Cell mass forming member, culture vessel, method for producing cultured cells, and cultured cells with cell mass forming member

Abstract

According to one embodiment of the present invention, provided are: a cell mass-forming member that is capable of easily forming a cell mass and superior in industrial mass productivity; a culture container equipped with the cell mass-forming member; a method for producing cultured cells using the cell mass-forming member; and cultured cells with a cell mass-forming member that are equipped with the cell mass-forming member.　The cell mass-forming member 1B according to one example of the present invention has a base material 2, wherein: an adhesion inhibition area 3A and a cell adhesion area 4B are formed on the surface 2a of the base material 2; a micro-concavo-convex structure area 6 including a plurality of convex portions 7 is formed in the cell adhesion area 4B; and a hydrophilic coating layer 5 is formed on both the adhesion inhibition area 3A and the cell adhesion area 4B. The culture container and the cultured cells with a cell mass-forming member according to one example of the present invention are equipped with the cell mass-forming member 1B. The method for producing cultured cells according to one example of the present invention comprises using the cell mass-forming member 1B.

Description

[Technical Field]

[0001] The present invention relates to a cell cluster forming member, a culture vessel, a method for producing cultured cells, and cultured cells with a cell cluster forming member. This application claims priority based on Patent Application No. 2021-058013, filed with the Japan Patent Office on March 30, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] In recent years, cell culture techniques have been used in various industrial fields (for example, Patent Documents 1 and 2). Patent Document 1 proposes a cell culture vessel that can be suitably used in biochemical experiments, clinical experiments, drug development research, etc. Patent Document 2 proposes a cell culture substrate for forming cell masses that have the morphological polarity and tissue motility characteristics observed in cancer tissues in vivo. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2007 / 125894 [Patent Document 2] International Publication No. 2018 / 182044 Summary of the Invention [Problem to be solved by the invention]

[0004] The bottom of the well of the cell culture vessel in Patent Document 1 is provided with a hydrophilic coating with a pattern of dot-shaped holes. This hydrophilic coating suppresses nonspecific adhesion of cells, and cells adhere to the dot-shaped holes. However, because the dot-shaped holes are dispersed within the well as a result of the patterning, cells cannot be cultured in a way that allows cell clumps to form.

[0005] In the cell culture substrate of Patent Document 2, cells are cultured on the rough surface portion that is not covered with the biocompatible polymer layer to form cell masses. However, the process of selectively applying the biocompatible polymer uses a photolithography technique, which is not suitable for mass production, so there is room for improvement in industrial mass production during manufacturing.

[0006] The present invention provides a cell cluster-forming member that can easily form cell clusters and is suitable for industrial mass production; a culture vessel equipped with the cell cluster-forming member; a method for producing cultured cells using the cell cluster-forming member; and cultured cells with the cell cluster-forming member equipped with the cell cluster-forming member. [Means for solving the problem]

[0007] The present invention has the following aspects. [1] A cell cluster forming member having a substrate; at least one adhesion-inhibiting region and at least one cell adhesion region are formed on the surface of the substrate; a fine uneven structure region including a plurality of convex portions or a plurality of concave portions is formed in at least a portion of the cell adhesion region; and a hydrophilic coating layer is formed on both the adhesion-inhibiting region and the cell adhesion region. [2] A cell cluster forming member having a substrate; at least one adhesion-inhibiting region and at least one cell adhesion region are formed on the surface of the substrate; a fine uneven structure region including a plurality of convex portions or a plurality of concave portions is formed in at least a portion of the cell adhesion region; and a hydrophilic coating layer is formed on one of the adhesion-inhibiting region and the cell adhesion region. [3] The cell cluster forming member of [1] or [2], wherein the average height of the plurality of convex portions or the average depth of the plurality of concave portions is 250 nm or more. [4] A cell cluster forming member according to any one of [1] to [3], wherein the aspect ratio of the average height of the plurality of convex portions to the average diameter of the plurality of convex portions, or the aspect ratio of the average depth of the plurality of concave portions to the average diameter of the plurality of concave portions, is 0.5 to 5.0. [5] The cell cluster forming member of any one of [1] to [4], wherein the average pitch between the plurality of convex portions or the plurality of concave portions is 50 nm to 1 μm. [6] The cell aggregate forming member of any one of [1] to [5], wherein the cell adhesion region is located at a position that provides a difference in height relative to the adhesion-inhibiting region in the thickness direction of the base material. [7] The cell aggregate forming member of any one of [1] to [5], wherein the difference in height between the cell adhesion region and the surface of the base material is 0 to 3 μm. [8] A cell aggregate forming member according to any one of [1] to [7], wherein a hydrophilic layer is formed in the recesses between the plurality of convex portions of the micro-relief structure region or on the bottom of the recesses between the plurality of convex portions. [9] The cell mass forming member of any of [1] to [8], wherein the base material contains at least one resin selected from the group consisting of polyethylene terephthalate, triacetyl cellulose, polycarbonate, cycloolefin polymer, cycloolefin copolymer, acrylic resin, polystyrene, and dimethylpolysiloxane.

[10] The cell cluster forming member of any of [1] to [9], wherein the ratio of the major axis of an ellipse having the smallest area circumscribing the cell adhesion region to the minor axis of the ellipse is 1 to 3.

[11] A cell-cluster-forming member according to any one of [1] to

[10] , wherein, in a planar view of the cell-cluster-forming member, the cell adhesion region is formed in an annular region surrounded by an outer circle and an inner circle, the diameter of the outer circle is 10 to 100 μm, the difference between the diameters of the outer circle and the inner circle is 200 nm to 30 μm, and a fine uneven structure including a plurality of convex portions or a plurality of concave portions is formed in at least a part of the annular region; and a hydrophilic coating layer is formed in both the adhesion-inhibiting region and the cell adhesion region.

[12] A culture vessel equipped with a cell mass forming member according to any one of [1] to

[11] .

[13] A method for producing cultured cells, comprising culturing cells using the cell cluster forming material according to any one of [1] to

[11] , and then detaching the cultured cells from the cell cluster forming material.

[14] A cultured cell system with a cell-cluster-forming member, comprising: a cell-cluster-forming member according to any one of [1] to

[11] ; and cultured cells attached to the cell-cluster-forming member.

[15] The cultured cells with a cell cluster forming member according to

[14] , wherein the cell clusters containing the cells form a three-dimensional structure. [Effects of the Invention]

[0008] According to the present invention, there are provided a cell cluster-forming member that can easily form cell clusters and is suitable for industrial mass production; a culture vessel equipped with the cell cluster-forming member; a method for producing cultured cells using the cell cluster-forming member; and cultured cells with the cell cluster-forming member equipped with the cell cluster-forming member. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a plan view schematically showing a cell aggregate forming member according to one embodiment. [Figure 2] FIG. 1 is a plan view schematically showing a cell aggregate forming member according to one embodiment. [Figure 3] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 4] FIG. 4 is a cross-sectional view taken along line IV-IV of FIG. [Figure 5] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 6] 6 is a cross-sectional view taken along the line VI-VI in FIG. 5. [Figure 7] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 8] FIG. 8 is a cross-sectional view taken along line VIII-VIII in FIG. 7. [Figure 9] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 10] XX cross-sectional view of FIG. 9. [Figure 11] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 12] 12 is a cross-sectional view taken along the line XII-XII of FIG. 11. [Figure 13] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 14] 14 is a cross-sectional view taken along the line XIV-XIV in FIG. 13. [Figure 15] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 16] 16 is a cross-sectional view taken along line XVI-XVI of FIG. 15. [Figure 17] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 18] 18 is a cross-sectional view taken along the line XVIII-XVIII in FIG. 17. [Figure 19] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 20] 20 is a cross-sectional view taken along the line XX-XX in FIG. 19. [Figure 21] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 22] 22-XXII cross-sectional view of FIG. 21. [Figure 23] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 24] 24-24 cross-sectional view of FIG. 23. [Figure 25] FIG. 2 is an enlarged plan view schematically showing an example of a cell cluster forming member. [Figure 26] 26 is a cross-sectional view taken along line XXVI-XXVI of FIG. 25. [Figure 27] 1 shows DIC observation images showing the results of culturing the human pancreatic ductal adenocarcinoma cell line PCI-55 after applying an MPC polymer to the cell cluster forming material of Example 1. [Figure 28] 10 is a DIC observation image showing the results of culturing the human pancreatic ductal adenocarcinoma cell line PCI-55 after applying an MPC polymer to the cell cluster forming material of Example 2. [Figure 29] 1 shows DIC observation images showing the results of culturing mouse myoblast cell line C2C12 using the cell cluster forming material of Example 1. [Figure 30] FIG. 4 is a cross-sectional view schematically showing a cell cluster forming member according to a modified example of FIG. 3. [Figure 31] FIG. 4 is a cross-sectional view schematically showing a cell cluster forming member according to a modified example of FIG. 3. [Figure 32] FIG. 4 is a cross-sectional view schematically showing a cell cluster forming member according to a modified example of FIG. 3. DETAILED DESCRIPTION OF THE INVENTION

[0010] In this specification, the use of "to" to indicate a range of values ​​means that the values ​​before and after it are included as the lower and upper limits. The lower and upper limits of the ranges disclosed in this specification can be combined in any way to create new ranges.

[0011] <Cell cluster forming material> A cell cluster forming member according to one embodiment will be described below with reference to the drawings as appropriate. The dimensional ratios in the drawings are for the sake of convenience and may differ from the actual ratios. In the following drawings, the same components are indicated by the same reference numerals, and descriptions of overlapping components may be omitted.

[0012] The cell cluster forming member according to one embodiment has a substrate. The material of the substrate is not particularly limited. For example, a substrate made of inorganic glass or a substrate made of organic polymer resin is included. Among these, a hydrophobic substrate is preferred as the substrate from the viewpoint of industrial mass productivity, and a resin substrate is more preferred from the viewpoint of moldability and manufacturing costs.

[0013] Examples of materials constituting the organic polymer resin substrate include thermoplastic resins, cured products of thermosetting resins, and cured products of photocurable resins. Among these, substrates containing at least one resin selected from the group consisting of polyethylene terephthalate (PET), triacetyl cellulose (TAC), polycarbonate (PC), cycloolefin polymer (COP), cycloolefin copolymer (COC), acrylic resin (such as polymethyl methacrylate (PMMA)), polystyrene (PS), and dimethylpolysiloxane (PDMS) are preferred. However, the materials constituting the organic polymer resin substrate are not limited to these examples.

[0014] Examples of materials constituting the inorganic glass substrate include quartz glass, various alkali glasses, and various alkali-free glasses. However, the materials constituting the inorganic glass substrate are not limited to these examples. When the fine relief structure is formed by a glass imprinting process, low-melting-point glass such as lead borosilicate glass or lead-free low-melting-point glass is preferably used. Another possible fabrication method is to apply a UV-curable resin to alkaline glass, and then expose the resin layer to UV imprinting, which involves pressing a microstructure plate against the resin coating. As shown in this example, the combination of an inorganic glass substrate and an organic polymer material is also effective.

[0015] The overall shape of the substrate is not particularly limited, and examples thereof include a film, a sheet, a plate, and a block. The overall shape of the substrate may be changed depending on the intended use of the cultured cells or cell aggregates containing the cultured cells. For example, when producing a cell sheet having cell aggregates formed on the surface, the overall shape of the substrate is preferably a sheet.

[0016] At least one adhesion-inhibiting region and at least one cell adhesion region are formed on the surface of the substrate. The adhesion-inhibiting region is a region on the surface of the cell-cluster-forming material that exhibits hydrophilicity. On the other hand, the cell adhesion region is a region on the surface of the cell-cluster-forming material that exhibits hydrophobicity. This difference in surface properties, hydrophilic and hydrophobic, results in differences in cell adhesiveness between the adhesion-inhibited region and the cell adhesion region. Therefore, with the cell cluster forming member according to one embodiment, cells can be selectively aggregated in the cell adhesion region, and cell clusters with a three-dimensional structure can be formed in the cell adhesion region.

[0017] In one embodiment of the cell cluster forming member, a hydrophilic coating layer is formed in the adhesion-inhibiting region. The hydrophilic coating layer is a layer of a hydrophilic material that imparts hydrophilic properties to the surface of the cell culture member. The hydrophilic material is not particularly limited as long as it has a hydrophilic functional group such as a hydroxyl group, a carbonyl group, or a carboxyl group. For example, a biocompatible polymer such as an MPC polymer with a phospholipid-like structure can be used. Biocompatible polymers are preferred because they have low cytotoxicity and can suppress nonspecific cell adhesion and the adsorption of cell adhesion factors such as proteins. The type of biocompatible polymer is not particularly limited. Examples include dimethylpolysiloxane (PDMS), polyethylene glycol (PEG), oligoethylene glycol (OED), 2-methacryloyloxyethyl phosphorylcholine (MPC), poly(MPC-co-butyl methacrylate) (PMB), and poly(MPC-co-dodecyl methacrylate) (PMD).

[0018] In one embodiment of the cell aggregate forming member, a hydrophilic coating layer is provided on a portion of the surface of the base material to form an adhesion-inhibiting region. When a hydrophilic coating layer is provided, the surface of the adhesion-inhibiting region is the surface of the hydrophilic coating layer. The thickness of the hydrophilic coating layer provided in the adhesion-suppressed region is preferably 0.005 to 0.500 μm, more preferably 0.005 to 0.300 μm, and even more preferably 0.010 to 0.100 μm. When the thickness of the hydrophilic coating layer is at least the lower limit of the above-mentioned numerical range, the adhesion-suppressed region tends to exhibit sufficient hydrophilicity. When the thickness of the hydrophilic coating layer is at most the upper limit of the above-mentioned numerical range, production costs tend to be reduced. The thickness of the hydrophilic coating layer is measured as the shortest distance between the surface of the substrate and the upper surface of the hydrophilic coating layer.

[0019] In one embodiment of the cell aggregate forming member, a hydrophilic coating layer is formed in both the adhesion-inhibited region and the cell adhesion region. In this case, the microrelief structure region in the cell adhesion region exhibits adhesiveness to cells. In this case, it is preferable that the average height of the plurality of convex portions or the average depth of the plurality of concave portions in the microrelief structure region is greater than the thickness of the hydrophilic coating layer, so that the thickness of the applied layer of the hydrophilicity-imparting liquid becomes thinner at the tops of the plurality of convex portions or the base surfaces of the plurality of concave portions, making it easier for cells to adhere. Furthermore, decreasing the thickness of the hydrophilic coating layer in the microrelief structure region increases the adhesive force between the cells and the cell-cluster-forming member, while increasing the thickness of the hydrophilic coating layer decreases the adhesive force between the cells and the cell-cluster-forming member. Thus, by controlling the thickness of the hydrophilic coating layer in the microrelief structure region, the adhesive force between the cells and the cell-cluster-forming member can be adjusted. Therefore, a state close to that of in vivo cell clusters corresponding to the microenvironment under set conditions can be reproduced.

[0020] In one embodiment of the cell cluster forming member, when the substrate is a resin substrate, the adhesion-inhibiting region may be formed by introducing hydrophilic functional groups into the surface of the resin substrate. For example, hydrophilic functional groups can be introduced into the surface of the resin substrate by irradiation treatment such as plasma irradiation, ion irradiation, radical irradiation, or ultraviolet irradiation. The portion of the surface of the resin substrate into which the hydrophilic functional groups have been introduced exhibits hydrophilicity.

[0021] In a cell cluster forming member according to one embodiment, the ratio of the major axis a of the ellipse with the smallest area circumscribing the cell adhesion region to the minor axis b of the ellipse is preferably 1 to 3, more preferably 1 to 2, and even more preferably 1 to 1.5. When the ratio of the major axis a of the ellipse to the minor axis b is within the above-mentioned range, cells are more likely to gather in the cell adhesion region during cell culture, promoting the formation of cell clusters with a three-dimensional structure in which multiple cells are aggregated. Here, for the lower limit of the above-mentioned range, the ratio of the major axis a to the minor axis b can be 1. When the ratio of the major axis a to the minor axis b is 1, the ellipse with the smallest area circumscribing the cell adhesion region is determined as the circumscribing circle of the cell adhesion region. However, in cell cluster forming members according to other embodiments, the ratio of the major axis a of the ellipse with the smallest area circumscribing the cell adhesion region to the minor axis b of the ellipse may exceed 3. In other embodiments, when this ratio exceeds 3, it becomes easier to obtain cell clusters in which the individual cells constituting the cell cluster are aligned in the direction of the major axis a.

[0022] The major axis a of the ellipse with the smallest area circumscribing the cell adhesion region is preferably 5 to 200 μm, more preferably 10 to 100 μm, and even more preferably 20 to 50 μm. When the major axis a of the ellipse with the smallest area circumscribing the cell adhesion region is equal to or greater than the lower limit of the aforementioned range, the formation of cell aggregates in the cell adhesion region is likely to be promoted. When the major axis a of the ellipse with the smallest area circumscribing the cell adhesion region is equal to or less than the upper limit of the aforementioned range, cells are likely to gather in the cell adhesion region during cell culture.

[0023] The minor axis b of the ellipse with the smallest area circumscribing the cell adhesion region is preferably 0.5 to 200 μm, more preferably 2 to 100 μm, and even more preferably 2 to 50 μm. When the minor axis b of the ellipse with the smallest area circumscribing the cell adhesion region is equal to or greater than the lower limit of the aforementioned numerical range, the formation of cell aggregates in the cell adhesion region is likely to be promoted. When the minor axis b of the ellipse with the smallest area circumscribing the cell adhesion region is equal to or less than the upper limit of the aforementioned numerical range, cells are likely to gather in the cell adhesion region during cell culture.

[0024] The major axis a and minor axis b of the ellipse with the smallest area circumscribing the cell adhesion region are determined as follows: A circumscribing ellipse that encompasses the cell adhesion region in a planar view of the cell aggregate forming member is determined, and the major and minor axes of the circumscribing ellipse are measured. If the shape of the cell adhesion region is irregular, there may be multiple circumscribing ellipses for one cell adhesion region. If there are multiple circumscribing ellipses for one cell adhesion region, the major and minor axes of the circumscribing ellipse with the smallest area among these multiple circumscribing ellipses are measured.

[0025] In a plan view of a cell aggregate forming member according to one embodiment, the shapes of the adhesion-inhibiting region and the cell adhesion region are not particularly limited. The shapes of the adhesion-inhibiting region and the cell adhesion region may be, for example, circular, elliptical, polygonal, or irregular, and are not limited to these examples.

[0026] Figure 1 is a plan view schematically showing a cell cluster forming member according to one embodiment. Four cell cluster forming regions 9, 9, 9, 9 are formed on the surface of the cell cluster forming member 1 shown in Figure 1. The four cell cluster forming regions 9, 9, 9, 9 are arranged in a square lattice pattern of 2 rows and 2 columns, and an adhesion-inhibiting region 3 is formed between each of the cell cluster forming regions 9. Each cell aggregate formation region 9 includes 25 circular cell adhesion regions 4 arranged in a square grid of 5 rows and 5 columns, and adhesion-inhibition regions 3 are formed between each of the 25 cell adhesion regions 4.

[0027] In a cell cluster forming member according to one embodiment, the arrangement of the cell adhesion regions 4 in each cell cluster forming region is not limited to a square lattice pattern. For example, the cell adhesion regions 4 may be arranged in a triangular lattice pattern, as in cell cluster forming regions 10, 10, 10, 10 shown in Figure 2. However, in a cell cluster forming member according to one embodiment, the formation modes of the adhesion-inhibiting regions, cell adhesion regions, and cell cluster forming regions are not limited to those shown in Figures 1 and 2. For example, although the cell cluster forming regions are regularly arranged on the surface of the cell cluster forming member in Figures 1 and 2, the cell cluster forming regions may also be arranged irregularly.

[0028] 1 and 2, four cell cluster formation regions are formed, but the number of cell cluster formation regions is not particularly limited. Similarly, the number of cell adhesion regions 4 included in each cell cluster forming member is not particularly limited. The geometric shape of the cell adhesion regions 4 is also not limited to the circular shape shown in FIGS. 1 and 2, and can be changed depending on the desired shape of the cell clusters. In this way, in a cell cluster forming member according to one embodiment, there can be an infinite number of configurations for the adhesion-inhibiting region, the cell adhesion region, and the cell formation region.

[0029] In one embodiment of the cell cluster forming member, a microrelief structure region including a plurality of convex portions or concave portions is formed in at least a portion of the cell adhesion region. In the microrelief structure region, a plurality of concave portions are formed between a plurality of convex portions. Alternatively, in the microrelief structure region, continuous convex portions are formed between a plurality of concave portions. This microrelief structure including a plurality of convex portions and a plurality of concave portions can serve as a structure that supports cells during culture. Therefore, the microrelief structure can function as a scaffold for cells, like an extracellular matrix. Therefore, the microrelief structure region exhibits relatively high cell adhesiveness among the cell adhesion regions, and can form a three-dimensional structure.

[0030] The microrelief structure region may be formed in at least a part of the cell adhesion region, or may be formed in the entire cell adhesion region. The proportion of the microrelief structure region in the cell adhesion region is not particularly limited and can be changed depending on the shape and properties of the desired cell aggregate. The shape of the microrelief structure region is not particularly limited and can be changed depending on the desired shape and properties of the cell cluster. For example, if the formation of a cell cluster with a tubular structure is desired, the microrelief structure region may be formed in a ring shape. The shape of the microrelief structure region may be changed depending on the desired shape of the cell cluster.

[0031] When a microrelief structure region is formed in part of the cell adhesion region, differences in cell adhesiveness may occur within the cell adhesion region depending on whether or not the microrelief structure region is present. In this case, it becomes easier to selectively gather cells in the microrelief structure region within the cell adhesion region, making it even easier to form cell aggregates with a three-dimensional structure.

[0032] The shapes of the convex and concave portions of the fine relief structure region are not particularly limited, and may be a cone shape, a cylindrical shape, a truncated cone shape, a pyramidal shape, or a bell shape, and are not limited to these exemplified shapes.

[0033] In the microrelief structure region, the average height of the multiple convex portions or the average depth of the multiple concave portions is preferably 250 nm or more, more preferably 300 nm to 2 μm, and even more preferably 400 nm to 1.2 μm. When the average height of the multiple convex portions or the average depth of the multiple concave portions is equal to or greater than the lower limit of the aforementioned range, the convex portions and concave portions of the microrelief structure region are easily formed, improving industrial mass productivity. In addition, when a cell aggregate forming member is manufactured by the method (α1) described below, after applying a hydrophilicity imparting liquid to the entire surface of the base material, the hydrophilicity imparting liquid in the coating layer flows into the adhesion-inhibited region, thereby reducing the thickness of the coating layer of the hydrophilicity imparting liquid at the tops of the multiple convex portions. Therefore, the productivity of the cell aggregate forming member is improved, improving industrial mass productivity. On the other hand, when the average height of the plurality of convex portions or the average depth of the plurality of concave portions is equal to or less than the upper limit of the above-mentioned numerical range, the adhesiveness of cells to the microrelief structure region is further improved.

[0034] The average height of the multiple convex portions and the average depth of the multiple concave portions are determined as follows: The cell aggregate forming member is cut perpendicular to the surface at any position using a microtome or CP processing (ion milling), and the cross section is observed under a scanning electron microscope (SEM). 20 convex portions or concave portions are randomly selected from multiple images taken at a magnification range in which 20 to 50 convex portions or concave portions are captured, and the height of the convex portions or the depth of the concave portions are measured. The arithmetic mean value is taken as the average height of the multiple convex portions or the average depth of the concave portions. Here, the height of each convex portion is measured as the shortest distance in the thickness direction of the substrate between the apex of the convex portion and the base surface of the fine concave-convex structure region, and the height of each concave portion is measured as the shortest distance in the thickness direction of the substrate between the apex of the concave portion and the surface of the substrate.

[0035] Instead of the average height of the plurality of convex portions, the average depth of the plurality of concave portions may be measured. The average depth of the plurality of concave portions is determined in the same manner as the average height of the convex portions, except that the depth of each concave portion is measured as the shortest distance between a concave portion at the same height as the apex of an adjacent convex portion and the base surface of the microrelief structure region. Details and preferred aspects of the average depth of the plurality of concave portions are the same as those described for the average height of the plurality of convex portions.

[0036] The average diameter of the plurality of convex or concave portions is preferably 50 nm to 1.2 μm, more preferably 100 nm to 1 μm, and even more preferably 200 nm to 900 nm. When the average diameter of the plurality of convex portions is equal to or greater than the lower limit of the aforementioned range, the formation of a microrelief structure region is facilitated, improving industrial mass productivity. When the average diameter of the plurality of convex or concave portions is equal to or less than the upper limit of the aforementioned range, the adhesiveness of cells to the microrelief structure region is further improved.

[0037] The average diameter of the plurality of convex portions is determined as follows: The cell aggregate forming member is observed in plan view using a scanning electron microscope (SEM), 20 convex portions are randomly selected from images within a magnification range in which 50 to 100 convex portions are captured, the major and minor axes of the circumscribed ellipses with the smallest areas in plan view of the 20 convex portions are measured, and the geometric mean values ​​of the major and minor axes are taken as the average diameter of the plurality of convex portions.

[0038] The average diameter of the plurality of recesses is determined in the same manner as the average diameter of the protrusions, except that the major and minor axes of the circumscribed ellipses with the smallest areas of the recesses in a plan view are measured. Details and preferred aspects of the average diameter of the plurality of recesses are the same as those described for the average diameter of the plurality of protrusions.

[0039] The aspect ratio of the average height of the plurality of convex portions to the average diameter of the plurality of convex portions, or the aspect ratio of the average depth of the plurality of concave portions to the average diameter of the plurality of concave portions, is preferably 0.5 to 5.0, more preferably 0.6 to 4.0, and even more preferably 0.8 to 3.0. When the aspect ratio is equal to or greater than the lower limit of the above-mentioned range, the adhesiveness of cells to the microrelief structure region is further improved.When the aspect ratio is equal to or less than the upper limit of the above-mentioned range, the microrelief structure region is easily formed, and industrial mass productivity is improved.

[0040] The average pitch between a plurality of convex portions or a plurality of concave portions is preferably 50 nm to 1 μm, more preferably 100 nm to 900 nm, and even more preferably 200 nm to 800 nm. When the average pitch between the plurality of convex portions is equal to or greater than the lower limit of the above-mentioned range, the formation of the microrelief structure region is facilitated, improving industrial mass productivity.When the average pitch between the plurality of convex portions is equal to or less than the upper limit of the above-mentioned range, the adhesiveness of cells to the microrelief structure region is further improved.

[0041] The average pitch of the multiple convex portions is determined as follows: The cell cluster forming member is observed in plan view using a scanning electron microscope (SEM), 20 convex portions are randomly selected from multiple images at a magnification range in which 50 to 100 multiple convex portions are captured, and the arithmetic mean value of the shortest distance between the centers of the circumscribed ellipses with the smallest areas in plan view of two adjacent convex portions is determined to be the average pitch of the multiple convex portions.

[0042] The average pitch of the plurality of recesses is determined in the same manner as the average pitch of the plurality of protrusions, except that the shortest distance between the centers of the circumscribed ellipses of the smallest areas of two adjacent recesses in a plan view is measured. Details and preferred aspects of the average pitch of the plurality of recesses are the same as those described for the average pitch of the plurality of protrusions.

[0043] In one embodiment, the cell adhesion region may be located at a position that has a height difference relative to the adhesion-inhibitory region in the thickness direction of the substrate. The "height difference between the adhesion-inhibitory region and the cell adhesion region" refers to the difference in height between the basal surfaces of the substrate in the adhesion-inhibitory region and the cell adhesion region. The "basal surface of the substrate" refers to the flat surface of the substrate itself when the surface of the substrate in the adhesion-inhibitory region or cell adhesion region is flat. When a microrelief structure is formed on the surface of the substrate in the adhesion-inhibitory region or cell adhesion region, the "basal surface of the substrate" refers to the surface from which all convex portions have been removed, or the surface formed by filling all concave portions of the surface.

[0044] In a cell aggregate forming member according to one embodiment, the positional relationship between the cell adhesion region and the adhesion-inhibiting region in the height direction (i.e., in the thickness direction of the substrate) is not particularly limited. That is, the cell adhesion region may be located higher or lower than the adhesion-inhibiting region in the thickness direction of the substrate, or the adhesion-inhibiting region and the cell adhesion region may be located at the same height. Here, the height position of the cell adhesion region is defined as the height position of the apex of the convex portion of the microrelief structure region. Therefore, when the adhesion-inhibiting region and the cell adhesion region are at the same height position, the height position of the apex of the convex portion of the microrelief structure region coincides with the height position of the surface of the adhesion-inhibiting region in the thickness direction of the base material.

[0045] If industrial mass production of the cell cluster forming member is desired, it is preferable that the cell adhesion region is located at a higher position in the thickness direction of the substrate than the adhesion-inhibition region. This is because, when producing the cell cluster forming member by method (α1) described below, after applying the hydrophilicity-imparting liquid to the entire surface of the substrate, the hydrophilicity-imparting liquid easily flows down from the cell adhesion region to the adhesion-inhibition region, making production easier.

[0046] In a cell cluster forming member according to one embodiment, the difference in height between the cell adhesion region and the surface of the substrate is preferably 0 to 3 μm, more preferably 100 nm to 2 μm, and even more preferably 300 nm to 1 μm. If the difference in height exceeds 0 μm and the cell adhesion region is located higher than the adhesion-inhibited region, when a cell cluster forming member is produced by the method (α1) described below, the hydrophilicity-imparting liquid is likely to flow down from the cell adhesion region to the adhesion-inhibited region after being applied to the entire surface of the substrate. This makes it easier to produce the cell cluster forming member, and industrial mass productivity is even better. If the difference in height is equal to or less than the upper limit of the aforementioned numerical range, cell migration to the cell adhesion region is less likely to be hindered.

[0047] In one embodiment of the cell aggregate forming member, a hydrophilic layer may be formed in the recesses between the multiple convex portions of the microrelief structure region in the cell adhesion region, or on the bottom of the recesses between the multiple convex portions. The hydrophilic layer is a layer of a hydrophilic material. Details and preferred aspects of the hydrophilic material are the same as those described for the hydrophilic coating layer. The hydrophilic material in the hydrophilic layer may be the same material as the hydrophilic material in the hydrophilic coating layer, or may be a different material. In consideration of industrial mass production, it is preferable that the hydrophilic material in the hydrophilic layer be the same material as the hydrophilic material in the hydrophilic coating layer.

[0048] In one embodiment of the cell cluster forming member, a hydrophilic layer may be formed in the recesses between the plurality of convex portions of the microrelief structure region or on the bottom of the recesses between the plurality of convex portions. In this case, the microrelief structure region in the cell adhesion region exhibits adhesiveness to cells.

[0049] In one embodiment of the cell cluster forming member, a layer of adhesion factors may be provided on the surface of the cell adhesion region to enhance cell adhesion. Examples of adhesion factors include extracellular matrices such as laminin, collagen, gelatin, fibronectin, polylysine (PDL, PLL), and hyaluronic acid, as well as polymers and gels.

[0050] (Action and effect) In the cell cluster forming member according to one embodiment described above, at least one adhesion-inhibiting region and at least one cell adhesion region are formed on the surface of the substrate, and a microrelief structure region is formed in at least a portion of the cell adhesion region. Therefore, cells adhere relatively more easily to the cell adhesion region compared to the adhesion-inhibiting region, and within the cell adhesion region, cells adhere even more easily to the microrelief structure region. Because there is thus a sufficient difference in cell adhesiveness between the adhesion-inhibiting region, the cell adhesion region, and the microrelief structure region, there is a strong tendency for cells to migrate from the adhesion-inhibiting region to the microrelief structure region within the cell adhesion region during cell culture. Therefore, the cell cluster forming member according to one embodiment makes it easier to form cell clusters.

[0051] (Manufacturing method) The method for producing the cell cluster forming member according to one embodiment is not particularly limited. For example, it can be produced by the following method (α). Method (α): A method in which the surface of a substrate is divided into at least one adhesion-inhibiting region and at least one cell adhesion region, a micro-relief structure region is formed in at least a portion of the cell adhesion region, and the adhesion-inhibiting region is subjected to a hydrophilic treatment.

[0052] In method (α), the division between the adhesion-inhibiting region and the cell adhesion region can be changed depending on the type of cell and the desired morphology and properties of the cell aggregate, and is not particularly limited. To form the fine relief structure region, for example, a monoparticulate film etching mask made of colloidal silica, as described in JP 2009-034630 A, may be used. The fine relief structure region can be formed on the substrate by vapor-phase etching using the monoparticulate film etching mask. Other methods for forming the fine relief structure region, such as colloidal lithography, anodic oxidation, and interference exposure, can also be used.

[0053] The hydrophilization treatment is not particularly limited as long as it is a treatment that enhances hydrophilicity. For example, a hydrophilicity-imparting liquid containing at least a hydrophilic material may be applied to the surface of the substrate. The applied layer of the hydrophilicity-imparting liquid may then be solidified to provide a hydrophilic coating layer on a portion of the surface of the substrate, thereby forming at least one adhesion-inhibited region and at least one cell adhesion region.

[0054] When a hydrophobic substrate is used, a part of the surface of the substrate may be subjected to a hydrophilization treatment to form at least one adhesion-inhibiting region and at least one cell adhesion region on the surface of the substrate. When a resin substrate is used, the surface of the resin substrate may be subjected to irradiation treatment such as plasma irradiation, ion irradiation, radical irradiation, ultraviolet irradiation, etc. as a hydrophilization treatment. As a result of the irradiation treatment, chemical bonds of molecules on the surface of the substrate are broken, hydrophilic functional groups according to the type of resin are generated, and hydrophilicity is improved compared to before the treatment. Among irradiation treatments, plasma irradiation is preferred because it causes less physical damage to the substrate surface.

[0055] To facilitate the detachment and recovery of cultured cells, a stimuli-responsive material may be applied to the surface of the cell aggregate forming member. As the stimuli-responsive material, a temperature-responsive polymer whose water affinity changes with temperature is preferred, and poly-N-isopropylacrylamide (PIPAAm) is preferred.

[0056] When the stimulus-responsive material is applied to the substrate, the microrelief region may be formed after the stimulus-responsive material is applied to the substrate, or the stimulus-responsive material may be applied to the substrate after the microrelief region is formed. However, when the stimulus-responsive material is applied to the substrate after the microrelief region is formed, it is preferable to apply the stimulus-responsive material to the substrate before the hydrophilization treatment.

[0057] From the viewpoint of industrial mass production, the following method (α1) is a preferred example of the method (α). Method (α1): A method in which the surface of a substrate is divided into an adhesion-inhibiting region and a cell adhesion region, a microrelief structure region is formed in the cell adhesion region, and then a hydrophilicity-imparting liquid is applied to the entire surface of the substrate.

[0058] In method (α1), when dividing the adhesion-inhibited region and the cell adhesion region, it is preferable to form the cell adhesion region at a position higher than the adhesion-inhibited region in the thickness direction of the substrate. This is because, after applying the hydrophilicity-imparting liquid to the entire surface of the substrate, the hydrophilicity-imparting liquid easily flows from the cell adhesion region to the adhesion-inhibited region, making production easier. When the cell adhesion region is located at a higher position in the thickness direction of the substrate than the adhesion-inhibited region, a height difference in the thickness direction may be provided on the surface of the substrate. This height difference may be provided on the substrate by a general photolithography method.

[0059] (Example of embodiment) Hereinafter, a cell aggregate forming member according to one embodiment will be described in detail by way of examples, but the present invention is not limited to these examples.

[0060] [Embodiment Example 1] Fig. 3 is an enlarged plan view schematically showing an example of a cell cluster forming member, and Fig. 4 is a cross-sectional view taken along line IV-IV in Fig. 3. The cell cluster forming member 1A shown in Figures 3 and 4 has a base material 2. Circular, spot-like cell adhesion regions 4A and adhesion-inhibiting regions 3A are formed on the surface 2a of the base material 2. This results in differences in the adhesiveness of cells in each region. Therefore, in the cell cluster forming member 1A, cells can be selectively gathered in the cell adhesion regions 4A, and cell clusters can be formed in the circular, spot-like cell adhesion regions 4A.

[0061] In the cell cluster forming member 1A, adhesion-inhibiting regions 3A are formed outside the circular, spot-like cell adhesion regions 4A. The adhesion-inhibiting regions 3A are covered with a hydrophilic coating layer 5 provided on the surface 2a of the base material 2. The adhesion-inhibiting regions 3A exhibit hydrophilicity due to this hydrophilic coating layer 5. The thickness of the hydrophilic coating layer 5 in the adhesion-inhibiting regions 3A is preferably 0.005 to 0.500 μm, more preferably 0.005 to 0.300 μm, and even more preferably 0.010 to 0.100 μm.

[0062] The spot-like cell adhesion regions 4A are circular in plan view. In this case, the ellipse with the smallest area circumscribing the cell adhesion regions 4A coincides with the outer circumscribing circle that separates the cell adhesion regions 4A from the adhesion-inhibition regions 3A. Therefore, in the cell aggregate forming member 1A, the major axis a of the ellipse with the smallest area circumscribing the cell adhesion regions 4A coincides with the minor axis b of the ellipse, which is equal to the diameter of the outer circumscribing circle that separates the cell adhesion regions 4A from the adhesion-inhibition regions 3A.

[0063] In the cell cluster forming member 1A, the entire cell adhesion region 4A is a microrelief structure region 6. The cell adhesion region 4A is hydrophobic due to the microrelief structure region 6 formed on the surface 2a of the base material 2. The microrelief structure region 6 includes a plurality of cone-shaped protrusions 7.

[0064] In the cell cluster forming member 1A, the cell adhesion region 4A is located higher than the adhesion-inhibited region 3A in the thickness direction of the base material 2. Therefore, when manufacturing a cell cluster forming member by method (α1), when the hydrophilicity-imparting liquid is applied to the entire surface 2a of the base material 2, the hydrophilicity-imparting liquid easily flows down from the cell adhesion region 4A to the adhesion-inhibited region 3A, making manufacturing easier.

[0065] The height difference h between the cell adhesion region 4A and the surface 2a of the base material 2 is preferably 0 to 3 μm, more preferably 100 nm to 2 μm, and even more preferably 300 nm to 1 μm. When the height difference h is equal to or greater than the lower limit of the aforementioned range, the cell aggregate forming member is easily produced, improving industrial mass productivity. When the height difference h is equal to or less than the upper limit of the aforementioned range, migration of cells to the cell adhesion region is less likely to be hindered.

[0066] In the cell cluster forming member 1A, there is a sufficient difference in adhesiveness to cells between the adhesion-inhibiting region 3A and the microrelief structure region 6. Therefore, during cell culture, there is a strong tendency for cells to migrate from the adhesion-inhibiting region 3A to the microrelief structure region within the cell adhesion region 4A. When the ratio of the major axis a to the minor axis b of the ellipse with the smallest area circumscribing the spot-shaped cell adhesion region 4A is sufficiently small, cells are more easily gathered in the spot-shaped cell adhesion region 4A during cell culture, and the formation of a cell mass with a three-dimensional structure in which multiple cells are aggregated is promoted. Therefore, cell clusters are easily formed using the cell cluster forming member 1A.

[0067] [Embodiment Example 2] Fig. 5 is an enlarged plan view schematically showing an example of the cell cluster forming member, and Fig. 6 is a cross-sectional view taken along line VI-VI of Fig. 5. The cell cluster forming member 1B shown in FIGS. 5 and 6 differs from the cell cluster forming member 1A in the following three points, but has a configuration in common with the cell cluster forming member 1A in other points. The hydrophilic coating layer 5 is also formed on the surfaces of the plurality of convex portions 7 of the microrelief structure region 6 of the cell adhesion region 4B. The hydrophilic layer 8 is formed on the bottom of the recesses between the plurality of protrusions 7 of the microrelief structure region 6B. The surface of the hydrophilic coating layer 5 formed on the microrelief structure region 6 of the cell adhesion region 4B conforms to the microrelief structure region 6.

[0068] In the cell cluster forming member 1B, the average height of the plurality of convex portions 7 or the average depth of the plurality of concave portions is greater than the thickness of the hydrophilic coating layer in the fine concave-convex structure region 6.

[0069] The cell cluster forming member 1B can be suitably produced by the method (α1). The hydrophilic coating layer 5 in the microrelief structure region 6 of the cell adhesion region 4B can be said to be a solidified liquid that remains on the surfaces of the multiple convex portions or multiple concave portions of the hydrophilicity imparting liquid that has been applied to the entire surface 2a of the base material 2. The thickness of the hydrophilic coating layer 5 in the microrelief structure region 6 of the cell adhesion region 4B is preferably 0.005 to 0.500 μm, more preferably 0.005 to 0.300 μm, and even more preferably 0.010 to 0.100 μm.

[0070] The hydrophilic layer 8 is a layer made of a hydrophilic material. Details and preferred embodiments of the hydrophilic material are the same as those described for the hydrophilic coating layer. The hydrophilic material in the hydrophilic layer 8 may be the same as or different from the hydrophilic material in the hydrophilic coating layer 5. However, considering industrial mass productivity, it is preferable that both layers use the same hydrophilic material.

[0071] The cell cluster forming member 1B also provides the same effects as the cell cluster forming member 1A. In addition, the cell cluster forming member 1B is also excellent in industrial mass productivity because the adhesion-inhibiting region 3A and the cell adhesion region 4B can be formed by applying a hydrophilicity-imparting liquid to the entire surface 2a of the base material 2 in the manufacturing process.

[0072] [Embodiment Example 3] Figure 7 is an enlarged plan view schematically showing an example of a cell cluster forming member. Figure 8 is a cross-sectional view taken along line VIII-VIII in Figure 7. Cell cluster forming member 1C shown in Figures 7 and 8 differs from cell cluster forming member 1A in that a microrelief structure region 6 is also formed in adhesion-inhibiting region 3B, but in other respects has a configuration common to cell cluster forming member 1A.

[0073] In the cell aggregate forming member 1C, the "height difference between the cell adhesion region and the surface of the substrate" refers to the difference in height between the base surface of the multiple convex portions of the micro-relief structure region 6 in the cell adhesion region 4A and the base surface of the multiple convex portions of the micro-relief structure region 6 in the adhesion-inhibition region 3B.

[0074] The details and preferred aspects of the fine concave-convex structure region 6 in the adhesion-inhibiting region 3B are the same as those of the fine concave-convex structure region 6 described for the cell cluster forming member 1A, except that the average height of the plurality of convex portions 7 is equal to or less than the thickness of the hydrophilic coating layer 5. The average height of the plurality of convex portions 7 in the adhesion-inhibiting region 3B is preferably 80% or less, and more preferably 50% or less, of the thickness of the hydrophilic coating layer 5.

[0075] The properties such as the average height, average diameter, and average pitch of the convex portions 7 of the microrelief structure region 6 in the adhesion-inhibiting region 3B may be the same as or different from the properties of the microrelief structure region 6 in the cell adhesion region 4A. The cell cluster forming member 1C also provides the same effects as those of the cell cluster forming member 1A.

[0076] [Embodiment Example 4] Figure 9 is an enlarged plan view schematically showing an example of a cell cluster forming member. Figure 10 is a cross-sectional view taken along line XX in Figure 9. The cell cluster forming member 1D shown in Figures 9 and 10 differs from the cell cluster forming member 1A in the following three points, but otherwise has a configuration in common with the cell cluster forming member 1A. The adhesion-suppressing region 3B has a fine uneven structure region 6 formed therein. The hydrophilic coating layer 5 is also formed on the surfaces of the plurality of convex portions 7 of the microrelief structure region 6 of the cell adhesion region 4B. The surface of the hydrophilic coating layer 5 formed on the microrelief structure region 6 of the cell adhesion region 4B conforms to the microrelief structure region 6.

[0077] In the cell cluster forming member 1D, the average height of the plurality of convex portions 7 or the average depth of the plurality of concave portions is greater than the thickness of the hydrophilic coating layer in the fine concave-convex structure region 6. The relationship between the average height of the plurality of convex portions 7 or the average depth of the plurality of concave portions and the thickness of the hydrophilic coating layer in the microrelief structure region 6 can be confirmed by observing the surface of the microrelief structure region 6 with an electron microscope and checking whether the microrelief structure appears on the surface. Alternatively, by preparing a cross-sectional sample of a cell aggregate forming member in which the microrelief structure region 6 is embedded in an embedding resin for cross-sectional observation and observing the cross-section with an electron microscope, the relationship between the average height of the plurality of convex portions 7 or the average depth of the plurality of concave portions and the thickness of the hydrophilic coating layer can be confirmed. The cell cluster forming member 1D also provides the same effects as the cell cluster forming member 1A. Furthermore, like the cell cluster forming member 1B, the cell cluster forming member 1D is also excellent in industrial mass productivity.

[0078] [Embodiment Example 5] Fig. 11 is an enlarged plan view schematically showing an example of a cell cluster forming member, and Fig. 12 is a cross-sectional view taken along line XII-XII of Fig. 11. The cell cluster forming member 1E shown in Figures 11 and 12 differs from the cell cluster forming member 1A in that the cell adhesion region 4A is located lower in the thickness direction of the substrate 2 than the surface 2a of the substrate 2, but otherwise has a configuration in common with the cell cluster forming member 1A. Even in cases such as cell cluster forming member 1E, where the cell adhesion region 4A is recessed and located lower than the adhesion-inhibition region 3A, there is a strong tendency for cells to move from the adhesion-inhibition region 3A to the cell adhesion region 4A during cell culture, and the same effect as that of cell cluster forming member 1A can be obtained.

[0079] [Embodiment 6] Fig. 13 is an enlarged plan view schematically showing an example of the cell cluster forming member, and Fig. 14 is a cross-sectional view taken along line XIV-XIV of Fig. 13. The cell cluster forming member 1F shown in Figures 13 and 14 differs from the cell cluster forming member 1B in that the cell adhesion region 4B is located lower in the thickness direction of the substrate 2 than the surface 2a of the substrate 2, but otherwise has a configuration in common with the cell cluster forming member 1B. The cell cluster forming member 1F also provides the same effects as those of the cell cluster forming member 1B.

[0080] [Embodiment Example 7] Fig. 15 is an enlarged plan view schematically showing an example of the cell cluster forming member, and Fig. 16 is a cross-sectional view taken along line XVI-XVI of Fig. 15. 15 and 16, an adhesion-inhibiting region 3A and a cell adhesion region 4C are formed on the surface 2a of the base material 2. In a plan view of the cell cluster forming member 1G, a microrelief structure region 6 is formed in a ring shape within the cell adhesion region 4C.

[0081] The outer periphery of the annular microrelief structure region 6 coincides with the outer edge of the cell adhesion region 4C. Furthermore, in the region inside the inner periphery of the annular microrelief structure region 6, the surface 2a of the base material 2 is exposed and forms part of the cell adhesion region 4C. In the cell cluster forming member 1G, the surface of the cell adhesion region 4C is configured to include the surface of the annular microrelief structure region 6 and the surface 2a of the base material 2.

[0082] The diameter R of the outer circle of the annular microrelief structure region 6 is preferably 10 to 100 μm, more preferably 10 to 60 μm, and even more preferably 20 to 45 μm. When the diameter R of the outer circle is equal to or greater than the lower limit of the aforementioned range, the annular microrelief structure region 6 is easily formed, improving industrial mass productivity. When the diameter R of the outer circle is equal to or less than the upper limit of the aforementioned range, cells are easily selectively gathered in the cell adhesion region 4C to form cell clusters.

[0083] The diameter r of the inner circle of the annular microrelief structure region 6 is preferably 500 nm or more, more preferably 1 μm or more, and even more preferably 10 μm or more. When the diameter r of the inner circle is equal to or greater than the lower limit of the above-mentioned range, the annular microrelief structure region 6 is easily formed, industrial mass productivity improves, and the formation of cell clusters having a three-dimensional structure in the annular microrelief structure region 6 is easily promoted.

[0084] The width W of the annular microrelief structure region 6, i.e., the difference between the outer diameter R and the diameter r of the inner circle, is preferably 200 nm to 30 μm, more preferably 1 to 20 μm, and even more preferably 3 to 15 μm. When the width W is equal to or greater than the lower limit of the aforementioned range, the annular microrelief structure region 6 is easily formed, improving industrial mass productivity. When the width W is equal to or less than the upper limit of the aforementioned range, the formation of cell clusters having a three-dimensional structure in the annular microrelief structure region 6 is easily promoted.

[0085] Within the cell adhesion region 4C, the surface of the convex portion 7 of the annular microrelief structure region 6 tends to exhibit relatively high adhesiveness to cells. Thus, in the cell cluster forming member 1G, differences in adhesiveness to cells are easily obtained not only between the adhesion-inhibiting region 3A and the cell adhesion region 4C, but also within the cell adhesion region 4C, depending on the presence or absence of the microrelief structure region 6.

[0086] Therefore, the adhesion-inhibiting region 3A in the cell cluster forming member 1G does not need to be made as hydrophilic as the adhesion-inhibiting region 3A in the cell cluster forming member 1A. As a result, the thickness of the hydrophilic coating layer 5 in the adhesion-inhibiting region 3A in the cell cluster forming member 1G may be thinner than the hydrophilic coating layer in the adhesion-inhibiting region 3A in the cell cluster forming member 1A.

[0087] From this perspective, the thickness of the hydrophilic coating layer 5 in the adhesion-inhibited region 3A in the cell cluster-forming member 1G is preferably 0.005 to 0.500 μm, more preferably 0.005 to 0.300 μm, and even more preferably 0.010 to 0.100 μm. When the thickness of the hydrophilic coating layer 5 in the adhesion-inhibited region 3A in the cell cluster-forming member 1G is at least the lower limit of the aforementioned numerical range, it is easy to impart sufficient hydrophilicity to the adhesion-inhibited region 3A in the cell cluster-forming member 1G. When the thickness of the hydrophilic coating layer 5 in the adhesion-inhibited region 3A in the cell cluster-forming member 1G is at most the upper limit of the aforementioned numerical range, it is easy to reduce production costs.

[0088] The cell cluster forming member 1G also provides the same effects as the cell cluster forming member 1A. In addition, in the cell cluster forming member 1G, during cell culture, cells tend to gather on the surface of the convex portions 7 of the microrelief structure region 6, even within the cell adhesion region 4C. As a result, cell clusters tend to form in a ring shape on the surface of the ring-shaped microrelief structure region 6, making it easier to form cell clusters with a tubular structure. Although further illustration is omitted, in other embodiments, the annular microrelief structure region 6 may be recessed and located lower than the adhesion-inhibiting region 3 A. In this case as well, the same effects as those of the cell cluster forming member 1G can be obtained.

[0089] [Embodiment 8] Figure 17 is an enlarged plan view schematically showing an example of a cell cluster forming member. Figure 18 is a cross-sectional view taken along line XVIII-XVIII of Figure 17. Cell cluster forming member 1H shown in Figures 17 and 18 differs from cell cluster forming member 1G in the following two points, but otherwise has a configuration in common with cell cluster forming member 1G. The hydrophilic coating layer 5 is also formed in the recesses between the plurality of protrusions 7 of the ring-shaped microrelief structure region 6. The adhesion-suppressing region 3A is also formed in the inner region of the ring-shaped micro-relief structure region 6.

[0090] The cell cluster forming member 1H also provides the same effects as the cell cluster forming member 1G. In addition, since the cell cluster forming member 1H has the hydrophilic coating layer 5, it can be suitably manufactured by the method (α1) in the same manner as the cell cluster forming member 1B, and is therefore suitable for industrial mass production. Although further illustration is omitted, in another embodiment, the ring-shaped microrelief structure region 6 may be recessed and located lower than the adhesion-inhibiting region 3A. In this case as well, the same effects as those of the cell cluster forming member 1H can be obtained.

[0091] [Embodiment Example 9] Figure 19 is an enlarged plan view schematically showing an example of a cell cluster forming member. Figure 20 is a cross-sectional view taken along line XX-XX of Figure 19. The cell cluster forming member 1I shown in Figures 19 and 20 differs from the cell cluster forming member 1A in that it has a concave microrelief structure region 6I formed therein that includes a plurality of recesses 11, but in other respects has a configuration that is common to the cell cluster forming member 1A. The cell cluster forming member 1I also provides the same effects as those of the cell cluster forming member 1A.

[0092] [Embodiment Example 10] Figure 21 is an enlarged plan view schematically showing an example of a cell cluster forming member. Figure 22 is a cross-sectional view taken along line XXII-XXII of Figure 21. The cell cluster forming member 1J shown in Figures 21 and 22 differs from the cell cluster forming member 1B in the following four points, but otherwise has a configuration in common with the cell cluster forming member 1B. A concave micro-relief structure region 6J is formed. The hydrophilic layer 8 is formed on the bottom of the plurality of recesses 11 in the microrelief structure region 6. The surface of the hydrophilic coating layer 5 formed on the microrelief structure region 6 of the cell adhesion region 4B conforms to the microrelief structure region 6. In the cell cluster forming member 1J, the average height of the plurality of convex portions 7J or the average depth of the plurality of concave portions is greater than the thickness of the hydrophilic coating layer in the microrelief structure region 6.

[0093] The cell cluster forming member 1J also provides the same effects as those of the cell cluster forming member 1B.

[0094] [Embodiment Example 11] Figure 23 is an enlarged plan view schematically showing an example of a cell cluster forming member. Figure 24 is a cross-sectional view taken along line XXIV-XXIV of Figure 23. The cell cluster forming member 1K shown in Figures 23 and 24 differs from the cell cluster forming member 1C in that it has a concave microrelief structure region 6K including a plurality of recesses 11, but in other respects has a configuration in common with the cell cluster forming member 1C. The cell cluster forming member 1K also provides the same effects as those of the cell cluster forming member 1C.

[0095] [Embodiment Example 12] Figure 25 is an enlarged plan view schematically showing an example of a cell cluster forming member. Figure 26 is a cross-sectional view taken along line XXVI-XXVI of Figure 25. The cell cluster forming member 1L shown in Figures 25 and 26 differs from the cell cluster forming member 1D in the following two points, but otherwise has a configuration in common with the cell cluster forming member 1D. The concave micro-relief structure region 6L is formed. The hydrophilic layer 8 is formed on the bottom of the plurality of recesses 11 in the microrelief structure region 6. The cell cluster forming member 1L also provides the same effects as those of the cell cluster forming member 1D.

[0096] [Embodiment Example 13] Figure 30 is a cross-sectional view corresponding to the cross section along line IV-IV in a modified example (1A') of the cell cluster forming member 1A shown in Figure 3. In this example, in the microrelief structure region 6 of the cell adhesion region 4A', the tips of the multiple convex portions 7 are exposed from the hydrophilic layer 8. The cell cluster forming member according to this example further enhances the adhesiveness of cells in the cell adhesion region 4A'.

[0097] [Embodiment Example 14] Figure 31 is a cross-sectional view corresponding to the cross section of line XX in a modified example (1D') of the cell cluster forming member 1D shown in Figure 9. In this example, in the microrelief structure region 6 of the cell adhesion region 4B', the tips of multiple convex portions are exposed from the hydrophilic layer 8. The cell cluster forming member according to this example further enhances the adhesiveness of cells in the cell adhesion region 4B'.

[0098] [Embodiment Example 15] Figure 32 is a cross-sectional view corresponding to the cross section along line XXII-XXII in a modified example (1J') of the cell cluster forming member 1J shown in Figure 21. In this example, in the cell adhesion region 4J', the tips of the multiple convex portions 7J are exposed from the hydrophilic layer 8. According to this example, the adhesiveness of cells in the cell adhesion region 4J' is further increased.

[0099] <Culture container> The culture vessel according to one embodiment includes the cell cluster forming member according to one embodiment, and therefore allows cell clusters to be easily formed, and is excellent in industrial mass productivity. An example of a culture vessel is one in which a cell cluster forming member is provided on the inner wall surface of a vessel body that contains a culture solution containing cells. The inner wall surface of the vessel body is not particularly limited as long as it is a surface that can come into contact with the culture solution, and may be the bottom surface of the vessel body or the inner side surface of the vessel body. The cell cluster forming member may be provided on the inner wall surface of the vessel body via an adhesive.

[0100] The shape and size of the culture vessel are not particularly limited, and examples of the culture vessel include a petri dish, a culture plate with one or more wells (holes), a culture flask, and a slide glass chamber. The number of wells in a culture plate is not particularly limited and is set depending on the purpose of the cultured cells, the analytical device to be used, etc. The shape of the wells in a plan view is not particularly limited. Examples include a perfect circle, ellipse, triangle, square, rectangle, and pentagon. The shape of the bottom of the well is also not particularly limited. Examples include a flat bottom, a round bottom, and an uneven bottom.

[0101] The material of the culture vessel is not particularly limited. Examples include polymeric materials, metal materials, and inorganic materials. Examples of polymeric resins include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymers, polyimide, polyamide, polyamideimide, (meth)acrylic resin, epoxy resin, and silicone. Examples of metal materials include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, and platinum. Examples of inorganic materials include silicon oxide (glass), aluminum oxide, titanium oxide, zirconium oxide, iron oxide, and silicon nitride.

[0102] <Cultured cell production method> In a method for producing cultured cells according to one embodiment, cells are cultured using a cell cluster-forming member according to one embodiment, and then the cultured cells are detached from the cell cluster-forming member. In the method for producing cultured cells, because the cells are cultured using the cell cluster-forming member according to one embodiment described above, cells tend to gather in the cell adhesion region, which tends to promote the formation of cell clusters with a three-dimensional structure.

[0103] In the method for producing cultured cells, it is preferable to seed cells onto the surface of the cell cluster forming member on which cell adhesion regions and adhesion-inhibiting regions are formed, and allow the cells to adhere to the surface of the cell cluster forming member. The cells are not particularly limited, and can be suitably selected from those capable of forming cell masses. Examples include hepatocytes, osteoblasts, chondrocytes, myoblasts, fibroblasts, cardiomyocytes, blood immune cells, and vascular endothelial cells. The cells may be stem cells with differentiation potential, such as iPS cells and ES cells, or differentiated cells induced from stem cells. Alternatively, tumor cells may be used. Epithelial tumor cells are preferred, including cervical cancer cells such as HeLa cells, pancreatic cancer cells, lung cancer cells, colon cancer cells, and head and neck cancer cells. The animal species of the cells, stem cells, differentiated cells, and tumor cells is not particularly limited, and examples include mammals such as humans, pigs, mice, rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, and goats.

[0104] The culture conditions are not particularly limited and can be changed depending on the cell type, the use of the cell mass, and the like. When detaching confluent cultured cells, a cell detachment agent may be used. The cell detachment agent is not particularly limited as long as it is a detachment agent commonly used in cell culture.

[0105] <Cultured cells with cell cluster forming material> A cultured cell with a cell cluster forming member according to one embodiment comprises: a cell cluster forming member according to one embodiment; and cultured cells adhered to the cell adhesion regions of the cell cluster forming member. Cultured cells with a cell cluster-forming member according to one embodiment are produced by using the above-described cell cluster-forming member and culturing cells in a state where the cells are attached to the surface of the cell cluster-forming member. Therefore, cell clusters of any three-dimensional structure, including cultured cells, can be attached to the surface of the cell cluster-forming member.

[0106] The type of cells is not particularly limited. Examples include the same cells as those exemplified in the section on the method for producing cultured cells. Cultured cells with cell mass-forming members can be suitably applied to regenerative medicine, research on viral and bacterial infections, and methods for screening therapeutic drugs for diseases. When applied to the development of therapeutic drugs for diseases, the drug will exert either or both of a preventive effect and a therapeutic effect for the disease. In the screening method, target cells of a disease or cell mass containing tumor cells are coexisted with a test substance, and the presence or absence of attenuation or loss of the morphology specific to the target cells in the presence of the test substance is observed, and the presence or absence of the preventive or therapeutic effect of the test substance on the disease is then determined.

[0107] Although one embodiment has been described above, the present invention is not limited to the embodiment disclosed in this specification and can be appropriately modified and implemented without departing from the spirit of the invention. The embodiment disclosed in this specification can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. [Example]

[0108] Hereinafter, one embodiment will be described in more detail using examples, but the present invention is not limited to these examples.

[0109] Example 1 A monolayer etching mask made of colloidal silica was fabricated on a Si wafer with a spot pattern (photoresist) using a conventional photolithography method, using the method described in JP 2009-034630 A. A fine uneven structure was formed on the Si substrate by vapor-phase etching using this monolayer etching mask, and the remaining photoresist was removed, leaving circular, spot-shaped fine uneven structure regions with a diameter of 30 μm, spaced 1 mm apart at intervals of a minimum of 40 μm.2 A convex Si surface microstructure master (1) was fabricated, with 100 convex convex portions arranged per square meter. The convex Si surface microstructure master (1) contained multiple convex portions with an average pitch of 200 nm and an average height of 530 nm within the microrelief structure region. The height difference h between the tips of the convex portions in the circular, spot-shaped microrelief structure region and the flat region outside the microrelief structure region was 270 nm. Next, a concave Ni electroformed stamper (1) was produced from a convex Si surface microstructure original plate (1). Then, using the concave Ni electroformed stamper (1), a convex structure serving as a mounting portion was transferred to the surface of a 188 μm-thick cycloolefin polymer film by thermal nanoimprinting, producing a convex resin transfer member (1). Specifically, the microstructure surface of the concave Ni electroformed stamper (1) was placed facing the surface of a 188 μm-thick cycloolefin polymer film, and the resulting film was set in the pressure unit of a nanoimprinting device. In this state, heating was initiated, and pressure was applied at 175°C for 5 minutes at 5 MPa. After 5 minutes had elapsed, the pressure unit was cooled to room temperature, and the concave Ni electroformed stamper (1) was released from the surface of the cycloolefin polymer film, yielding a convex resin transfer member (1). This convex resin transfer member (1) was used as the convex cell mass-forming member of Example 1.

[0110] <Example 2> A concave resin original plate (1) was produced by transferring a convex structure to serve as a mounting portion onto the surface of a 188 μm-thick cycloolefin polymer film by thermal nanoimprinting from the convex Si surface microstructure original plate (1) produced in Example 1. A convex Ni electroformed stamper (1) was produced from the concave resin original plate (1), and a concave cell cluster forming member was obtained in the same manner as in Example 1, except that the convex Ni electroformed stamper (1) was used.

[0111] Example 3 Using the method described in JP 2009-034630 A, a monolayer etching mask made of colloidal silica was fabricated on a Si wafer with a ring pattern (photoresist) using a general photolithography method. A circular fine relief structure region was formed on the Si substrate by a vapor-phase etching method using this monolayer etching mask (the remaining photoresist was removed). The diameter R of the outer circle of the circular fine relief structure was 40 μm, the diameter r of the inner circle was 30 μm, and the width W was 5 μm. The circular fine relief structure region was spaced 1 mm apart at a minimum interval of 30 μm. 2 A convex Si surface microstructure master (2) was fabricated, with 100 convex portions arranged per square meter. The convex Si surface microstructure master (2) contained multiple convex portions with an approximately conical shape within the annular microrelief structure region, with an average pitch of 400 nm and an average height of 1100 nm. The height difference h between the tips of the convex portions in the annular microrelief structure region and the flat region outside the microrelief structure region was 500 nm. Thereafter, a convex cell cluster forming member was obtained in the same manner as in Example 1, except that a concave Ni electroformed stamper (2) made from a convex Si surface microstructure original plate (2) was used.

[0112] Example 4 A concave resin original plate (2) made of cycloolefin polymer was produced by transferring a convex structure to serve as a mounting portion onto the surface of a 188 μm-thick cycloolefin polymer film by thermal nanoimprinting from the convex Si surface microstructure original plate (2) produced in Example 3. A concave cell cluster forming member was obtained in the same manner as in Example 1, except that a convex Ni electroformed stamper (2) produced from the concave resin original plate (2) was used.

[0113] <Culture of human pancreatic ductal adenocarcinoma cell line PCI-55> After the surface of each cell-cluster-forming member obtained in Examples 1 to 4 was subjected to hydrophilization treatment by UV ozone method, an MPC polymer solution was applied by spin coating, and an MPC polymer coating layer with an average thickness of 40 nm was provided on the surface of the base material. The coating layer was dried to obtain a cell-cluster-forming member with adhesion-inhibiting regions and cell adhesion regions formed on its surface. The cell-cluster-forming member was washed with distilled water, air-dried, cut into pieces of 20 mm x 20 mm, and fixed to the inner bottom of a 40 mm diameter x 13.5 mm deep culture dish (Aznol Petri dish, AS ONE Corporation) with petrolatum. Next, the cell-cluster-forming member was washed three times by adding DMEM (Dulbecco's Modified Eagle's Medium), and then 2 x 10 cells suspended in 3 mL of DMEM were added. 6 PCI-55 cells were seeded onto each cell cluster forming member and cultured at 37°C overnight for 48 hours.

[0114] Figure 27 is a DIC observation image showing the results of culturing the human pancreatic ductal adenocarcinoma cell line PCI-55 after applying an MPC polymer to the cell cluster forming material of Example 1. Multiple polypoid cell clusters were formed that straddled two or more cell adhesion regions and firmly adhered to the cell adhesion regions. Figure 28 is a DIC observation image showing the results of culturing the human pancreatic ductal adenocarcinoma cell line PCI-55 after applying an MPC polymer to the cell cluster forming material of Example 2. Multiple polypoid cell clusters were formed that were elongated in the height direction of one cell adhesion region. From the above culture results, it was confirmed that the cell cluster forming materials of Examples 1 to 4 formed self-organized, anchorage-dependent, non-spheroid-like cancer cell clusters that possessed morphological polarity and tissue movement polarity.

[0115] <Culture of mouse myoblast cell line C2C12> An MPC polymer solution was applied to the entire surface of each of the cell-cluster-forming members obtained in Examples 1 to 4 by spin coating, and an MPC polymer coating layer with an average thickness of 40 nm was provided on the surface of the base material. The coating layer was dried to obtain a cell-cluster-forming member with adhesion-inhibiting regions and cell-adhesion regions formed on its surface. The cell-cluster-forming member was washed with distilled water, air-dried, cut into pieces of 20 mm x 20 mm, and fixed to the inner bottom of a 40 mm diameter x 13.5 mm deep culture dish (Aznol Petri dish, AS ONE Corporation) with petrolatum. The cell-cluster-forming member was washed three times with DMEM, and then 5 x 10 cells suspended in 3 mL of DMEM containing 10% FBS were added. 5 C2C12 cells were seeded onto each cell cluster forming member and cultured at 37°C overnight to 48 hours. After the culture was completed, the cell aggregates were fixed with paraformaldehyde, actin was stained with phalloidin, and cell nuclei were stained with DAPI. Two-dimensional images were then obtained using an all-in-one fluorescence microscope (BZ-X800, Keyence Corporation).

[0116] 29 is a DIC observation image showing the results of culturing mouse myoblast cell line C2C12 using the cell cluster forming material of Example 1. The formation of non-spheroid cell clusters spanning two or more spots was observed. Similarly, when the mouse myoblast cell line C2C12 was cultured using the cell cluster forming materials of Examples 2 to 4, the formation of non-spheroid cell clusters spanning two or more spots was observed. From the above culture results, it was confirmed that in the cell cluster forming materials of Examples 1 to 4, self-organized, anchorage-dependent, non-spheroid-like cell clusters possessing morphological polarity and tissue movement polarity were formed. [Industrial Applicability]

[0117] According to one embodiment, there are provided a cell cluster forming member that can easily form cell clusters and is suitable for industrial mass production; a culture vessel equipped with the cell cluster forming member; a method for producing cultured cells using the cell cluster forming member; and cultured cells with the cell cluster forming member equipped with the cell cluster forming member. [Explanation of symbols]

[0118] 1...cell aggregate forming member, 2...substrate, 3...adhesion-inhibiting region, 4...cell adhesion region, 5...hydrophilic coating layer, 6...fine uneven structure region, 7...convex portion, 8...hydrophilic layer, 9, 10...cell formation region, 11...concave portion

Claims

1. A cell cluster forming member having a substrate, At least one adhesion-inhibiting region and at least one cell adhesion region are formed on the surface of the substrate, a microrelief structure region including a plurality of convex portions or a plurality of concave portions is formed in at least a part of the cell adhesion region, a hydrophilic coating layer is formed on both the adhesion-inhibited region and the cell adhesion region; the hydrophilic coating layer is a layer formed of a hydrophilic material, the hydrophilic material is a biocompatible polymer; A cell aggregate forming member, wherein the surface of the hydrophilic coating layer formed on the microrelief structure region of the cell adhesion region conforms to the microrelief structure region.

2. A cell cluster forming member having a substrate, At least one adhesion-inhibiting region and at least one cell adhesion region are formed on the surface of the substrate, a microrelief structure region including a plurality of convex portions or a plurality of concave portions is formed in at least a part of the cell adhesion region, a hydrophilic coating layer is formed only on the adhesion-suppressed region; the hydrophilic coating layer is a layer formed of a hydrophilic material, the hydrophilic material is a biocompatible polymer; a hydrophilic layer is formed in the recesses between the plurality of protrusions in the microrelief structure region; A cell cluster forming member, wherein the plurality of convex portions protrude from the hydrophilic layer.

3. The cell cluster forming member according to claim 1 or 2, wherein the average height of the plurality of convex portions or the average depth of the plurality of concave portions is 250 nm or more.

4. The cell cluster forming member according to any one of claims 1 to 3, wherein the aspect ratio of the average height of the plurality of convex portions to the average diameter of the plurality of convex portions, or the aspect ratio of the average depth of the plurality of concave portions to the average diameter of the plurality of concave portions, is 0.5 to 5.

0.

5. The cell cluster forming member according to any one of claims 1 to 4, wherein an average pitch between the plurality of convex portions or the plurality of concave portions is 50 nm to 1 µm.

6. The cell cluster forming member according to any one of claims 1 to 5, wherein the cell adhesion region is located at a position that is different in elevation from the adhesion-inhibiting region in the thickness direction of the base material.

7. The cell cluster forming member according to any one of claims 1 to 5, wherein the height difference between the cell adhesion region and the surface of the substrate is 0 to 3 µm.

8. The cell cluster forming member according to any one of claims 1 to 7, wherein the base material comprises at least one resin selected from the group consisting of polyethylene terephthalate, triacetyl cellulose, polycarbonate, cycloolefin polymer, cycloolefin copolymer, acrylic resin, polystyrene, and dimethylpolysiloxane.

9. The cell cluster forming member according to any one of claims 1 to 8, wherein the ratio of the major axis of an ellipse having the smallest area circumscribing the cell adhesion region to the minor axis of the ellipse is 1 to 3.

10. In a plan view of the cell cluster forming member, the cell adhesion region is formed in an annular region surrounded by an outer diameter circle and an inner diameter circle, the diameter of the outer diameter circle is 10 to 100 μm, the difference between the diameter of the outer diameter circle and the diameter of the inner diameter circle is 200 nm to 30 μm, and a fine uneven structure including a plurality of convex portions or a plurality of concave portions is formed in at least a part of the annular region, The cell cluster forming member according to claim 1 , wherein a hydrophilic coating layer is formed on both the adhesion-inhibiting region and the cell adhesion region.

11. A culture vessel comprising the cell cluster forming member according to any one of claims 1 to 10.

12. A method for producing cultured cells, comprising culturing cells using the cell cluster forming member according to any one of claims 1 to 10, and then detaching the cultured cells from the cell cluster forming member.

13. The cell cluster forming member according to any one of claims 1 to 10, cultured cells attached to the cell cluster forming member; A cultured cell comprising a cell cluster forming member.

14. The cultured cells with cell cluster-forming member according to claim 13, wherein the cell clusters containing the cells form a three-dimensional structure.

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