JP2019024350A - Cell culture vessel, cell sheet production method, and cell culture vessel production method - Google Patents

Abstract

To provide a cell culture vessel that can stably preserve a cell sheet after a stimulus has been applied to a stimulus responsive polymer and with which a cell sheet can be easily recovered.SOLUTION: The present embodiment is a cell culture vessel comprising a sheet formation region that includes a stimulus responsive region in which the degree of cell adhesiveness changes in response to a stimulus and at least one cell adhesiveness region having cell adhesiveness arranged so as to surround and be contiguous with the stimulus responsive region. There is at least one adhesion boundary at the boundary where the stimulus responsive region is contiguous with the cell adhesion region, and the stimulus responsive region is a region that is in continuous contact with each adhesion boundary.SELECTED DRAWING: Figure 11

Description

  The present disclosure relates to a cell culture container, a method for producing a cell sheet, and a method for producing a cell culture container.

  One technique for regenerative medicine is to transplant cells. Examples of the cell form for transplantation include a cell sheet obtained by culturing cells in a sheet form. The cell sheet is a sheet-like cell aggregate in which cells are connected to each other by intercellular bonding. The cell sheet can be formed by culturing cells on a substrate such as a petri dish.

  As a method for forming a cell sheet, a method using a stimulus-responsive layer containing a stimulus-responsive polymer in which the degree of adhesion to cells is changed by stimulation such as light or temperature is known. Examples of the stimulus responsive polymer include a temperature responsive polymer. This temperature-responsive polymer has the property that its conformation changes when the temperature is lowered. In a method for forming a cell sheet using a temperature-responsive polymer, a container in which a layer containing a temperature-responsive polymer is formed on a substrate is used as a culture layer for culturing cells on the surface. A cell sheet can be formed by culturing cells on the layer containing the temperature-responsive polymer while adhering them at a cell culture temperature (for example, 37 ° C.). Then, after the cells are cultured to form a cell sheet, when the temperature of the layer containing the temperature-responsive polymer is lowered, the conformation of the temperature-responsive polymer changes, and the cell sheet cannot adhere to the surface. As a result, the cell sheet can be peeled off and collected from the layer containing the temperature-responsive polymer.

  Examples of a document disclosing a method for forming a cell sheet using a stimulus-responsive polymer include the following patent documents.

  In Patent Document 1, skin cells are placed below the upper critical solution temperature or higher than the lower critical solution temperature on the cell culture support in which the substrate surface is coated with a polymer having an upper or lower critical solution temperature of 0 to 80 ° C. with respect to water. A skin cell culturing method is disclosed, wherein the cultured skin cells are peeled off by culturing at a temperature of not less than the upper critical solution temperature or lower than the lower critical solution temperature.

  In patent document 2, the cell addition process which adds the culture solution containing a cell to the culture container which has an inner bottom face which can change to cell non-adhesiveness, and the cell added to the culture container toward the inner bottom face direction. A cell culture step in which cell culture is performed under the condition that cell-cell adhesion is formed while acting a centrifugal force, and a tissue is formed by adhering cells, and the tissue obtained in the cell culture step is the inner bottom surface A method for producing a tissue is disclosed, which includes a peeling step of peeling and recovering from a tissue.

  In Patent Document 3, cells are cultured on a cell culture substrate containing an iron-crosslinked alginate gel, and the cell culture substrate containing an iron-crosslinked alginate gel is dissolved using a chelating agent that specifically binds to iron. And a method for producing a cell sheet, comprising a step of obtaining cultured cells as a cell sheet.

  Patent Documents 4 and 5 disclose cell culture substrates that specify the arrangement of the stimulus-responsive region, the cell adhesive region, and the cell non-adhesive region.

  In Patent Document 4, a stimulation-responsive region in which the degree of cell adhesion changes by stimulation, a cell-adhesive region having cell adhesion, and a cell non-adhesion region having cell non-adhesion are provided, and the stimulation An adhesive boundary that is a boundary portion between the responsive region and the cell adhesive region, and is a boundary portion between the stimulus responsive region and the cell non-adhesive region at least at a part of the outer periphery of the stimulus responsive region A cell culture substrate characterized by having a non-adhesive boundary is disclosed.

  In Patent Document 5, a stimulation-responsive region in which cell adhesion changes by stimulation, a cell adhesion region having cell adhesion properties, and a cell non-adhesion region having cell non-adhesion properties, the stimulation The responsive region is adjacent to the cell adhesive region in at least two places, and has at least two separated adhesion boundaries that are boundary portions of the stimulus responsive region and the cell adhesive region, and the stimulus responsive region However, there is disclosed a cell culture container characterized in that it is arranged so as to connect the adhesion boundaries, and the non-cell-adhesive region is arranged around the stimulus-responsive region.

JP 05-192138 A JP 2014-23540 A JP 2012-65632 A JP 2012-105608 A JP 2012-105609 A

  In Patent Documents 1 to 3, cell sheets are formed using various stimulus-responsive polymers. However, in these methods, when a predetermined stimulus is applied to the stimulus-responsive polymer, the cell sheet is completely detached from the culture surface. Since the cell sheet is fragile, once it is peeled off from the culture surface, the cell sheet is likely to be broken, and the shape is easily deformed by contraction due to tension. Therefore, the cell sheet needs to be held by another support immediately after being peeled from the base material. However, this operation is complicated, and in some cases, the cell sheet may be torn or deformed by this operation. Therefore, there is a demand for the development of a cell culture container that can stably hold the cell sheet on the culture surface even after stimulating the stimulus-responsive polymer and that can easily collect the cell sheet after the stimulus is applied. .

  In Patent Documents 4 and 5, it has not been studied from the viewpoint of the shape stability of the cell sheet after imparting a stimulus and the ease of collection of the cell sheet, and it does not sufficiently answer the above requirements.

  Then, this indication aims at providing the cell culture container which can hold | maintain a cell sheet stably on a culture surface even after provision of a stimulus, and can collect | recover a cell sheet easily.

  Thus, one embodiment of the present invention is described below.

(1) a stimulus-responsive region in which the degree of cell adhesion changes by stimulation;
At least one cell adhesive region having cell adhesion disposed adjacent to the stimulus responsive region so as to surround the stimulus responsive region;
A cell culture container having a sheet-forming region comprising:
There is at least one adhesion boundary that is a boundary portion where the stimulus-responsive area and the cell adhesion area are adjacent;
The stimulus responsive region is a continuous region in contact with each of the adhesion boundaries.
(2) The cell culture container according to (1), wherein the adhesion boundary is disposed in the vicinity of the outer periphery of the stimulus responsive region.
(3) The cell culture container according to (1) or (2), wherein a plurality of the adhesion boundaries exist.
(4) The cell culture container according to any one of (1) to (3), wherein there are at least four adhesion boundaries.
(5) An angle formed by two straight lines connecting the center of gravity of the stimulus responsive region and each of two points of one adhesion boundary, and the two points are set so that the angle becomes maximum. The cell culture container according to (3) or (4), wherein the sum of the angles θ corresponding to all of the adhesion boundaries is 90 ° or more when the selected angle is an angle θ.
(6) The cell culture container according to (5), wherein the sum of the angles θ is 180 ° or more.
(7) A region surrounded by two straight lines connecting the center of gravity of the stimulus-responsive region and each of two points of one adhesive boundary and the one adhesive boundary, and the area of the region is maximized. When the area of the region where the two points are selected is defined as area T, the total of the areas T corresponding to all of the adhesion boundaries is 0.2 times or more than the area of the stimulus-responsive region. The cell culture container according to any one of (3) to (6).
(8) The cell culture according to any one of (3) to (7), wherein the total length of the adhesion boundary is 0.2 times or more of the outer circumference of the stimulus-responsive region. container.
(9) The sheet forming region is a straight line extending from the center of gravity of the stimulus responsive region of the total number of the adhesion boundaries, and the stimulus responsive region is the total number of the adhesion boundaries so that the respective areas after division are equal. When divided into regions, the cell culture container according to any one of (3) to (8), which has an arrangement shape in which at least one adhesion boundary is in contact with each of the divided stimulus-responsive regions.
(10) The cell culture container according to (9), wherein the sheet forming region has an arrangement shape in which only one adhesion boundary is in contact with each of the divided stimulus-responsive regions.
(11) A straight line connecting one point of one adhesive boundary and one point of the adhesive boundary adjacent to the one adhesive boundary, and the one of the points so that the straight line is the shortest When each selected straight line is a straight line L, the area of the region surrounded by the straight line L corresponding to all the bonding boundaries and all the bonding boundaries is 0.6 times or more than the area of the sheet forming region. The cell culture container according to any one of (3) to (10).
(12) In (1) or (2), there is only one adhesive boundary, and the length of the adhesive boundary is not less than 0.7 times the outer peripheral length of the stimulus-responsive region. The cell culture container described.
(13) The cell culture container according to (12), wherein the cell adhesive region is disposed in contact with the entire outer periphery of the stimulus responsive region.
(14) The cell culture container according to any one of (1) to (13), wherein the ratio of the area of the stimulus responsive region to the area of the sheet forming region is 0.6 or more.
(15) The cell culture container according to any one of (1) to (14), wherein a cell non-adhesive region having cell non-adhesive properties is disposed outside the sheet forming region.
(16) The stimulus responsive region is composed of a stimulus responsive layer containing a stimulus responsive polymer,
The cell culture container according to any one of (1) to (15), wherein the stimulus-responsive layer is formed on a substrate.
(17) The cell culture container according to (16), wherein the stimulus-responsive polymer includes a temperature-responsive polymer having a lower critical solution temperature.
(18) The cell culture container according to (16) or (17), wherein the base material has cell adhesiveness, and the cell adhesive region is composed of a surface of the base material.
(19) The cell culture container according to (18), wherein the base material contains glass or plastic.
(20) A method for producing a cell sheet, comprising a step of culturing cells in the cell culture container according to any one of (1) to (19) to form a cell sheet.
(21) A method for producing the cell culture container according to (18) or (19),
Forming the stimulus-responsive layer on the cell-adhesive substrate;
Partially removing the stimulus responsive layer to expose the substrate;
A method for producing a cell culture container.

  According to the present disclosure, it is possible to provide a cell culture container that can stably hold the cell sheet on the culture surface even after the stimulus is applied and can easily collect the cell sheet.

It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is a schematic plan view for demonstrating the structural example of the sheet | seat formation area | region which the cell culture container of this embodiment has. It is the schematic for demonstrating the structural example of the cell culture container of this embodiment. It is the schematic for demonstrating the structural example of the cell culture container of this embodiment. It is the schematic for demonstrating the structural example of the cell culture container of this embodiment.

  Hereinafter, embodiments of the present invention will be described.

  The present embodiment includes a stimulus-responsive region in which the degree of cell adhesion changes due to stimulation, and at least one cell-adhesive region having cell adhesion disposed adjacent to the stimulus-responsive region. A cell culture container having a sheet-forming region, wherein there is at least one adhesion boundary which is a boundary portion where the stimulus-responsive region and the cell-adhesive region are adjacent to each other, and the stimulus-responsive region is It is a cell culture vessel, which is a continuous area in contact with each of the boundaries.

  According to this embodiment, it is possible to provide a cell culture container that can stably hold a cell sheet even after giving a stimulus to a stimulus-responsive region and can easily collect the cell sheet. In the cell culture container according to this embodiment, when a predetermined stimulus is applied to the stimulus responsive region, the stimulus responsive region becomes non-cell-adhesive and the cell sheet is peeled off from the stimulus responsive region. At this time, the cell sheet is not completely removed from the sheet forming region, and a part of the cell sheet is attached to the cell adhesive region. Thereby, a cell sheet can be stably hold | maintained on a culture surface. In addition, since the cell sheet is partially attached to the culture surface, it can be easily removed from the culture surface and recovered by a water flow such as a peeling operation using tweezers or pipetting.

  Stimulus responsive region is a region where the degree of cell adhesion changes due to stimulation, is a region that can be made cell-adhesive during cell culture, and can be made non-cell-adhesive by applying stimulus during cell sheet peeling operations It is. The stimulus responsive region is formed to be continuous with each of the adhesion boundaries.

  The cell adhesion region is a region having cell adhesion. Note that the cell adhesion region or the cell non-adhesion property of the cell adhesion region is not substantially changed by the stimulation. The cell adhesion region is disposed adjacent to the stimulus responsive region so as to surround the stimulus responsive region.

  The adhesion boundary is a boundary portion where the stimulus responsive region and the cell adhesive region are adjacent to each other. The adhesion boundary is preferably arranged near the outer periphery of the stimulus responsive region. There may be at least one adhesion boundary. Moreover, in this embodiment, it is preferable that there are at least two, that is, a plurality of adhesion boundaries, more preferably at least three, and even more preferably at least four. When there are a plurality of bonding boundaries, the plurality of bonding boundaries are separated from each other.

  In this specification, the outer periphery refers to an outer edge that defines a boundary with an outer region in one continuous region. Further, in this specification, the inner periphery refers to an inner outer edge that defines a boundary with an inner region in one continuous region, that is, a region surrounded by the region.

  The sheet forming region is a region where a cell sheet is formed by cell culture, and includes a stimulus responsive region and a cell adhesive region. A sheet non-forming portion may be disposed outside the sheet forming region. The sheet non-formation part is not particularly limited as long as the cell sheet connected to the cell sheet formed in the sheet formation region is not formed. For example, as the sheet non-forming part, a cell non-adhesive region having cell non-adhesiveness may be arranged. That is, the cell non-adhesive region may be disposed so as to contact the outer periphery of the sheet forming region. Further, as the sheet non-forming part, a side wall forming a culture space for holding the culture solution may be arranged. That is, the outer periphery of the sheet forming region may be in contact with the side wall that forms the culture space. Further, as the sheet non-forming part, for example, a culture space is present outside the sheet forming region due to the presence of a step or the like, and the cell sheet itself connected to the cell sheet formed in the sheet forming region is not formed. You can also

  At least one sheet forming region can be formed on the culture surface of the cell culture container, and a plurality of sheet forming regions may be formed on the culture surface. When a plurality of sheet forming areas are arranged on the culture surface, it is preferable that a cell non-adhesive area is arranged between the sheet forming areas.

  Hereinafter, the cell culture container of the present embodiment will be described with reference to the drawings.

  Note that the “center of gravity”, “angle”, or “area” described below can be obtained using image analysis software. The image analysis software is not particularly limited as long as these can be obtained, and examples thereof include “ImageJ”.

  One embodiment of the present invention has a cell adhesive region that surrounds a stimulus responsive region, and there is only one adhesive boundary that is a boundary portion adjacent to the stimulus responsive region and the cell adhesive region. An example of a configuration in which only one adhesion boundary exists is shown in FIG. In FIG. 1, the cell adhesive region is disposed in contact with the entire outer periphery of the stimulus responsive region, and there is only one adhesion boundary. The present embodiment is not limited to the case where the cell adhesive region is disposed in contact with the entire outer periphery of the stimulus responsive region. For example, in a part of the outer periphery of the stimulus responsive region, the cell adhesive region is It may not be arranged in contact.

  In FIG. 1, the code | symbol 1 shows a stimulus responsive area | region and the code | symbol 2 shows a cell-adhesive area | region. The cell adhesive region 2 is disposed adjacent to the stimulus responsive region 1 so that the entire outer periphery of the stimulus responsive region 1 is in contact with the cell adhesive region 2. Reference numeral 3 denotes an adhesion boundary which is a boundary portion where the stimulus responsive region 1 and the cell adhesive region 2 are adjacent to each other. In FIG. 1, there is one bonding boundary.

  In FIG. 2, there is one adhesion boundary, and the cell adhesion region is not disposed in contact with part of the outer periphery of the stimulus responsive region. In the case of this form, it is preferable that the cell adhesive region is disposed in contact with 70% or more of the entire outer periphery of the stimulus responsive region. That is, it is preferable that the length of the adhesion boundary is 0.7 times or more with respect to the length of the outer periphery of the stimulus responsive region. Further, the length of the adhesion boundary is more preferably 0.8 times or more, and further preferably 0.9 times or more. By providing a portion of the outer periphery of the stimulus-responsive region where the cell adhesive region is not in contact, the water flow caused by pipetting enters under the cell sheet, facilitating the peeling operation.

  A sheet formation region where a cell sheet is formed by cell culture includes a stimulus responsive region 1 and a cell adhesive region 2. The shape of the sheet forming region is not particularly limited. For example, FIG. 1A shows an example in which the shape of the sheet forming region is circular, and FIG. An example is shown.

  As shown in FIG. 3, the cell non-adhesive area | region which has cell non-adhesiveness may be arrange | positioned on the outer side of a sheet | seat formation area so that it may adjoin to a sheet | seat formation area. That is, the cell non-adhesive region may be disposed so as to contact the outer periphery of the sheet forming region. Moreover, the outer periphery of the sheet forming region may be in contact with a side wall that forms a culture space for holding a culture solution. Further, a step or the like may exist outside the sheet forming region, and a culture space may exist.

  Moreover, as shown in FIG. 4, in addition to the stimulus responsive region 1 surrounded by the cell adhesive region 2, a second stimulus responsive region (also referred to as an outer stimulus responsive region) 11 is a cell adhesive region. You may arrange | position adjacent to the outer side of 2. For example, the outer stimulus responsive region 11 may be disposed so as to partially contact the outer periphery of the cell adhesive region 2 or may be disposed so as to contact the entire outer periphery of the cell adhesive region 2. By providing this outer stimulus responsive region 11, when a stimulus is applied, a portion that rises at the end of the cell sheet is formed, so that the cell sheet can be easily peeled off. The area of the outer stimulus responsive region 11 is preferably 0.1% or more and 30% or less with respect to the area of the sheet forming region. Further, as shown in FIG. 5, in addition to the cell adhesive region 2 disposed adjacently so as to surround the stimulus responsive region 1, a second cell adhesive region (also referred to as an outer cell adhesive region) 12. May be arranged adjacent to the outside of the outer stimulus responsive region 11. For example, the outer cell adhesive region 12 may be disposed so as to partially contact the outer periphery of the outer stimulus responsive region 11 or may be disposed so as to contact the entire outer periphery of the outer stimulus responsive region 11. . By providing the outer cell adhesive region 12, the end of the cell sheet can be strongly adhered to the culture surface when a stimulus is applied. The area of the outer cell adhesive region 12 is preferably 10% or less with respect to the area of the sheet forming region. The outer stimulus responsive region 11 and the outer cell adhesive region 12 may be repeatedly provided outside the cell adhesive region 2.

  In addition, the shapes of the stimulus responsive region 1 and the cell adhesive region 2 are not particularly limited. In the present embodiment, the cell adhesive region 2 may be disposed in contact with the outer periphery of the stimulus responsive region 1. That's fine. 1A shows an example in which the shape of the stimulus responsive region 1 is circular, and FIG. 1B shows an example in which the shape of the stimulus responsive region 1 is square. 6 may be atypical.

  Further, in addition to the cell adhesive region 2 disposed adjacently so as to surround the stimulus responsive region 1, a further cell adhesive region may be disposed in the stimulus responsive region. By providing a further cell adhesion region, the adhesion of the cell sheet can be improved as necessary. In addition, a cell non-adhesive region may be disposed in the stimulus responsive region. Under the present circumstances, these cell adhesion area | regions and cell non-adhesion area | regions can be arrange | positioned in the range which does not inhibit the effect of this invention.

  One embodiment of the present invention has a cell adhesive region that surrounds a stimulus responsive region, and there are at least two adhesive boundaries that are border portions where the stimulus responsive region and the cell adhesive region are adjacent to each other. Moreover, it is preferable that there are at least three adhesion boundaries, and it is more preferable that there are at least four adhesion boundaries. The presence of a plurality of adhesion boundaries makes it easy to balance the stability of the cell sheet after applying the stimulus and the adhesive force with the sheet formation region. An example of the configuration of the sheet forming region of the embodiment in which at least two adhesion boundaries exist is shown in FIGS.

  FIG. 7 is a schematic plan view for explaining a configuration example of a sheet forming region included in the cell culture container of the present embodiment, and shows a form in which two adhesion boundaries exist. In FIG. 7, reference numeral 1 indicates a stimulus responsive area, and reference numerals 2a and 2b indicate cell adhesive areas. The cell adhesive regions 2a and 2b are arranged adjacent to each other so as to surround the stimulus responsive region 1. In the form of FIG. 7, the outer periphery of the stimulus responsive region 1 is partially in contact with the cell adhesive region. Reference numerals 3a and 3b denote adhesion boundaries which are boundary portions where the stimulus responsive region 1 and the cell adhesive regions 2a and 2b are adjacent to each other. In the form of FIG. 7, there are two adhesion boundaries, and one adhesion boundary is formed in each of two individual cell adhesion regions.

  FIG. 8 is a schematic plan view for explaining a configuration example of the sheet forming region included in the cell culture container of the present embodiment, and shows a form in which three adhesion boundaries exist. In FIG. 8, reference numeral 1 indicates a stimulus-responsive area, and reference numerals 2a, 2b, and 2c indicate cell adhesive areas. The cell adhesive regions 2a, 2b, and 2c are arranged adjacent to each other so as to surround the stimulus responsive region 1. In the form of FIG. 8, the outer periphery of the stimulus responsive region 1 is partially in contact with the cell adhesive region. Moreover, the code | symbol 3a, 3b, 3c shows the adhesion boundary which is a boundary part which the stimulus responsive area | region 1 and the cell adhesiveness area | region 2a, 2b, 2c adjoin. In the form of FIG. 8, there are three adhesion boundaries, and one adhesion boundary is formed in each of three individual cell adhesion regions.

  FIG. 9 is a schematic plan view for explaining a configuration example of a sheet forming region included in the cell culture container of the present embodiment, and shows a form in which four adhesion boundaries exist. In FIG. 9, reference numeral 1 indicates a stimulus-responsive area, and reference numerals 2a, 2b, 2c, and 2d indicate cell adhesion areas. The cell adhesive regions 2a, 2b, 2c, and 2d are arranged adjacent to each other so as to surround the stimulus responsive region 1. In the form of FIG. 9, the outer periphery of the stimulus responsive region 1 is partially in contact with the cell adhesive region. Reference numerals 3a, 3b, 3c, and 3d denote adhesion boundaries that are boundary portions where the stimulus-responsive region 1 and the cell adhesive regions 2a, 2b, 2c, and 2d are adjacent to each other. In the form of FIG. 9, there are four adhesion boundaries, and one adhesion boundary is formed in each of four individual cell adhesion regions.

  FIG. 10 is a schematic plan view for explaining a configuration example of a sheet forming region included in the cell culture container of the present embodiment, and shows a form in which eight adhesion boundaries exist. In FIG. 10, reference numeral 1 indicates a stimulus responsive area, and reference numerals 2a, 2b, 2c, 2d, 2e, 2f, 2g, and 2h indicate cell adhesive areas. The cell adhesion regions 2 a, 2 b, 2 c, 2 d, 2 e, 2 f, 2 g and 2 h are arranged adjacent to each other so as to surround the stimulus responsive region 1. In the form of FIG. 10, the outer periphery of the stimulus responsive region 1 is partially in contact with the cell adhesive region. In addition, the reference signs 3a, 3b, 3c, 3d, 3e, 3f, 3g, and 3h are adjacent to the stimulus-responsive region 1 and the cell adhesive regions 2a, 2b, 2c, 2d, 2e, 2f, 2g, and 2h. The adhesion boundary which is a boundary part is shown. In the form of FIG. 10, there are eight adhesion boundaries, and one adhesion boundary is formed in each of the eight individual cell adhesion regions.

  The plurality of cell adhesive regions are arranged adjacent to each other so as to surround the stimulus responsive region. In addition, the plurality of cell adhesive regions are arranged near the outer periphery of the stimulus responsive region. By arranging the cell adhesion region near the periphery of the stimulus responsive region, the cell sheet can be more stably held on the culture surface after the stimulus is applied.

  7 to 10, the plurality of cell adhesive regions 2 are arranged in contact with a part of the outer periphery of the stimulus responsive region 1. In other words, the plurality of cell adhesive regions 2 are arranged outside the stimulus responsive region 1 so as to be adjacent to the stimulus responsive region 1. In addition, this embodiment is not limited to the form in which such a cell adhesive region is disposed in contact with a part of the outer periphery of the stimulus responsive region, and as shown in FIG. The cell adhesive region may be arranged inside the stimulus responsive region and near the outer periphery of the stimulus responsive region. Moreover, although the form where the space | interval between adjacent cell-adhesive area | regions is respectively equal is described in FIGS. 7-10, this embodiment is not specifically limited to such a form, Adjacent cell The spacing between the adhesive regions may be different.

  In the present embodiment, the two points are formed by two straight lines connecting the center of gravity of the stimulus responsive region and each of two points of one adhesive boundary, and the two points are maximized. Is the angle θ, the sum of the angles θ corresponding to all of the adhesion boundaries is preferably 90 ° or more. This form will be described with reference to FIGS. FIG. 12 is a diagram illustrating the angle θ for the sheet forming region illustrated in FIGS. 9 and 10. FIG. 13 is a diagram for explaining the concept of the angle θ. Two points selected from the adhesion boundary are selected so that the angle θ is maximized. As shown in FIG. 13A, in the case where the cell adhesive region spreads away from the center of gravity of the stimulus responsive region, the above two points can usually be the end of the adhesion boundary. On the other hand, as shown in FIG. 13B, when the cell adhesive region becomes narrower as it becomes farther from the center of gravity of the stimulus responsive region, the above two points can be places other than the end of the adhesion boundary. In the present embodiment, the sum of the angles θ corresponding to all of the adhesion boundaries is preferably 90 ° or more, more preferably 120 ° or more, further preferably 150 ° or more, and 180 ° or more. It is particularly preferred. As the total sum of the angles θ increases, the length of the adhesion boundary increases, and the cell sheet can be stably held on the culture surface even after the stimulus is applied. Further, the sum of the angles θ corresponding to all of the adhesion boundaries is preferably 270 ° or less, more preferably 240 ° or less, and further preferably 210 ° or less. The smaller the sum of the angle θ, the shorter the length of the adhesion boundary, and the cell sheet can be easily detached from the culture surface after the stimulus is applied. When there are a plurality of sets of two points at which the angle θ is maximum, the two points at which the area T described later is maximized are selected with priority.

  Note that the center of gravity of the stimulus responsive region is a position where the sum of the first moments of the stimulus responsive region is 0, and a simple figure can be obtained by calculation. Even a complicated figure can be easily calculated by using image analysis software.

  The method for observing the adhesion boundary is not particularly limited, but for example, the following method can be used. For example, i) a method of coloring and observing the cell adhesive region by adding a staining component to the cell sheet, or ii) giving a stimulus after the cell adheres and peeling only the cell adhered to the stimulus responsive region There is a method of observing a region where cells are adhered after removing cells detached by stimulation.

  The angle θ can be measured, for example, by marking the two points specified by the above method with a marker, drawing a straight line from the center of gravity to the mark, visualizing the angle θ, and then using image analysis software. it can.

  In the present embodiment, the area surrounded by two straight lines connecting the center of gravity of the stimulus-responsive area and each of two points of one adhesive boundary and one adhesive boundary, and the area of the area is maximized. When the area of the region where the two points are selected is defined as area T, the total of the areas T corresponding to all of the adhesion boundaries is 0.2 times or more than the area of the stimulus-responsive region. It is preferable. This form will be described with reference to FIG. FIG. 14 is a diagram showing an area T for the sheet forming region illustrated in FIGS. 9 and 10. Two points selected from the adhesion boundary are selected so that the area T is maximized. In the present embodiment, the total sum of the areas T corresponding to all of the adhesion boundaries is preferably 0.2 times or more, more preferably 0.3 times or more with respect to the area of the stimulus responsive region. , 0.4 times or more is more preferable, and 0.5 times or more is particularly preferable. The greater the sum of the areas T, the more stably the cell sheet can be held on the culture surface even after stimulation. Further, the total sum of the areas T corresponding to all of the adhesion boundaries is preferably 0.9 times or less, more preferably 0.8 times or less with respect to the area of the stimulus responsive region, and More preferably, it is 7 times or less. The smaller the total area T, the easier it is to peel the cell sheet from the cell adhesion area after applying the stimulus. In the present embodiment, when there is a cell adhesive region that is included in the stimulus responsive region and is arranged near the outer periphery of the stimulus responsive region, the center of gravity of the stimulus responsive region is between the two points. On the other hand, an adhesion boundary located inside and an adhesion boundary located outside the center of gravity of the stimulus responsive region are formed. In FIG. 15, the adhesion boundary 3 ′ located inside the centroid of the stimulus responsive area and the adhesion boundary 3 ″ located outside the centroid of the stimulus responsive area are indicated by bold lines. In such a case, an adhesive boundary 3 ′ located inside the center of gravity of the stimulus responsive region is used to form the region constituting the area T.

  The area T can also be measured using, for example, image analysis software.

  In this embodiment, it is preferable that the total length of the adhesion boundary is 0.2 times or more the outer peripheral length of the stimulus responsive region. This form will be described with reference to FIG. FIG. 16 shows the outer periphery and adhesion boundary of the stimulus-responsive region in the sheet forming region shown in FIG. In FIG. 16, adhesion boundaries 3a, 3b, 3c, and 3d are indicated by thick lines, and portions other than the adhesion boundary in the outer periphery of the stimulus responsive region are indicated by arrows. In FIG. 16, the length of the outer periphery of the stimulus responsive region is the total length of the adhesion boundary indicated by a bold line and the total length of the portion other than the adhesion boundary of the outer periphery of the stimulus responsive region indicated by an arrow. Is the sum of In FIG. 16, the total length of the adhesion boundary is the sum of the lengths of the plurality of adhesion boundaries 3a, 3b, 3c, and 3d. In the present embodiment, the total length of the adhesion boundary is preferably 0.2 times or more, more preferably 0.3 times or more with respect to the length of the outer periphery of the stimulus responsive region. More preferably, it is 4 times or more. As the total length of the adhesion boundary increases, the cell sheet can be more stably held on the culture surface even after the stimulus is applied. Further, the total length of the adhesion boundary is preferably 0.9 times or less, more preferably 0.8 times or less, and 0.7 times or less with respect to the outer peripheral length of the stimulus responsive region. More preferably. As the total length of the adhesion boundary becomes smaller, the cell sheet can be easily peeled off from the culture surface after the stimulus is applied.

  Note that the total length of the adhesion boundary can also be measured using, for example, image analysis software. Similarly, the length of the outer periphery of the stimulus responsive region can also be measured using image analysis software.

  In the present embodiment, the sheet formation area is a straight line extending from the center of gravity of the stimulation-responsive area of the total number of adhesion boundaries, and the stimulation-responsive area is divided into the total number of adhesion boundaries so that the respective areas after division are equal. In this case, it is preferable to have an arrangement shape in which at least one adhesion boundary is in contact with each of the divided stimulus-responsive regions. FIG. 17 shows a state in which the stimulus-responsive region is divided into the total number of adhesion boundaries so that the divided areas of the sheet forming regions shown in FIGS. 9 and 10 are equal. In addition, the method / form which divides | segments a stimulus responsive area | region into the area | region of the total number of adhesion boundaries so that each area after a division | segmentation may become equal in the straight line extended from the gravity center of the stimulation responsive area of the total number of adhesion boundaries. However, in this embodiment, in any of the equally divided forms, it is only necessary to obtain an arrangement shape in which at least one adhesion boundary is in contact with each of the stimulus-responsive areas after division. Moreover, in any of the equal division forms, it is preferable to have an arrangement shape in which at least one adhesion boundary is in contact with each of the stimulus-responsive regions after division. In addition, as shown in FIG. 17, in any of the equal division forms, it is preferable to have an arrangement shape in which only one adhesion boundary is in contact with each of the stimulus-responsive regions after division. In FIGS. 17A and 17B, one adhesion boundary is in contact with each of the stimulus-responsive regions after division.

  In the present embodiment, the one line between one point of one bonding boundary and one point of the bonding boundary adjacent to the one bonding boundary, and the one line is the shortest. When a straight line with each point selected is defined as a straight line L, the area of the region surrounded by the straight lines L corresponding to all the bonding boundaries and all the bonding boundaries (hereinafter referred to as area S) is the sheet forming region. It is preferably 0.6 times or more with respect to the area. This form will be described with reference to FIG. FIG. 18 shows the concept of the straight line L for the sheet forming region shown in FIG. In FIG. 18, for example, one adhesion boundary 3a exists in one cell adhesive region 2a. The adhesion boundary adjacent to the adhesion boundary is an adhesion boundary arranged adjacent to the direction along the outer periphery of the stimulus responsive region, and the direction along the outer periphery is opposite to one direction. Both directions can be considered. In FIG. 18A, the adhesive boundary adjacent to the adhesive boundary 3a is the adhesive boundary 3b and the adhesive boundary 3d. In FIG. 18B, the adhesive boundary adjacent to the adhesive boundary 3a is the adhesive boundary. 3b and adhesion boundary 3h. In FIG. 18A, the shortest straight line connecting the bonding boundary 3a and the bonding boundary 3b is a straight line indicated by the symbol L1, and the shortest straight line connecting the bonding boundary 3a and the bonding boundary 3d is a straight line indicated by the symbol L4. . In FIG. 18B, the shortest straight line connecting the bonding boundary 3a and the bonding boundary 3b is a straight line indicated by the symbol L1, and the shortest straight line connecting the bonding boundary 3a and the bonding boundary 3h is a straight line indicated by the symbol L8. . The straight line L corresponding to all the adhesion boundaries is a straight line indicated by symbols L1 to L8. In FIG. 18, the region surrounded by the straight lines L corresponding to all the bonding boundaries and all the bonding boundaries is a region surrounded by the straight lines L1 to L8 and the bonding boundaries 3a to 3h. In the present embodiment, the area S of the region surrounded by the straight lines L corresponding to all the adhesion boundaries and all the adhesion boundaries is preferably 0.6 times or more the area of the sheet formation region, The ratio is more preferably 0.7 times or more, further preferably 0.8 times or more, and particularly preferably 0.9 times or more. As the area S relative to the area of the sheet forming region increases, the cell sheet can be more stably held on the culture surface even after the stimulus is applied. The upper limit value of the area S is not particularly limited, but is, for example, 1 time or less. Further, the area S is preferably 0.8 times or more, more preferably 0.9 times or more with respect to the area of the stimulus responsive region. In the present embodiment, when there is a cell adhesive region that is included in the stimulus responsive region and is arranged near the outer periphery of the stimulus responsive region, the stimulus responsive layer region is between two adjacent straight lines L. An adhesive boundary located on the inner side with respect to the center of gravity of the (or sheet forming region) and an adhesive boundary positioned on the outer side with respect to the center of gravity of the stimulus responsive layer region (or sheet forming region) are formed. In FIG. 19, the adhesion boundary 3 ′ located on the inner side with respect to the center of gravity of the stimulus responsive layer region (or sheet forming region) and the adhesion located on the outer side with respect to the center of gravity of the stimulus responsive layer region (or sheet forming region). The boundary 3 ″ is indicated by a bold line. In such a case, the formation of the region uses an adhesive boundary 3 ′ located inside the center of gravity of the stimulus responsive layer region (or sheet forming region). In the present embodiment, when there are a plurality of shortest straight lines between two adjacent adhesion boundaries, the straight line having the smallest area S is selected to form the region.

  The area S can also be measured using image analysis software, for example.

  In the present embodiment, the cell non-adhesive region may be disposed outside the sheet forming region. That is, the cell non-adhesive region may be disposed in contact with the outer periphery of the sheet forming region. Specifically, the cell non-adhesive region may be disposed in contact with the outer periphery of the cell adhesive region and / or the stimulus responsive region. Further, for example, as shown in FIG. 20A, in the present embodiment, the cell non-adhesive region can be disposed in contact with the outer periphery of the cell adhesive region and the stimulus-responsive region. In FIG. 20A, the cell non-adhesive region 4 is disposed in contact with a portion other than the adhesion boundary in the outer periphery of the cell adhesive region and a portion other than the adhesion boundary in the outer periphery of the stimulus responsive region. For example, as shown in FIG. 20B, the cell non-adhesive region can be disposed in contact with the outer periphery of the stimulus responsive region. Further, the cell non-adhesive region can be disposed in contact with the outer periphery of the cell adhesive region. Moreover, the side wall which forms the culture | cultivation space holding a culture solution may be arrange | positioned outside the sheet | seat formation area. That is, the outer periphery of the sheet forming region may be in contact with a side wall that forms a culture space for holding the culture solution. Further, a step or the like may exist outside the sheet forming region, and a culture space may exist.

  In the present embodiment, the stimulus responsive region can be formed by, for example, a stimulus responsive layer having a surface having stimulus responsiveness, and the cell adhesive region has a cell adhesive layer having a surface having cell adhesiveness. The cell non-adhesive region can be formed by a cell non-adhesive layer having cell non-adhesiveness.

In the present embodiment, a sheet non-forming part can be arranged outside the sheet forming region.
FIG. 21 is a diagram for explaining a configuration example of the cell culture container in a form in which the sheet forming region is in contact with the side wall forming the culture space for holding the culture solution. FIG. 21A is a schematic top view of a cell culture container having the sheet forming region shown in FIG. The stimulus responsive region is formed from the stimulus responsive layer 101, and the cell adhesive region is formed from the cell adhesive layer 102. 21B to 21E are schematic cross-sectional views showing a configuration example of the cell culture container in a vertical cross section taken along line AA ′ in FIG. In FIG. 21A, the base material 105 shown in FIGS. 21B to 21E is omitted. In FIG. 21B, a stimulus responsive layer 101 and a cell adhesive layer 102 are disposed on a base material 105, respectively. The stimulus responsive layer 101 and the cell adhesive layer 102 are disposed on the same plane. In FIG. 21C, the cell adhesive layer 102 is disposed on the base material 105, and the stimulus responsive layer 101 is disposed on the cell adhesive layer 102. The surface of the cell adhesive layer 102 exposed around the stimulus responsive layer 101 forms a cell adhesive region. In FIG. 21D, the stimulus-responsive layer 101 is disposed on the substrate 105 having a cell-adhesive surface, and the surface of the substrate 105 having cell-adhesive properties exposed around the stimulus-responsive layer 101. Forms a cell adhesion region. In FIGS. 21C and 21D, the heights of the stimulus-responsive region and the cell adhesion region are such that a continuous cell sheet is formed between them. In FIG. 21E, the stimulus responsive layer 101 is disposed on the base material 105, and the cell adhesive layer 102 is disposed on the stimulus responsive layer 101. The cell adhesive layer 102 is disposed along the outer peripheral edge of the stimulus responsive layer 101.
Moreover, the figure for demonstrating the structural example of a cell culture container about the form by which the cell non-adhesion area | region is formed in the outer side of a sheet | seat formation area in FIG. FIG. 22A is a schematic top view of a cell culture container having the sheet forming region shown in FIG. 20A and a cell non-adhesive region disposed outside the sheet forming region. The stimulus responsive region is formed from the stimulus responsive layer 101, the cell adhesive region is formed from the cell adhesive layer 102, and the cell non-adhesive region is formed from the cell non-adhesive layer 104. 22B to 22D are schematic cross-sectional views showing a configuration example of the cell culture container in a vertical cross section taken along line BB ′ in FIG. In FIG. 22A, the base material 105 shown in FIGS. 22B to 22D is omitted. In FIG. 22B, a stimulus responsive layer 101, a cell adhesive layer 102, and a cell non-adhesive region 104 are disposed on a substrate 105, respectively. The stimulus-responsive layer 101, the cell adhesive layer 102, and the cell non-adhesive region 104 are arranged on the same plane. In FIG. 22C, a cell adhesive layer 102 is disposed on a base material 105 having a cell non-adhesive surface, and a stimulus responsive layer 101 is disposed on the cell adhesive layer 102. . The surface of the cell adhesion layer 102 exposed around the stimulation-responsive layer 101 is formed by the surface of the cell non-adhesive substrate 105 exposed around the cell adhesion layer 102 forming a cell non-adhesion region. Forms a cell adhesion region. In FIG. 22D, the stimulus responsive layer 101 is disposed on the base material 105 having a cell non-adhesive surface, and the cell adhesive layer 102 is disposed on the stimulus responsive layer 101. . The cell adhesive layer 102 is disposed along the outer peripheral edge of the stimulus responsive layer 101. The surface of the substrate 105 having cell non-adhesiveness exposed around the cell adhesive layer 102 forms a cell non-adhesive region. In the present embodiment, if necessary, another layer having no exposed surface or another layer having an exposed surface serving as another region may be disposed.
FIG. 23 is a diagram for explaining a configuration example of the cell culture container in a form in which a culture space exists outside the sheet formation region. FIG. 23A is a schematic top view of the cell culture container having the sheet forming region shown in FIG. FIG. 23B is a schematic cross-sectional view showing a configuration example of the cell culture container in a vertical cross section taken along line AA ′ in FIG. In FIG. 23 (B), the stimulus-responsive layer 101 and the cell adhesive layer 102 are respectively disposed on the base material 105 ′, and the substrate 105 ′ is disposed on the bottom surface of the cell container. A culture space exists outside the cell adhesive layer 102.

[Stimulus responsive area]
The stimulus-responsive region is a region in which the degree of cell adhesion is changed by stimulation, and can be made cell-adhesive during cell culture and non-cell-adhesive by applying stimulus during cell sheet peeling operation. The stimulus responsive region can be formed by, for example, a stimulus responsive layer including a stimulus responsive polymer. The stimulus responsive region is the surface of the stimulus responsive layer. When cells are seeded in a stimulus-responsive area, the cells adhere to the stimulus-responsive area when cell adhesion is expressed, but the cells adhere to the stimulus-responsive area when cell non-adhesive is expressed. Therefore, the cell sheet adhered to the stimulus responsive region can be peeled off. The type of stimulation is not particularly limited, and examples thereof include temperature, pH, ions, light, electric potential, or magnetic field.

  The stimulus-responsive polymer is a material whose degree of adhesion to cells changes according to a predetermined stimulus. The stimulus-responsive layer containing the stimulus-responsive polymer has a surface that can change its property from cell adhesiveness to cell nonadhesiveness by changing the degree of cell adhesion by a predetermined stimulus. Examples of the stimulus responsive polymer include a temperature responsive polymer, a pH responsive polymer, an ion responsive polymer, and a photoresponsive polymer. Of these, a temperature-responsive polymer is preferably used from the viewpoint of easy application of stimulation.

  As the temperature-responsive polymer, for example, a polymer that exhibits cell adhesion at a temperature for culturing cells and exhibits cell non-adhesion at a temperature at which the formed cell sheet is peeled can be used. For example, a temperature-responsive polymer has improved affinity to surrounding water at a temperature below the lower critical solution temperature, and the polymer takes up water and swells to make it difficult for cells to adhere to the surface (cell non-adhesiveness) At a temperature equal to or higher than the lower critical solution temperature, those exhibiting a property (cell adhesion) that facilitates adhesion of cells to the surface due to shrinkage of the polymer due to water desorption from the polymer are preferably used. It is preferable to use a temperature-responsive polymer having a lower critical solution temperature (T) of 0 ° C. or higher and 80 ° C. or lower, preferably 0 ° C. or higher and 50 ° C. or lower, more preferably 37 ° C. or lower.

  Suitable temperature-responsive polymers include, for example, acrylic polymers or methacrylic polymers. More specifically, poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propylacrylamide (T = 21 ° C), poly-Nn-propyl methacrylamide (T = 32 ° C), poly-N-ethoxyethylacrylamide (T = about 35 ° C), poly-N-tetrahydrofurfurylacrylamide (T = about 28 ° C) ), Poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.), poly-N, N-diethylacrylamide (T = 32 ° C.) and the like. Further, it may be a copolymer obtained by combining two or more monomers for forming these polymers.

  As a monomer for forming these polymers, a monomer that can be polymerized by irradiation with radiation can be used. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, vinyl ether derivatives, and the like. A monomer may be used individually by 1 type or may be used in combination of 2 or more type. When one type of monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple types of monomers are used in combination, the polymer formed on the substrate is a heteropolymer. Both forms are encompassed by the present invention.

  Further, when it is necessary to control T depending on the type of proliferating cells, other monomers other than those described above may be further added for copolymerization.

  The pH-responsive polymer or ion-responsive polymer can be appropriately selected from those suitable for the cell sheet to be formed.

Cell adhesion refers to the property that cells adhere easily. Cell adhesion is determined by whether or not cell adhesion or extension is likely to occur depending on the chemical or physical properties of the surface. As an index for determining the cell adhesion of the stimulus-responsive layer, the cell adhesion extension rate when the cells are actually cultured can be used. The cell adhesive surface is preferably a surface having a cell adhesion extension rate of 60% or more, and more preferably a surface having a cell adhesion extension rate of 80% or more. If the cell adhesion extension rate is high, cells can be cultured efficiently. The cell adhesion spreading rate is determined by seeding target cells to be cultured in a seeding density range of 4000 cells / cm ² or more and less than 30000 cells / cm ² on a measurement target surface and storing them in an incubator at 37 ° C. and 5% CO ₂ concentration. Then, it can be defined as the ratio of the cells that have adhered and extended at the time of culturing for 14.5 hours ({(number of adherent cells) / (number of cells seeded)} × 100 (%)). Cell seeding is suspended in DMEM medium containing 10% FBS, seeded on the measurement object, and then slowly shaken the measurement object on which the cells are seeded so that the cells are distributed as uniformly as possible. To do. Furthermore, the measurement of the cell adhesion extension rate is performed after exchanging the medium immediately before the measurement to remove the non-adhered cells. When measuring cell adhesion spread rate, measure the location excluding the location where the cell density tends to be specific (for example, the center of the predetermined area where the density is likely to be high, the periphery of the predetermined area where the density is likely to be low) A place.

  On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether or not cell adhesion or extension is unlikely to occur due to chemical or physical properties of the surface. The cell adhesion spreading rate of the non-cell-adhesive region is preferably less than 60%, more preferably less than 40%, still more preferably 5% or less, and particularly preferably 2% or less. preferable.

  One kind of stimulus-responsive polymer may be used alone, or a plurality of kinds may be used in combination.

  The stimulus responsive layer contains the stimulus responsive polymer as a main component. The content of the stimulus-responsive polymer in the stimulus-responsive layer is preferably 50% by mass or more, more preferably 70% by mass or more, and 90% by mass or more in the total solid content of the stimulus-responsive layer. More preferably, it is 95% by mass or more, and particularly preferably 100% by mass. The stimulus responsive layer may contain an additive in addition to the stimulus responsive polymer. Examples of the additive include other polymers and plasticizers.

  The thickness of the stimulus responsive layer is not particularly limited and can be appropriately selected. The thickness of the stimulus responsive layer is, for example, 1 nm or more, preferably 5 nm or more, and more preferably 10 nm or more. Further, the thickness of the stimulus responsive layer is, for example, 200 nm or less, and preferably 100 nm or less.

  The stimulus-responsive layer is preferably formed on the substrate.

  The method for forming the stimulus-responsive layer on the substrate surface is not particularly limited. For example, after placing the polymer solution on the substrate surface by dipping, spraying, spin coating, etc., the solvent is added. The method of removing (drying) is mentioned. The polymer liquid is preferably applied using a bar coater from the viewpoint of uniformity. In addition, as a method for forming a stimulus-responsive layer, a monomer for forming a polymer is applied to the surface of a substrate, and the monomer is polymerized by electron beam irradiation, ultraviolet irradiation, plasma irradiation, polymerization reaction, or the like. preferable. If necessary, chemical treatment such as corona treatment, plasma treatment, ozone treatment, or silane coupling agent treatment may be performed as a pretreatment of the substrate surface before forming the stimulus-responsive layer. By this treatment, the stimulus-responsive layer can be more stably immobilized on the substrate surface.

  The material of the substrate is not particularly limited, and for example, glass or plastic can be used. Examples of the plastic include polystyrene, polypropylene, polyvinyl chloride, polyethylene, cyclic polyolefin, polyethylene terephthalate, polycarbonate, silicone resin, and acrylic resin. As the acrylic resin, polymethyl methacrylate (PMMA) is preferable from the viewpoint of versatility.

  A base material may be used as a material which comprises the outer wall of a cell culture container. The shape of the cell culture vessel is not particularly limited, and can be appropriately selected in consideration of the desired shape of the cell sheet. The shape of the cell culture container may be, for example, a petri dish, a flask, a beaker, a well plate, or the like. The base material may comprise, for example, the side wall and the bottom wall of the outer wall of the cell culture container. In addition, the cell culture container can have an appropriately sized lid. The cell culture container is preferably liquid-tight when closed with the lid.

  The shape of the stimulus responsive region is not particularly limited as long as it is a continuous shape in contact with each of the adhesion boundaries. For example, a substantially circular shape such as shown in FIG. 1 or a substantially polygonal shape such as a substantially quadrangular shape can be used, and a non-symmetrical shape such as non-linear symmetry or non-point symmetry is also included (for example, FIG. 6). Further, the ratio of the area of the stimulus responsive area to the area of the sheet forming area is, for example, 0.6 or more, more preferably 0.8 or more, and further preferably 0.9 or more.

  When the shape of the stimulus responsive region is a substantially polygon, the cell adhesive region is preferably arranged near the vertex of the polygon. The presence of the cell adhesive region near the apex of the polygon of the stimulus responsive region allows the cell sheet to be more stably held on the culture surface even after the stimulus is applied.

  The adhesion boundary is a boundary between the stimulus-responsive region and the cell adhesion region. The adhesion boundary can form a continuous cell sheet in which cells are bound to each other on the stimulus-responsive region and the cell-adhesive region. After the stimulus is applied to the stimulus-responsive region, the adhesion boundary is formed on the stimulus-responsive region and the cell-adhesive region. As long as a continuous cell sheet can be maintained, there is no particular limitation. That is, the adhesion boundary not only indicates a place where the stimulus-responsive region and the cell-adhesive region are in contact with each other in plan view, but also between the stimulus-responsive region and the cell-adhesive region. There may be other regions such as a non-cell-adhesive region arranged with a width that allows cells cultured on the adhesive region to adhere to each other.

  The adhesion boundary is not limited to the one located on the outer periphery of the stimulus responsive region in plan view, and may be one located on the inside of the stimulus responsive region in plan view as illustrated in FIG. The cell adhesion region is disposed adjacent to the stimulus responsive region, and the term “adjacent” includes the concept of being disposed inside the stimulus responsive region as described above.

  The number of adhesion boundaries is not particularly limited as long as it is at least one, and can be appropriately set according to the application. In the present embodiment, the number of adhesion boundaries is preferably at least two, preferably at least three, and preferably at least four. The plurality of adhesive boundaries exist apart from each other.

  One adhesive boundary may be formed in one continuous cell adhesive region, or a plurality of adhesive boundaries may be formed in one continuous cell adhesive region. In the present embodiment, the adhesion boundary is preferably one that forms one adhesion boundary in one continuous cell adhesion region.

  It is preferable that the stimulus-responsive region and / or the cell adhesive region is surrounded by a cell non-adhesive region. That is, it is preferable that the cell non-adhesive region is disposed outside the stimulus-responsive region and / or the cell adhesive region. Moreover, it is preferable that the cell non-adhesive region is disposed around both the stimulus-responsive region and the cell adhesive region.

  In addition, the fact that the cell non-adhesive region is arranged around the stimulus responsive region means that there is a non-adhesive boundary on the outer periphery of the stimulus responsive region. At the non-adhesive boundary, it is preferable that the cell sheet floats in the medium along the non-adhesive boundary when a stimulus is applied. Such a non-adhesive boundary is usually in contact with the cell non-adhesive region in plan view, but for example, a stimulus responsive region is stimulated between the stimulus responsive region and the cell non-adhesive region. In this case, it also includes locations where other regions such as a cell adhesive region formed with such a width that the cell sheet can be suspended in the medium along the non-adhesive boundary. As a positional relationship between the stimulus responsive region and the cell non-adhesive region, it is preferable that a part of the outer periphery of the stimulus responsive region is in contact with the cell non-adhesive region. If the stimulus responsive region is arranged in contact with the cell non-adhesive region, it becomes easy to give a water flow from the non-adhesive boundary by pipetting or the like, and the cell sheet is easily peeled off.

[Cell adhesion area]
The cell adhesive region is a surface having cell adhesiveness, and the cell adhesive region can be formed from a layer having a cell adhesive surface (cell adhesive layer).

  The cell adhesive region may be formed from one region or may be formed from two separate regions.

  As the number, shape, and arrangement of the cell adhesive regions in the present embodiment, the cell adhesive regions may be composed of one continuous region as shown in FIGS. 1 to 6, as shown in FIGS. In addition, it may be composed of a plurality of divided areas.

  The shape of the cell adhesive region is not particularly limited as long as the cell sheet can be stably held after the stimulus is applied to the stimulus responsive region. Further, the area per cell adhesive region is not particularly limited as long as the cell sheet can be stably held after the stimulus is applied to the stimulus responsive region. Further, the ratio of the total area of the cell adhesive region to the area of the sheet forming region is, for example, 0.4 or less, preferably 0.2 or less, and more preferably 0.1 or less. Further, the ratio of the total area of the cell adhesive region to the area of the sheet forming region is preferably 0.01 or more, more preferably 0.05 or more.

  In the present embodiment, when the maximum width of the cell adhesive region when the line is drawn from the center of gravity of the stimulus responsive region so as to pass through an arbitrary point of the cell adhesive region is the adhesive region width H, the adhesive region width H Is, for example, 10% or less, preferably 5% or less, with respect to the maximum diameter of the sheet forming region. The adhesion region width H is preferably 0.05% with respect to the maximum diameter of the sheet formation region. The maximum diameter of the sheet forming region refers to the diameter of the smallest circle that can include the entire sheet forming region.

  As a kind of cell adhesive region, only one type may be sufficient and the cell adhesive region containing a different cell adhesive material may be included.

  The cell adhesion region may or may not be in contact with the cell non-adhesion region. The cell adhesion region is preferably in contact with the cell non-adhesion region.

  The height of the cell adhesive region relative to the stimulus responsive region is not particularly limited as long as a cell sheet can be formed. For example, the cell adhesion region may be higher, the same or lower than the stimulus responsive region. The same applies to the height of the cell adhesive region relative to the non-cell adhesive region, and is not particularly limited.

  The degree of cell adhesion of the cell adhesion region is not particularly limited as long as it exhibits the desired cell adhesion, and cell adhesion when the stimulus-responsive region expresses cell adhesion. And can be similar.

  The cell adhesive layer can be formed by a layer containing a cell adhesive material, for example. The cell adhesive layer may have cell adhesion due to, for example, biochemical characteristics, or may have cell adhesion due to physicochemical characteristics. As the cell adhesion material, for example, a cell adhesion material used for a general cell culture substrate or the like can be used. For example, as materials that adhere to cells due to physicochemical properties, for example, hydrophilic polymers such as hydrophilic polystyrene and polylysine, aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxy Examples thereof include basic compounds such as silane and condensates containing them. Examples of biochemically adhesive materials for cells include fibrin, fibronectin, laminin, tenascin, vitronectin, RGD (arginine-glycine-aspartic acid) sequence-containing peptide, YIGSR (tyrosine-isoleucine-glycine-serine-arginine). ) Sequence-containing peptides, collagen, atelocollagen, gelatin, and mixtures thereof, such as Matrigel.

  Further, as the cell adhesive layer, for example, various glasses, polystyrene subjected to plasma treatment, polypropylene, and the like can be used.

  When the surface of a base material has cell adhesiveness, a base material can be utilized as a cell adhesive layer. That is, the substrate surface having cell adhesiveness can be used as the cell adhesive region. For example, after forming a stimulus-responsive layer containing a stimulus-responsive polymer on a substrate having cell adhesion, the stimulus-responsive layer is exposed by removing a predetermined portion of the stimulus-responsive layer and exposing the substrate surface. Can be easily formed. Further, as a method other than the method of partially removing the stimulus responsive layer, for example, a method of forming the stimulus responsive layer only partially on the substrate surface from the beginning using a technique such as photolithography is also used. be able to. In addition, the cell adhesiveness of a base material can be judged by seed | inoculating a target cell on a base material and investigating whether a target cell adheres to a base material and proliferates, for example.

  The base material is preferably configured to include a plastic having a hydrophilic treatment surface from the viewpoint of cell adhesiveness. Examples of the plastic include polystyrene, polypropylene, polyvinyl chloride, polyethylene, cyclic polyolefin, polyethylene terephthalate, polycarbonate, polyimide, silicone, and acrylic. The hydrophilic treatment is not particularly limited as long as the treatment can modify the surface of the plastic to be hydrophilic. Examples of such surface treatment include corona discharge treatment, plasma treatment, UV ozone treatment, electron beam irradiation treatment, and laser treatment. A commercially available substrate having a hydrophilic treatment surface may be used. For example, a Nunc ™ cell culture dish (trade name, manufactured by Thermo Fisher Scientific), a Falcon cell culture dish (manufactured by Corning), cell culture, and the like. It is commercially available as a petri dish (manufactured by Sumitomo Bakelite Co., Ltd.) or a tissue culture dish (manufactured by Asahi Glass Co., Ltd.). Moreover, the base material which has a hydrophilic treatment surface can also be prepared by performing a hydrophilic treatment to the base material made from an untreated plastic which has not been subjected to the hydrophilic treatment, for example. Examples of the untreated substrate include Nunc ™ Petri dish (trade name, manufactured by Thermo Fisher Scientific), Falcon Petri dish (Corning), suspension culture dish (Sumitomo Bakelite), or untreated dish (Asahi Glass). Can be used. It is considered that the hydrophilic functional group (for example, a hydroxy group or a carbonyl group) present on the plastic surface is increased by the hydrophilic treatment. Cell adhesion can be improved by treating the surface of the plastic substrate with a hydrophilic treatment. When the surface of the base material is treated with hydrophilicity, nutrients that become the material of the intercellular matrix such as proteins contained in the culture solution are likely to gather on the adhesive surface, and as a result, when an untreated base material is used It is considered that the adhesiveness of the cells is improved as compared with the above.

  You may add an additive, binder resin, etc. to a cell adhesion layer.

  As a method for forming the cell adhesive layer, a method similar to the method for forming the stimulus-responsive layer can be used.

[Cell non-adhesive area]
The cell non-adhesive region is a surface having cell non-adhesion and can be formed from a layer having a cell non-adhesive surface (cell non-adhesive layer).

  The degree of cell adhesion of the cell non-adhesive region is not particularly limited as long as it exhibits a desired cell non-adhesive property, and the case where the stimulus responsive region expresses cell non-adhesive property. It can be similar to cell adhesion.

  The cell non-adhesive layer can be formed by a layer containing a cell non-adhesive material, for example. As such a cell non-adhesive material, any material having non-adhesiveness with cells may be used. For example, a material having high hydration ability can be used. Examples of materials that have high hydration ability and are used as cell non-adhesive materials include polyethylene glycol, zwitterionic materials having a betaine structure, phospholipid-containing materials, and the like.

  Further, as the cell non-adhesive material, a material having water repellency or oil repellency or a material having super hydrophilicity can also be used. As a material having water repellency or oil repellency, for example, a material having an organic substituent of water repellency or oil repellency can be used. Specifically, hydrolysis or polycondensation of chloro or alkoxysilane by sol-gel reaction or the like. It is also possible to use organopolysiloxanes exhibiting great strength, organopolysiloxanes cross-linked with reactive silicones, and surfactants having water-repellent or oil-repellent organic substituents. Further, examples of the material having super hydrophilicity include those obtained by decomposing organic substituents such as the above organopolysiloxane by the action of a photocatalyst accompanying energy irradiation.

  The cell non-adhesive layer may contain an additive, a binder resin, and the like.

  As a method for forming the cell non-adhesive layer, a method similar to the method for forming the stimulus-responsive layer can be used.

[Method for producing cell sheet]
One embodiment of the present invention relates to a method for producing a cell sheet, comprising the step of culturing cells in a cell culture container to form a cell sheet in the above-described embodiment.

  After the cells are cultured using the cell culture container according to the above-described embodiment to form a cell sheet, the cell sheet emerges in a form attached to the cell adhesive region by applying a stimulus. Since the cell sheet is attached to the cell adhesive region, its shape is stable. In addition, since the cell sheet can be easily peeled off from the cell adhesive region from the surface of the cell sheet, it can be easily applied to the intended use.

  Examples of the method for peeling the cell sheet include the following methods. For example, the cell sheet can be peeled off by applying a shear stress such as pipetting to a site where the cell sheet is attached to the cell adhesive region. Further, the cell sheet can be peeled off by cutting off the cells adhering to the cell adhesion region from the cell sheet by cutting with a tweezers or pipette tip at a site where the cell sheet is adhered. In addition, when a cut is made, it is preferable to use a member with a thin tip, but the cell sheet is cut with a member softer than the substrate or with a round tip so that impurities are not generated by scratching the substrate. It is desirable. Alternatively, the cell sheet may be peeled off from the substrate by applying physical force directly to the cell sheet with tweezers or pipette tips to remove the adhesive seal. It is desirable to do.

  In the formation of the cell sheet, it is preferable that the cells are seeded on a culture surface including a stimulus-responsive region and a cell adhesion region, and then the cells are grown by culturing to reach confluence. As used herein, “confluent” refers to a state in which cells cover the culture surface.

  In this embodiment, it is preferable that the surfaces of the stimulus-responsive layer and the cell adhesive region become a culture surface that is a surface on which cells are cultured. That is, it is preferable that the cells adhere to the surfaces of the stimulus-responsive layer and the cell adhesive region.

  The culture time of the cells is not particularly limited, and the cells can be cultured until a cell sheet is formed. In order to form a cell sheet, it is necessary for the seeded cells to form an extracellular matrix between adjacent cells.

  The target cell is not particularly limited, and any cell that can form a cell sheet can be used without particular limitation. The target cell is, for example, an adherent cell. Adhesive cells include, for example, liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermis Epidermal cells such as keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, myocytes such as smooth muscle cells and cardiomyocytes, myoblasts Examples include cells, kidney cells, pancreatic Langerhans islet cells, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, or bone cells. These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are embryonic stem cells that are undifferentiated cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation is completed. Any of the cells obtained may be used. In addition, the cells may be cultured as a single species, or two or more cells may be co-cultured.

  The culture solution is not particularly limited, and for example, a cell culture medium generally used in the art can be used. Examples of the medium include MEM medium, BME medium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, and RPMI1640 medium, depending on the type of cells used. A basal medium can be used. The basal medium is described, for example, on page 581 of “Tissue Culture Technology 3rd Edition” edited by Asakura Shoten. Furthermore, serum (such as fetal bovine serum), various growth factors, antibiotics, amino acids, etc. may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used. Considering the clinical application of the finally obtained cell sheet, it is preferable to use a medium that does not contain animal-derived components.

  In addition, this embodiment is not limited to the said embodiment. The above embodiment is an exemplification, and any structure having substantially the same configuration as the technical idea described in the claims of the present embodiment and having the same function and effect can be used. It is included in the technical scope of this embodiment. In addition, the contents described in the present embodiment, such as preferred examples, ranges, numerical values, materials, conditions, and the like, can be understood as one of the present embodiments in combination, and the present embodiment can be any combination thereof. It is included.

  Hereinafter, the present embodiment will be described using examples. In this example, an example in which a cell culture container having the sheet forming region shown in FIG. 9 was produced is shown.

Example 1
Poly-N-isopropylacrylamide (PNIPAAm) was coated on the entire surface of a circular polystyrene substrate having a diameter of 2 cm to form a stimulus-responsive layer having a surface that can be changed between cell adhesion and cell non-adhesion by temperature stimulation. Next, the stimulus-responsive layer was decomposed and removed by ozone treatment in a range from the outer periphery to the inner side of 1 mm to expose the cell-adhesive base material, thereby forming a cell-adhesive region. At this time, the cell adhesive region had a shape having four regions as shown in FIG. Further, the four gaps between the adjacent cell adhesive regions (corresponding to the straight lines L1 to L4 shown in FIG. 18A) were each 1 mm. The substrate on which the stimulus responsive region and the cell adhesive region were formed in this manner was placed on the bottom surface of the container to produce a cell culture container E1.

(Comparative Example 1)
As a comparative example, a cell culture vessel C1 was produced in the same manner as in Example 1 except that the stimulus-responsive layer was not partially removed.

(Evaluation)
Mouse myoblasts were seeded at a concentration of 1 × 10 ⁶ cells / cm ² on the substrate of the prepared cell culture container C1, and cultured at 37 ° C. for 1 day to form a cell sheet. Next, when the temperature of the cell culture container was lowered to 20 ° C. and the stimulus responsive layer was given a temperature stimulus, the cell sheet was completely detached from the substrate after 15 minutes.

  On the other hand, when the cell culture vessel E1 was similarly evaluated, the cell sheet did not completely peel off from the base material even after 1 hour from the application of temperature stimulation, and the cell adhesive region was not confirmed without contraction of the cell sheet. It was attached to. In addition, the cell sheet could be easily recovered from the substrate by lightly pipetting.

(Example 2)
Poly-N-isopropylacrylamide (PNIPAAm) was coated on the entire surface of a circular polystyrene substrate having a diameter of 2 cm to form a stimulus-responsive layer having a surface that can be changed between cell adhesion and cell non-adhesion by temperature stimulation. Next, collagen was pipetted in a range of 0.5 mm from the outer periphery to the inner side, gelled, and then dried to form a cell adhesive region.
At this time, the cell adhesive region has a shape having 8 regions as shown in FIG. 10, the gaps at 8 locations (corresponding to L1 to L8 in FIG. 18B) are 6 mm, respectively, and the cell culture vessel E2 is Produced.
Similarly, the cell adhesive region has a shape having four regions as shown in FIG. 9, and the four gaps (corresponding to the straight lines L1 to L4 shown in FIG. 18 (A)) are each 14 mm. A culture vessel E3 was prepared.
Further, similarly, as shown in FIG. 1 (A), the cell adhesive region was formed so that there was no gap in one region, and a cell culture container E4 was produced. Note that the ratio of the area of the cell adhesion region to the sheet formation region is about 0.2.

(Comparative Example 2)
As a comparative example, a cell culture vessel C2 was produced in the same manner as in Example 2 except that the cell adhesive layer containing the collagen was not formed.

(Evaluation)
Mouse myoblasts were seeded at a concentration of 1 × 10 ⁶ cells / cm ² on the substrate of the produced cell culture vessel C2, and cultured at 37 ° C. for 1 day to form a cell sheet. Next, when the temperature of the cell culture container was lowered to 20 ° C. and the stimulus responsive layer was given a temperature stimulus, the cell sheet was completely detached from the substrate after 15 minutes.

  On the other hand, when the cell culture vessels E2, E3, and E4 were similarly evaluated, the cell sheet was not completely detached from the substrate even after 1 hour from the application of the temperature stimulus, and adhered to the cell adhesive region. . Moreover, the contraction of the cell sheet was not confirmed at all in the E4 cell sheet, and the contraction of the cell sheet was more effectively suppressed in the order of E4, E3, and E2. Then, the cell sheet could be easily recovered from the base material by peeling the adhesion site with pipetting or tweezers. At this time, the cell sheet could be more easily recovered from the base material in the order of E2, E3, and E4.

DESCRIPTION OF SYMBOLS 1 Stimulus response area | region 2 Cell adhesion area | region 3 Adhesion boundary 4 Cell non-adhesion area | region 11 Outer stimulus response area | region 12 Outer cell adhesion area | region 101 Stimulation response layer 102 Cell adhesion layer 104 Cell non-adhesion layer 105,105 ' Base material

Claims (21)

A stimulus-responsive region in which the degree of cell adhesion changes due to a stimulus,
At least one cell adhesive region having cell adhesion disposed adjacent to the stimulus responsive region so as to surround the stimulus responsive region;
A cell culture container having a sheet-forming region comprising:
There is at least one adhesion boundary that is a boundary portion where the stimulus-responsive area and the cell adhesion area are adjacent;
The stimulus responsive region is a continuous region in contact with each of the adhesion boundaries.   The cell culture container according to claim 1, wherein the adhesion boundary is disposed near an outer periphery of the stimulus responsive region.   The cell culture container according to claim 1, wherein a plurality of the adhesion boundaries are present.   The cell culture container according to any one of claims 1 to 3, wherein at least four adhesion boundaries are present.   The two points were selected to be an angle formed by two straight lines connecting the center of gravity of the stimulus-responsive region and each of two points of one adhesive boundary, and the angle is maximized The cell culture container according to claim 3 or 4, wherein the sum of the angles θ corresponding to all of the adhesion boundaries is 90 ° or more when the angle is an angle θ.   The cell culture container according to claim 5, wherein the sum of the angles θ is 180 ° or more.   Two straight lines connecting the center of gravity of the stimulus-responsive region and each of two points of one adhesive boundary and a region surrounded by the one adhesive boundary, and the area of the region is maximized When the area of the region in which two points are selected is defined as the area T, the sum of the areas T corresponding to all of the adhesion boundaries is 0.2 times or more the area of the stimulus-responsive region. The cell culture container according to any one of claims 3 to 6.   The cell culture container according to any one of claims 3 to 7, wherein a total length of the adhesion boundary is 0.2 times or more with respect to a length of an outer periphery of the stimulus responsive region.   The sheet formation region is a straight line extending from the center of gravity of the stimulus responsive region of the total number of the adhesion boundaries, and the stimulus responsive region is divided into the total number of the adhesion boundaries so that the respective areas after division are equal. When it does, the cell culture container of any one of Claims 3-8 which has the arrangement | positioning shape which at least 1 adhesion boundary touches each of the stimulation-responsive area | region after a division | segmentation.   The cell culture container according to claim 9, wherein the sheet forming region has an arrangement shape in which only one adhesion boundary is in contact with each of the stimulus-responsive regions after the division.   Each of the points is a straight line connecting one point of one adhesive boundary and one point of the adhesive boundary adjacent to the one adhesive boundary so that the straight line is the shortest. When the straight line formed is a straight line L, the area of the region surrounded by the straight line L corresponding to all the bonding boundaries and all the bonding boundaries is 0.6 times or more the area of the sheet forming region. Item 11. The cell culture container according to any one of Items 3 to 10.   The cell culture container according to claim 1 or 2, wherein there is only one adhesion boundary, and the length of the adhesion boundary is 0.7 times or more of the outer circumference of the stimulus-responsive region. .   The cell culture container according to claim 12, wherein the cell adhesive region is disposed in contact with the entire outer periphery of the stimulus responsive region.   The cell culture container according to any one of claims 1 to 13, wherein the ratio of the area of the stimulus-responsive region to the area of the sheet forming region is 0.6 or more.   The cell culture container according to any one of claims 1 to 14, wherein a cell non-adhesive region having cell non-adhesive properties is disposed outside the sheet forming region. The stimulus responsive region is comprised of a stimulus responsive layer comprising a stimulus responsive polymer;
The cell culture container according to any one of claims 1 to 15, wherein the stimulus-responsive layer is formed on a substrate.   The cell culture vessel according to claim 16, wherein the stimulus-responsive polymer comprises a temperature-responsive polymer having a lower critical solution temperature.   The cell culture container according to claim 16 or 17, wherein the substrate has cell adhesiveness, and the cell adhesive region is composed of a surface of the substrate.   The cell culture container according to claim 18, wherein the substrate comprises glass or plastic.   The manufacturing method of a cell sheet including the process of culturing a cell with the cell culture container of any one of Claims 1-19, and forming a cell sheet. A method for producing the cell culture container according to claim 18 or 19,
Forming the stimulus-responsive layer on the cell-adhesive substrate;
Partially removing the stimulus responsive layer to expose the substrate;
A method for producing a cell culture container.