Patent Document

CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application is a divisional application of U.S. application Ser. No.11/441,235, filed May 26, 2006, the contents of which are incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    The present invention relates to a cell culture vessel having excellent cell separatability, a production process thereof, and cells cultured in such a culture vessel. 
         [0003]    The recent years have seen remarkable progress of the techniques for culture of cells used for medical purposes, and the cultured cells are actually used for grafting of the skin. There has also been seen progress in cell culturing techniques to be applied to autologous transplantation and heterologous transplantation, which application is not limited to grafting of simple tissues such as skin but extends to transplantation of more complex organs such as cornea, tooth, bone and viscera. 
         [0004]    Various vessels such as glass or resin-made Petri dish are used for culture of cells. For instance, the Petri dishes for cell culture made by the following methods have been disclosed. In one method, a collagen solution of a specified concentration is pipetted to a culture vessel for coating such as to form a uniform surface and then dried for 15 minutes to 72 hours. In another method, a collagen solution of a specified concentration is coated on a flexible culture base material such as silicone membrane and polymerized in a 15-42° C. incubator for 20-120 minutes, then the flexible culture substrate is left under UV lamp irradiation for 15 minutes to 72 hours, and after collagen has been dried away, the substrate is again wetted with a phosphate butter solution, then extended 10-40% and fixed (see JP-A-2002-142751, e.g., Example A). 
         [0005]    According to the first method, although it is possible to culture a cell on collagen having affinity for the cell, difficulties are involved in separating the cell because of strong adhesion between the cell and the culture vessel. If the cell is separated by mechanical means, physical damage may be given to the cell, and when a chemical treatment with an enzyme such as trypsin is applied on the cell, the membrane protein in the cell surface may be destroyed to reduce the cell fixing rate to the tissue after grafting. 
         [0006]    The present inventors previously disclosed, in the below-mentioned patent, a cell culture vessel free of the above problem. In this patent, the cell is cultured on a functional substrate provided with organic polymer-made columnar microprojections capable of shape controlling, whereby the culture solution can be readily placed below the cell and also cell separatability is improved (see JP-A-2004-170935, e.g., Example 5). 
         [0007]    According to the second method mentioned above, the problem relating to separation of the cell is solved by lowering adhesion between the cell and the culture vessel by reducing the area of the culture vessel surface where the cell is brought into contact. This method, however, had the problem that the cell would be adsorbed even to the sheet end or the portion of the dish bottom where the functional substrate is absent, causing a drop of cell culturing efficiency. Further, in case the cells cultured on the microprojections exhibit the properties different from those shown when the cells are cultured in an ordinary culture vessel, the cells cultured on the microprojections may be mixed with the cells cultured on a flat portion having no microprojections. 
         [0008]    It is therefore an object of the present invention to provide a cell culture vessel which is simple in structure and easy to handle, and is capable of preventing damage to the cells when separated, promoting transport of nutrients and excretion of effete matter, and elevating the culturing efficiency improving effect by the structural features. 
       SUMMARY OF THE INVENTION 
       [0009]    In order to solve the above problems, the present invention provides, in a first embodiment thereof, a cell culture vessel comprising a culture section provided with a plurality of projections having an equivalent diameter smaller than the cells to be cultured and the culture section side walls enclosing said culture section, wherein the space between any portion of said side walls and the projection located nearest thereto on the culture section is smaller than the diameter of the cells to be cultured. In this cell culture vessel, there is eliminated any projection-absent portion near the edge of the culture section so as to maximize the effect of the projections on the cells. The term “equivalent diameter” is used in the present invention to accommodate all possible sectional shapes of the projections which are not necessarily circular but may be elliptical, polygonal, asymmetric or other shapes. In the present invention, the equivalent diameter is the diameter of a section at the bottom of the projections. 
         [0010]    In some cell lines with strong adhesiveness to the vessel, adsorption of the cells to the culture section side walls is unnegligible, and there are the cell lines in which it is undesirable that the cells cultured on the side wall be mixed with those on the culture section. For such lines, it is the best to provide the similar projections on the culture section side walls, too, but in case it is difficult to provide such projections, the culture section side walls may be made non-adhesive to the cells by adopting a specific structure or using a specific material for the side walls. By forming a buffer section, positioned at a lower level than the culture section, between the culture section having the microprojections and the side walls, it is possible to prevent the cells adsorbed to the side walls from moving to transfer to the culture section having the projections. In this case, too, it is possible to maximize the effect of the microprojections on the cells by setting the distance between the microprojection closest to the buffer section and the edge of the culture section to be smaller than the diameter of the cells to be cultured. 
         [0011]    In order to allow the culture solution to pervade over the culture section, the surface of the culture section of the cell culture vessel may be treated to be hydrophilic to accelerate the flow of the culture solution. However, for certain kinds of cells to be cultured, it may be required to treat the vessel surface to be hydrophobic or to coat the vessel surface with a metal or a protein. In the present invention, therefore, a hydrophobicity treatment or other treatments such as metal or protein coating necessary for the cell culture may be applied only on the top end portions of the microprojections where the cells are brought into contact in the cell culture vessel. 
         [0012]    In case where it is desired to afford specific properties, such as orientation, to the cells to be cultured, a specific treatment such as hydrophobicity treatment may be applied partially on the bottom surface of the cell culture vessel according to the properties to be afforded to the cells. In the present invention, a treatment necessary for cell culture, such as hydrophobicity treatment or metal or protein coating, can be applied selectively to the top ends of the microprojections on the surface of the cell culture vessel. 
         [0013]    Applying the present invention has the effects of preventing damage to the cells when separated, promoting transport of nutrients and excretion of effete matter and enhancing the cultivation effect by the projections. It is also possible to derive the effect of preventing the cells cultured at the no-projection area from being mixed with the cells on the projections. 
         [0014]    Other objects, features and advantages of the invention will become apparent from the following description of the embodiments of the invention taken in conjunction with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0015]      FIG. 1  is a schematic illustration showing a perspective view of one example of cell culture vessel according to the present invention. 
           [0016]      FIG. 2  is schematic illustrations showing the sectional views of one example of cell culture vessel according to the present invention. 
           [0017]      FIG. 3  is a schematic illustration showing a perspective view of another example of cell culture vessel according to the present invention. 
           [0018]      FIG. 4  is a schematic illustration showing a sectional view of another example of cell culture vessel according to the present invention. 
           [0019]      FIG. 5  is a schematic illustration showing a perspective view of a third example of cell culture vessel according to the present invention. 
           [0020]      FIG. 6  is a schematic illustration showing a sectional view of the third example of cell culture vessel according to the present invention. 
           [0021]      FIG. 7  is schematic illustrations showing the production process of a cell culture vessel according to the present invention. 
           [0022]      FIG. 8  is schematic illustrations showing the production process of another cell culture vessel according to the present invention. 
           [0023]      FIG. 9  is schematic illustrations showing the production process of a third cell culture vessel according to the present invention. 
           [0024]      FIG. 10  is a schematic illustration showing a mode of use of a cell culture vessel according to the present invention. 
           [0025]      FIG. 11  is a schematic illustration showing cell culture in a cell culture vessel having a region devoid of projections in the culture section. 
           [0026]      FIG. 12  is a schematic illustration showing a scanning electron microphotograph of the projections in the present invention. 
           [0027]      FIG. 13  is schematic illustrations showing the production process of a mold in the first embodiment. 
           [0028]      FIG. 14  is a schematic illustration showing a perspective view of the culture section side walls in the second embodiment. 
           [0029]      FIG. 15  is schematic illustrations showing the production process of a culture section side wall in the second embodiment. 
           [0030]      FIG. 16  is a schematic illustration showing a perspective view of the cell culture vessel in the fourth embodiment. 
           [0031]      FIG. 17  is schematic illustrations showing scanning electron microphotographs of the projections in the fourth embodiment. 
       
    
    
     DESCRIPTION OF REFERENCE NUMERALS 
       [0032]      100 : cell culture vessel;  101 : projection;  102 : culture section;  103 : culture section side wall;  104 : buffer section;  105 : vessel material;  106 ,  117 : mold;  107 : culture solution;  108 : cell;  109 : cell adhering to the flat portion;  110 : culture section substrate;  111 : first mold matrix component;  112 : second mold matrix component;  113 : mold matrix;  114 : replica material;  115 : mold replica;  116 : nickel sheet. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0033]    The cell culture vessel of the present invention will be described in detail below with reference to the accompanying drawings. 
         [0034]      FIG. 1  is a perspective view showing a first embodiment of cell culture vessel  100  according to the present invention. In the culture section  102  of the cell culture vessel  100  containing the cells and their culture solution, there are formed the projections  101  having an equivalent diameter smaller than the cells to be cultured. The equivalent diameter of these projections is 10 nm to 10 μm and their height is 10 nm to 1 mm. For reducing the contact area with the cells, the equivalent diameter of these projections is set to be sufficiently smaller than the cell diameter, for instance ⅕ or less of the cell diameter. The interval between the projections  101  needs to be less than the cell diameter. Thus, the projections are designed such that their sectional area will be smaller than the sectional area of the cells, and a plurality of these projections will exist within a section of the cell. Also, because of the necessity to let the culture solution infiltrate sufficiently into the lower portions of the projections  101 , these projections are designed to have a sufficient height, such as equal to the equivalent diameter or greater, more preferably 5 or more times the equivalent diameter. From the viewpoint of structural strength, however, the height is preferably not more than 100 times the equivalent diameter. As for the culture section  102 , although only one such culture section may be provided for one cell culture vessel  100 , the section may be divided into two or more portions by the side walls  103  as shown in  FIG. 1 , which makes it possible to culture the cells under varied conditions with one cell culture vessel  100 . 
         [0035]      FIG. 2(   a ) is a schematic illustration showing the section cut along the line A-B in  FIG. 1  in the first embodiment of cell culture vessel  100  according to the present invention, and  FIG. 2(   b ) is a schematic illustration showing in particular the boundary between the culture section  102  and its side wall  103 . The projections  101  are provided to a point close to the side wall  103  and substantially cover up the whole span of the culture section  102 . The culture vessel is designed such that the distance I between an arbitrary position on the culture section/side wall boundary line and the nearest projection  101  will be not greater than the equivalent diameter of the cells to be cultured. The culture section  102  including the projections  101  is integral with the side walls  103 , and they are made of a same material. It is most preferable to provide the projections  101  on the side walls  103 , too, as this arrangement can further raise the effect of the projections  101 . When such working is difficult, there may be provided a different surface structure to which the cells are harder to adhere than to the culture section  102 , or a non-cell-adherent surface treatment may be applied. The culture section side wall  103  is preferably of the height that can hold the culture solution without allowing it to overflow from the surface of the culture section  102 . This height can be properly determined in accordance with the shape and area of the culture section  102 , its hydrophilicity and the amount of the culture solution supplied to the culture section  102 , but preferably the height h of the side wall  103  from the culture section  102  is selected to be 0.05 to 100 mm. Also, the side wall  103  is preferably made higher than the projections  101  on the culture section  102 , more preferably 10 or more times the height of the projections  101 . This height depends on the configuration of the culture section  102 , but its lower threshold is decided according to whether a proper amount of culture solution can be held on the culture section  102  having the projections  101 , while the upper threshold is decided by the operatability with the culture solution or cells. The angle of inclination θ made by the side wall  103  with the culture section  102  is preferably selected to be not less than 45 degrees for preventing adhesion of the cells to the side walls  103 . It is also possible where necessary to provide the stepped side walls to make it easier to form a specific surface structure for the side walls  103 . 
         [0036]      FIG. 3  is a perspective view of the second embodiment of cell culture vessel  100  according to the present invention. The geometry of the projections  101  on the culture section  102  is the same as that in the first embodiment, but the side walls  103  are made of a material with lower adhesiveness to the cells than the culture section  102  and are bonded to the culture section  102 . Bonding between the side walls  103  and the culture section  102  is made such that they can be detached from each other without harming the projections  101 , which can facilitate post-culture observation. 
         [0037]      FIG. 4  is a schematic illustration showing a sectional view taken along the line A-B in  FIG. 1  in the second embodiment of cell culture vessel  100  according to the present invention. Like in the first embodiment, the projections  101  are formed to a point close to the side wall  103  and cover up substantially the entirety of the culture section  102 . The vessel is designed such that the distance I between an arbitrary position on the culture section/side wall boundary line and the nearest projection  101  is not greater than the equivalent diameter of the cell to be cultured. 
         [0038]      FIG. 5  is a perspective view showing the third embodiment of cell culture vessel  100  according to the present invention. The geometry of the projections  101  on the culture section  101  are the same as those in the first and second embodiments, but in the instant embodiment a buffer section  104  is provided between the culture section  102  and the side walls  103 , said buffer section  104  surrounding the culture section  102  and disposed at a lower position than the culture section  102 . Even when the cells are adsorbed on the side walls  103 , it is possible, because of the presence of said buffer section  104 , to prevent the adsorbed cells from transferring to the culture section  102 . 
         [0039]      FIG. 6  is a schematic illustration showing a sectional view taken along the line A-B in  FIG. 1  in the third embodiment of cell culture vessel according to the present invention. The projections are provided to a point close to the buffer section  104  and cover up substantially the entirety of the culture section  102 . The vessel is designed such that the distance between the buffer section side edge of the culture section  102  and the nearest projection  101  is not greater than the equivalent diameter of the cell to be cultured. The buffer section  104  is of a depth sufficient for preventing the cells which moved onto the buffer section  104  from further transferring to the culture section  102 . This depth, although variable depending on the type of the cells to be cultured and the culturing conditions, has a height of 0.01 mm or greater from the culture section  102  and is smaller than the height of the side wall  103  for allowing the culture section  102  to be submerged in the culture solution. The width d of the buffer section (distance from the side wall to the edge of the culture section) is preferably made greater than the equivalent diameter of the cells to be cultured for preventing the cells from transferring to the culture section  102 . 
         [0040]    The material of the cell culture vessel  100  according to the present invention is not specifically defined, but it is selected in consideration of desired working precision, surface properties, optical properties, strength and other factors of the vessel to be made. Specifically, there can be used thermoplastic resins such as polyethylene, polypropylene, polyvinyl alcohol, polyvinylidene chloride, polyethylene terephthalate, polyvinyl chloride, polystyrene, ABS resins, AS resins, acrylic resins, polyamides, polyacetal, polybutyrene terephthalate, glass-reinforced polyethylene terephthalate, polycarbonates, modified polyphenylene ethers, polyphenylene sulfide, polyether ether ketone, liquid crystalline polymers, fluorine resins, polyarylates, polysulfone, polyether sulfone, polyamide-imide, polyether-imide and thermoplastic polyimides, thermosetting resins such as phenol resins, melamine resins, urea resins, epoxy resins, unsaturated polyester resins, alkyd resins, silicone resins, diallyl phthalate resins, polyamide-bismaleimide and polybisamide-triazole, and mixtures of two or more of these resins. It is also possible to use inorganic materials such as glass. Since the material used for the cell culture vessel  100  is usually required to have affinity for the cells, it needs to confirm affinity for the cells to be cultured before selecting the material. Also, in order to facilitate culture evaluation, it is preferable to use a material which can be regarded as transparent to the light of a wavelength used for the observation and which substantially does not emit fluorescent light in the same wavelength region. For instance, by using biodegradable resins including aliphatic polyesters or polyacid anhydrides such as polylactic acid and polycaprolactone, synthetic materials such as synthetic polypeptide and natural materials such as chitosan and cellulose, it is possible to make a cell culture vessel which allows easy biodegradation of the culture. 
         [0041]    The material of the projections  101  in the present invention is also not specifically defined, but as in the case of vessel material, the above-mentioned resin compositions or inorganic materials such as glass may be used in consideration of desired working precision, surface properties, optical properties, strength and other factors. These projections are preferably formed integral with the culture section  102 . 
         [0042]    A process for forming the first embodiment of cell culture vessel  100  according to the present invention is shown in  FIG. 7 . In this embodiment, so-called nano-imprinting techniques are used for forming the vessel  100 . The vessel base material  105  is softened by heating, and a mold  106  having a geometry defining the projections  101 , culture section  102  and its side walls  103  is pressed against the softened vessel base material to transfer the geometry of the mold to the vessel base material, thereby obtaining a cell culture vessel  100  having the projections  101 , culture section  102  and its side walls  103 . 
         [0043]      FIG. 8  shows a process for making the second embodiment of cell culture vessel according to the present invention by using nano-imprinting techniques. The vessel base material  105  is softened by heating, to which a mold  106  having a geometry defining the projections  101  and culture section  102  is pressed to transfer the geometry of the mold  106  to the vessel base material  105 . Then the separately formed culture section side walls  103  are joined to the culture section  102  to make a cell culture vessel. 
         [0044]      FIG. 9  shows a process for forming the third embodiment of cell culture vessel  100  according to the present invention by using nano-imprinting techniques. The vessel base material  105  is softened by heating, and a mold  106  having a geometry defining the projections  101 , culture section  102 , its side walls  103  and buffer section  104  is pressed to said softened base material  105  to transfer the mold geometry to the vessel base material  105  to obtain a cell culture vessel  100  having the projections  101 , a culture section  102 , its side walls  103  and a buffer section  1   
         [0045]    In the above processes for constituting the first to third embodiments of cell culture vessel, it is possible to employ a high aspect ratio nano-imprinting method which makes it possible to form the projections  101  with a shape of a higher aspect ratio than the mold  106  by drawing the vessel base material  105  filled in the shape corresponding to the projections  101  in the mold when the mold is separated from the vessel base material  105  after pressing. 
         [0046]    The molds  106  used for forming the respective cell culture vessels  100  described above are required to have a shape corresponding to the projections  101  to be formed on the vessel, that is, they need to be have on their surface a configuration which is 10 nm to 10 μm in equivalent diameter and 100 nm to 1 mm in height. To meet this requirement, the molds contain at least one of metal, inorganic material such as carbon and silicon, and resin composition, and their surface geometry is formed by fine working such as photo-lithography, electron-beam direct writing, corpuscular beam working and scanning probe working, self-organization of fine particles, etc., or formed by conducting shape transfer, by such method as nano-imprinting, injection molding or electroless plating, from the master made by using the above-said techniques. The shape defining the culture section side walls  103  is of macro-scale as compared with the fine geometry of the projections  101 , and in such a case, it is possible to form the molds  106  with high precision by employing machine working jointly with the techniques for joining the plural molds. It is also possible to afford non-cell-adhesiveness to the culture section side walls by applying fine working on them by combining the macro-shape defining technique with photo-lithography with large depth of focus, laser working or self-organization of corpuscles. In  FIGS. 7 to 9 , as the method for forming the projections  101 , there is used a so-called nano-imprinting method in which the mold  106  is pressed to the softened vessel base material  105 , but obviously it is also possible to employ other methods, for example, resin molding such as injection molding or direct working such as laser working without using the mold. 
         [0047]    In the present invention, the cell culture vessel is subjected as desired to a surface treatment necessary for cell culture, for example, dipping in a solvent containing an oxidizing agent such as hydrogen peroxide or ozone, ultraviolet irradiation, hydrophilicity treatment such as vapor-phase plasma treatment, coating with a protein such as polylysine, albumin, collagen, fibronectin, fibrinogen, vitronectin and laminin by dipping in a solution or other means, plating, metal coating by vapor phase deposition, or surface modification by irradiation with light, electron rays or corpuscular beams. Further, it is possible to apply a surface treatment to the top end portions of the projections  101  or the culture section side walls  103  alone, if necessary with an in-plane distribution, by conducting such treatments as stamping with an elastic material such as silicone rubber, resin film or metallic film, heating by use of a partial dispensing method, or partial coating with a solvent containing a hydrophobic agent such as resin paste, silicone grease or fluorine coating agent or a protein. 
         [0048]    The culture section side walls in the embodiments shown in  FIGS. 3 ,  4  and  8  are formed by machining, molding or other means separately from the culture section  102  described above. The material of these side walls is selected from those mentioned above for use for the cell culture vessel  100 . If the side walls  103  are formed with the same material as used for the culture section  102 , there can be obtained a structure with high geometrical precision and strength after joining and a visual appearance shown in  FIG. 1 . For joining these side walls  103  and culture section  102 , there can be used high-precision joining means such as pressing under heating, heat fusion such as vibratory fusion or infrared laser fusion, and use of an adhesive. In heat fusion, however, it is necessary to select the conditions that would not cause deformation of the projections  101  on the culture section  102  beyond the allowable range. In case of using an adhesive, it is essential to select the conditions under which the adhesive is not allowed to transfer to the surface of the culture section  102  during the joining operation, the cells won&#39;t be adsorbed on the surface after curing, and the elute does not show cytotoxicity. By forming the culture section side walls  103  with a material having elasticity and high adhesiveness to the cell culture vessel  100 , there can be obtained a sufficient adhesive strength by merely attaching the side walls  103  tightly on the cell culture vessel  100 . Also, by forming the side walls  103  in this way, it is possible to obtain a structure in which the side walls  103  can be detached without harming the projections  101  and the cells cultured thereon, which can facilitate the observation work after culture. In order to narrow down the space between the culture section side walls  103  and the projections  101  on the culture section  102 , it needs to do in advance high-precision positioning of the projections  101  and the side walls  103 . For this purpose, as shown in  FIG. 8(   d ), the region where the projections  101  are formed is made larger than the culture section  102  so that only the projections  101  immediately below the side walls  103  will be smashed when joined, or the projections  101  will be covered up with the lower portions of the side walls  103 , whereby it is possible to reduce the distance between the projections  101  remaining on the culture section  102  and the culture section side walls  103 . 
         [0049]    The cell culture vessel  100  may have two or more culture sections  102 , and in use thereof, such culture sections  102  may be separated from each other and used for different modes of culture. Also, obviously a part of the cell culture vessel  100  which may hinder the culture operation can be cut away. Such cutoff is facilitated if the vessel  100  is provided in advance with a means that is helpful for making such cutoff, for example providing a line of notches or cutouts along which an unnecessary vessel part can be cut away with ease as by bending. 
         [0050]    The cell culture vessel  100  of the present invention has to be transported, after production, to a site where the cells are actually cultured. During such transport, it is required to prevent the vessel from being contaminated with foreign matter such as dust and bacteria. For this purpose, it is necessary to incorporate a vessel packaging step for isolating the culture section  102  from the outside, by for instance putting the whole vessel  100  in a pouch and heat sealing its opening, or attaching a cover film to cover only those portions of the culture section  102  and its side walls  103  which will be brought into contact with the culture solution in the culturing operation. It is also desirable to conduct a sterilization treatment for killing the microorganisms remaining on the culture section  102  after packaging. Exemplary of such sterilization treatment is autoclaving using high-temperature steam, but this method involves a possibility to cause deformation or denaturation of the product by high-temperature steam depending on the material of the vessel  100  or configuration of the projections  101 . Therefore, it is advisable to apply a sterilization treatment, such as ultraviolet-light irradiation, ethylene oxide gas sterilization or gamma-ray sterilization, which can be carried out at a temperature lower than the glass transition point of the material composing the vessel. Of course, autoclaving can be employed in case a heat-resist resin or glass is used as vessel material. The sterilization treatment is usually conducted after packaging, and vapor-permeable type packaging is adopted depending on the sterilization method used. In such packaging, it is also recommendable to adapt an indicator which can indicate whether the necessary treatment has been done or not by reacting to the particular treatment. 
         [0051]      FIG. 10  illustrates a mode of use of a cell culture vessel  100  according to the present invention. The culture section  102  is filled with a culture solution, and the cells  108  are placed on the projections  101  and cultured in the ordinary way. In the cell culture vessel  100  of the present invention, the mode of contact between each cell  108  and the culture section  102  is point contact because of the presence of the projections  101 , so that it is possible to prevent the cells  108  from being damaged when separated. Also, by varying the geometry and surface treatment of the projections  101  depending on the type of cell to be cultured, it is possible to form the high-quality cultured cells without causing damage during separation. Further, in the cell culture vessel according to the present invention, since the projections  101  are arranged so as to cover up substantially the entirety of the culture section  102 , there exist no cells adhering to the flat portion of the culture section  102  unlike in the type of the vessel having the flat portion along the edge as shown in  FIG. 11 , so that the cell culture improving effect of the projections  101  is enhanced. Moreover, by substantially eliminating the region of flat portion where the cells  108  are liable to adhere to the culture section  102 , and, in some cases, by incorporating a structure which can prevent the cells  108  from being adsorbed to the culture section  102  and, should they be adsorbed, can prevent the adsorbed cells from being positioned on the culture section  102 , there can be obtained an additional effect to prevent mixing of the cells cultured at the area having no projections when the projections  101  give a specific influence to the cells  108 . In the present invention, the “cell  108 ” is a concept including the organized cultured cells represented by cultured tissue and organ cells. 
         [0052]    In use of the cell culture vessel  100  according to the present invention, culture is carried out with the culture solution  107  and cells  108  being placed on the culture section  102  in a thermostatic culture tank in the conventional way, and there are conducted as required such operations as changing of the culture solution  107 , taking-out of the cells  108 , supply of the reagents, shaking, observation by a microscope, and in some cases freezing in a freezer. During these unit operations, the culture vessel must be transferred from one operating unit to another. It is necessary to prevent the culture vessel from being deflected to cause biasing of the culture solution  107  or cells  108  or their leakage from the culture section  102  during the above-said operations or transfer, so that it is desirable that the culture vessel  100  has sufficient rigidity as a whole. Therefore, at least the peripheral portion of the vessel and part of the region other than the culture section  102  are preferably constructed to have rigidity with a thickness of not less than 0.5 mm. In order to alleviate extinction or fluorescence in the members composing the culture section  102  and encourage conduction of temperature from the outside, the culture section  102  may be designed to be smaller in thickness than the peripheral portion. Also, for providing a lightweight and high-rigidity structure, the portions having no bearing on strength or handling qualities may be excluded by, for instance, forming a region for reinforcing strength by bending an edge of the vessel, or making hollow the side wall  103  on the side opposite from the culture section  102 . Further, in case the culture vessel does not have enough rigidity in itself, a separately prepared member or members having rigidity may be adapted to the vessel after forming the culture section  102  to thereby afford desired rigidity to the vessel. 
       DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0053]    The present invention is further illustrated by the following embodiments. 
       Example 1  
       [0054]    An embodiment of the present invention is described below.  FIG. 12  is a schematic illustration of s scanning electron microphotograph of the culture section  102  made according to the instant embodiment. The culture section  102  is furnished with a plurality of projections  101  which are made of polystyrene having a molecular weight of 2,000 to 3,840,000. The upper limit of the molecular weight can be extended up to 6,000,000. 
         [0055]    These projections  101  are of a columnar configuration measuring 500 nm in diameter at the bottom and 1 μm in height. So, the ratio of height to one side of the columnar projection is 2, or greater than 1. The interval between the adjoining projections is 1 μm, which is smaller than the diameters of the ordinary cells. The projections  101  are arrayed in a two-dimensional square arrangement as shown in  FIG. 12 . 
         [0056]    The diameter at the top end of each projection is 400 nm, that is, the projection is diminished in diameter toward the top end. In this embodiment, each projection tapers off toward its top end, but the same effect can be obtained by designing each projection as a column having no reduced-diameter portions (constrictions) or to have a mushroom-like shape which tapers off toward the top end but has a bulge at the top end. 
         [0057]    Each projection  101  is made of polystyrene same as the culture section substrate  110 , and they are connected to each other to form an integral structure. 
         [0058]    The shape of the projections  101  tapering off toward the top end from the bottom end has the effect of securing the projections against coming off the culture section substrate  110 . As the projections  101  are made of the same material as and integrated with the substrate  110 , they are additionally ensured against coming off the substrate. 
         [0059]    While polystyrene is used as the material of both projections  101  and culture section substrate  110  in the present embodiment, it is possible to select other material from those exemplified above as usable materials of the cell culture vessel  100  in accordance with the type of the cells to be cultured and the way of use of the vessel  100 . In the embodiments shown later, the projections  101  and the substrate  110  of culture section can be produced through the similar process by properly changing the conditions in accordance with the material used. 
         [0060]      FIG. 1  is a schematic illustration of the cell culture vessel  100  produced in the instant embodiment. This cell culture vessel  100  is made, in its entirety, of polystyrene having a molecular weight in the above-defined range, and comprises a culture section  102  provided with the projections  101  and the culture section side walls  103 . The culture section  102  is square-shaped with its one side measuring 1 cm. The culture section side walls  103  are erected substantially vertically to enclose the culture section  102  and measure 0.7 mm in height. There are provided two culture sections  102  separated at the center of the culture vessel  100  and spaced apart 5 mm from each other. The above cell culture vessel  100  was made in the following way.  FIG. 7  illustrates a production process of the cell culture vessel  100  in the instant embodiment. In this process, the cell culture vessel  100  was formed by the nano-imprinting method. The vessel base material  105  (20 mm×40 mm, 1 mm thick) comprising polystyrene mentioned above was heated to 150° C. and thereby softened, and a nickel-made mold  106  having a geometry defining the projections  101 , culture section  102  and culture section side walls  103  was pressed to the softened vessel base material  105  under a pressure of 4 MPa for 180 seconds. Then, with the pressure of the press unreleased, the whole assembly was cooled to 35° C., after which the mold  106  and vessel base material  105  were taken out and the mold  106  was separated by lifting it up vertically from the vessel base material  105  to thereby obtain a cell culture vessel  100  having the projections  101 , culture section  102  and culture section side walls  103 . 
         [0061]    A production process of the mold  106  is shown in  FIG. 13 . The first mold matrix component  111  shown in  FIG. 13(   a ) is a both-side polished silicon wafer with 1 cm square crystal orientation ( 100 ) having a geometry defining the projections  101 , culture section  102  and culture section side walls  103 , and the second mold matrix component  112  is a flat 20 mm×40 mm anode-joining glass plate defining the shape of the topmost portions of the culture section side walls  103  and other regions. In the first mold matrix component  111 , a fine concave and convex configuration corresponding to the projections  101  on the cell culture vessel  100  is formed by photo-lithography. This component was cut out with the specified size from a 20 mm-diameter silicon wafer and cleaned. Then, the first and second mold matrix components  111  and  112  were joined by the anode joining method (400° C., 1 kV) as shown in  FIG. 12(   b ) to make a mold matrix  113 . The height of the culture section side walls  103  is decided by the height of the mold matrix component  111 , but its height can be varied by suitable means such as placing a specified spacer between the first and second mold matrix components  111  and  112 . Adjustment of height can be made over a wider range if the second mold matrix component  112  is provided with a geometry defining at least a part of the culture section side walls  103 . Various mold matrix component joining methods other than said anode joining method, such as methods for forming metallic or inorganic or organic adhesive layers and welding, can be selected depending on the material of said spacer or mold matrix components, flatness and configuration of the interface of joining to enhance working precision. A fluorine type release agent was applied to the mold matrix  113 , and as shown in  FIG. 13(   c ), a 20 mm×40 mm, 2 mm thick polystyrene-made replica material  114  was pressed under 10 MPa at 150° C. and, after cooling, the mold matrix  113  was separated to obtain a mold replica  115 . A nickel coating film was formed on said mold replica  115  by electroless plating, and then electroplating was carried out thereon to increase the nickel coating thickness to 3 mm, thereby fabricating a mold  106 . Thereafter, fluorine type release treatment was conducted on the region including the portions defining the projections  101 , culture section  102  and culture section side walls  103  in the mold. 
         [0062]    It is preferable to make the mold  106  with a resin having high heat resistance and strength by using nano-imprinting or casting techniques used for forming the replica, instead of said plating method, as the mold making process can be simplified as compared with when using the plating method. In case of making a trial product in a small quantity, the mold matrix  113  may be used directly as the mold  106 . 
         [0063]    In this embodiment, the mold  106  is additionally provided with a shape corresponding to the buffer section  104  between the culture section  102  and its side walls  103 , said buffer section  104  surrounding the culture section  102  and disposed at a lower position than the culture section  102 , to provide the structure presented as the third embodiment of the present invention. 
         [0064]    In the present invention, a 1 mm thick vessel base material  105  comprising polystyrene is used, but it is necessary to provide an optimal thickness in accordance with the desired area of the culture section  102  and the desired height of the culture section side walls  103 . For instance, in the case of a cell culture vessel having a larger culture section than in the instant embodiment, the thickness of the peripheral portion of the vessel or the culture section must be made greater than 0.5 mm for maintaining rigidity of the whole vessel in handling thereof. Actually, however, the thickness of the vessel base material  105  needs be selected to be optimal in balance with rigidity by taking into account the space held by the whole vessel and its operatability. In case the culture sections  102  are designed the mutually separated  16  mm diameter circles (corresponding to 24-well microplate), 8 mm diameter circles (corresponding to 96-well microplate), 4 mm diameter circles (corresponding to 384-well microplate) or 2 mm diameter circles (corresponding to 1,536-well microplate) as in the widely used microplates, the thickness of the culture section  102  and the thickness of the culture section side walls  103  can be made smaller than 0.5 mm. Even in these microplates, as it is required to maintain sufficient rigidity of the whole vessel, it is preferable to have a region, for example an edge of the microplate or a portion between the culture sections, where the thickness is not less than 0.5 mm. In order to afford such rigidity, a reinforcement such as outer frame or backing may be separately provided on the culture vessel  100  after forming the culture sections  102 . Particularly when the culture vessel is designed to have a configuration like a microplate, the height of the side walls  103  in each culture section  102  is made around 1 cm in many cases, but it can be changed according to the area of the culture sections  102 . Especially in case the area of the culture section  102  is small as when it was designed correspondent with a 384-well microplate or 1,536-well microplate such as mentioned above, the height of the culture section side walls  103  can be made even smaller as the required amount of the culture solution is lessened to about 1 to 100 μl. 
         [0065]    The shape of the culture section  102  is most preferably circular for preventing stagnation of the culture solution in the culture section  102 , but in the present embodiment, the culture section  102  was shaped square for facilitating dicing of the first mold matrix component  111  on the mold  106 . The shape of the culture section, however, is not specified in the present invention; it may be elliptical, polygonal or otherwise shaped according to the culturing method, production process and arrangement of the culture section  102  on the cell culture vessel  100 . The size of the culture section  102 , its depth and the number of the culture sections on one cell culture vessel  100  may be decided to be optimal according to the cell line to be cultured and the purpose of use of the culture. For instance, the culture section  102  is preferably enlarged in case the cell culture vessel  100  is used for obtaining a relatively large culture organization or a plurality of cultured cells of the same condition, but the culture section  102  needs be rather reduced in size in case the culture vessel is used for culturing the cells for making experimental assays under multiple conditions. If the culture vessel has a geometry like a microplate having many separated culture sections in one vessel, it is possible to lessen the amount of the reagents or the number of the cells used for the cultured. Also, by changing the surface treatment of the projections  101  in the culture section  102 , shape of the top ends of the projections and their size (diameter, height, etc.) and arrangement for each of the separated regions according to the purpose of use of the culture, it is possible to test the change of condition of the cultured cells by the shape and arrangement of the projections  101  when a same type of cells are cultured under the same conditions, and to decide the optimal pattern of projections  101  for each type of cells when different types of cells are cultured. Also, by changing the shape and arrangement of the projections  101  in a culture section  102 , it is possible to use the device for such purpose as search for the projection pattern optimal for the cells to be cultured by testing the change of condition of the cultured cells by using one culture section  102  alone. 
       Example 2 
       [0066]    Another embodiment of the present invention is described below.  FIG. 2  is schematic illustrations of the cell culture vessel  100  produced in the instant embodiment. In this cell culture vessel  100 , the culture section  102  including the projections  101  is composed of polystyrene of the same molecular weight as that used in the first embodiment, and the culture section side walls  103  are made of PDMS (poly(dimethyl siloxane) produced by Dow Corning Co., Ltd.) and have the geometry shown in  FIG. 14  with a height of 0.7 mm. The culture section  102  is square with each side measuring 0.90 cm, and the side walls  103  are erected substantially vertically enclosing the culture section  102 . There are provided two culture sections  102  divided at the center of the culture vessel  100  and separate 5 mm from each other. The projections  101  formed on each culture section  102  are of a cylindrical shape measuring 500 nm in diameter at the lower end and 1 μm in height and have the same construction as those in the first embodiment. 
         [0067]    The above cell culture vessel  100  was produced in the following way.  FIG. 8  illustrates a production process of the cell culture vessel  100  in the present embodiment. The aforementioned vessel base material  105  (20 mm×40 mm×1 mm thick) comprising polystyrene was softened by heating to 150° C., and a nickel-made mold  106  having a geometry defining the projections  101  and culture sections  102  (1 cm square) was pressed to the softened vessel base material under pressure of 4 MPa for 180 seconds. Then, with the pressure kept unreleased, the whole unit was cooled to 35° C., the mold  106  and the vessel base material  105  were taken out, and the mold  106  was separated from the vessel base material  105  lifting the mold vertically from the vessel base material, thus forming the projections  101  and the culture sections  102 . Then the PDMS-made side walls  103  shown in  FIG. 14 , which had been formed by casting from a separate mold, were attached tightly to the culture sections  102  to obtain a cell culture vessel  100 . 
         [0068]    The mold production process is the same as in the first embodiment, but since the culture section side walls  103  are joined later to form the vessel, there is no need of providing a corresponding configuration to the related part of the mold  106 . It is however possible to give to the mold a configuration corresponding to the lower part of the side wall  103  for the purpose of supplementing the height of the side wall. A mold is also used for forming the side walls  103 , and this mold is in many cases made of such metal as iron or nickel, but it is possible to form the mold from an organic resin, silicon wafer or an inorganic material such as glass to meet the requirements for working precision, simplicity or visibility. For making the mold for forming the culture section side walls  103 , a suitable method is selected from those shown above for producing the mold  106  used for making the cell culture vessel  100  according to working precision required and the mold material used. 
         [0069]    A process for forming the culture section side walls  103  in the instant embodiment is illustrated in  FIG. 15 . In this embodiment, a 2 mm thick nickel plate was machined into a mold  117  with a geometry having the convex portions measuring 0.90 cm in one side and 1 mm in height corresponding to the side wall  103 . A fluorine type mold release treatment was conducted on the surface of the mold, then the non-cured PDMS was mixed with a curing agent, and an amount thereof for providing a height of 0.7 mm was cast into the mold  117 , followed by a heat treatment at 120° C. for 10 minutes to cure PDMS, after which the molding was separated from the mold to obtain the culture section side walls  103 . The thus formed PDMS-made culture section side walls  103  were low in adhesiveness to the cells and suited for use in the cell culture vessel  100 . Also, because of high adhesiveness of the PDMS-made culture section side walls  103  to the culture section  102 , there can be obtained air tightness required for culture by correctly disposing and tightly attaching the side walls  103  at the prescribed positions on the culture section  102 . The projections  101  on the culture section  102  are formed in a 1 cm 2  square region as mentioned above, so that by arranging the side walls  103  to cover a portion of 0.05 cm at the edge of the culture section  102 , it is possible to make the distance between each side wall  103  and the nearest projection  101  1 μm same as the interval between the projections. Also, after culturing the necessary cells, the culture section side walls  103  can be removed with ease by detaching them from the culture section  102  after removing the culture solution. Thus, in the cell culture vessel according to the present invention, it is possible to remove the culture section side walls  103  which may become an obstacle to the cultured cell observation, so that the cell culture vessel of the present invention is suited for high-resolution observation by an erect optical microscope or form evaluation of the cultured cells by a scanning electron microscope. 
         [0070]    In this embodiment, as in the first embodiment, the size and depth of the culture sections  102  and the number of the culture sections on one cell culture vessel can be optimized according to the cell line to be cultured and the purpose of use of the culture. For instance, the culture sections  102  are preferably enlarged in case the cell culture vessel  100  is used as production means for obtaining plenty of cultured cells under the same condition, but the culture sections  102  are preferably reduced in size in case the culture vessel is used for culturing the cells for the purpose of making experimental assays under multiple conditions. If the cell culture vessel has a geometry like a microplate having many culture sections in one vessel, it is possible to reduce the amount of the reagents and the number of the cells used for culture. 
       Example 3 
       [0071]    Described here is an instance where a surface treatment suited for cell culture has been applied on a cell culture vessel according to the present invention. An oxygen plasma treatment (100 W, 30 sec.) was applied as a hydrophilicity treatment on the cell culture vessel  100  having the projections  101  formed by the method of Example 1. A surface-hydrophilized PDMS-made flat stamp coated with 50 μg/mL of a collagen I solution (trade name: Cultrex, a bovine collagen I; solvent: 0.02M acetic acid solution) to a coating thickness smaller than the height of the projections  101  was attached tightly to the top ends of the projections  101  in the cell culture vessel  100  so that the collagen I solution would be applied to the top ends alone of the projections  101 . After removing the flat stamp, the vessel was kept at room temperature for one hour and washed with PBS (phosphate buffer solution), thus modifying the top ends alone of the projections  101  with collagen I to effectuate a surface treatment suited for cell culture. 
         [0072]    In the instant embodiment, after forming the projections  101 , modification is conducted with collagen which is a kind of protein, but it is possible to apply any other suitable surface treatments according to the type the cells to be cultured and the material of the cell culture vessel  100  and the projections  101 , such treatments including ultraviolet-light irradiation, hydrophilicity treatments such as dipping in hydrogen peroxide or ozone solution, introduction of functional groups such as amino group, carboxyl group, methyl group and CF3 group, and coating with proteins such as polylysine, albumin, collagen, fibronectin, fibrinogen, vitronectin and laminin. Such a surface treatment may be applied to a part alone of the projections  101  to control the form of the cells to be cultured. 
       Example 4 
       [0073]    An exemplification of the improvement of cell separatability after culturing the cells by using a cell culture vessel according to the present invention is shown below. 
         [0074]      FIG. 16  is a perspective view of a cell culture vessel  100  made in the instant embodiment according to the method of Example 1, and  FIG. 17  shows an example of arrangement of the projections  101  formed on the cell culture vessel  100 . In this embodiment, in order to examine the relationship between separatability of the cultured cells and configuration of the projections  101  on the culture sections  102 , there were formed in the vessel  100  four culture sections  102  in which the diameter R of the projections  101  was (a) 180 nm, (b) 240 nm, (c) 500 mm and (d) 2 μm, respectively. The height H of the projections  101  was 1 μm in all of the culture sections. Arrangement of the projections  101  was two-dimensional square lattice-like array as shown in  FIG. 17 , with the distance D between the adjoining projections  101  being twice the diameter R. These projections  101  were formed on each culture section  102  successively to a point close to the culture section side wall  103 . 
         [0075]    For the evaluation of cell separatability, the human mesenchymal stem cells (Cyro hMSC No. 2051 available from Cambrex Co., Ltd.) were seeded in a medium (Bullet Kit MSCGM (mesenchymal stem cell growth medium) available from Cambrex Co., Ltd.)) on each culture section  102 , and cultured in a 37° C. and 5% CO 2  incubator for 5 days. By way of comparison, the human mesenchymal stem cells were cultured under the same conditions in a commonly used animal cell culture dish (available from Corning Co., Ltd.). 
         [0076]    Cell separatability was evaluated by separating the human mesenchymal stem cells on each culture section  102  by a water flow generated in the medium after 5-day incubation. The water flow was generated by ejecting the medium used for the culture from a 200 μl pipette tip attached to a 250 μl pipette (EDP plus EP-250) at an angle of 70° to the bottom surface from a spot close to each culture section  102 . 
         [0077]    The medium was ejected once and the condition of separation of the cells on each culture section  102  was observed under a microscope, obtaining the results shown in Table 1. In any of the culture sections  102  formed according to the present invention, betterment of separatability of the human mesenchymal stem cells was confirmed in comparison to the animal cell culture dish used for comparison. It was also found that separatability of the human mesenchymal stem cells from the cell culture vessel  100  depended on the diameter R of the projections  101  and was maximized when the diameter was large (R=2 μm). This indicates that adhesiveness of the human mesenchymal stem cells, which are the adhesive cells, is lessened on the projections  101 , which attests to the excellent cell separating characteristics of the projections  101 . Also, this effect was observed over the entirety of any of the culture sections  102 , from which it could be assumed that there substantially existed no human mesenchymal stem cells adhering to the culture sections  102  without receiving the effect of the projections  101 . 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                 Comparative 
               
               
                 Amount 
                 Ejection 
                 Example 
                 Example 
                 Example 
                 Example 
                 Example 
               
               
                 ejected 
                 rate 
                 A 
                 B 
                 C 
                 D 
                 Animal cell 
               
               
                 (μl) 
                 (μl/sec) 
                 R = 180 nm 
                 R = 240 nm 
                 R = 500 nm 
                 R = 2 μm 
                 culture dish 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 50 
                 56 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 112 
                 − 
                 ± 
                 ± 
                 + 
                 − 
               
               
                   
                 192 
                 + 
                 + 
                 + 
                 + 
                 − 
               
               
                 100 
                 56 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 112 
                 ± 
                 ± 
                 + 
                 ++ 
                 − 
               
               
                   
                 192 
                 ++ 
                 ++ 
                 ++ 
                 ++ 
                 − 
               
               
                 200 
                 56 
                 − 
                 − 
                 − 
                 − 
                 − 
               
               
                   
                 112 
                 ± 
                 + 
                 + 
                 ++ 
                 − 
               
               
                   
                 192 
                 ++ 
                 ++ 
                 ++ 
                 +++ 
                 − 
               
               
                   
               
               
                 +++: Could be separated very well. 
               
               
                 ++: Could be separated well. 
               
               
                 +: Could be separated. 
               
               
                 ±: Could be separated to a limited degree. 
               
               
                 −: Could not be separated at all. 
               
             
          
         
       
     
         [0078]    It should be further understood by those skilled in the art that although the foregoing description has been made on embodiments of the invention, the invention is not limited thereto and various changes and modifications may be made without departing from the spirit of the invention and the scope of the appended claims.

Technology Category: c