Carrier for determining conditions for forming cell aggregates on amorphous carbon film

Abstract

To determine a shape of an amorphous carbon film suitable for cells forming cell aggregates when culturing the cells on an amorphous carbon film.SOLUTION: The problem is solved by a carrier for determining conditions under which cells form cell aggregates on an amorphous carbon (DLC) film. The carrier has a region where independent DLC films are deposited on a surface of the carrier, the deposition region being substantially triangular and / or substantially trapezoidal. By using an instrument including the carrier as part of its structure, and by seeding and maintaining cells on the surface of the amorphous carbon film within the instrument, it becomes possible to identify the width of the amorphous carbon film deposition region that is suitable for the cells frequently forming cell aggregates on the deposited amorphous carbon film. That is, based on the knowledge of the deposition region width thus obtained, frequent formation of cell aggregates of the cells can be achieved by applying this knowledge to an instrument equipped with a deposition region having the deposition region width in a wide range, using the same type of cells and the same type of amorphous carbon film as those in the system where the knowledge was obtained.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a carrier for finding conditions under which cells form cell aggregates on an amorphous carbon film. [Background technology]

[0002] Diamond-like carbon (DLC) film, one of the amorphous carbon films, has sp 3 carbon with bonds and sp corresponding to the graphite structure 2 It is an amorphous film in which bonded carbon atoms are randomly mixed, with some carbon atoms hydrogen-terminated. Because of its properties of high hardness, low friction, and inertness, it is known that when used as a coating material for the surface of substrates made of inorganic materials such as metals and ceramics, or organic materials such as polymer resins, it can provide properties such as wear resistance, corrosion resistance, and sliding properties to the substrate surface.

[0003] Because amorphous carbon films also have properties such as biocompatibility and chemical stability, they are expected to be useful as a means of surface modification for medical devices. In addition to these properties, as mentioned above, coating various materials such as metals, ceramics, and polymer resins with them improves surface hardness and sliding properties, and their application to implants (Patent Document 1), stents (Patent Document 2), and catheters (Patent Document 3) has been proposed.

[0004] Although it is generally called an amorphous carbon film, the basic physical properties (sp 3 Structure: Diamond structure derived constituents, sp 2The structure (graphite structure-derived constituent amount, H: hydrogen content) differs. Specifically, based on these fundamental physical properties, amorphous carbon films are classified into ta-C, ta-C:H, aC, aC:H, polymer, and graphitic, which determine their basic surface functions. Furthermore, while amorphous carbon films have been evaluated by macroscopic analysis of the entire film, it has become clear that the film properties of individual amorphous carbon films vary depending on the deposition method in the surface, bulk, and substrate interface layers that make up the film. Furthermore, doping amorphous carbon films with third elements such as fluorine, nitrogen, and silver can achieve even more complex film property variations. Thus, amorphous carbon films can be formed with a variety of properties depending on the deposition method and whether or not they are doped. Therefore, if it were possible to easily and reproducibly determine the degree to which a given amorphous carbon film possesses certain properties, particularly its properties as a biointerface based on its affinity with biological substances, it would be extremely useful for industrial applications of amorphous carbon films.

[0005] Until now, the main indicators of the properties of amorphous carbon films as biointerfaces have been their biocompatibility and bioaffinity, i.e., whether they can provide good bonding or adhesion with biological materials. However, in recent years, a new concept, cell aggregation, has been proposed that is distinct from such cell affinity. Cell aggregation is the ability to form cell aggregates. A cell aggregate is a mass formed by the aggregation of dispersed cells, in which the cells adhere to each other. Cell aggregates also include cell clusters, embryoid bodies, spheres, and spheroids. Recently, pluripotent stem cells, such as ES cells and iPS cells, have been attracting attention. It is known that when artificially propagating these stem cells, the stem cells also form cell aggregates. The ability to induce the formation of such cell aggregates is gaining attention as a new feature required for biointerfaces, distinguishing them from the previously utilized cell affinity.

[0006] Pluripotent stem cells have the ability to proliferate indefinitely and differentiate into various cell types, making them a promising treatment for intractable diseases and lifestyle-related diseases in regenerative medicine. However, cell production is a major obstacle to their practical application. Organ regeneration generally requires a large number of cells, and conventional two-dimensional culture on a flat substrate is not a practical method for supplying cells. To solve this problem, methods have been developed for actively aggregating cells in two-dimensional culture and transferring them to three-dimensional culture in the form of cell aggregates, as well as dedicated equipment, etc. However, most conventional equipment for forming cell aggregates is simply a molded product made of a material to which cells have difficulty adhering (Patent Documents 4 and 5), which has led to the problem that efficient formation of cell aggregates cannot be achieved due to cell death caused by apoptosis resulting from excessively low cell adhesiveness.

[0007] To date, the inventors have conducted extensive research into the correlation between the cell aggregation properties of amorphous carbon films, using various types of amorphous carbon films formed by different deposition methods, evaluating the cell aggregation properties of each carbon film and measuring indices that reflect the basic structure of the amorphous carbon film and are likely to be related to cell aggregation properties (Patent Document 8). Furthermore, the inventors have established a system that enables easy and highly reproducible evaluation of differences in the effects of amorphous carbon films on cells, including cell aggregation properties, by qualitative shape observation and / or quantitative counting, without being affected by accidental differences in the culture environment of the cells, i.e., enables evaluation in multiple amorphous carbon film environments simultaneously under the same culture conditions (Patent Document 9).

[0008] Specifically, an amorphous carbon film was formed in a lattice pattern with regular intervals on the surface of a cover glass made of glass or the like, and then a single type of cell was uniformly seeded on the surface, and the cells were cultured for a certain period of time in a single-well cell culture vessel with the cover glass. Furthermore, by constructing the lattice-like film formation region using multiple types of amorphous carbon films formed under different film formation conditions, it became possible to compare the behavior of adherent cells due to the differences in the properties of various carbon films within the same cell culture vessel (Patent Document 9). By using this device, amorphous carbon films formed under different film formation conditions are formed in adjacent regions in a lattice pattern, making it possible to simultaneously observe static differences between cultured cells in nearly identical culture environments except for the different carbon films. Furthermore, by observing cultured cells in this device over time, the inventors have discovered dynamic behavior in which cells migrate in search of a more favorable film environment during the process of repeated adhesion and / or proliferation. This makes it possible to more clearly determine which amorphous carbon film surface under which film formation conditions provides a more favorable environment for the target cells in terms of adhesion and / or proliferation. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2014-4166 [Patent Document 2] Patent Publication No. 2010-280636 [Patent Document 3] Patent Publication No. 2008-245883 [Patent Document 4] International Publication No. 2013 / 099901 Brochure [Patent Document 5] International Publication No. 2013 / 022085 Brochure [Patent Document 6] Patent Publication No. 2019-030260 [Patent Document 7] Special Publication 2007-508816 [Patent Document 8] Patent Publication No. 2022-77545 [Patent Document 9] Patent Publication No. 2023-119382 [Non-patent literature]

[0010] [Non-Patent Document 1] Naoto Otake and two others, 2012, NEW DIAMOND Vol. 28, No. 3, pp. 12-18 Summary of the Invention [Problem to be solved by the invention]

[0011] The inventors used the above-mentioned device to form various amorphous carbon films with different cell aggregation properties, as described in Patent Document 8, in a lattice pattern with regular intervals, and then uniformly seeded a single type of cell on the surface, thereby observing the process of cell aggregate formation on each carbon film.As a result, they found that amorphous carbon films with high cell aggregation properties produced cell aggregates more frequently than carbon films with low cell aggregation properties, but that the frequency did not necessarily depend solely on the properties of the amorphous carbon films as carbon films. When an amorphous carbon film with high cell aggregation properties was uniformly formed on the surface of a carrier such as glass or plastic, and cultured cells were uniformly seeded on the film, the cells that were uniformly dispersed immediately after seeding migrated in search of a more favorable membrane environment as the culture period passed, and did not necessarily form cell aggregates uniformly and efficiently.On the other hand, when amorphous carbon films of various shapes were formed on carriers and cells were cultured on the carbon films, it was found that the frequency of cell aggregate formation was affected by the shape of the carbon film. That is, it has been found that the efficient formation of cell aggregates from cultured cells seeded on an amorphous carbon film formed on a carrier is greatly influenced not only by the physical properties of the carbon film but also by the shape of the carbon film on which the cells are cultured, but it has been unclear what shape is appropriate.

[0012] In view of the above problems, an object of the present invention is to find an amorphous carbon film shape suitable for forming cell aggregates when cells are cultured on the amorphous carbon film. [Means for solving the problem]

[0013] In order to find a shape suitable for cells to form cell aggregates when cultured on an amorphous carbon film, the inventors formed amorphous carbon films of various shapes on the surface of a cover glass made of a material such as glass, and then uniformly seeded a single type of cell on the surface. They then cultured the cells for a certain period of time in a single-well cell culture vessel, along with the cover glass, and investigated what shape would result in the formation of cell aggregates with high efficiency. Through intensive research, we have found that, because there is no significant change in the foothold of attached cells on the surface of an amorphous carbon film that has been formed over a wide area and uniformly, in addition to the differences in the surface conditions of various amorphous carbon films with different physical properties, deviations in the expression of cell function and differentiation characteristics, particularly cell aggregation, are likely to occur due to local cell distribution (dense or coarse cell density), and that the film is susceptible to the influence of minute cell density in the cell culture environment; that the cell aggregation properties on amorphous carbon films vary greatly depending on the width of the film-formed region sandwiched on both sides by non-film-formed regions; and that, because the cell aggregation function differs depending on the type of cell seeded on the amorphous carbon film, the optimal width of the film-formed region for inducing cell aggregates also differs depending on the cell.

[0014] To summarize the above findings, it has become clear that the cell aggregation properties of cells expected to form cell aggregates vary depending on the size of the deposition region pattern of the amorphous carbon film on which the cells are cultured, particularly the width of the deposition region sandwiched on both sides by non-deposition regions, and that the optimal deposition region width for cell aggregate formation varies depending on the cell type. For this reason, it has been difficult to comprehensively evaluate the cell aggregation function of cells cultured on the deposition region pattern of an amorphous carbon film consisting of a fixed deposition region width, such as a lattice pattern with a fixed interval, as previously reported. Therefore, the inventors conducted further research and discovered that by patterning an amorphous carbon film into a shape in which the width of the film-forming region changes continuously, for example, into an approximately triangular and / or approximately trapezoidal shape, it is possible to continuously observe the relationship between cell coagulation and the width of the film-forming region.

[0015] Based on the above findings, the present invention has been completed. That is, the present invention relates to the following (1) to (4). (1) A carrier for finding conditions under which cells form cell aggregates on an amorphous carbon (DLC) film, characterized in that the carrier has an area on its surface where an independent DLC film is formed, and the formed area is approximately triangular and / or approximately trapezoidal. (2) The carrier according to (1), characterized in that the component of the carrier is glass and / or resin. (3) An instrument comprising the carrier according to any one of (1) to (2) as part of its configuration. (4) A method for finding conditions under which cells form cell aggregates on an amorphous carbon (DLC) film, comprising: Culturing cells that induce cell aggregate formation on a DLC film formed in a substantially triangular and / or substantially trapezoidal shape; and Identifying the area where cell aggregates form after a certain period of time from the start of culture; The method comprising: [Effects of the Invention]

[0016] According to the present invention, it is possible to continuously observe the relationship between the aggregability of a desired amorphous carbon film and / or cells of a desired cell type and the width of the film-forming region, thereby finding the film-forming region width that is optimal for the formation of cell aggregates on a specific amorphous carbon film by the desired cells, and by using a carrier on which a large number of amorphous carbon films having that region width are formed, it becomes possible to efficiently produce the cell aggregates. [Brief explanation of the drawings]

[0017] [Figure 1]Amorphous carbon film deposition pattern (left image; right-angled triangle with opposite sides of 1 mm and adjacent sides of 10 mm) and F2 cell aggregation formation within the deposition pattern area (right image; 1500 cells / mm2, cultured for 3 days). [Figure 2] Various amorphous carbon film deposition patterns (triangles with opposite sides of 0.5 mm and lengths of 11 / 10 / 8 / 5 mm, left: overall view, right: enlarged view). Note that "length" refers to the shortest distance between the vertex and the opposite side. [Figure 3] F2 cells in the deposition area of ​​an amorphous carbon film (CH4 + H2 (H2:0%)) (cultured for 72 hours). Deposition area width: 100 μm and 400-500 μm (left image), deposition area width: 200-300 μm (right image). Cell aggregates can be seen in the dashed area. [Figure 4] F2 cells in the deposition area of ​​an amorphous carbon film (CH4 + H2 (H2: 20%)) (cultured for 72 hours). Deposition area width: 100 μm and 400-500 μm (left image), deposition area width: 200-300 μm (right image). Cell aggregates can be seen in the dashed line area. [Figure 5] F2 cells in the deposition area of ​​an amorphous carbon film (CH4 + H2 (H2: 60%)) (cultured for 72 hours). Deposition area width: 100 μm and 400-500 μm (left image), deposition area width: 200-300 μm (right image). Cell aggregates can be seen in the dashed line area. [Figure 6] Deposition pattern of amorphous carbon film with a constant deposition area width (rectangle with short side 0.1 mm and long sides 11 / 10 / 9 / 8 / 7 / 6 mm, left image: overall view, right image: enlarged view). [Figure 7] F2 cells in the deposition region of an amorphous carbon film with a fixed deposition region width (0.1 mm) (72 hours of culture). Upper left: amorphous carbon film (CH4 + H2 (H2: 0%)), upper right: amorphous carbon film (CH4 + H2 (H2: 20%)), lower left: amorphous carbon film (CH4 + H2 (H2: 40%)), lower right: amorphous carbon film (CH4 + H2 (H2: 60%)). [Figure 8]NIH3T3 cells in the deposition areas of various amorphous carbon films (cultured for 72 hours). Top left: amorphous carbon film (CH4 + H2 (H2: 0%)), top right: amorphous carbon film (CH4 + H2 (H2: 20%)), bottom left: amorphous carbon film (CH4 + H2 (H2: 40%)), bottom right: amorphous carbon film (CH4 + H2 (H2: 60%)). DETAILED DESCRIPTION OF THE INVENTION

[0018] The first aspect of the present invention is a carrier for finding conditions under which cells form cell aggregates on an amorphous carbon (DLC) film, characterized in that the carrier has an area on its surface where an independent DLC film is formed, and the formed area is approximately triangular and / or approximately trapezoidal.

[0019] Amorphous carbon films are produced by various deposition methods and have a wide range of applications, but the physical properties of amorphous carbon films have not been defined, and there are no quantitative evaluation indices, which hinder their production and use. Therefore, in order to properly utilize amorphous carbon films suited to their intended applications, classification and standardization of the applications and physical properties of the carbon films have been required. To date, more than 75 types of amorphous carbon films have been classified into sp 2 / sp 3 A total of 14 analyses were performed, including bond ratio, hydrogen content, and film density, and sp 2 / sp 3 Based on the bonding ratio and hydrogen content, amorphous carbon films are classified into ta-C, ta-C:H, aC, and aC:H films with a tolerance of ±5% (Non-Patent Document 1). In addition to the above, amorphous carbon films are also classified into PLC (Polymer-like carbon), which has a high hydrogen content and exhibits polymeric properties, and GLC (Graphite-like carbon), which exhibits graphite-like properties, and these are collectively classified as amorphous carbon films. The amorphous carbon film according to the present invention is a graphite structure-derived sp 2 and sp derived from diamond structure 3Any carbon film having a mixed hybrid orbital and a hydrogenated structure may be used, and it may belong to any of the above categories, without any particular limitation.

[0020] The method for forming the amorphous carbon film according to the present invention is not particularly limited and may be any existing film-forming method and / or a new method, such as ionization deposition, sputtering, chemical vapor deposition (CVD), DC plasma CVD, high-frequency plasma CVD, plasma ion implantation, superimposed high-frequency plasma ion implantation, ion plating, arc ion plating, ion beam deposition, or laser ablation, or a combination thereof, such as ionization deposition, sputtering, chemical vapor deposition (CVD), DC plasma CVD, high-frequency plasma CVD, plasma ion implantation, superimposed high-frequency plasma ion implantation, ion plating, arc ion plating, ion beam deposition, or laser ablation, or a combination thereof, with sputtering and CVD being preferred, and CVD being particularly preferred, although the present invention is not limited to amorphous carbon films formed by these film-forming methods.

[0021] Furthermore, the raw material for the amorphous carbon film according to the present invention is not particularly limited, but a hydrocarbon-based raw material gas (a raw material gas composed of hydrocarbons, which is a general term for compounds made up of only carbon atoms and hydrogen atoms) may be used, and specifically, methane (CH), ethane (C2H6), propane (C3H8), butane (C4H 10 ), pentane (CH 12 ), hexane (CH 14 ), heptane (C7H 16 ), octane (C8H 18 ), Nonane (C9H 20 ), Decane (C 10 H 22 ), ethylene (C2H4), propylene (C3H6), butene (C4H8), pentene (C5H 10 ), hexene (C6H 12), acetylene (C2H2), propyne (C3H4), benzene (C6H6), toluene (C6H5CH3), dimethylbenzene (C6H4C2H6), trimethylbenzene (C6H3C3H9), etc. In addition, hydrocarbons that may contain multiple double bonds, multiple triple bonds, and multiple benzene rings, or combinations of these, may also be used. In addition, these hydrocarbons may be used alone or in combination.

[0022] Furthermore, by adjusting the content of nitrogen, fluorine, oxygen, silicon, silver, etc. in the gas flowing in during film formation in addition to the composition of the source gas, it is also possible to form amorphous carbon films with different surface properties. Although not particularly limited, such amorphous carbon films may be used as the amorphous carbon film according to the present invention.

[0023] The thickness of the amorphous carbon film to be formed may be any thickness as long as it is not easily detached from the support, and is not particularly limited, but may preferably be 100 nm to 300 nm.

[0024] A support is a substance that serves as a base for immobilizing other substances, and although it may exhibit adsorption or catalytic activity, it is generally desirable that the support itself is chemically stable and does not interfere with the intended operation. The support in the present invention may be any substance that stably immobilizes an amorphous carbon film when the film is formed, and may have any physical properties depending on the application based on the function of the formed amorphous carbon film. The material is not particularly limited, but examples include metals such as iron, aluminum, copper, stainless steel, nickel, tungsten, chromium, and titanium; precious metals such as gold, silver, platinum, palladium, rhodium, iridium, ruthenium, and osmium; thermoreversible resins such as polyethylene, polypropylene, polyethylene terephthalate, vinyl chloride, polystyrene (styrene resin), ABS resin, acrylic (Methaclear), polyamide (nylon resin), polycarbonate, tetrafluoroethylene (Teflon®), and ethylene diamine phosphate copolymer; thermosetting resins such as phenol resin, bakelite, melamine (Decora), unsaturated polyester, and epoxy thermosetting resin; and glasses such as soda-lime glass, lead glass, borosilicate glass, alkali-free glass, quartz glass, and phosphate glass. In particular, in the present invention, in order to evaluate the affinity and aggregability of biological substances, such as cells, it is desirable to be able to directly observe the biological substances. Therefore, the carrier is preferably made of a material that can transmit light, such as thermoplastic resins, thermosetting resins, and glasses, and particularly preferably glasses.

[0025] As described in

[0005] , the cell aggregate according to the present invention is an aggregate formed by the aggregation of dispersed cells, in which the cells are adhered to one another. In one embodiment, the cell aggregate according to the present invention includes cells that are surface-adhered to one another, and in another embodiment, cells that form cell-cell junctions and / or cell adhesions, such as adherence junctions, in part or all of the aggregate. However, the present invention is not limited to a specific embodiment and includes various cell aggregates. Therefore, the phrase "cells form cell aggregates on an amorphous carbon film" according to the present invention means that, when cells are cultured on the carbon film, the cells that were cultured in a two-dimensionally dispersed state on the surface of the substrate immediately after the start of culture aggregate over time, forming the cell aggregate.

[0026] To identify the conditions for cells to form cell aggregates on amorphous carbon films, particularly the shape of the amorphous carbon film that is suitable for cell aggregate formation, the inventors formed amorphous carbon films of various shapes on the surface of cover glasses made of glass or other materials, uniformly seeded a single type of cell on the surface, and cultured the cells for a certain period of time in a single-well cell culture vessel with each cover glass. They then investigated the shape of the amorphous carbon film that efficiently forms cell aggregates. As a result, they found that even in the limited area of ​​a cover glass, cell aggregates do form on the surface of a uniformly formed amorphous carbon film with a certain extent, but the frequency of formation is extremely low and the variation is large. This is presumably because the scaffold (amorphous carbon film) for adhered cells is uniform and exhibits little change in physical properties, which makes it easy for variations in cell aggregation to occur due to local cell distribution (dense or coarse cell density), resulting in inefficient formation of cell aggregates. On the other hand, when an amorphous carbon film was sandwiched between non-film-formed regions on both sides, the cell aggregation properties of cells localized on the carbon film varied significantly depending on the width of the film-formed region. For example, when an amorphous carbon film in the shape of a right triangle with a 1 mm opposite side and a 10 mm adjacent side was formed on a support and F2 cells were seeded, cell aggregates with a diameter of approximately 100 μm were uniformly formed only in the region with a film-formed region width ranging from 100 to 200 μm (Figure 1).

[0027] To confirm whether the cell aggregation of cells cultured on amorphous carbon films is affected by the width of the film-formed region sandwiched between non-film-formed regions, various triangular film-formed patterns were created in which the width of the region varied continuously (Figure 2). F2 cells were seeded on the film-formed patterns and cultured for 72 hours. When cell aggregates were formed only in the region where the width of the film-formed region was in the range of 100 to 200 μm, regardless of the amorphous carbon films with different CH4:H2 composition ratios (Figures 3 to 5).

[0028] Using the previously mentioned amorphous carbon film deposition pattern with a continuously varying width (triangle), we found that cell aggregates form only in the region between two non-deposited regions with a width of 100-200 μm. Utilizing this finding, we deposited four types of amorphous carbon films with different CH4:H2 composition ratios on a support as rectangular films with a 100 μm width (Figure 6). We then cultured the same cell species (F2 cells) used in the previous evaluation. Good cell aggregates were formed with all of the amorphous carbon films (Figure 7). This confirms that the use of a continuously varying width amorphous carbon film deposition pattern can identify conditions for cell aggregate production suitable for a specific cell type (F12 cells).

[0029] The inventors also conducted similar cell experiments using cultured cell types other than F2 cells (NIH-3T3 cells). The results are shown in Figure 8. Unlike the F12 cells, the width of the film-formed region sandwiched between non-film-formed regions, where cell aggregates are likely to form, varied for each of the four types of amorphous carbon films with different CH:H composition ratios. This suggests that by using a shape deposition pattern in which the width of the deposition region of the amorphous carbon film continuously changes, it is possible to find suitable conditions for producing cell aggregates for each case in which the cell type and / or physical properties of the amorphous carbon film differ.

[0030] In view of the above results, in the present invention, the regions of the support surface on which the amorphous carbon film is formed must be independent regions. Here, "independent" means that the regions on which the amorphous carbon film is formed are present in independent positions on the same surface of the support, and the amorphous carbon film may be surrounded by regions on which the film is not formed. Furthermore, particularly when the purpose is to confirm the differences in the properties of different amorphous carbon films, it is desirable to make the environments as similar as possible except for the differences. Therefore, the areas on which the amorphous carbon films are formed may be similar in size and shape, and the areas may be adjacent or spaced apart at a fixed interval. The length of the gap separating the film-forming regions is not particularly limited, but it is desirable that the environments on the surfaces of adjacent amorphous carbon film regions are substantially identical except for the differences in the properties of the carbon film, and that the surfaces of both regions are close enough to be easily compared and observed. Therefore, the length of the gap separating the film-forming regions is preferably 500 μm to 1 mm, and more preferably 500 μm.

[0031] Since cell aggregates frequently form in the deposition region of an amorphous carbon film where cells are cultured on its surface when the carbon film is sandwiched at a certain limited distance by non-deposition regions on both sides, a deposition pattern with a shape in which the width of the deposition region continuously varies is desirable in order to find this limited distance. Therefore, to achieve this goal, a shape (triangle) connecting both ends of a certain point (vertex) and a line segment (base) or a shape (trapezoid) connecting the ends of two line segments (the shorter line segment is the upper base and the longer line segment is the lower base) so that they do not intersect is desirable as the deposition region of the amorphous carbon film. That is, the approximately triangular and / or approximately trapezoidal deposition region of the amorphous carbon film of the present invention is defined as a region in which cell aggregates are frequently formed, and the lines connecting the vertices and bases of the triangle and / or the lines connecting the upper and lower bases do not need to be straight. As long as the width of the deposition region in which cell aggregate formation is promoted can be clearly determined, the approximately triangular and / or trapezoidal deposition region of the present application is included. Specifically, in the approximately triangular shape, one or both sides connecting the vertex and base may be curved, preferably with the width of both sides increasing simply from the vertex to the base, and more preferably with both sides connecting the vertex and base being straight and the width of both sides increasing at a constant rate from the vertex to the base. Furthermore, in the approximately trapezoidal shape, one or both sides connecting the upper and lower bases may be curved, preferably with the width of both sides increasing simply from the upper base to the lower base, and more preferably with both sides connecting the upper and lower bases being straight and the width of both sides increasing at a constant rate from the upper base to the lower base. Although not particularly limited, the base of the approximate triangle is preferably 1 mm or less, particularly preferably 500 μm or less, the lower base of the approximate trapezoid is preferably 1 mm or less, particularly preferably 500 μm or less, and the upper base of the approximate trapezoid is preferably 200 μm or less, particularly preferably 100 μm or less.

[0032] The cells of the present invention may be derived from any organism, including humans, and include any cells present in the body of such organisms. Furthermore, as long as they contain cells derived from an organism, they are not particularly limited and may be any of a single cell, a cell group, a tissue fragment, etc., and may contain a single species or multiple species. Furthermore, the cells may be directly collected from tissue or may be already established as a cell line or cell type. In particular, when the cells are artificially cultured ex vivo, i.e., so-called cultured cells, the cultured cells may be cultured in a manner not particularly limited, such as organ culture (organ culture), or may be cells individually isolated from tissue by enzymatic and / or mechanical means before culturing, or cells derived from a cell line or cell type (cell culture). Pluripotent stem cells, such as ES cells and iPS cells, are preferred, with ES cells and / or iPS cells being particularly preferred.

[0033] The second aspect of the present invention is an apparatus that includes, as part of its configuration, the carrier of the first aspect of the invention. Any apparatus may be used as long as it has a region on the surface of a carrier where the amorphous carbon film defined in the first aspect is formed. Preferably, the apparatus is used for the purpose of comparing the cell aggregability of the amorphous carbon film. When such a comparison of cell aggregability is envisioned, the apparatus may be an apparatus related to cell culture. Examples of such an apparatus include slides, cover glasses, flasks, tissue culture flasks, culture dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, microcarriers, beads, stack plates, spinner flasks, and roller bottles, but are not limited to these. Furthermore, a person skilled in the art can select an appropriate apparatus depending on the scale, conditions, and duration of the culture. Although not particularly limited, a preferred tool is a cover glass. A cover glass is a thin, transparent plate that covers a specimen placed on a glass slide during microscopic observation. While it is originally a tool used to cover a specimen on a glass slide, in the present invention, it may be used alone without a glass slide. That is, the cover glass can be used in any situation as long as an independent amorphous carbon film having the predetermined shape defined in the first embodiment can be formed on the surface of the cover glass. Although the name includes the word "glass," it does not necessarily have to be made of glass; it can also be made of a transparent material, such as resin. Furthermore, the size and thickness are not particularly limited, and a person skilled in the art can select the appropriate size, thickness, material, etc., depending on the conditions of use.

[0034] A third aspect of the present invention is a method for finding conditions under which cells form cell aggregates on an amorphous carbon (DLC) film, the method comprising culturing cells that induce cell aggregate formation on a DLC film formed in an approximately triangular and / or approximately trapezoidal shape, and identifying areas in which cell aggregates are formed after a certain period of time from the start of culturing.

[0035] The conditions for forming cell aggregates according to the present invention include any conditions related to the formation of cell aggregates by cells when cultured on an amorphous carbon film, and are not particularly limited, but may be conditions related to the shape of the carbon film formed, preferably the shortest distance between the two sides of the carbon film sandwiched between non-film-formed regions, and more preferably the shortest distance between both sides of an amorphous carbon film formed in an approximately triangular and / or approximately trapezoidal shape, if the shape is approximately triangular, or between both sides connecting the upper and lower bases if the shape is approximately trapezoidal.

[0036] The cell culture method according to the present invention is not particularly limited, and may be any method that a person skilled in the art considers appropriate for determining cell aggregation properties as a culture method for each cell to be evaluated when culturing the cells according to the present invention described in

[0032] . Accordingly, in the present invention, the period after the start of culture during which the formation of cell aggregates is confirmed is also not particularly limited, and an appropriate period may be set depending on each cell to be evaluated. [Example]

[0037] Examples are shown below. These are merely illustrative examples and do not limit the scope of the present invention, and various improvements and design changes may be made without departing from the spirit of the present invention.

[0038] 1. Experiment 1: Formation of right-angled triangular amorphous carbon films on cover glass surfaces and preliminary investigation of cell aggregation properties with F2 cells Overview In order to study the effect of the shape of the amorphous carbon film on the cell aggregation of cells cultured on the film, and particularly to evaluate the effect on the cultured cells from the non-film-formed regions on both sides of the amorphous carbon film-formed region, amorphous carbon films were repeatedly formed in identical triangular shapes on the surface of a cover glass, and cells were cultured on the surface of the film-formed regions, and the state of cell aggregate formation was observed.

[0039] 1-2. Experimental method In this experiment, a circular glass substrate (cover glass) was used as the substrate on which the amorphous carbon film was formed. The circular cover glass substrate was placed in a plasma CVD apparatus, and Ar plasma treatment (power frequency: 29.6 kHz, power supply power: 50 W, gas pressure: 30 Pa, gas flow rate: 0.835 × 10) was performed as substrate pretreatment. -7 m 3 / s) for 15 minutes, and then film formation was carried out. The power supply frequency and power supply conditions during film formation were the same as those for Ar plasma treatment. At this time, CH4 gas was used as the source gas, the gas pressure was 30 Pa, and the total gas flow rate was 1.67 × 10 -6 m 3The DLC was fabricated at / s with a thickness of 400 nm. The deposition pattern consisted of adjacent right-angled triangles with opposite sides of 1 mm and adjacent sides of 10 mm, spaced 1 mm apart (Figure 1, left). The cover glass with the film formed was placed in the well of a 24-well polystyrene multi-plate, and F2 cells (seeding density: 1500 cells / mm 2 ) was cultured (37°C, 5% CO2), and the presence or absence of cell aggregate formation was observed for 1 to 3 days after seeding.

[0040] 1-3.Results Figure 1 shows the state of F2 cells three days after the start of culture. On the third day of culture, almost no cells had adhered to the uncoated cover glass surface. In contrast, in the amorphous carbon film-coated area, cells from the surrounding area (uncoated area) flowed into the coated area one day after cell seeding and showed a tendency to proliferate. Furthermore, three days after cell seeding, aggregates formed within the coated area (Figure 1, right). At this time, cell aggregates with a diameter of approximately 100 μm were uniformly formed only in the area of ​​the coated area sandwiched between the non-coated areas, where the width (distance between the hypotenuse and the adjacent side) was 100 to 200 μm (Figure 1, right).

[0041] 2. Experiment 2: Deposition of amorphous carbon films of various compositions into various triangular shapes and investigation of the relationship between the width of the deposited area between non-deposited areas in each carbon film and cell aggregation in F2 cells 2-1. Overview The results of Experiment 1 above suggested that when an amorphous carbon film is sandwiched between non-film-formed regions on both sides, the aggregability of cells localized on the carbon film may vary significantly depending on the width of the film-formed region. To explore this possibility, amorphous carbon films with different hydrogen (H2) contents were formed on cover glasses in various triangular shapes, and cells were cultured on the surface of the film-formed region, after which the formation of cell aggregates was observed.

[0042] 2-2. Experimental method In this experiment, a circular glass substrate (cover glass) was used as the substrate on which the amorphous carbon film was formed. The circular cover glass substrate was placed in a plasma CVD apparatus, and Ar plasma treatment (power frequency: 29.6 kHz, power supply power: 50 W, gas pressure: 30 Pa, gas flow rate: 0.835 × 10) was performed as substrate pretreatment. -7 m 3 / s) for 15 minutes, and then film formation was performed. The power supply frequency and power supply conditions during film formation were the same as those for Ar plasma treatment. At this time, a mixture of CH4 and H2 was used as the source gas, with a gas pressure of 30 Pa and a total gas flow rate of 1.67 × 10 -6 m 3 / s. For the CH4 and H2 mixed gas, the H2 mixture ratio (H2 / (CH4+H2)) was set to 0, 20, and 60% of the total flow rate, and DLC was fabricated to a film thickness of 400 nm for each. The deposition pattern consisted of triangular shapes with opposite sides of 500 μm and lengths of 11, 10, and 8.5 mm, spaced 500 μm apart (Figure 2). Note that "length" refers to the shortest distance between the vertex and the opposite side. The cover glass with each of the above-mentioned films formed thereon was placed in the well of a 24-well polystyrene multi-plate, and F2 cells (seeding density: 1500 cells / mm 2 ) was cultured (37°C, 5% CO2), and the presence or absence of cell aggregate formation was observed 3 days after seeding.

[0043] 2-3.Results The state of F2 cells after 3 days of culture on amorphous carbon films with H2 mixture ratios of 0, 20, and 60% during film formation is shown in Figures 3, 4, and 5, respectively. For all of the carbon films, regardless of the shape of the triangular film formation region, a tendency for cell aggregates to form in the 100-200 μm-wide portions of the film formation region sandwiched between non-film formation regions was confirmed.

[0044] 3. Experiment 3: Deposition of amorphous carbon films of various compositions onto various rectangular shapes and confirmation of frequent cell aggregate formation in F2 cells in the deposition area (area width: 100 μm) of each carbon film. Overview The results of Experiment 2 above confirmed that, for any amorphous carbon film with a different H content, when sandwiched between non-film-forming regions, cell aggregates tend to form in the film-forming region with a width of 100 to 200 μm. Utilizing this finding, we confirmed whether cell aggregates can be formed frequently by providing a wide film-forming region of the amorphous carbon film, with the film-forming region sandwiched between non-film-forming regions having a width of approximately 100 to 200 μm.

[0045] 3-2. Experimental method In this experiment, a circular glass substrate (cover glass) was used as the substrate on which the amorphous carbon film was formed. The circular cover glass substrate was placed in a plasma CVD apparatus, and Ar plasma treatment (power frequency: 29.6 kHz, power supply power: 50 W, gas pressure: 30 Pa, gas flow rate: 0.835 × 10) was performed as substrate pretreatment. -7 m 3 / s) for 15 minutes, and then film formation was performed. The power supply frequency and power supply conditions during film formation were the same as those for Ar plasma treatment. At this time, a mixture of CH4 and H2 was used as the source gas, with a gas pressure of 30 Pa and a total gas flow rate of 1.67 × 10 -6 m 3 / s. For the CH4 and H2 mixed gas, the H2 mixture ratio (H2 / (CH4 + H2)) was set to 0, 20, 40, and 60% of the total flow rate, and DLC was fabricated to a thickness of 400 nm for each. The film formation pattern consisted of rectangular shapes with short sides of 100 μm and long sides of 11, 10, 9, 8, 7, and 6 mm, arranged adjacently at 500 μm intervals (Figure 6). The cover glass with each of the above-mentioned films formed thereon was placed in the well of a 24-well polystyrene multi-plate, and F2 cells (seeding density: 1500 cells / mm 2 ) was cultured (37°C, 5% CO2), and the presence or absence of cell aggregate formation was observed 3 days after seeding.

[0046] 3-3.Results Figure 7 shows the state of F2 cells 3 days after the start of culture on amorphous carbon films with H2 mixture ratios of 0, 20, 40, and 60% during film formation. It was confirmed that cell aggregates were frequently formed in rectangular film formation regions of any length (short side was always 100 μm) on all carbon films. From the above, by using the triangular deposition area pattern of the amorphous carbon film in Experiment 2, the deposition area width exhibiting high cell aggregation, which was found in cells cultured on the surface of the deposition area, can be applied to an instrument equipped with a wide area of ​​deposition areas having this area width, and can be used as a system for forming cell aggregates of the cells at a high frequency.

[0047] 4. Experiment 4: Deposition of amorphous carbon films of various compositions into various triangular shapes and investigation of the relationship between the width of the deposited area between non-deposited areas in each carbon film and the cell aggregation properties of NIH3T3 cells Overview The results of Experiment 2 above confirmed that, for any amorphous carbon film with a different H2 mixing ratio during film formation, when the film was sandwiched between non-film-formed regions, F12 cells tended to form cell aggregates in the film-formed region where the width was 100 to 200 μm. To confirm whether this finding also applies to other cell types, a similar experiment was conducted using another cell type, NIH3T3 cells.

[0048] 4-2. Experimental method In this experiment, the film was formed using the same method as in Experiment 2 (

[0042] ). The film formation pattern consisted of adjacent triangular shapes with opposite sides of 500 μm and lengths of 11, 10, and 8.5 mm, spaced 500 μm apart (Figure 2). Note that "length" refers to the shortest distance between the vertex and the opposite side. The cover glass with each of the above-mentioned films formed thereon was placed in the well of a 24-well polystyrene multi-plate, and NIH3T3 cells (seeding density: 1500 cells / mm 2 ) was cultured (37°C, 5% CO2), and the presence or absence of cell aggregate formation was observed 3 days after seeding.

[0049] 4-3.Results Figure 8 shows the state of NIH3T3 cells after 3 days of culture on amorphous carbon films with H2 mixture ratios of 0, 20, 40, and 60% during deposition. For amorphous carbon films with a 0% H2 mixture ratio during deposition, aggregates were observed in the deposition region widths of 100 μm and 500 μm. For amorphous carbon films with a 20% H2 mixture ratio during deposition, aggregates were observed in the deposition region widths of 200 to 300 μm. For amorphous carbon films with a 40% H2 mixture ratio during deposition, cell aggregates in the pre-aggregate stage were observed in the deposition region widths of 300 to 400 μm. Furthermore, for amorphous carbon films with a 60% H2 mixture ratio during deposition, aggregates were observed in the deposition region widths of 100 to 500 μm. The above results are significantly different from those of F12 cells, in which cell aggregates were found to form in the 100 to 200 μm-wide portions of the deposition region sandwiched between non-deposition regions, regardless of the shape of the triangular deposition region of any carbon film. This demonstrates that the deposition region width of an amorphous carbon film that enhances cell aggregation differs depending on the cell type, and that the support characterized by having a deposition region according to the present invention, as exemplified in the above examples, makes it possible to search for a deposition region width of the carbon film that is suitable for cell aggregate formation in various cells and / or various amorphous carbon films. [Industrial Applicability]

[0050] The present invention provides a carrier for identifying conditions under which cells form cell aggregates on an amorphous carbon (DLC) film. The carrier has a region on its surface where a separate DLC film is formed, and the region is approximately triangular and / or trapezoidal. The carrier can be used as a device for cell culture, such as a cover glass. By seeding and maintaining cells on the surface of the cover glass, it is possible to identify the width of the carbon film formation region on the amorphous carbon film that is suitable for the cells to form cell aggregates at high frequency. Based on this knowledge of the film formation region width, the device can be applied to a wide-area film formation region with the same cells and amorphous carbon film as the system in which the knowledge was discovered, thereby enabling the cells to form cell aggregates at high frequency. This allows for the mass production of pluripotent stem cells, such as ES cells and iPS cells, which are expected to have various medical uses, and has high industrial applicability.

Claims

1. A carrier for finding conditions under which cells form cell aggregates on an amorphous carbon (DLC) film, characterized in that the carrier has an area on its surface where an independent DLC film is formed, and the formed area is approximately triangular and / or approximately trapezoidal.

2. 2. The carrier according to claim 1, wherein the component of the carrier is glass and / or resin.

3. A device comprising the carrier according to any one of claims 1 and 2 as a part of its configuration.

4. 1. A method for identifying conditions under which cells form cell aggregates on an amorphous carbon (DLC) film, comprising: Culturing cells that induce cell aggregate formation on a DLC film formed in a substantially triangular and / or substantially trapezoidal shape; and Identifying the area where cell aggregates form after a certain period of time from the start of culture; The method comprising:

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