the second region 222 is shown by a virtual line (broken line).

[0047] The first region 221 is the region of the bottom surface 209 that is covered by the mask bottom portion 105 shown in FIG. 2(a). The first region 221 indicates a region that is adjacent to the second region 222 from the outside of the second region 222. In the example of FIG. 2(b), the first region 221 surrounds the second region 222. That is, the first region 221 has a substantially annular shape. In the example of FIG. 2(b), the first region 221 has a substantially circular ring shape. In this case, the second region 222 is substantially circular. Each of the first region 221 and the second region 222 is a continuous plane.

[0048] FIG. 3(a) is a cross-sectional view taken along line IIIA-IIIA of FIG. 2(a). As shown in FIG. 3(a), the mask portion 103 opens upward. That is, the first opening 115 is an opening on the upper end side of the mask portion 103. The first opening 115 is defined by the upper end portion of the mask wall portion 107. Also, the mask portion 103 opens downward. That is, the second opening 117 is an opening on the lower end side of the mask portion 103. The second opening 117 is defined by the mask bottom portion 105. The second opening 117 faces the first opening 115 in the vertical direction. Also, the mask wall portion 107 has an outer wall surface 111. The outer wall surface 111 is the outer peripheral surface of the mask wall portion 107.

[0049] Figure 3(b) is a cross-sectional view taken along line IIIB-IIIB of Figure 2(b). As shown in Figure 3(b), the processing target container body 203 is open upward. That is, the opening 215 is an opening on the upper end side of the processing target container body 203. The opening 215 is defined by the upper end portion of the wall portion 207. The opening 215 faces the bottom portion 205 in the vertical direction.

[0050] In addition, the processing target container body 203 further has an inner wall surface 211. The inner wall surface 211 is the inner peripheral surface of the wall portion 207. The inner wall surface 211 extends from the bottom surface 209 to the container base portion 201.

[0051] Here, as shown in Figure 3(a), the length d10 from the inner side surface to the outer side surface of the mask bottom portion 105 is smaller than the length d20 from the center line 1 of the mask portion 103 to the inner side surface of the mask bottom portion 105. The center line 1 passes through the center of the mask portion 103 and extends in the vertical direction. Although not limited, as an example, d20÷d10 is 1.5 or more and 8 or less. It is more preferable that d20÷d10 is 2 or more and 7 or less.

[0052] On the other hand, as shown in Figure 3(b), the length d1 from the inner edge to the outer edge of the first region 221 is smaller than the length d2 from the center line 2 of the processing target container body 203 to the outer edge of the second region 222. The center line 2 passes through the center of the processing target container body 203 and extends in the vertical direction. The inner edge of the first region 221 coincides with the outer edge of the second region 222. Although not limited, as an example, d2÷d1 is 1.5 or more and 8 or less. It is more preferable that d2÷d1 is 2 or more and 7 or less.

[0053] The first region 221 is the region of the bottom surface 209 of the processing target container body 203 that is covered by the mask bottom portion 105 when the mask portion 103 is fitted into the processing target container body 203. Therefore, the length d1 substantially coincides with the length d10. Also, the second region 222 is the region of the bottom surface 209 of the processing target container body 203 that is exposed from the second opening 117 when the mask portion 103 is fitted into the processing target container body 203. Therefore, the length d2 substantially coincides with the length d20.

[0054] Figures 4 to 6 are cross-sectional views showing the container manufacturing method according to the present embodiment. FIG. 4 is a cross-sectional view showing step S1 of the container manufacturing method. FIG. 5 is a cross-sectional view showing step S2 of the container manufacturing method. FIG. 6 is a cross-sectional view showing step S3 of the container manufacturing method.

[0055] As shown in FIGS. 4 to 6, the container manufacturing method according to the present embodiment includes step S1, step S2, and step S3. Note that steps S1 and S3 are executed by, for example, an actuator or an operator.

[0056] First, as shown in FIG. 4, in step S1, the mask portion 103 is fitted into the container body 203 to be processed. That is, the mask portion 103 is disposed in the space 213 (FIG. 3(b)) of the container body 203 to be processed.

[0057] Specifically, in step S1, the first region 221 of the bottom surface 209 of the container body 203 to be processed is covered with the mask member 100. That is, in step S1, each of the plurality of mask portions 103 covers the first region 221 of the corresponding container body 203 to be processed. Specifically, in step S1, the mask bottom portion 105 covers the first region 221 of the corresponding container body 203 to be processed.

[0058] Preferably, in step S1, the mask member 100 is in surface contact with the first region 221. Specifically, in step S1, it is preferable that the mask bottom portion 105 is in surface contact with the first region 221.

[0059] In the example of FIG. 4, in step S1, the mask portion 103 covers not only the first region 221 of the container body 203 to be processed but also the inner wall surface 211 of the container body 203 to be processed, and the mask base portion 101 covers the container base portion 201. In this case, it is preferable that the surface 109 of the mask bottom portion 105 facing the first region 221 has a shape along the first region 221. That is, it is preferable that the surface 109 is a flat surface. Further, it is preferable that the outer wall surface 111 of the mask wall portion 107 has a shape along the inner wall surface 211 extending from the bottom surface 209 of the container body 203 to be processed. In this preferred example, since the adhesion of the mask member 100 to the container 200 to be processed is improved, the first region 221 can be accurately covered by the mask bottom portion 105, and the hydrophilic treatment can be accurately performed on the second region 222 in step S2. Further, in the container 200 to be processed, it is possible to suppress the hydrophilic treatment from being performed on the regions other than the second region 222 in step S2. Further, the mask portion 103 can be accurately positioned with respect to the container body 203 to be processed. Furthermore, it is preferable that the outer wall surface 111 of the mask wall portion 107 is in contact with the inner wall surface 211 of the container body 203 to be processed. More preferably, the outer wall surface 111 is in surface contact with the inner wall surface 211.

[0060] Further, in step S1, the second opening 117 of the mask portion 103 exposes the second region 222.

[0061] Next, as shown in FIG. 5, after the first region 221 is covered by the mask member 100 (after step S1), in step S2, the hydrophilic treatment apparatus 300 performs a hydrophilic treatment on the second region 222 of the bottom surface 209 of the mask portion 103 through the second opening 117. That is, the mask portion 103 does not prohibit the hydrophilic treatment on the second region 222. The hydrophilic treatment refers to a treatment that makes the water contact angle of the second region 222 smaller than the water contact angle of the second region 222 before the execution of the hydrophilic treatment. In other words, the hydrophilic treatment refers to a treatment that makes the water contact angle of the second region 222 smaller than the water contact angle of the first region 221.

[0062] Specifically, in step S2, it is preferable to perform a hydrophilic treatment so that the water contact angle of the second region 222 is 10 degrees or more and less than 60 degrees.

[0063] In some cases, the property regarding the wettability of a substance after performing the hydrophilic treatment may be described as "hydrophilicity". Also, in some cases, the property of a substance regarding the wettability of a substance before performing the hydrophilic treatment, or the property regarding the wettability of a substance on which the hydrophilic treatment has not been performed may be described as "hydrophobicity". For example, a substance with a water contact angle of 10 degrees or more and less than 60 degrees can be defined as having hydrophilicity, and a substance with a water contact angle of 60 degrees or more and 80 degrees or less can be defined as having hydrophobicity.

[0064] Here, the water contact angle refers to the static contact angle of water (typically pure water) on the solid surface. In other words, the water contact angle refers to the angle formed by the liquid surface and the solid surface when the surface of stationary water (typically pure water) contacts the wall surface of the solid. For example, the water contact angle is measured by the sessile drop method described in JIS R3257:1999, but the measurement method is not particularly limited.

[0065] Also, in step S2, it is preferable that the hydrophilic treatment apparatus 300 performs the hydrophilic treatment with a non-liquid. That is, it is preferable to perform the hydrophilic treatment by a dry process. "Non-liquid" indicates an electromagnetic wave or a substance that is not a liquid. "Electromagnetic wave" is, for example, light. "Substance that is not a liquid" is, for example, plasma, electrons, or gas. According to this preferable example, after performing the hydrophilic treatment, cleaning of the container 2000 and the mask member 100 becomes unnecessary, so the manufacturing man-hours of the container 2000 can be reduced. Also, since no liquid is used for the hydrophilic treatment, no liquid for the hydrophilic treatment enters between the mask bottom 105 and the first region 221. As a result, it is possible to suppress the hydrophilic treatment from being performed on the first region 221.

[0066] For example, the hydrophilization treatment device 300 performs a hydrophilization treatment on the second region 222 by irradiating plasma through the second opening 117 to the second region 222 of the container body 203 to be treated. In this case, as the plasma treatment, vacuum plasma or atmospheric pressure plasma can be used, but it is preferable to use atmospheric pressure plasma from the viewpoints of cost and convenience.

[0067] Alternatively, for example, the hydrophilization treatment device 300 performs a hydrophilization treatment on the second region 222 by irradiating ultraviolet rays through the second opening 117 to the second region 222. As the ultraviolet light source, the type of lamp and the like are not particularly limited, and ultraviolet A wave (UV-A), ultraviolet B wave (UV-B), etc. can be used.

[0068] Alternatively, for example, the hydrophilization treatment device 300 performs a hydrophilization treatment on the second region 222 by performing a corona discharge treatment through the second opening 117 to the second region 222. The corona discharge treatment is a treatment in which electrons generated using a high-frequency high voltage are made to collide with the second region 222.

[0069] Note that the treatment area is smaller for plasma treatment and ultraviolet irradiation treatment than for corona discharge treatment. Therefore, from the viewpoint of the treatment area, plasma treatment and ultraviolet irradiation treatment have greater advantages.

[0070] Alternatively, for example, the hydrophilization treatment device 300 performs a hydrophilization treatment on the second region 222 by supplying oxygen or an allotrope of oxygen through the second opening 117 to the second region 222. The allotrope of oxygen is, for example, ozone.

[0071] Note that in step S2, the hydrophilization treatment device 300 or the operator may perform the hydrophilization treatment with a liquid. That is, the hydrophilization treatment may be performed by a wet process. For example, the hydrophilization treatment device 300 or the operator performs a hydrophilization treatment on the second region 222 by supplying a liquid for hydrophilization treatment through the second opening 117 to the second region 222 of the container body 203 to be treated.

[0072] For example, a hydrophilic treatment may be performed on the second region 222 with a solution containing a protein. The solution containing a protein is, for example, a BSA (Bovine serum albumin) solution. In this case, the hydrophilic treatment is performed by utilizing the amphiphilic property of BSA, which is a kind of protein.

[0073] Alternatively, for example, a hydrophilic treatment may be performed on the second region 222 with a surfactant. Alternatively, for example, a hydrophilic treatment may be performed on the second region 222 with a liquid containing a silane-based compound.

[0074] Also, in step S2, the hydrophilic treatment is not performed on the first region 221 and the inner wall surface 211 of the container body 203 to be processed, nor on the container base portion 201. This is because the mask portion 103 covers the first region 221 and the inner wall surface 211, and the mask base portion 101 covers the container base portion 201. That is, the mask portion 103 prohibits the hydrophilic treatment of the first region 221 and the inner wall surface 211, and the mask base portion 101 prohibits the hydrophilic treatment of the container base portion 201.

[0075] Note that it is preferable to select a hydrophilic treatment that has a small influence on the transmittance and haze value of the bottom surface 209 of the container body 203 to be processed. This is for effectively performing image acquisition by imaging technology.

[0076] Next, as shown in FIG. 6, after the hydrophilic treatment is performed (after step S2), in step S3, the mask member 100 is removed from the container 200 to be processed. As a result, the container 2000 is completed. Since the hydrophilic treatment is performed on the second region 222 in step S2, a hydrophilic layer 2010 having hydrophilicity is formed on the second region 222. The water contact angle of the hydrophilic layer 2010 is smaller than the water contact angle of the second region 222 before the hydrophilic treatment is performed. That is, the water contact angle of the hydrophilic layer 2010 is smaller than the water contact angle of the first region 221. The first region 221 has hydrophobicity. After step S3, the container manufacturing method ends.

[0077] As described above with reference to FIGS. 4 to 6, according to the present embodiment, a hydrophilic treatment is performed on the second region 222 of the container body 203 to be processed. Therefore, in the container body 301 which is the container body 203 to be processed after the hydrophilic treatment is performed, the water contact angle of the second region 222 becomes smaller as compared with that before the hydrophilic treatment is performed. That is, hydrophilicity is imparted to the second region 222. Therefore, the sample disposed in the second region 222 spreads out and wets in the second region 222, and the thickness of the sample in the vertical direction becomes smaller as compared with the case where the sample is disposed on a hydrophobic surface. As a result, the container 2000 suitable for the imaging technique can be provided. That is, according to the container manufacturing method according to the present embodiment, the container 2000 suitable for the imaging technique can be manufactured.

[0078] The imaging technique is a technique for measuring information of a sample and imaging or visualizing it. In the imaging technique, for example, information of a sample is measured by light (including electromagnetic waves, radiation, visible light, and invisible light), magnetism, or ultrasonic waves. Therefore, if the thickness of the sample is large, for example, it may take time for measurement or the accuracy of image analysis may decrease. For this reason, when acquiring information of a sample by the imaging technique, it is effective to reduce the thickness of the sample by imparting hydrophilicity to the second region 222 where the sample is disposed.

[0079] Examples of the imaging technique include a phase contrast microscope, a cell imaging device, and an optical coherence tomography (OCT).

[0080] Further, in the present embodiment, the bottom portion 2050 of the container body 2030 having the hydrophilic second region 222 is formed without using a plurality of parts. Therefore, the structure of the container body 2030 is simpler as compared with the case of using a plurality of parts.

[0081] As a result, according to this embodiment, a container 2000 with a simple structure suitable for imaging technology can be provided. That is, according to the container manufacturing method according to this embodiment, a container 2000 with a simple structure suitable for imaging technology can be manufactured.

[0082] Furthermore, in this embodiment, with respect to the first region 221 adjacent to the second region 222 from the outside, since it is covered by the mask bottom 105 during the hydrophilization treatment, the hydrophilization treatment is not performed. Therefore, the first region 221 has hydrophobicity. As a result, even when the container 2000 is inclined with respect to the horizontal plane, it is possible to suppress the sample from adhering to the inner wall surface 211 of the container body 2030.

[0083] Preferably, in each of the container body 203 to be processed and the container body 2030, the first region 221 surrounds the second region 222. According to this preferred example, even when the container 2000 is inclined with respect to the horizontal plane, it is possible to more effectively suppress the sample from adhering to the inner wall surface 211 of the container body 2030. In particular, when the mask bottom 105 is substantially annular, the first region 221 surrounding the second region 222 can be easily formed.

[0084] Also, preferably, the mask member 100 includes a plurality of mask portions 103, and the container 200 to be processed includes a plurality of container bodies 203 to be processed. According to this preferred example, in a plurality of container bodies 203 to be processed, the hydrophilization treatment can be performed simultaneously on a plurality of second regions 222. Also, preferably, the container 2000 includes a plurality of container bodies 301. According to this preferred example, a plurality of samples can be processed simultaneously. For example, when the sample is a cell suspension, a plurality of samples can be cultured simultaneously.

[0085] In addition, in the present embodiment, in the mask member 100, each of the plurality of mask portions 103 covers the first region 221 of the bottom surface 209 of the corresponding processing target container body 203, thereby prohibiting the hydrophilic treatment of the first region 221. The first region 221 indicates a region adjacent to the second region 222 where a sample can be placed on the bottom surface 209 from the outside of the second region 222. Further, the mask portion 103 does not prohibit the hydrophilic treatment of the second region 222 by exposing the second region 222 through the second opening 117. As a result, by masking the processing target container 200 with the mask member 100, in the container 2000 as a product, a hydrophobic first region 221 and a hydrophilic second region 222 can be easily formed.

[0086] Furthermore, according to the present embodiment, it is preferable that the mask member 100 has elasticity. According to this preferable example, the mask bottom portion 105 is more likely to adhere to the first region 221 of the processing target container body 203 than when it does not have elasticity. As a result, when performing the hydrophilic treatment, it is possible to prevent hydrophilicity from being imparted to the first region 221.

[0087] Furthermore, in the present embodiment, in step S2, it is preferable to perform the hydrophilic treatment so that the water contact angle of the second region 222 is 10 degrees or more and less than 60 degrees. According to this preferable example, since the water contact angle is 10 degrees or more, it is possible to suppress the sample from peeling off from the second region 222. In particular, if the water contact angle is too small, depending on the environment such as the container 2000 being tilted and the characteristics of the sample, the sample may peel off from the second region 222. In contrast, in the present embodiment, by setting the water contact angle of the second region 222 to 10 degrees or more, it is possible to suppress the sample from peeling off from the second region 222.

[0088] In the present embodiment, for example, a substance with a water contact angle of 10 degrees or more and less than 60 degrees can be defined as having hydrophilicity, and a substance with a water contact angle of 60 degrees or more and 80 degrees or less can be defined as having hydrophobicity.

[0089] Also, as described with reference to FIG. 3(a), regarding the length d10 of the first region 221 and the length d20 of the second region 222, as an example, d20÷d10 is 1.5 or more and 8 or less. For example, when the radius of the bottom surface 209 of the container body 203 to be processed is 4 mm, d10 = 1.5 mm and d20 = 2.5 mm. Also, when the radius of the bottom surface 209 is 4 mm, for example, d10 is preferably 1.5 mm or less. More preferably, d20÷d10 is 2 or more and 7 or less. Even when the area of the bottom surface 209 of the container body 203 to be processed is relatively small and the mask bottom 105 is also relatively small, by setting "d20÷d10" to 7 or less, the strength of the mask bottom 105 can be ensured and the adhesion between the mask bottom 105 and the first region 221 can be ensured. For example, when the radius of the bottom surface 209 of the container body 203 to be processed is 4 mm, d10 = 0.5 mm and d20 = 3.5 mm. Also, by setting "d20÷d10" to 1.5 or more or 2 or more, in the container body 203 shown in FIG. 3(b), it is possible to suppress the first region 221 from becoming too large with respect to the second region 222 where the sample is placed.

[0090] FIG. 7 is a plan view showing the container 2000 according to the present embodiment. The container 2000 shown in FIG. 7 is, for example, a well plate. In this case, for example, the container body 301 is a well. Hereinafter, the differences between the container 2000 and the container 200 to be processed will be mainly described.

[0091] The container 2000 includes a container base portion 201 and at least one container body 2030. In the example of FIG. 7, the container 2000 includes a plurality of container bodies 2030. Since the container body 2030 is generated from the container body 203 to be processed (FIGS. 4 to 6), the shape and number of the container body 2030 are the same as the shape and number of the container body 203 to be processed, respectively.

[0092] FIG. 8(a) is a cross-sectional view taken along line VIIIA-VIIIA of FIG. 7. As shown in FIG. 8(a), the container body 2030 includes a bottom portion 2050 and a wall portion 207. The shape of the bottom portion 2050 is the same as the shape of the bottom portion 205 (FIGS. 1 to 6) of the container body 203 to be processed. In the container body 2030, the wall portion 207 extends from the bottom portion 2050 to the container base portion 201. Specifically, the wall portion 207 extends from the outer edge of the bottom portion 2050 to the container base portion 201.

[0093] As shown in FIGS. 7 and 8(a), the container body 2030 has a bottom surface 209. The bottom surface 209 is the upper surface of the bottom portion 2050. The bottom surface 209 has a first region 221 and a second region 222. Also in the container body 2030, each of the first region 221 and the second region 222 is a continuous plane. A hydrophilic layer 2010 is formed in a substantially circular shape in the second region 222. In FIGS. 7 and 8(a), for ease of understanding, the hydrophilic layer 2010 is hatched.

[0094] The length d1 from the inner edge to the outer edge of the first region 221 is smaller than the length d2 from the center line 2 of the container body 2030 to the outer edge of the second region 222. The center line 2 passes through the center of the container body 2030 and extends in the vertical direction. The center line 2 of the container body 2030 coincides with the center line 2 (FIG. 3(b)) of the container body 203 to be processed. The inner edge of the first region 221 coincides with the outer edge of the second region 222. Although not limited, as an example, d2÷d1 is 1.5 or more and 8 or less. It is more preferable that d2÷d1 is 2 or more and 7 or less. The length d1 in the container body 2030 substantially coincides with the length d10 (FIG. 3(a)) in the mask portion 103. Also, the length d2 in the container body 2030 substantially coincides with the length d20 (FIG. 3(a)) in the mask portion 103.

[0095] Also, the container body 2030 has a space 213 and is open upward. That is, the container body 2030 has an opening 215. The opening 215 is an opening on the upper end side of the container body 2030. The opening 215 faces the bottom portion 2050 in the vertical direction. Further, the container body 2030 has an inner wall surface 211.

[0096] Next, with reference to FIGS. 8(a) and 8(b), the water contact angles before and after the hydrophilic treatment will be described. FIG. 8(b) is a cross-sectional view showing the container body 203 to be treated before the hydrophilic treatment is performed.

[0097] In FIG. 8(a), water 10 is shown in the second region 222 for explaining the water contact angle θ1 of the second region 222 after the hydrophilic treatment is performed. Also, in FIG. 8(b), water 10 is shown in the second region 222 for explaining the water contact angle θ2 of the second region 222 before the hydrophilic treatment is performed.

[0098] As shown in FIGS. 8(a) and 8(b), the water contact angle θ1 of the second region 222 of the container body 2030 is smaller than the water contact angle θ2 of the second region 222 of the container body 203 to be treated. Also, since the hydrophilic treatment is not performed on the first region 221 (FIG. 4), the water contact angle θ3 of the first region 221 is the same as the water contact angle θ2 of the second region 222 of the container body 203 to be treated. Therefore, the water contact angle θ1 of the second region 222 of the container body 2030 is smaller than the water contact angle θ3 of the first region 221 of the container body 2030.

[0099] The water contact angle θ1 of the second region 222 of the container body 2030 is preferably, for example, 10 degrees or more and less than 60 degrees. Note that, for example, each of the water contact angle θ3 of the first region 221 of the container body 2030, the water contact angle θ3 of the first region 221 of the container body 203 to be treated, and the water contact angle θ2 of the second region 222 of the container body 203 to be treated is, for example, 60 degrees or more and 80 degrees or less.

[0100] Next, with reference to FIG. 9, the state of the cells 20 in the sample 15 will be described. FIG. 9(a) is a view showing the state of the cells 20 included in the sample 15 disposed in the container body 2030. FIG. 9(b) is a view showing the state of the cells 20 included in the sample 15 disposed in the container body 301 according to the comparative example. FIG. 9(c) is a view showing the fluorescence attenuation from the cells 20 included in the sample 15 disposed in the container body 301 according to the comparative example.

[0101] As shown in Fig. 9(a), the sample 15 is placed in the container body 2030 of the container 2000. Specifically, the sample 15 is placed in the second region 222 of the container body 2030. That is, the second region 222 is the region of the bottom surface 209 where the sample 15 can be placed.

[0102] The container 2000 is, for example, a cell culture container. In this case, the sample 15 includes, for example, a plurality of cells 20. The cells can be single cells or cell aggregates. The type of cells is not particularly limited. For example, the cells are animal cells. The animal cells are, for example, human, mouse, or rat cells.

[0103] For example, the sample 15 is a cell suspension. The cell suspension is, for example, a substance in which the cells 20 are suspended in a gel, or a substance in which the cells 20 are suspended in a gel and mixed with a culture medium. The gel is, for example, agarose, collagen, alginic acid, Matrigel (registered trademark), or Vitrigel (registered trademark). The gel is, for example, a hydrogel. The culture medium is, for example, a liquid culture medium. When the container 2000 is a cell culture container, the second region 222 of the container body 2030 is a culture surface.

[0104] In the container body 2030 of the present embodiment, a hydrophilic layer 2010 is formed in the second region 222. Therefore, the water contact angle θ1 of the second region 222 is smaller than the water contact angle θ3 of the first region 221. As a result, the second region 222 is easily wetted, and the sample 15 spreads in the horizontal direction. Thus, since the height H1 of the apex of the sample 15 with respect to the second region 222 is relatively low, the plurality of cells 20 are dispersed inside the sample 15 with a relatively low height.

[0105] In Fig. 9(a), although the drawing is simplified, the cells 20 in the sample 15 placed in the second region 222 of the container body 2030 are, for example, three-dimensionally cultured. Three-dimensional culture is different from culturing cells in a single layer in a planar manner. It is a culture in which cells are grown while adhering to each other to form a three-dimensional cell mass or spheroid.

[0106] On the other hand, as shown in FIG. 9(b), the sample 15 is disposed on the bottom surface 309 of the container body 301 according to the comparative example. The amount of the sample 15 shown in FIG. 9(b) is the same as the amount of the sample 15 shown in FIG. 9(a). No hydrophilic treatment is performed on the bottom surface 309 of the comparative example. Therefore, the water contact angle of the bottom surface 309 of the comparative example is larger than the water contact angle θ1 of the second region 222 of the present embodiment. As a result, the bottom surface 309 is difficult to wet, and the sample 15 is in a dome shape. The dome shape indicates a substantially hemispherical shape.

[0107] In particular, when the sample 15 (cell suspension) in which the cells 20 are suspended in the gel solution is disposed on the bottom surface 309 of the container body 301, the sample 15 becomes dome-shaped. This is because the cell suspension containing the gel has high viscosity. Culturing the cells 20 in the dome-shaped sample 15 is a kind of three-dimensional culture.

[0108] For example, when dropping the sample 15 containing the gel into the container body 301, the shape of the sample 15 on the bottom surface 309 is determined by the composition, concentration, surface tension, temperature, and liquid volume of the gel, the discharge rate of the sample 15, and the state of the bottom surface 309 of the container body 301. Generally, when culturing the cells 20 by encapsulating them with a gel containing collagen or laminin such as Matrigel, the gel concentration is cultured at 20 (v / v)% or more and 100 (v / v)% or less. In this case, the shape of the sample 15 depends on the properties of the gel, but often becomes dome-shaped as shown in FIG. 9(b). For example, in the case of Matrigel, it becomes solid at -20°C, becomes liquid around 0°C to 4°C, and becomes gel-like and solidifies again at 37°C.

[0109] When culturing the cells 20 in the dome-shaped sample 15 and observing and analyzing the cells during the culture process using imaging technology, it may be difficult to acquire an image of the cells 20. The reasons are as follows.

[0110] That is, as shown in Fig. 9(b), since the sample 15 is dome-shaped, the height H2 of the apex of the sample 15 with respect to the bottom surface 309 of the container body 301 is relatively high. Therefore, the plurality of cells 20 are dispersed inside the sample 15 with a relatively high height. That is, the plurality of cells 20 are dispersed in the vertical direction. As a result, for example, an overlap may occur between the cells 20 in the vertical direction, making it difficult to acquire an image and perform image analysis using imaging technology. Also, for example, when imaging or visualizing the cells 20 cultured three-dimensionally by imaging technology, it is required to measure and image the cells 20 at different positions in the vertical direction within the dome-shaped sample 15. As a result, it may take time to acquire an image. In particular, the larger the number of container bodies 301, the more time is required for image acquisition.

[0111] In contrast, in the present embodiment, as shown in Fig. 9(a), the sample 15 is disposed in the second region 222 where a hydrophilic treatment is performed on the container body 2030. Therefore, the height H1 of the sample 15 in the second region 222 is lower than the height H2 of the dome-shaped sample 15 according to the comparative example. Therefore, it is difficult for an overlap to occur between the cells 20 in the vertical direction. As a result, it becomes easier to acquire an image using imaging technology, and the accuracy of image analysis also improves. Also, since the height H1 of the sample 15 in the second region 222 is low, the number of measurement times and imaging times at each position in the vertical direction by imaging technology is reduced. As a result, the time required for measuring and imaging the cells 20 by imaging technology can be shortened. Also, in the present embodiment, since the height H1 of the sample 15 in the second region 222 is low, the shape of the sample 15 is stable on the container body 2030.

[0112] Also, in the present embodiment, a hydrophilic treatment is not performed on the first region 221 of the container body 2030. That is, the first region 221 has hydrophobicity. Therefore, for example, even when the container 2000 is tilted during the culturing operation, it is possible to prevent the sample 15 from adhering to the inner wall surface 211 of the container body 2030.

[0113] Here, when observing the form of gel culture or evaluating the localization of a protein that appears to be specifically expressed, for example, when visually evaluating a protein specific to cells, techniques such as immunostaining are used. Generally, in immunostaining, a primary antibody that binds to the protein is reacted with a secondary antibody to which a fluorescent substance that recognizes the primary antibody is attached. Then, the fluorescent substance is irradiated with excitation light or the like, and the emitted fluorescence is detected. In this case, if the sample 15 has a certain thickness, attenuation and scattering of the excitation light occur, resulting in weaker detectable fluorescence.

[0114] For example, as shown in FIG. 9(c), in the dome-shaped sample 15 disposed on the bottom surface 309 of the comparative example, for the cells 20 with a low vertical height, the attenuation of fluorescence is small. On the other hand, for the cells 20 with a high vertical height, the attenuation of fluorescence is large. Therefore, the detection accuracy by immunostaining decreases.

[0115] In contrast, in the present embodiment, as shown in FIG. 9(a), the sample 15 is disposed in the second region 222 where a hydrophilic treatment is performed in the container body 2030. Therefore, the height H1 of the sample 15 in the second region 222 is lower than the height H2 of the dome-shaped sample 15 according to the comparative example. Therefore, the attenuation of fluorescence is small in a large number of cells 20 included in the sample 15. As a result, a decrease in the detection accuracy by immunostaining can be suppressed.

[0116] As described above with reference to FIG. 9, according to the present embodiment, hydrophilicity is imparted to the second region 222 of the container body 2030. Therefore, since the height H1 of the sample 15 disposed in the second region 222 is relatively low, the vertical dispersion of a plurality of cells 20 can be reduced within the sample 15.

[0117] As a result, for example, when evaluating the three-dimensional culture using the sample 15 containing the gel by image analysis using imaging technology, it leads to an improvement in the accuracy of image analysis by shortening the imaging time and avoiding the overlap of the cells 20, and a stable culture method can be provided. That is, in the present embodiment, a culture suitable for imaging technology becomes possible. Therefore, for example, in observing the culture process of organoids, it is possible to shorten the imaging time while reducing the running cost. Further, for example, in imaging technology, it becomes easier to acquire an image with the foci coinciding, contributing to an improvement in the accuracy of image analysis.

[0118] (First Modification Example) Referring to FIG. 10, a first modification example of the present embodiment will be described. The first modification example is mainly different from the above-described embodiment described with reference to FIGS. 1 to 9 in that the mask bottom portion 105 and the mask wall portion 107 are substantially flush. Hereinafter, the points in which the first modification example differs from the above-described embodiment will be mainly described.

[0119] FIG. 10 is a cross-sectional view showing a mask member 100 used in the container manufacturing method according to the first modification example. As shown in FIG. 10, the mask bottom portion 105 and the mask wall portion 107 are substantially flush. Specifically, the inner surface 132 of the mask bottom portion 105 and the inner wall surface 131 of the mask wall portion 107 are substantially flush.

[0120] That is, in the first modification example, one wall portion 130 is formed by the mask bottom portion 105 and the mask wall portion 107. The wall portion 130 has a substantially cylindrical shape. In the example of FIG. 10, the wall portion 130 has a substantially cylindrical shape. Note that, for example, the wall portion 130 may have a substantially rectangular cylindrical shape. Further, the wall portion 130 may have a substantially cylindrical shape and a tapered shape that tapers downward.

[0121] (Second Modification Example) Referring to FIGS. 11 and 12, a second modification of the present embodiment will be described. In the second modification, the processing target container body 203 and the container body 2030 have a curved surface 217 continuous with the bottom surface 209, which is mainly different from the above-described embodiment described with reference to FIGS. 1 to 9. Hereinafter, the differences between the second modification and the above-described embodiment will be mainly described.

[0122] FIG. 11 is a cross-sectional view showing a mask member 100 and a processing target container 200 used in the container manufacturing method according to the second modification. FIG. 11 shows a state corresponding to step S2 of FIG. 5.

[0123] As shown in FIG. 11, in the second modification, the mask wall portion 107 of the mask portion 103 includes a curved portion 121. The curved portion 121 is curved. The curved portion 121 is located at the lower end portion of the mask wall portion 107. The curved portion 121 is connected to the mask bottom portion 105.

[0124] On the other hand, the inner wall surface 211 of the processing target container body 203 has a curved surface 217. The curved surface 217 is curved. The curved surface 217 is located at the lower end portion of the inner wall surface 211. The curved surface 217 is connected to the bottom surface 209. Specifically, the curved surface 217 is connected to the first region 221.

[0125] FIG. 12 is a cross-sectional view showing a container body 2030 manufactured by the container manufacturing method according to the second modification. As shown in FIG. 12, the inner wall surface 211 of the container body 2030 has a curved surface 217. The curved surface 217 is curved. The curved surface 217 is located at the lower end portion of the inner wall surface 211. The curved surface 217 is connected to the bottom surface 209. Specifically, the curved surface 217 is connected to the first region 221.

[0126] In the second modification, the curved surface 217 of the container body 2030 is located outside the first region 221. In addition, the first region 221 has hydrophobicity. Therefore, even when the container 2000 is inclined with respect to the horizontal plane, it is possible to suppress the sample 15 from adhering to the curved surface 217.

[0127] (Third Modification) Referring to FIG. 13, a third modification of the present embodiment will be described. The third modification is mainly different from the above-described embodiment described with reference to FIGS. 1 to 9 in that the mask bottom 105 and the first region 221 are not substantially annular. Hereinafter, the differences between the third modification and the above-described embodiment will be mainly described.

[0128] FIG. 13(a) is a plan view showing a mask member 100 used in the container manufacturing method according to the third modification. As shown in FIG. 13(a), the mask bottom 105 includes a plurality of bottom elements 108. The bottom element 108 has a substantially arc shape in plan view. Also, a gap 119 is formed between the bottom elements 108 adjacent to each other in the circumferential direction 151. The gap 119 constitutes a part of the second opening 117. Note that there may be one gap 119 for one mask portion 103.

[0129] FIG. 13(b) is a plan view showing a container 200 to be processed used in the container manufacturing method according to the third modification. As shown in FIG. 13(b), in the container body 203 of the container 200 to be processed, the first region 221 of the bottom surface 209 includes a plurality of first region elements 225. The first region element 225 has a substantially arc shape in plan view. Also, a second region element 223 is disposed between the first region elements 225 adjacent to each other in the circumferential direction 151. The second region element 223 constitutes a part of the second region 222. Note that there may be one second region element 223 for one container body 203 to be processed.

[0130] Although not shown, in the third modification, in the container body 2030 of the container 2000, the same first region 221 and second region 222 as those shown in FIG. 13(b) are also formed.

[0131] (Fourth Modification) Referring to FIGS. 14 to 16, a fourth modification of the present embodiment will be described. In the fourth modification, each of the mask portion 103, the container body 203 to be processed, and the container body 2030 mainly differs from the above-described embodiment described with reference to FIGS. 1 to 9 in that each has a tapered shape. Hereinafter, the points in which the fourth modification differs from the above-described embodiment will be mainly described.

[0132] FIG. 14 is a perspective view showing a mask member 100 and a container 200 to be processed according to the fourth modification. As shown in FIG. 14, the mask portion 103 has a tapered shape. Specifically, the mask wall portion 1107 of the mask portion 103 has a tapered shape. That is, the mask wall portion 1107 has a hollow truncated inverted cone shape.

[0133] Further, the container body 203 to be processed has a tapered shape. Specifically, the wall portion 231 of the container body 203 to be processed has a tapered shape. That is, the wall portion 231 has a hollow truncated inverted cone shape.

[0134] FIG. 15 is a cross-sectional view showing a mask member 100 and a container 200 to be processed used in the container manufacturing method according to the fourth modification. FIG. 15 shows a state corresponding to step S2 in FIG. 5.

[0135] As shown in FIG. 15, in the fourth modification, in a cross-sectional view, the mask wall portion 1107 has a tapered shape from the mask base portion 101 toward the mask bottom portion 105. Further, in a cross-sectional view, the wall portion 231 of the container body 203 to be processed has a tapered shape from the container base portion 201 toward the bottom portion 205. Further, the outer wall surface 111 of the mask wall portion 1107 has a shape along the inner wall surface 211 of the container body 203 to be processed.

[0136] FIG. 16 is a cross-sectional view showing a container 2000 manufactured by the container manufacturing method according to the fourth modification. As shown in FIG. 16, in a cross-sectional view, the wall portion 231 of the container body 2030 has a tapered shape from the container base portion 201 toward the bottom portion 2050.

[0137] In addition, in FIGS. 14 to 16, each of the mask wall portion 1107, the wall portion 231 of the container body 203 to be processed, and the wall portion 231 of the container body 2030 may have, for example, a hollow inverted truncated pyramid shape.

[0138] (Fifth Modification Example) Referring to FIGS. 17 and 18, a fifth modification example of the present embodiment will be described. In the fifth modification example, the main difference from the above-described embodiment described with reference to FIGS. 1 to 9 is that each of the mask wall portion 1117, the wall portion 241 of the container body 203 to be processed, and the wall portion 241 of the container body 2030 has a substantially rectangular tube shape. Hereinafter, the differences between the fifth modification example and the above-described embodiment will be mainly described.

[0139] FIG. 17 is a perspective view showing a mask member 100 and a container 200 to be processed used in the container manufacturing method according to the fifth modification example. As shown in FIG. 17, the mask portion 103 has a substantially rectangular tube shape. Specifically, the mask wall portion 1117 of the mask portion 103 has a substantially rectangular tube shape. Further, the mask bottom portion 1051 has a substantially rectangular ring shape.

[0140] In addition, the container body 203 to be processed has a substantially bottomed rectangular tube shape. Specifically, the wall portion 241 of the container body 203 to be processed has a substantially rectangular tube shape.

[0141] FIG. 18 is a plan view showing the mask member 100 used in the container manufacturing method according to the fifth modification example. As shown in FIG. 18, the bottom portion 245 of the container body 203 to be processed has a substantially square shape in plan view. Further, the bottom surface 209 of the container body 203 to be processed has a substantially square shape in plan view. The first region 221 has a substantially rectangular ring shape. The second region 222 has a substantially square shape.

[0142] Next, the present invention will be specifically described based on examples, but the present invention is not limited by the following examples.

Examples

[0143] Referring to FIGS. 19 and 20, examples and comparative examples of the present invention will be described. In the examples, a hydrophilic treatment was performed on the container, while in the comparative examples, no hydrophilic treatment was performed on the container. Hereinafter, for the sake of easy understanding, the reference numerals used in the above embodiments and the above comparative examples will be used to describe the examples and comparative examples.

[0144] FIG. 19(a) is a photograph showing the sample 15 disposed on the bottom surface 309 of the container body 301 according to the comparative example. The outer edge of the sample 15 on the bottom surface 309 is indicated by a circle 31. FIG. 19(b) is a photograph showing the sample 15 disposed in the second region 222 of the container body 2030 according to the example of the present invention. The outer edge of the sample 15 in the second region 222 is indicated by a circle 41.

[0145] It was observed that the sample 15 according to the example (FIG. 19(b)) spread more than the sample 15 according to the comparative example (FIG. 19(a)). Also, it was observed that the plurality of cells 20 included in the sample 15 according to the example were more horizontally dispersed than the plurality of cells 20 included in the sample 15 according to the comparative example.

[0146] FIG. 20(a) is a diagram showing the cells 20 included in the sample 15 disposed on the bottom surface 309 of the container body 301 according to the comparative example. The upper limit in the vertical direction of the sample 15 is indicated by a dashed line 7. FIG. 20(b) is a diagram showing the cells 20 included in the sample 15 disposed in the second region 222 (hydrophilic layer 2010) of the container body 2030 according to the example of the present invention. The upper limit in the vertical direction of the sample 15 is indicated by a dashed line 8.

[0147] It was observed that the sample 15 according to the example (FIG. 20(b)) had a lower height in the vertical direction than the sample 15 according to the comparative example (FIG. 20(a)). Specifically, when the thickness of the sample 15 was measured with an OCT observation device "Cell3iMager Estier", the maximum thickness was 300 μm in the comparative example, while it was 100 μm in the example.

[0148] Also, it was observed that the plurality of cells 20 included in the sample 15 according to the example had less vertical dispersion than the plurality of cells 20 included in the sample 15 according to the comparative example. Also, it was observed that the plurality of cells 20 included in the sample 15 according to the example had less vertical overlap than the plurality of cells 20 included in the sample 15 according to the comparative example.

[0149] Next, the experimental conditions of the examples and comparative examples will be described. The sample 15 was prepared as follows. That is, as the sample 15, HCT116 cells were suspended in Matrigel (#356234, Corning) and seeded in a 96-well plate (#3596, Corning), and the gel shape was confirmed. The 96-well plate was used as the processing target container 200 of the example and the container of the comparative example. Therefore, the processing target container 200 of the example had 96 processing target container main bodies 203 (wells), and the container of the comparative example had 96 container main bodies 301 (wells).

[0150] In the 96-well plate of the example, the mask member 100 was fitted, and the first region 221 of each well (the processing target container main body 203 of the example) was masked. Then, a bovine serum albumin (BSA) solution (#015-23871, Fujifilm Wako Pure Chemical Corporation) diluted with phosphate-buffered saline (PBS: #14249-24, Nacalai Tesque) was added to each well (the processing target container main body 203 of the example) at a predetermined concentration (2%) in an amount of 100 μL, and the treatment was performed at room temperature for 1 hour. Then, the BSA solution and the mask member 100 were removed. That is, in the example, the second region 222 of each well (the processing target container main body 203 of the example) was hydrophilized with the BSA solution. As a result, a hydrophilic layer 2010 was formed in the second region 222, and the container 2000 of the example was manufactured from the processing target container 200. That is, a 96-well plate having a hydrophilic layer 2010 was manufactured and used as the container 2000 of the example.

[0151] On the other hand, in the comparative example, an untreated 96-well plate without hydrophilic treatment with a BSA solution was used as the container.

[0152] In the examples and comparative examples, the 96-well plate was placed on a block heater set at 37°C, and 2 μL of sample 15, which was a cell suspension, was dropped into each well. The number of cells was adjusted to 4000 cells per well. After dropping sample 15, it was left standing in a 37°C CO2 incubator for 10 minutes, and then 100 μL of "DMEM + 1% Penicillin-Streptomycin + 10% FBS" was added per well, and observations were made the next day. DMEM is Dulbecco's Modified Eagle Medium. FBS is fetal bovine serum.

[0153] For the observations, a digital single-lens camera (α6000, SONY), "Cell3iMager NX" (CC-100, SCREEN), and "Cell3iMager Estier" (CC-9000, SCREEN) were used.

[0154] The photographs in FIGS. 19(a) and 19(b) were photographs taken by a digital single-lens camera of the observation results of cells 20 by "Cell3iMager NX".

[0155] The images in FIGS. 20(a) and 20(b) were the observation results of cells 20 by "Cell3iMager Estier".

[0156] The embodiments of the present invention have been described above with reference to the drawings. However, the present invention is not limited to the above embodiments, and can be implemented in various forms without departing from the gist thereof. Also, the plurality of components disclosed in the above embodiments can be modified as appropriate. For example, a certain component among all the components shown in one embodiment may be added to the components of another embodiment, or some of the components among all the components shown in one embodiment may be deleted from the embodiment.

[0157] In addition, for the purpose of facilitating the understanding of the invention, the drawings schematically show each component mainly. The thickness, length, number, interval, etc. of each illustrated component may be different from the actual ones for the convenience of drawing preparation. Also, the configuration of each component shown in the above embodiments is an example and is not particularly limited. Needless to say, various modifications are possible without substantially departing from the effects of the present invention.

[0158] (1) In the embodiments and modified examples described with reference to FIGS. 1 to 18, the mask member 100 may be formed by one mask portion 103. In this case, the mask member 100 may not include the mask base portion 101. One processing target container 200 may be formed by one processing target container main body 203. In this case, the processing target container 200 may not include the container base portion 201. One container 2000 may be formed by one container main body 2030. In this case, the container 2000 may not include the container base portion 201.

[0159] Also, in the embodiments and modified examples described with reference to FIGS. 1 to 18, the mask member 100 may be formed by one mask bottom portion 105. In this case, the mask member 100 does not include the mask base portion 101 and the mask wall portion 107.

[0160] (2) An example of the method for preparing and seeding the cell suspension used as the sample 15 will be described. For the cells 20 included in the sample 15, various cell lines, primary cultures, etc. can be used, and the type of the cells 20 is not particularly limited. The seeding number of the cells 20 is not particularly limited, but considering the observation, for example, it is preferably about 100 cells or more and 30,000 cells or less per 1 cm 2 ². Regarding the gel included in the sample 15, Matrigel, laminin, collagen, etc. manufactured by Corning can be used and can be appropriately mixed with the culture medium for use.

[0161] The liquid volume of the cell suspension (sample 15) in which the cells 20, the gel, and the culture medium are mixed is 1 cm 2It is preferably 200 μL or less per area. This is to surely prevent the sample 15 from contacting the inner wall surface 211 of the container body 2030 beyond the hydrophobic region (first region 221). Regarding the preparation of the cells 20, enzymatic treatment such as trypsin is performed on the container 2000 for subculture, the cells 20 are collected, and they can be suspended in a gel and a medium for use.

[0162] A micropipette or a pipette can be used for seeding the cells 20. Also, it is possible to use a dispenser (automatic pipetting device). When using Matrigel or the like, it is preferable to seed while heating with a horizontal hot plate from below the container 2000. It is preferable to solidify the gel while heating so that the cells 20 do not adhere to the culture surface (second region 222). Note that when the thickness of the sample 15 is reduced by the hydrophilic treatment, the cells 20 cultured in the three-dimensional culture may adhere to the culture surface (second region 222). This is because the specific gravity of the cells 20 is heavier than that of the gel and the medium, and they settle over time. Therefore, it is preferable to heat.

Industrial Applicability

[0163] The present invention relates to a container manufacturing method, a container, and a mask member, and has industrial applicability.

Explanation of Signs

[0164] 100 Mask member 101 Mask base part 103 Mask part 105 Mask bottom 107, 1107, 1117 Mask wall part 109 Surface 111 Outer wall surface 200 Container to be processed 201 Container base part 203 Container body to be processed 209 Bottom surface 211 Inner wall surface 221 First region 222 Second region 2000 Container 2030 Container body

Claims

1. A method for manufacturing a container, which manufactures a container including at least one container body in which a sample is disposed, by performing a hydrophilic treatment on a container to be treated including at least one container body to be treated, the method including a step of covering a first region of the bottom surface of the container body to be treated with a mask member, and a step of performing the hydrophilic treatment on a second region of the bottom surface after the first region is covered with the mask member, wherein the hydrophilic treatment is a treatment for making the water contact angle of the second region smaller than the water contact angle of the second region before the hydrophilic treatment is performed, and the first region is a region adjacent to the second region from the outside of the second region. A method for manufacturing a container.

2. The mask member has elasticity. The method for manufacturing a container according to Claim 1.

3. The heat-resistant temperature of the mask member is 120° C. or higher. The method for manufacturing a container according to Claim 1 or Claim 2.

4. In the step of performing the hydrophilic treatment, the hydrophilic treatment is performed so that the water contact angle of the second region is 10° or more and less than 60°. The method for manufacturing a container according to Claim 1 or Claim 2.

5. In the step of performing the hydrophilic treatment, the hydrophilic treatment is performed by a non-liquid. The method for manufacturing a container according to Claim 1 or Claim 2.

6. The first region surrounds the second region. The method for manufacturing a container according to Claim 1 or Claim 2.

7. The container to be treated includes a plurality of the container bodies to be treated, and a container base portion in which the plurality of container bodies to be treated are disposed, wherein the mask member includes a plurality of mask portions provided corresponding to the plurality of container bodies to be treated respectively, and a mask base portion on which the plurality of mask portions are disposed, wherein in the step of covering the first region with the mask member, each of the plurality of mask portions covers the first region of the corresponding container body to be treated. The method for manufacturing a container according to Claim 1 or Claim 2.

8. Each of the plurality of mask portions protrudes downward from the mask base portion, and each of the plurality of mask portions includes a mask bottom portion that covers the first region of the corresponding container body to be treated, and a mask wall portion that extends from the mask bottom portion to the mask base portion. The method for manufacturing a container according to Claim 7.

9. The surface of the mask bottom portion facing the first region has a shape along the first region. ​ The method for manufacturing a container according to claim 8, wherein an outer wall surface of the mask wall portion has a shape along an inner wall surface extending from the bottom surface of the container body to be processed.

10. The method for manufacturing a container according to claim 8, wherein the mask bottom portion has an annular shape.

11. Comprising at least one container body in which a sample is disposed, The container body, A bottom surface, And an inner wall surface extending from the bottom surface And has, The bottom surface, A first region, And a second region in which the sample is disposed And has, The first region indicates a region adjacent to the second region from the outside of the second region with respect to the second region, A container in which a water contact angle of the second region is smaller than a water contact angle of the first region.

12. The container according to claim 11, wherein the first region surrounds the second region.

13. The container according to claim 11 or claim 12, further comprising a container base portion on which a plurality of the container bodies are disposed.

14. The container according to claim 11 or claim 12, wherein the water contact angle of the second region is 10 degrees or more and less than 60 degrees.

15. A mask member that covers a part of a container to be processed including a plurality of container bodies to be processed, A plurality of mask portions provided corresponding to the plurality of container bodies to be processed, respectively, And a mask base portion on which the plurality of mask portions are disposed And comprises, Each of the plurality of mask portions prohibits hydrophilization treatment of the first region by covering the first region of the bottom surface of the corresponding container body to be processed, The first region indicates a region adjacent to the second region from the outside of the second region with respect to a second region of the bottom surface where a sample can be disposed, The hydrophilization treatment indicates a treatment that makes the water contact angle of the second region smaller than the water contact angle of the second region before the execution of the hydrophilization treatment, The mask member that does not prohibit the hydrophilization treatment of the second region by exposing the second region.

Images