Culture base material for production, assessment, and functional regulation of regenerative medical cells, and method for using same

Abstract

[Problem] To provide a stripe structure that is applied to cells. [Solution] According to one aspect, the present disclosure provides an article comprising a stripe structure. In the present disclosure, the stripe structure is intended to be brought into contact with cells, and therefore, the stripe structure is arranged on a surface of the article at which the stripe structure can make contact with cells. The stripe structure may be disposed on a plurality of surfaces of the article, or disposed at a plurality of positions on a single surface of the article. The present disclosure also provides cell separation and / or culturing using the article comprising the stripe structure. The present disclosure further provides: cells that are separated and / or cultured using the article comprising the stripe structure; or a tissue comprising the cells.

Description

Culture substrate for producing, evaluating and regulating the function of cells for regenerative medicine and method of using the same

[0001] The present disclosure provides an article having a striped structure. The present disclosure also provides the isolation and / or culture of cells using an article having a striped structure. The present disclosure further provides cells or tissues containing the cells isolated and / or cultured using an article having a striped structure.

[0002] Regenerative medicine has made remarkable progress in recent years, and is on the verge of realizing fundamental cures for a variety of diseases that have been considered difficult to treat until now. The main players in the development of regenerative medicine are iPS cells, ES cells, various somatic cells, and somatic stem cells. Various cells are used for a variety of purposes, including medical applications and experiments for drug development. However, there are still many areas for improvement in the handling of cells, such as the acquisition of target cells and their efficient culture.

[0003] As a result of extensive research, the present inventors have found that a striped structure is useful for separating and / or culturing cells. Based on this finding, the present disclosure provides an article having a striped structure and uses thereof. The present disclosure also provides cells separated and / or cultured using an article having a striped structure, or tissues containing such cells.

[0004] Accordingly, the present invention provides the following: (Item 1) An article comprising a cell adhesive surface, wherein the cell adhesive surface has a striped structure comprising a repeating plurality of recesses extending in a specific direction. (Item 2) Any of the articles above, wherein the recesses have a depth of about 5 μm to 50 μm. (Item 3) Any of the articles above, wherein the recesses have a depth of about 10 μm to 40 μm. (Item 4) Any of the articles above, wherein the recesses have a width of about 1 μm to 100 μm. (Item 5) Any of the articles above, wherein the recesses have a width of about 3 μm to 5 μm. (Item 6) Any of the articles above, wherein the distance between adjacent recesses is about 1 μm to 50 μm. (Item 7) Any of the articles above, wherein the distance between adjacent recesses is about 2 μm to 10 μm. (Item 8) Any of the above-mentioned articles, wherein the recesses have a width of about 3 μm to 5 μm and a depth of about 10 μm to 40 μm, and the distance between adjacent recesses is about 2 μm to 10 μm. (Item 9) Any of the above-mentioned articles for adhesion of fibroblasts, hepatocytes, or adipose stem cells (ADSCs). (Item 10) Any of the above-mentioned articles, which are petri dishes. (Item 11) Any of the above-mentioned articles, which are implants. (Item 12) Any of the above-mentioned articles, which are made of a flexible material. (Item 13) Any of the above-mentioned articles, wherein multiple surfaces of the article are provided with the striped structure. (Item 14) Any of the above-mentioned articles, wherein the article includes a flat-plate-shaped part, and both surfaces of the flat-plate-shaped part are provided with the striped structure. (Item 15) A method for isolating a cell of interest, comprising: placing a cell population or cell-containing tissue containing the cell of interest in a first region of any of the articles described above; and incubating the article under conditions in which the cell of interest can survive, causing the cell of interest to migrate from the first region along the cell adhesive surface. (Item 16) The method of any of the above items, further comprising, after the incubating step, detecting and / or recovering the cell of interest from a second region on the cell adhesive surface that is different from the first region.(Item 17) A method for culturing cells, comprising a step of incubating the cells on the article of any of the above items under conditions that allow the cells to survive. (Item 18) The method of any of the above items, wherein the incubation step is carried out under conditions that allow the cells to form a sheet. (Item 19) A set of petri dishes comprising: a first petri dish having a cell adhesive surface, wherein the cell adhesive surface has a first striped structure comprising a repeat of a plurality of first recesses extending in a specific direction; and a second petri dish having a cell adhesive surface, wherein the cell adhesive surface has a second striped structure comprising a repeat of a plurality of second recesses extending in a specific direction, wherein the first striped structure and the second striped structure differ in at least one of: (i) the widths of the first recesses and the second recesses, (ii) the depths of the first recesses and the second recesses, and (iii) the distance between adjacent plurality of first recesses and the distance between adjacent plurality of second recesses. (Item 20) A set of petri dishes according to any of the above items, comprising petri dishes having at least five different striped structures. (Item 21) A method for selecting a petri dish for culturing cells, comprising: incubating cells seeded in each of a set of petri dishes of any of the above items under conditions that allow the cells to survive, observing the cells in each of the petri dishes after the incubating step, and selecting a petri dish from the set of petri dishes based on the observation results. (Item 22) The method of any of the above items, in which a petri dish is selected based on at least one of colony shape, cell density, tissue thickness, and fiber orientation. (Item 23) The article of any of the above items, wherein the cell adhesive surface comprises: (i) a second region adjacent to the striped structure in the direction in which the plurality of recesses extends, and / or (ii) a second striped structure comprising a repetition of a plurality of second recesses extending in a direction different from the striped structure.(Item 24) Any of the above-mentioned articles, comprising a first region and a second region on either end of the striped structure, sandwiching the striped structure. (Item 25) Any of the above-mentioned articles, wherein the surface height of the second region is the same as the bottom surface of the recess. (Item 26) Any of the above-mentioned articles, comprising a striped structure arranged to surround the first region. (Item 27) Any of the above-mentioned articles, wherein the second region has a peelable coating. (Item 28) A laminate comprising a plurality of any of the above-mentioned articles and a cell supply channel, wherein the plurality of articles are stacked in an arrangement in which the first region of each of the articles overlaps, and the cell supply channel is arranged so as to contact the first region of each of the articles. (Item 29) Any of the above-mentioned laminates, further comprising a cell recovery channel arranged so as to contact the second region of each of the articles. (Item 30) A method for recovering cells of interest, comprising the steps of: placing a cell population or cell-containing tissue containing the cells of interest in the first region of any of the articles described above; incubating the article under conditions in which the cells of interest can survive, causing the cells of interest to migrate from the first region along the cell adhesion surface to the second region; and recovering the cells of interest from the second region.

[0005] It is contemplated that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] The article having the striped structure of the present disclosure can provide easy isolation of target cells and improved cell culture, and can contribute to the development of cell-based medicines and medical devices (such as implants).

[0007] Schematic diagram of a striped structure. A microscopic image of the striped structure is shown. The scale bar represents 100 μm. A photograph of human adipose tissue cultured on a Petri dish having the striped structure of a, b, d, e, and k and a Petri dish having a flat surface (φ35 mm). A microscopic photograph showing ADSCs migrated from human adipose tissue seeded in specific areas on a Petri dish having the striped structure of a, b, d, e, and k and a Petri dish having a flat surface (φ35 mm). The scale bar represents 300 μm. The results of the WST-1 assay of Example 4 are shown. * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. A fluorescent microscopic photograph of actin-stained and nuclear-stained ADSCs migrated on a Petri dish having the striped structure of a, b, d, e, and k and a Petri dish having a flat surface. The scale bar represents 200 μm. a, b, d, e, k are fluorescence micrographs of adipose tissue cultured on a Petri dish with a striped structure, taken from the cross section of the striped structure. The scale bar indicates 40 μm. A microscopic image of a cell migrating separator chip is shown. The image on the bottom left shows the overall image, with enlarged views of the positions of each symbol shown. This shows the migration of ADSCs when a cell migrating separator chip is used. The upper panel shows a microscopic image. The lower panel shows the relationship between the distance from the seeded adipose tissue region and the number of cells (nuclei) present there. This shows the migration of ADSCs when a flat surface is used. The upper panel shows a microscopic image. The lower panel shows the relationship between the distance from the seeded adipose tissue region and the number of cells (nuclei) present there. This is a photograph of osteoblasts induced to differentiate from ADSCs on a striped structure with a recess width of 3 μm and a recess-to-recess distance of 2 μm. These are microscopic images of adipose tissue cultured on a Petri dish with a striped structure (k, a, b, d, and e) and a Petri dish with a flat surface (φ35 mm). For each dish, a photograph from the top (upper panel) and a photograph from the side (lower panel) are shown. In the lower panel, dotted and solid lines indicate the outline of the cell sheet and the top and bottom surfaces of the striped structure. The maximum thickness from the bottom surface to the top surface of the cell sheet and the maximum thickness of the cell sheet are shown with numerical values.

[0008] The present invention will now be described with reference to the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0009] (Definitions) The definitions of terms particularly used in this specification and / or basic technical content will be explained as appropriate below.

[0010] As used herein, the term "striped structure" refers to a solid structure having a repetition of multiple recesses (which can also be expressed as grooves) extending in a specific direction (see FIG. 1). The portion between adjacent recesses can be described as a protrusion (which can also be expressed as a ridge). The distance between the bottom surface of a recess and the top surface of a protrusion can be described as the depth (which can also be expressed as the height) of the recess. The distance between recesses can also be the width of the protrusion. A recess angle can be formed between the side surface of a recess and the bottom surface of the recess. The bottom surface, side surface, and / or top surface are typically flat, but may also be curved. The extension of the recesses in a specific direction can be linear or curved (typically, the width of the recesses and the distance between the recesses are constant). Typically, in one striped structure, the extension direction, the width of the recesses, the distance between the recesses, and the depth of the recesses are constant. If there is a portion where the extension direction is not continuous, it can be described as having multiple striped structures. The striped structure may be on a curved surface; for example, a flexible member having a striped structure may be fixed to the curved surface of an implant to produce an article having a striped structure on a curved surface.

[0011] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., an article having a striped structure, additional components, instructions, etc.) are provided, typically separated into two or more compartments. This kit form is preferred when the purpose is to provide articles that should not be provided in contact with each other for reasons of stability, etc., but are preferably brought into contact immediately before use. Such kits advantageously include instructions or manuals describing how to use or process the provided parts (e.g., an article having a striped structure, additional components, etc.). When the term "kit" is used herein, the kit typically includes instructions describing how to use the article having a striped structure of the present disclosure, etc.

[0012] As used herein, the term "about" refers to the indicated value plus or minus 10% unless otherwise specified. When used in reference to temperature, "about" may refer to the indicated value plus or minus 5°C.

[0013] (Description of Preferred Embodiments) Preferred embodiments of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present invention in light of the description in this specification. Regarding these embodiments, those skilled in the art can combine any embodiments as appropriate.

[0014] Although the present disclosure will be described with a focus on an article having a striped structure, various aspects, such as a system including such an article, a method of using such an article, and a product obtained by using such an article, are also contemplated as the invention. A description of one aspect, such as an article, applies equally to other aspects. For example, it is understood that any feature described with respect to the article can also be applied to a method of using the article.

[0015] (Article with Striped Structure) In one aspect, the present disclosure provides an article with a striped structure. In the present disclosure, it is intended that the striped structure will come into contact with cells, and therefore the striped structure is arranged on a surface of the article that can be contacted by cells. The striped structure may be arranged on multiple surfaces of the article, or the striped structure may be arranged in multiple locations on one surface.

[0016] (Striped structure) The striped structure can be mainly characterized by the width of the recesses, the distance between the recesses, and the depth of the recesses, and these factors may particularly affect cells in contact with the striped structure. Typically, the width of the recesses, the distance between the recesses, and the depth of the recesses in one striped structure are constant, but this does not have to be the case. Typically, the recesses are linear, but may be curved. Typically, the pattern of the recesses in one striped structure is regular.

[0017] In one embodiment, the recesses of the striped structure have a thickness of about 1 μm to 200 μm, for example, about 2 μm to 200 μm, about 3 μm to 200 μm, about 4 μm to 200 μm, about 5 μm to 200 μm, about 7 μm to 200 μm, about 10 μm to 200 μm, about 15 μm to 200 μm, about 20 μm to 200 μm, about 1 μm to 150 μm, about 2 μm to 150 μm, about 3 μm to 150 μm, about 4 μm to 150 μm, about 5 μm to 150 μm, about 7 μm to 150 μm, about 10 μm to 150 μm, about 15 μm to 150 μm, about 20 μm to 150 μm, approximately 1 μm to 100 μm, approximately 2 μm to 100 μm, approximately 3 μm to 100 μm, approximately 4 μm to 100 μm, approximately 5 μm to 100 μm, approximately 7 μm to 100 μm, approximately 10 μm to 100 μm, approximately 15 μm to 100 μm, approximately 20 μm to 100 μm, approximately 1 μm to 70 μm, approximately 2 μm to 70 μm, approximately 3 μm to 70 μm, approximately 4 μm to 70 μm, approximately 5 μm to 70 μm, approximately 7 μm to 70 μm, approximately 10 μm to 70 μm, approximately 15 μm to 70 μm, approximately 20 μm to 70 μm, approximately 1 μm to 50 μm, approximately 2 μm to 50 μm, about 3 μm to 50 μm, about 4 μm to 50 μm, about 5 μm to 50 μm, about 7 μm to 50 μm, about 10 μm to 50 μm, about 15 μm to 50 μm, about 20 μm to 50 μm, about 1 μm to 40 μm, about 2 μm to 40 μm, about 3 μm to 40 μm, about 4 μm to 40 μm, approximately 5 μm to 40 μm, approximately 7 μm to 40 μm, approximately 10 μm to 40 μm, approximately 15 μm to 40 μm, approximately 20 μm to 40 μm, approximately 1 μm to 30 μm, approximately 2 μm to 30 μm, approximately 3 μm to 30 μm, approximately 4 μm to 30 μm, approximately 5 μm to 30 μm, The width is about 7 μm to 30 μm, about 10 μm to 30 μm, about 15 μm to 30 μm, about 1 μm to 20 μm, about 2 μm to 20 μm, about 3 μm to 20 μm, about 4 μm to 20 μm, about 5 μm to 20 μm, about 7 μm to 20 μm, about 10 μm to 20 μm, about 1 μm to 15 μm, about 2 μm to 15 μm, about 3 μm to 15 μm, about 4 μm to 15 μm, about 5 μm to 15 μm, about 7 μm to 15 μm, about 1 μm to 10 μm, about 2 μm to 10 μm, about 3 μm to 10 μm, about 4 μm to 10 μm, or about 5 μm to 10 μm.

[0018] In one embodiment, the distance between adjacent recesses of the striped structure is about 1 μm to 50 μm, for example, about 2 μm to 50 μm, about 3 μm to 50 μm, about 4 μm to 50 μm, about 5 μm to 50 μm, about 7 μm to 50 μm, about 10 μm to 50 μm, about 15 μm to 50 μm, about 20 μm to 50 μm, about 1 μm to 40 μm, about 2 μm to 40 μm, about 3 μm to 40 μm, about 4 μm to 40 μm, about 5 μm to 40 μm, about 7 μm to 40 μm, about 10 μm to 40 μm, about 15 μm to 40 μm, about 20 μm to 40 μm, about 1 μm to 30 μm, about 2 μm to 30 μm, about 3 μm to 4 ... m ~ 30 μm, approximately 4 μm ~ 30 μm, approximately 5 μm ~ 30 μm, approximately 7 μm ~ 30 μm, approximately 10 μm ~ 30 μm, approximately 15 μm ~ 30 μm, approximately 1 μm to 20 μm, approximately 2 μm to 20 μm, approximately 3 μm to 20 μm, approximately 4 μm to 20 μm, approximately 5 μm to 20 μm, approximately 7 μm to 20 μm, approximately 10 μm m ~ 20 μm, approximately 1 μm ~ 15 μm, approximately 2 μm ~ 15 μm, approximately 3 μm ~ 15 μm, approximately 4 μm ~ 15 μm, approximately 5 μm ~ 15 μm, approximately 7 μm ˜15 μm, about 1 μm to 10 μm, about 2 μm to 10 μm, about 3 μm to 10 μm, about 4 μm to 10 μm, or about 5 μm to 10 μm. A distance between adjacent recesses in a striped structure of 30 μm or more may be detrimental to cell migration or proliferation. Without wishing to be bound by a particular theory, as the distance between recesses increases, the efficiency of cell and / or tissue adhesion, migration, and / or proliferation per area may decrease.

[0019] In one embodiment, the recesses of the striped structure have a thickness of about 1 μm to 200 μm, for example, about 2 μm to 200 μm, about 3 μm to 200 μm, about 4 μm to 200 μm, about 5 μm to 200 μm, about 7 μm to 200 μm, about 10 μm to 200 μm, about 15 μm to 200 μm, about 20 μm to 200 μm, about 1 μm to 150 μm, about 2 μm to 150 μm, about 3 μm to 150 μm, about 4 μm to 150 μm, about 5 μm to 150 μm, about 7 μm to 150 μm, about 10 μm to 150 μm, about 15 μm to 150 μm, about 20 μm to 150 μm, approximately 1 μm to 100 μm, approximately 2 μm to 100 μm, approximately 3 μm to 100 μm, approximately 4 μm to 100 μm, approximately 5 μm to 100 μm, approximately 7 μm to 100 μm, approximately 10 μm to 100 μm, approximately 15 μm to 100 μm, approximately 20 μm to 100 μm m, approximately 1 μm to 70 μm, approximately 2 μm to 70 μm, approximately 3 μm to 70 μm, approximately 4 μm to 70 μm, approximately 5 μm to 70 μm, approximately 7 μm to 70 μm, approximately 10 μm to 70 μm, approximately 15 μm to 70 μm, approximately 20 μm to 70 μm, approximately 1 μm to 50 μm, approximately 2 μm to 5 0 μm, approximately 3 μm to 50 μm, approximately 4 μm to 50 μm, approximately 5 μm to 50 μm, approximately 7 μm to 50 μm, approximately 10 μm to 50 μm, approximately 15 μm to 50 μm, approximately 20 μm to 50 μm, approximately 1 μm to 40 μm, approximately 2 μm to 40 μm, approximately 3 μm to 40 μm, approximately 4 μm m to 40 μm, about 5 μm to 40 μm, about 7 μm to 40 μm, about 10 μm to 40 μm, about 15 μm to 40 μm, about 20 μm to 40 μm, about 1 μm to 30 μm, about 2 μm to 30 μm, about 3 μm to 30 μm, about 4 μm to 30 μm, about 5 μm to 30 μm, about The depth is 7 μm to 30 μm, about 10 μm to 30 μm, about 15 μm to 30 μm, about 1 μm to 20 μm, about 2 μm to 20 μm, about 3 μm to 20 μm, about 4 μm to 20 μm, about 5 μm to 20 μm, about 7 μm to 20 μm, about 10 μm to 20 μm, about 1 μm to 15 μm, about 2 μm to 15 μm, about 3 μm to 15 μm, about 4 μm to 15 μm, about 5 μm to 15 μm, about 7 μm to 15 μm, about 1 μm to 10 μm, about 2 μm to 10 μm, about 3 μm to 10 μm, about 4 μm to 10 μm, or about 5 μm to 10 μm. For the strength of the stripe structure, it may be preferable that the recesses have a depth of about 40 μm or less.

[0020] In one embodiment, the porosity of the striped structure (which is the ratio of the area occupied by recesses when the striped structure is viewed two-dimensionally, and can be calculated as (recess width) / (recess width+distance between recesses)) is about 0.05 to 0.95, for example, about 0.1 to 0.95, about 0.15 to 0.95, about 0.2 to 0.95, about 0.3 to 0.95, about 0.4 to 0.95, about 0.5 to 0.95, about 0.6 to 0.95, about 0.7 to 0.95, about 0.8 to 0.95, 0.95, about 0.05-0.9, about 0.1-0.9, about 0.15-0.9, about 0.2-0.9, about 0.3-0.9, about 0.4-0.9, about 0.5-0.9, about 0.6-0.9, about 0.7-0.9, about 0.8-0.9, about 0.05-0.85, about 0.1-0.85, about 0.15-0.85, about 0.2-0.85, about 0.3-0.85, about 0.4-0.85, about 0.5-0.85, about 0.6-0.85, about 0.7-0.8 5, about 0.05 to 0.8, about 0.1 to 0.8, about 0.15 to 0.8, about 0.2 to 0.8, about 0.3 to 0.8, about 0.4 to 0.8, about 0.5 to 0.8, about 0.6 to 0.8, about 0.7 to 0.8, about 0.05 to 0.7, about 0.1 to 0.7, about 0.15 to 0.7, about 0.2 to 0.7, about 0.3 to 0.7, about 0.4 to 0.7, about 0.5 to 0.7, about 0.6 to 0.7, about 0.05 to 0.6, about 0.1 to 0.6, about 0.15 to 0. 0.6, about 0.2-0.6, about 0.3-0.6, about 0.4-0.6, about 0.5-0.6, about 0.05-0.5, about 0.1-0.5, about 0.15-0.5, about 0.2-0.5, about 0.3-0.5, about 0.4-0.5, about 0.05-0.4, about 0.1-0.4, about 0.15-0.4, about 0.2-0.4, about 0.3-0.4, about 0.05-0.3, about 0.1-0.3, about 0.15-0.3, or about 0.2-0.3.

[0021] In one embodiment, the number of recesses in one stripe structure is, but is not particularly limited to, about 10 to 1,000,000. In one embodiment, the extension length of the recesses in one stripe structure is, but is not particularly limited to, about 100 μm to 10,000,000 μm. In one embodiment, the angle between the bottom and side of the recesses in the stripe structure (recess angle) is, for example, about 70° to 110°, for example, about 90°.

[0022] The striped structure of the present disclosure can be used with any cells, including stem cells (such as ADSCs, ES cells, and iPS cells), cancer cells (especially for diagnostic purposes), and any known cultured cells.

[0023] Differences in the stripe structure can have different effects on cells and can affect the types of cells that can be suitably attached. Therefore, the preferred stripe structure may vary depending on the purpose of the cells used, but some suitable examples are shown below.

[0024] A striped structure with a recess width of 1 μm to 20 μm, a recess spacing of 2 μm to 20 μm, and a recess depth of 10 μm to 40 μm may be effective for adhesion and / or migration of fibroblasts (NIH3T3 cells), adipose stem cells (ADSCs), bone marrow mesenchymal stem cells (BMMSCs), kidney cells (COS-1 cells), adrenal medulla cells (PC12 cells), vascular endothelial cells (HUVECs), keratinocytes (HaCaT cells), and hepatocytes (HepG2 cells, HepaRG cells). This striped structure may also be effective for other cells with properties similar to these cells (including cells differentiated from embryonic stem cells, somatic stem cells, etc.).

[0025] A striped structure (particularly with a porosity of about 0.5 or more) with a recess width of 3 μm to 20 μm, a recess spacing of 2 μm to 20 μm, and a recess depth of 10 μm to 40 μm may be effective for the rapid proliferation of fibroblasts (NIH3T3 cells), adipose tissue stem cells (ADSCs), bone marrow mesenchymal stem cells (BMMSCs), kidney cells (COS-1 cells), adrenal medulla cells (PC12 cells), vascular endothelial cells (HUVECs), keratinocytes (HaCaT cells), and hepatocytes (HepG2 cells, HepaRG cells), and may also be effective in inducing the differentiation of adipose tissue stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMMSCs) into chondrocytes or vascular endothelial-like cells. This striped structure may also be effective in the differentiation-inducing culture of somatic stem cells and embryonic stem cells.

[0026] A striped structure having a recess width of 1 μm to 9 μm, a spacing between recesses of 2 μm to 10 μm, a recess depth of 10 μm to 40 μm, and a porosity of about 0.5 or more can be effective in promoting the formation of cell aggregates (spheroids) such as hepatocytes. A striped structure having a recess width of 3 μm to 5 μm, a spacing between recesses of 2 μm to 10 μm, a recess depth of 10 μm to 40 μm, and a porosity of about 0.5 can be effective in promoting the migration of cells with slow migration speeds, such as hepatocytes (HepG2 cells, etc.). This striped structure can be particularly effective on cells of a size comparable to HepG2 cells.

[0027] A striped structure with a recess width of 10 μm to 100 μm, a recess spacing of 10 μm to 20 μm, and a recess depth of 10 μm to 40 μm can be effective for isolating stem cells (ADSCs, UCMSCs) from adipose tissue and umbilical cord tissue. This striped structure can also be used to separate specific cells from somatic tissues (bone marrow tissue, vascular tissue, skin tissue, cartilage tissue, synovial tissue, dental pulp tissue, etc.) and bodily fluids (blood, bone marrow fluid, umbilical cord blood, etc.). When these tissues are seeded on the striped structure, the one-dimensionally restricted migration direction makes contact inhibition less likely, facilitating cell proliferation. Furthermore, the one-dimensionally continuous grooves improve cell or tissue adhesion, potentially favoring cell extraction from the tissue.

[0028] In addition to the cell types specifically listed above, cells with similar cell sizes, cell nuclei, and / or cell adhesion to the edges of the convex portions may have similar types of suitable stripe structures. For example, cells with similar cell sizes may have a volume (average) ratio or area (average) ratio within a range of about 10-1000%, about 20-500%, about 30-300%, or about 50-200% of each other. For example, cells with similar nuclear sizes may have nuclei (which may be determined based on nuclear staining) within a volume (average) ratio or area (average) ratio within a range of about 10-1000%, about 20-500%, about 30-300%, or about 50-200% of each other. For example, cells with similar cell adhesion to the edges of the convex portions may have adhesion strength (e.g., the force (N) required to detach the cells) to the striped structure within a range of about 10-1000%, about 20-500%, about 30-300%, or about 50-200% of each other.

[0029] The striped structure can be fabricated by photolithography or injection molding using a microfabricated mold. An article having a striped structure does not necessarily need to be molded as a single unit; for example, a member having the striped structure may be fabricated and then combined (e.g., glued) with other members to form an article having the striped structure. The striped structure can be fabricated from any known resin or other material, such as polystyrene, PMEA, polypropylene, polybutadiene, polyurethane, PET, Teflon (registered trademark) (polytetrafluoroethylene), or polysilicon. The striped structure may also be made of a biodegradable plastic material, such as PLLA, PLGA, or PCL. The striped structure may be made of a flexible or rigid material.

[0030] The article having the striped structure may also be made of a resin such as polystyrene, PMEA, polypropylene, polybutadiene, polyurethane, PET, Teflon (registered trademark) (polytetrafluoroethylene), polysilicon, or a biodegradable plastic material such as PLLA, PLGA, or PCL, and may be made of a flexible material or a rigid material.

[0031] The shape of the article may be any shape, such as a flat plate (including structures with holes such as plates and meshes), a rectangular parallelepiped, or a sphere, and those skilled in the art will understand the shape appropriate for the intended application. The article may have a striped structure on only a specific surface, or on multiple surfaces. In particular, when the purpose is to form tissues or three-dimensional cultures by culture, the surface of the article with the striped structure may have holes that allow fluids such as culture medium to pass through. Cells may not adhere to the entire bottom surface of the recesses with the striped structure, and fluid communication may be maintained near the bottom surface even after cell culture, but the presence of holes may make it easier for cultured cells to come into contact with fluids.

[0032] The cell adhesion surface or striped structure of the article of the present disclosure may be subjected to any surface modification for cell culture. Examples of surface modifications include hydrophilization treatments such as UV irradiation and plasma treatment (e.g., treatments that result in a water contact angle of 20 to 80 degrees), coatings with polylysine and cell adhesive proteins (e.g., collagen), and coatings with agents for adhering and releasing feeder cells (e.g., substances that denature depending on conditions such as heating, isopropyl acrylamide (NIPAM)). Cells cultured on the striped structure can also be used as feeder cells.

[0033] In one embodiment, the article having a striped structure is a Petri dish. As will be understood by those skilled in the art, a Petri dish (also called a dish) is a container for culturing cells, is typically circular, and is provided with a lid. Typically, a striped structure is disposed on at least a portion of the bottom of the Petri dish. In one embodiment, the article having a striped structure is a plate, which is placed in the Petri dish. By placing this plate on the bottom of an existing Petri dish, it can be used in the same way as the Petri dish having the striped structure described above. Alternatively, after cells are seeded on the bottom of the Petri dish, this plate can be placed to cover the cells, and further cells can be seeded on the plate. The plate may have a striped structure on only one side or on both sides. The striped structure of the present disclosure can have high cell adhesiveness, and therefore, even cells that normally float during culture can be cultured in an adherent manner (for example, by contacting the striped structure with the cells so that they are placed on top of the cells).

[0034] In one embodiment, the article having a striped structure is a transplant. Examples of implants include dental implants and stents placed in blood vessels. Examples of implants include articles combining an article having a striped structure with a transplantable organ, and articles for use with a transplantable organ. The overall shape and material of the implantable article can mimic known implants and can be appropriately selected by those skilled in the art. The implant may have a striped structure on multiple surfaces. The implant may have a non-flat structure with holes, such as a mesh or stripe, and the sides of the holes may have a striped structure. The striped structure on the cell adhesion surface of the implant may have advantageous effects such as fixing the implant at a specific location, increasing adhesion between the implant and cells, promoting the formation of desired tissue (e.g., with a specific orientation) around the implant, and reducing host immunity to the implant. The preferred striped structure may vary depending on the site (organ, tissue) into which the implant is to be implanted. For example, when the implant is a stent placed in a coronary artery to support the vascular wall, a striped structure that promotes adhesion of vascular endothelial cells is arranged on the cell adhesion surface that contacts the vascular wall, and if necessary, a striped structure that promotes adhesion of vascular endothelial cells may be arranged on the cell adhesion surface that contacts the blood flow, and if necessary, the implant may have a structure with holes such as a mesh to increase the contact area between the blood flow and the vascular wall. In order to fix the implant at specific locations, striped structures may be provided in multiple locations like tire cleats.

[0035] In one embodiment, the article having a striped structure is an article for cell separation, and is a plate or an article including a plate. The article for cell separation typically includes a seeding area (first area) and / or a collection or detection area (second area). Cells may be cultured on the article for cell separation. The article for cell separation of the present disclosure can provide target cells without the need for enzymatic treatment or the like. The article for cell separation of the present disclosure is also intended for use in sequentially separating cells induced to differentiate from seeded cells. The article for cell separation of the present disclosure can provide target cells without the need for enzymatic treatment or the like. Different striped structures can also be combined to separate multiple types of cells.

[0036] The seeding region (first region) is intended to be the region of the article that first contacts cells. It is not necessary to seed cells or tissues onto the seeding region; an embodiment is also possible in which the article is placed over the seeded cells or tissues so that the seeded cells or tissues contact the seeding region. The seeding region (first region) may be a portion comprising a striped structure or a portion adjacent to the striped structure (particularly, an adjacent portion positioned in the direction in which the recesses extend). In some embodiments, the seeding region (first region) and the recovery or detection region (second region) may be arranged adjacent to each other on opposite sides of the striped structure (e.g., in the direction in which the recesses of the striped structure extend). When the seeding region (first region) is a portion adjacent to the striped structure, the surface of the seeding region (first region) is preferably lower than the top surface of the striped structure. If the surface of the seeding region (first region) is higher than the top surface of the striped structure, the seeded cells may naturally spread over the striped structure, making it difficult to distinguish between seeded cells and migrated cells. Even in this case, seeded cells and migrated cells can be easily distinguished if the recesses in the striped structure have sufficient length. Cell migration can be made more efficient by arranging the seeding region (first region) and the striped structure so that the seeded cells or tissues can contact a larger number of recesses (or their openings) (e.g., the cuts in the cell migrating separator in Figure 8). In some embodiments, the striped structure (particularly the openings of the recesses) is arranged to surround the seeding region (first region). In some embodiments, a release agent (e.g., a substance that denatures depending on conditions such as heating, such as NIPAM) is applied to the recovery or detection region (second region).

[0037] The collection or detection region (second region) may be a portion comprising the striped structure or a portion adjacent to the striped structure (particularly, an adjacent portion positioned in the extending direction of the recesses). Because cells can migrate on the striped structure in the extending direction of the recesses, in many embodiments, migrated cells are collected or detected. To collect or detect migrated cells, the seeding region (first region) may be different from the collection or detection region (second region). However, in some embodiments, the seeding region (first region) may be the collection or detection region (second region) to collect or detect non-migrated cells. In many cases, cells migrate on the striped structure with only a portion of the cell placed on the protrusions, and the cell nucleus positioned in the recesses (not necessarily below the top surface of the protrusions). Therefore, when the collection or detection region (second region) is adjacent to the striped structure, the surface of the collection or detection region (second region) preferably has a height equal to or lower than the bottom surface of the striped structure, which can facilitate the cells' arrival in the collection or detection region (second region). Since the cells may reach the collection or detection region (second region) sequentially, a wall may be provided in the collection or detection region (second region) to prevent the cells from spreading excessively. A striped structure (a second striped structure having a repetition of multiple recesses extending in different directions) oriented so that the openings of the recesses do not contact the cells can function as a wall.

[0038] The purpose of cell separation is primarily to obtain and / or detect specific cells. The usefulness of detecting specific cells, such as diagnosing a subject's condition (such as the presence or absence of a disease) using a sample obtained from the subject and assessing the quality of cultured cells, is generally understood by those skilled in the art. In some embodiments, the article having a striped structure is an article for detecting specific cells after cell separation, enabling the determination of the presence or absence of specific cells in a sample and / or their quantification, and is contemplated to be in the form of a kit. The detection region (second region) is configured to enable detection of specific cells, and can be configured, for example, using a transparent member to enable optical detection, or to have a detection reagent (such as an antibody) disposed therein, or to have an inlet for adding the detection reagent disposed therein. The collection or detection region (second region) and / or the seeding region (first region) may be provided with an indicator.

[0039] In some embodiments, an article with a striped structure is an article for recovering specific cells after cell separation (and optionally after further cell detection), and may be useful for supplying cellular materials. For efficient supply of cellular materials, articles with striped structures on both sides of a flat plate and articles in which flat plates with striped structures are stacked are contemplated. For example, a stack including multiple flat plate-like parts, each of which is an article for cell separation described herein, and a cell collection channel connecting collection or detection regions (second regions) and / or a cell supply channel connecting seeding regions (first regions) is contemplated. In some embodiments, multiple flat plate-like parts may be arranged so that the collection or detection regions (second regions) and / or seeding regions (first regions) overlap each other.

[0040] (Method) In one embodiment, the present disclosure provides a method for culturing cells, the method comprising a step of incubating cells on an article described herein under conditions in which the cells are viable. In one embodiment, the incubation step is carried out under conditions in which the cells form a sheet. The incubation conditions may be conditions normally used for the cells or tissue used, or may be modified, and can be appropriately set by a person skilled in the art.

[0041] In one embodiment, the present disclosure provides a method for isolating a cell of interest, the method comprising the steps of placing a cell population or cell-containing tissue containing the cell of interest in a seeding region (first region) of an article described herein, and incubating the article under conditions conducive to the survival of the cell of interest, wherein the cell of interest migrates from the first region along the cell adhesive surface. In one embodiment, after the incubating step, the method further comprises detecting and / or recovering the cell of interest from a recovery or detection region (second region) on the cell adhesive surface that is different from the first region.

[0042] (Multiple Types of Stripe Structures) Because differences in stripe structure can affect cells and the types of cells that can be favorably adhered, a set of multiple types of stripe structures is preferably provided to allow selection of a stripe structure suited to the intended cell type. In one embodiment, a plate or petri dish is provided having multiple types of stripe structures described herein on one surface. In one embodiment, the present specification provides a set of petri dishes comprising: a first petri dish having a cell adhesion surface, the first petri dish having a first stripe structure comprising a repeating plurality of first recesses extending in a specific direction; and a second petri dish having a cell adhesion surface, the second petri dish having a second stripe structure comprising a repeating plurality of second recesses extending in a specific direction, wherein the first stripe structure and the second stripe structure differ in at least one of (i) the width of the first recesses and the second recesses, (ii) the depth of the first recesses and the second recesses, and (iii) the distance between adjacent first recesses and the distance between adjacent second recesses. The set of petri dishes may also include petri dishes with three, four, five, six, seven, eight, nine, ten, or more different stripe structures. It is believed that in many cases, a generally suitable stripe structure can be selected by culturing the target cells on about five different stripe structures.

[0043] In one embodiment, the present disclosure provides a method for selecting a dish for culturing cells, the method comprising the steps of: incubating cells seeded in each dish of a set of dishes described herein under conditions that allow the cells to survive; observing the cells in each dish after the incubation step; and selecting a dish from the set of dishes based on the observations. In one embodiment, the dish is selected based on at least one of colony shape, cell density, tissue thickness, and fiber orientation.

[0044] (Cultures) In one aspect, the present disclosure provides cultures cultured using articles having the striped structure described herein. Such cultures may have characteristics such as exhibiting orientation in a specific direction (e.g., the direction of cytoskeleton extension). When cells come into contact with each other, contact inhibition, in which they inhibit each other, can occur. However, cells cultured on the striped structure described herein can be predominantly present in the recesses, separated only by the inter-recess spacing, forming a culture (or culture surface) with reduced direct cell-to-cell contact. The striped structure described herein can form thick cell (tissue) sheets with fewer defects. Such cultures can be used as transplants (e.g., for regenerative therapy) or as feeder layers for culturing stem cells (e.g., ES cells, iPS cells).

[0045] The present invention has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0046] (Example 1: Various stripe structure designs) A number of stripe structures with different recess widths, recess-to-recess distances, and recess depths were investigated.

[0047] A stripe structure was fabricated having the recess width, recess-to-recess distance, and recess depth shown in the table below. *The stripe structures Nos. 1 to 46 were fabricated on a silicon wafer by photolithography. *The stripe structures Nos. 47 to 54 were formed on a petri dish.

[0048] (Example 2: Use of striped petri dishes) Petri dishes were produced as articles having striped structures (FIG. 2) of the types a, b, d, e, and k patterns designed in Example 1 on their surfaces, and these were compared with conventional petri dishes.

[0049] Human adipose tissue collected subcutaneously by liposuction at the University of the Ryukyus Hospital was washed three times with D-PBS containing 1% penicillin-streptomycin. The washed human adipose tissue was then cut with scissors into approximately 2 mm pieces, which were then seeded at 0.05 g per 35 mm striped dish. Care was taken to ensure that the adipose tissue fragments were dispersed as uniformly as possible throughout the dish. After seeding, 500 μl of medium was added to initiate culture. ADSC-4 (Kohjin Bio, Japan) or ADSC-GM (Lonza, US) medium was used. Medium was replaced every 24 hours by removing 200 μl of medium and adding 200 μl of fresh medium. Cell proliferation was assessed 21 days after seeding by WST-1 absorbance measurement (n = 4). WST-1 was purchased from Takara Bio (Japan). Twenty-one days after seeding, phase-contrast images of the morphology of adipose stem cells (ADSCs) grown on striped dishes were observed using an inverted microscope CKX53 (Olympus, Japan). A CACHN10x IPC objective (Olympus, Japan) was used for this observation. On day 21 of culture, cells on the striped dishes were fixed with 4% paraformaldehyde phosphate buffer (Nacalai, Japan) (room temperature, 20 minutes) and then fluorescently stained with Phalloidin Alexa Fluor 546 (Thermo Fisher Scientific, US) and DAPI (Dojindo, Japan). The fluorescently stained cells were observed using a BZ-9000 fluorescence microscope (Keyence, Japan). This study was approved by the University of the Ryukyus' Ethics Committee for Human Research in Life Sciences and Medicine (Permission No. 810).

[0050] The results are shown in Figure 3. In all striped dishes, superior adhesion of adipose tissue was observed compared to the conventional flat dish.

[0051] Example 3: Study on combinations of stripes and cell types For the various stripe structures designed in Example 1, suitable combinations with cells (tissues) were studied.

[0052] Differences in effectiveness were generally observed based on the following classifications (1) to (5): (1) Recess width: 1 μm to 2 μm, spacing between recesses: less than 10 μm (2) Recess width: 3 μm to 5 μm, spacing between recesses: less than 10 μm (3) Recess width: 6 μm to 9 μm, spacing between recesses: less than 10 μm (4) Recess width: 10 μm to 20 μm, spacing between recesses: 10 μm to 20 μm (5) Recess width: 21 μm to 100 μm, spacing between recesses: 10 μm to 20 μm *The depth of the recesses is 10 μm or more and less than 40 μm.

[0053] In all of (1) to (5), the adhesion, spreading, and migration of seeded NIH3T3 cells, human ADSCs, human BMMSCs, COS-1 cells, PC12 cells, HUVECs, HaCaT cells, HepG2 cells, and HepaRG cells were observed to be restricted in one dimension (the direction of extension of the recesses). This effect was similarly reproduced with various cells with properties such as fibroblasts, mesenchymal stem cells, neurons, vascular endothelial cells, keratinocytes, and hepatocytes, suggesting that this effect is also widely effective for cells with similar properties to these cells (including cells differentiated from embryonic stem cells and somatic stem cells). This effect was particularly strong with the striped structures (1) to (4).

[0054] Among (1) to (3), when the porosity (the ratio of the area occupied by recesses when the striped structure is viewed two-dimensionally) is approximately 0.5 or higher, the formation of cell aggregates (spheroids) is promoted. Spheroid formation is expected to improve the liver function of cells such as HepG2 and HepaRG cells. Meanwhile, research into cell therapy, in which cells are formed into spheroids and then transplanted, is also progressing. This method can also be used to produce spheroids for such cell therapy.

[0055] Among the (2) materials, those with a porosity of around 0.5 were confirmed to significantly improve the migration speed of slow-migrating cells (such as HepG2) cultured thereon (see also Example 4). In this case, HepG2 cells placed their bodies containing cell nuclei on the top of the wells, extended long protrusions to one or both of the adjacent wells, and migrated at high speed as if pulled by the protrusions. It is believed that the same striped structure can be expected to produce similar effects on cells of a similar size to HepG2.

[0056] The proliferation rates of NIH3T3 cells, human ADSCs, human BMMSCs, COS-1 cells, PC12 cells, HUVECs, HaCaT cells, HepG2 cells, and HepaRG cells seeded on (2), (3), and (4) surfaces were higher than on flat surfaces. For all cell types, cell states, and culture conditions tested, porosity of approximately 0.5 or higher was advantageous for improving proliferation rates. This effect was similarly reproduced for various cells with properties such as fibroblasts, mesenchymal stem cells, neurons, vascular endothelial cells, keratinocytes, and hepatocytes. Therefore, structures (2), (3), and (4) are also considered advantageous for improving proliferation rates for cells with properties similar to these cells (including cells differentiated from embryonic stem cells and somatic stem cells).

[0057] When human ADSCs and human BMMSCs were cultured on (2), (3), and (4) to induce differentiation into osteoblast-like cells, chondrocytes, vascular endothelial-like cells, etc., it was found that the differentiation induction efficiency was higher than that on a flat surface (see also Example 6 and Figure 11). It is thought that such a striped structure is advantageous not only for somatic stem cells, but also for similar differentiation induction culture of embryonic stem cells.

[0058] Experiments were conducted on (4) and (5) to extract and culture ADSCs from human adipose tissue and umbilical cord-derived mesenchymal stem cells (UCMSCs) from human umbilical cord tissue. The results showed that culturing on (4) and (5) resulted in greater numbers of ADSCs and UCMSCs than culturing on flat surfaces. This is likely due to the greater adhesion of seeded tissue to the striped structures of (4) and (5) than to other striped structures and flat surfaces (see Example 4 and Figure 7 for the adhesion of seeded human adipose tissue to the striped structure). The striped structure of (4) was also used in the cell migrating separator (CMS) of Example 5. Various body tissues and fluids can be seeded on (4) and (5). Body tissues include bone marrow tissue, vascular tissue, skin tissue, cartilage tissue, synovial tissue, and dental pulp tissue. Body fluids include blood, bone marrow fluid, and umbilical cord blood. Regardless of which material is seeded on the striped structure, the increased surface area and one-dimensionally restricted migration direction of the striped structure make contact inhibition less likely, facilitating cell proliferation. Furthermore, when somatic tissue is seeded, the one-dimensionally continuous grooves facilitate adhesion of the seeded tissue, which is advantageous for cell extraction from the tissue.

[0059] It has been found that the preferred stripe structure may vary depending on the cell or tissue or purpose, and therefore it is believed that a preferred stripe structure can be selected by examining the compatibility of cells or tissues with multiple types of stripe structures.

[0060] (Example 4: Cell separation using striped structure) Depending on the type of cell or tissue, the adhesiveness to and mobility on the striped structure may differ. Therefore, by incubating a mixture containing multiple types of cells on the striped structure, it is possible to separate and obtain specific types of cells.

[0061] The tissues were cultured using five types of Petri dishes with striped structures as shown in the table below, and flat Petri dishes made of the same material.

[0062] Human adipose tissue was placed in a portion of each dish and cultured in ADSC-4 medium for 21 days, after which it was observed under a microscope. The results are shown in Figure 4. On the striped dish, ADSCs were observed to migrate preferentially, reaching areas distant from the tissue fragments. Migration was also prominent in the same direction as the stripes. ADSCs migrated significantly, particularly on the striped structure 4 (recess width: 100 μm, recess distance: 20 μm, recess depth: 20 μm) shown in Figure e. On the other hand, on the flat dish, few cells were observed at locations distant from the tissue fragments. This demonstrates that ADSCs can be rapidly extracted and cultured from adipose tissue without enzymatic treatment.

[0063] When comparing cell numbers across the entire dish using the WST-1 assay, higher activity was observed in the striped dish than in the flat dish (Figure 5). Because the same amount of adipose tissue was seeded, the amount of ADSCs contained in the adipose tissue was also expected to be the same. However, because ADSCs are thought to be unable to proliferate within adipose tissue and have low metabolic activity, the results of this WST-1 assay suggest that ADSCs were rapidly extracted from the adipose tissue, detached from it, and subsequently proliferated in the striped dish. When different culture media (ADSC-4 and ADSC-GM) were used, differences in the extraction level of ADSCs were observed depending on the type of striped dish, but both achieved better extraction culture than the flat dish.

[0064] Observation of the extracted ADSCs on each dish confirmed differences in cell adhesion pattern and cell shape depending on the striped structure (actin staining and nuclear staining images, Figure 6). Furthermore, when adipose tissue was stained with DAPI (1:400, 20 min) and Lipi Dye and observed from the cross section of the striped structure, it was observed that the shape of the adipose tissue also differed depending on the type of striped structure (Figure 7). It can be seen that in striped dishes k and a, with narrow groove widths, the seeded adipose tissue did not penetrate the grooves very much, whereas in striped dishes d and e, with wide groove widths, it penetrated the grooves extensively. From the perspective of increasing the surface area (seeding surface), a smaller groove width and spacing in the striped dish is advantageous, but a certain groove width is considered advantageous for ensuring firm tissue contact within the grooves. It is believed that firm tissue contact with the grooves of the striped dish improves the efficiency of extraction and culture from the seeded tissue.

[0065] Furthermore, HepG2 cells were tested for migration when cultured on striped structures.

[0066] Specifically, the fabricated stripe structure (recess width: 3 μm, recess distance: 3 μm or 9 μm, recess depth: 20 μm) was treated with O ２ The surface was made hydrophilic by plasma treatment and sterilized, and then immersed in a culture medium and degassed under reduced pressure. HepG2 cells were cultured at 1 × 10 ４ cells / cm ２The HepG2 cells were seeded on the striped silicon wafer and fluorescently stained with PKH26. The striped silicon wafer with the HepG2 cells attached was transferred to a 35 mm glass base dish filled with culture medium, and the cell adhesion surface was placed facing downwards on the glass window at the bottom of the dish. At this time, the four vertices of each substrate were hooked onto the frame of the glass window, leaving a sufficient gap between the cell adhesion surface and the glass. Four hours after seeding, time-lapse observation of cells was initiated at designated fixed points on each striped structure and on the flat surface. Time-lapse observation was performed for 72 hours at 30-minute intervals using a fluorescence microscope Observer Z1 (ZEISS AG, Germany) equipped with a Stage Top® Incubator INU-ZILCS (Tokai Hit Co., Ltd., Japan). A 10x objective lens was used, and the reflected image of the striped structure and the fluorescent image of the cells were captured with a CCD camera. The movement of cells (cell migration) on each of the obtained striped structures was manually tracked using AxioVision 4.8 (ZEISS GmbH, Germany). The average migration speed [μm / h] of cells migrated on each striped structure was calculated from the obtained trajectory of each cell migration. This method for observing cell migration is described in Hiroshi Sunami et al., J. Surf. Sci. Nanotech. Vol. 12 (2014) 289-298.

[0067] The results are shown in the table below.

[0068] Although HepG2 cells hardly migrate when cultured on a flat surface, their motility speed was improved when cultured on a striped structure.

[0069] (Example 5: Development of a Cell-Migrating Separator) We further developed a cell-migrating separator with a striped structure specialized for cell separation. Figure 8 shows an example of a cell-migrating separator chip. This was fabricated from silicon wafer material by photolithography for the purpose of separating ADSCs. The striped structure was fabricated to have a recess width of 10 μm, a recess-to-recess distance of 10 μm, and a recess depth of 10 μm. The chip has a central area (seeding area 7000 μm × 7000 μm) with the same depth as the bottom of the recess, where the tissue to be separated is placed. The chip has four striped structures (the orientation of the recesses is the same within each area) surrounding the seeding area, with the openings of the grooves of the recesses facing the seeding area. The striped structures have cuts that are spaced apart and do not touch each other except at the ends, and the bottoms of the cuts are the same depth as the seeding area. The presence of the notches increases the contact area between the seeded tissue (cells) and the openings of the grooves in the recesses of the striped structure, which is expected to improve the extraction efficiency of the target cells. The target cells are intended to migrate from the seeding area through the striped structure to the outer recovery region. The bottom of the recovery region is designed to be the same depth as the bottom of the recesses.

[0070] The performance of the cell migration separator was tested using adipose tissue similar to that used in Example 1. Results were compared with those of a control chip with a flat surface. The cell migration separator was placed over human adipose tissue placed in a petri dish, cultured for 17 days, and then microscopically observed. Cells were stained with Phalloidin 488 (1:200, 4 h) and DAPI (1:400, 2 h). Images were taken using a confocal laser microscope FV1000D equipped with an UPlanSApo 20x objective, spanning 16 images at 2.0 μm intervals along the Z axis from the surface of the dish to the bottom of the striped structure, to a depth of 30 μm. The adipose tissue was divided into seven sections, each 400 μm apart, and the number of cells (nuclei) in each section was counted.

[0071] The results are shown in Figure 9 (cell migrating separator) and Figure 10 (flat surface). When the cell migrating separator chip was used, ADSCs that migrated out of the adipose tissue actively crawled along the striped pattern structure and migrated long distances of 2-5 mm toward the four sides of the cell migrating separator, i.e., the outer edge, while actively proliferating. Furthermore, we observed that cells with different sizes, morphologies, and levels of cytoskeletal development were distributed depending on the migration distance.

[0072] (Example 6: Culture on striped structure) Since it was found that the striped structure has various effects on cell or tissue culture, the characteristics of cell culture on the striped structure were investigated from several aspects.

[0073] The effect of the striped structure on the differentiation of ADSCs into osteoblasts was examined. Specifically, ADSCs were seeded onto silicon wafers with striped structures numbered 1 to 25 in Table 1, and differentiation was induced for 16 days in Osteoblast Differentiation Medium (ZEN-BIO, INC., US). After staining with alizarin red, the cells were observed under a microscope.

[0074] As a result, it was found that the striped structures numbered 1 to 5 (recess width: 3 to 7 μm, recess distance: 2 μm, recess depth: 10 to 40 μm) were strongly stained with alizarin red, and differentiation into osteoblasts in particular was promoted (Figure 11).

[0075] Adipose tissue was also cultured on the striped structure to form a cell sheet. The striped structure used is shown in the table below.

[0076] 0.15 g of finely cut adipose tissue pieces were seeded onto one 35 mm Petri dish containing each surface structure, and cultured in ADSC-GM medium (LONZA, US) for 15 days under ceiling culture conditions (37°C, 5% CO ２ After that, the plates were inverted and cultured upright for another 20 days (37°C, 5% CO ２The medium was changed every 3 days, and on day 35 of culture, the cells were fixed with 4% paraformaldehyde phosphate buffer (Nacalai, Japan). Then, cells were stained for Talin with anti-Talin-Alexa 488 (Thermo Fisher Scientific, US) (1:200, 4°C, overnight), followed by F-actin staining with Phalloidin 546 (Thermo Fisher Scientific, US) (1:200, RT, 60 min). Finally, nuclei were stained with DAPI (Dojindo, Japan) (1:400, RT, 10 min). After staining, an appropriate amount of PBS was added to each of the 35 mm dishes, and the dishes were observed using a confocal laser microscope FV1000D (Olympus, Japan) equipped with a water immersion objective lens LUMFL 60XW NA: 1.10 (Olympus, Japan).

[0077] The results are shown in Figure 12. It was found that the cell sheet on the striped dish was thicker than the cell sheet on the flat dish. The thickness of the cell sheet on the flat dish was a maximum of 10 µm despite being confluent, whereas the thickness of the cell sheet on the striped dish was a maximum of k = 18.0 µm, a = 21.0 µm, b = 19.5 µm, d = 17.5 µm, and e = 20.0 µm. It was found that thicker cell sheets could be produced using the striped dish than the flat dish.

[0078] An article having a striped structure according to the present disclosure can provide easy isolation of target cells and improved cell culture, and can contribute to the development of cell-based medicines and medical devices (such as implants).

Claims

1. An article comprising a cell adhesion surface, said cell adhesion surface having a striped structure comprising a repeating plurality of recesses extending in a particular direction.

2. The article of claim 1, wherein said recesses have a depth of about 5 μm to 50 μm.

3. The article of claim 1, wherein said recesses have a depth of about 10 μm to 40 μm.

4. The article of claim 1, wherein the recesses have a width of about 1 μm to 100 μm.

5. The article of claim 1, wherein the recesses have a width of about 3 μm to 5 μm.

6. The article of claim 1, wherein the distance between adjacent recesses is between about 1 μm and 50 μm in width.

7. The article of claim 1, wherein the distance between adjacent recesses is between about 2 μm and 10 μm in width.

8. The article of claim 1, wherein said recesses have a width of about 3 μm to 5 μm and a depth of about 10 μm to 40 μm, and the distance between adjacent recesses is about 2 μm to 10 μm in width.

9. The article of claim 1 for adhesion of fibroblasts, hepatocytes, or adipose stem cells (ADSCs).

10. The article of claim 1 which is a petri dish.

11. The article of claim 1 which is an implant.

12. The article of claim 11, constructed from a flexible material.

13. The article of claim 1, wherein multiple faces of the article are provided with said striped structure.

14. The article of claim 1, wherein the article comprises a flat part, both sides of the flat part being provided with the stripe structure.

15. A method for isolating a cell of interest, comprising the steps of: placing a cell population or cell-containing tissue containing the cell of interest in a first region of an article of claim 1; and incubating the article under conditions in which the cell of interest is viable, wherein the cell of interest migrates from the first region along the cell adhesive surface.

16. The method of claim 15, further comprising the step of detecting and / or recovering the cells of interest from a second region on the cell adhesion surface that is different from the first region after the incubating step.

17. A method of culturing cells, comprising the step of incubating said cells on the article of claim 1 under conditions in which said cells are viable.

18. The method of claim 1, wherein the incubating step is carried out under conditions whereby the cells form a sheet.

19. A set of petri dishes comprising: a first petri dish having a cell adhesion surface, the cell adhesion surface having a first striped structure comprising a repeat of a plurality of first recesses extending in a specific direction; and a second petri dish having a cell adhesion surface, the cell adhesion surface having a second striped structure comprising a repeat of a plurality of second recesses extending in a specific direction, wherein the first striped structure and the second striped structure differ in at least one of: (i) the widths of the first recesses and the second recesses; (ii) the depths of the first recesses and the second recesses; and (iii) the distance between adjacent plurality of the first recesses and the distance between adjacent plurality of the second recesses.

20. The set of petri dishes according to claim 19, comprising at least five different types of petri dishes with different stripe structures.

21. A method for selecting a dish for culturing cells, comprising the steps of: incubating the cells seeded in each dish of a set of dishes according to claim 19 or 20 under conditions in which the cells can survive; observing the cells in each of the dishes after the incubation step; and selecting a dish from the set of dishes based on the observation results.

22. The method of claim 21, wherein the dish is selected based on at least one of colony shape, cell density, tissue thickness, and fiber orientation.

23. The article of claim 1, wherein the cell adhesive surface comprises: (i) a second region adjacent to the striped structure in the direction in which the plurality of recesses extends; and / or (ii) a second striped structure comprising a repeating plurality of second recesses extending in a different direction than the striped structure.

24. The article according to claim 23, comprising a first region and a second region on either side of the striped structure.

25. The article of claim 23, wherein the surface of the second region is flush with the bottom surface of the recess.

26. The article of claim 23, comprising a striped structure disposed so as to surround the first region.

27. The article of claim 23, wherein the second region has a strippable coating.

28. A laminate comprising a plurality of articles according to claim 23 and a cell supply channel, the plurality of articles being stacked in an overlapping arrangement with the first regions of each of the articles, and the cell supply channel being positioned so as to contact the first regions of each of the articles.

29. The laminate of claim 28, further comprising a cell collection channel disposed adjacent the second region of each of said articles.

30. A method for recovering a cell of interest, comprising the steps of: placing a cell population or cell-containing tissue containing the cell of interest in the first region of the article of claim 24; incubating the article under conditions in which the cell of interest is viable, wherein the cell of interest migrates from the first region along the cell adhesive surface to the second region; and recovering the cell of interest from the second region.

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