Production and evaluation of cells for regenerative medicine, culture substrates for functional regulation, and methods for using them.

Abstract

To provide a stripe structure applied to cells.SOLUTION: In one aspect, the present disclosure provides an article having a stripe structure. In the present disclosure, since it is intended that the stripe structure contacts cells, the stripe structure is arranged on a surface of the article that can be contacted by cells. The stripe structure may be arranged on multiple surfaces of the article, or may be arranged at multiple locations on one surface. The present disclosure also provides the separation and / or culture of cells using an article having a stripe structure. The present disclosure further provides cells, or tissues containing the same separated and / or cultured using the article having a stripe structure.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure provides an article having a stripe structure. The present disclosure also provides a method for separating and / or culturing cells using an article having a stripe structure. The present disclosure further provides cells separated and / or cultured using an article having a stripe structure or tissues containing the same.

Background Art

[0002] In recent years, regenerative medicine has made remarkable progress and aims to achieve radical treatment of various diseases that have been 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. Although various cells are used for various purposes such as use in medical applications and experiments for drug development, there are still many points for improvement in handling cells such as obtaining target cells and efficiently culturing cells.

Summary of the Invention

Means for Solving the Problems

[0003] As a result of intensive research, the present inventors have found that a stripe structure is useful in cell separation and / or culture. Based on such findings, the present disclosure provides an article having a stripe structure and its use. The present disclosure also provides cells separated and / or cultured using an article having a stripe structure or tissues containing the same.

[0004] Therefore, the present invention provides the following. (Item 1) An article comprising a cell adhesion surface, wherein the cell adhesion surface has a stripe structure comprising a repetition of a plurality of recesses extending in a specific direction. (Item 2) The article according to any one of the above items, wherein the recess has a depth of about 5 μm to 50 μm. (Item 3) An article of any of the above items, wherein the recess has a depth of approximately 10 μm to 40 μm. (Item 4) An article of any of the above items, wherein the recess has a width of approximately 1 μm to 100 μm. (Item 5) An article of any of the above items, wherein the recess has a width of approximately 3 μm to 5 μm. (Item 6) An article of any of the above items, wherein the distance between adjacent recesses is approximately 1 μm to 50 μm. (Item 7) An article of any of the above items, wherein the distance between adjacent recesses is approximately 2 μm to 10 μm wide. (Item 8) Any of the above items, wherein the recess has a width of approximately 3 μm to 5 μm and a depth of approximately 10 μm to 40 μm, and the distance between adjacent recesses is approximately 2 μm to 10 μm. (Item 9) Any of the above items for the adhesion of fibroblasts, hepatocytes, or adipose-derived stem cells (ADSCs). (Item 10) A petri dish, or any of the items listed above. (Item 11) Any of the above items that are transplanted. (Item 12) Any of the above items, made of a flexible material. (Item 13) An article of any of the above items, wherein multiple surfaces of the article have the stripe structure described above. (Item 14) Any of the above items, wherein the article includes a flat plate-shaped part, and both sides of the flat plate-shaped part have the stripe structure. (Item 15) A method for isolating target cells, A step of placing a cell population or cell-containing tissue containing the aforementioned target cells in a first region of any of the above items, A step of incubating the article under conditions in which the target cells can survive, wherein the target cells migrate from the first region along the cell adhesion surface. Methods that include... (Item 16) Any method of the above item, further comprising the step of detecting and / or recovering the target cells from a second region on the cell adhesion surface different from the first region, after the incubation step. (Item 17) A method for culturing cells, The step of incubating the cells on any of the above items under conditions in which the cells can survive. Methods that include... (Item 18) Any of the above methods, wherein the incubation step is carried out under conditions that allow the cells to form a sheet. (Item 19) A first petri dish comprising a cell adhesion surface, wherein the cell adhesion surface comprises a first stripe structure having a plurality of repeating first recesses extending in a specific direction, A second petri dish comprising a cell adhesion surface, wherein the cell adhesion surface comprises a second stripe structure having a plurality of repeating second recesses extending in a specific direction. A set of petri dishes equipped with, The first stripe structure and the second stripe structure are, (i) the width of the first recess and the second recess, (ii) the depth of the first recess and the second recess, and (iii) the distance between a plurality of adjacent first recesses and the distance between a plurality of adjacent second recesses A set of petri dishes that differ in at least one of their features. (Item 20) A set of petri dishes from any of the above items, including petri dishes with at least five different stripe patterns. (Item 21) A method for selecting a petri dish for culturing cells, comprising: incubating the cells seeded in each petri dish of a set of petri dishes of any of the above items under conditions where the cells are viable; after the incubating step, observing the cells in each of the petri dishes; and selecting a petri dish from the set of petri dishes based on the observation results. (Item 22) Any of the methods above, wherein the petri dish is selected based on at least one of colony shape, cell density, tissue thickness, and fiber orientation. (Item 23) where the cell adhesion surface (i) in the direction in which the plurality of recesses extend, a second region adjacent to the stripe structure, and / or (ii) a second stripe structure having a repetition of a plurality of second recesses extending in a direction different from the stripe structure Any of the articles above comprising. (Item 24) Any of the articles above, comprising a first region and the second region at both ends of the stripe structure so as to sandwich the stripe structure. (Item 25) Any of the articles above, wherein the height of the surface of the second region is the same as the bottom surface of the recess. (Item 26) Any of the articles above, comprising a stripe structure arranged to surround the first region. (Item 27) Any of the articles above, wherein the second region has a peelable coating. (Item 28) A laminate comprising a plurality of any of the articles above and a cell supply channel, where the plurality of articles are laminated in an arrangement where the first regions of each of the articles overlap, and the cell supply channel is arranged to contact the first region of each of the articles. Laminate. (Item 29) A laminate of any of the above items, further comprising cell retrieval channels arranged in contact with each of the second regions of the article. (Item 30) A method for collecting target cells, A step of placing a cell population or cell-containing tissue containing the aforementioned target cells in the first region of any of the above items, A step of incubating the article under conditions in which the target cells can survive, wherein the target cells migrate from the first region along the cell adhesion surface to the second region. A step of recovering the target cells from the second region. Methods that include...

[0005] In the present invention, one or more of the above features may be provided in combination with those explicitly stated. Further embodiments and advantages of the present invention will be apparent to those skilled in the art by reading and understanding the detailed description below as needed. [Effects of the Invention]

[0006] Articles having the stripe structure of this disclosure may provide convenient isolation of target cells and improved cell culture. Articles having the stripe structure of this disclosure may contribute to the development of cell-based pharmaceuticals and medical devices (such as implants). [Brief explanation of the drawing]

[0007] [Figure 1] This is a schematic diagram of the stripe structure. [Figure 2] The image shows a microscopic observation of the stripe structure. The scale bar represents 100 μm. [Figure 3] These are photographs of human adipose tissue cultured in petri dishes with a striped structure (a, b, d, e, k) and in a petri dish with a flat surface (φ35 mm). [Figure 4]These are micrographs showing ADSCs migrating from human adipose tissue seeded in specific areas on a petri dish with a striped structure a, b, d, e, and k, and a petri dish (φ35 mm) with a flat surface. The scale bar represents 300 μm. [Figure 5] The results of the WST-1 assay in Example 4 are shown. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001. [Figure 6] These are fluorescence micrographs of ADSCs migrating on petri dishes with stripe structures a, b, d, e, and k, and on petri dishes with flat surfaces, stained with actin and nuclei. The scale bar represents 200 μm. [Figure 7] This is a fluorescence micrograph of adipose tissue cultured on a petri dish with stripe structures a, b, d, e, and k, taken from a cross-section of the stripe structure. The scale bar represents 40 μm. [Figure 8] Microscopic images of a cell migrating separator tip are shown. The lower left image shows the overall view, and magnified views of the positions of each symbol indicated therein are also shown. [Figure 9] This shows the migration of ADSCs when using a cell migrating separator tip. The upper panel shows microscopic images. The lower panel shows the relationship between the distance from the seeded adipose tissue area and the number of cells (nuclei) present there. [Figure 10] This shows the migration of ADSCs using a flat surface. The upper panel shows microscopic observations. The lower panel shows the relationship between the distance from the seeded adipocyte region and the number of cells (nuclei) present there. [Figure 11] This is a photograph of osteoblasts differentiated from ADSCs on a striped structure with a recess width of 3 μm and a distance between recesses of 2 μm. [Figure 12]These are microscopic images of adipose tissue cultured in petri dishes with striped structures k, a, b, d, and e, and in petri dishes with a flat surface (φ35 mm). For each petri dish, a photograph from above (upper panel) and a photograph from the side (lower panel) are shown. In the lower panel, dotted and solid lines indicate the contours of the cell sheet, the top and bottom surfaces of the striped structure, and the cell sheet's maximum thickness from the bottom surface to the top surface of the cell sheet, respectively, are shown with numerical values. [Modes for carrying out the invention]

[0008] The present invention will be described below in best mode. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Accordingly, singular articles (for example, "a," "an," and "the" in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0009] (definition) The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0010] In this specification, “stripe structure” refers to a solid structure comprising a repeating set of recesses (which can also be described as grooves) extending in a particular direction (see Figure 1). The portion between adjacent recesses may be described as a convex portion (which can also be described as a ridge). The distance between the bottom surface of a recess and the top surface of a convex portion may be described as the depth (which can also be described as the height) of the recess. The distance between recesses may also be the width of the convex portion. A recess angle may be formed between the side surface of a recess and the bottom surface of a recess. The bottom surface, side surface, and / or top surface are typically planar, but may be curved. The extension of the recesses in a particular direction may be linear or curvilinear (typically, the width of the recesses and the distance between recesses are constant). Typically, in a single stripe structure, the direction of extension, the width of the recesses, the distance between recesses, and the depth of the recesses are constant. If there are portions where the direction of extension is not continuous, it may be described as the presence of multiple stripe structures. The stripe structure may be on a curved surface; for example, an article with a stripe structure on a curved surface can be manufactured by fixing a flexible member with a stripe structure to the curved surface of a transplanted object.

[0011] In this specification, “kit” means a unit in which the parts to be provided (e.g., an article having a striped structure, additional components, instructions, etc.) are provided, usually divided 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 stability or other reasons, and which are preferably brought into contact immediately before use. Such a kit is preferably advantageous to include instructions or a manual describing how to use or handle the provided parts (e.g., an article having a striped structure, additional components, etc.). When a kit is used in this specification, the kit usually includes instructions describing how to use the article having a striped structure, etc. of this disclosure.

[0012] In this specification, the term "approximately" refers to a range of plus or minus 10% of the indicated value unless otherwise specified. When used in reference to temperature, "approximately" may refer to a range of plus or minus 5°C of the indicated value.

[0013] (Description of preferred embodiments) Preferred embodiments of the present invention are described below. The embodiments provided below are provided for a better understanding of the present invention, and it should be understood that the scope of the present invention should not be limited to the following description. Accordingly, it will be clear to those skilled in the art that they can make appropriate modifications within the scope of the present invention by taking into consideration the description herein. Those skilled in the art may combine any of these embodiments as appropriate.

[0014] This specification primarily describes articles having a striped structure, but various embodiments are also contemplated as inventions, such as systems including such articles, methods of using such articles, and products obtained using such articles. Descriptions of one embodiment, such as an article, apply similarly to other embodiments. For example, any feature described in relation to an article may be understood to apply to methods of using the article.

[0015] (Articles with a striped structure) In one aspect, the present disclosure provides an article having a striped structure. In the present disclosure, since the striped structure is intended to come into contact with cells, the striped structure is arranged on a surface of the article that can come into contact with cells. The striped structure may be arranged on multiple surfaces of the article, or it may be arranged at multiple locations on a single surface.

[0016] (Striped structure) Stripe structures can be characterized primarily by the width of the depressions, the distance between depressions, and the depth of the depressions, and these elements can particularly affect cells adjacent to the stripe structure. Typically, the width of the depressions, the distance between depressions, and the depth of the depressions in a single stripe structure are constant, but they may not be. Typically, the depressions are linear, but they may also be curved. Typically, the pattern of depressions in a single stripe structure is regular.

[0017] In one embodiment, the recesses of the stripe structure are approximately 1 μm to 200 μm, for example, approximately 2 μm to 200 μm, approximately 3 μm to 200 μm, approximately 4 μm to 200 μm, approximately 5 μm to 200 μm, approximately 7 μm to 200 μm, approximately 10 μm to 200 μm, approximately 15 μm to 200 μm, approximately 20 μm to 200 μm, approximately 1 μm to 150 μm, approximately 2 μm to 150 μm, approximately 3 μm to 150 μm, approximately 4 μm to 150 μm, approximately 5 μm to 150 μm, approximately 7 μm to 150 μm, approximately 10 μm to 150 μm, approximately 15 μm to 150 μm, approximately 20μm~150μm, approx. 1μm~100μm, approx. 2μm~100μm, approx. 3μm~100μm, approx. 4μm~100μm, approx. 5μm~100μm, approx. 7μm~100μm, approx. 10μm~100μm, approx. 15μm~100μm, approx. 20μm~100 μm, approximately 1μm~70μm, approximately 2μm~70μm, approximately 3μm~70μm, approximately 4μm~70μm, approximately 5μm~70μm, approximately 7μm~70μm, approximately 10μm~70μm, approximately 15μm~70μm, approximately 20μm~70μm, approximately 1μm~50μm, approximately 2μm~ 50μm, approximately 3μm~50μm, approximately 4μm~50μm, approximately 5μm~50μm, approximately 7μm~50μm, approximately 10μm~50μm, approximately 15μm~50μm, approximately 20μm~50μm, approximately 1μm~40μm, approximately 2μm~40μm, approximately 3μm~40μm, approximately 4 μm ~ 40 μm, approximately 5 μm ~ 40 μm, approximately 7 μm ~ 40 μm, approximately 10 μm ~ 40 μm, approximately 15 μm ~ 40 μm, approximately 20 μm ~ 40 μm, approximately 1 μm ~ 30 μm, approximately 2 μm ~ 30 μm, approximately 3 μm ~ 30 μm, approximately 4 μm ~ 30 μm, approximately 5 μm ~ 30 μm, They have widths of approximately 7 μm to 30 μm, approximately 10 μm to 30 μm, approximately 15 μm to 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 to 20 μm, approximately 1 μm to 15 μm, approximately 2 μm to 15 μm, approximately 3 μm to 15 μm, approximately 4 μm to 15 μm, approximately 5 μm to 15 μm, approximately 7 μm to 15 μm, approximately 1 μm to 10 μm, approximately 2 μm to 10 μm, approximately 3 μm to 10 μm, approximately 4 μm to 10 μm, or approximately 5 μm to 10 μm.

[0018] In one embodiment, the distance between adjacent recesses in the stripe structure is approximately 1 μm to 50 μm, for example, approximately 2 μm to 50 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 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 m~30μm, approx. 4μm~30μm, approx. 5μm~30μm, approx. 7μm~30μm, approx. 10μm~30μm, approx. 15μm~30μm, approx. 1 μm~20μm, approx. 2μm~20μm, approx. 3μm~20μm, approx. 4μm~20μm, approx. 5μm~20μm, approx. 7μm~20μm, approx. 10μm m~20μm, approx. 1μm~15μm, approx. 2μm~15μm, approx. 3μm~15μm, approx. 4μm~15μm, approx. 5μm~15μm, approx. 7μm ~15 μm, approximately 1 μm to 10 μm, approximately 2 μm to 10 μm, approximately 3 μm to 10 μm, approximately 4 μm to 10 μm, or approximately 5 μm to 10 μm. The distance between adjacent recesses in stripe structures larger than 30 μm may be detrimental to cell migration or proliferation. While we do not wish to be bound by any particular theory, larger distances between recesses may reduce the efficiency per unit area of ​​cell and / or tissue adhesion, migration, and / or proliferation.

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

[0020] In one embodiment, the porosity of the stripe structure (the ratio of the area occupied by recesses when the stripe structure is viewed two-dimensionally, and can be calculated as (recess width) / (recess width + distance between recesses)) is approximately 0.05 to 0.95, for example, approximately 0.1 to 0.95, approximately 0.15 to 0.95, approximately 0.2 to 0.95, approximately 0.3 to 0.95, approximately 0.4 to 0.95, approximately 0.5 to 0.95, approximately 0.6 to 0.95, approximately 0.7 to 0.95, approximately 0.8 to 0.95, approximately 0.05-0.9, approximately 0.1-0.9, approximately 0.15-0.9, approximately 0.2-0.9, approximately 0.3-0.9, approximately 0.4-0.9, approximately 0.5-0.9, approximately 0.6-0.9, approximately 0.7-0.9, approximately 0.8-0.9, approximately 0.05-0.85, approximately 0.1-0.85, approximately 0.15-0.85, approximately 0.2-0.85, approximately 0.3-0.85, approximately 0.4-0.85, approximately 0.5-0.85, approximately 0.6-0.85, approximately 0.7-0.8 5. Approximately 0.05~0.8, approximately 0.1~0.8, approximately 0.15~0.8, approximately 0.2~0.8, approximately 0.3~0.8, approximately 0.4~0.8, approximately 0.5~0.8, approximately 0.6~0.8, approximately 0.7~0.8, approximately 0.05~0.7, approximately 0.1~0.7, approximately 0.15~0.7, approximately 0.2~0.7, approximately 0.3~0.7, approximately 0.4~0.7, approximately 0.5~0.7, approximately 0.6~0.7, approximately 0.05~0.6, approximately 0.1~0.6, approximately 0.15~0. 6. Approximately 0.2-0.6, approximately 0.3-0.6, approximately 0.4-0.6, approximately 0.5-0.6, approximately 0.05-0.5, approximately 0.1-0.5, approximately 0.15-0.5, approximately 0.2-0.5, approximately 0.3-0.5, approximately 0.4-0.5, approximately 0.05-0.4, approximately 0.1-0.4, approximately 0.15-0.4, approximately 0.2-0.4, approximately 0.3-0.4, approximately 0.05-0.3, approximately 0.1-0.3, approximately 0.15-0.3, or approximately 0.2-0.3 is possible.

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

[0022] The stripe structure of this disclosure can be used in any cell, such as stem cells (ADSCs, ES cells, iPS cells, etc.) and cancer cells (especially for diagnostic purposes), and can be applied to any known cultured cell.

[0023] Because the effects on cells and the types of cells to which the stripes can adhere can differ depending on the stripe structure, the optimal stripe structure may vary depending on the cells used and the purpose. However, some suitable examples are shown below.

[0024] A striped structure with recess widths of 1 μm to 20 μm, spacing between recesses of 2 μm to 20 μm, and recess depths of 10 μm to 40 μm may be effective for the adhesion and / or migration of fibroblasts (NIH3T3 cells), adipose-derived stem cells (ADSCs), bone marrow mesenchymal stem cells (BMMSCs), renal 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 be equally effective for other cells with similar properties to these cells (including cells differentiated from embryonic stem cells and somatic stem cells).

[0025] A striped structure with a recess width of 3 μm to 20 μm, a spacing between recesses of 2 μm to 20 μm, and a recess depth of 10 μm to 40 μm (especially with a porosity of approximately 0.5 or higher) may be effective in the rapid proliferation of fibroblasts (NIH3T3 cells), adipose-derived stem cells (ADSCs), bone marrow mesenchymal stem cells (BMMSCs), renal cells (COS-1 cells), adrenal medulla cells (PC12 cells), vascular endothelial cells (HUVECs), keratinocytes (HaCaT cells), and hepatocytes (HepG2 cells, HepaRG cells). It may also be effective in inducing differentiation of adipose-derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMMSCs) into chondroid cells or vascular endothelial cells. This striped structure may be similarly effective in the differentiation induction culture of somatic stem cells and embryonic stem cells.

[0026] A striped structure with recesses 1 μm to 9 μm wide, 2 μm to 10 μm apart, and 10 μm to 40 μm deep, with a porosity of approximately 0.5 or higher, may be effective in promoting the formation of cell aggregates (spheroids) such as hepatocytes. A striped structure with recesses 3 μm to 5 μm wide, 2 μm to 10 μm apart, and 10 μm to 40 μm deep, with a porosity of approximately 0.5, may be effective in promoting the migration of slow-migrating cells such as hepatocytes (HepG2 cells, etc.). This striped structure may have a similar effect, especially on cells of a similar size to HepG2 cells.

[0027] A striped structure with recess widths of 10 μm to 100 μm, spacing between recesses of 10 μm to 20 μm, and recess depths of 10 μm to 40 μm may be effective in separating stem cells (ADSCs, UCMSCs) from adipose tissue and umbilical cord tissue. This striped structure can similarly separate specific cells when applied to somatic tissues (bone marrow, vascular tissue, skin tissue, cartilage tissue, synovial tissue, dental pulp tissue, etc.) and body fluids (blood, bone marrow fluid, umbilical cord blood, etc.). When these tissues are seeded on a striped structure, the one-dimensionally restricted migration direction makes contact inhibition less likely, thus promoting cell proliferation. Furthermore, the one-dimensionally continuous grooves improve cell or tissue adhesion, which may be advantageous for cell extraction from the tissue.

[0028] In addition to the cell types specifically mentioned above, cells with similar cell size, cell nucleus size, and / or cell adhesion to the edges of protrusions may have similar types of preferred stripe structures. For example, cells with similar cell size may have a volume (average) ratio or area (average) ratio of about 10–1000%, about 20–500%, about 30–300%, or about 50–200% relative to one another. For example, cells with similar nuclear size may have nuclei (which may be determined based on nuclear staining) within a volume (average) ratio or area (average) ratio range of about 10–1000%, about 20–500%, about 30–300%, or about 50–200% relative to one another. For example, cells with similar adhesion to the edges of protrusions may have an adhesion force (e.g., the force (N) required to detach cells) to each other in the range of approximately 10–1000%, approximately 20–500%, approximately 30–300%, or approximately 50–200% to the stripe structure.

[0029] The stripe structure can be fabricated by photolithography or injection molding using a microfabricated mold. Articles with a stripe structure do not necessarily need to be molded as a single piece; for example, a component with a stripe structure may be fabricated and then combined with other components (e.g., bonded) to form an article with a stripe structure. The stripe structure can be fabricated from any known material such as resin, for example, polystyrene, PMEA, polypropylene, polybutadiene, polyurethane, PET, Teflon® (polytetrafluoroethylene), polysilicon, etc. The stripe structure may also be made of biodegradable plastic material such as PLLA, PLGA, or PCL. The stripe structure may be made of a flexible material or a rigid material.

[0030] Articles having a stripe structure may be made of resins such as polystyrene, PMEA, polypropylene, polybutadiene, polyurethane, PET, Teflon® (polytetrafluoroethylene), polysilicon, or biodegradable plastic materials such as PLLA, PLGA, and PCL, and may be made of flexible materials or rigid materials.

[0031] The shape of the article can be arbitrary, such as a flat plate (including structures with holes such as plates and meshes), a rectangular prism, or a sphere, and a shape suitable for the application will be understood by those skilled in the art. The article may have a striped structure on only certain surfaces, or it may have a striped structure on multiple surfaces. In particular, when the purpose is to form tissue or a three-dimensional culture by culture, the surface of the article having a striped structure may have holes that allow fluids such as culture media to pass through. Cells do not adhere to the entire bottom surface of the recesses of the striped structure, and fluid communication can be maintained near the bottom surface even after cell culture, but the presence of holes may allow cultured cells to come into contact with the fluid more easily.

[0032] The cell adhesion surface or stripe structure of the articles of this disclosure may be subjected to any surface modification for cell culture. Examples of surface modifications include hydrophilization treatment by UV irradiation and plasma treatment (e.g., treatment that results in a water contact angle of 20 to 80 degrees), coating with polylysine and cell adhesion proteins (e.g., collagen), adhesion of feeder cells, and coating with a release agent (e.g., a substance that denatures in a condition-dependent manner such as heating, isopropyl acrimide (NIPAM)). Cells cultured on the stripe structure can also be used as feeder cells.

[0033] In one embodiment, the article having the 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, and is typically round and comes with a lid. Typically, the striped structure is positioned on at least a portion of the bottom of the petri dish. In one embodiment, the article having the striped structure is a plate, which is used in place of a petri dish. By placing this plate at the bottom of an existing petri dish, it can be used in the same way as a petri dish having the striped structure described above. Alternatively, after seeding cells at the bottom of the petri dish, the plate can be placed to cover the cells, and cells can be seeded on the plate. The plate may have the striped structure on one side only, or on both sides. The striped structure of the present disclosure may have high cell adhesion, so that even cells that normally float during culture can be allowed to adhere to the striped structure (for example, by bringing the cells into contact with the striped structure so that they rest on top of the cells).

[0034] In one embodiment, the article having a striped structure is a graft. Examples of grafts include dental implants and intravascular stents. Grafts also include articles combining the article with the striped structure with an organ for transplantation, and articles applied to the organ for transplantation. The overall shape and material of the graft article can mimic known grafts and can be appropriately selected by those skilled in the art. The graft may have striped structures on multiple surfaces. The graft may have a structure that is not flat but has holes, such as a mesh or stripe, and the sides of the holes may have striped structures. Striped structures on the cell adhesion surface of a graft may have beneficial effects such as fixing the graft in a specific location, increasing adhesion between the graft and cells, promoting the formation of desired tissue around the graft (e.g., with a specific orientation), and reducing host immunity to the graft. The preferred striped structure may vary depending on the site (organ, tissue) where the graft is to be transplanted. For example, if the implant is a stent placed in a coronary artery to support the vessel wall, a stripe structure that promotes the adhesion of vascular endothelial cells may be arranged on the cell adhesion surface in contact with the vessel wall, and if necessary, a stripe structure that promotes the adhesion of vascular endothelial cells may be arranged on the cell adhesion surface in contact with 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 vessel wall. Multiple stripe structures may be provided, like anti-slip features on tires, to fix the implant in a specific location.

[0035] In one embodiment, the article having a stripe structure is a cell separation article, which is a plate or an article containing a plate. The cell separation article typically comprises a seeding region (first region) and / or a recovery or detection region (second region). Cells may be cultured on the cell separation article. The cell separation article of this disclosure can provide target cells without requiring enzymatic treatment or the like. The cell separation article of this disclosure is also intended for use in sequentially separating differentiated cells from seeded cells. It can provide target cells without requiring enzymatic treatment or the like. Multiple types of cells can also be separated by combining different stripe structures.

[0036] The seeding region (first region) is intended to be the region of the article that first comes into contact with cells, and it is not necessary to seed cells or tissue on the seeding region; embodiments are also possible in which the article is placed over seeded cells or tissue so that the seeded cells or tissue come into contact with the seeding region. The seeding region (first region) may be a portion comprising a stripe structure or a portion adjacent to a stripe structure (particularly an adjacent portion located in the direction in which the recesses extend). In some embodiments, the seeding region (first region) and the retrieval or detection region (second region) may be arranged adjacent to each other on opposite sides of the stripe structure (for example, in the direction in which the recesses of the stripe structure extend). If the seeding region (first region) is a portion adjacent to a stripe structure, the surface of the seeding region (first region) preferably has a height lower than the top surface of the stripe structure. If the surface of the seeding region (first region) is higher than the top surface of the stripe structure, seeded cells may naturally diffuse onto the stripe structure, making it difficult to distinguish between seeded cells and migratory cells. Even in this case, if the recesses in the stripe structure are of sufficient length, seeded cells and migrating cells can be easily distinguished. Cell migration can be made more efficient by arranging the seeding region (first region) and the stripe structure so that seeded cells or tissue can come into contact with a larger number of recesses (or openings thereof) (e.g., the notches in the cell migrating separator in Figure 8). In some embodiments, the stripe structure (particularly the openings of the recesses) is arranged to surround the seeding region (first region). In some embodiments, the collection or detection region (second region) is coated with a release agent (e.g., a substance that denatures in a condition-dependent manner, such as by heating, such as NIPAM).

[0037] The retrieval or detection region (second region) may be a portion comprising the stripe structure or a portion adjacent to the stripe structure (particularly an adjacent portion located in the direction in which the recesses extend). Since cells can migrate on the stripe structure in the direction in which the recesses extend, in many embodiments, migrating cells are retrieved or detected. To retrieve or detect migrating cells, the seeding region (first region) may differ from the retrieval or detection region (second region), but in some embodiments, the seeding region (first region) may be the retrieval or detection region (second region) to retrieve or detect non-migrating cells. Often, cells move along the stripe structure with only a portion of the cell on the convex portion and the cell nucleus located in the recess (not necessarily below the top surface of the convex portion). Therefore, if the retrieval or detection region (second region) is a portion adjacent to the stripe structure, the surface of the retrieval or detection region (second region) preferably has a height equal to or lower than the bottom surface of the stripe structure, thereby facilitating cell access to the retrieval or detection region (second region). Since cells can sequentially reach the collection or detection area (second area), a wall may be provided in the collection or detection area (second area) to prevent excessive diffusion of cells. A stripe structure (a second stripe structure comprising repeating recesses extending in different directions) arranged in such a way that the openings of the recesses do not come into contact with the cells can function as a wall.

[0038] The primary purpose of cell separation is the acquisition and / or detection of specific cells. The usefulness of detecting specific cells is generally understood by those skilled in the art, such as diagnosing the condition of a subject (e.g., presence or absence of disease) using a sample obtained from the subject, and evaluating the quality of cultured cells. In some embodiments, articles having a stripe structure are articles 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 are intended in the form of a kit, etc. The detection area (second area) is configured to enable the detection of specific cells and can be configured, for example, by using a transparent member to enable detection by light, by arranging a detection reagent (e.g., an antibody), or by arranging an inlet for adding the detection reagent. The recovery or detection area (second area) and / or seeding area (first area) may be equipped with indicators.

[0039] In some embodiments, articles having a stripe structure are articles for recovering specific cells after cell separation (and, if necessary, after further detection of cells), and may be useful for supplying cell material. For efficient supply of cell material, articles having stripe structures on both sides of a plate and articles consisting of stacked plates having stripe structures are envisioned. For example, a stack is envisioned that includes a plurality of plate-like parts, each of which is a cell separation article as described herein, and cell recovery channels connecting recovery or detection areas (second areas) and / or cell supply channels connecting seeding areas (first areas). In some embodiments, the plurality of plate-like parts may be arranged so that the recovery or detection areas (second areas) and / or seeding areas (first areas) overlap.

[0040] (method) In one embodiment, the Disclosure provides a method for culturing cells, comprising the step of incubating the 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 those commonly used for the cells or tissues used, or may be modified, and can be appropriately set by those skilled in the art.

[0041] In one embodiment, the present disclosure provides a method for isolating cells of interest, comprising the steps of: placing a cell population or cell-containing tissue containing cells of interest in a seeding area (first area) of an article described herein; and incubating the article under conditions in which the cells of interest are viable, such that the cells of interest migrate from the first area along a cell adhesion surface. In one embodiment, the method further comprises, after the incubation step, detecting and / or recovering the cells of interest from a recovery or detection area (second area) on a cell adhesion surface different from the first area.

[0042] (Multiple types of stripe structures) Since the effects on cells and the types of cells to which the stripe structures can be suitably adhered may differ depending on the stripe structure, a set of multiple types of stripe structures is preferably provided in order to select a stripe structure suitable for the purpose for the cells to be used. In one embodiment, a plate or petri dish having multiple types of stripe structures described herein on a single surface is provided. In one embodiment, a set of petri dishes is provided herein, comprising: a first petri dish including a cell adhesion surface, the first petri dish having a first stripe structure comprising a repeating plurality of first recesses extending in a particular direction; and a second petri dish including a cell adhesion surface, the second petri dish having a second stripe structure comprising a repeating plurality of second recesses extending in a particular direction, wherein the first stripe structure and the second stripe structure differ in at least one of (i) the width of the first and second recesses, (ii) the depth of the first and second recesses, and (iii) the distance between adjacent plurality of first recesses and the distance between adjacent plurality of second recesses. A set of petri dishes may contain three, four, five, six, seven, eight, nine, ten, or more types of stripe structures, each with a different stripe pattern. By culturing the target cells on approximately five different stripe structures, it is generally possible to select a generally suitable stripe structure.

[0043] In one embodiment, the Disclosure provides a method for selecting petri dishes for culturing cells, comprising the steps of: incubating cells seeded in each of the set of petri dishes described herein under conditions in which the cells can survive; observing the cells in each petri dish after the incubation step; and selecting petri dishes from the set of petri dishes based on the observation results. In one embodiment, the petri dishes are selected based on at least one of colony shape, cell density, tissue thickness, and fiber orientation.

[0044] (culture) In one aspect, this disclosure provides a culture cultured using an article having the stripe structure described herein. Such a culture may have characteristics such as exhibiting orientation in a specific direction (e.g., direction of cytoskeleton elongation). When cells come into contact with each other, contact inhibition may occur, but cells cultured on the stripe structure described herein may mostly reside in the recesses and be separated only by the spacing between the recesses, thus forming a culture (or culture surface) with reduced direct contact between cells. The stripe structure described herein may form a thick cell (tissue) sheet with fewer defects. Such a culture can be used as a transplant (e.g., for regenerative medicine) or as a feeder layer for culturing stem cells (e.g., ES cells, iPS cells).

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

[0046] (Example 1: Design of various stripe structures) We investigated multiple stripe structures with different recess widths, inter-recess distances, and recess depths.

[0047] A striped structure with the recess width, distance between recesses, and recess depth shown in the table below was fabricated. [Table 1-1] [Table 1-2] *The stripe structures numbered 1-46 were fabricated on a silicon wafer using photolithography. *The striped structures numbered 47-54 were formed on a petri dish.

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

[0049] Human adipose tissue, harvested subcutaneously by liposuction at Ryukyu University Hospital, was washed three times with D-PBS containing 1% penicillin-streptomycin. Next, the washed human adipose tissue was cut with scissors into pieces approximately 2 mm in size, and 0.05 g of these pieces were seeded into each Φ35 mm striped petri dish. Care was taken to ensure that the adipose tissue pieces were distributed as uniformly as possible throughout the petri dish. After seeding, 500 μl of culture medium was added to start culturing. ADSC-4 (Kojin Bio, Japan) or ADSC-GM (LONZA, US) was used as the culture medium. Culture medium was changed every 24 hours by removing 200 μl of medium and adding 200 μl of fresh medium. Cell proliferation was evaluated 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-derived stem cells (ADSCs) proliferated on striped petri dishes were observed using an inverted microscope CKX53 (Olympus, Japan). A CACHN10×IPC objective lens (Olympus, Japan) was used for this observation. On day 21 of culture, the cells on the striped petri 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 fluorescence microscope BZ-9000 (Keyence, Japan). This study was approved by the Life Science and Medical Research Ethics Committee of the University of the Ryukyus (Approval Number 810).

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

[0051] (Example 3: Examination of combinations of stripes and cell types) We investigated the optimal cell (tissue) combinations for the various stripe structures designed in Example 1.

[0052] Generally, differences in effectiveness were observed based on the following classifications (1) to (5). (1) Width of recess: 1 μm to 2 μm, spacing between recesses: less than 10 μm (2) Width of recess: 3 μm to 5 μm, spacing between recesses: less than 10 μm (3) Width of recess: 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 recess is 10 μm or more and less than 40 μm.

[0053] In all of (1) to (5), an effect was observed that restricted the adhesion, extension, and migration of seeded NIH3T3 cells, human ADSCs, human BMMSCs, COS-1 cells, PC12 cells, HUVECs, HaCaT cells, HepG2 cells, HepaRG cells, etc., in one dimension (in the direction in which the concave parts extend). Since this effect was similarly reproduced in various cells with properties such as fibroblasts, mesenchymal stem cells, nerve cells, vascular endothelial cells, keratinocytes, and hepatocytes, it is thought that this effect is broadly 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 in the stripe structure of (1) to (4).

[0054] Of the three types (1) to (3), when the porosity (the ratio of the area occupied by recesses when the stripe structure is viewed two-dimensionally) was approximately 0.5 or higher, the formation of cell aggregates (spheroids) was promoted. Along with spheroid formation, improvement in liver function of cells such as HepG2 cells and HepaRG cells can be expected. Meanwhile, research into cell therapy using spheroids for transplantation is also progressing. This technology can also be used to produce spheroids for such cell therapy.

[0055] (2) Of these, those with a porosity of around 0.5 were found to significantly improve the migration speed of slow-migrating cells (such as HepG2) cultured on them (see also Example 4). At this time, HepG2 cells placed their bodies, including the cell nucleus, on top of the depressions, extended long protrusions to both or one of the continuous depressions, and migrated at high speed as if being pulled by them. We believe that cells of a similar size to HepG2 can be expected to have a similar effect using the same stripe structure.

[0056] In (2), (3), and (4), the proliferation rate of seeded NIH3T3 cells, human ADSCs, human BMMSCs, COS-1 cells, PC12 cells, HUVECs, HaCaT cells, HepG2 cells, and HepaRG cells was higher than on a flat surface. In all the cells, cell states, and culture conditions tested, a porosity of approximately 0.5 or higher was advantageous for improving the proliferation rate. This effect was similarly reproduced in various cells with properties such as fibroblasts, mesenchymal stem cells, nerve cells, vascular endothelial cells, keratinocytes, and hepatocytes, suggesting that the structures in (2), (3), and (4) are also advantageous for improving the proliferation rate of cells with similar properties (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, chondroid cells, vascular endothelial cells, etc., it was found that the differentiation induction efficiency was higher than on a flat surface (see also Example 6, Figure 11). It is thought that such stripe structures are advantageous not only when performing similar differentiation induction culture from somatic stem cells but also when performing the same from embryonic stem cells.

[0058] Experiments were conducted in which ADSCs were extracted from human adipose tissue and umbilical cord-derived mesenchymal stem cells (UCMSCs) were extracted and cultured on (4) and (5). The results showed that when cultured on (4) and (5), more ADSCs and UCMSCs were obtained earlier compared to cultured on a flat surface. This is presumed to be because the seeded tissue adheres more easily to the striped structure of (4) and (5) than to other striped structures and flat surfaces (see also Example 4, Figure 7 for how seeded human adipose tissue adheres to the striped structure). Note that this striped structure of (4) is used in the cell migrating separator (CMS) of Example 5. Various body tissues and bodily 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. Bodily fluids include blood, bone marrow fluid, and umbilical cord blood. Regardless of which type of seeding is performed on the striped structure, the increased surface area and one-dimensionally restricted migration direction due to the striped structure make contact inhibition less likely, thus facilitating cell proliferation. Furthermore, when somatic tissue is seeded, the one-dimensionally continuous grooves facilitate the adhesion of the seeded tissue, which is advantageous for cell extraction from the tissue.

[0059] Since it has been found that the preferred stripe structure may differ depending on the cell or tissue, or depending on the purpose, it is thought 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 a stripe structure) Different cell or tissue types may exhibit different levels of adhesion to and mobility on striped structures. Therefore, specific cell types can be isolated and obtained by incubating a mixture containing multiple cell types on a striped structure.

[0061] Tissue was cultured using five types of petri dishes with the striped structure shown in the table below, as well as a flat petri dish made of the same material. [Table 3]

[0062] Human adipose tissue was placed in a portion of each petri 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 petri dishes with a striped structure, ADSCs were observed to migrate preferentially and reach areas far from the tissue fragment. Migration was also most pronounced in the same direction as the stripes. ADSCs migrated particularly extensively in stripe structure 4 (recess width: 100 μm, distance between recesses: 20 μm, recess depth: 20 μm). On the other hand, on flat petri dishes, few cells were observed at locations far from the tissue fragment. It was found that ADSCs can be rapidly extracted and cultured from adipose tissue without enzymatic treatment.

[0063] When comparing the total number of cells in each petri dish using the WST-1 assay, higher activity was observed in petri dishes with a striped structure compared to flat petri dishes (Figure 5). Since the amount of seeded adipose tissue was the same, the amount of ADSCs contained in the adipose tissue is also considered to be the same. However, ADSCs are thought to have low metabolic activity and can hardly proliferate in adipose tissue. Therefore, the results of this WST-1 assay suggest that ADSCs were rapidly extracted from the adipose tissue in petri dishes with a striped structure, detached from it, and then proliferated. 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 structure, but in all cases, superior extraction culture was achieved compared to flat petri dishes.

[0064] Observation of extracted ADSCs on each petri dish revealed differences in cell adhesion patterns and cell morphology due to differences in the stripe structure (actin-stained and nuclear-stained images, Figure 6). Furthermore, observation of adipose tissue from cross-sections of the stripe structure after DAPI staining (1:400, 20 min.) and Lipi Dye staining revealed that the shape of the adipose tissue also differed depending on the type of stripe structure (Figure 7). In striped petri dishes k and a, with narrow groove widths, the seeded adipose tissue did not penetrate the grooves very well, whereas in striped petri dishes d and e, with wider groove widths, the seeded adipose tissue penetrated the grooves significantly. From the viewpoint of increasing the surface area (seeding surface), smaller groove widths and spacing in striped petri dishes are advantageous, but it is thought that grooves of a certain width are advantageous for tissue to make firm contact with the grooves. It is thought that firm contact of tissue with the grooves of the striped petri dish improves the efficiency of extraction and culture from seeded tissue.

[0065] Furthermore, we tested the migration of HepG2 cells when cultured on a stripe structure.

[0066] Specifically, the fabricated stripe structure (recess width: 3 μm, distance between recesses: 3 μm or 9 μm, recess depth: 20 μm) was hydrophilized by O2 plasma treatment before cell culture, sterilized, and then immersed in culture medium and degassed under reduced pressure. HepG2 cells were then placed on this striped silicon wafer in a 1 x 10⁶ layer. 4 cells / cm 2HepG2 cells were seeded and fluorescently stained with PKH26. The silicon wafer with the attached HepG2 cells and stripe structure was transferred to a 35mm / Glass Base Dish filled with culture medium, and the cell-adhering surface was placed face down in the glass window at the bottom of the dish. At this time, the four vertices of each substrate were positioned to catch on the frame of the glass window, ensuring that there was sufficient space between the cell-adhering surface and the glass. Four hours after seeding, time-lapse observation of cells was started at designated fixed points on each stripe structure and on the plane. Time-lapse observation was performed for 72 hours at 30-minute intervals using an Observer Z1 fluorescence microscope (ZEISS Inc., Germany) equipped with a Stage Top® Incubator INU-ZILCS (Tokai Hit Co., Ltd., Japan). During this time, a 10x objective lens was used to capture the reflected image of the stripe structure and the fluorescence image of the cells using a CCD camera. The movement of cells on each stripe structure (cell migration) was manually tracked using AxioVision 4.8 (ZEISS Inc., Germany). From the trajectories of individual cell migrations obtained, the average migration velocity [μm / h] of cells migrating on each stripe structure was determined. This method of 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. [Table 4]

[0068] HepG2 cells hardly migrated when cultured on a flat surface, but their motility improved when cultured on a striped structure.

[0069] (Example 5: Development of a cell migrating separator) Furthermore, we developed a cell migrating separator equipped with a stripe structure specifically designed for cell separation. An example of a cell migrating separator chip is shown in Figure 8. This was fabricated from a silicon wafer material using photolithography for the purpose of separating ADSCs. The stripe structure was fabricated to have a recess width of 10 μm, a 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 stripe structures at four locations surrounding the seeding area, with the openings of the grooves of the recesses facing the seeding area (the orientation of the recesses is the same within each location). The stripe structures are spaced apart so that they do not touch each other except at the endpoints, and the bottom of the cut sections is the same depth as the seeding area. The presence of cuts is expected to increase the contact area between the seeded tissue (cells) and the openings of the grooves of the recesses in the stripe structure, thereby improving the extraction efficiency of the target cells. The target cells are intended to migrate from the seeding area through the stripe structure to the outer perimeter harvesting area. The bottom of the harvesting area is designed to be the same depth as the bottom surface of the recess.

[0070] The performance of the cell migrating separator was tested using adipose tissue similar to that in Example 1. The results were compared with those of a control chip with a flat surface. The cell migrating separator was placed over human adipose tissue on a petri dish and cultured for 17 days, followed by microscopic observation. Cells were stained with Phalloidin 488 (1:200, 4h) and DAPI (1:400, 2h), and 16 images were taken 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, using a confocal laser microscope FV1000D equipped with an UPlanSApo 20x objective lens, with a depth of 30 μm. Additionally, the adipose tissue was divided into 7 sections every 400 μm from the edge, and the number of cells (number of nuclei) in each section was counted.

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

[0072] (Example 6: Culture on a striped structure) Since stripe structures have been found to have various effects on cell or tissue culture, the characteristics of cell culture on stripe structures were investigated from several perspectives.

[0073] The effect of stripe structure on the differentiation of ADSCs into osteoblasts was investigated. Specifically, ADSCs were seeded on silicon wafers with stripe structures numbered 1-25 in Table 1, differentiated in Osteoblast Differentiation Medium (ZEN-BIO, INC., US) for 16 days, stained with alizarin red, and then observed under a microscope.

[0074] As a result, stripe structures numbered 1-5 (recess width: 3-7 μm, distance between recesses: 2 μm, recess depth: 10-40 μm) stained strongly with alizarin red, and it was found that differentiation into osteoblasts was particularly promoted (Figure 11).

[0075] Furthermore, adipose tissue was cultured on a striped structure to form cell sheets. The striped structures used are shown in the table below. [Table 5]

[0076] Finely cut adipose tissue fragments (0.15 g each) were seeded into one 35 mm petri dish containing each surface structure, and cultured in ADSC-GM medium (LONZA, US) on the ceiling for 15 days (37°C, 5% CO2). Afterward, they were inverted and cultured upward for another 20 days (37°C, 5% CO2). The medium was changed every 3 days, and on day 35 of culture, the tissue was fixed with 4% paraformaldehyde phosphate buffer (Nacalai, Japan). Talin was then stained 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), and finally the nuclei were stained with DAPI (Dojindo, Japan) (1:400, RT, 10 min). Appropriate amounts of PBS were added to each of the 35mm petri dishes after staining, and these 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 cell sheets on striped petri dishes were thicker than cell sheets on flat petri dishes. The maximum thickness of cell sheets on flat petri dishes was 10 μm even in a confluent state, while the maximum thickness of cell sheets on striped petri dishes was 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 can be produced using striped petri dishes than using flat petri dishes. [Industrial applicability]

[0078] Articles having the stripe structure of this disclosure may provide convenient isolation of target cells and improved cell culture, and may contribute to the development of cell-based pharmaceuticals and medical devices (such as implants).

Claims

1. A method for separating target cells from a cell population or cell-containing tissue that includes target cells having migratory ability, A step of preparing an article including a cell adhesion surface, wherein the cell adhesion surface has a stripe structure comprising a plurality of repeating recesses extending in a specific direction, and the stripe structure has a constant width of the recesses and a constant distance between the recesses, A step of placing a cell population or cell-containing tissue containing the aforementioned target cells in a first region of the article, The process includes the steps of incubating the article under conditions in which the target cells can survive, causing the target cells to migrate from the first region along the cell adhesion surface to a second region different from the first region, thereby separating the target cells from the cell population or cell-containing tissue, The extended length of the recess in the aforementioned stripe structure is approximately 100 μm to 10,000,000 μm, which is a length sufficient to separate the target cells. The aforementioned stripe structure has a recess width of approximately 1 μm to 100 μm, a recess depth of approximately 5 μm to 50 μm, and a distance between adjacent recesses of approximately 1 μm to 50 μm. method.

2. The method according to claim 1, further comprising the step of detecting and / or recovering the target cells from the second region after incubation.

3. A step of separating the target cells by the method described in claim 1, and A step of incubating the target cells on the article under conditions in which the target cells can survive. A method for culturing the cells, including the above.

4. The method according to claim 3, wherein the step of incubating the cells on the article is carried out under conditions that cause the cells to form a sheet.

5. The method according to claim 1, wherein the stripe structure has a recess width of about 10 μm to 100 μm, a recess depth of about 10 μm to 40 μm, and a distance between adjacent recesses of about 10 μm to 50 μm.

6. An article for separating, detecting, and / or recovering target cells from a cell population or cell-containing tissue containing migratory target cells, the cell adhesion surface comprising a stripe structure having a repeating plurality of recesses extending in a specific direction, the stripe structure having a constant recess width and a constant distance between recesses, The cell adhesion surface is (i) In the direction in which the plurality of recesses extend, the first region and the second region adjacent to both ends of the stripe structure so as to sandwich the stripe structure, and (ii) A second stripe structure comprising a plurality of repeating second recesses extending in a direction different from the stripe structure between the first region and the second region. Equipped with, The first region is for arranging a cell population or cell-containing tissue containing the target cells, and the second region is for detecting and / or recovering the target cells. The extended length of the recesses in the aforementioned stripe structure and the second stripe structure is approximately 100 μm to 10,000,000 μm, which is a length that allows for the separation of target cells having migratory ability. An article wherein the stripe structure and the second stripe structure each have a recess width of approximately 1 μm to 100 μm, a recess depth of approximately 5 μm to 50 μm, and a distance between adjacent recesses of approximately 1 μm to 50 μm.

7. The article according to claim 6, wherein the height of the surface of the second region is the same as the bottom surface of the recess.

8. The article according to claim 6, comprising a stripe structure arranged to surround the first region.

9. The article according to claim 6, wherein the second region has a peelable coating.

10. A laminate for separating target cells from a cell population or cell-containing tissue containing target cells having migratory ability, comprising a plurality of articles according to claim 6 and cell supply channels, The multiple articles are stacked in such an arrangement that the first regions of each article overlap. The cell supply channels are arranged so as to be in contact with each of the first regions of the article. Laminates.

11. The laminate according to claim 10, further comprising cell retrieval channels arranged in contact with each of the second regions of the article.

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