JP6695810B2 - Method for separating undifferentiated cells and substrate for separating undifferentiated cells - Google Patents

Description

  The present invention relates to a method for separating an undifferentiated cell from a cell mixture containing an undifferentiated cell and a differentiated cell, and a substrate used for separating the undifferentiated cell.

  Stem cells typified by somatic stem cells, iPS cells, ES cells and the like have the ability to differentiate into cells having various functions such as nerve cells, osteoblasts, vascular endothelial cells and cardiomyocytes. Therefore, it is expected to be applied to the field of regenerative medicine that reconstructs cells, tissues, organs, etc. of a living body using nerve cells, osteoblasts, vascular endothelial cells, cardiomyocytes obtained by inducing differentiation of stem cells. There is.

  In the application research of stem cells to the field of regenerative medicine, undifferentiated stem cells are selected from a cell mixture containing stem cells and differentiated cells derived from the stem cells (neural cells, osteoblasts, vascular endothelial cells, cardiomyocytes, etc.). It may be desirable to separate / acquire. When differentiating cells coexist when inducing differentiation of undifferentiated cells, the efficiency of inducing differentiation of undifferentiated cells may decrease. Further, when the cells thus induced to differentiate are used for, for example, tissue formation, tissue formation failure may occur. Therefore, it is important to remove the differentiated cells from the cell mixture and to separate and obtain the undifferentiated cells.

  On the other hand, there is also a report that it is possible to form a tissue by using a cell mixture containing undifferentiated cells and differentiated cells (see Non-Patent Document 1). In this case, it is important that the cell density of undifferentiated cells in the cell mixture is adjusted to an appropriate range.

  Conventionally, as a method for separating the target cells from a cell mixture containing predetermined target cells, focusing on the size difference between the target cells and other cells contained in the cell mixture, a filter capable of capturing the target cells is provided. A method of separating using an equipped separation device (see Patent Document 1), a method of adding an identification label or the like to the separation target cells, and separating the target cells by detecting the identification label or the like (Patent Documents 2 and 3) , Non-Patent Document 2) and the like are known.

JP, 2003-274923, A JP, 2005-27579, A JP, 2011-87475, A

Takabe et al., "Vascularized and Complex Organ Buds from Diverse Tissues via Mesenchymal Cell-Driven Condensation", Cell Stem Cell, Vol. 16, pp. 556-565, 2015 Kuanyin K. Lin et al., "Detection of Hematopoietic Stem Cells by Flow Cytometry", METHODS IN CELLS BIOLOGY, Vol. 103, 2011

  The separation method described in Patent Document 1 uses a filter capable of capturing target cells according to the size of the target cells contained in the cell mixture and the size of other cells contained in the cell mixture. This is a method of separating cells. Therefore, when the size of the predetermined target cell contained in the cell mixture is different from the size of other cells contained in the cell mixture, the target cell can be separated. However, since the sizes of the undifferentiated cells and the differentiated cells are substantially the same, the undifferentiated cells cannot be completely separated and obtained from the cell mixture even when the above-mentioned separation method is used. For example, it is difficult to improve the cell density of undifferentiated cells).

  Further, in order to separate undifferentiated cells in a cell mixture using the separation methods described in Patent Document 2, Patent Document 3 and Non-Patent Document 2, an identification label or the like is attached to the undifferentiated cells in the cell mixture. It is necessary and is a method capable of separating undifferentiated cells by detecting the identification label. However, in order to perform tissue formation using the differentiated cells obtained by inducing differentiation of the separated undifferentiated cells, it may be necessary to remove the identification label etc. from the undifferentiated cells after separation. There is a risk that the undifferentiated cells may be damaged by applying and removing the label or the like to the undifferentiated cells. There is also a problem that the work of applying and removing the identification label and the like is complicated.

  In view of such a problem, the present invention provides a method and a method for separating undifferentiated cells selectively from a cell mixture containing undifferentiated cells and differentiated cells, easily and minimally invasively. An object is to provide a substrate for separating undifferentiated cells used.

In order to solve the above problems, the present invention is a method for separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells, wherein a plurality of pillar-shaped patterns are arranged with regularity. Using a nano-relief structure consisting of, comprising the step of contacting the cell mixture to the plurality of pillar-shaped pattern, the undifferentiated cells are mouse-derived mesenchymal stem cells, the differentiated cells, a osteoblasts differentiated from differentiated cells, and the gap G and the size D of the pillar-shaped pattern, easily the undifferentiated cells adhered to the pillar-like pattern, wherein the differentiated cells are adhered to the pillar-like pattern A method for separating undifferentiated cells , which has a difficult relationship and has any of the following relationships (1) to (7) .
100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D <162.5 nm or 187.5 nm ≦ D ≦ 250 nm (3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <112.5 nm or 237.5 nm ≦ D ≦ 250 nm (6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D <137.5 nm or 162.5 nm ≦ D <237.5 nm (7)

  According to the earnest research of the present inventors, a plurality of pillar-shaped patterns are regularly arranged on the nano-relief structure, and thus a region where the plurality of pillar-shaped patterns of the nano-relief structure is arranged is arranged. , It was found that the undifferentiated cells and the differentiated cells differ in adhesiveness to the pillar pattern. Therefore, according to the above invention (Invention 1), undifferentiated cells can be selectively, easily and minimally invasively separated from a cell mixture containing undifferentiated cells and differentiated cells.

  In the present invention, the interval of the pillar-shaped pattern means "the dimension of the pillar-shaped pattern from the length of a straight line connecting the center of the one pillar-shaped pattern in a plan view and the center of another pillar-shaped pattern adjacent to the pillar-shaped pattern in a plan view. The length of the pillar-shaped pattern means the length of one side of the square if the pillar-shaped pattern is substantially square in plan view, and the pillar-shaped pattern is substantially circular in plan view. For example, "the diameter of the circle". Further, in the present invention, “separation” means that any one cell (eg, undifferentiated cell) is completely separated and obtained from a cell mixture containing at least two types of cells (eg, undifferentiated cell and differentiated cell). Not only that, but also means increasing the cell density of any one of the cells (for example, undifferentiated cells) in the cell mixture.

The present invention is a method of separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells, using a nano-relief structure in which a plurality of pillar-shaped patterns are arranged with regularity. The step of contacting the cell mixture with the plurality of pillar-shaped patterns, wherein the undifferentiated cell is a human-derived mesenchymal stem cell, and the differentiated cell is an osteoblast differentiated from the undifferentiated cell. , A preadipocyte or a chondrocyte, wherein the gap G and the dimension D of the pillar-shaped pattern are such that one of the undifferentiated cell and the differentiated cell easily adheres to the pillar-shaped pattern, and the other is the Provided is a method for separating undifferentiated cells, which has a relationship that is difficult to adhere to a pillar-shaped pattern and has any one of the following relationships (1) to (4).
112.5 nm ≦ G <137.5 nm; 187.5 nm ≦ D <212.5 nm (1)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <137.5 nm (2)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D <212.5 nm (3)
237.5 nm ≦ G ≦ 250 nm; 237.5 nm ≦ D ≦ 250 nm (4)

Oite above onset bright, the uneven structure, said to plurality of the pillar-shaped patterns may be arranged on the grid points of a regular triangle lattice, the plurality of pillar-shaped patterns each lattice point of the square lattice but it may also have been arranged in the above.

The present invention also provides a substrate for separating undifferentiated cells used for separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells, the first surface facing the first surface and the first surface. Mesenchyme comprising a base material having a second surface, and a plurality of pillar-shaped patterns formed on the first surface of the base material with regularity, wherein the undifferentiated cell is derived from a mouse. a system stem cells, wherein the differentiated cells, the a osteoblast cells differentiated from undifferentiated cells, and the gap G and the size D of the pillar-shaped pattern, easily the undifferentiated cells adhered to the pillar-like pattern The present invention provides a substrate for separating undifferentiated cells, characterized in that the differentiated cells have a relationship in which they are unlikely to adhere to the pillar-shaped pattern and have one of the following relationships (1) to (7) .
100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D <162.5 nm or 187.5 nm ≦ D ≦ 250 nm (3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <112.5 nm or 237.5 nm ≦ D ≦ 250 nm (6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D <137.5 nm or 162.5 nm ≦ D <237.5 nm (7)

The present invention relates to a substrate for separating undifferentiated cells used for separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells, the first surface and a second surface facing the first surface. A mesenchymal stem cell derived from a human, wherein the undifferentiated cell comprises a substrate having a surface and a plurality of pillar-like patterns formed on the first surface of the substrate with regularity And the differentiated cells are osteoblasts, preadipocytes or chondrocytes differentiated from the undifferentiated cells, and the interval G and the dimension D of the pillar-shaped pattern are the undifferentiated cells and the differentiated cells. One of them is easily bonded to the pillar-shaped pattern and the other is hard to be bonded to the pillar-shaped pattern, and has a relationship of any of the following (1) to (4). A substrate for separating undifferentiated cells is provided.
112.5 nm ≦ G <137.5 nm; 187.5 nm ≦ D <212.5 nm (1)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <137.5 nm (2)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D <212.5 nm (3)
237.5 nm ≦ G ≦ 250 nm; 237.5 nm ≦ D ≦ 250 nm (4)

Oite above onset bright, the plurality of pillar-shaped pattern may be arranged on the grid points of a regular triangle lattice, but it may also have been arranged on each lattice point of the square lattice.

  According to the present invention, a method for selectively, easily and minimally invasively separating differentiated cells from a cell mixture containing undifferentiated cells and differentiated cells, and for undifferentiated cell separation used in the separation method A substrate can be provided.

FIG. 1 is a cut end view showing a schematic configuration of a substrate for separating undifferentiated cells according to an embodiment of the present invention. FIG. 2 is a plan view schematically showing an aspect of an arrangement configuration of a plurality of pillar-shaped patterns on the undifferentiated cell separation substrate according to the embodiment of the present invention. FIG. 3 is a plan view schematically showing another aspect of the arrangement configuration of a plurality of pillar-shaped patterns of the undifferentiated cell separation substrate according to the embodiment of the present invention. FIG. 4 is a process flow diagram showing, in a cut end view, a manufacturing process of the undifferentiated cell separation substrate according to the embodiment of the present invention. FIG. 5: is a top view which shows each area | region in which the pillar-shaped pattern of the test | inspection undifferentiated cell separation substrate used in Test Examples 1 and 2 was formed. FIG. 6 is an image of mouse-derived mesenchymal stem cells in the vicinity of the area PA ₃₄ imaged in the evaluation test of Test Example 1 and a micrograph showing the locus of migration of mouse-derived mesenchymal stem cells. FIG. 7 is an image of a mouse-derived osteoblast in the vicinity of the area PA ₃₄ imaged in the evaluation test of Test Example 2 and a micrograph showing a locus of movement of the mouse-derived osteoblast. FIG. 8 is an image of human-derived mesenchymal stem cells in the vicinity of the area PA ₃₄ imaged in the evaluation test of Test Example 3 and a micrograph showing the locus of migration of human-derived mesenchymal stem cells. FIG. 9 is an image of human-derived osteoblasts in the vicinity of the area PA ₃₄ imaged in the evaluation test of Test Example 4 and a micrograph showing the locus of movement of human-derived osteoblasts. FIG. 10 is an image of a human-derived preadipocyte in the vicinity of the area PA ₂₄ imaged in the evaluation test of Test Example 5 and a micrograph showing a movement trajectory of the human-derived preadipocyte. FIG. 11 is an image of human-derived chondrocytes in the vicinity of the area PA ₂₄ imaged in the evaluation test of Test Example 6 and a micrograph showing the movement trajectory of human-derived chondrocytes.

Embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a cut end view showing a schematic configuration of an undifferentiated cell separation substrate according to the present embodiment, and FIG. 2 is an arrangement configuration of a plurality of pillar-shaped patterns of the undifferentiated cell separation substrate according to the present embodiment. FIG. 3 is a plan view schematically showing one aspect, and FIG. 3 is a plan view schematically showing another aspect of the arrangement configuration of the plurality of pillar-shaped patterns of the undifferentiated cell separation substrate according to the present embodiment. is there.

  As shown in FIG. 1, the undifferentiated cell separation substrate 1 according to the present embodiment is composed of a nano-relief structure having a base 2 and a pillar-shaped pattern 3 formed on a main surface 21 of the base 2. , Is used for separating undifferentiated cells from a cell mixture containing differentiated cells and undifferentiated cells.

  The undifferentiated cells and differentiated cells contained in the cell mixture are not particularly limited as long as they have adhesive properties, and examples of the undifferentiated cells include somatic stem cells, iPS cells, ES cells and the like. Examples of the differentiated cells include nerve cells, osteoblasts, vascular endothelial cells and cardiomyocytes that are induced to differentiate from the above undifferentiated cells. Among these, in particular, when the undifferentiated cell is a mesenchymal stem cell derived from a mouse and the differentiated cell is an osteoblast differentiated from the undifferentiated cell, when the undifferentiated cell is derived from a human If the differentiated cells are osteoblasts, preadipocytes or chondrocytes that have been induced to differentiate from the undifferentiated cells, by using the undifferentiated cell separation substrate 1 according to the present embodiment, Undifferentiated cells can be separated more efficiently.

  As the material forming the base 2, glass materials such as quartz glass, synthetic quartz glass, soda glass, fluorite, calcium fluoride, magnesium fluoride, acrylic glass, borosilicate glass; polycarbonate, polypropylene, polyethylene, polystyrene, etc. A transparent material such as a resin material such as polyolefin can be used. In particular, it is preferable to use the transparent material as the material forming the base 2, because the undifferentiated cells can be separated while observing the adhesion state of the undifferentiated cells to the pillar-shaped pattern 3 with a microscope or the like.

  In the present embodiment, “transparent” means that when light having a wavelength of 190 to 800 nm is irradiated from one side of the object (base 2 in the present embodiment), the light reaches the side opposite to the irradiated side. Means that. If a suitable standard is represented by the transmittance, it is 20% or more, preferably 50% or more, particularly preferably 80% or more.

  The shape of the base 2 is not particularly limited, and examples thereof include a substantially rectangular shape in plan view and a substantially circular shape in plan view. Further, the size of the base portion 2 in plan view and the thickness of the base portion 2 are not particularly limited, and may be appropriately set according to the application of the undifferentiated cell separation substrate 1. For example, in order to separate the undifferentiated cells from the cell mixture, when the undifferentiated cell separation substrate 1 is housed in a dish or the like and the undifferentiated cell separation substrate 1 and the cell mixture are contacted in the dish, The size of the cell separation substrate 1 in plan view can be set to a size that can be accommodated in the dish. Similarly, the thickness of the undifferentiated cell separation substrate 1 is also set to such a thickness that the pillar-shaped pattern 3 and the cell mixture can contact each other when the undifferentiated cell separation substrate 1 is placed in the dish. obtain.

  The pillar-shaped pattern 3 has a substantially circular shape or a substantially rectangular shape in a plan view, and is arranged as a structure integrated with the base 2 in a predetermined area PA on the main surface 21 of the base 2 with a predetermined regularity. Is formed.

  Specifically, as shown in FIG. 2, pillar-shaped patterns 3 each having a substantially rectangular shape in plan view may be arranged on each lattice point of the equilateral triangular lattice Gr, or as shown in FIG. It may be arranged on each grid point of the square grid Gr.

  In the present embodiment, the predetermined area PA where the pillar-shaped pattern 3 is formed is such that the size of the area NPA where the pillar-shaped pattern 3 is not formed on the main surface 21 of the base 2 is as small as possible. It is preferably set on the main surface 21. As will be described later, the pillar-shaped pattern 3 according to the present embodiment has a nano-level spacing G and a dimension D, and in particular, because they have a predetermined relationship, the undifferentiated cells and the differentiated cells in the cell mixture are separated. Due to the difference in adhesiveness, one of the undifferentiated cells and the differentiated cells is relatively easy to adhere to the pillar-shaped pattern 3, and the other is relatively difficult to adhere to the pillar-shaped pattern 3. On the other hand, both undifferentiated cells and differentiated cells adhere to the region NPA where the pillar-shaped pattern 3 is not formed. That is, if the area NPA where the pillar-shaped pattern 3 is not formed is large, there is a possibility that the undifferentiated cells cannot be efficiently separated. Therefore, the undifferentiated cells can be efficiently separated by making the area NPA where the pillar-shaped pattern 3 is not formed as small as possible.

  In the present embodiment, both the spacing G and the dimension D of the pillar-shaped pattern 3 are preferably in the range of 50 to 500 nm, which is the same size as the cell adhesion complex, and more preferably in the range of 100 to 250 nm. preferable.

The combination of undifferentiated cells and differentiated cells contained in the cell mixture, mouse-derived mesenchymal stem cells and osteoblasts, human-derived mesenchymal stem cells and osteoblasts, human-derived mesenchymal stem cells and preadipocytes, or In the case of human-derived mesenchymal stem cells and chondrocytes, preferably the interval G and the dimension D have any one of the following relationships (1-1) to (1-7).
100 nm ≦ G <112.5 nm; 100 nm ≦ D ≦ 250 nm (1-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D ≦ 250 nm (1-2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D ≦ 250 nm (1-3)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D ≦ 250 nm (1-4)
187.5 nm ≦ G <212.5 nm; 100 nm ≦ D <187.5 nm or 212.5 nm ≦ D ≦ 250 nm (1-5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <137.5 nm or 212.5 nm ≦ D ≦ 250 nm (1-6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D ≦ 250 nm (1-7)

More preferably, the spacing G and the dimension D have any one of the following relationships (1-1 ′) to (1-7 ′).
100 nm ≦ G ≦ 110 nm; 100 nm ≦ D ≦ 250 nm (1-1 ′)
115 nm ≤ G ≤ 135 nm; 115 nm ≤ D ≤ 250 nm (1-2 ')
140 nm ≦ G ≦ 160 nm; 100 nm ≦ D ≦ 250 nm (1-3 ′)
165 nm ≦ G ≦ 185 nm; 115 nm ≦ D ≦ 250 nm (1-4 ′)
190 nm ≦ G ≦ 210 nm; 100 nm ≦ D ≦ 185 nm or 215 nm ≦ D ≦ 250 nm (1-5 ′)
215 nm ≦ G <235 nm; 100 nm ≦ D ≦ 135 nm or 215 nm ≦ D ≦ 250 nm (1-6 ′)
240 nm ≦ G ≦ 250 nm; 100 nm ≦ D ≦ 250 nm (1-7 ′)

More preferably, the distance G and the dimension D have any one of the following relationships (1-1 ″) to (1-7 ″).
100 nm ≦ G ≦ 105 nm; 100 nm ≦ D ≦ 250 nm (1-1 ″)
120 nm ≦ G ≦ 130 nm; 120 nm ≦ D ≦ 250 nm (1-2 ″)
145 nm ≦ G ≦ 155 nm; 100 nm ≦ D ≦ 250 nm (1-3 ″)
170 nm ≦ G ≦ 180 nm; 120 nm ≦ D ≦ 250 nm (1-4 ″)
195 nm ≦ G ≦ 205 nm; 100 nm ≦ D ≦ 180 nm or 220 nm ≦ D ≦ 250 nm (1-5 ″)
220 nm ≦ G ≦ 230 nm; 100 nm ≦ D ≦ 130 nm or 220 nm ≦ D ≦ 250 nm (1-6 ″)
245 nm ≦ G ≦ 250 nm; 100 nm ≦ D ≦ 250 nm (1-7 ″)

A combination of undifferentiated cells and differentiated cells contained in the cell mixture, mouse-derived mesenchymal stem cells and osteoblasts, and human-derived mesenchymal stem cells and osteoblasts, human-derived mesenchymal stem cells and adipose precursor cells, Alternatively, in the case of human-derived mesenchymal stem cells and chondrocytes, it is preferable that the interval G and the dimension D have any one of the following relationships (2-1) to (2-7).
100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (2-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (2-2)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <162.5 nm or 187.5 nm ≦ D <237.5 nm (2-3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (2-4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (2-5)
212.5 nm ≦ G <237.5 nm; 237.5 nm ≦ D ≦ 250 nm (2-6)
237.5 nm ≦ G ≦ 250 nm; 162.5 nm ≦ D <237.5 nm (2-7)

More preferably, the spacing G and the dimension D have any one of the following relationships (2-1 ′) to (2-7 ′).
100 nm ≦ G ≦ 110 nm; 115 nm ≦ D ≦ 250 nm (2-1 ′)
115 nm ≦ G ≦ 135 nm; 115 nm ≦ D ≦ 135 nm (2-2 ′)
140 nm ≦ G ≦ 160 nm; 115 nm ≦ D ≦ 160 nm or 190 nm ≦ D <235 nm (2-3 ′)
165 nm ≤ G ≤ 185 nm; 165 nm ≤ D ≤ 210 nm or 240 nm ≤ D ≤ 250 nm (2-4 ')
190 nm ≦ G ≦ 210 nm; 215 nm ≦ D ≦ 235 nm (2-5 ′)
215 nm ≦ G ≦ 235 nm; 240 nm ≦ D ≦ 250 nm (2-6 ′)
240 nm ≦ G ≦ 250 nm; 165 nm ≦ D ≦ 235 nm (2-7 ′)

More preferably, the distance G and the dimension D have any one of the following relationships (2-1 ″) to (2-7 ″).
100 nm ≤ G ≤ 105 nm; 120 nm ≤ D ≤ 250 nm (2-1 ")
120 nm ≦ G ≦ 130 nm; 120 nm ≦ D ≦ 130 nm (2-2 ″)
145 nm ≦ G ≦ 155 nm; 120 nm ≦ D ≦ 155 nm or 195 nm ≦ D ≦ 230 nm (2-3 ″)
170 nm ≦ G ≦ 180 nm; 170 nm ≦ D ≦ 205 nm or 245 nm ≦ D ≦ 250 nm (2-4 ″)
195 nm ≦ G ≦ 205 nm; 220 nm ≦ D ≦ 230 nm (2-5 ″)
220 nm ≦ G ≦ 230 nm; 245 nm ≦ D ≦ 250 nm (2-6 ″)
245 nm ≦ G ≦ 250 nm; 170 nm ≦ D ≦ 230 nm (2-7 ″)

When the combination of undifferentiated cells and differentiated cells contained in the cell mixture is mouse-derived mesenchymal stem cells and osteoblasts, preferably the interval G and the dimension D are the following (3-1) to (3). -7) Has any one of the relationships.
100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (3-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (3-2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D <162.5 nm or 187.5 nm ≦ D ≦ 250 nm (3-3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (3-4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (3-5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <112.5 nm or 237.5 nm ≦ D ≦ 250 nm (3-6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D <137.5 nm or 162.5 nm ≦ D <237.5 nm (3-7)

More preferably, the distance G and the dimension D have any one of the following relationships (3-1 ') to (3-7').
100 nm ≤ G ≤ 110 nm; 115 nm ≤ D ≤ 250 nm (3-1 ')
115 nm ≦ G ≦ 135 nm; 115 nm ≦ D ≦ 135 nm (3-2 ′)
140 nm≤G≤160 nm; 100 nm≤D≤160 nm or 190 nm≤D≤250 nm (3-3 ')
165 nm ≤ G ≤ 185 nm; 165 nm ≤ D ≤ 210 nm or 240 nm ≤ D ≤ 250 nm (3-4 ')
190 nm ≦ G ≦ 210 nm; 215 nm ≦ D ≦ 235 nm (3-5 ′)
215 nm ≦ G ≦ 235 nm; 100 nm ≦ D ≦ 110 nm or 240 nm ≦ D ≦ 250 nm (3-6 ′)
240 nm ≤ G ≤ 250 nm; 100 nm ≤ D ≤ 135 nm or 165 nm ≤ D ≤ 235 nm (3-7 ')

More preferably, the distance G and the dimension D have any one of the following relationships (3-1 ″) to (3-7 ″).
100 nm ≤ G ≤ 105 nm; 120 nm ≤ D ≤ 250 nm (3-1 ")
120 nm ≦ G ≦ 130 nm; 120 nm ≦ D ≦ 130 nm (3-2 ″)
145 nm ≦ G ≦ 155 nm; 100 nm ≦ D ≦ 155 nm or 195 nm ≦ D ≦ 250 nm (3-3 ″)
170 nm ≤ G ≤ 180 nm; 170 nm ≤ D ≤ 205 nm or 245 nm ≤ D ≤ 250 nm (3-4 '')
195 nm ≦ G ≦ 205 nm; 220 nm ≦ D ≦ 230 nm (3-5 ″)
220 nm ≦ G ≦ 230 nm; 100 nm ≦ D ≦ 105 nm or 245 nm ≦ D ≦ 250 nm (3-6 ″)
245 nm ≦ G ≦ 250 nm; 100 nm ≦ D ≦ 130 nm or 170 nm ≦ D ≦ 230 nm (3-7 ″)

The combination of undifferentiated cells and differentiated cells contained in the cell mixture is human-derived mesenchymal stem cells and osteoblasts, human-derived mesenchymal stem cells and preadipocytes, or human-derived mesenchymal stem cells and chondrocytes In this case, preferably, the distance G and the dimension D have any one of the following relationships (4-1) to (4-7).
100 nm ≦ G <112.5 nm; 100 nm ≦ D ≦ 250 nm (4-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D ≦ 250 nm (4-2)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D ≦ 250 nm (4-3)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D ≦ 250 nm (4-4)
187.5 nm ≦ G <212.5 nm; 100 nm ≦ D <187.5 nm or 212.5 nm ≦ D ≦ 250 nm (4-5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <137.5 nm or 212.5 nm ≦ D ≦ 250 nm (4-6)
237.5 nm ≦ G ≦ 250 nm; 137.5 nm ≦ D ≦ 250 nm (4-7)

More preferably, the spacing G and the dimension D have any one of the following relationships (4-1 ′) to (4-7 ′).
100 nm ≦ G ≦ 110 nm; 100 nm ≦ D ≦ 250 nm (4-1 ′)
115 nm ≦ G ≦ 135 nm; 115 nm ≦ D ≦ 250 nm (4-2)
140 nm ≦ G ≦ 160 nm; 115 nm ≦ D ≦ 250 nm (4-3 ′)
165 nm ≦ G ≦ 185 nm; 115 nm ≦ D ≦ 250 nm (4-4 ′)
190 nm ≦ G ≦ 210 nm; 100 nm ≦ D ≦ 185 nm or 215 nm ≦ D ≦ 250 nm (4-5 ′)
215 nm ≦ G ≦ 235 nm; 100 nm ≦ D ≦ 135 nm or 215 nm ≦ D ≦ 250 nm (4-6 ′)
240 nm ≦ G ≦ 250 nm; 140 nm ≦ D ≦ 250 nm (4-7 ′)

More preferably, the distance G and the dimension D have any one of the following relationships (4-1 ″) to (4-7 ″).
100 nm ≤ G ≤ 105 nm; 100 nm ≤ D ≤ 250 nm (4-1 ")
120 nm ≦ G ≦ 130 nm; 120 nm ≦ D ≦ 250 nm (4-2)
145 nm ≦ G ≦ 155 nm; 120 nm ≦ D ≦ 250 nm (4-3 ″)
170 nm ≦ G ≦ 180 nm; 120 nm ≦ D ≦ 250 nm (4-4 ″)
195 nm ≦ G ≦ 205 nm; 100 nm ≦ D ≦ 180 nm or 220 nm ≦ D ≦ 250 nm (4-5 ″)
220 nm ≦ G ≦ 230 nm; 100 nm ≦ D ≦ 130 nm or 220 nm ≦ D ≦ 250 nm (4-6 ″)
245 nm ≦ G ≦ 250 nm; 145 nm ≦ D ≦ 250 nm (4-7 ″)

The combination of undifferentiated cells and differentiated cells contained in the cell mixture is human-derived mesenchymal stem cells and osteoblasts, human-derived mesenchymal stem cells and preadipocytes, and human-derived mesenchymal stem cells and chondrocytes In this case, preferably, the distance G and the dimension D have any one of the following relationships (5-1) to (5-4).
112.5 nm ≦ G <137.5 nm; 187.5 nm ≦ D <212.5 nm (5-1)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <137.5 nm (5-2)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D <212.5 nm (5-3)
237.5 nm ≦ G ≦ 250 nm; 237.5 nm ≦ D ≦ 250 nm (5-4)

More preferably, the distance G and the dimension D have any one of the following relationships (5-1 ') to (5-4').
115 nm ≤ G ≤ 135 nm; 190 nm ≤ D ≤ 210 nm (5-1 ')
140 nm ≤ G ≤ 160 nm; 115 nm ≤ D ≤ 135 nm (5-2 ')
165 nm ≦ G ≦ 185 nm; 115 nm ≦ D ≦ 210 nm (5-3 ′)
240 nm ≦ G ≦ 250 nm; 240 nm ≦ D ≦ 250 nm (5-4 ′)

More preferably, the distance G and the dimension D have any one of the following relationships (5-1 ″) to (5-4 ″).
120 nm ≦ G ≦ 130 nm; 195 nm ≦ D ≦ 205 nm (5-1-1)
145 nm ≦ G ≦ 155 nm; 120 nm ≦ D ≦ 130 nm (5-2-2)
170 nm ≦ G ≦ 180 nm; 120 nm ≦ D ≦ 205 nm (5-3 ″)
245 nm ≦ G ≦ 250 nm; 245 nm ≦ D ≦ 250 nm (5-4 ″)

  The interval G and the dimension D of the pillar-shaped pattern 3 are in any one of the above relationships (1-1) to (1-7), and preferably in any one of the above (1-1 ′) to (1-7 ′). More preferably, by providing any one of the relationships (1-1 ″) to (1-7 ″), one of the cells (undifferentiated cell or differentiated cell) has a pillar-shaped pattern 3. It is relatively easy to adhere, and the other cell (differentiated cell or undifferentiated cell) becomes relatively difficult to adhere to the pillar-shaped pattern 3.

  The distance G and the dimension D of the pillar-shaped pattern 3 are in any of the above relationships (2-1) to (2-7), and preferably in any of the above (2-1 ') to (2-7'). More preferably, the undifferentiated cells and the differentiated cells contained in the cell mixture are derived from a mouse by providing any one of the relationships (2-1 '') to (2-7 ''). Whether or not it is of human origin, one of the cells (undifferentiated cell or differentiated cell) relatively easily adheres to the pillar-shaped pattern 3, and the other cell (differentiated cell or undifferentiated cell) Is relatively difficult to adhere to the pillar-shaped pattern 3 and the versatility is enhanced.

  When the undifferentiated cells and the differentiated cells contained in the cell mixture are derived from a mouse, the interval G and the dimension D of the pillar-shaped pattern 3 are in any one of the relationships (3-1) to (3-7), It preferably has any one of the above relationships (3-1 ′) to (3-7 ′), and more preferably one of the above (3-1 ″) to (3-7 ″). Thus, the undifferentiated cells are likely to adhere to the pillar-shaped pattern 3 relatively easily, and the differentiated cells are relatively difficult to adhere to the pillar-shaped pattern 3.

  If the undifferentiated cells contained in the cell mixture are human-derived mesenchymal stem cells, and the differentiated cells are osteoblasts, preadipocytes or chondrocytes that have been differentiated therefrom, the spacing G and dimension of the pillar-shaped pattern 3 D is any one of the above relationships (4-1) to (4-7), preferably one of the above (4-1 ′) to (4-7 ′), and more preferably the above (4-1). By having any one of the relations ″) to (4-7 ″), one of the cells (undifferentiated cell or differentiated cell) easily adheres to the pillar-shaped pattern 3 relatively, and the other It becomes relatively difficult for cells (differentiated cells or undifferentiated cells) to adhere to the pillar-shaped pattern 3.

  In particular, the interval G and the dimension D of the pillar-shaped pattern 3 are in any one of the above relationships (5-1) to (5-4), and preferably in any one of the above (5-1 ') to (5-4'). And more preferably any one of the above (5-1 ″) to (5-4 ″), the undifferentiated cells contained in the cell mixture are human-derived mesenchymal stem cells. Therefore, whether the differentiated cell is an osteoblast, a preadipocyte or a chondrocyte, the differentiation of which is induced, one of the cells (undifferentiated cell or differentiated cell) relatively adheres to the pillar-shaped pattern 3. And the other cell (differentiated cell or undifferentiated cell) becomes relatively difficult to adhere to the pillar-shaped pattern 3 and the versatility becomes high.

  In this way, by utilizing the difference in the adhesiveness of the undifferentiated cells and the differentiated cells contained in the cell mixture with respect to the pillar-shaped pattern 3, the pillar-shaped pattern 3 is simply contacted with the cell mixture. The cells that are relatively difficult to adhere to can be suspended in the culture solution. When the target cell is a cell that is relatively easy to adhere to the pillar-shaped pattern 3, the cell that remains on the pillar-shaped pattern 3 can be easily separated. On the other hand, when the target cell is a cell that is relatively difficult to adhere to the pillar-shaped pattern 3, the cell can be separated by collecting the culture solution.

  In the present embodiment, the interval G between the pillar-shaped patterns 3 means “a straight line connecting the center C of one pillar-shaped pattern 3 in plan view and the center C of another closest pillar-shaped pattern 3 in plan view”. The length D is the length obtained by subtracting the dimension D of the pillar-shaped pattern from the length L, and the dimension D of the pillar-shaped pattern 3 is "the length of one side of the square if the pillar-shaped pattern 3 has a substantially square shape in plan view. If the pillar-shaped pattern 3 is substantially circular in a plan view, it means "the diameter of the circle".

  The height (aspect ratio) of the pillar-shaped pattern 3 is not particularly limited, and can be appropriately set according to the dimension D of the pillar-shaped pattern 3. Usually, the height of the pillar-shaped pattern 3 can be set so that the aspect ratio of the pillar-shaped pattern 3 is about 0.5 to 5.

  In the present embodiment, the predetermined area PA in which the pillar-shaped pattern 3 is formed is set on substantially the entire main surface 21 of the base portion 2, but the present invention is not limited to this mode. For example, the main surface 21 of the base portion 2 may be divided into first to Nth regions (N is an integer of 2 or more), and the pillar-shaped pattern 3 may be formed in each region. In this case, the gap G and the dimension D of the pillar-shaped pattern 3 may be the same or different in each region.

  According to the undifferentiated cell separation substrate 1 having the above-described configuration, when the cell mixture containing the differentiated cells and the undifferentiated cells and the pillar-shaped pattern 3 are brought into contact with each other, as is clear from Examples described later, One of the undifferentiated cell and the differentiated cell is relatively easy to adhere to the pillar-shaped pattern 3, and the other is relatively difficult to adhere to the pillar-shaped pattern 3. Therefore, undifferentiated cells can be selectively and easily separated from the cell mixture. For example, the undifferentiated cells remaining on the pillar-shaped pattern 3 are removed by using the undifferentiated cell separation substrate 1 having the pillar-shaped pattern 3 to which the undifferentiated cells relatively easily adhere in the relationship of the interval G and the dimension D. It can be selectively and easily separated. On the other hand, by using the undifferentiated cell separation substrate 1 having the pillar-shaped pattern 3 in which the undifferentiated cells are relatively difficult to adhere in the relationship of the interval G and the dimension D, the undifferentiated cells floating in the culture solution are selectively selected. And can be easily separated. Further, unlike the conventional method for separating the undifferentiated cells by detecting the identification label or the like attached to the undifferentiated cells, it is not necessary to perform the operation of attaching / removing the identification labels or the like, and therefore, according to the present embodiment. According to the undifferentiated cell separation substrate 1, the undifferentiated cells can be separated from the cell mixture with minimal invasion.

  In the field of regenerative medicine in which mesenchymal stem cells that are undifferentiated cells are administered to patients, it may be desired to selectively culture only mesenchymal stem cells. In the process of culturing the mesenchymal stem cells, there is a possibility that some of them will be differentiated into osteoblasts and the like, and unnecessary cells (osteoblasts that are differentiated cells and the like) will be mixed. However, if the undifferentiated cell separation substrate 1 according to the present embodiment is used, unnecessary cells mixed in such a manner (such as osteoblasts that are differentiated cells) are removed, and the target mesenchymal stem cells are selected. Since it can be easily and easily separated, it becomes possible to culture mesenchymal stem cells at a high cell density.

  In the field of regenerative medicine in which chondrocytes obtained by differentiating mesenchymal stem cells that are undifferentiated cells are transplanted to a patient, it is desirable to selectively culture and proliferate mesenchymal stem cells in order to differentiate into chondrocytes. There are cases. In the process of culturing the mesenchymal stem cells, some of the mesenchymal stem cells may be differentiated into osteoblasts. However, if the undifferentiated cell separation substrate 1 according to the present embodiment is used, unintentionally differentiated osteoblasts can be removed. Therefore, mesenchymal stem cells are cultured at a high cell density. , Can be propagated.

  The undifferentiated cell separation substrate 1 having the above-described configuration can be manufactured, for example, as follows. FIG. 4 is a process flow diagram showing, in a cut end view, a manufacturing process of the undifferentiated cell separation substrate 1 according to the present embodiment.

  As shown in FIG. 4A, first, a substrate 20 having a hard mask layer 30 is prepared, a resist film 40 is formed on the hard mask layer 30 of the substrate 20, and a resist is formed using an electron beam drawing apparatus or the like. A pattern latent image is formed on the film 40.

  As the substrate 20, a constituent material of the undifferentiated cell separation substrate 1 according to the present embodiment (for example, quartz glass, synthetic quartz glass, soda glass, fluorite, calcium fluoride, magnesium fluoride, acrylic glass, borosilicate glass). A glass material such as; a transparent material such as a resin material such as polycarbonate, polypropylene, polyethylene, polystyrene, or polyolefin), and the like.

  As a material forming the hard mask layer 30, for example, metal chromium, chromium oxide, chromium nitride, chromium oxynitride, or the like can be used. The hard mask layer 30 is a layer for forming a hard mask pattern 31 used for etching the substrate 20 in a step (see FIG. 4D) described later. Therefore, it is made of a material in consideration of the etching selection ratio with the material of the substrate 20. For example, when the substrate 20 is made of quartz glass, the hard mask layer 30 is preferably made of metallic chrome or the like.

  The resist material forming the resist film 40 is not particularly limited, and a conventionally known energy ray sensitive resist material (eg, electron beam sensitive resist material, ultraviolet ray sensitive resist material, etc.) can be used. it can.

  The pattern latent image formed on the resist film 40 is formed at substantially the same intervals and dimensions as the intervals G and the dimensions D of the pillar-shaped patterns 3 on the undifferentiated cell separation substrate 1.

  Next, as shown in FIG. 4B, the resist film 40 is developed to form a resist pattern 41. As shown in FIG. 4C, the hard mask layer 30 is formed using the resist pattern 41 as a mask. The hard mask pattern 31 is formed by etching.

  Thereafter, as shown in FIG. 4D, the substrate 20 is etched using the hard mask pattern 31 as a mask to form the pillar-shaped pattern 3 in a predetermined area PA of the main surface 21 of the substrate 20, and finally the hard mask. By peeling off the pattern 31, as shown in FIG. 4E, the undifferentiated cell separation substrate 1 having the base 2 and the pillar-shaped pattern 3 formed on the main surface 21 of the base 2 is manufactured. You can

  In the substrate 1 for separating undifferentiated cells having the above-described configuration, when the pillar-shaped pattern 3 is brought into contact with a cell mixture containing undifferentiated cells and differentiated cells, one of the undifferentiated cells and the differentiated cells becomes pillar-shaped. It is easy to adhere to the pattern 3, and the other is difficult to adhere to the pillar-shaped pattern 3. By utilizing this difference in adhesiveness, the undifferentiated cell separation substrate 1 according to the present embodiment has the effect of separating undifferentiated cells selectively, easily, and with minimal invasiveness. It can be suitably used for a method for separating differentiated cells.

  In such an undifferentiated cell separation method, as a method of bringing the pillar-shaped pattern 3 of the undifferentiated cell separation substrate 1 into contact with the cell mixture, for example, the undifferentiated cell separation substrate 1 is placed in a dish, and the dish Examples include a method in which the pillar-shaped pattern 3 is brought into contact with the cell mixture by seeding the cell mixture therein.

  In the method of bringing the pillar-shaped pattern 3 of the undifferentiated cell separation substrate 1 into contact with the cell mixture in the dish, the time of bringing the pillar-shaped pattern 3 into contact with the cell mixture selectively separates the undifferentiated cells. There is no particular limitation as long as it is sufficient time.

  As described above, according to the present embodiment, by bringing the pillar-shaped pattern 3 of the undifferentiated cell separation substrate 1 into contact with the cell mixture, one of the undifferentiated cells and the differentiated cells has the pillar-shaped pattern. 3 is easy to adhere to, and the other is difficult to adhere to the pillar-shaped pattern 3. Therefore, the undifferentiated cells can be selectively, easily and minimally invasively separated from the cell mixture containing the undifferentiated cells and the differentiated cells.

  The embodiments described above are described to facilitate the understanding of the present invention, and are not described to limit the present invention. Therefore, each element disclosed in the above-described embodiment is intended to include all design changes and equivalents within the technical scope of the present invention.

  In the above-described embodiment, the undifferentiated cell separation substrate 1 having the pillar-shaped pattern 3 formed as an integral concavo-convex structure on the main surface 21 of the base 2 has been described as an example, but the present invention is as described above. It is not limited to the embodiment. For example, the undifferentiated cell separation substrate 1 includes an uneven structure having a pillar-shaped pattern 3 formed as a member separate from the base 2 on the main surface 21 of the base 2 (for example, a sheet-shaped structure having the pillar-shaped pattern 3). (Members and the like) may be attached.

  In the above embodiment, the pillar-shaped pattern 3 is formed by etching, and then the hard mask pattern 31 is peeled off to manufacture the undifferentiated cell separation substrate 1. However, the present invention is not limited to this mode. Not something. For example, the base 2 on which the pillar-shaped pattern 3 is formed may be thinned without changing the size and height of the pillar-shaped pattern 3. Alternatively, the undifferentiated cell separation substrate 1 may be manufactured by forming pillar-shaped patterns 3 in a plurality of regions on the base 2 and cutting (separating) each region.

  Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited to the following Examples and the like.

[Production Example 1 of test undifferentiated cell separation substrate]
A quartz glass substrate (152 mm × 152 mm, thickness: 6.35 mm) as a substrate 20 having a hard mask layer 30 made of Cr and having a thickness of 6 nm provided on the main surface 21 is prepared, and an electron beam sensitive resist (product (Name: SEBP-90012, manufactured by Shin-Etsu Chemical Co., Ltd.) was applied on the hard mask layer 30 to form a resist film 40. A pillar-shaped resist pattern 41 was formed on the resist film 40 by using an electron beam drawing apparatus. The pillar-shaped resist pattern 41 is formed so as to be located on each lattice point of the equilateral triangular lattice Gr, as shown in FIG. Further, ₄₉ divided areas PA _{01 to} PA ₄₉ (size of each area PA _{01 to} PA ₄₉ : 450 μm × 450 μm) are set in a 14 mmφ area PA on the main surface 21 of the quartz glass substrate 20, and each area is set. Pillar-shaped resist patterns 41 having different intervals G and different dimensions D were formed (see FIG. 5).

Next, the hard mask layer 30 was dry-etched (etching gas: Cl ₂ + O ₂ ) using the resist pattern 41 as a mask, and the remaining resist pattern 41 was removed to form a hard mask pattern 31.

The quartz glass substrate 20 is etched by using the hard mask pattern 31 formed as described above as a mask, and the pillar-shaped pattern 3 having a predetermined gap G and dimension D is formed in each region of the main surface 21 of the quartz glass substrate 20. Formed. Finally, the hard mask pattern 31 was peeled off to manufacture a test undifferentiated cell separation substrate (see FIG. 5). The spacing G and the dimension D of the pillar-shaped pattern 3 formed in each region PA _{01 to} PA ₄₉ of the test undifferentiated cell separation substrate were measured by using a scanning electron microscope (manufactured by Vistec, product name: LWM9000). The measurement results were as shown in Table 1 below.

[Test Example 1]
A perforated dish (35 mmφ) having holes of 14 mmφ formed on the bottom surface was prepared, and each test area PA _{01 to} PA ₄₉ in which the pillar-shaped pattern 3 was formed was exposed from the holes of the bottom surface. The substrate for separating differentiated cells was attached to the outside of the bottom surface of the perforated dish.

  Next, mouse-derived mesenchymal stem cells (RIKEN BRC, undifferentiated cells) were seeded in the perforated dish, and the undifferentiated cells were brought into contact with the pillar-shaped pattern 3 of the undifferentiated cell separation substrate for testing. It was POWERED BY10 (manufactured by Glyco Technica) was used as a culture solution.

An evaluation test was performed to evaluate the relationship between the spacing G and the dimension D of the pillar-shaped pattern 3 and the state of cell adhesion, based on the images obtained by imaging the vicinity of each area PA _{01 to} PA ₄₉ 36 hours after cell seeding.

In the evaluation test, the cell number ratio P _MU in each area PA _{01 to} PA ₄₉ (P _MU = N _in / (N _in + N _out ), N _in is “in each area PA _{01 to} PA ₄₉ (pillar shape. a pattern 3 above) and the number of cells present in the boundary of each region PA ₀₁ ~PA ₄₉ ", N _out is a region of the same area as the" respective regions PA ₀₁ ~PA _49, each region PA ₀₁ ~PA ₄₉ The number of cells existing in the area surrounding the outer circumference of the cell is calculated.), And the cell number ratio P _MU was evaluated as an index of the affinity for the pillar-shaped pattern 3.

It can be evaluated that the larger the value of the cell number ratio P _{MU is,} the easier it is to adhere to the pillar-shaped pattern 3 (higher affinity). Low).

The evaluation results are shown in Table 2. Further, FIG. 6 shows an image of the mesenchymal stem cells in the vicinity of the area PA ₃₄ imaged in the above evaluation test and a movement trajectory of the mesenchymal stem cells. The arrow lines shown in FIG. 6 represent the loci of movement of mesenchymal stem cells.

[Test Example 2]
The cell number ratio P _MD in each region PA _{01 to} PA ₄₉ was the same as in Test Example 1 except that osteoblasts (differentiated cells) in which mouse-derived mesenchymal stem cells were induced to differentiate were seeded in the dish. _{P MD = N in / (N} in + N out), N in the "number of cells present in the boundary of each region PA ₀₁ ~PA within ₄₉ (pillar-shaped pattern 3 above) and each area PA ₀₁ ~PA _49" , N _out is by calculating the.) representing "an area of the same area as each area PA ₀₁ ~PA _49, the number of cells present in the area surrounding the periphery of each region PA ₀₁ ~PA _49" An evaluation test was performed to evaluate the relationship between the spacing G and the dimension D of the pillar-shaped pattern 3 and the state of cell adhesion. The evaluation results are also shown in Table 2. Further, FIG. 7 shows an image of the osteoblasts in the vicinity of the area PA ₃₄ imaged in the evaluation test and the movement trajectory of the osteoblasts. The arrow lines shown in FIG. 7 represent the locus of movement of osteoblasts.

[Test Example 3]
In the same manner as in Test Example 1 except that human-derived mesenchymal stem cells (undifferentiated cells) were seeded in the dish, the cell number ratio P _HU (P _HU = N _in / ( _in each region PA _{01 to} PA ₄₉ . _{N in + N out), N} in the "number of the regions PA ₀₁ ~PA within ₄₉ (pillar-shaped pattern 3 above) and cells present in the boundary of each region PA ₀₁ ~PA _49", N _out is "each area a region of the same area as the PA ₀₁ ~PA _49, representing the number "of cells present in the area surrounding the periphery of each region PA ₀₁ ~PA _49.) by calculating the spacing of the pillar-shaped patterns 3 An evaluation test was carried out to evaluate the relationship between G and dimension D and the state of cell adhesion. The evaluation results are also shown in Table 2. Further, FIG. 8 shows an image of mesenchymal stem cells in the vicinity of the area PA ₃₄ imaged in the evaluation test and a locus of movement of the mesenchymal stem cells. The arrow line shown in FIG. 8 represents the locus of movement of mesenchymal stem cells.

[Test Example 4]
In the same manner as in Test Example 1, except that osteoblasts (differentiated cells) obtained by differentiating human-derived mesenchymal stem cells were seeded in the dish, the cell number ratio P _HO (in each region PA _{01 to} PA ₄₉ ) _{P HO = N in / (N} in + N out), N in the "number of cells present in the boundary of each region PA ₀₁ ~PA within ₄₉ (pillar-shaped pattern 3 above) and each area PA ₀₁ ~PA _49" , N _out is by calculating the.) representing "an area of the same area as each area PA ₀₁ ~PA _49, the number of cells present in the area surrounding the periphery of each region PA ₀₁ ~PA _49" An evaluation test was performed to evaluate the relationship between the spacing G and the dimension D of the pillar-shaped pattern 3 and the state of cell adhesion. The evaluation results are also shown in Table 2. In addition, FIG. 9 shows an image of the osteoblasts in the vicinity of the area PA ₃₄ imaged in the evaluation test and the movement trajectory of the osteoblasts. The arrow lines shown in FIG. 9 represent the locus of movement of osteoblasts.

[Test Example 5]
In the same manner as in Test Example 1, except that adipose progenitor cells (differentiated cells) obtained by inducing differentiation of human-derived mesenchymal stem cells were seeded in a dish and Adipogenic Maintenance Medium (Lonza) was used as a medium, The cell number ratio P _HA in each area PA _{01 to} PA ₄₉ (P _HA = N _in / (N _in + N _out ), N _in is “in each area PA _{01 to} PA ₄₉ (on the pillar pattern 3) and each area PA. the number "of cells present in the boundary of _{01 ~PA 49,} N _out is a region of the same area as the" respective regions PA ₀₁ ~PA _49, present in the region surrounding the periphery of each region PA ₀₁ ~PA ₄₉ The number of cells to be treated "is calculated.), And an evaluation test for evaluating the relationship between the spacing G and the dimension D of the pillar-shaped pattern 3 and the state of cell adhesion was performed. The evaluation results are also shown in Table 2. Further, FIG. 10 shows an image of a fat progenitor cell in the vicinity of the area PA ₂₄ imaged in the evaluation test and a movement trajectory of the fat progenitor cell. In addition, the arrow line shown in FIG. 10 represents the movement track of a fat precursor cell.

[Test Example 6]
Chondrocytes (differentiated cells) obtained by inducing differentiation of human-derived mesenchymal stem cells were seeded in a dish, and Chondrocyte Growth Medium (PromoCell) was used as a medium, in the same manner as in Test Example 1 area PA ₀₁ cells ratio in _{~PA 49 P HC (P HC =} N in / (N in + N out), N in the "each area PA ₀₁ ~PA within ₄₉ (pillar-shaped pattern 3 above) and each area PA ₀₁ the number "of cells present at the boundary of ~PA _49, N _out is a region of the same area as the" respective regions PA ₀₁ ~PA _49, present in the region surrounding the periphery of each region PA ₀₁ ~PA ₄₉ By expressing "the number of cells"), an evaluation test for evaluating the relationship between the spacing G and the dimension D of the pillar-shaped pattern 3 and the state of cell adhesion was performed. The evaluation results are also shown in Table 2. Further, FIG. 11 shows an image of chondrocytes in the vicinity of the area PA ₂₄ imaged in the evaluation test and a movement trajectory of the chondrocytes. The arrow lines shown in FIG. 11 represent the locus of movement of chondrocytes.

From the evaluation results of Test Example 1 and Test Example 2, the undifferentiated cell capture efficiency E _MU and the differentiated cell capture efficiency E _MD in each region PA _{01 to} PA ₄₉ were calculated by the following formulas. The results are shown in Table 3.
E _MU = P _MU / (P _MU + P _MD )
E _MD = P _MD / (P _MU + P _MD )

From the evaluation results of Test Example 3 and Test Example 4, the undifferentiated cell capture efficiency E _HOU and the differentiated cell capture efficiency E _HOD in each of the regions PA _{01 to} PA ₄₉ were calculated by the following formulas. The results are also shown in Table 3.
E _HOU = P _HU / (P _HU + P _HO )
E _HOD = P _HO / (P _HU + P _HO )

From the evaluation results of Test Example 3 and Test Example 5, the undifferentiated cell capture efficiency E _HAU and the differentiated cell capture efficiency E _HAD in each region PA _{01 to} PA ₄₉ were calculated by the following formulas. The results are also shown in Table 3.
E _HAU = P _HU / (P _HU + P _HA )
E _HAD = P _HA / (P _HU + P _HA )

From the evaluation results of Test Example 3 and Test Example 6, the undifferentiated cell capture efficiency E _HCU and the differentiated cell capture efficiency E _HCD in each region PA _{01 to} PA ₄₉ were calculated by the following formulas. The results are also shown in Table 3.
E _HCU = P _HU / (P _HU + P _HC )
E _HCD = P _HC / (P _HU + P _HC )

A cell mixture containing mouse-derived mesenchymal stem cells (undifferentiated cells) and osteoblasts (differentiated cells) from the calculation results of undifferentiated cell capture efficiency E _MU and differentiated cell capture efficiency E _MD shown in Table 3. Evaluation of Selective Separability of Mouse-Derived Mesenchymal Stem Cells Estimated by Contacting Pillar Pattern 3 with A _mu and Osteoblast Selective Separability A _MD It shows in Table 4.

A cell mixture containing human-derived mesenchymal stem cells (undifferentiated cells) and osteoblasts (differentiated cells) from the calculation results of the undifferentiated cell capture efficiency E _HOU and the differentiated cell capture efficiency E _HOD shown in Table 3. A comprehensive evaluation A _HOU for the selective separability of human-derived mesenchymal stem cells and a comprehensive evaluation A _HOD for the selective separability of osteoblasts, which are estimated when the cells are contacted with pillar-shaped pattern 3 It is also shown in Table 4.

From the calculation results of the undifferentiated cell capture efficiency E _HAU and the differentiated cell capture efficiency E _HAD shown in Table 3, a cell mixture containing human-derived mesenchymal stem cells (undifferentiated cells) and adipose precursor cells (differentiated cells) A comprehensive evaluation of the selective separability of human-derived mesenchymal stem cells A _HAU and a comprehensive evaluation of the selective separability of adipose progenitor cells A _HAD estimated when the cells are contacted with pillar-shaped pattern 3 It is also shown in Table 4.

From the calculation result of undifferentiated cell capture efficiency E _{HCU and} the calculated result of differentiated cell capture efficiency E _HCD shown in Table 3, a cell mixture containing human-derived mesenchymal stem cells (undifferentiated cells) and chondrocytes (differentiated cells) was obtained. Table 4 shows a comprehensive evaluation A _HCU for the selective separability of human-derived mesenchymal stem cells and a general evaluation A _HCD for the selective separability of chondrocytes, which are estimated when they are contacted with the pillar-shaped pattern 3. Shown together with.

In Table 4, “∘”, “◯”, “Δ” and “x” in the comprehensive evaluation are based on the following indexes.
⊚: E _MU , E _HOU , E _HAU , E _HCU >0.8; E _MD , E _HOD , E _HAD , E _HCD > 0.8
○: 0.6 <E _MU , E _HOU , E _HAU , E _HCU ≦ 0.8; 0.6 <E _MD , E _HOD , E _HAD , E _HCD ≦ 0.8
Δ: 0.5 <E _MU , E _HOU , E _HAU , E _HCU ≦ 0.6; 0.5 <E _MD , E _HOD , E _HAD , E _HCD ≦ 0.6
×: E _MU , E _HOU , E _HAU , E _HCU ≦ 0.5; E _MD , E _HOD , E _HAD , E _HCD ≦ 0.5

From the results shown in Tables 2 to 4 and FIGS. 6 to 11, if the nano-relief structure, particularly the spacing G and the dimension D are all pillar-shaped patterns 3 within the range of 100 to 250 nm, the cell adhesiveness is improved. It has been found that there is a tendency to change. Furthermore, it was revealed that the change in adhesiveness depends on the differentiation state of cells. In the region having the pillar-shaped pattern 3 in which the capture efficiency E _MD , E _{HOD of} mesenchymal stem cells is relatively high (the region where the comprehensive evaluation shown in Table 4 is “◎” or “◯”), the mesenchymal stem cells are Then, the mesenchymal stem cells in the cell mixture containing the osteoblasts that have been induced to differentiate relatively easily adhere to the pillar-shaped pattern 3, so that the cell mixture is simply contacted with the pillar-shaped pattern 3. It is considered that the mesenchymal stem cells can be selectively and easily separated and obtained, or the cell density of the mesenchymal stem cells can be increased. On the other hand, a pillar-shaped pattern 3 having a relatively high capture efficiency E _MD , E _HOD , E _HAD , and E _{HCD of} the differentiated cells (osteoblasts, preadipocytes, chondrocytes, etc.) differentiated from the mesenchymal stem cells is obtained. In the region (the region where the comprehensive evaluation shown in Table 4 is “⊚” or “∘”), the differentiated cells in the cell mixture containing the mesenchymal stem cells and the differentiated cells differentiated from the mesenchymal stem cells are relative to the pillar-shaped pattern 3. Since it becomes easier to adhere to the cells, it is possible to selectively and easily separate and obtain mesenchymal stem cells from the cell mixture only by contacting the cell mixture with the pillar-shaped pattern 3, or the mesenchymal system. It is thought that the cell density of stem cells can be increased.

  As is clear from the results of the above-described test example, a nano-concavo-convex structure in which a plurality of pillar-shaped patterns 3 having a dimension D (D = 100 to 250 nm) are regularly arranged at intervals G (G = 100 to 250 nm). When the body is used, by simply contacting a cell mixture containing undifferentiated cells and differentiated cells with the pillar-shaped pattern 3, the undifferentiated cells are selectively selected from the cell mixture based on the difference in cell adhesiveness. And it is considered that they can be easily separated and acquired.

  Adhesion of cells is preferably achieved by using the undifferentiated cell separation substrate 1 on which the pillar-shaped pattern 3 having the interval G and the dimension D having the relations (1-1) to (1-7) below is formed. Based on the sex difference, undifferentiated cells can be selectively and easily separated / acquired from the cell mixture, or the cell density of undifferentiated cells can be increased.

100 nm ≦ G <112.5 nm; 100 nm ≦ D ≦ 250 nm (1-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D ≦ 250 nm (1-2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D ≦ 250 nm (1-3)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D ≦ 250 nm (1-4)
187.5 nm ≦ G <212.5 nm; 100 nm ≦ D <187.5 nm or 212.5 nm ≦ D ≦ 250 nm (1-5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <137.5 nm or 212.5 nm ≦ D ≦ 250 nm (1-6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D ≦ 250 nm (1-7)

  Particularly preferably, by using the undifferentiated cell separation substrate 1 on which the pillar-shaped pattern 3 having the interval G and the dimension D having the relationship shown in (2-1) to (2-7) below is formed, It is possible to selectively and easily separate or obtain undifferentiated cells from a cell mixture or to increase the cell density of undifferentiated cells based on the difference in cell adhesiveness, regardless of the type.

100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (2-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (2-2)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <162.5 nm or 187.5 nm ≦ D <237.5 nm (2-3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (2-4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (2-5)
212.5 nm ≦ G <237.5 nm; 237.5 nm ≦ D ≦ 250 nm (2-6)
237.5 nm ≦ G ≦ 250 nm; 162.5 nm ≦ D <237.5 nm (2-7)

  When the undifferentiated cell contained in the cell mixture is a mouse-derived mesenchymal stem cell and the differentiated cell is an osteoblast differentiated from it, preferably the following (3-1) to (3-7) are shown. By using the undifferentiated cell separation substrate 1 on which the pillar-shaped pattern 3 having the relationship G and the dimension D is formed, the undifferentiated cells are selectively selected from the cell mixture based on the difference in the adhesiveness of the cells. In addition, it is possible to easily separate and obtain, or to increase the cell density of undifferentiated cells.

100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (3-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (3-2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D <162.5 nm or 187.5 nm ≦ D ≦ 250 nm (3-3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (3-4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (3-5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <112.5 nm or 237.5 nm ≦ D ≦ 250 nm (3-6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D <137.5 nm or 162.5 nm ≦ D <237.5 nm (3-7)

  When the undifferentiated cells contained in the cell mixture are human-derived mesenchymal stem cells, and the differentiated cells are osteoblasts, preadipocytes or chondrocytes that have been induced to differentiate, preferably (4-1) to By using the undifferentiated cell separation substrate 1 on which the pillar-shaped pattern 3 having the interval G and the dimension D having the relationship shown in (4-7) is formed, it is possible to remove the cell mixture from the cell mixture based on the difference in cell adhesiveness. Undifferentiated cells can be selectively and easily separated / acquired, or the cell density of undifferentiated cells can be increased.

100 nm ≦ G <112.5 nm; 100 nm ≦ D ≦ 250 nm (4-1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D ≦ 250 nm (4-2)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D ≦ 250 nm (4-3)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D ≦ 250 nm (4-4)
187.5 nm ≦ G <212.5 nm; 100 nm ≦ D <187.5 nm or 212.5 nm ≦ D ≦ 250 nm (4-5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <137.5 nm or 212.5 nm ≦ D ≦ 250 nm (4-6)
237.5 nm ≦ G ≦ 250 nm; 137.5 nm ≦ D ≦ 250 nm (4-7)

  Particularly preferably, by using the undifferentiated cell separation substrate 1 on which the pillar-shaped pattern 3 having the interval G and the dimension D having the relationships shown in the following (5-1) to (5-4) is formed, a cell mixture is obtained. Whether the undifferentiated cells contained in are human-derived mesenchymal stem cells, and whether the differentiated cells are osteoblasts, preadipocytes or chondrocytes that have been induced to differentiate from them, differences in cell adhesion Based on the above, it is possible to selectively and easily separate and obtain undifferentiated cells from a cell mixture, or to increase the cell density of undifferentiated cells.

112.5 nm ≦ G <137.5 nm; 187.5 nm ≦ D <212.5 nm (5-1)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <137.5 nm (5-2)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D <212.5 nm (5-3)
237.5 nm ≦ G ≦ 250 nm; 237.5 nm ≦ D ≦ 250 nm (5-4)

  As described above, only by contacting the pillar-shaped pattern 3 of the undifferentiated cell separation substrate 1 with the cell mixture containing the undifferentiated cells and the differentiated cells, the undifferentiated cells are selectively extracted from the cell mixture, and It can be easily separated and obtained, or the cell density of undifferentiated cells can be increased.

  INDUSTRIAL APPLICABILITY The present invention is useful in the technical field of regenerative medicine using undifferentiated cells and differentiated cells.

1 ... Undifferentiated cell separation substrate 2 ... Base (base material)
21 ... Main surface (first surface)
3 ... Pillar pattern

Claims (8)

A method for separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells,
Using a nano-relief structure formed by a plurality of pillar-shaped patterns are arranged with regularity, including a step of contacting the cell mixture to the plurality of pillar-shaped patterns,
The undifferentiated cells are mesenchymal stem cells derived from mouse,
The differentiated cells are osteoblasts differentiated from the undifferentiated cells,
Wherein the gap G and the size D of the pillar-shaped patterns, easily the undifferentiated cells adhered to the pillar-like pattern, wherein the differentiated cell is an adhesive hardly related to the pillar-like pattern, the following (1) to A method for separating undifferentiated cells, which has any one of the relationships (7) .
100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D <162.5 nm or 187.5 nm ≦ D ≦ 250 nm (3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <112.5 nm or 237.5 nm ≦ D ≦ 250 nm (6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D <137.5 nm or 162.5 nm ≦ D <237.5 nm (7) A method for separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells,
Using a nano-relief structure formed by a plurality of pillar-shaped patterns are arranged with regularity, including a step of contacting the cell mixture to the plurality of pillar-shaped patterns,
The undifferentiated cells are human-derived mesenchymal stem cells,
The differentiated cells are osteoblasts differentiated from the undifferentiated cells, preadipocytes or chondrocytes,
The spacing G and the dimension D of the pillar-shaped pattern are such that one of the undifferentiated cells and the differentiated cells is likely to adhere to the pillar-shaped pattern and the other is difficult to adhere to the pillar-shaped pattern. And a method for separating undifferentiated cells, which has any one of the following relationships (1) to (4).
112.5 nm ≦ G <137.5 nm; 187.5 nm ≦ D <212.5 nm (1)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <137.5 nm (2)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D <212.5 nm (3)
237.5 nm ≦ G ≦ 250 nm; 237.5 nm ≦ D ≦ 250 nm (4) The method for separating undifferentiated cells according to claim 1 or 2 , wherein the concavo-convex structure has the plurality of pillar-shaped patterns arranged on respective lattice points of an equilateral triangle lattice. The method for separating undifferentiated cells according to claim 1 or 2 , wherein the concavo-convex structure has the plurality of pillar-shaped patterns arranged on respective lattice points of a rectangular lattice. A substrate for separating undifferentiated cells used for separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells,
A base material having a first surface and a second surface facing the first surface;
A plurality of pillar-shaped patterns formed in an array with regularity on the first surface of the substrate,
The undifferentiated cells are mesenchymal stem cells derived from mouse,
The differentiated cells are osteoblasts differentiated from the undifferentiated cells,
Wherein the gap G and the size D of the pillar-shaped patterns, easily the undifferentiated cells adhered to the pillar-like pattern, wherein the differentiated cell is an adhesive hardly related to the pillar-like pattern, the following (1) to A substrate for separating undifferentiated cells, which has any one of the relationships (7) .
100 nm ≦ G <112.5 nm; 112.5 nm ≦ D ≦ 250 nm (1)
112.5 nm ≦ G <137.5 nm; 112.5 nm ≦ D <137.5 nm (2)
137.5 nm ≦ G <162.5 nm; 100 nm ≦ D <162.5 nm or 187.5 nm ≦ D ≦ 250 nm (3)
162.5 nm ≦ G <187.5 nm; 162.5 nm ≦ D <212.5 nm or 237.5 nm ≦ D ≦ 250 nm (4)
187.5 nm ≦ G <212.5 nm; 212.5 nm ≦ D <237.5 nm (5)
212.5 nm ≦ G <237.5 nm; 100 nm ≦ D <112.5 nm or 237.5 nm ≦ D ≦ 250 nm (6)
237.5 nm ≦ G ≦ 250 nm; 100 nm ≦ D <137.5 nm or 162.5 nm ≦ D <237.5 nm (7) A substrate for separating undifferentiated cells used for separating the undifferentiated cells from a cell mixture containing undifferentiated cells and differentiated cells,
A base material having a first surface and a second surface facing the first surface;
A plurality of pillar-shaped patterns formed on the first surface of the base material with regularity;
Equipped with
The undifferentiated cells are human-derived mesenchymal stem cells,
The differentiated cells are osteoblasts differentiated from the undifferentiated cells, preadipocytes or chondrocytes,
The spacing G and the dimension D of the pillar-shaped pattern are such that one of the undifferentiated cells and the differentiated cells is likely to adhere to the pillar-shaped pattern and the other is difficult to adhere to the pillar-shaped pattern. And a substrate for separating undifferentiated cells, which has any one of the following relationships (1) to (4).
112.5 nm ≦ G <137.5 nm; 187.5 nm ≦ D <212.5 nm (1)
137.5 nm ≦ G <162.5 nm; 112.5 nm ≦ D <137.5 nm (2)
162.5 nm ≦ G <187.5 nm; 112.5 nm ≦ D <212.5 nm (3)
237.5 nm ≦ G ≦ 250 nm; 237.5 nm ≦ D ≦ 250 nm (4) The substrate for separating undifferentiated cells according to claim 5 or 6 , wherein the plurality of pillar-shaped patterns are arranged on each lattice point of an equilateral triangle lattice. The substrate for separating undifferentiated cells according to claim 5 or 6 , wherein the plurality of pillar-shaped patterns are arranged on each lattice point of a rectangular lattice.