JP2017060428A - Substrate for cell behavior assessment, container for cell behavior assessment, and cell behavior assessment method - Google Patents

Abstract

A cell behavior evaluation substrate, a cell behavior evaluation container, and a cell behavior evaluation method capable of accurately evaluating the behavior of cells on a fine uneven pattern.
A cell behavior evaluation substrate for evaluating the behavior of cells on a fine concavo-convex pattern includes a base portion having a first surface and a second surface facing the first surface, and is set on a part of the first surface of the base portion. In each of the first to Nth (N is an integer greater than or equal to 2) evaluation regions that are sequentially arranged along a predetermined direction, first to Nth uneven patterns as fine uneven patterns are formed. The Mth unevenness pattern formed in the Mth evaluation region (M is an integer of 1 to N-1) and the M + 1th unevenness pattern formed in the M + 1 evaluation region are 100 to 500 nm. And have different dimensions so that the behavior of the cells can be evaluated.
[Selection] Figure 1

Description

  The present invention relates to a cell behavior evaluation substrate and a cell behavior evaluation container used for evaluating the behavior of cells, and a method for evaluating the behavior of cells.

  In recent years, in biotechnology-related fields such as drug discovery development and regenerative medicine, cells isolated from living tissues, cell components (eg, membrane vesicles (exosomes) secreted from cultured cells, cell small cells) Organs (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), granules (melanosomes, microbodies, glyoxysomes, bibel-parade bodies, etc.), cytoskeletons, central bodies, flagella, cilia, ribosomes Etc.) has been actively conducted. In general, a test / research using cultured cells is advantageous in that it is less expensive than a test / research using animals, requires only a small amount of reagent to be evaluated, and easily obtains homogeneous experimental data. In such tests and researches, it is important to suppress cell death of cells isolated from living tissue, and to stably culture and proliferate the cells.

  In order to stably cultivate and proliferate cells, developments related to cell culture devices for culturing cells have been underway. 2. Description of the Related Art Conventionally, a cell culture container of a multiwell plate type, in which a culture sheet having a pillar-shaped uneven structure is attached to the bottom surface of each well, has been proposed (see Patent Document 1).

International Publication No. 2013/030940

  In the cell culture container described in Patent Document 1, when cells to be cultured are seeded on the pillar-shaped uneven structure of the culture sheet, the cells aggregate on the pillar-shaped uneven structure to form spheroids. Is done.

  By the way, in order to stably culture cells using the cell culture vessel described in Patent Document 1, it is necessary that the pillar-shaped uneven structure has dimensions suitable for the cell type to be cultured. It becomes. This is because the size or the like of the concavo-convex structure showing suitable adhesiveness varies depending on the cell type or the like. Therefore, depending on the size of the concavo-convex structure, etc., the behavior of the cell type etc. to be cultured must be evaluated and the size of the concavo-convex structure of the cell culture vessel must be determined. The equipment to be evaluated is currently not proposed.

  In view of such problems, an object of the present invention is to provide a cell behavior evaluation substrate, a cell behavior evaluation container, and a cell behavior evaluation method capable of accurately evaluating the behavior of cells on a fine uneven pattern. .

  In order to solve the above-mentioned problems, the present invention is a cell behavior evaluation substrate for evaluating the behavior of cells on a fine unevenness pattern, and has a first surface and a second surface facing the first surface. Each of the first to Nth (N is an integer of 2 or more) evaluation regions that are provided with a base and are sequentially arranged along a predetermined direction set on a part of the first surface of the base. , The first to Nth concavo-convex patterns as the fine concavo-convex patterns are formed, and the Mth concavo-convex pattern formed in the Mth (M is an integer of 1 to N-1) evaluation region; A cell behavior evaluation characterized in that the M + 1 uneven pattern formed in the M + 1 evaluation region has a dimension within a range of 100 to 500 nm and has a dimension different from each other to the extent that the behavior of the cell can be evaluated. A substrate is provided (Invention 1)

  According to the said invention (invention 1), several evaluation area | regions (1st-Nth (N> = 2) evaluation are arranged in order along a predetermined direction on a part of the first surface of the cell behavior evaluation substrate. Region) is set, and an Mth (1 ≦ M ≦ N−1) concavo-convex pattern among the concavo-convex patterns (first to Nth concavo-convex patterns) formed in each evaluation region, and an M + 1-th concavo-convex pattern adjacent thereto By having different dimensions (within a range of 100 to 500 nm) to such an extent that the behavior of the cells can be evaluated, the behavior of the seeded cells can be accurately evaluated.

  In the said invention (invention 1), the identification mark containing the information regarding the dimension of the said 1st-Nth uneven | corrugated pattern is provided in the vicinity of each of the said 1st-Nth evaluation area | region in the said 1st surface. Preferred (Invention 2).

  Since the first to Nth uneven patterns formed on the cell behavior evaluation substrate have dimensions in the range of 100 to 500 nm, when observing cells on the fine uneven pattern with an optical microscope, It is difficult to grasp the dimensions of the fine concavo-convex pattern in which the cells exhibit good adhesion. However, according to the said invention (invention 2), the identification mark containing the information regarding the dimension of the uneven | corrugated pattern currently formed in each evaluation area | region is provided in the vicinity of each evaluation area | region. Even in the case of observing, it is possible to easily grasp the dimension of the fine concavo-convex pattern in which the cell exhibits good adhesiveness.

  In the said invention (invention 1 and 2), it is preferable that the dimension of the said 1st-Nth uneven | corrugated pattern changes in steps in parallel order of the said 1st-Nth evaluation area | region (invention 3). According to this invention (invention 3), since each concavo-convex pattern is formed so that the dimensions change (increase / decrease) stepwise, the cells show good adhesiveness (or the cells hardly show adhesiveness). The dimension range of the fine uneven pattern can be easily determined.

  In the said invention (invention 1-3), the said Mth uneven | corrugated pattern currently formed in the said Mth evaluation area | region and the said M + 1 uneven | corrugated pattern currently formed in the said M + 1 evaluation area are continuing. Preferred (Invention 4).

  If there is a structure different from the fine concavo-convex pattern between adjacent evaluation areas, it is difficult to ignore the influence of the structure on the behavior of the cell. According to the above invention (Invention 4), Since there is no structure that may affect the cell behavior because the micro concavo-convex patterns formed on each other are continuous with each other, the effect of the size of the micro concavo-convex pattern on the cell behavior can be accurately determined. Can be evaluated.

  In the said invention (invention 1-4), it is preferable that the mark which shows the boundary of the said Mth evaluation area | region and the said M + 1 evaluation area | region in the said 1st surface is provided in the vicinity of the said boundary (invention 5). .

  Since the first to Nth uneven patterns have a size of 100 to 500 nm, it is difficult to determine the boundary between adjacent fine uneven patterns in the observation using an optical microscope. According to the present invention, it is possible to accurately evaluate the size of the fine concavo-convex pattern in which cells located in the vicinity of the boundary between the adjacent evaluation regions exhibit good adhesion by providing the mark indicating the boundary between the adjacent evaluation regions. it can.

  In the said invention (invention 1-5), it is preferable that the top part of the said 1st-Nth uneven | corrugated pattern is located on the same plane as area | regions other than the said 1st-Nth evaluation area | region in the said 1st surface (invention). 6).

  When cells are seeded on the first to Nth evaluation regions, the cells themselves may be present on regions other than the evaluation region. When the cells present in the region other than the evaluation region move into the evaluation region, it becomes possible to accurately evaluate the size of the fine uneven pattern in which the cells exhibit good adhesion, but the fine uneven pattern in the evaluation region If the height of the top and the area other than the evaluation area are different, there is a risk of inhibiting cell migration. However, according to the said invention (invention 6), since the top part of a fine unevenness | corrugation pattern and the area | regions other than an evaluation area | region are located on the same plane, a cell tends to move into an evaluation area | region from areas other than an evaluation area | region. Therefore, the dimension of the fine concavo-convex pattern in which cells exhibit good adhesion can be accurately evaluated.

  In the said invention (invention 1-6), the said 1st-Nth uneven | corrugated pattern can be made into a pillar shape or a line and space shape (invention 7).

  In the above inventions (Inventions 1 to 7), each of the first to Nth evaluation regions is large enough to fit within a field of view of an optical microscope for observing the behavior of the cells on the first to Nth uneven patterns. It is preferable (Invention 8).

  According to the said invention (invention 8), the magnitude | size of the 1st-Nth evaluation area | region is a grade which is settled in the visual field of the optical microscope for observing the behavior of a cell, and moves the visual field of an optical microscope. It is possible to observe and evaluate the behavior of cells.

  In addition, the present invention provides a container body having a bottom part formed with a through-hole and a peripheral wall part continuous with an outer peripheral edge of the bottom part, and a cell behavior according to the inventions (Inventions 1 to 8) attached to the bottom part. An evaluation substrate, and the cell behavior evaluation substrate is arranged so that the first to Nth uneven patterns overlap the through-hole in a plan view of the container body and is positioned in the container body. Provided is a cell behavior evaluation container, which is attached to the bottom portion, and wherein the through hole is closed by the cell behavior evaluation substrate (Invention 9).

  In the above invention (Invention 9), the cell behavior evaluation substrate is attached to the bottom so that the first to Nth uneven patterns are physically included in the through hole in a plan view of the container body. (Invention 10)

  In the said invention (invention 9 and 10), the said cell behavior evaluation board | substrate is attached to the outer surface of the said bottom part of the said container main body so that the said 1st-Nth uneven | corrugated pattern may be exposed from the said through-hole. (Invention 11)

  In the said invention (invention 9-11), it is preferable that the said board | substrate for cell behavior evaluation is attached to the said bottom part through the adhesive agent (invention 12).

  Furthermore, this invention seed | inoculates the evaluation object cell in the container for cell behavior evaluation which concerns on the said invention (invention 9-12), The said evaluation object cell on the said 1st-Nth uneven | corrugated pattern is said cell behavior evaluation. A cell behavior evaluation method characterized by observing from the second surface side of the substrate for evaluation and evaluating the behavior of the cell (Invention 13).

  In the said invention (invention 13), it is preferable to observe the said evaluation object cell using an inverted microscope (invention 14).

  According to the present invention, it is possible to provide a cell behavior evaluation substrate, a cell behavior evaluation container, and a cell behavior evaluation method capable of accurately evaluating the behavior of cells on a fine uneven pattern.

FIG. 1A is a plan view showing a schematic configuration of a cell behavior evaluation substrate according to one embodiment of the present invention, and FIG. 1B is a schematic configuration of each evaluation region in one embodiment of the present invention. FIG. FIG. 2 is a cross-sectional end view taken along line II in FIG. 1 (B), showing a schematic configuration of the cell behavior evaluation substrate according to the embodiment of the present invention. FIG. 3 is a plan view showing a schematic configuration of identification marks and boundary marks in one embodiment of the present invention. FIG. 4 is a process flow diagram showing the manufacturing process of the cell behavior evaluation substrate according to the embodiment of the present invention in a cut end view. FIG. 5 is a manufacturing process of a cell behavior evaluation substrate according to an embodiment of the present invention, and each process (each process of the transparent substrate polishing method) continued from the process shown in FIG. FIG. FIG. 6 is a plan view showing a schematic configuration of the evaluation container according to the embodiment of the present invention. 7 is a sectional view taken along line II-II in FIG. 6, showing a schematic configuration of the evaluation container in one embodiment of the present invention. FIG. 8 is a schematic view showing an evaluation method using the evaluation container in one embodiment of the present invention. FIG. 9 is a perspective view schematically showing another aspect of the evaluation region in one embodiment of the present invention. 10A and 10B are photomicrographs on a cell behavior evaluation substrate after cell culture in Test Example 1. FIG. FIG. 11 is a photomicrograph on the cell behavior evaluation substrate after cell culture in Test Example 2.

Embodiments of the present invention will be described with reference to the drawings.
[Cell behavior evaluation substrate]
FIG. 1A is a plan view showing a schematic configuration of a cell behavior evaluation substrate according to the present embodiment, and FIG. 1B is a plan view showing a schematic configuration of each evaluation region in the present embodiment. FIG. 2 is a cross-sectional end view taken along the line II in FIG. 1 (B), showing a schematic configuration of the cell behavior evaluation substrate according to the present embodiment.

As shown in FIGS. 1 and 2, the cell behavior evaluation substrate 10 according to the present embodiment includes a base 11 having a first surface 11a and a second surface 11b facing the first surface 11a. On the first surface 11a side of the base 11, the first to Nth (N is an integer of 2 or more. In the example shown in FIG. 1B, N = 12) evaluation regions ER _{1 to} ER _N (ER ₁₂ ) Including the evaluation region group GR, and the fine concavo-convex pattern 12 is formed in each of the evaluation regions ER _{1 to} ER _N in the evaluation region group GR.

  The cell behavior evaluation substrate 10 according to the present embodiment is used for observing cells as evaluation objects existing on the fine concavo-convex pattern 12 from the second surface 11b side using an inverted microscope. The evaluation object is not limited to cells. For example, membrane vesicles (exosomes) secreted from cultured cells, organelles (organelles), vesicles (lysosomes, endosomes, phagosomes, vacuoles, etc.), Also included are cell components such as granules (melanosomes, microbodies, glyoxisomes, bibel-parade bodies, etc.), cytoskeleton, central bodies, flagella, cilia, ribosomes, etc. Hereinafter, in this embodiment, a substrate for evaluating the behavior of a cell as an evaluation object will be described as an example.

  Materials constituting the base 11 include quartz glass, synthetic quartz glass, soda glass, fluorite, calcium fluoride, magnesium fluoride, acrylic glass, borosilicate glass, and the like; polycarbonate, polypropylene, polyethylene, polystyrene, and others A transparent material such as a resin material such as polyolefin can be used.

  In the present embodiment, “transparent” means light having a wavelength of 190 to 800 nm from one side (the first surface 11a side or the second surface 11b side) of the object (the cell behavior evaluation substrate 10 in the present embodiment). When irradiated, it means that light reaches the opposite side (the second surface 11b side or the first surface 11a side) from the irradiated side. If a suitable standard is expressed by transmittance, it is 20% or more, preferably 50% or more, particularly preferably 80% or more.

  The shape (plan view shape) of the base 11 is not particularly limited, and examples thereof include a substantially rectangular shape in plan view and a substantially circular shape in plan view. In addition, the size of the base 11 in plan view is not particularly limited, but as described later, the cell behavior evaluation substrate 10 according to the present embodiment is preferably a container body 2 having a through hole 2c. Is used by being attached to the bottom portion 2a of the base plate 2 so as to close the through hole 2c (see FIGS. 6 and 7). Therefore, it is preferable that the size of the base 11 is set to such an extent that the through hole 2c can be closed according to the size of the through hole 2c of the container body 2.

The thickness T _{11 of the} base 11 is 0.5 mm or less, preferably 0.03 to 0.5 mm, and particularly preferably 0.1 to 0.2 mm. The cell behavior evaluation substrate 10 according to the present embodiment evaluates the behavior of the cell by observing the cell as the evaluation object existing on the fine concavo-convex pattern 12 from the second surface 11b side using an inverted microscope. Used for. When observing cells on the fine concavo-convex pattern 12, it is desirable that observation is possible at an extremely high magnification (20 times or more, preferably about 50 times or more), but when the thickness T _{11 of the} base 11 exceeds 0.5 mm. The distance between the cells on the fine concavo-convex pattern 12 and the objective lens of the inverted microscope located on the second surface 11b side so as to face each other with the base 11 in between is increased, and at a high magnification (about 20 times or more) Observation becomes difficult. In the present embodiment, the thickness T ₁₁ of the base 11 can be measured using a micrometer or the like.

In this embodiment, the evaluation region group in GR, is provided with N number of evaluation regions ER ₁ to Er _N in parallel in one direction, the evaluation region group GR is integrally fine uneven pattern 12 and the base 11 The first evaluation region group GR ₁ formed as a general structure and the second concave / convex pattern 12 having a shape different from the fine concave / convex pattern 12 are formed as a structure integral with the base 11. It is divided into a rating region group GR _2.

In the present embodiment, the fine concavo-convex pattern 12 formed in each evaluation region ER _{2 to} ER ₆ of the first evaluation region group GR ₁ has a pillar shape, and each evaluation region ER of the second evaluation region group GR _{2. The} fine concavo-convex pattern 12 formed in _{8 to} ER ₁₂ has a line and space shape. The fine concavo-convex patterns 12 formed in the respective evaluation regions ER _{2 to} ER ₆ and ER _{8 to} ER ₁₂ of the first evaluation region group GR ₁ and the second evaluation region group GR ₂ are of different types. What is necessary is just to have a shape, and the shape of these fine uneven | corrugated patterns is not limited to the said aspect.

In the evaluation regions ER _{2 to} ER ₆ and ER _{8 to} ER ₁₂ included in each of the first evaluation region group GR ₁ and the second evaluation region group GR ₂ , fine uneven patterns 12 having different dimensions are formed. In the present embodiment, fine concavo-convex patterns 12 having dimensions of 100 nm, 150 nm, 200 nm, 300 nm, and 500 nm are formed in the evaluation regions ER _{2 to} ER ₆ and ER _{8 to} ER ₁₂ so as to be arranged in that order. That is, the size of each evaluation area ER ₂ ~ER _6, ER ₈ fine uneven pattern 12 formed on the to Er ₁₂ is graded in parallel order of the evaluation region _{ER 2 ~ER 6, ER 8 ~ER} 12 To do. Note that the evaluation regions ER ₁ and ER ₇ adjacent to the evaluation regions ER ₂ and ER ₈ where the fine concavo-convex pattern 12 having a dimension of 100 nm is formed (the left side in FIG. 1B) are regions where the fine concavo-convex pattern 12 does not exist. However, the cell behavior evaluation substrate 10 according to the present embodiment may not have the evaluation regions ER ₁ and ER ₇ .

The dimensions of each evaluation area ER ₂ ~ER _6, ER ₈ fine uneven pattern 12 formed on the to Er ₁₂ is in the range of 100 to 500 nm. When the dimension of the fine concavo-convex pattern 12 is within the above range, it is possible to accurately evaluate the adhesiveness of the cell to be evaluated according to the dimension of the fine concavo-convex pattern 12.

  Note that the dimension of the fine concavo-convex pattern 12 is, for example, the length of one side of the square if the fine concavo-convex pattern 12 is in a substantially square pillar shape in plan view, and the diameter of the circle in the case of a substantially circular pillar shape in plan view. In the case of line and space, it means the width in the short direction of the line pattern. Further, when the plan view shape of the fine concavo-convex pattern 12 is not substantially square or substantially circular (in the case of an indeterminate shape), the cut passes through a height that is ½ of the height (pillar height) of the fine concavo-convex pattern 12. The diameter of a circle having the same area as that of the cut surface parallel to the first surface 11 a of the base 11 is defined as the dimension of the fine concavo-convex pattern 12.

  Further, if the fine concavo-convex pattern 12 is a hole-shaped pattern having a substantially circular shape in plan view, the diameter of the circle may be the dimension of the fine concavo-convex pattern 12, and the fine concavo-convex pattern 12 may be a hole-shaped pattern having an irregular shape in plan view. For example, the cut surface passing through half the depth (hole depth) of the fine concavo-convex pattern 12 and having the same area as the cut surface parallel to the first surface 11 a of the base 11. The diameter of the circle is the dimension of the fine uneven pattern 12.

The fine unevenness pattern 12 formed in the Mth (M is an integer between 1 and N-1) evaluation region ER _M and the fine unevenness pattern formed in the M + 1th evaluation region ER _{M + 1} adjacent thereto Each of 12 has dimensions that are so different that the behavior of the cell can be evaluated. Specifically, the dimensional difference (absolute value) of the fine concavo-convex pattern 12 formed in the adjacent evaluation regions ER _M and ER _{M + 1} is preferably 10 nm or more, and particularly preferably 50 nm or more. . If the dimensional difference is less than 10 nm, it may be difficult to accurately evaluate the adhesion of the cells to be evaluated depending on the size of the fine concavo-convex pattern 12.

  The shape of the fine concavo-convex pattern 12 is preferably a substantially circular, substantially rectangular or substantially polygonal pillar shape in a plan view, a line and space shape, etc., but is not limited to these shapes. It may be a substantially circular, substantially rectangular or substantially polygonal hole shape. Moreover, the fine uneven | corrugated pattern 12 may have the side wall shape of a taper shape or a reverse taper shape.

The size of each evaluation area ER ₁ to Er _N is preferably each evaluation area ER ₁ to Er _N is a size that fits within the field of view of the inverted microscope. If the size of each evaluation region ER is large enough to protrude from the field of view of the inverted microscope, the cell may move out of the field of view of the inverted microscope when observing the cell as the evaluation object. The observation of cells over time may be difficult. On the other hand, when the size of the evaluation area ER ₁ to Er _N is too small, the evaluation cell is not fit in the area ER ₁ to Er _N, to accurately evaluate the adhesion that depends on the dimensions of the fine concavo-convex pattern 12 May become difficult. From such a viewpoint, the size of each of the evaluation regions ER _{1 to} ER _N is preferably about 1.5 mm × 0.5 mm, for example.

  In addition, as described later, the cell behavior evaluation substrate 10 according to the present embodiment preferably has an outer surface of the bottom 2a (the container main body 2) of the container main body 2 in which a through hole 2c is formed in the approximate center of the bottom 2a. Outside), the nano uneven pattern 12 is attached so as to be exposed from the through hole 2c (see FIGS. 6 and 7). Therefore, the size of the evaluation region group GR is set to such an extent that the evaluation region group GR can be exposed from the through hole 2c according to the size of the through hole 2c of the container body 2, that is, the cell behavior evaluation substrate 10 is attached. The evaluation region group GR is appropriately set to such an extent that the evaluation region group GR can be physically contained in the through hole 2c in the plan view of the container body 2 (see FIG. 6).

A vicinity of the substantially rectangular one short side of each evaluation region ER ₁ to Er _N, in the example shown in the outside (Figure 1 evaluation region ER ₁ to Er _N underside of each evaluation region ER ₁ to Er _N ) Is formed with an identification mark IM indicating the type (shape) and dimension of the fine uneven pattern 12 formed in the evaluation regions ER _{1 to} ER _N. In the example shown in FIG. 1B, “D100” means that a pillar-shaped fine uneven pattern 12 having a dimension of 100 nm is formed in the evaluation region ER ₂ , and “L100” means a line-and-line having a dimension of 100 nm. It means that the space-like fine uneven pattern 12 is formed in the evaluation region ER ₈ .

Further, on the outside of each evaluation area ER _{1 to} ER _N (in the example shown in FIG. 1B, the upper side and the lower side of each evaluation area ER _{1 to} ER ₁₂ ), adjacent evaluation areas ER _M and ER _{M + A} boundary mark BM indicating the boundary of ₁ is formed. In the present embodiment, it is almost impossible to discriminate the type (shape) and size of the fine concavo-convex pattern 12 formed in the evaluation region ER with an inverted microscope simultaneously with cell observation. However, according to the cell behavior evaluation substrate 10 according to the present embodiment, information on the type (shape) and size of the fine concavo-convex pattern 12 in which the cells exhibit good adhesion can be obtained simultaneously with the observation of the cells by the identification mark IM and the boundary. It can be acquired from the mark BM. Therefore, the type (shape) and dimension of the fine concavo-convex pattern 12 contributing to cell adhesion can be grasped while observing the behavior of the cell to be evaluated.

In the present embodiment, as shown in FIG. 3, the identification mark IM and the boundary mark BM are configured by a line-and-space concavo-convex structure representing characters and triangles as a whole, and outside the respective evaluation regions ER _{1 to} ER _N. Is provided.

  In the present embodiment, the overall size of each identification mark IM and each boundary mark BM is a size that can be discriminated with an inverted microscope used for cell observation. For example, the size of the identification mark IM is about 100 μm. The size of the boundary mark BM can be set to about 50 μm.

Moreover, it is preferable that the dimension of the uneven structure of the line and space shape which comprises each identification mark IM and each boundary mark BM is about 1-10 micrometers. Since the identification mark IM and the boundary mark BM are configured by the concavo-convex structure having such a size range, the marks IM and BM hardly affect the adhesiveness of the cell to be evaluated. It is possible to accurately evaluate the behavior of cells depending on the size of the fine concavo-convex pattern 12 formed in the regions ER _{1 to} ER _N.

Distance L _IM from the identification mark IM to evaluation area ER may be any grade which is not a barrier to the movement of cells towards the evaluation region ER from outside evaluation area ER, for example, it may be set to the extent more than 100 [mu] m. The distance L _BM from the boundary mark BM to evaluation area ER, for example, may be set to about 30 [mu] m. Incidentally, in cell behavior evaluation substrate 10 according to this embodiment, the adjacent evaluation area ER _M, a portion of the ER M + _1, identification mark IM and boundary marks corresponding to each evaluation area ER _M, ER _M + ₁ It is preferable that the identification mark IM and the boundary mark BM are provided so that the BM is within the field of view of the inverted microscope at a high magnification. A part of the evaluation areas ER _M and ER _{M + 1} adjacent to each other in the field of view of the inverted microscope at a high magnification, and the identification mark IM and the boundary mark BM are accommodated, so that the fine concavo-convex pattern 12 having different dimensions and intervals can be formed. The behavior of the cells in the formed evaluation areas ER _M and ER _{M + 1} can be observed simultaneously and in detail. Specifically, the size of the field of view of the inverted microscope at a high magnification (objective X10) is 0.5 mm × 0.5 mm, a part of two evaluation areas and two identification marks IM in the field of view. The identification mark IM and the boundary mark BM are preferably provided so that at least one boundary mark BM is included.

  As shown in FIG. 2, it is preferable that the top part 12a of the fine concavo-convex pattern 12 is located on the same plane as the first surface 11a of the base part 11 outside the evaluation region group GR. When the top portion 12a of the fine concavo-convex pattern 12 is located on a plane different from the first surface 11a of the base portion 11, the cell moves to the evaluation region group GR (inside the evaluation region ER) when it exists outside the evaluation region group GR. It may be difficult to evaluate the behavior of the cells.

  The height (aspect ratio) of the fine concavo-convex pattern 12 is not particularly limited, and can be appropriately set according to the dimensions of the fine concavo-convex pattern 12. Usually, the height of the fine concavo-convex pattern 12 can be set so that the aspect ratio of the fine concavo-convex pattern 12 is about 0.1 to 5, preferably about 0.3 to 3.

  The region outside the evaluation region ER on the first surface 11a of the base portion 11 is an adhesive when attached to the outer surface of the bottom portion 2a of the container body 2 in which a through hole 2c is formed in the approximate center of the bottom portion 2a described later. Is configured as a region to be coated.

According to the cell behavior evaluation substrate 10 according to the present embodiment having the above-described configuration, the fine concavo-convex pattern 12 having different dimensions is formed in each of the evaluation regions ER _{1 to} ER _N. It is possible to accurately evaluate the behavior of the cells according to the dimensions, that is, the dimensions of the fine concavo-convex pattern 12 where the cells exhibit good adhesion.

[Method for producing substrate for cell behavior evaluation]
The cell behavior evaluation substrate 10 having the above-described configuration can be manufactured as follows. FIG. 4 is a process flow diagram showing a manufacturing process of the cell behavior evaluation substrate according to this embodiment in a cut end view, and FIG. 5 is a manufacturing process of the cell behavior evaluation substrate according to this embodiment. FIG. 5 is a process flow diagram showing each step (each step of the transparent substrate polishing method) following the step shown in FIG. 4 in a cut end view.

  First, a transparent substrate 31 having a first surface 31a and a second surface 31b opposite to the first surface 31a and having a hard mask layer 41 and a resist film 42 formed in that order on the first surface 31a is prepared. (See FIG. 4A).

As the transparent base material 31, the constituent material of the cell behavior evaluation substrate 10 according to this embodiment (for example, quartz glass, synthetic quartz glass, soda glass, fluorite, calcium fluoride, magnesium fluoride, acrylic glass, borosilicate A substrate made of glass material such as glass; transparent material such as resin material such as polycarbonate, polypropylene, polyethylene, polystyrene, and other polyolefins) can be used. The transparent substrate 31 has a thickness exceeding 0.5 mm, and usually has a thickness of about 1.0 to 6.35 mm.
In the present embodiment, the transparent base material 31 is a multi-faced transparent base material in which regions corresponding to the plurality of cell behavior evaluation substrates 10 are allocated on the first surface 31a side, and will be described later (FIG. 5 ( Each cell behavior evaluation substrate 10 is manufactured by being cut in step D).

  As a material constituting the hard mask layer 41, for example, metal chromium, chromium oxide, chromium nitride, chromium oxynitride, or the like can be used. The hard mask layer 41 is a layer for forming a hard mask pattern 44 used for etching the transparent substrate 31 in a process (see FIG. 4C) described later. Therefore, it is comprised by the material which considered the etching selection ratio with the material which comprises the transparent base material 31, and the aspect ratio of the nano uneven | corrugated pattern 12 in the board | substrate 10 for cell behavior evaluation. For example, when the transparent base material 31 is made of quartz glass, the hard mask layer 41 is preferably made of metal chromium or the like.

  The resist material constituting the resist film 42 is not particularly limited, and a conventionally known energy ray sensitive resist material (for example, an electron beam sensitive resist material, an ultraviolet sensitive resist material, etc.) or the like may be used. it can.

Next, the resist film 42 is patterned to form a resist pattern 43 corresponding to the fine uneven pattern 12 formed in each of the evaluation regions ER _{1 to} ER _N of the cell behavior evaluation substrate 10 on the first surface of the transparent substrate 31. in 31a side is formed in a region corresponding to each evaluation area ER ₁ to Er _N of cell behavior evaluation board 10 (see FIGS. 1 and 2) (see FIG. 4 (B)).

  The method for forming the resist pattern 43 is not particularly limited. For example, the resist pattern 43 can be formed by an electron beam lithography method, a photolithography method, a nanoimprint lithography method, or the like.

  Subsequently, the hard mask layer 41 is etched by dry etching using the resist pattern 43 as a mask to form a hard mask pattern 44 (see FIG. 4C). Then, using the hard mask pattern 44 as a mask, the first surface 31a of the transparent substrate 31 is etched to form the fine concavo-convex pattern 12, and then the hard mask pattern 44 is removed (see FIG. 4D).

  Thus, the transparent base material 31 in which the fine uneven pattern 12 is formed on the first surface 31a side is polished from the second surface 31b side so that the thickness of the transparent base material 31 is 0.5 mm or less. The method for polishing the transparent substrate 31 can be carried out as follows. FIG. 5 is a process flow schematically showing the steps of the method for polishing the transparent substrate 31 (the transparent substrate 31 having the fine concavo-convex pattern 12 formed on the first surface 31a side) in this embodiment in a cut end view. FIG.

  First, the metal-containing film 51 is formed on the first surface 31a so as to cover the entire surface of the first surface 31a of the transparent substrate 31 on which the fine uneven pattern 12 is formed on the first surface 31a (FIG. 5 ( A)).

  Examples of the material constituting the metal-containing film 51 include metal materials such as chromium and copper, oxides and nitrides of the metals, and the like. Considering that the metal-containing film 51 is peeled off by an etching process after a polishing step (see FIG. 5C) described later (see FIG. 5D), when using quartz glass as the transparent substrate 31, From the standpoint of selectivity, etc., it is preferable to use chromium, chromium oxide or the like that can be easily peeled as a material constituting the metal-containing film 51.

  The thickness of the metal-containing film 51 (thickness on the top 12a of the fine concavo-convex pattern 12) is preferably 50 nm or more, and preferably 50 to 200 nm. In the present embodiment, the transparent substrate 31 is polished from the second surface 31b side until the thickness of the transparent substrate 31 becomes 0.5 mm or less (see FIG. 5C). As the thickness of the material 31 decreases, the strength of the transparent substrate 31 decreases. Further, as will be described later, the substrate for cell behavior evaluation 10 is manufactured by cutting the transparent base material 31 polished to a thickness of 0.5 mm or less into individual pieces (FIG. 5D, (See (E)). Therefore, if the thickness of the metal-containing film 51 is less than 50 nm, the reinforcing effect of the transparent base material 31 becomes insufficient, and the transparent base material 31 may be damaged in a polishing process or a cutting process.

  The method for forming the metal-containing film 51 on the first surface 31a is not particularly limited, and for example, a known film forming method such as a sputtering method, a CVD method, a PVD method, or a vacuum evaporation method may be employed. it can.

  Next, an organic film 52 is formed over the metal-containing film 51 (see FIG. 5B). In a polishing step (see FIG. 5C), which will be described later, fine particles and the like are generated. By forming the organic film 52 on the metal-containing film 51, the particles and the like are removed in the process of removing the organic film 52. Since the particles can be removed at the same time, the particles can be prevented from adhering to the fine concavo-convex pattern 12.

  The material constituting the organic film 52 is not particularly limited, and for example, a material similar to the material constituting the resist film 42 (see FIG. 4A) described above can be used.

  The method for forming the organic film 52 on the metal-containing film 51 is not particularly limited. For example, after applying a material constituting the organic film 52 by a known coating method such as a spin coating method, the material It is possible to employ a method of curing

  The thickness of the organic film 52 is not particularly limited, and it is possible to prevent fine particles or the like that may be generated in a polishing step (see FIG. 5C) described later from adhering to the fine uneven pattern 12. Any thickness may be used. For example, the thickness of the organic film 52 is about 200 to 500 nm.

  Subsequently, the transparent substrate 31 on which the organic film 52 is formed is polished from the second surface 31b side until the thickness becomes 0.5 mm or less (see FIG. 5C). Since the metal-containing film 51 is formed on the first surface 31a side, the transparent substrate 31 can be polished until the thickness becomes 0.5 mm or less without damaging the transparent substrate 31. In addition, since the organic film 52 is formed on the metal-containing film 51, it is possible to prevent particles or the like generated in the polishing process from adhering to the fine concavo-convex pattern 12.

  The method for polishing the transparent substrate 31 is not particularly limited, and examples thereof include chemical mechanical polishing (CMP) and mechanical polishing using a rotary polishing disk. Preferably, after polishing (primary polishing) to a thickness of more than 0.5 mm to about 1 mm or less by mechanical polishing treatment, polishing (secondary polishing) is performed to a thickness of 0.5 mm or less by CMP.

  The organic film 52 and the metal-containing film 51 are peeled from the first surface 31a side of the transparent substrate 31 that is polished in this way and has a thickness of 0.5 mm or less. Thereby, the cell behavior evaluation substrate 10 according to the present embodiment can be manufactured (see FIG. 5D).

  The method for removing the organic film 52 and the metal-containing film 51 is not particularly limited. For example, the method for dissolving and removing the organic film 52 with a cleaning solution containing sulfuric acid or the like, the metal constituting the metal-containing film 51 Examples include a method of removing the metal-containing film 51 using an etchant that can etch the material.

  According to the polishing method and the manufacturing method of the cell behavior evaluation substrate 10 including the same in the present embodiment, the thickness 0 is obtained without damaging the transparent substrate 31 in which the nano uneven pattern 12 is formed on the first surface 31a side. The substrate 10 for cell behavior evaluation that can be polished to 5 mm or less and can be observed from the second surface 11b side using an inverted microscope can be easily manufactured.

[Evaluation container]
The cell behavior evaluation substrate 10 having the configuration described above observes cells to be evaluated, which are present on the fine concavo-convex pattern 12 on the first surface 11a side, from the second surface 11b side using an inverted microscope. Although used for evaluating the behavior of cells, it is preferably used in the form of an evaluation container described later. FIG. 6 is a plan view (top view) showing a schematic configuration of the evaluation container in the present embodiment, and FIG. 7 shows a schematic configuration of the evaluation container in the present embodiment, taken along the line II-II in FIG. It is an end view.

  As shown in FIGS. 6 and 7, the evaluation container 1 in the present embodiment has a bottom 2a and a peripheral wall 2b continuous to the outer peripheral edge of the bottom 2a, and a through hole 2c is formed at substantially the center of the bottom 2a. The container main body 2 thus formed, and the cell behavior evaluation substrate 10 according to this embodiment attached to the bottom 2a of the container main body 2 are provided.

  In the present embodiment, the container body 2 is a petri dish-like container having a through hole 2c formed in the approximate center of the bottom 2a and opening upward. However, as long as the through hole is formed in the bottom, such a mode It is not limited to. For example, a flask-like container having a through-hole formed in the bottom may be used.

  The size of the container body 2 in plan view is not particularly limited, and is preferably the same diameter as a petri dish generally used in cell culture or the like, for example, about 30 to 60 mm in diameter. Moreover, the thickness of the bottom part 2a of the container main body 2 is about 0.8-1.5 mm.

  The container body 2 may be made of synthetic resin or glass. Further, the peripheral wall 2b and the bottom 2a may be made of different materials (for example, the peripheral wall 2b is made of synthetic resin and the bottom 2a is made of glass or the like).

  The size of the through hole 2c is not particularly limited, but is preferably a size that can physically include the evaluation region group GR of the first surface 11a of the cell behavior evaluation substrate 10. For example, The diameter is about 10 to 30 mm.

In the present embodiment, the substrate for cell behavior evaluation 10 is exposed so that the evaluation region group GR is positioned in the container body 2, that is, the fine uneven patterns 12 in all the evaluation regions ER _{1 to} ER _N are exposed from the through holes 2 c. In this way, the container body 2 is attached to the outer surface of the bottom 2b. In the plan view (top view) of the evaluation container 1 (container body 2), the cell behavior evaluation substrate 10 is such that the evaluation region group GR (all the evaluation regions ER _{1 to} ER _N ) overlaps the through hole 2c. In other words, the evaluation region group GR (all evaluation regions ER _{1 to} ER _N ) is physically attached to the through hole 2 and attached to the outer surface of the bottom 2 a of the container body 2.

  The cell behavior evaluation substrate 10 is attached to the outer surface of the bottom 2a of the container body 2 so as to close the through hole 2c. That is, the bottom surface of the container body 2 is configured by the bottom portion 2a in which the through hole 2c is formed and the cell behavior evaluation substrate 10 that closes the through hole 2c.

  The cell behavior evaluation substrate 10 is preferably attached by adhering to the vicinity of the periphery of the through hole 2c on the outer surface of the bottom 2a of the container body 2 via an adhesive (not shown). Such an adhesive is not particularly limited, and has a high adhesive force to the cell behavior evaluation substrate 10 and the constituent material of the bottom 2a of the container body 2, and is accommodated in the evaluation container 1. It is preferable that it has a sealing property (liquid tightness) with respect to a culture solution and the like and does not adversely affect cells (no toxicity etc.). As the adhesive, for example, a deoxime-type silicone-based adhesive can be used.

According to the evaluation container 1 in the present embodiment having the above-described configuration, the cell behavior evaluation substrate 10 having a thickness T _{11 of} 0.5 mm or less is placed on the outer surface of the bottom 2a of the container body 2 in the entire evaluation region ER _1. The cells existing on the fine concavo-convex pattern 12 are observed at high magnification using an inverted microscope by attaching ~ ER _N so as to be physically included in the through hole 2c of the container body 2 Can do. In addition, since the cells exhibit good adhesion to the fine concavo-convex pattern 12 of any size formed in any of the evaluation regions ER, the adhesion depending on the size of the fine concavo-convex pattern 12 is evaluated. can do.

[Method for producing evaluation container]
The evaluation container 1 described above prepares a container body 2 having a bottom 2a and a peripheral wall 2b continuous to the outer peripheral edge of the bottom 2a, and having a through hole 2c formed in the approximate center of the bottom 2a. Manufactured by attaching the cell behavior evaluation substrate 10 to the outer surface of the bottom 2a of the main body 2 via an adhesive applied to the outer peripheral region of the evaluation region group GR of the first surface 11a of the cell behavior evaluation substrate 10. Can be done.

At this time, the evaluation region group GR (all evaluation regions ER _{1 to} ER _N ) in the cell behavior evaluation substrate 10 is exposed from the through hole 2c so as to block the through hole 2c of the container body 2. The cell behavior evaluation substrate 10 is attached to the bottom 2 a of the container body 2. Thus, all of the evaluation area ER ₁ to Er fine uneven pattern 12 formed on the _N can be positioned in the container body 2.

  According to the present embodiment, the evaluation container 1 can be manufactured simply by adhering the cell behavior evaluation substrate 10 so as to block the through hole 2c formed at the approximate center of the bottom 2a of the container body 2. The evaluation container 1 can be easily manufactured.

[Evaluation method]
The above-described evaluation container 1 is used for observing cells to be evaluated and evaluating the behavior of the cells. Hereinafter, a method for observing cells using the evaluation container 1 and evaluating the behavior of the cells will be described. FIG. 8 is a schematic diagram showing an evaluation method in the present embodiment.

  As shown in FIG. 8, in this embodiment, the cells 71 are seeded in the evaluation container 1, and the evaluation container 1 is placed on the stage 61 of an inverted microscope (not shown). Since the opening 61 having a predetermined size is formed in the stage 61 of the inverted microscope, the evaluation container 1 is placed on the stage 61 so that the evaluation region group GR of the evaluation container 1 is positioned in the opening 62. Placed on.

  Then, the objective lens 63 of the inverted microscope is brought close to the second surface 11b side of the cell behavior evaluation substrate 10 bonded to the bottom 2a of the evaluation container 1, and the cells 71 are placed on the second surface at a desired magnification. Observe from the 11b side.

At this time, the cells 71 in the evaluation container 1 are formed in the evaluation regions ER _{1 to} ER _N of the evaluation region group GR, and the fine concavo-convex pattern having a predetermined size among the fine concavo-convex patterns 12 having different dimensions. 12 shows good adhesion. Therefore, as in this embodiment, by observing the cells 71 from the second surface 11b side using an inverted microscope, it is possible to easily evaluate the adhesiveness of the cells 71 according to the dimensions of the fine concavo-convex pattern 12. it can.

Moreover, according to the evaluation method in the present embodiment, the thickness T _{11 of the} cell behavior evaluation substrate 10 attached to the bottom 2a of the evaluation container 1 is 0.5 mm or less, so that the fine uneven pattern 12 is formed. The distance between the existing cell 71 and the objective lens 63 can be observed with an extremely short distance. Therefore, the cell 71 can be observed at an extremely high magnification.

  The embodiment described above is described for facilitating understanding of the present invention, and is not described for limiting the present invention. Therefore, each element disclosed in the above embodiment is intended to include all design changes and equivalents belonging to the technical scope of the present invention.

In the above embodiment, although the evaluation area ER ₁ to Er _N of cell behavior evaluation substrate 10 is parallel to the dimension order of the fine concavo-convex pattern 12 formed in each evaluation area ER ₁ to Er _N, the present invention Is not limited to such an embodiment.

In the above embodiment, the boundary between the adjacent evaluation regions ER _M and ER _{M + 1} of the cell behavior evaluation substrate 10 is indicated by the boundary mark BM. However, the present invention is not limited to such a mode. Absent. For example, as shown in FIG. 9, there may be a wall 13 that separates adjacent evaluation regions ER _M and ER _{M + 1} . In this case, the short dimension W ₁₃ of the wall portion 13 may be set to recognize the wall portion 13 at an inverted microscope used during cell observation (discrimination) can be sized (e.g., about 10 [mu] m).

In the above embodiment, although the manner in which the pillar-like and line-and-space-shaped fine uneven pattern 12 to each evaluation area ER ₁ to Er _N evaluation region group GR are formed has been described, the present invention is the It is not limited to such an aspect. For example, a mode in which pillar-shaped fine concavo-convex patterns 12 or line-and-space fine concavo-convex patterns 12 having different dimensions are formed in each of the evaluation regions ER _{1 to} ER _N of the evaluation region group GR may be employed.

In the above embodiment, the boundary mark BM indicating the boundary between the adjacent evaluation regions ER _M and ER _{M + 1} has a triangular shape as a whole, and the fine concavo-convex pattern 12 formed in each of the evaluation regions ER _{1 to} ER _N. Although the identification mark IM indicating the dimension has a character (alphanumeric) shape, the present invention is not limited to such a mode, as long as the functions of the boundary mark BM and the identification mark IM can be achieved. Any shape is possible.

  In the above embodiment, the evaluation container 1 has the cell behavior evaluation substrate 10 attached to the outer surface of the bottom 2a of the container body 2, but the present invention is not limited to such an embodiment. For example, an adhesive is applied to the second surface 11b side of the cell behavior evaluation substrate 10, and the cell behavior evaluation substrate 10 is adhered to the outer peripheral edge region of the through hole 2c of the bottom 2a of the container body 2. The evaluation substrate 10 may be attached in the container body 2.

  In the said embodiment, although the board | substrate 10 for cell behavior evaluation was demonstrated and mentioned as an example using the aspect attached to the said bottom part 2a of the container main body 2 by which the through-hole 2c is formed in the bottom part 2a, this invention is this. It is not limited to such an aspect. The cell behavior evaluation substrate 10 may be used for observation / evaluation of cells to be evaluated as they are.

  In the above embodiment, after forming the fine uneven pattern 12 on the first surface 31a side of the transparent substrate 31, the metal-containing film 51 is formed after removing the hard mask pattern 44. It is not limited to such an aspect. For example, the metal-containing film 51 may be formed without removing the hard mask pattern 44. In this case, since it is preferable that the hard mask pattern 44 can be removed together with the metal-containing film 51 in the step of peeling the metal-containing film 51 (see FIG. 5D), the hard mask pattern 44, the metal-containing film 51, and Are preferably made of a material that can be etched with the same etching solution.

  In the above-described embodiment, an example in which one cell behavior evaluation substrate 10 is manufactured using a transparent base material having a size corresponding to one cell behavior evaluation substrate 10 has been described. Is not limited to such an embodiment. For example, the cell behavior evaluation substrate 10 may be manufactured using the multi-faced transparent base material 31 in which regions corresponding to the plurality of cell behavior evaluation substrates 10 are allocated on the first surface 31a side. In this embodiment, the metal-containing film 51 may be peeled after the transparent substrate 31 after polishing is cut into individual pieces (see FIG. 5D).

  EXAMPLES Hereinafter, although an Example etc. are given and this invention is demonstrated further in detail, this invention is not limited at all by the following Example etc.

[Production example of substrate for cell behavior evaluation]
A quartz glass substrate (152 mm × 152 mm, thickness: 6.35 mm) is prepared as a transparent base material 31 having a hard mask layer 41 made of Cr having a thickness of 6 nm provided on the first surface 31a. A resist (product name: SEBP-9902, manufactured by Shin-Etsu Chemical Co., Ltd.) was applied on the hard mask layer 41 to form a resist film 42. A pillar-shaped resist pattern 43 was formed on the resist film 42 using an electron beam drawing apparatus. Further, five evaluation regions ER _{2 to} ER ₆ (size of each evaluation region ER _{2 to} ER ₆ : 500 μm × 1500 μm) are set in a 14 mmφ region on the first surface 31 a of the quartz glass substrate 31. Pillar-shaped resist patterns 43 having different intervals and dimensions in the regions (intervals and dimensions of the resist pattern 43: evaluation region ER ₂ = 100 nm, evaluation region ER ₃ = 150 nm, evaluation region ER ₄ = 200 nm, evaluation region ER ₅ = 300 nm, Evaluation region ER ₆ = 500 nm), the resist pattern of the identification mark IM corresponding to each of the evaluation regions ER _{2 to} ER ₆ (character string of each identification mark IM: 100, 150, 200, 300, 500) and each boundary mark BM A resist pattern was formed (see FIG. 4B). The size of the identification mark IM is 100 μm, the size of the boundary mark BM is 50 μm, and the dimension of the concavo-convex structure of the line and space shape constituting each identification mark IM and each boundary mark BM is 2 μm.

Next, the hard mask layer 41 was dry-etched (etching gas: Cl ₂ + O ₂ ) using the resist pattern 43 as a mask, and the remaining resist pattern 43 was removed to form a hard mask pattern 44.

The quartz glass substrate 31 is etched using the hard mask pattern 44 formed as described above as a mask, and predetermined intervals and dimensions are set in the evaluation regions ER _{2 to} ER ₆ of the first surface 31 a of the quartz glass substrate 31. Pillar-shaped fine concavo-convex pattern 12 (interval and dimension of fine concavo-convex pattern 12 (measured using a scanning electron microscope (product name: LWM9000)): evaluation region ER ₂ = 100 nm, evaluation region ER ₃ = 150 nm, evaluation region ER ₄ = 200 nm, evaluation region ER ₅ = 300 nm, evaluation region ER ₆ = 500 nm) and each identification mark IM and each boundary mark BM. The height of the pillar-shaped fine concavo-convex pattern 12 was 100 to 200 nm although a measurement error was expected. Finally, the hard mask pattern 44 was peeled off to manufacture the cell behavior evaluation substrate 10.

[Test Example 1]
A perforated dish (35 mmφ) having a 14 mmφ hole formed on the bottom surface is prepared, and cell behavior is performed so that each evaluation region ER _{2 to} ER ₆ on which the fine uneven pattern 12 is formed is exposed from the hole on the bottom surface. The evaluation substrate 10 was attached to the outside of the bottom surface of the perforated dish.
Next, 2 × 10 ⁵ human fibroblasts (NHDF) were seeded in the perforated dish, and the cells were brought into contact with the fine concavo-convex pattern 12 of the cell behavior evaluation substrate 10. Then, the cells were cultured for 24 hours in a 37 ° C. incubator. As a culture solution, Dulbecco's modified Eagle medium (DMEM medium) supplemented with kanamycin was used.

After the culture, an evaluation test for evaluating the relationship between the interval and size of the fine uneven pattern 12 and the state of cell adhesion was performed based on an image obtained by imaging the vicinity of each of the evaluation regions ER _{2 to} ER ₆ . The results are shown in FIG.

  FIG. 10A is a photomicrograph of the vicinity of the boundary between the evaluation region a and the evaluation region b in which the interval and dimensions of the pillar-shaped fine unevenness pattern 12 are different. As shown in FIG. 10 (A), it was clear that the cell density in the evaluation region b was larger than the cell density in the evaluation region a. FIG. 10B is a photomicrograph of the vicinity of the boundary between the evaluation region c and the evaluation region d different from the evaluation region a and the evaluation region b in FIG. As shown in FIG. 10B, it was clear that the cell density of the evaluation region c was larger than the cell density of the evaluation region d even at the boundary of another evaluation region.

[Test Example 2]
A perforated dish (35 mmφ) having a 14 mmφ hole formed on the bottom surface is prepared, and cell behavior is performed so that each evaluation region ER _{2 to} ER ₆ on which the fine uneven pattern 12 is formed is exposed from the hole on the bottom surface. The evaluation substrate 10 was attached to the outside of the bottom surface of the perforated dish.

Next, 1 × 10 ⁴ human fibroblasts (NHDF) were seeded in the perforated dish, and the cells were brought into contact with the fine concavo-convex pattern 12 of the cell behavior evaluation substrate 10. Then, the cells were cultured for 24 hours in a 37 ° C. incubator. As a culture solution, a DMEM medium supplemented with kanamycin was used. The results are shown in FIG.

FIG. 11 is a photograph of the evaluation regions ER _{3 to} ER ₅ captured using an inverted microscope.
As shown in FIG. 11, a boundary mark BM indicating the boundary between adjacent evaluation regions ER _M and ERM _{+ 1} , and an identification mark indicating the dimensions of the fine uneven pattern 12 formed in each of the evaluation regions ER _{3 to} ER ₅ It was confirmed that IM _{3 to} IM ₅ can be identified.

From the test examples 1 and 2, the intervals and dimensions of the fine unevenness pattern 12 formed in each of the evaluation regions ER _{1 to} ER _{N of} the cell behavior evaluation substrate 10 are set within an appropriate range, and have different intervals and dimensions. It was confirmed that the behavior of cells can be accurately evaluated by forming the fine uneven patterns 12 adjacent to each other.

  The present invention is useful for evaluating cell behavior in biotechnology-related fields such as drug development and regenerative medicine.

DESCRIPTION OF SYMBOLS 1 ... Evaluation container 2 ... Container main body 2a ... Bottom part 2b ... Peripheral wall part 2c ... Through-hole 10 ... Substrate 11 for cell behavior evaluation ... Base 11a ... First surface 11b ... Second surface 12 ... Fine uneven pattern ER ... Evaluation region

Claims (14)

A cell behavior evaluation substrate for evaluating cell behavior on a fine uneven pattern,
A base having a first surface and a second surface opposite the first surface;
The fine unevenness is formed in each of the first to Nth (N is an integer of 2 or more) evaluation regions, which are set in a part of the first surface of the base and are sequentially arranged along a predetermined direction. The first to Nth uneven patterns as patterns are formed,
The Mth concavo-convex pattern formed in the Mth (M is an integer from 1 to N-1) evaluation region and the M + 1th concavo-convex pattern formed in the M + 1 evaluation region are 100 to 500 nm. A cell behavior evaluation substrate having dimensions within a range and different from each other to such an extent that the behavior of the cells can be evaluated.   The identification mark including the information regarding the dimension of the said 1st-Nth uneven | corrugated pattern is provided in the vicinity of each of the said 1st-Nth evaluation area | region in the said 1st surface. Substrate for cell behavior evaluation.   3. The cell behavior evaluation substrate according to claim 1, wherein the dimensions of the first to Nth uneven patterns change stepwise in the order in which the first to Nth evaluation regions are arranged in parallel.   4. The M-th uneven pattern formed in the M-th evaluation region and the M + 1-th uneven pattern formed in the M + 1 evaluation region are continuous. The cell behavior evaluation substrate according to claim 1.   The cell according to any one of claims 1 to 4, wherein a mark indicating a boundary between the Mth evaluation region and the M + 1th evaluation region on the first surface is provided in the vicinity of the boundary. Behavior evaluation board.   The top of the first to Nth uneven patterns is located on the same plane as a region other than the first to Nth evaluation regions on the first surface. Substrate for cell behavior evaluation.   The cell behavior evaluation substrate according to any one of claims 1 to 6, wherein the first to Nth uneven patterns are in the form of pillars or lines and spaces.   The first to Nth evaluation regions are all sized to fit within a field of view of an optical microscope for observing the behavior of the cells on the first to Nth uneven patterns. The cell behavior evaluation substrate according to any one of 1 to 7. A container body having a bottom formed with a through-hole and a peripheral wall continuous to the outer periphery of the bottom;
The cell behavior evaluation substrate according to any one of claims 1 to 8, wherein the substrate is attached to the bottom.
The cell behavior evaluation substrate is attached to the bottom so that the first to Nth uneven patterns overlap the through-hole in a plan view of the container body and are positioned in the container body. ,
The cell behavior evaluation container, wherein the through hole is closed with the cell behavior evaluation substrate.   The cell behavior evaluation substrate is attached to the bottom so that the first to Nth uneven patterns are physically included in the through-hole in a plan view of the container body. The container for evaluating cell behavior according to claim 9.   11. The cell behavior evaluation substrate is attached to an outer surface of the bottom portion of the container body so as to expose the first to Nth uneven patterns from the through holes. The container for cell behavior evaluation described in 1.   The cell behavior evaluation container according to any one of claims 9 to 11, wherein the cell behavior evaluation substrate is attached to the bottom through an adhesive. Seeding cells to be evaluated in the cell behavior evaluation container according to any one of claims 9 to 12,
A cell behavior evaluation method, comprising: observing the evaluation target cells on the first to Nth uneven patterns from the second surface side of the cell behavior evaluation substrate and evaluating the behavior of the cells.   The cell behavior evaluation method according to claim 13, wherein the cell to be evaluated is observed using an inverted microscope.