Cell culture construct

Abstract

To provide a cell-culturing structure that contributes to more efficiency of cell culture, and can make a damage caused on the cell sheet when removing a cell sheet smaller.SOLUTION: A cell-culturing structure 100 which is used by being immersed in culture solution and has a cell attaching face S on which a cell C to be cultured can be attached, comprises a planar substrate, and a plurality of projection parts PR protruded from one surface of the substrate, where at least an apex A of each of the plurality of projection parts is the cell attaching face, when the cell-culturing structure is seen in a plan view from one front side of the substrate, the plurality of projection parts are constituted only of a plurality of lines of a limited length or constituted of the plurality of lines of limited length and a plurality of points in combination, and regardless of whether the plurality of projection parts are constituted of any case, a closed space is not formed by the plurality of lines of a limited length and the a plurality of points.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority from Japanese Patent Application No. 2023-061465, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a cell culture construct. More specifically, the present invention relates to a cell culture structure for obtaining a cell sheet formed by connecting a plurality of cells. [Background technology]

[0003] BACKGROUND ART Conventionally, it is known to culture cells using a cell culture construct in order to obtain a cell sheet composed of a plurality of cells connected together (for example, Patent Document 1 listed below).

[0004] The following Patent Document 1 discloses a cell culture sheet as the cell culture structure, which comprises a thin film (sheet) and a plurality of columnar microprotrusions provided on one surface of the thin film (sheet) so as to extend in a direction away from the surface. In addition, in the following Patent Document 1, the aggregate of the plurality of columnar minute projections is referred to as a columnar minute projection group.

[0005] Patent Document 1 listed below describes that normal human epidermal keratinocytes, which are skin cells, are grown in a sheet-like form on a cell culture sheet constructed as described above. That is, Patent Document 1 below describes that a cell sheet is obtained by connecting a plurality of normal human epidermal keratinocytes on a cell culture sheet constructed as described above.

[0006] Furthermore, Patent Document 1 below discloses that a cross-shaped gap is provided in the group of columnar micropillar projections so that the center of the gap substantially coincides with the center of the group of columnar micropillar projections. Furthermore, Patent Document 1 below also discloses that the cross-shaped gaps are formed by removing a portion of the columnar minute projections from the group of columnar minute projections. That is, in the cell culture sheet disclosed in Patent Document 1 below, the distance between the cross-shaped gaps is wider than the distance between the adjacent columnar microprojections.

[0007] The above Patent Document 1 describes that by providing the cross-shaped gaps in the group of columnar microprojections as described above, the culture solution (liquid medium) can flow more easily within the group of columnar microprojections, and waste products generated during cell culture can be more easily discharged outside the group of columnar microprojections. Furthermore, the above-mentioned Patent Document 1 describes that by making the culture solution (liquid medium) flow more easily within the columnar microprojection group, nutrients contained in the culture solution (liquid medium) can be efficiently supplied to cells attached to the columnar microprojections that make up the columnar microprojection group. As described above, if nutrients contained in the culture solution (liquid medium) can be efficiently supplied to cells in the cell culture structure, cell culture can be carried out efficiently. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-170935 Summary of the Invention [Problem to be solved by the invention]

[0009] Incidentally, although there has been a demand in recent years for even greater efficiency in cell culture within the cell culture structure, it is difficult to say that this demand has yet been fully met. Furthermore, although it is desirable to be able to remove (peel off) the cell sheet obtained in the cell culture structure from the cell culture structure without damaging it as much as possible, it is difficult to say that this demand has yet been fully met.

[0010] Therefore, an object of the present invention is to provide a cell culture structure that contributes to further improving the efficiency of cell culture and can reduce damage to the cell sheet when the cell sheet is removed. [Means for solving the problem]

[0011] After extensive research, the present inventors have found that in a cell culture structure comprising a flat substrate and a plurality of protrusions protruding from one surface of the substrate, cell culture can be carried out more efficiently by making the protrusions have a specific shape. Furthermore, after extensive research, the inventors have discovered that by making the cross-sectional shape of the multiple protrusions in a cell culture structure into a predetermined shape, it is possible to reduce damage to the cell sheet when removing the cell sheet from the cell culture structure. As a result, the present invention was conceived.

[0012] That is, the cell culture structure according to the present invention is A cell culture structure that is used by being immersed in a culture solution and has a cell attachment surface to which cells to be cultured can attach, a flat substrate and a plurality of protrusions protruding from one surface of the substrate, at least a top of each of the plurality of protrusions being the cell adhesion surface; When the cell culture structure is viewed from one surface side of the substrate in a plan view, the plurality of protrusions are formed only of a plurality of lines having a finite length, or are formed by a combination of a plurality of lines having a finite length and a plurality of dots, In either case where the plurality of protrusions are configured as described above, a closed space is not formed by the plurality of lines of finite length and the plurality of points, When the plan view of the adjacent protrusions is a line, the distance between the lines is I A When the plan view of the adjacent protrusions is a line and a point, the distance between the line and the point is I B When the plan view of the adjacent protrusions is a point, the distance between the points is I CThe diameter of the largest circle contained in the widest area of ​​the cultured cells when viewed in a plane is S. C When I A , I B , and I C is S C shows a value smaller than Each of the first cut surfaces obtained by cutting the multiple lines in a direction perpendicular to the extension direction of these lines, and each of the second cut surfaces obtained by cutting the multiple points in a direction perpendicular to the surface direction of the substrate, have a shape that narrows in width in a direction away from one surface of the substrate. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a cell culture structure that contributes to further improving the efficiency of cell culture and can reduce damage to the cell sheet when the cell sheet is removed. [Brief explanation of the drawings]

[0014] [Figure 1A] 1 is a plan view of the overall configuration of a cell culture structure according to an embodiment of the present invention, viewed from the protrusion side. [Figure 1B] FIG. 1B is a perspective view showing a portion of the cell culture construct shown in FIG. 1A. [Figure 2] FIG. 1 is a plan view of a state in which cells are being cultured on a cell culture structure according to one embodiment of the present invention, viewed from above a protrusion. [Figure 3A] 3 is a cross-sectional view showing the cell culture structure shown in FIG. 2 taken along line IIIA-IIIA. [Figure 3B] 3 is a cross-sectional view showing the cell culture structure shown in FIG. 2 cut at another portion. [Figure 4A] FIG. 10 is a plan view of a part of a cell culture structure according to another embodiment of the present invention, viewed from the protrusion side. [Figure 4B] FIG. 4B is a plan view of another part of the cell culture structure shown in FIG. 4A viewed from the protrusion side. [Figure 4C] FIG. 4B is a plan view of another part of the cell culture structure shown in FIG. 4A viewed from the protrusion side. [Figure 5] FIG. 10 is a plan view of a cell culture structure according to still another embodiment of the present invention, viewed from the protrusion side. [Figure 6] FIG. 10 is a plan view of a part of a cell culture structure according to yet another embodiment of the present invention, viewed from the protrusion side. [Figure 7] FIG. 10 is a plan view of a part of a cell culture structure according to still another embodiment of the present invention, viewed from the protrusion side. [Figure 8] FIG. 10 is a plan view of a part of a cell culture structure according to yet another embodiment of the present invention, viewed from the protrusion side. [Figure 9] FIG. 10 is a plan view of a part of a cell culture structure according to still another embodiment of the present invention, viewed from the protrusion side. [Figure 10] FIG. 10 is a plan view of a part of a cell culture structure according to yet another embodiment of the present invention, viewed from the protrusion side. [Figure 11] FIG. 10 is a plan view of a part of a cell culture structure according to yet another embodiment of the present invention, viewed from the protrusion side. [Figure 12] 10 is a diagram of a Turing pattern showing the arrangement pattern of protrusions when viewed from the protrusion side of a cell culture structure according to yet another embodiment of the present invention, in which black lines and dots correspond to the protrusions. [Figure 13] 10 is a diagram of a Turing pattern showing the arrangement pattern of protrusions when viewed from the protrusion side of a cell culture structure according to yet another embodiment of the present invention, in which black lines and dots correspond to the protrusions. [Figure 14] 10 is a diagram of a Turing pattern showing the arrangement pattern of protrusions when viewed from the protrusion side of a cell culture structure according to yet another embodiment of the present invention, in which black lines and dots correspond to the protrusions. [Figure 15] 10 is a diagram of a Turing pattern showing the arrangement pattern of protrusions when viewed from the protrusion side of a cell culture structure according to yet another embodiment of the present invention, in which black lines and dots correspond to the protrusions. DETAILED DESCRIPTION OF THE INVENTION

[0015] (Cell culture structure) The cell culture structure according to the present invention is a cell culture structure that is used while immersed in a culture medium and has a cell attachment surface onto which cells to be cultured can be attached. The cell culture structure according to the present invention comprises a flat substrate and a plurality of protrusions protruding from one surface of the substrate. In the cell culture structure according to the present invention, at least the top of each of the plurality of protrusions serves as the cell attachment surface. In the cell culture structure of the present invention, when the cell culture structure is viewed in plan from one surface side of the substrate, the multiple protrusions are composed of only multiple lines of a finite length, or are composed of a combination of multiple lines of a finite length and multiple points. In the cell culture structure of the present invention, regardless of whether the multiple protrusions are configured in any of the above ways, no closed space is formed by the multiple lines of finite length and the multiple points. In the cell culture structure according to the present invention, when the planar view of the adjacent protrusions is a line, the distance between the lines is I A When the plan view of the adjacent protrusions is a line and a point, the distance between the line and the point is I B When the plan view of the adjacent protrusions is a point, the distance between the points is I C The diameter of the largest circle contained in the widest area of ​​the cultured cell C when viewed in a plane is S. C When I A , I B , and I C is S C indicates a value smaller than In the cell culture structure of the present invention, each of the first cut surfaces obtained by cutting the multiple lines in a direction perpendicular to the extension direction of these lines, and each of the second cut surfaces obtained by cutting the multiple points in a direction perpendicular to the surface direction of the substrate, have a shape that narrows in width in a direction away from one surface of the substrate.

[0016] In the cell culture structure of the present invention, since a closed space is not formed by the multiple lines of finite length and the multiple points, the culture medium can be distributed thoroughly throughout the grooves formed between the multiple protrusions formed by at least one of the multiple lines and the multiple points. In addition, in the cell culture structure according to the present invention, A , I B , and I C is S C Since the value is smaller than , the cells can be attached to the cell attachment surfaces of the plurality of protrusions at positions spaced apart from the grooves. This allows the culture medium to be distributed thoroughly over the cells attached to the cell attachment surfaces of the plurality of protrusions to be cultured. It also promotes the excretion of waste products produced during the culture. As described above, the cell culture structure according to the present invention contributes to further improving the efficiency of cell culture. Furthermore, since the first cut surface and the second cut surface have a narrow shape as described above, when cells proliferate (grow) on each of the plurality of protrusions and adhere to each other to form a cell sheet, the contact area between the cell sheet and each of the plurality of protrusions can be reduced. As a result, when the cell sheet is detached (separated) from each of the plurality of protrusions, damage to the cell sheet can be reduced.

[0017] Hereinafter, a cell culture structure 100 according to one embodiment of the present invention will be described with reference to FIGS. 1A to 3A and 3B. In the following, the cell culture structure 100 according to one embodiment of the present invention may be simply referred to as the cell culture structure 100 according to this embodiment. Figure 1A shows a plan view of a cell culture structure 100 in which multiple protrusions PR are composed of a combination of multiple lines LI of finite length and multiple points PO, as viewed from the protrusion side; Figure 1B shows an oblique view of a portion of the cell culture structure 100; Figure 2 shows a plan view of the cell culture structure 100 in the state in which cells are being cultured on the cell culture structure 100, as viewed from above the protrusions; Figure 3A shows a cross-sectional view of the cell culture structure 100 cut along line IIIA-IIIA in Figure 2; and Figure 3B shows a cross-sectional view of the cell structure 100 cut at another part. Figure 3A shows only protrusions PR that are classified as lines when viewed in a plane, while Figure 3B shows both protrusions PR that are classified as lines and protrusions PR that are classified as points when viewed in a plane. 2, 3A, and 3B, the cells are indicated by the symbol C. In the cell culture structure 100 according to this embodiment, the shape of the cell C when viewed from above is irregular as shown in FIG. Specifically, the shape of cell C is such that the center is wide and narrows toward both ends. Therefore, the diameter of the largest perfect circle, S C This refers to the diameter of the largest circle contained in the wide central region (see Figure 2). If cell C is a nerve cell having a cell body and an axon, the diameter of the largest perfect circle, S C means the diameter of the largest circle contained in the cell body region. Furthermore, in the cell culture structure 100 according to this embodiment shown in Figures 1A to 3A and 3B, when the cell culture structure 100 is viewed in plan from one surface side of the substrate 10, the arrangement pattern of the multiple protrusions PR is a Turing pattern. The Turing patterns are described in more detail below.

[0018] In this specification, a line refers to a figure having a length and a width, where the length is 1.2 times or more larger than the width, i.e., where L is the length and D is the width, the ratio L / D is 1.2 or more. Also, a point means a shape where L / D is less than 1.2. Furthermore, when the line has a bent shape (for example, an L-shape or a V-shape) or a meandering shape, the length L means the length of the line segment connecting one end of the line to the other end in the bent or meandering state. For example, an ellipse having a major axis and a minor axis, in which the size of the major axis is 1.2 times or more the size of the minor axis, is classified as a line figure, and in which the size of the major axis is less than 1.2 times the size of the minor axis, is classified as a point figure. A perfect circle is a figure in which the length and width are equal, and L / D is 1, so it is classified as a figure belonging to a point. Furthermore, the concept of a line includes not only a single line consisting of a single line, but also a branch line that has a single main line and at least one or more secondary lines branching off from that line. In the branched lines, the main line means the line with the longest length between both ends.

[0019] Furthermore, the diameter of the largest circle contained in the widest area of ​​the cultured cell C when viewed in plan can be determined by observation using a microscope. For example, it can be determined by analyzing a photograph of the cells using any software, etc. Examples of such measurement methods include a method using an all-in-one fluorescence microscope, BZ-H3C Hybrid (manufactured by Keyence Corporation), and image processing software such as dedicated cell counting software (Keyence).

[0020] As described above, the cell culture structure 100 according to this embodiment is used while immersed in a culture solution. More specifically, the cell culture structure 100 according to this embodiment is used by being immersed in a culture solution placed in a container such as a petri dish. When cells are cultured to obtain a cell sheet using the cell culture structure 100 according to this embodiment, waste products are produced. When waste products accumulate in the culture solution, cell culture using the cell culture structure 100 becomes difficult to proceed satisfactorily. Therefore, in cell culture using the cell culture structure 100, it is preferable to remove a portion of the culture medium from the container after a predetermined period of time has elapsed, and then refill the container with new culture medium in an amount equivalent to the amount removed.

[0021] The culture medium is a solution containing components necessary for cell culture (hereinafter also referred to as culture medium components). The culture medium is not particularly limited as long as it is suitable for the cells to be cultured. Examples of culture medium components contained in the culture medium include sugars, amino acids, vitamins, inorganic salts, trace metals, and additives. The culture medium may contain one of the culture medium components alone, or may contain two or more of the culture medium components in combination. The culture medium components may be appropriately selected from known components depending on the cells to be cultured, etc. Among the culture medium components, sugars, amino acids, vitamins, inorganic salts, and trace metals are classified as nutritional components.

[0022] Examples of the sugars include monosaccharides such as glucose, fructose, mannose, and galactose; disaccharides such as sucrose, sucralose, trehalose, maltose, and lactose; trisaccharides such as glucosylsucrose, lactosucrose, and raffinose; tetrasaccharides such as acarbose and maltotetraose; and oligosaccharides such as cyclodextrin.

[0023] Examples of the amino acid include L-glutamic acid, L-glutamine, L-arginine, L-cystine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-alanine, L-asparagine, L-aspartic acid, L-cysteine, and L-hydroxyproline.

[0024] Examples of the vitamins include sodium ascorbate, choline, folic acid, niacin, biotin, pantothenic acid, pyridoxine, riboflavin, thiamine, thymidine, and vitamin B12.

[0025] Examples of the inorganic salt include various sodium salts such as sodium chloride, sodium hydroxide, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium carbonate, and sodium hydrogen carbonate; various potassium salts such as potassium chloride, potassium hydroxide, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium carbonate, and potassium hydrogen carbonate; various calcium salts such as calcium chloride, calcium sulfate, calcium nitrate, calcium phosphate, and calcium carbonate; and various magnesium salts such as magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium phosphate, and magnesium carbonate. Examples of the trace metals include iron sulfate, iron nitrate, copper sulfate, copper nitrate, and zinc sulfate.

[0026] Examples of the additives include serum such as fetal bovine serum, horse serum, and human serum; growth factors (growth-promoting components) such as fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF); proteins such as albumin, antioxidants such as glutathione, ascorbic acid, and ascorbic acid derivatives; antibiotics such as penicillin and streptomycin; pH adjusters such as HEPES; organic acids such as lactic acid and propionic acid; lipids such as cholesterol; fatty acids such as linolenic acid; amines such as ethanolamine and putrescine; reducing agents such as mercaptoethanol and 3-mercapto-1,2-propanediol; thickeners such as sodium alginate, polyvinylpyrrolidone, carboxymethylcellulose, and pullulan; and pH indicators such as phenol red. The serum generally contains proteins such as albumin and the growth factors.

[0027] Examples of culture media containing the above-mentioned culture medium components include equilibrated buffers such as AIM V medium, HFDM-1 Medium, Dulbecco's phosphate buffered saline (D-PBS) and Hank's balanced salt solution (HBSS); Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimal Essential Medium (EMEm), α-MEM (Minimum Essential Medium alpha Modification), Iscove's Modified Dulbecco's Medium (IMDM), Glasgow's MEM (GMEM), Ham's F-10 medium, Ham's F-12 medium, Ham's F-12K medium, RPMI medium 1640, M-199 medium, L-15 medium, McCoy's 5A Medium, MCDB105 medium, MCDB107 medium, MCDB131 medium, MCDB153 medium, MCDB201 medium, NCTC109 medium, NCTC135 medium, Waymouth's Examples of basal culture media include MB752 / 1 medium, CMRL-1066 medium, Williams' medium E, Brinster's BMOC-3 Medium, and E8 medium. The above-mentioned basal culture medium may be used alone or in combination of two or more. Furthermore, the basal culture medium may contain or remove culture medium components depending on the type and state of the cells. The above-mentioned basal culture medium may be appropriately selected from known ones depending on the cells to be cultured.

[0028] As described above, the cell culture structure 100 according to this embodiment has a cell adhesion surface S to which cells to be cultured can adhere. The type of the cells is not particularly limited. The cell is selected from the group consisting of an animal cell, an insect cell, a plant cell, a yeast cell, and a bacterium. The animal cells are broadly divided into cells derived from animals belonging to the phylum Vertebrate and cells derived from invertebrates (animals other than those belonging to the phylum Vertebrate). In the present specification, the origin of the animal cells is not particularly limited, but cells derived from animals belonging to the phylum Vertebrate are preferred. The phylum Vertebrate includes the classes Agnathostomata and Gnathostomata, which include the classes Mammalia, Aves, Amphipods, and Reptilia. The cells derived from an animal belonging to the phylum Vertebrate are preferably cells derived from an animal belonging to the class Mammalia, which is called a mammal. The mammal is not particularly limited. Examples of the mammals include mice, rats, humans, monkeys, pigs, dogs, sheep, and goats.

[0029] In the present specification, the origin of the plant cells is not particularly limited, but cells of plants such as mosses, ferns, and spermatophytes are particularly preferred. Plants from which seed plant cells are derived include both monocotyledonous and dicotyledonous plants. The monocotyledonous plants include orchids, grasses (rice, corn, barley, wheat, sorghum, etc.), and sedges. The dicotyledonous plants include plants belonging to many subclasses such as the subclass Chrysanthemum, the subclass Magnolia, and the subclass Rosa.

[0030] Algae can also be considered as organisms derived from cells. The algae include cyanobacteria (blue-green algae), which are true bacteria; unicellular eukaryotic organisms (such as diatoms, xanthophytes, and dinoflagellates); and multicellular marine algae (such as red algae, brown algae, and green algae).

[0031] Archaea and bacteria can also be considered as organisms derived from cells. Examples of the archaea include methanogens, extreme halophiles, thermoacidophiles, and hyperthermophiles. Examples of the bacteria include lactic acid bacteria, Escherichia coli, and Bacillus subtilis. Cyanobacteria, which are true bacteria, belong to the algae and also to the bacteria.

[0032] The types of animal or plant cells that can be used in the cell culture structure 100 of this embodiment are not particularly limited, but are preferably selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, and germ cells.

[0033] As used herein, the term "pluripotent stem cells" refers to a general term for stem cells that have the ability to differentiate into cells of any tissue (pluripotency). Although not particularly limited, the pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), germ stem cells (GS cells), and the like. The pluripotent stem cells are preferably ES cells or iPS cells. Furthermore, in consideration of the absence of ethical issues, it is particularly preferable that the pluripotent stem cells are iPS cells. As the pluripotent stem cells, various known ones can be used, for example, those described in WO 2009 / 123349.

[0034] As used herein, "tissue stem cells" refer to stem cells that have the ability to differentiate into a variety of cell types (differentiation diversity), even though the cell lineage they can differentiate into is limited to a specific tissue. For example, hematopoietic stem cells in bone marrow differentiate into blood cells (red blood cells, white blood cells, platelets, etc.), and neural stem cells differentiate into neurons. The tissue stem cells are preferably selected from the group consisting of mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, and hematopoietic stem cells.

[0035] As used herein, the term "somatic cells" refers to cells that constitute a multicellular organism, other than germ cells. Note that somatic cells are not passed on to the next generation through sexual reproduction. The somatic cells are preferably selected from the group consisting of hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, kidney cells, lung cells, and blood cells. The blood cells include lymphocytes, erythrocytes, leukocytes, monocytes, macrophages, megakaryocytes, and the like.

[0036] As used herein, the term "germ cells" refers to cells that play a role in transmitting genetic information to the next generation during reproduction. Examples of the reproductive cells include gametes for sexual reproduction and spores for asexual reproduction. The gametes include eggs, egg cells, sperm, sperm cells, and the like.

[0037] The cells may be selected from the group consisting of sarcoma cells, cell lines, and transformed cells. The sarcoma cells are cells derived from a sarcoma. "Sarcoma" refers to a malignant tumor (cancer) that develops in connective tissue cells derived from non-epithelial cells such as bone, cartilage, fat, muscle, and blood. Examples of sarcoma include soft tissue sarcoma and malignant osteosarcoma. The aforementioned cell line is a cell that has become capable of growing indefinitely without being restricted by the cell cycle. The established cell line may be a cell that has been maintained in vitro and has acquired certain stable properties, that is, a cell line. The established cell lines include cell lines derived from various tissues of various species, including humans, such as PC12 cells (derived from rat adrenal medulla), CHO cells (derived from Chinese hamster ovary), HEK293 cells (derived from human fetal kidney), HL-60 cells (derived from human leukocytes), HeLa cells (derived from human cervical cancer), Vero cells (derived from African green monkey kidney epithelial cells), MDCK cells (derived from canine kidney tubular epithelial cells), and HepG2 cells (a cell line derived from human liver cancer). The transformed cells refer to cells whose genetic properties have been changed by introducing nucleic acid (such as DNA) from outside the cells. For the transformation of animal cells, plant cells, and bacteria, suitable methods are known.

[0038] The culture conditions for culturing the cells using the culture medium are not particularly limited as long as they allow the cultured cells to reach the desired state. Typical culture conditions include, for example, using an adjusted basal culture medium in an environment of 95% relative humidity, 37°C temperature, and 5% CO2. Culture conditions may be appropriately set depending on the cells to be cultured. The period of cell culture is not particularly limited as long as it allows the cultured cells to reach the desired state. The cell culture period is, for example, 28 days or less, 21 days or less, 14 days or less, 7 days or less, 5 days or less, or 3 days or less.

[0039] The number of cells seeded in the cell culture structure 100 is not particularly limited. The number of cells seeded in the cell culture structure 100 is, for example, 1 × 10 4 cells / mL or more 1×10 10 cells / mL or less. The number of cells seeded on the cell culture structure 100 is 2 × 10 5 Preferably, the cell density is 5 x 10 cells / mL or higher. 5 More preferably, 1 x 10 cells / mL or more 6 More preferably, it is equal to or greater than cells / mL. The number of cells seeded on the cell culture structure 100 is 1×10 9 Preferably less than 1 x 10 cells / mL 8 cells / mL or less is more preferable, and 1 x 10 7 More preferably, it is less than cells / mL.

[0040] The flat substrate 10 may be made of any of various known materials. Examples of the substrate 10 include those made of inorganic materials such as glass, quartz, and sapphire, and those made of organic materials such as polyethylene resin, polypropylene resin, polyvinyl alcohol resin, polyvinylidene chloride resin, polyvinyl chloride resin, polyethylene terephthalate (PET) resin, polyurethane resin, polystyrene resin, ABS resin, AS resin, acrylic resin, polyamide resin, polyacetal resin, and fluororesin. Among these, it is preferable to use a glass plate made of glass. In the above, each of the resins exemplified as organic materials is a resin with low cytotoxicity. Furthermore, the material constituting the base material 10 and the material constituting the plurality of protrusions PR may be the same.

[0041] As described above, at least the top A of each of the plurality of protrusions PR serves as a cell attachment surface S onto which cells to be cultured can be attached. Here, the cells to be cultured are attached to the cell attachment surface S of each of the plurality of protrusions PR via a protein called the extracellular matrix. In order to allow sufficient attachment of the extracellular matrix, which is a protein, the cell attachment surface S is preferably made of a material with an appropriate balance of hydrophilicity and hydrophobicity.

[0042] Whether or not the material has an appropriate balance of hydrophilicity and hydrophobicity can be determined by evaluating the water wettability of the material that forms the cell adhesion surface S. Specifically, it can be determined by evaluating the wettability of water to a plate-shaped product formed from the material for forming the cell adhesion surface S. The water wettability of the material can be evaluated by measuring the contact angle of water with the material. Specifically, if a material has a water contact angle of 50° or more and 120° or less, it can be said that the material constituting the cell attachment surface S of the multiple protrusions PR has an appropriate balance of hydrophilicity and hydrophobicity. In addition, if the cell attachment surface S is to be made more hydrophobic, it is preferable to use a material with a water contact angle of 90° or more, more preferably 100° or more, and even more preferably 110° or more. Furthermore, if the cell attachment surface S is to be made more hydrophilic, it is preferable to use a material with a water contact angle of 80° or less, more preferably 70° or less, and even more preferably 60° or less. By constructing the cell attachment surface S using a material whose water contact angle falls within the above numerical range, the cell attachment surface S becomes one to which the extracellular matrix can be suitably attached.

[0043] In the technical field of cell culture, a material with a water contact angle of 60° to 70° is considered to be hydrophobic, a material with a water contact angle of less than 60° is considered to be hydrophilic, and a material with a water contact angle of more than 70° is considered to be superhydrophobic. A typical cell culture vessel (e.g., a petri dish) having a flat cell attachment surface S is generally made of a material classified as superhydrophobic in the field of cell culture technology, specifically polystyrene (which, as will be described later, has a water contact angle of 100°). Therefore, in cell culture vessels made of polystyrene or the like, the cell attachment surface S is subjected to an oxidation treatment such as corona discharge so that the cell attachment surface S exhibits a water contact angle within the range of 60° to 70°. In this way, when the cell attachment surface S exhibits a water contact angle within the above range, the cell adhesion rate to the cell attachment surface S will be maximized (cells will adhere to the cell attachment surface S with the highest probability). However, in the cell culture structure 100 of this embodiment, as described above, the cross-sections of the multiple protrusions PR have a narrow shape, and the cell adhesion surface S is formed mainly near the tops of the multiple protrusions PR, so the formation area of ​​the cell adhesion surface S can be made smaller. That is, since the contact area of ​​the cells with the cell adhesion surface S can be made small, it is not necessary to select a material for forming the cell adhesion surface S that maximizes the adhesion rate. Therefore, as described above, the cell adhesion surface S can be made of materials having various contact angles.

[0044] The water contact angle can be determined using a contact angle measuring device (trade name "DM300" manufactured by Kyowa Interface Science Co., Ltd.) and evaluation and analysis software "FAMAS" (manufactured by Kyowa Interface Science Co., Ltd.). Specifically, the water is dropped in a 10 μm droplet onto one surface of the plate-like molding, and then the static contact angle between the water droplet and the plate-like molding is determined within 10 seconds using the contact angle measuring device and the evaluation and analysis software. The water contact angle is a value calculated by the θ / 2 method.

[0045] As a material having a water contact angle of 50° or more and 120° or less, it is preferable to use the organic materials exemplified above as the organic material constituting the substrate 10. Specifically, it is preferable to use, as the organic material, polyethylene resin, polypropylene resin, polyvinyl alcohol resin, polyvinylidene chloride resin, polyvinyl chloride resin, polyethylene terephthalate (PET) resin, polyurethane resin, polystyrene resin, ABS resin, AS resin, acrylic resin, polyamide resin, polyacetal resin, fluororesin, etc. Among these organic materials, it is preferable to use fluororesin, polystyrene resin, acrylic resin, polyethylene resin, or polypropylene resin. Furthermore, the fluororesin may be either a crystalline fluororesin or a non-crystalline (amorphous) fluororesin, but it is more preferable to use an amorphous fluororesin. The crystalline fluororesin may be FEP (perfluoroethylene propene copolymer), and the amorphous fluororesin may be one represented by the following formula (1): The amorphous fluororesin represented by the following formula (1) has terminal functional groups such as -COOH and -CONHSi(OR) n or may have —CF3, where R is a hydrocarbon group. Commercially available amorphous fluororesin products represented by the following formula (1) include the CYTOP (registered trademark) series manufactured by Asahi Kasei Corporation.

[0046] [ka]

[0047] When the polypropylene resin is used, the water contact angle is 91°, when the polyethylene resin is used, the water contact angle is 83°, when the acrylic resin is used, the water contact angle is 64°, when the polystyrene resin is used, the water contact angle is 100°, when the FEP is used, the water contact angle is 115°, and when the amorphous fluororesin represented by the above formula (1) and having -COOH as a terminal functional group is used, the water contact angle is 112°.

[0048] Furthermore, the material for forming the cell adhesion surface S may be a thermosetting resin. As the thermosetting resin, it is preferable to use polyimide resin, phenol resin, epoxy resin, melamine resin, urea resin, unsaturated polyester resin, alkyd resin, or the like. Furthermore, as the thermosetting resin, a polyurethane resin having thermosetting properties (thermosetting polyurethane resin), a polyimide resin having thermosetting properties (thermosetting polyimide resin), or a fluororesin having thermosetting properties (thermosetting fluororesin) may be used. The thermosetting fluororesin may be, for example, an amorphous fluororesin represented by the above formula (1). The amorphous fluororesin represented by the above formula (1) is polymerized to have a three-dimensional network structure by heating at a temperature of about 80°C. For example, the amorphous fluororesin represented by the above formula (1) can be polymerized in a form having a three-dimensional network structure by dissolving the amorphous fluororesin represented by the above formula (1) in a fluorine-based solvent or the like to form a liquid composition, and then heating the liquid composition at a temperature of about 80°C. The CYTOP (registered trademark) series is an amorphous fluororesin represented by the above formula (1) dissolved in a fluorine-based solvent, and the amorphous fluororesin represented by the above formula (1) includes a low molecular weight one with a mass average molecular weight of about 200,000 and a standard molecular weight one with a mass average molecular weight of about 300,000.

[0049] The organic material preferably has optical transparency. In this specification, the term "light transmittance" refers to the degree to which transmitted light is transmitted relative to incident light, or the degree to which fluorescence emitted by excitation light passes through and is scattered less. In addition, in this specification, the expression "having optical transparency" means, for example, that the ratio of light emitted from an object to light incident on the object is high (that is, transmitted light). The light transmittance can be expressed as a percentage, for example, where the ratio of transmitted light to incident light is 0% for a light-blocking substance (e.g., thick black plastic or metal) and 100% for a transparent liquid such as water. When light transmittance is expressed as a percentage as above, "having light transmittance" means that the light transmittance (the ratio of transmitted light to incident light) measured using a spectrophotometer is 40% or more.

[0050] In the cell culture structure 100 according to this embodiment, it is preferable that the entirety of each of the plurality of protrusions PR is made of a material that has a contact angle with water of 50° or more and 120° or less. Specifically, in the cell culture structure 100 of this embodiment, it is preferable that the entirety of the multiple lines LI constituting the multiple protrusion portions PR and the entirety of the multiple points PO are made of a material having a water contact angle of 50° or more and 120° or less. That is, it is preferable that the entirety of each of the plurality of protrusions PR is formed from one of the materials exemplified as the material for forming the cell adhesion surface S.

[0051] The organic material preferably has a refractive index of 1.4 or less at 20°C. In the cell culture structure 100 according to this embodiment, each of the plurality of protrusions PR is preferably made of an organic material having a refractive index at 20° C. of 1.4 or less. By constructing each of the multiple protrusions PR from the organic material described above, the difference between the refractive index of each of the multiple protrusions PR and the refractive index of water (1.3334 at 20°C), which is contained in large amounts (98% by mass or more) in the culture medium, can be made sufficiently small. This makes it possible to prevent the light incident on each of the plurality of protrusions PR (incident light) from being scattered by each of the plurality of protrusions PR, and as a result, it becomes possible to observe the cells being cultured under a microscope with much clearer images than before. Furthermore, during culture, the presence of each of the multiple protrusions PR can be made less noticeable in the culture medium, which also allows the cells being cultured to be observed under a microscope with much clearer images than before. As a result, it is possible to easily inspect the growing cells and then efficiently culture the cells.

[0052] Among the various organic materials described above, the amorphous fluororesin exhibits high light transmittance due to its amorphous nature, and due to its high light transmittance, it exhibits a refractive index of 1.4 or less at 20°C. In particular, as described above, the terminal functional groups are -COOH, -CONHSi(OR) nAlternatively, amorphous fluororesin having -CF3 exhibits a refractive index at 20°C of approximately 1.33 to 1.34. Therefore, from the viewpoint of easily inspecting the growing cells and culturing the cells efficiently, it is particularly preferable to use the amorphous fluororesin among the various organic materials mentioned above.

[0053] Here, since the serums described above, such as fetal bovine serum, horse serum, and human serum, usually contain extracellular matrices such as fibronectin, by adding the serum to the culture medium, the extracellular matrix can be present in the cell culture structure 100. Furthermore, when the culture medium does not contain the serum, the cell attachment surface S of each of the multiple protrusions PR may be coated with the extracellular matrix before cell culture is performed using the cell culture structure 100. In addition, in cell culture using the cell culture structure 100, if the cells being cultured are unable to attach to the cell attachment surfaces S of the multiple protrusions PR that serve as scaffolds for proliferation (growth), the cells will undergo apoptosis and die.

[0054] As described above, in the cell culture structure 100 according to this embodiment, when the planar view of adjacent projections PR is a line LI, the distance between the lines LI is I. A When the plan view of the adjacent protrusions PR is a line LI and a point PO, the distance between the line LI and the point PO is I B When the plan view of the adjacent protrusions PR is at points PO, the distance between the points PO is I C The size of the cultured cells when viewed in a plane is S C When I A , I B , and I C is S C indicates a value smaller than

[0055] Distance I A , distance I B , and distance IC is preferably 50 nm or more and 500 μm or less. Furthermore, distance I A means the distance between the opposite edges of the lines LI, and the distance I B means the distance between the center of the point PO and the edge of the line LI opposite to the point PO, and the distance I C means the distance between the centers of the points PO.

[0056] The line width of the line LI and the diameter of the point PO are preferably 10 nm or more and 500 μm or less. In addition, the diameter S of the largest circle contained in the widest area of ​​the cultured cell C when viewed in a plane is C is preferably 100 nm or more and 5 mm or less.

[0057] Furthermore, the height of the protrusions PR from one surface of the flat substrate 10 (the surface on which the protrusions PR are arranged) is preferably 50 nm or more and 5 mm or less.

[0058] In the cell culture structure 100 according to this embodiment, the flat substrate 10 has openings O1 and O2 at one end and the other end, respectively, as shown in FIGS. 1A and 2. In the cell culture structure 100 according to this embodiment, as shown in FIGS. In this way, since the flat substrate 10 has openings O1 and O2 at one end and the other end, respectively, it becomes easier to supply culture medium to the entire surface of the flat substrate 10 through the grooves D formed between the multiple protrusions PR. Furthermore, through the grooves D, metabolites produced by cell culture can be more easily discharged to the outside of the cell culture structure 100. This allows cell culture to be carried out in the cell culture structure 100 more efficiently.

[0059] In the cell culture structure 100 according to this embodiment, the adjacent protrusions PR do not come into contact with each other regardless of the configuration of the protrusions PR, as shown in Figures 1A, 1B, and 2. That is, in the cell culture structure 100 according to this embodiment, the areas occupied by the adjacent protrusions PR in plan view do not overlap with each other, even if the adjacent protrusions are lines in plan view, a line and a point in plan view, or point in plan view. In this way, since adjacent protrusions PR are not in contact with each other, the culture medium can flow more smoothly in the grooves D formed between the protrusions PR, making it possible to more efficiently supply the culture medium to the entire surface of the flat substrate 10 and discharge it to the outside of the cell culture structure 100. This allows cell culture to be carried out in the cell culture structure 100 more efficiently.

[0060] In the cell culture structure 100 of this embodiment, as shown in Figures 3A and 3B, each of the first cut surfaces S1 obtained by cutting multiple lines LI in a direction perpendicular to the extension direction of these lines LI, and each of the second cut surfaces S2 obtained by cutting multiple points PO in a direction perpendicular to the surface direction of the substrate 10, have a shape that narrows in width in a direction away from one surface of the substrate 10. In the cell culture structure 100 according to this embodiment, as shown in FIGS. 3A and 3B, the narrowed shape has at least an arc-shaped apex. Specifically, in the cell culture structure 100 shown in FIGS. 3A and 3B, the first cut surface S1 and the second cut surface S2 have a semicircular shape with rounded apexes. Because the first cut surface S1 and the second cut surface S2 have such a shape, the damage that the cells receive from the protrusion PR can be reduced compared to when the tops of the first cut surface S1 and the second cut surface S2 have a sharp, narrow shape with an acute angle. Furthermore, as explained above, when cells proliferate (grow) on each of the multiple protrusions PR and these cells adhere to each other to form a cell sheet body, the contact area between the cell sheet body and each of the multiple protrusions PR can be reduced. As a result, when the cell sheet is detached (separated) from each of the plurality of protrusions PR, damage to the cell sheet can be reduced. That is, the cell sheet can be efficiently detached while minimizing damage caused by each of the plurality of protrusions PR.

[0061] Here, when the cell sheet is obtained using a cell culture structure consisting only of a flat substrate, most of the surface of the cell sheet is attached to the surface of the flat substrate via proteins such as extracellular matrix. Therefore, when removing (peeling) the cell sheet from the flat substrate, it is necessary to use a protease to decompose proteins such as the extracellular matrix. However, if a large amount of the protease is used to sufficiently decompose proteins such as the extracellular matrix, the adhesion between cells in the cell sheet will be broken down, making the individual cells more likely to separate. As a result, it may become impossible to detach the cells in a sheet state. Furthermore, if the amount of protease used is reduced in order to prevent the cells in the cell sheet from losing their adhesion, the adhesive strength between the cells and the substrate via the extracellular matrix is ​​strong, and it is not easy to adjust the amount of force used to detach the cells in sheet form without damaging the cells. From the above viewpoint, when using the protease to remove (peel) sheet-like cells (cell sheet) from the cell culture structure, the amount of the protease used and the peel operation require the proficiency of the worker. However, in the cell culture structure 100 of this embodiment, cell culture is mainly carried out near the tops A of the multiple protrusions PR, and the resulting cell sheet body is supported by the tops A of the multiple protrusions PR. Furthermore, the plurality of protrusions PR have a shape that narrows toward the apex A on the cut surface, so that the contact area between the cell sheet body and the apex A of the plurality of protrusions PR is extremely small. Therefore, in the cell culture structure 100 according to this embodiment, the obtained cell sheet can be removed (peeled off) without using the protease. That is, with the cell culture structure 100 according to this embodiment, the obtained cell sheet can be easily removed (peeled off) regardless of the skill level of the operator.

[0062] In the cell culture structure 100 according to this embodiment, the cell adhesion surface S is made of an organic material having optical transparency. In the cell culture structure 100 according to this embodiment, all of the exposed surfaces of the plurality of protrusions PR formed by the plurality of lines LI and the plurality of points PO are made of the optically transparent organic material. If the entirety of each of the multiple protrusions PR formed by the multiple lines LI and multiple points PO is made of the organic material having optical transparency, then, as described above, the entire exposed surface will be made of the organic material having optical transparency. In the cell culture structure 100 according to this embodiment, it is preferable to use an organic material having optical transparency with a refractive index of 1.4 or less, as described above. Therefore, it is preferable to use the amorphous fluororesin as the light-transmitting organic material, and among the amorphous fluororesins, it is preferable to use the amorphous fluororesin having a terminal functional group such as -COOH, -CONHSi(OR) n Alternatively, it is preferable to use an amorphous fluororesin having —CF3.

[0063] In the cell structure 100 of this embodiment, when the cell culture structure 100 is viewed in a plane from one surface side of the substrate 10, the arrangement pattern of the multiple protrusions is preferably a Turing pattern that shows the concentration distribution of two types of substances on a two-dimensional plane derived from a reaction-diffusion equation.

[0064] The Turing pattern is a periodic pattern that spontaneously emerges from a chemical reaction system that satisfies certain conditions, and its theoretical existence was proven by British mathematician Alan Turing. With the development of computers, the Turing patterns have been applied to research into the manifestation of patterns found in the natural world, such as life and geology. More specifically, the Turing pattern refers to a periodic pattern (design) that is drawn when two chemical substances satisfy certain conditions and are in a relationship in which they control each other's synthesis, such that the concentration distribution of the two chemical substances is not uniform in space (for example, a two-dimensional planar space), and areas of high concentration (hereinafter referred to as high-concentration areas) and areas of low concentration (hereinafter referred to as low-concentration areas) exist in the space, and the high-concentration areas and the low-concentration areas form a repeating pattern in the space and are stabilized. It should be noted that the "spontaneously generated periodic pattern" refers to, for example, a periodic pattern that is formed in a self-organizing manner.

[0065] The mechanism by which the Turing pattern is formed in the space can be mathematically expressed using the Gray-Scott model shown in the following equation (1) which represents reaction-diffusion.

[0066]

number

[0067] The Gray-Scott model is a model of a reaction-diffusion system. Here, a reaction-diffusion system is a mathematical model that describes how the concentration of one or more substances distributed in space changes due to the influence of two processes: a local chemical reaction process in which one or more substances change into each other in space, and a diffusion process in which one or more substances spread throughout space. In other words, a reaction-diffusion system is a model of how the concentration of one or more substances distributed in a space changes over time due to a chemical reaction process involving the one or more substances and a diffusion process of the one or more substances into the space. When the substances involved in the reaction-diffusion system are two types, one chemical substance U and another chemical substance V, the reaction-diffusion system can be expressed by solving the reaction-diffusion equation shown in the following formula (1').

[0068]

number

[0069] In the reaction-diffusion equation shown above in equation (1'), when the reaction terms g(u,v) and h(u,v) satisfy certain conditions, a spatial pattern spontaneously emerges in the distribution of the concentration u of one chemical substance U and the distribution of the concentration v of another chemical substance V. The spatial patterns that arise in this way are collectively called Turing patterns.

[0070] Here, the Gray-Scott model shown in the above formula (1) originally models the following reaction, which is an autocatalytic chemical reaction in a gel medium, which is represented by one chemical substance U as a raw material, another chemical substance V as an intermediate product and also as an autocatalytic substance (hereinafter referred to as the autocatalytic substance), and an inert substance P as a chemically stable final product. U+2V→3V (a) V→P (b)

[0071] As explained above, the inactive substance P is chemically stable and therefore does not react with the raw material chemical substance (one chemical substance U) and the autocatalytic substance (another chemical substance V). Furthermore, since the raw material chemical substance (one chemical substance U) is periodically supplied from the outside, the above chemical reaction can proceed perpetually. In this way, when the above-mentioned chemical reaction is continuously progressing, and the diffusion and reaction of the raw material chemical substance (one chemical substance U) and the autocatalytic substance (another chemical substance V) proceed simultaneously, in a space such as a two-dimensional plane, differences in concentration occur depending on the location in the space, and the concentration changes over time. This allows a pattern to appear in a space such as a two-dimensional plane space that changes over time.

[0072] Here, the reaction of the formula (a) in the above autocatalytic chemical reaction is expressed by the formula uv, which is expressed by the concentration u of one chemical substance U and the concentration v of another chemical substance V. 2 It is shown as follows. On the other hand, the reaction of equation (b) can be expressed as kv, which is expressed in terms of the concentration v of another chemical substance V, which is also an intermediate product, and the reaction coefficient k. Here, in order to continue the above autocatalytic chemical reaction, it is necessary to not only replenish one chemical substance U consumed in the reaction, but also to remove another chemical substance V that becomes excessive in the reaction. Therefore, in addition to the above equation, after determining the inflow / outflow coefficient f, one chemical substance U is inserted as an inflow (supply) term in the equation f(1-u) using its concentration u, and another chemical substance V is inserted as an outflow (discharge) term in the equation fv using its concentration v. Then, by expressing the reaction terms g(u,v) and h(u,v) shown in the above equation (1') using the equations shown above, the Gray-Scott model equation shown in the above equation (1) is obtained.

[0073] In recent years, the use of the Gray-Scott model shown in the above formula (1) has been proposed to simplify and reproduce cell culture-related phenomena such as cell proliferation. When the above-described cell culture is expressed by the Gray-Scott model shown in equation (1), one chemical substance U is a nutrient such as sugars, amino acids, vitamins, inorganic salts, etc., and another chemical substance V is a growth factor (growth-promoting component) such as FGF, EGF, HGF, VEGF, PDGF, etc., and cell growth is considered to be a reaction dynamics. In order to achieve a state in which cells grow continuously in the place where the cell culture is performed (for example, a scaffold material such as a culture vessel), it is assumed that the nutrient (chemical substance U) is inflowed with a coefficient f, and growth factors (chemical substance V) that become excessive as a result of the culture are discharged with a coefficient f that is the same as the inflow coefficient f of the nutrient, and further that growth factors (chemical substance V) that have lost their growth-promoting function are discharged with a coefficient k. Under this assumption, the above-mentioned autocatalytic chemical reaction represented by equation (a) can be interpreted as a cell growing by consuming one unit of a nutrient component (U), which is a chemical substance, using two units of a growth factor (growth-promoting component) as a catalyst, and producing and secreting physiologically active substances such as hormones and cytokines equivalent to one unit of the growth factor (growth-promoting component). The physiologically active substance can be considered as another chemical substance V since it is classified as a growth factor (growth-promoting component). On the other hand, equation (b) in the above autocatalytic chemical reaction assumes that another chemical substance, V, a growth factor (growth-promoting component), is converted by cell growth into a physiologically inactive substance with no growth-promoting function with a coefficient k.

[0074] From these facts, equation (a) in the above autocatalytic chemical reaction means that when cells use two units of growth factor (growth-promoting component) as a catalyst to consume one unit of nutritional component, one new unit of growth factor (growth-promoting component) is produced, resulting in three units of growth factor (growth-promoting component) being present in the culture medium. Furthermore, equation (b) in the above autocatalytic chemical reaction means that one unit of another chemical substance V, a growth factor (growth-promoting component), is converted with reaction coefficient k into a physiologically inactive substance that has lost its growth-promoting function, i.e., into a chemically stable final product as an inactive substance P. Here, the unit amount means the ratio of each chemical substance in a state where an autocatalytic chemical reaction (self-reproduction in the case of cell culture) ideally proceeds.

[0075] When applying the Gray-Scott model shown in the above formula (1) to cell culture, u is the concentration of the nutrient, v is the concentration of the growth factor, and D u is the diffusion coefficient of the nutrient, and D vis the diffusion coefficient of the growth factor, and f is the inflow / outflow coefficient. In the first equation of the above formula (1), f is the inflow coefficient of nutrients, and in the second equation of the above formula (1), f is the outflow coefficient of excess growth factors. k is the reaction coefficient at which growth factors are converted into substances that lose their growth-promoting function.

[0076] In actual cell culture operations, the medium is refreshed to an ideal state by periodically replacing the medium with a new one containing the nutritional components and growth factors at optimal concentrations. By periodically exchanging the culture medium as described above, the nutrients and growth factors flow in and out with a coefficient f, and the chemically stable final product as an inactive substance P flows out (excretes) with a coefficient k. In the above explanation, the term "medium" is a concept that includes not only liquids such as culture solutions but also jelly-like substances.

[0077] Furthermore, u and v are functions of time t and spatial position coordinates x and y, and are expressed as u(t,x,y) and v(t,x,y). Furthermore, Δ is the Laplacian, and if the space has two dimensions (x and y coordinates), then Δ=∂ 2 / ∂x 2 +∂ 2 / ∂y 2 Represents. The Laplacian is an expression for eliminating the difference in concentration between the concentration u1 of one chemical substance U at an arbitrary point in the space and the concentration u2 of one chemical substance U in the vicinity of the arbitrary point, or for eliminating the difference in concentration between the concentration v1 of another chemical substance V at an arbitrary point in the space and the concentration v2 of another chemical substance V in the vicinity of the arbitrary other point. For example, if the concentration u1 of the one chemical substance U at the arbitrary location is higher than the concentration u2 of the one chemical substance U in the vicinity thereof, the one chemical substance U will diffuse from the arbitrary location toward the vicinity thereof. On the other hand, if the concentration u1 of the one chemical substance U at the arbitrary location is lower than the concentration u2 of the one chemical substance U in the vicinity thereof, the one chemical substance U diffuses from the vicinity toward the arbitrary location. As explained above, in cell culture, one chemical substance U is a nutrient component, and another chemical substance V is a growth factor (growth-promoting component).

[0078] The growth factors (growth-promoting components) act as intercellular signal transducing substances by specifically binding to receptor proteins present on the surface of target cells. The growth factors (growth-promoting components) act to regulate various cellular and physiological processes. As explained above, the growth factors (growth-promoting components) are also produced and secreted as physiologically active substances such as hormones and cytokines during cell culture. As described above, the cell culture structure 100 according to this embodiment is used by being immersed in a culture solution placed in a container such as a petri dish. In order to prevent the accumulation of the final product (e.g., waste product) which is the inactive substance P in the container during cell culture, as described above, in cell culture using the cell culture structure 100, after a predetermined period of time has elapsed, a portion of the culture medium may be removed from the container, and an amount of new culture medium equivalent to the amount removed may be replenished into the container. In such cases, as described above, it is preferable to remove a portion of the culture medium from the container so that the physiologically active substances produced and secreted during cell culture remain in the container as the growth factors (growth-promoting components).

[0079] In the Gray-Scott model equation shown in equation (1) above, the first term on the right-hand side of the equation for ∂u / ∂t represents the diffusion of one chemical substance U (nutrient) in space, and the second term on the right-hand side is a reaction term, representing the change in concentration of both the one chemical substance U (nutrient) and another chemical substance V (growth factor) in space due to an autocatalytic chemical reaction (here, the self-proliferation of cells). The third term on the right side is a perturbation term for continuing the reaction, and means that one chemical substance U (nutrient component) consumed in the reaction term is replenished by flowing it into the space. That is, the first term on the right side of the equation for ∂u / ∂t represents the diffusion term, the second term represents the reaction term, and the third term represents the inflow term. The lower the concentration u of the one chemical substance U (nutrient component) in the space, the greater the inflow of the one chemical substance U (nutrient component) into the space, while the higher the concentration u of the one chemical substance U (nutrient component) in the space, the smaller the inflow of the one chemical substance U (nutrient component) into the space. Then, when the maximum concentration reaches 1, the inflow of the one chemical substance U (nutrient component) into the space stops.

[0080] Furthermore, in the Gray-Scott model equation shown in equation (1) above, the first term on the right-hand side of the equation for ∂v / ∂t also represents a diffusion term, just as in the equation for ∂u / ∂t. The second term on the right-hand side is a reaction term, which represents the change in concentration of one chemical substance U (nutrient component) and another chemical substance V (growth factor) in the space due to an autocatalytic chemical reaction (here, the self-renewal of cells). The third term on the right-hand side is a perturbation term to continue the reaction, as in the equation for ∂u / ∂t, and means that other chemical substances V (in the case of cell culture, excess growth factors and growth factors that have lost their growth function) produced in the reaction term are discharged (exhausted) from the space. In other words, the second term on the right hand side of the equation for ∂v / ∂t also represents a reaction term, and the third term represents an outflow term. It should be noted that the higher the concentration v of the other chemical substance V in the space, the greater the amount of the other chemical substance V that flows out of the space. Then, when the concentration v of the other chemical substance V becomes 0, the outflow of the other chemical substance V from the space stops.

[0081] Incidentally, patterns drawn on the surface of the body of animals and the like also belong to the Turing patterns. For example, the patterns (markings) drawn on the body surfaces of cheetahs, leopards, giraffes, zebras, etc. all belong to the Turing pattern and can be expressed using the Gray-Scott model shown in equation (1) above. The pattern on the surface of a cheetah's body is a spotted pattern, the pattern on the surface of a leopard's body is a leopard print, the pattern on the surface of a giraffe's body is a mesh pattern, and the pattern on the surface of a zebra's body is a striped pattern.

[0082] The Turing pattern is preferably a pattern that indicates a region where the concentration of one of the substances is high in the concentration distribution of one chemical substance U and another chemical substance V, which is obtained by computer simulation using a finite difference method of the Gray-Scott model shown in the above formula (1), which represents the reaction-diffusion between one chemical substance U and another chemical substance V on a two-dimensional plane. The computer simulation begins with an initial state in which one chemical U and another chemical V are randomly distributed in equal amounts on the two-dimensional plane, and ends when the reaction-diffusion reaches equilibrium.

[0083] The difference method is preferably carried out using the recurrence formulas expressed by the following formulas (2) and (3).

[0084]

number

[0085]

number

[0086] The Turing pattern is preferably generated by a simulation in which a periodic boundary condition is applied to the outermost boundary of a two-dimensional plane divided into a finite number of elements. A periodic boundary condition is a condition that is set on the boundary surface of a single area pattern when a pattern (design) is formed by the periodic repetition of multiple area patterns and only one area pattern is modeled to perform a simulation. That is, it is preferable that the Turing pattern is generated by a simulation in which a periodic boundary condition is applied to the outermost boundary of the one area pattern located outermost on the two-dimensional plane.

[0087] In the Gray-Scott model equation (1), the coefficient D u is in the range of 0.002 to 0.01, and the coefficient D v It is preferable that the coefficient f is in the range of 0.0003 or more and 0.0015 or less, the coefficient f is in the range of 0.03 or more and 0.18 or less, and the coefficient k is in the range of 0.03 or more and 0.07 or less. Also, coefficient D u is more preferably in the range of 0.003 to 0.004, and the coefficient D v More preferably, coefficient f is in the range of 0.0008 to 0.0010, more preferably coefficient f is in the range of 0.16 to 0.17, and more preferably coefficient k is in the range of 0.04 to 0.05.

[0088] The Turing patterns shown in Figures 1A and 1B are based on the coefficient D in the Gray-Scott model shown in equation (1) above. u is set to 0.004, and coefficient D v is 0.0009, coefficient f is 0.167, and coefficient k is 0.0425, and this is a pattern of the region where the concentration of chemical substance V (growth factor) is higher than the concentration of chemical substance U (nutrient component) when the reaction reaches equilibrium.

[0089] The simulation can be performed, for example, by using a computer and executing a program using JavaScript as a programming language within a limited space of 200 x 200 pixels. Here, since a periodic boundary condition is applied to the simulation, it is possible to connect finite spaces of 200 x 200 pixels together to obtain Turing patterns of any size. Specifically, if 200 x 200 pixels are squares with sides of 100 μm (in this case, one pixel is a square with sides of 0.5 μm), by connecting 10 x 10 of these 100 μm squares in a matrix, the patterns formed in each cell (squares with sides of 100 μm) can be connected naturally (without appearing unnatural) at the boundaries between them. This allows a Turing pattern to be formed within an area of ​​a square with sides of 1 mm (10 x 10 squares with sides of 100 μm connected in a matrix).

[0090] Here, when a simulation using the Gray-Scott model expressed by the above formula (1) is performed over a long period of time, the pattern change depicted by one chemical substance U and another chemical substance V converges. In other words, the reaction-diffusion carried out by one chemical substance U and another chemical substance V reaches an equilibrium state. In this way, when reaction-diffusion reaches equilibrium, the entire cell attachment surface S is covered with proliferated cells, and cell proliferation no longer progresses. Then, by reflecting a pattern on the flat substrate 1 in which protrusions PR are provided at locations where the concentration of the growth factor (growth-promoting component), which is another chemical substance V, is higher than the concentration of the nutrient component, which is one chemical substance U, at the point when the reaction-diffusion reaches equilibrium, a cell culture structure 100 having multiple protrusions PR based on the Turing pattern can be obtained. Furthermore, in a static state, such as when the cell culture structure 100 having the protrusions PR formed in this manner is immersed in a culture medium, other chemical substances V, such as growth factors, can be distributed over a wider area in the cell culture structure 100 than in a cell culture structure that does not have the protrusions formed as described above. This allows cell culture to be carried out more efficiently. In addition, in dynamic conditions such as when the culture medium is replaced, the culture medium flows through the gaps between the multiple protrusions PR, so that the culture medium components contained in the culture medium can be supplied from below to the cells attached to the cell attachment surface S of the protrusions PR. This also allows cell culture to be carried out more efficiently.

[0091] As described above, when the Turing pattern is obtained by simulation using the condition of adding a periodic boundary condition to the Gray-Scott model equation, the results of the simulation are calculated as concentrations u and v of chemical substances U and V for each pixel, and therefore the Turing pattern is usually expressed in a raster format. The raster format is a format that uses multiple pixels to represent objects in an image, so if a simple object represented by, for example, a curve or a circle is represented in the raster format, the edges of the curve or circle in the object will be blurred, making it impossible to represent the object smoothly. Another problem is that the data size of objects displayed in the raster format becomes large. In contrast, the vector format stores and reproduces the positions of multiple points and lines connecting multiple points as numerical data, making it suitable for smoothly displaying simple objects represented by curves, circles, etc. Therefore, when the Turing pattern obtained by simulation is expressed in a raster format, it is preferable to convert the Turing pattern expressed in the raster format so that it is expressed in the vector format. This allows the contour of the Turing pattern (an object including lines and circles) obtained by simulation to be expressed smoothly.

[0092] (Method of manufacturing a cell culture structure) The cell culture structure of this embodiment can be manufactured using a laminate for a cell culture structure in which a resin layer for forming protrusions is laminated on a flat substrate, and a formwork having multiple recesses corresponding to the multiple protrusions described above. The mold form can be obtained by preparing a master mold on a substrate on which protrusions having the same shape as the protrusions described above are formed, forming a metal layer to cover the protrusions on the master mold, and then removing the metal layer from the master mold. The method for producing the cell culture structure will be described in detail below.

[0093] [Master mold creation] The master mold can be produced by photolithography using a master mold laminate in which a layer formed of photoresist (hereinafter also referred to as a photoresist layer) is laminated on a substrate, and a photomask on which a pattern corresponding to the pattern to be formed on one surface of the flat substrate is formed. An example of how to fabricate a master mold will be described below.

[0094] <Preparation of laminate for master mold> The photoresist is applied to a substrate to a predetermined thickness to form a coating layer, and then the coating layer is dried. This makes it possible to obtain a master mold laminate in which a photoresist layer is laminated on a substrate. As the photoresist, a positive photoresist is preferably used. The positive photoresist may be a composition containing a base resin and a positive photosensitizer. Examples of the base resin include novolac resins such as cresol novolac resins and acrylic resins, and examples of the positive photosensitizer include naphthoquinone diazide sulfonic acid esters such as 1,2-naphthoquinone diazide sulfonic acid esters, and polyhydroxybenzophenones such as 2,3,4-trihydroxybenzophenone and 2,3,4,4'-tetrahydroxybenzophenone. The positive photoresist preferably contains the acrylic resin as the base resin and the naphthoquinone diazide sulfonic acid ester as the positive photosensitive agent. By using the above-mentioned positive photoresist, the sensitivity of the positive photoresist to light such as ultraviolet light can be improved, and in addition, the positive photoresist can be made to be easily thermally flowable (easily flowable by heat). This makes it easier for the unexposed portion to have a shape that narrows in the direction away from one surface of the base material after the development process described below is performed, and also makes it easier for the narrow shape of the unexposed portion to be an arc shape. The substrate is preferably a glass plate.

[0095] <Exposure processing> Next, the photomask is placed above the photoresist layer, and then ultraviolet light is irradiated from above the photomask. When the thickness of the photoresist layer is 1 μm to 20 μm, the irradiation is performed using ultraviolet light having a wavelength of 200 nm to 450 nm, and the cumulative light amount is 50 mJ / cm 2 2 ~1800mJ / cm 2 It is preferable to carry out the process so that In particular, when the thickness of the photoresist layer is 10 μm, the irradiation is performed using ultraviolet light (I-ray) with a wavelength of 365 nm, with an integrated light amount of 500 mJ / cm 2 When irradiating with ultraviolet light, it is preferable to use a high-pressure mercury lamp as the light source. This allows the pattern formed on the photomask to be reflected in the photoresist layer in the thickness direction as well.

[0096] <Developing and baking process> Next, a development process is carried out on the photoresist layer onto which a predetermined pattern (the pattern of the photomask) has been transferred by exposure to light. As a result, the exposed portions (portions irradiated with ultraviolet light) of the photoresist layer can be removed, and the unexposed portions (portions not irradiated with ultraviolet light) can remain. The development treatment can be carried out by immersing the master mold laminate in a developer containing an alkaline component (e.g., tetramethylammonium hydroxide (TMAH)) at a predetermined concentration for a predetermined period of time, rinsing the entire master mold laminate taken out of the developer with pure water, and then drying the master mold laminate. For example, when the thickness of the photoresist layer is 10 μm, the development treatment is preferably carried out by immersing the master mold laminate in a developer containing 2.38% TMAH for 60 seconds, rinsing the entire master mold laminate taken out of the developer with pure water, lightly blowing air onto the master mold laminate, and then drying the master mold laminate after air blowing at 120° C. for 5 minutes using a hot plate. By carrying out the development treatment as described above, the unexposed portion has a shape that narrows in width in the direction away from the one surface of the base material. Next, the photoresist layer after the development process is subjected to a baking process. The firing treatment can be carried out, for example, by heating at a temperature of 150° C. or higher and 200° C. or lower for a predetermined period of time. When the thickness of the photoresist layer is 10 μm, the baking treatment is preferably carried out by heating at a temperature of 180° C. for 10 minutes. By carrying out the baking treatment as described above, the narrowed shape of the unexposed portion can be made into an arc shape. As described above, by carrying out the development process and the baking process on the photoresist layer, the photoresist layer comes to have a three-dimensional shape corresponding to the predetermined pattern. That is, the photoresist layer has a plurality of protrusions having the same shape as the protrusions suitable for cell culture. In this way, a master mold can be produced.

[0097] [Creating the formwork] The mold frame can be fabricated by forming a metal layer so as to cover the plurality of protrusions on the master mold, and then removing the metal layer from the master mold.

[0098] The metal layer can be produced by forming a thin film of a first metal by sputtering to cover the plurality of protrusions, and then growing a layer of a second metal on the thin film of the first metal to a predetermined thickness by electroforming. The metal layer can be produced, for example, according to the following procedure. (1) A thin film (for example, 10 nm to 30 nm thick) of a first metal is formed by sputtering so as to cover the plurality of protrusions on the master mold. (2) An electrolytic solution containing nickel chloride and boric acid and nickel sulfamate at a concentration of 300 g / L is placed in an electrodeposition bath, and then the electrolytic solution is heated to a temperature in the range of 50 to 60°C. (3) After immersing the master mold on which the thin film of the first metal has been formed in the electrolyte heated to the above temperature, a cathode current density of 100 mA / cm 2 The electrodeposition is carried out for a predetermined time while adjusting the applied voltage so that

[0099] Examples of the first metal and the second metal include copper, nickel, and iron. The first metal and the second metal may be the same kind of metal or different kinds of metal, but are preferably the same kind of metal. When the first metal and the second metal are the same type of metal, the first metal and the second metal are preferably nickel.

[0100] The thickness of the metal layer is preferably 1.5 times or more, and more preferably 2.0 times or more, the height of the protrusions of the master mold. The upper limit of the thickness of the metal layer is usually 3.0 times the height of the protrusions of the master mold. The thickness of the metal layer can be, for example, in the range of 200 μm to 500 μm.

[0101] By removing the metal layer from the master mold, the metal layer is provided with a plurality of depressions corresponding to a plurality of protrusions suitable for cell culture. This makes it possible to obtain a mold having a plurality of depressions corresponding to a plurality of protrusions suitable for cell culture. In addition, if the metal layer is thinner than the intended thickness, a metal plate may be attached by welding or the like to the surface opposite to the side on which the multiple recesses are formed so that the metal layer has the specified thickness.

[0102] The plurality of recesses in the metal layer may be cleaned. By cleaning the plurality of recesses, foreign matter (for example, residues of the photoresist layer that constituted the master mold) adhering to the plurality of recesses can be sufficiently removed. Examples of the cleaning liquid for the plurality of recesses include organic solvents and surfactants. The plurality of recesses can be cleaned by ultrasonic cleaning or high-pressure cleaning.

[0103] [Cell culture construct fabrication] The cell culture structure can be manufactured using a laminate for a cell culture structure in which a resin layer for forming protrusions is laminated on a flat substrate, and a mold having multiple depressions as described above. Specifically, the cell culture structure can be manufactured by covering the resin layer for forming the protrusions in the cell culture structure laminate with the formwork from the side where the multiple recesses are formed, forming multiple protrusions in the resin layer corresponding to the multiple recesses, and then removing the formwork from the resin layer for forming the protrusions.

[0104] The cell culture structure laminate can be obtained by applying a resin composition for forming protrusions to a substrate in a predetermined thickness to form a coating layer, and then drying the coating layer. The resin contained in the resin composition for forming the protrusions is preferably a thermosetting resin. As described above, examples of the thermosetting resin include polyimide resin, phenol resin, epoxy resin, melamine resin, urea resin, unsaturated polyester resin, alkyd resin, thermosetting polyurethane resin, thermosetting polyimide resin, and thermosetting fluororesin. Among the thermosetting resins, it is preferable to use the thermosetting fluororesin, and among the thermosetting fluororesin, it is preferable to use an amorphous fluororesin such as the above-mentioned CYTOP (registered trademark). Furthermore, the resin contained in the resin composition may be a photocurable resin that can be cured by irradiation with light. The photocurable resin preferably generates a small amount of radicals upon irradiation with light. Radicals are highly cytotoxic, so cell culture can be performed favorably by generating a small amount of radicals. In addition, when the resin layer for forming the protrusions contains a thermosetting resin or a photocurable resin as the resin, it is preferable that the resin layer of the protrusion formation layer is heat-cured or cured by light irradiation after the multiple protrusions are formed and before the formwork is removed. By carrying out thermal curing or curing by light irradiation at the timing described above, the shape of the protrusion formed by removing the mold from the resin layer for forming the protrusion can be sufficiently maintained.

[0105] As described above, by removing the mold from the resin layer for forming protrusions, the resin layer for forming protrusions has a plurality of protrusions corresponding to the plurality of recesses of the mold. This makes it possible to obtain a cell culture structure having a plurality of protrusions suitable for cell culture on the substrate.

[0106] In the above, we have described an example in which multiple protrusions are made of the same material, i.e., the multiple protrusions and the cell adhesion surface are made integrally from the same material, but the multiple protrusions and the cell adhesion surface may also be made of different materials. For example, when a fluororesin such as amorphous fluororesin is used as the resin that forms the cell adhesion surface and a resin other than fluororesin is used as the resin that forms the multiple protrusions, multiple protrusions can be formed on a substrate using a resin other than fluororesin according to the procedure described above, and then the fluororesin can be deposited at predetermined locations, including the tops of the multiple protrusions, by radio frequency (RF) sputtering, to form the cell adhesion surface at the predetermined locations.

[0107] The matters disclosed by this specification include the following.

[0108] (1) A cell culture structure that is used by being immersed in a culture solution and has a cell attachment surface to which cells to be cultured can attach, a flat substrate and a plurality of protrusions protruding from one surface of the substrate, each of the plurality of protrusions having at least a top surface serving as the cell adhesion surface; When the cell culture structure is viewed from one surface side of the substrate in a plan view, the plurality of protrusions are formed only of a plurality of lines having a finite length, or are formed by a combination of a plurality of lines having a finite length and a plurality of dots, In any of the above configurations of the plurality of protrusions, a closed space is not formed by the plurality of lines of finite length and the plurality of points, When the plan view of the adjacent protrusions is a line, the distance between the lines is I A When the plan view of the adjacent protrusions is a line and a point, the distance between the line and the point is I B When the plan view of the adjacent protrusions is a point, the distance between the points is I C The diameter of the largest circle contained in the widest area of ​​the cultured cells when viewed in a plane is S. C When I A , I B , and I C is S C shows a value smaller than Each of the first cut surfaces obtained by cutting the plurality of lines in a direction perpendicular to the extending direction of the lines, and each of the second cut surfaces obtained by cutting the plurality of points in a direction perpendicular to the surface direction of the base material, have a shape that narrows in width in a direction away from one surface of the base material. Cell culture construct.

[0109] (2) The flat substrate has an open portion on each of one end side and the other end side. The cell culture structure according to (1) above.

[0110] (3) In any of the above configurations of the plurality of protrusions, adjacent protrusions are not in contact with each other. The cell culture structure according to (1) or (2) above.

[0111] (4) The narrowed shape has at least an arc-shaped top. A cell culture structure according to any one of (1) to (3) above.

[0112] (5) The plurality of protrusions are made of an organic material having a refractive index of 1.4 or less at 20°C. A cell culture structure according to any one of (1) to (4) above.

[0113] (6) When a plate-shaped molded product is formed using the organic material, The contact angle of water with the plate-shaped molded product is 50° or more and 120° or less. The cell culture structure according to (5) above.

[0114] (7) The organic material is an amorphous fluororesin. The cell culture structure according to (5) or (6) above.

[0115] (8) The arrangement pattern of the plurality of protrusions when the cell culture structure is viewed in plan from one surface side of the substrate is a Turing pattern that represents the concentration distribution of two types of substances on a two-dimensional plane derived from a reaction-diffusion equation. A cell culture structure according to any one of (1) to (7) above.

[0116] (9) The Turing pattern is a pattern that indicates a region where the concentration of one of the substances U and the other chemical substance V is high in the concentration distribution of the one chemical substance U and the other chemical substance V, which is obtained by computer simulation using a finite difference method of the Gray-Scott model shown in the following formula (1), which represents the reaction-diffusion between the one chemical substance U and the other chemical substance V on a two-dimensional plane: The computer simulation starts with an initial state in which the one chemical substance U and the other chemical substance V are randomly distributed in equal amounts on the two-dimensional plane, and ends when the reaction-diffusion reaches an equilibrium state. The cell culture structure according to (8) above.

[0117]

number

[0118] (10) The difference method is carried out using the recurrence formula expressed by the following formulas (2) and (3): The cell culture structure according to (9) above.

[0119]

number

[0120]

number

[0121] (11) The Turing pattern was generated by a simulation in which a two-dimensional plane divided into a finite number of elements was given a periodic boundary condition on the outermost boundary. The cell culture structure according to any one of (8) to (10) above.

[0122] (12) In the Gray-Scott model equation (1), the coefficient D u is in the range of 0.002 to 0.01, and the coefficient D v is in the range of 0.0003 to 0.0015, coefficient f is in the range of 0.03 to 0.18, and coefficient k is in the range of 0.03 to 0.07. The cell culture structure according to any one of (8) to (11) above.

[0123] The cell culture structure according to the present invention is not limited to the above-described embodiment. Furthermore, the cell structure according to the present invention is not limited by the above-mentioned effects. The cell culture structure according to the present invention can be modified in various ways without departing from the gist of the present invention.

[0124] For example, when the cell culture structure 100 is viewed in plan from one surface side of the substrate 10, the multiple protrusions PR may be configured as multiple lines LI of finite length and multiple points PO in a manner as shown in Figures 4A to 11. Furthermore, the cell culture structure 100 may have a plurality of protrusions PR that are formed only from a plurality of lines LI of finite length in a form similar to the Turing pattern shown in FIGS. Furthermore, the cell culture structure 100 may be configured such that the multiple lines LI constituting the multiple protrusions PR are relatively long as shown in FIG. 13, or may be configured such that the multiple lines LI are relatively short as shown in FIGS. 14 and 15. Furthermore, the cell culture structure 100 may include a relatively large number of points PO that form a plurality of protrusions PR, as shown in FIG. 4 to 15, the arrangement pattern of the plurality of protrusions PR is also a Turing pattern. [Example]

[0125] The present invention will be further clarified by the following specific examples, but the present invention is not limited to the following examples.

[0126] The cell culture structures of Examples 1 to 12, each having a protrusion arrangement pattern based on the Turing pattern, were produced by the method described below.

[0127] [Turing pattern generation] As described herein, Turing patterns were generated by computer simulation of the Gray-Scott model equation shown in the above equation (1) using a JavaScript program with a finite difference method using the recurrence formulas expressed by the above equations (2) and (3). In each example, the values ​​shown in Table 1 below were used as parameters Du, Dv, f, and k used in these equations.

[0128] The computer simulation was performed in a finite space of 200 × 200 pixels with periodic boundary conditions. The computer simulation started with an initial state in which one chemical substance U and another chemical substance V were randomly distributed in equal amounts on the two-dimensional plane, and ended when the reaction-diffusion reached equilibrium.

[0129] The 200 x 200 pixel Turing pattern thus obtained was treated as a single square cell having the side length shown in Table 1 below, and a plurality of such cells were connected in a matrix to create a larger Turing pattern in the shape of a square with a side length of 132 mm or more. The larger Turing pattern thus obtained was then subjected to vector conversion to form protrusion arrangement patterns based on the Turing patterns of Examples 1 to 12.

[0130] Regarding the concentration distribution of one chemical substance U and another chemical substance V that appeared in these Turing patterns, the pattern showing the area where the concentration of one substance was high was taken as the Turing pattern representing the arrangement pattern of the protrusions of the cell culture structure.

[0131] Examples 1 to 4 The protrusion arrangement patterns of Examples 1 to 4 were all formed based on the same computer simulation using the parameter values ​​shown in the following Table 1. Here, the arrangement patterns of Examples 2 to 4 were obtained by multiplying the length of one side of the cell included in the above-mentioned larger Turing pattern obtained by this computer simulation by 0.8 times, 0.6 times, and 0.4 times, respectively, relative to the arrangement pattern of Example 1, as shown in the following Table 1. 4A to 4C show three different portions of the arrangement pattern obtained in Example 1. Parts of the arrangement patterns obtained in Examples 2 to 4 are shown in FIGS. The arrangement patterns of the protrusions in the cell culture structures shown in Figures 1A, 1B, and 2, which were referred to in the description of the above embodiments, are also based on Turing patterns obtained by computer simulations performed in these examples.

[0132] Examples 5 and 6 The protrusion arrangement patterns of Examples 5 and 6 were both formed based on the same computer simulation using the parameter values ​​shown in the following Table 1. Here, the arrangement pattern of Example 6 was obtained by multiplying the planar scale of the larger Turing pattern obtained by this computer simulation by 0.75 relative to the arrangement pattern of Example 5. Some of the arrangement patterns obtained in Examples 5 and 6 are shown in FIGS.

[0133] Examples 7 to 12 The arrangement patterns of the protrusions in Examples 7 to 12 were formed based on computer simulations using the parameter values ​​shown in the following Table 1. Some of the arrangement patterns obtained in Examples 7 to 12 are shown in Figures 10 to 15.

[0134] [Creating a mold with Turing patterns] First, a master mold was fabricated by photolithography using a master mold laminate in which a photoresist layer was laminated on a flat substrate, and a quartz glass photomask on which the arrangement patterns of the protrusions obtained in Examples 1 to 12 were formed, according to the method described in this specification. The photomask used in each example had an effective area of ​​132.4 mm x 132.4 mm, and a protrusion arrangement pattern based on the Turing pattern shown in the figure corresponding to each example was formed therein.

[0135] Next, a metal mold onto which the pattern of the master mold was transferred was produced by the method described herein.

[0136] [Cell culture construct fabrication] Using the method described in this specification, the cell culture structures of Examples 1 to 12 were manufactured using the mold prepared as described above and a laminate for a cell culture structure in which a resin layer for forming protrusions was laminated on a flat substrate.

[0137] Specifically, first, an amorphous fluororesin, CYTOP (registered trademark) (refractive index at 20°C = 0.134, water contact angle = 110°, solid content = 9%, dry peak and curing temperature = 180°C), was applied as a resin composition for forming protrusions onto a flat substrate made of transparent alkali-free glass to form a coating layer with a thickness of 62 μm. The coating layer on the substrate was then dried to form a resin layer for forming protrusions with a thickness of 5.6 μm, made of the dried coating layer, on the substrate. In this way, a laminate for a cell culture structure was obtained in which a resin layer for forming protrusions was laminated on a flat substrate.

[0138] Next, a mold was placed on the resin layer for forming the protrusions in the cell culture structure laminate from the side on which the Turing pattern had been transferred, and multiple protrusions corresponding to the Turing pattern were formed on the resin layer.The mold was then removed from the resin layer, yielding a cell culture structure in which multiple protrusions, each 5.0 μm high, protruded from a 132.4 mm × 132.4 mm area on one surface of the flat substrate in an arrangement pattern represented by the Turing patterns of Examples 1 to 12. [Table 1]

[0139] [Cell culture using cell culture constructs] The cell culture constructs produced in Examples 1 to 12 were cut into circles with a diameter of 92 mm and placed on the bottom of circular sterile polystyrene Petri dishes with a diameter of 96 mm and a depth of 18 mm. Cells were cultured using the cell culture constructs arranged in this manner, resulting in roughly circular cell sheets with a diameter of approximately 60 mm. Here, the cells to be cultured are those with the largest diameter of a circle contained in the widest area (i.e., S in the above explanation). C The size of the cell culture structure is determined by the distance between the protrusions formed on the cell culture structure (i.e., the size of the cell culture structure in the above description). A , I B , and I C(the distance corresponding to

[0140] In the cell culture carried out in this manner, the cells could be cultured more efficiently than in the case where a conventional cell culture structure was used.

[0141] Furthermore, when the cell sheet thus obtained was removed from the cell culture construct, almost no damage to the cell sheet was observed. [Explanation of symbols]

[0142] 10 substrates, 100 cell culture structures, A apex, C cell, D groove, LI line, O1 opening, O2 opening, PO point, PR protrusion, S cell attachment surface, S1 first cut surface, S2 second cut surface.

Claims

1. A cell culture structure that is used by being immersed in a culture solution and has a cell attachment surface to which cells to be cultured can attach, a flat substrate and a plurality of protrusions protruding from one surface of the substrate to a height of 50 nm or more and 5 mm or less, wherein at least a top of each of the plurality of protrusions serves as the cell adhesion surface; When the cell culture structure is viewed from one surface side of the substrate in a plan view, the plurality of protrusions are composed of only a plurality of lines of a finite length that are mainly curved, or are composed of a combination of a plurality of lines of a finite length that are mainly curved and a plurality of points, and the area ratio occupied by the protrusions composed of the plurality of lines of a finite length is larger than the area ratio occupied by the protrusions composed of the plurality of points, the width of the plurality of lines of finite length and the diameter of the plurality of dots are 10 nm or more and 500 μm or less; In any of the above configurations of the plurality of protrusions, a closed space is not formed by the plurality of lines of finite length and the plurality of points, When the plan view of the adjacent protrusions is a line, the distance between the lines is I Ａ When the plan view of the adjacent protrusions is a line and a point, the distance between the line and the point is I Ｂ When the planar view of the adjacent protrusions is a point, the distance between the points is I Ｃ The diameter of the largest circle contained in the widest area of ​​the cultured cells when viewed in plan is S. Ｃ When I Ａ , I Ｂ , and I Ｃ is 50 nm or more and 500 μm or less, and S Ｃ shows a value smaller than Each of the first cut surfaces obtained by cutting the plurality of lines in a direction perpendicular to the extending direction of the lines, and each of the second cut surfaces obtained by cutting the plurality of points in a direction perpendicular to the surface direction of the base material, have a shape that narrows in width in a direction away from one surface of the base material. Cell culture construct.

2. A cell culture structure as described in claim 1, wherein the multiple lines of finite length that constitute at least a portion of the multiple protrusions include branch lines having a single main line and at least one or more secondary lines branching off from the single line.

3. The flat substrate has an open portion on each of one end side and the other end side. The cell culture structure according to claim 1 or 2.

4. In any of the above configurations of the plurality of protrusions, adjacent protrusions are not in contact with each other. The cell culture structure according to claim 1 or 2.

5. The narrowed shape has at least an arc-shaped top. The cell culture structure according to claim 1 or 2.

6. The plurality of protrusions are made of an organic material having a refractive index of 1.4 or less at 20°C. The cell culture structure according to claim 1 or 2.

7. When a plate-shaped molded product is formed using the organic material, The contact angle of water with the plate-shaped molded product is 50° or more and 120° or less. The cell culture structure of claim 6 .

8. The organic material is an amorphous fluororesin. The cell culture structure of claim 6 .

9. A cell culture structure that is used by being immersed in a culture medium and has a cell attachment surface to which cells to be cultured can attach, a flat substrate and a plurality of protrusions protruding from one surface of the substrate, at least a top of each of the plurality of protrusions being the cell adhesion surface; When the cell culture structure is viewed from one surface side of the substrate in a plan view, the plurality of protrusions are formed only of a plurality of lines having a finite length, or are formed by a combination of a plurality of lines having a finite length and a plurality of dots, In any of the above configurations of the plurality of protrusions, a closed space is not formed by the plurality of lines of finite length and the plurality of points, When adjacent protrusions are lines in a planar view, the distance between the lines is I A , when adjacent protrusions are a line and a point in a planar view, the distance between the line and the point is I B , when adjacent protrusions are points in a planar view, the distance between the points is I C , and when the diameter of the largest perfect circle contained in the widest area of ​​the cultured cell in a planar view is S C , I A , I B , and I C are smaller than S C , each of first cut surfaces obtained by cutting the plurality of lines in a direction perpendicular to the extending direction of the lines, and each of second cut surfaces obtained by cutting the plurality of points in a direction perpendicular to the surface direction of the base material, have a shape that narrows in width in a direction away from one surface of the base material, The cell culture structure, wherein the arrangement pattern of the plurality of protrusions when the cell culture structure is viewed in a plane from one surface side of the substrate is a Turing pattern that represents the concentration distribution of two types of substances on a two-dimensional plane derived from a reaction-diffusion equation.

10. The Turing pattern is a pattern that indicates an area where the concentration of one of the substances is high in the concentration distribution of one chemical substance U and another chemical substance V, which is obtained by computer simulation of a reaction-diffusion equation that represents the reaction-diffusion between the one chemical substance U and another chemical substance V on a two-dimensional plane using a finite difference method, The computer simulation is started from an initial state in which the one chemical substance U and the other chemical substance V are randomly distributed in equal amounts on the two-dimensional plane, and is terminated when the reaction-diffusion reaches an equilibrium state. The cell culture structure of claim 9 .

11. The Turing pattern is a pattern that indicates a region where the concentration of one of the substances U and the other chemical substance V is high in the concentration distribution of the one chemical substance U and the other chemical substance V, which is obtained by computer simulation using a finite difference method of the Gray-Scott model shown in the following formula (1), which represents the reaction-diffusion between the one chemical substance U and the other chemical substance V on a two-dimensional plane: The computer simulation starts with an initial state in which the one chemical substance U and the other chemical substance V are randomly distributed in equal amounts on the two-dimensional plane, and ends when the reaction-diffusion reaches an equilibrium state. The cell culture structure of claim 9 . [Equation 1] where u is the concentration of the nutrient, v is the concentration of the growth factor, and D ｕ is the diffusion coefficient of the nutrient, and D ｖ is the diffusion coefficient of the growth factor, and f is the inflow / outflow coefficient. In the first formula of the above formula (1), f is the inflow coefficient of the nutrient component, and in the second formula of the above formula (1), f is the outflow coefficient of the growth factor that becomes excessive in quantity. k is the reaction coefficient at which the growth factor changes into a substance that loses its growth-promoting function.

12. The difference method is carried out using the recurrence formulas expressed by the following formulas (2) and (3): The cell culture structure of claim 11. [Equation 2] where δ is the time difference. [Equation 3] where ε is the spatial difference.

13. The Turing pattern was generated by a simulation in which a two-dimensional plane divided into a finite number of elements was given a periodic boundary condition on the outermost boundary. The cell culture structure of claim 12.

14. In the Gray-Scott model represented by the above formula (1), the coefficient D ｕ is in the range of 0.002 or more and 0.01 or less, and the coefficient D ｖ is in the range of 0.0003 or more and 0.0015 or less, the coefficient f is in the range of 0.03 or more and 0.18 or less, and the coefficient k is in the range of 0.03 or more and 0.07 or less. The cell culture structure of claim 11.

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