Abstract:
Provided is a culture sheet which enables a technique for forming a three-dimensional tissue having uniform diameter without applying any chemical on the surface of a culture substrate. On the culture sheet ( 150 ) of the culture substrate, a plurality of holes ( 152 ) are formed and nanopillars ( 153 ), which are capable of controlling the adhesiveness and migration ability of cells, are formed on the bottom surface of each hole ( 152 ), said bottom face serving as a culture surface. The culture surface of each hole ( 151 ) is provided with a partition wall ( 152 ) and the internal nanopillars ( 153 ) are formed in the vicinity of the center of the hole ( 151 ). Owing to this configuration, the interaction among the disseminated cells can be restricted so that uniformly sized three-dimensional structures of the cells can be formed.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a technique of culturing animal and plant cells using a culture substrate, and graphically forming spheroids (3D tissues), and monolayer tissues (2D tissues) of the cells. 
       BACKGROUND ART 
       [0002]    In the process of developing pharmaceuticals, in vitro assays using cells instead of animal experiments have been required. In particular, the demand for applying such in vitro assays to the screening and toxicity and metabolism tests of candidate pharmaceutical substances has been increasing. 
         [0003]    In such a background, approaches of alternative methods using cells in place of conventional animal experiments has been actively attempted, but many of such approaches have limitations in predicting their clinical reactions. This assumedly because the forms of the cells are not mimicking their actual in vivo structures in these culture methods (Non-Patent Literature 1). Therefore, the construction of 3D tissues which exhibits functions more similar to those of living bodies has been attempted so far, and 3D tissues has been successfully formed for various cell strains. 
         [0004]    As a substrate for forming 3D tissues of cells, a sheet (nanopillar sheet) for culture in which regularly arranged ultrafine pillar structures or protrusions are formed on the surface of a sheet has been developed, the 3D tissues formed has the problems that they have high release properties from the substrate (Patent Literature 1), and that they are lost in the process of medium change. Moreover, since it is impossible to control the diameter of formed 3D tissues, it entails the problem that their sizes are not uniform, and therefore the performance of each of the 3D tissues is varied. It is thus still premature as a practical formation method. 
         [0005]    To this end, a technique of providing minute cavity structures in a culture substrate, and forming a single 3D tissue per cavity (cellular tissue microchip) has been developed so far (Patent Literature 2, Non-Patent Literature 2). A feature of this technique is that by applying a substance having adhesion to a predetermined region around the center of at the bottom of the cavity, a cell adhesive region and a cell non-adhesive region are defined, and the cavity itself is rotated by a rotation drive apparatus or the like to perform rotation culture, so that cultured cells are retained around the center of the bottom of the cavity which is the cell adhesive region. 
       CITATION LIST 
     Patent Literature 
       [0006]    Patent Literature 1: Japanese Unexamined Patent Application Publication No. 2005-312343 
         [0007]    Patent Literature 2: Japanese Unexamined Patent Application Publication No. 2006-121991 
       Non-Patent Literature 
       [0008]    Non-Patent Literature 1: “The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model” Leoni A. Kunz-Schughart, James P. Freyer, Ferdinand Hofstaedter, and Reinhard Ebner J Biomol Screen, 9: 273-285 (2004) 
         [0009]    Non-Patent Literature 2: “Orderly arrangement of hepatocyte spheroids on a microfabricated chip.” J Fukuda and K Nakazawa Tissue Eng, 11:1254-62 (2005) 
         [0010]    Non-Patent Literature 3: “Formation of Hepatocyte Spheroids with Structural Polarity and Functional Bile Canaliculi Using Nanopillar Sheets.” R Takahashi, H Sonoda, Y Tabata and A Hisada, Tissue Eng Part A, 1-45 (Mar. 4, 2010) 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0011]    While cellular tissue microchips have such features, in order to compulsorily adhere cells onto specific portions on the surface of the substrate, the cell adhesive region and cell non-adhesive region need to be defined by applying a chemically synthesized substance on the surface of the substrate, which entails some problems. 
         [0012]    First, these chemicals applied may adversely affect the growth of cells, but also this operation requires application or adhesion of chemicals in the hyperfine region, which greatly complicates the operation and requires production costs. 
         [0013]    Moreover, when inoculated cells fall into non-adhesive regions, they are inevitably disposed of along with the medium when the medium is changed during culture, which is hardly considered as an efficient culture method. Furthermore, it is suspected that the cells which have fallen into the adhesion region are caused to form tissues compulsorily by rotation culture, and therefore stress is exerted on cells, which leads to a lowered activity. 
         [0014]    Meanwhile, known nanopillar sheets also have the problems that it is difficult to control the cell movement on the substrate plane, and that it is impossible to control the dimension and diameter of the 3D tissues formed. At the same time, it also has the problem that it is impossible to retain the formed 3D tissues in a target position. 
         [0015]    An object of the present invention is to provide a culture sheet, a culture substrate, and a cell culture method using the same which enable forming 3D tissues having a uniform diameter without applying chemicals on the surface of the culture substrate, and further retaining the 3D tissues in a target position. 
       Solution to Problem 
       [0016]    In order to achieve the above-mentioned object, the present invention provides a culture substrate and a culture sheet in which a culture region is provided, a plurality of projections are formed in the culture region, a partition which partitions the culture region and is taller than the projections around the culture region form, and the constitutional proportion of the projections in the culture region is in the range from 20% to 75%. 
         [0017]    Moreover, in order to achieve the above-mentioned object, the present invention provides a culture substrate and a culture sheet in which a culture region is provided, a plurality of projections are formed in the culture region, a partition which partitions the culture region and is taller than the projections around the culture region is formed, and the constitutional proportion of the projections in the culture region is in the range from 40% to 50%. 
       Advantageous Effects of Invention 
       [0018]    By applying the present invention, formation of 3D tissues can be realized under circumstances with little stress while using only a single material and maintaining their activities by promoting cell movement which is a function inherent to cells. 
         [0019]    Moreover, by integrally providing a limited region, i.e., a partition, from the same material, cells inoculated within the limited region are all involved in the formation of a single 3D tissue. This achieves a very efficient culture method, and also leads to the expectation that the sizes of a plurality of 3D tissues formed for the respective limited regions are uniform and homogenous, which is effective in cell assays. 
         [0020]    Furthermore, it is expected that the 3D tissues are retained in a target position within the limited region, i.e., the partition. Furthermore, 2D tissues can be formed depending on the purpose. Similar effects are also expected on the 2D tissues. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0021]      FIG. 1  is a drawing which shows the culture sheet according to Example 1 and the hole structure in the culture sheet. 
           [0022]      FIG. 2  is an enlarged view which shows the nanopillar structure according to Example 1. 
           [0023]      FIG. 3  is a drawing which shows a chamber slide according to Example 1, with the culture sheet affixed thereto. 
           [0024]      FIG. 4  is a drawing which shows the constitution of a plate frame body according to Example 2. 
           [0025]      FIG. 5  is a drawing for illustrating the flow of the ultrasonic welding of the plate and culture sheet according to Example 2. 
           [0026]      FIG. 6  is a drawing which shows the flowchart of hepatocyte culture according to Example 3. 
           [0027]      FIG. 7  is a drawing which shows a photograph of a hepatocyte 3D tissue by the culture sheet by the hepatocyte culture flow according to Example 3. 
           [0028]      FIG. 8  is a drawing which shows a two-stage and multi-stage nanopillar culture sheet according to Example 4. 
           [0029]      FIG. 9  is a drawing which shows the types of arrangement patterns of the nanopillars of Examples. 
           [0030]      FIG. 10A  is a drawing which shows the results of the cell culture (the state of cells) when culture sheets having different pillar diameters shown in  FIG. 9  are used. 
           [0031]      FIG. 10B  is a drawing which shows the results of the cell culture (number of cells formed) when culture sheets having different pillar diameters shown in  FIG. 9  are used. 
           [0032]      FIG. 11  is a drawing which shows an inclined nanopillar culture sheet which is a variant of Example 4 of culture sheets. 
           [0033]      FIG. 12  is a drawing which shows a well of the culture sheet having a surface tension avoiding pattern, which is variant 4 of Example of the culture sheet. 
           [0034]      FIG. 13A  is a drawing which shows an appearance perspective view, top view, upper and lower side view of the culture substrate in Example 1. 
           [0035]      FIG. 13B  is a partially enlarged view of the culture substrate in Example 1, which shows an A-A, B-B partially enlarged view and a C-C, D-D partially enlarged view. 
           [0036]      FIG. 13C  is a partially enlarged view and end view of the culture substrate in Example 1, and is a drawing which shows an E-E, F-F partially enlarged view, line G-G end view. 
           [0037]      FIG. 14A  is a drawing which shows a perspective view and bottom view of the appearance of the culture substrate in Example 2. 
           [0038]      FIG. 14B  is a drawing which shows a top view, upper and lower side view of the culture substrate in Example 2. 
           [0039]      FIG. 14C  is a partially enlarged view and partial cross-sectional view of the culture substrate in Example 2, which shows an A-A, B-B partially enlarged view, a C-C, D-D partially enlarged view, and an H-H cross-sectional view. 
           [0040]      FIG. 14D  is a partially enlarged view, and an end view of the culture substrate in Example 2, which shows an E-E, F-F partially enlarged view, and a line G-G end view. 
           [0041]      FIG. 15A  is a drawing which shows the culture sheet and the hole structure in the culture sheet according to Examples 5 and 6. 
           [0042]      FIG. 15B  is a schematic diagram which shows an assembly of projection portions having different diameters according to Examples 5 and 6. 
           [0043]      FIG. 15C  is a drawing which shows an SEM image of the culture sheet of the assembly of projection portions having a diameter of 80 μm according to Examples 5 and 6. 
           [0044]      FIG. 15D  is a drawing which shows an SEM image of the culture sheet and the assembly of projection portions having a diameter of 20 μm according to Examples 5 and 6. 
           [0045]      FIG. 16A  is a drawing which shows an example of the distance between the center of a hepatocyte 3D tissue and the center of the hole structure by the culture sheet by the flow of hepatocyte culture according to Example 7. 
           [0046]      FIG. 16B  is a drawing which shows another example of the distance between the center of the hepatocyte 3D tissue and the center of the hole structure by the culture sheet by the flow of hepatocyte culture according to Example 7. 
           [0047]      FIG. 16C  is a drawing which shows another example of the distance between the center of the hepatocyte 3D tissue and the center of the hole structure by the culture sheet by the flow of hepatocyte culture according to Example 7. 
           [0048]      FIG. 16D  is a drawing which shows another example of the distance between the center of the hepatocyte 3D tissue and the center of the hole structure by the culture sheet by the flow of hepatocyte culture according to Example 7. 
           [0049]      FIG. 17  is a drawing which shows an example of a photograph of the hepatocyte 3D tissue by the culture sheet by the flow of hepatocyte culture according to Example 7. 
           [0050]      FIG. 18  is a drawing which shows an example of a photograph of the hepatocyte 3D tissue by the culture sheet by the flow of hepatocyte culture. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0051]    The best mode for realizing a method for culturing cells using a culture sheet, and forming the 3D tissues which is a cell cluster, or 2D tissues will be described below in detail. 
       Example 1 
       [0052]    Example 1 shows an example in which the culture sheet is applied to the chamber slide which is a culture sheet retaining member. Hereinafter, a sheet which has a partition structure which forms the culture region in the present invention on a known nanopillar sheet, and on which a plurality of projections are formed inside the partition structure is referred to as a culture sheet. 
         [0000]    The culture sheet is formed from a material which has no adverse effect on cells, in this example, it is polystyrene. 
         [0053]    However, it goes without saying that the material is not limited to polystyrene. 
         [0054]      FIG. 1  is a schematic diagram of a scanning electron micrograph of a culture sheet  100  prepared in this Example. Simultaneously, it shows the structure of one of holes  101  (hereinafter referred to as hole) constituted by a plurality of partition structures  102  existing in a single culture sheet. The inside of the hole  101  constitutes a culture region by cell tissue formation unit. 
         [0055]    A plurality of projections  102  retained at the bottom of the hole  101  includes a plurality of microprojections  103  (hereinafter also referred to as projections, pillars or nanopillars). Moreover, the diameter of this hole  101  is a hole diameter  105 . In the culture sheet  100 , the hole  101  including the above-mentioned partition wall  102  and a plurality of projections  103  formed inside the hole  101  are formed from the same material integrally. It should be noted that the shape of this hole  101  is not limited to round, but may have other shapes such as a square shape. 
         [0056]    In this manner, the hole  101  and the plurality of projections  103  formed inside the hole  101  including the partition wall  102  are formed integrally as the culture sheet  100  from a single material which has no adverse effects on cells, whereby cells can be grown without foreign substances bonding to the cells in the culture steps. Furthermore, since cells are grown in each of the partitions, cells of a homogeneous size can be formed. 
         [0057]    Moreover, a plurality of projections are provided within the partition wall  102  arranged in a surrounding manner, and therefore the cell movement which is the ability inherent to the cells is promoted, and cells are grown by the movement so that cell culture which can maintain the cell activity is possible with no influence of disturbance (stress) by rotation culture or the like. 
         [0058]    When a culture region is to be formed while these holes  101  and projection assembly  103  are provided separately, they need to be joined by adhesion or welding. 
         [0059]    For example, when these are joined by adhesion, adhesive components enter into the culture region, which may adversely affect generated cells. In joining by welding, the inner diameter of the hole  101  is a hyperfine region diameter on the cell formation level, and therefore it is very difficult to perform welding while forming a target cell region and not damaging the partitions and projections. When the partitions and projections have damages and deformations, unwanted stress may be applied on the cells in the process of cell formation, and the movement of the cells themselves may be impaired. 
         [0060]    Therefore, the hole bottom  104 , partition wall  102  and projections  103  constituting the holes  101  which forms the culture region are preferably formed integrally. By forming integrally in such a manner, it is preferable because culture excluding the influence of unwanted components other than those required for cell culture can be performed. 
         [0061]    Subsequently, an enlarged view of the projection  103  is shown in  FIG. 2 . A pillar diameter indicates a diameter  106  of the tip of the projection. A pillar pitch indicates a distance  107  from the center of the tip of the projection to the center of the tip of the adjacent projection. A pillar height indicates a height  108  from the tip of a nanopillar to the bottom thereof.  FIG. 2(   a ) and  FIG. 2(   b ) indicate a square arrangement and a triangle arrangement, respectively, of nanopillars of this Example. 
         [0062]    In this Example, culture sheets in which the pillar diameter, pillar pitch and pillar height are 2.0 μm, 4.0 μm, and 1.0 μm, respectively, were used, but as will be described later, such culture sheets are not necessarily used. The height of the partition structure is 70 μm in this Example, but this value is not necessarily used, and suitably the height may be such that the formed cells do not get over the partition. 
         [0063]    The culture sheet  100  in this Example is produced by the method described below. A mold in which round holes each having a diameter of 200 μm and depth of 70 μm are arranged in the form of squares, and micropores each having a diameter of 2.0 μm and a depth of 1.0 μm are formed at the bottom at a pitch of 4.0 μm was pressed against a polystyrene film having a thickness of 400 μm at 135° C. and a pressure of 2 MPa. The film was took out from a press machine after being cooled to room temperature, and the mold was peeled off from the polystyrene film, whereby a culture sheet retaining a plurality of holes each having a hole diameter of 200 μm and having a plurality of projections at the bottom thereof can be produced. 
         [0064]    Herein, a mold material is silicon wafer, and in order to prevent adhesion with the polystyrene film during the production of the culture sheet, a mold releasing process is performed in advance with a fluorine-based mold releasing agent. Silicon wafer was used as the mold material in this Example, but a mold made from other metal materials and the like may be also used. 
         [0065]    As shown in  FIG. 3 , the culture sheet  100  produced by integral molding from the single material in this manner was cut into 2-cm square pieces in this example, and a surgical glue  110  was applied onto the glass bottom of the chamber slide  109  to adhere the chamber slide  109  and the culture sheet  100 , whereby the chamber slide  109  with the culture sheet  100  affixed thereto is produced. It should be noted that in  FIG. 3 ,  109   a  represents a frame for partitioning the culture sheets  100 . This frame  109   a  is formed from, for example, a plastic material or the like. It should be noted that the shape of a frame body such as this frame  109   a  is not limited to square, but may be other shapes such as a round shape. 
         [0066]      FIGS. 13  A,  13 B,  13 C, show the overall constitution diagram and principal part cross-sectional view of the chamber slide with the culture sheet of this Example affixed thereto. 
         [0067]      FIG. 13A  is an appearance perspective view, top view, and upper and lower side views of the culture substrate in this Example. Illustration of left and right side elevational views is omitted since its form is obvious from the perspective view. 
         [0068]      FIG. 13B  is a partially enlarged view, which shows an A-A, B-B partially enlarged view, and a C-C, D-D partially enlarged view. 
         [0069]      FIG. 13C  is a partially enlarged view and end view, which shows an E-E, F-F partially enlarged view, and a line G-G end view. 
         [0070]    The article shown in  FIGS. 13A to 13C  is a culture device (culture containers) for culturing cells of humans, animals, plants and others, and are each constituted by the culture sheet  100  and a retaining member (chamber slide)  109  which retains the culture sheet  100 . A plurality of partition portions  102  are formed on the surface of the culture sheet  100 , and is provided at the bottom of the inside of a cylindrical hole portion  109   a  formed on the retaining member  109 . 
         [0071]    Furthermore, culture regions having a plurality of minute projection portions  103  within the partition portion are formed respectively. When target cells to be cultured are added to the inside of the hole portion  109   a,  as added to the sheet surface forming the culture regions within the partition portion  102 , the target cell is retained in the plurality of minute projection portions  103  and cultured. 
       Example 2 
       [0072]    Subsequently, Example 2 will be described with reference to  FIGS. 4 and 5 . In Example 2, the constitution of a multiwell plate with a culture sheet and a production example thereof will be shown.  FIG. 4(   a ) is a bottom view of a frame body  111  constituting the multiwell plate. The frame body  111  which is a culture sheet retaining member is such that has  24  cylindrical hole portions  111   a  in total, arranged in 4 rows and 6 columns, formed in an area measuring about 125 mm in width, about 80 mm in length, and about 20 mm in height. The material used is polystyrene. 
         [0073]    The number of holes formed on the frame body normally ranges from 6 to 1536, varied depending on the use, and therefore the number of holes on this frame body is not limited to 24. The material of the frame body is not limited to polystyrene either. 
         [0074]    In producing the culture substrate, the frame body  111  and the culture sheet  100  is joined by ultrasonic welding. 
         [0075]    The following processes are performed on the frame body  111  in advance. As the first process, a projection for fixing film  112  is processed at the bottom of the frame body  111  for the purpose of preventing the cell culture sheet and the plate from being shifted due to the vibration of ultrasonic waves provided when the frame body  111  and the culture sheet  100  are welded. As the second process, a rib structure  113  is provided to weld the culture sheet by ultrasonic waves. 
         [0076]      FIGS. 4(   b ) and  4 ( c ) are shows the cross-sectional views at lines B-B′ and A-A′, respectively, in  FIG. 4(   a ). Moreover, holes  114  having the same diameter are provided in the culture sheet in the same position when both are overlapped so that the projection engages with the projection for fixing film. Successively, this frame body and the culture sheet  100  are adhered by ultrasonic welding. 
         [0077]    The step of the welding is shown in  FIG. 5 . First, the holes of the projection for fixing film of the frame body and of the culture sheet are placed together and stacked ( FIG. 5(   a )). Subsequently, ultrasonic waves are produced from the culture sheet side from an ultrasonic wave oscillator via a converter, a booster, or further a horn, and both are welded ( FIG. 5(   b )). A horn is an apparatus for welding by irradiating an appropriate position with ultrasonic waves of an appropriate energy. A specific apparatus designed so that ultrasonic waves are generated appropriately along the position of the rib structure was produced and used.  115  shows a top view of the thus-produced plate. 
         [0078]    The frame body and the culture sheet were joined by using ultrasonic welding in this Example, but it goes without saying that the joining is not limited to this method. Formation of a plate can be realized without any intervention of organic matters such as adhesives which affects cells by ultrasonic welding. Therefore, no adverse effects are caused on cells. Needless to say, this Example is a culture sheet which is applicable and useful not only to toxicity and metabolism tests in new drug development processes, but also to the formation of organizations intended for regenerative medicine. 
         [0079]    It is needless to say that by providing a plurality of the rib structures at the bottom of the frame  109   a  also in the culture substrate of the chamber slide shape shown as an example in Example 1, and performing welding with the culture sheet  100  by the rib structures, the culture substrate can be produced by a joining method similar to this Example. 
         [0080]    In the culture substrate prepared in this manner, a plurality of the holes  101  are formed on the culture sheet  100  formed at the bottom of the frame body  111 , and a plurality of projections constituted at the bottom  104  of the hole include a plurality of microprojections  103  (hereinafter also referred to as projections, pillars or nanopillars). Moreover, the diameter of this hole  101  is used as a hole diameter  105 . In the culture sheet  100 , the hole  101  including the above-mentioned partition wall  102  and the plurality of projections  103  formed inside the hole  101  are formed from the same material integrally. It should be noted that the shape of this hole  101  is not limited to round, but may have other shapes such as a square shape. 
         [0081]    In this manner, the hole  101  including the partition wall  102  and the plurality of projections  103  formed inside the hole  101  are formed integrally from a single material which has no adverse effects on cells as a culture sheet, whereby cells can be grown with no foreign substances adhering to cells in the culture step. Furthermore, since cells are grown in each of the partitions, cells of a homogeneous size can be formed. 
         [0082]    Moreover, a plurality of projections are provided within the partition arranged in a surrounding manner, and therefore cell movement, which is the ability inherent to the cells, is promoted, and cells are grown by the movement so that cell culture which can maintain the cell activity is possible with no influence of disturbance (stress) by rotation culture or the like. 
         [0083]    When a culture region is to be formed while these holes  101  and projection assembly  103  are provided separately, they need to be joined by adhesion or welding. For example, when joined by adhesion, adhesive components enter into the culture region, which may adversely affect generated cells. 
         [0084]    Moreover, when welding is to be performed, the inner diameter of the hole  101  is a hyperfine region diameter on the cell formation level, and therefore it is very difficult to perform welding while forming a target cell region and not damaging the partitions and projections. When the partitions and projections have damages and deformations, unwanted stress may be applied on the cells in the process of cell formation, and the movement of the cells themselves may be impaired. 
         [0085]    Therefore, the hole  101  which forms the culture region and the projections  103  are preferably formed integrally by forming integrally in such a manner, it is preferable because culture excluding the influence of unwanted components other than those required for cell culture can be performed. 
         [0086]    Herein, in  FIGS. 14A ,  14 B,  14 C, and  14 D, an overall constitution diagram and a principal part cross-sectional view of a multiwell plate with the culture sheet of this Example are shown. 
         [0087]      FIG. 14A  shows an appearance perspective view and a bottom view of the culture substrate in this Example. 
         [0088]      FIG. 14B  shows a top view and upper and lower side views of the culture substrate. Herein, illustration of left and right side elevational views is omitted since its form is obvious from the appearance perspective view. 
         [0089]      FIG. 14C  is a partially enlarged view and a partial cross-sectional view, which show an A-A, a B-B partially enlarged view, a C-C, D-D partially enlarged view, and an H-H cross-sectional view. 
         [0090]      FIG. 14D  is a partially enlarged view, and an end view, which show an E-E, F-F partially enlarged view, and a line G-G end view. 
         [0091]    The article shown in  FIGS. 14A ,  14 B,  14 C,  14 D is a culture device (culture container) for culturing cells of humans, animals, plants and others, and is constituted by the culture sheet  100  and a retaining member (frame body)  111  which retains the culture sheet  100 . 
         [0092]    A plurality of the holes  101  are formed on the surface of the culture sheet  100 , and is provided at the bottom of the inside of a cylindrical hole portion  111   a  formed in the retaining member. 
         [0000]    Furthermore, culture regions having a plurality of minute projection portion  103  within the partition portion are formed respectively. When target cells to be cultured are added to the inside of the hole portion  111   a,  as added to the sheet surface forming the culture regions within the hole  101 , the target cell is retained in the plurality of minute projection portions  103  and cultured. 
         [0093]    Moreover, the culture substrate of this example shows an example in which the culture sheet is welded from the back side of the frame body  111 , and the frame body  111  which is a retaining member and the culture sheet  100  are welded via a joint  1112 . 
         [0000]    The joint  1112  is provided on the outside of the hole portion  111   a,  and the culture region is not affected by the welding.
 
Therefore, although welding was shown as an example in this example, the joining method is not limited to this, and other joining methods can be also employed since joining does not affect the culture region itself with other joining methods.
 
         [0094]    In addition, in the substrate of this example, the frame body  111  has the form of a square, and at least of the four apexes is cut off. The formation of this cut surface  1113  facilitates specification of the position of the hole portion of the substrate by the operator who performs culture. 
         [0000]    This cut face is not essential, and of course may be or may not be present. A non-slip portion  1111  is formed on the culture substrate, which can prevent the operator from unexpectedly shaking and dropping the substrate and prevent other accidents during the operation. 
       Example 3 
       [0095]    Example 3 shows an example of application of cells to tissue culture using the culture substrates produced in Examples 1 and 2. In the development of new drugs, the construction of the 3D tissues which reflects biological functions has demands for various evaluations utilizing cells substituting animal experiments. In addition, when the thus-formed 3D tissues are subjected to various tests in screening of pharmaceuticals or development of new drugs, it is necessary to verify in advance whether the 3D tissues retain activities to withstand the tests. In this case, if the formed spheroids are held in the predetermined position with high reproducibility, it is expected that they are suitable for high through-put screening and various tests. 
         [0096]    Moreover, before culturing induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) to cause them to differentiate into target cells, 3D tissues need to be formed. Therefore, a technique of easily constructing 3D tissues has been demanded also in the field of regenerative medicine. From such a background, an example of forming 3D tissues using the chamber slide in particular is shown herein, but the essential part of cell culture is not especially different even for a multiwell plate. In this example, an example using rat hepatocytes is shown, but as mentioned above, it is applicable to cell strains of various animals and plants, and cell strains are not especially limited. 
         [0097]    Preparation of hepatocytes is performed according to in situ collagenase perfusion technique. The detail is as follows: The abdomen of a Fisher 344 male rat (7 to 10 weeks old) is opened under pentobarbital anesthesia, and a catheter is inserted into the portal vein to inject a pre-perfusate (Hanks&#39; solution not including Ca 2+  or Mg 2+  and containing EGTA). 
         [0098]    The postcaval vein in the lower liver is simultaneously incised to discharge blood. Next, the thorax is opened, the postcaval vein which goes into the right atrium is incised, and the postcaval vein in the lower liver is clipped with a clamp to perform perfusion. Perfusion is stopped after it is confirmed that the blood removal from the liver has been fully conducted. The perfusate is exchanged to a collagenase solution to perform perfusion. 
         [0099]    Perfusion is performed using the Hanks&#39; solution containing 0.05% of collagenase in this example, but this solution is not necessarily used. Perfusion is stopped after it is confirmed that intercellular tissues have been digested by collagenase. The liver is separated, cut into small pieces in a cooled Hanks&#39; solution, and is dispersed into cells by pipetting. Subsequently, undigested tissues are removed by gauze filtration. The cell suspension is repeatedly centrifuged at 50 G for 1 minute several times to remove nonparenchymal cells. Subsequently, damaged hepatocytes are removed by centrifugal separation at 500 G for 5 minutes using an isotonic Percoll solution. The survival rate of the obtained hepatocytes is measured by the trypan blue exclusion method, and the hepatocytes with a survival rate of 85% or higher are used for culture. Herein, the hepatocytes with a survival rate of 85% or higher are used for culture, but it goes without saying that this condition is not necessarily used. Preparation of the hepatocytes is not necessarily limited to the in situ collagenase perfusion technique. 
         [0100]    A flowchart of the culture using the thus-obtained hepatocytes is shown in  FIG. 6 . 
         [0101]    In the flowchart of  FIG. 6 , first, type I collagen  116  is applied to the culture sheet of the chamber slide type produced in Example 1. A 1 to 1.5-ml portion of a diluted solution which has been produced by diluting type I collagen dissolved in a weakly acidic solution with sterile water to a predetermined concentration is added to the chamber slide mentioned above ( FIG. 6(   a )). Next, a decompression operation is performed in order to cause the added type I collagen to be adsorbed onto the nanopillar sheet  100  completely ( FIG. 6(   b )). The decompression operation is performed at 0.04 atmosphere or lower using a decompression container  117  and a decompression pump  118 . The decompression time is not particularly limited, but the decompression is performed for 10 minutes in this Example. The constitution of the apparatus used for decompression is not particularly limited. Herein, the range of the predetermined concentration of the diluted solution is 100 (ng/ml) or higher and 10 μg/ml) or lower. The concentration is not necessarily limited to this range, but this range is suitable for spherical 3D tissues to form. Finally, an excess of type I collagen is removed, and PBS(−)  119  is added thereto ( FIG. 6(   c )). This operation is performed three times, and an excess of type I collagen is washed. 
         [0102]    Hepatocytes  120  prepared by the in situ collagenase perfusion technique as above-mentioned are suspended in a medium  121 , and the suspension is inoculated on the NP sheet with Type I collagen prepared as stated above applied thereto similarly ( FIG. 6(   d )). The medium is not particularly limited, but, a Williams E medium including a medium containing serum (FCS), insulin, and dexamethasone (hereinafter referred to as medium (including 10% FCS)) is used. In this Example, a Williams E medium containing 10% FCS, 8.6 nM insulin, and 255 nM dexamethasone is particularly used. After inoculation, culture is started using a CO 2  incubator under the conditions of 5% CO 2  and 37° C., the first medium exchange is performed after 18 hours or more has elapsed, and medium exchange is performed every 24 hours henceforth. Although the medium used for the culture after the 18th hour after the inoculation is not particularly limited, in this example, a medium (hereinafter referred to as medium (containing no FCS) with FCS removed from a medium (containing 10% FCS) is used. 
         [0103]    Moreover, the inoculation density of hepatocytes was set to 1×10 5  cells/ml in this Example, but is not limited to this concentration. Herein, the culture sheet  100  used for culture has a pillar height, pillar diameter and pillar pitch of 1.0 μm, 2.0 μm, and 4.0 μm, respectively, but the values are not limited to these. 
         [0104]    Moreover, the concentration of Type I collagen added to the culture sheet is set to 100 (ng/ml) in this Example, but may be a concentration other than this. Spheroids may be formed at a concentration other than this concentration depending on the conditions of the cells. The cells are cultures for 96 hours in total, whereby 3D tissues  122  are formed ( FIG. 6(   e )). 
         [0105]      FIG. 7  shows a photograph of the results of actual culture of hepatocytes using the above-mentioned culture sheet having a hole diameter of 200 μm. As can be seen from  FIG. 7 , spherical 3D tissues  71  having such similar sizes are formed in the holes  70  with no special chemical applied onto the surface of the culture sheet and by stationary culture having little stress on cells. This culture method supposedly does not deteriorate the activity of the cells originally retained, and is therefore effective for cell assays and the like. 
       Example 4 
       [0106]      FIG. 8  shows a variant of Example of the culture sheet  100  mentioned above as Example 4. First, an example is shown in which in a culture sheet  123 , by arranging the arrangement pattern of projections which provides differences in the migration and adhesion of cells in two stages, as in  FIG. 8(   a ), in a manner of surrounding a first arrangement pattern  125   a  with a second arrangement pattern  125   b,  3D tissues or 2D tissues are formed on the first arrangement pattern  125   a  (for example, near the center of the hole). 
         [0107]    Contrarily, an example is shown in which, as in  FIG. 8(   b ), by arranging in two stages in a manner of surrounding the second arrangement pattern  125   b  with the first arrangement pattern  125   a,  3D tissues or 2D tissues are formed on the second arrangement pattern  125   b  (for example, the periphery of the hole). It should be noted that  124  represents a hole as in the preceding Examples. The dotted line indicates the boundary of the patterns. 
         [0108]    By arranging the first arrangement pattern not only in the central portion of the hole  124 , but also arranging the same as in the culture sheet  126  of  FIG. 8(   c ), by surrounding, for example, 4 portions of the first arrangement patterns  127   b  with the second arrangement pattern  127   a,  tissues having similar sizes can be formed on the first arrangement pattern. In this manner, the combination of the pillar diameter, pillar pitch, and arrangement pattern can be an optimum pattern of arrangement depending on the purpose to perform culture. Similarly,  FIG. 8(   d ) shows a culture sheet  128  in which the arrangement pattern is set to be multi-stage patterns  129   c,    129   b,    129   a.    
         [0109]    Next, using  FIG. 9 , the types of the arrangement patterns (hereinafter referred to as pillar patterns) of the projections in the above-mentioned Example will be described. As shown in  FIG. 9 , 11 types of arrangement patterns have been shown as examples. As can be seen from the same figure, there are 11 types of arrangement patterns with the pillar diameter and pillar pitch ranging from 0.18 to 20.0 μm and from 0.36 to 40.0 μm, respectively, but the pillar diameter and pillar pitch are not limited to these. 
         [0110]    An example of hepatocytes cultured under these pillar patterns is shown in  FIGS. 10A and 10B . 
         [0111]    It should be noted that in the culture on a flat plane with no pillar pattern, many cells are discharged along with the medium when the medium is changed during the culture, and therefore desired cultured cells cannot be effectively obtained. Accordingly, no illustration is provided in  FIG. 10A . 
         [0112]      FIG. 10A  are figures which show the states of the cells when culture is performed using the culture sheet  100  with the double pitch relative to the pillar diameter. As a result, when the pillar diameter is 0.18 μm, 0.5 μm, and 1.0 μm, flat tissues which are not spherical are adhered at the bottom of the substrate, while, when it is 2.0 μm and 5.0 μm, 3D tissues which are spherical are formed on the substrate. 
         [0113]    Comparing the spherical cells formed in the substrate with the pillar diameter of 2.0 μm and 5.0 μm, the substrate with the pillar diameter of 2.0 μm had more cells adhered onto the substrate, indicating that it is in a stable state. That is, it can be seen that as for the cell adhesion, the greater the pillar diameter, the lower the adhesion and the more promoted the movement by cells. 
         [0114]      FIG. 10B  is a graph which shows, as for the number of 3D tissues (spheroids) of the hepatocytes formed on the sheets with each of the pillar diameters, the results grouped by diameter of the spheroids formed. The area of the sheet is 4 square cm (2 cm×2 cm). 
         [0115]    In the 3D tissues of hepatocytes, in cell assays intended for drugs screening, and the toxicity and metabolism tests which can substitute animal experiments in the innovative drug development field, cells having diameters of 50 to 100 microns are preferable. In this example, it can be seen that the number of the formed cells of this size is the most in the case of the substrate with a pillar diameter of 2.0 μm, indicating that this pillar diameter is preferable. 
         [0116]    However, under the above-mentioned examination, it was stated that the case where the pillar diameter is 2.0 μm is preferable in order to form cells having diameters of 50 to 100 microns, but the pillar diameter is not limited to this, and for all the pillar diameters used in this examination, it was found that a greater number of cells with stable shapes are formed compared to the flat state with no pillar formed. Thus, the form or adhesion to the substrate of cells or tissues formed from cells can be freely changed by the difference in pillar pattern. 
         [0117]    By applying the results stated above, as explained in Example of  FIG. 8 , by arranging in two stages in a manner of surrounding the first arrangement pattern with a small pillar diameter (pillar pitch) by the second arrangement pattern with a large pillar diameter (pillar pitch), or arranging in multiple stages, tissues having target shapes can be formed in target positions within the holes utilizing cell adhesion and the motion characteristics of cells themselves. 
         [0118]    Moreover, by decreasing the heights of the nanopillars having the same size of the pillar diameter from the periphery toward the central portion of the hole, it is possible to provide a difference in height gradually in a manner of inclining, to promote the movement of cells so that they gather in the central portion by gravity and form tissues. 
         [0119]      FIG. 11(   a ) shows a culture sheet  130  which is a variant in which a difference is provided in the heights of the nanopillars gradually. At this time, unlike in a normal U-shaped culture container, there is produced an effect that the cells are retained in the center by the presence of the pillars. In addition, as in the culture sheet  131  of  FIG. 11(   b ), it is also possible to promote the effect stated above by providing a difference in pillar diameter even in the inclination. 
         [0120]    In the variant of  FIG. 11 , the height is changed gradually to smoothen the inclination, but a constitution in which the height is sequentially changed stepwise may be also employed. 
         [0121]    Moreover, a plurality of holes gather to form a culture surface (square shape in the case of the chamber slide, round shape in the case of and the plate), but in the culture, a difference occurs in how 3D tissues are formed in the central portion and peripheral portion of the culture surface by the influence of the surface tension. That is, although 3D tissues are formed in the central portion of the culture surface, 3D tissues may not be formed in some events for the reason that the amount of the medium is increased of the portion by the surface tension in the peripheral portion, the amount of oxygen supplied is lowered, or the high water pressure is applied. In order to avoid this phenomenon, the culture sheets  132 ,  133  retaining the hole structure may be produced only in the central portion of the culture surface as shown in  FIGS. 12  ( a ), ( b ). 
         [0122]    By forming the culture sheet in this manner, the culture substrate having high culture efficiency and little production load can be achieved. 
       Example 5 
       [0123]      FIG. 15A ,  FIG. 15B ,  FIG. 150 , and  FIG. 15D  show the culture sheet of Example 5. In Example 5, among the various variants shown in Example 4 of  FIG. 8 , an example is shown in which the first arrangement pattern  125   a  is a flat structure, and a culture sheet having a pattern in which projections are arranged in the central portion of the hole is applied to the chamber slide which is a culture sheet retaining member. That is, in this Example, by providing a culture sheet having a structure in which the culture regions consist of the first region and the second region surrounding the same, projections are arranged on in the first region, and projections are not formed in the second region, spheroids which are 3D tissues having similar diameters are retained in the central portion of the culture region corresponding to the first region, whereby the spheroids can be retained in the target position. 
         [0124]    Herein, an example in which projections are arranged near the center in the culture region is shown, but the center need not be necessarily included, and it goes without saying that projections may be arranged in a desired region in the culture region. Moreover, although an example in which the projection region of an approximate rhombus shape and circle shape is formed is shown, it goes without saying that the projection region may be in the shape of a square or a polygon. 
         [0125]      FIG. 15A  shows an example of the culture sheet prepared in this Example.  FIG. 15A  shows one structure of holes  151  constituted by a plurality of partition structures  152  in a single culture sheet. The configuration that the inside of the hole  101  constitutes a culture region by a cell tissue formation unit partitioned by a partition wall is the same as in the above-mentioned Example. A plurality of projections  153  retained at the bottom  154  of the hole  151  includes a plurality of microprojections. Moreover, the diameter of this hole  151  is set tube a hole diameter  155 . Preferably, in the culture sheet  150 , the hole  151  including the above-mentioned partition wall  152  and a plurality of projections  153  formed within the hole  151  are formed from the same material integrally. It should be noted that the shape of this hole  151  is not limited to round, but may be another shape such as a square, as in the above-mentioned Example. 
         [0126]    In a suitable aspect of this Example, as shown in the culture sheet  150   a  of  FIG. 15B , culture sheets having a pillar height, pillar diameter, and pillar pitch of 1.0 μm, 1.0 μm, 2.0 μm and, 1.0 μm, 2.0 μm, 4.0 μm, respectively, and a diameter of the assembly of projection portions of 200 μm (nanopillars on the entire surface), 150 μm, 120 μm, 100 μm, 80 μm, 60 μm, 40 μm, 20 μm can be used. 
         [0127]    As shown in an enlarged portion  150   b  of  FIG. 15B , providing a culture substrate in which the formation region (constitutional proportion) of projections by in the hole, that is, a cell tissue formation unit partitioned by a partition wall constituted in multi-stages is effective as a test substrate for grasping an optimum formation rate of projection regions in Example 7 described later. An optimum constitutional proportion may vary depending on the cell strains and desired size intended for culture, and therefore performing a culture test in advance using such a culture substrate is useful since it affects the culture efficiency thereafter. It should be noted that the hole  151   a  indicates a hole in which no projection is formed. 
         [0128]    The culture sheet  150   a  is formed from a material which does not adversely affect cells, and it is, in this example, polystyrene. However, it goes without saying that the material is not limited to polystyrene. 
         [0129]    As a typical example, a SEM image in which the diameter of the assembly of projection portions is 80 μm is shown in  FIG. 15C , and an SEM image in which the diameter is 20 μm is shown in  FIG. 15D . In each of  FIG. 15C , and  FIG. 15D ,  156  and  158  represent a hole, while  157  and  159  represent a projection assembly. 
         [0130]    In this manner, the hole  151  including the partition wall  152  and the plurality of projections  153  formed inside the hole  151  are formed integrally from a single material which has no adverse effects on cells as culture sheets  150 ,  150   a,  whereby cells can be grown with no foreign substances adhering to cells in the culture step. 
         [0000]    Furthermore, since cells are grown in each of the partitions, cells of a homogeneous size can be formed. 
         [0131]    Moreover, a plurality of projections are provided within the partition wall  152  arranged in a surrounding manner. Therefore, cell movement, which is the ability inherent to the cells, is promoted, and cells are grown by the movement so that a cell culture which can maintain the cell activity is possible with no influence of disturbance (stress) by rotation culture or the like. 
         [0132]    When a culture region is to be formed while these holes  151  and projection assembly  153  are provided separately, they need to be joined by adhesion or welding. For example, when these are joined by adhesion, adhesive components enter into the culture region, which may adversely affect generated cells. 
         [0000]    In joining by welding, the inner diameter of the hole  151  is a hyperfine region diameter on the cell formation level, and therefore it is very difficult to perform welding while forming a target cell region and not damaging the partitions and projections. When the partitions and projections have damages and deformations, unwanted stress may be applied on the cells in the process of cell formation, and the movement of the cells themselves may be impaired. 
         [0133]    Therefore, also in this Example, as stated above, the hole bottom  154 , partition wall  152  and projections  153  constituting the holes  151  which form the culture region are preferably formed integrally by forming integrally in such a manner, it is preferable because culture excluding the influence of unwanted components other than those required for cell culture can be performed. 
         [0134]    In this Example, a culture sheet in which the pillar diameter, pillar pitch and pillar height are 1.0 μm or 2.0 μm, 2.0 μm or 4.0 μm, 1.0 μm, respectively, was used, but as will be described later, the culture sheet may be one with other specifications than these. The height of the partition structure is 70 μm in this Example, but this value is not necessarily used, and suitably the height may be such that the formed cells do not get over the partition. 
         [0135]    The culture sheets  150 ,  150   a  in this Example are produced by a method similar to that in Example  1 , and therefore detailed description of the production method will be omitted herein. In addition, also in this Example, the chamber slide  109  with the culture sheet  150  affixed as shown in  FIG. 3  can be produced, and it goes without saying that a chamber slide having an overall constitution and a principal part cross section similar to those in  FIG. 13A ,  FIG. 13B ,  FIG. 13C  can be obtained, and therefore explanation will be omitted herein. 
       Example 6 
       [0136]    Subsequently, Example 6 will be described with reference to  FIGS. 4 and 5 . This Example shows the constitution of a multiwell plate with a culture sheet using the culture sheets  150 ,  150   a  described in Example 5, and a production example thereof. The constitution of the multiwell plate and a production example of the same have been described in  FIGS. 4 and 5 , but this Example is basically similar to Example 2 except that the culture sheets  150 ,  150   a  are used in place of the culture sheet  100  used in Example 2. 
         [0137]      FIG. 4(   a ) is a bottom view of the frame body  111  constituting the multiwell plate. The frame body  111  which is a culture sheet retaining member is such that has 24 cylindrical hole portions  111   a  in total, arranged in 4 rows and 6 columns, are formed in an area measuring about 125 mm in width, about 80 mm in length, and about 20 mm in height. The material used is polystyrene. 
         [0138]    The number of holes formed on the frame body normally ranges from 6 to 1536, varied depending on the use, and therefore the number of holes on this frame body is not limited to 24. The material of the frame body is not limited to polystyrene either. 
         [0139]    In producing the culture substrate, the frame body  111  and the culture sheets  150 ,  150   a  in  FIGS. 15A and 15B  are joined by ultrasonic welding. The process and constitution mentioned above are the same as those in Example 2, and their explanation will be therefore omitted herein. 
         [0140]    In the culture substrate prepared in this manner, a plurality of holes  151  are formed on the culture sheets  150 ,  150   a  used in place of the culture sheet  100  formed at the bottom of the frame body  111 , and a plurality of projections constituted at the bottom  154  of the hole are constituted by a plurality of microprojections  153 . In the culture sheets  150 ,  150   a,  the holes  151  including the above-mentioned partition wall  152  and the plurality of projections  153  formed within the holes  151  are formed from the same material integrally. It should be noted that the shape of this hole  151  is not limited to round, and may have other shapes such as a square shape. 
         [0141]    In this manner, the hole  151  including the partition wall  152  and the plurality of projections  153  formed inside the hole  151  are formed integrally from a single material which has no adverse effects on cells as a culture sheet, whereby cells can be grown with no foreign substances adhering to cells in the culture step. 
         [0000]    Furthermore, since cells are grown in each of the partitions, cells of a homogeneous size can be formed. 
         [0142]    Moreover, a plurality of projections are provided within the partition arranged in a surrounding manner. Therefore, cell movement, which is the ability inherent to the cells, is promoted, and cells are grown by the movement so that a cell culture which can maintain the cell activity is possible with no influence of disturbance (stress) by rotation culture or the like. 
         [0143]    As stated above, also in this Example, the holes  151  which form the culture region and the projections  153  are preferably formed integrally. By forming integrally in such a manner, culture excluding the influence of unwanted components other than those required for cell culture can be favorably performed. 
         [0144]    The overall constitution diagram and principal part cross-sectional view of the multiwell plate with the culture sheet of this Example are also as shown in  FIGS. 14A ,  14 B,  14 C,  14 D as in Example 2, and explanation will be therefore omitted herein. 
       Example 7 
       [0145]    Subsequently, Example 7 shows an example of application of cells to tissue culture using the culture substrate produced in Examples 5 and 6. An example of application of cells to tissue culture using the culture substrates produced in Examples 1 and 2 was shown previously as Example 3 using  FIGS. 6 and 7 . The difference between this Example and Example 3 is that a culture substrate in which the culture sheets  150 ,  150   a  are used in place of the culture sheet  100  is used. Since explanation is common for other point, explanation will be therefore omitted herein. 
         [0146]    It should be noted that in this Example, the inoculation density of hepatocytes is set to 5×10 5  cells/ml, but is not limited to this concentration. Herein, the culture sheets  150 ,  150   a  used for culture as previously explained, have a pillar height, pillar diameter and pillar pitch of 1.0 μm, 1.0 μm, 2.0 μm and, 1.0 μm, 2.0 μm, 4.0 μm, respectively, but the values are not limited to these. 
         [0147]    Moreover, the concentration of Type I collagen added to the culture sheets  150 ,  150   a  was set to Example 100 (ng/ml) in this Example, but may be a concentration other than this. Spheroids may be formed at a concentration other than this concentration depending on the conditions of the cells. In this Example, as shown in  FIG. 15B , culture sheets having a pillar height, pillar diameter and pillar pitch of 1.0 μm, 1.0 μm, 2.0 μm and, 1.0 μm, 2.0 μm, 4.0 μm, respectively, a hole diameter of 200 μm, a diameter of the assembly of projection portions of 200 μm (nanopillars on the entire surface), 150 μm, 120 μm, 100 μm, 80 μm, 60 μm, 40 μm, 20 μm, respectively was used. Needless to say, the hole diameter and the diameter of the assembly of projection portions are not limited to these values. 
         [0148]      FIGS. 16A ,  16 B,  16 C,  16 D show the results of the cell culture for 96 hours in total using the culture sheets having these patterns. That is, the graphs of the results of measuring the distance between the center of the hole and the center of the spheroid and verifying the hole center retention rate of the spheroids are shown.  FIGS. 16A ,  16 B,  16 C,  16 D, as illustrated, correspond to a square arrangement with a pillar diameter of 2.0 μm, a triangle arrangement with a pillar diameter of 2.0 μm, a square arrangement with a pillar diameter of 1.0 μm, and a triangle arrangement with a pillar diameter of 1.0 μm, respectively. 
         [0149]    The distance between the centers of the hole center and spheroid is indicated in 3 steps of 0 to 19 μm, 20 to 39 μm, 40 μm or more on the horizontal axis of each graph, and the proportion of the number of spheroids occupying each range in the total number of spheroids is indicated on the vertical axis. As a result, in all of the patterns examined at this time, the sheets having a diameter of the assembly of projection portions of 100 μm or 80 μm had higher rates that spheroids are retained closer to the center. 
         [0150]    It has been shown that an optimum rate of the diameter of the assembly of projection portions relative to the hole diameter is 40% to 50%, but it is not limited to this value depending on the cell strain and culture conditions. According to the experiment results, when the rate is from 20% to 75%, more than half of the spheroids also fell within the range of the distance between the hole center and the center of spheroid from 20 to 39 μm. The rate may be therefore within this range from 20% to 75%. 
         [0151]      FIGS. 17 and 18  show the results of the culture sheets in which a pillar height, pillar diameter, and a pillar pitch of 1.0 μm, 2.0 μm, 4.0 μm, respectively, and a diameter of the assembly of projection portions of 80 μm and 20 μm, respectively, as typical examples of phase-contrast micrographs of the results of culture of the culture sheet of this Example. The numbers  170 ,  180  in the holes  156 ,  158  in the figures represent spheroids. As shown in  FIG. 17 , when the diameter of the assembly of projection portions is 80 μm, the spheroids  170  having almost the same diameter were retained in the central portions of the holes  156 . In contrast, as shown in  FIG. 18 , in the sheet having a diameter of the assembly of projection portions of 20 μm the spheroids  180  were not retained in the central portions. 
         [0152]    As can be clearly seen from the results described above, it was found that according to the culture substrate and culture sheet of the present invention, spherical 3D tissues having such similar sizes are formed without applying any special chemical on the surface of the culture sheet and by stationary culture which causes little stress on cells, and spheroids having similar sizes are retained in the central portions of the holes by appropriately setting the hole diameter and the diameter of the assembly of projection portions. 
       REFERENCE SIGNS LIST 
       [0000]    
       
           100 ,  123 ,  126 ,  128 ,  130 ,  131 ,  132 ,  133 ,  150 ,  150   a . . .  Culture sheet 
           101 ,  124 ,  151 ,  156 ,  158  . . . Hole 
           102 ,  152  . . . Partition wall 
           103 ,  153 ,  157 ,  159  . . . Projection/projection assembly 
           104 ,  154  . . . Hole bottom 
           105 ,  155  . . . Hole diameter 
           106  . . . Pillar diameter 
           107  . . . Pillar pitch 
           108  . . . pillar height 
           109  . . . Chamber slide 
           110  . . . Surgical glue 
           111  . . . Frame body 
           111   a . . .  Hole portion formed on frame body 
           112  . . . Projection for fixing film 
           113  . . . Rib structure 
           114  . . . Culture sheet hole 
           115  . . . Cell culture plate 
           116  . . . Type I collagen solution 
           117  . . . Decompression container 
           118  . . . Decompression pump 
           119  . . . Saline for washing (PBS(−)) 
           120  . . . Medium 
           121  . . . Hepatocyte 
           122  . . . Hepatocyte spheroid 
           125   a,    125   b,    127   a,    127   b,    129   a,    129   b,    129   c . . .  Projection arrangement pattern 
           1111  . . . Non-slip portion 
           1112  . . . Joint 
           1113  . . . Cut face