JP2005168494A - Cell culture vessel and cultured cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture vessel capable of preventing damage to the cell, when the cell is detached therefrom, by a convenient and simple structure, and capable of promoting transportation of nutriments and discharge of a waste product. <P>SOLUTION: This cell culture vessel has a group of projections formed on its surface, wherein the projections each has an equivalent diameter of not less than 10 nm and not more than 10 μm and a height of not less than 10 nm and not more than 1 mm. Thus, a culture medium is made to permeate through a space under the cell, so as to promote supply of the nutriment required by the cell and the discharge of the waste product released from the cell. Further, the damage to the cell caused by detaching the cell therefrom is prevented, by bringing the cell into contact with the vessel in a pointedly contacted manner. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell culture container and cultured cells cultured in the container.

  In recent years, cell culture technology used for medical purposes has advanced and is actually used for skin transplantation and the like. In addition, culture techniques for autotransplantation and allogeneic transplantation are seen from small amounts of cells to complex organs such as the cornea, teeth, bones, and organs as well as tissues such as skin.

  Containers such as glass or resin petri dishes are used for cell culture. For example, the following petri dishes are disclosed. A collagen solution having a predetermined concentration is dispensed and applied to a culture vessel with a pipette so that the surface is uniform, and then dried for 15 minutes to 72 hours. Alternatively, a collagen solution having a predetermined concentration is applied on a culture substrate having elasticity such as a silicon film, polymerized in an incubator at 15 to 42 ° C. for 20 to 120 minutes, and then the elasticity of this silicone film or the like. The cell culture medium is allowed to stand under a UV lamp for 15 minutes to 72 hours, and after collagen has dried, it is again moistened with a phosphate buffer, and then stretched and fixed by 10 to 40%. There is a method of creating a petri dish (see Patent Document 1).

  Moreover, the following culture containers are disclosed as another cell culture container. A culture vessel suitable for detachment of cells after culturing can be prepared by applying a polymer whose hydrophilicity and hydrophobicity are switched depending on the temperature to the bottom surface of the culture vessel and fixing it by ultraviolet irradiation or the like (see Patent Document 2).

JP 2002-142751 A (Example A) Japanese Patent Laid-Open No. 5-244938 (Example 1)

  According to the first method, cells can be cultured on collagen having affinity for the cells, but on the other hand, the adhesion between the cells and the culture vessel is strong, so that it was difficult to detach the cells. . When the cells are mechanically peeled against this adhesion, the cells are physically damaged, and when chemically treated with an enzyme such as trypsin, the membrane proteins on the cell surface are destroyed and the tissue of the cells after transplantation. There was a problem that the fixing rate to the lowering.

  The second method can solve the peeling problem by reducing the adhesion of the cell surface by adjusting the hydrophilicity of the culture vessel surface. However, the condition of the surface of the cell culture vessel is limited to a specific substance. In addition, for example, an arbitrary surface state corresponding to various cells can be created, the time taken to make the culture vessel can be reduced, the center of the sheet-like cell Issues such as transportation of nutrients to the department and promotion of waste discharge were still left.

  An object of the present invention is to provide a cell culture container that prevents damage to cells during detachment with a simple and simple structure, and promotes transport of nutrients and discharge of waste products.

  In order to solve the above problems, a first aspect of the present invention is characterized by a cell culture container in which a projection group having an equivalent diameter of 10 nm to 10 μm and a height of 10 nm to 1 mm is formed on the bottom surface of the cell culture container. And This spreads the culture solution to the lower part of the cell, promotes the supply of nutrients required by the cell and the discharge of waste products released by the cell, and makes the cell-container contact a point contact. It is an object of the present invention to provide a cell culture container that can prevent damage to cells that sometimes occurs. Moreover, in order to further promote the transport of nutrients and the discharge of waste products, it is preferable that the projection group has a gap through which the culture solution flows. The term equivalent diameter is used because the cross section of the protrusion is not necessarily circular but may be an ellipse, a polygon, an asymmetric shape, etc. In the present invention, the equivalent diameter is used to encompass all of these. ing. In the present invention, the equivalent diameter is the diameter of the cross section of the bottom surface of the protrusion.

  It is necessary to treat the surface of the cell culture vessel to be hydrophilic in order to spread the culture solution. However, depending on the type of cells to be cultured, it may be necessary to treat the surface of the container to be hydrophobic, or to cover the surface of the container with metal or protein. For this reason, the present invention provides a cell that has been subjected to treatments necessary for cell culture, such as a hydrophobic treatment necessary for cell culture, a metal coating, a protein coating, etc., only at the tip portion where the projection group formed in the cell culture container contacts the cell A culture vessel is provided.

  In addition, when a specific shape such as orientation is required for the cells to be cultured, a specific treatment such as a hydrophobic treatment may be partially processed on the bottom surface of the cell culture container according to the shape of the cells to be cultured. The present invention provides a cell culture container in which treatment necessary for cell culture, such as hydrophobicity, metal coating, and protein coating, is applied to the tip of the projection group on the surface of the cell culture container. It is.

  By applying the present invention, it is possible to provide a cell culture container that prevents damage to cells at the time of detachment with a simple and simple structure, and promotes transport of nutrients and waste discharge. Since the above effect is achieved by the shape effect using the projection group, the formation process can be simplified, and there is no introduction of new chemicals or processing equipment compared to the conventional method, so it is necessary to consider a new disposal method It was also possible to obtain the effect that there was no.

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

FIG. 1 is a bird's-eye view showing a cell culture container 100 according to the present invention. The equivalent diameter is 10 nm or more and 10 μm or less on the bottom surface of the cell 100 and the container 100 in which the culture solution is placed, and the height is 10
A projection aggregate 101 having a thickness of not less than nm and not more than 1 mm is formed. Further, a gap 103 for facilitating the flow of the culture solution is formed in the projection aggregate 101.

  The equivalent diameter of the protrusion assembly 101 in FIG. 1 is sufficiently smaller than the diameter of the cell to reduce the contact area with the cell, and is set to, for example, one fifth or less of the diameter of the cell. Further, since it is necessary to sufficiently infiltrate the culture solution into the lower part of the protrusions, it is preferable that the height of these protrusions is sufficiently larger than the equivalent diameter, for example, more preferably five times the equivalent diameter. However, 100 times or less is preferable from the viewpoint of structural strength.

  FIG. 2 is a bird's-eye view showing a state after the surface treatment of the tip of the projection group is performed on the cell culture container 100 according to the present invention. A protrusion assembly 101 having an equivalent diameter of 10 nm or more and 10 μm or less and a height of 10 nm or more and 1 mm or less is formed on the bottom surface of the container 100 in which the cells and the culture solution are placed. Only the tip of the protrusion is formed. Further, a treatment 102 necessary for cell culture, such as a hydrophobic treatment, a metal coat, and a protein coat, is applied.

  FIG. 3 is a bird's eye view showing a state after subjecting the cell culture container 100 according to the present invention to surface treatment on a part of the tip of the protrusion. A protrusion assembly 101 having an equivalent diameter of 10 nm or more and 10 μm or less and a height of 10 nm or more and 1 mm or less is formed on the bottom surface of the container 100 in which the cells and the culture solution are placed. The tip portion is subjected to a treatment 102 necessary for cell culture, such as a hydrophobic treatment necessary for cell culture, a metal coat, and a protein coat.

  The material of the cell culture container 100 in the present invention is not particularly limited, but is selected according to desired processing accuracy, surface characteristics, optical characteristics, strength, and the like. Specifically, polyethylene, polypropylene, polyvinyl alcohol, polyvinylidene chloride, polyethylene terephthalate, polyvinyl chloride, polystyrene, ABS resin, AS resin, acrylic resin, polyamide, polyacetal, polybutylene terephthalate, glass reinforced polyethylene terephthalate, polycarbonate, modified Thermoplastic resins such as polyphenylene ether, polyphenylene sulfide, polyether ether ketone, liquid crystalline polymer, fluororesin, polyarate, polysulfone, polyethersulfone, polyamideimide, polyetherimide, thermoplastic polyimide, phenol resin, melamine resin, urea Resin, epoxy resin, unsaturated polyester resin, alkyd resin, silicone resin, diallyl phthalate resin, poly Bromide bismaleimide, poly bisamide thermosetting resin triazole and the like, and it is possible to use two or more kinds of these blended material. In addition, inorganic materials such as quartz and glass can also be used.

  The material of the projection aggregate 101 in the present invention is not particularly limited, but it is also made of the above-described resin composition or an inorganic substance such as quartz or glass depending on desired processing accuracy, surface characteristics, optical characteristics, strength, and the like. When the strength of the protrusion group becomes a problem, it is preferable that these protrusion groups are made of the same material as the cell culture container and are integrated.

  FIG. 4 shows a method for forming a protrusion group on the bottom surface of the cell culture container in the present invention. A cell culture in which a container 401 is heated and softened, and a mold 402 having a fine concavo-convex pattern formed on the container 401 is pressed to transfer the fine shape 403 of the mold 402 to the container 401, thereby forming a projection group. A container 100 can be obtained.

  The fine shape 403 on the mold 402 has a size necessary for providing the above-mentioned effects on the projection group on the cell culture vessel 400, an equivalent diameter of 10 nm to 10 μm, and a height of 10 nm to 1 mm. Is required. Therefore, the mold includes at least one of a metal, an inorganic material such as carbon and silicon, and a resin composition, and the surface shape thereof is an optical lithography method, an electron beam direct writing method, a particle beam beam processing method, a scanning probe processing method. It is formed by transferring a shape by a nano-printing method, an injection molding method, an electroless plating method or the like from a fine processing method such as self-organization of fine particles, or a master formed by these methods. In FIG. 4, the so-called nano-printing method in which a mold is pressed against a softened container is used as a method for forming the protrusion group. However, other resin molding methods such as injection molding methods and molds are not used. Obviously, it is also possible to directly process by a laser processing method or the like.

  In the present invention, the cell culture vessel may be hydrophilized by immersion in a solvent using a solvent containing an oxidizing agent such as hydrogen peroxide or ozone, or vapor phase treatment such as ultraviolet irradiation or plasma treatment, or a solution as necessary. Surface modification necessary for cell culture is performed by coating with protein by dipping in a metal or by surface modification by plating, metal coating by vapor deposition, light, electron beam, particle beam or the like.

  Next, the surface modification method for only the tips of the projection groups in the present invention will be described with reference to FIG. A stamp 502 having a treatment agent 503 attached to the surface thereof is brought into close contact with the cell culture vessel 100 in which the protrusions are formed and the necessary surface treatment is applied to the entire surface, and if necessary, the stamp is applied by heating, light irradiation, or the like. A desired surface treatment 501 can be applied only to the tip of the projection assembly 101 in contact with 502.

  The stamp 502 needs to be in close contact with many protrusion assemblies 101 on the surface of the cell culture container 100. Therefore, the stamp 502 is preferably made of an elastic body such as a silicone rubber, a resin film, or a metal thin film having necessary elasticity. Further, as shown in FIG. 6, by forming the unevenness 601 on the stamp 502, it is possible to form the distribution of processing to the tip end of the projection group.

  Examples of the treatment agent 503 include a hydrophobic agent such as resin paste, silicone grease, and fluorine-based coating agent, and a solvent containing protein. By applying an in-plane distribution based on the presence / absence, concentration, etc. of the processing agent when applying the processing agent 503 to the stamp 502, the distribution of processing to the tip end of the projection group can also be formed. Further, the treatment agent 503 can be omitted when the tip of the projection aggregate 101 is directly changed by heating or the like.

  FIG. 7 shows how to use the cell culture container 100 in the present invention. The inside of the cell culture vessel 100 is filled with the culture solution 701, and the cell 702 is placed on the protrusion assembly 101. Thereafter, the cells 702 are cultured by a conventional method. In the cell culture container 100 of the present invention, since the contact between the cell 702 and the container is a point contact, damage that occurs when the cell 702 is detached can be prevented. In addition, by changing the shape of the protrusion assembly 101 and the surface treatment of the protrusion assembly 101 according to the cells 702 to be cultured, high-quality cultured cells can be formed without damage during peeling. The cultured cell is a concept that includes organized cultured cells represented by cultured tissues and organs.

  Hereinafter, the present invention will be described in more detail by way of examples.

  An embodiment of the present invention will be described below. FIG. 8 is a schematic diagram of a scanning electron micrograph of the protrusion assembly 101 produced in this example. The projection aggregate 101 is composed of a plurality of columnar microprojections 801. The material of the columnar microprojections 801 is polystyrene, the molecular weight is 2000 to 600,000, and the upper limit of the molecular weight can be extended to 6 million.

FIG. 9 is a schematic diagram of a scanning electron micrograph in which the projection aggregate 101 is enlarged. The height of the columnar microprojection 801 is 3 μm, and the length of one side is 300 nm at the root. The columnar microprojection 801 has a smooth surface state in the upper portion of about 1 μm, and the surface of the portion of about 2 μm from the root has a striped pattern.

  In addition, since the columnar microprojection 801 has one side of the bottom surface of 300 nm and a height of 3 μm, the ratio of the height to one side is 10 and is larger than 1.

  Further, it can be seen that the columnar microprojection 801 has a tip end portion smaller than the bottom surface portion, and has a divergent shape. In this embodiment, the columnar microprojection 801 has a shape that narrows from the root to the tip, but may be a mushroom shape that narrows from the root to the tip and has a thick portion at the tip. One feature of the columnar microprojection 801 of the present invention is that it has a portion that narrows from the root to the tip.

  The columnar microprojection 801 is made of the same polystyrene as the base 802.

  Further, it can be seen that the columnar microprojections 801 are connected to the base 802 and are integrated.

  In addition, since the tip of the protrusion is smaller than the bottom surface of the protrusion and has a divergent shape, an effect that the protrusion is difficult to remove from the substrate can be obtained. Further, since the protrusion is the same as the base material, it is possible to obtain an effect that the protrusion is difficult to remove from the base. Moreover, since the protrusion is integrated with the substrate, it is difficult to remove the protrusion from the substrate.

  In this embodiment, polystyrene is used as the material for the columnar microprojections 801 and the bottom surface 802, but the materials exemplified above may be used as the material for the cell culture container.

The columnar microprojections 801 described above were produced by the method described below. FIG. 10 shows a manufacturing process of the columnar microprojection 801. The container 401 was heated to 100 ° C., and a die 402 having micropores with a diameter of 0.5 μm and a depth of 1.0 μm formed on the entire surface at a pitch of 1.0 μm was pressed at a pressing pressure of 4 MPa for 300 s. After cooling to 70 ° C. in the press apparatus, the mold 402 and the container 401 are taken out of the press apparatus and peeled off by pulling the mold 402 vertically from the container 401 to produce the cell culture container 100 having the columnar microprojections 801. did. Mold 402 has crystal orientation
(100), a silicon wafer having a diameter of 25 mm, and the columnar microprojection 801 having a mold release treatment with a fluorine-based mold release agent to prevent adhesion to the container 401 during molding has an aspect ratio of the mold 402 The aspect ratio of the unevenness is about 3 times. Such a high aspect ratio structure can also be formed on the cell culture vessel 100 by forming irregularities having a large aspect ratio on the mold 402, but if the method of this embodiment is used, a columnar microprojection having a high aspect ratio. The effect of being able to form 801 from the low aspect ratio mold 402 that is relatively easy to produce can be obtained.

  In this embodiment, polystyrene is used for the container 401. However, the container 401 is not limited to polystyrene, but may be an organic substance such as polycarbonate, an inorganic substance such as glass, or a laminated structure thereof.

  In this embodiment, a silicon wafer having a crystal orientation (100) and a diameter of 25 millimeters was used for the mold 402, but it is not particularly necessary that the crystal orientation is (100) or the material is single crystal silicon. It may be a metal thin film such as nickel or an organic material such as PDMS (polydimethylsiloxane). Further, in order to prevent damage to the container 401 and the columnar microprojections 801 when the mold 402 is released from the heated container 401, the mold 402 is coated with a release agent such as fluorine or silicone. It is desirable that

Further, the diameter and height of the columnar microprojection 801 can be controlled by adjusting the depth of the concave portion of the mold 402 and the material of the container 401.

  Further, the size of the bottom of the columnar microprojection 801 can be controlled by increasing the opening area of the concave portion of the mold 402.

  Furthermore, the position where the columnar microprojections 801 are formed can be controlled by controlling the position of the concave portion of the mold 402.

  Further, by making the material of the columnar microprojections 801 thermoplastic, it is possible to obtain an effect that the shape of the columnar microprojections 801 can be easily controlled by adjusting the temperature at the time of forming the columnar microprojections 801. it is obvious.

  In addition, by making the material of the columnar microprojections 801 photocurable, it is possible to obtain an effect that the shape of the columnar microprojections 801 can be easily controlled by making light incident when the columnar microprojections 801 are formed. it is obvious.

Hereinafter, other embodiments of the present invention will be described. FIG. 1 is a bird's-eye view showing a cell culture vessel 100 produced in this example. The cell culture container 100 has a petri dish-shaped shape with a diameter of 35 mm mainly composed of polystyrene having a thickness of 2 mm. On the bottom surface of the cell culture vessel 100, the projection aggregate 101 is formed in a region having a diameter of 30 mm by using the method shown in the first embodiment. Further, after the formation of the projection aggregate 101, oxygen plasma treatment (100 W, 30 s) was performed as a hydrophilic treatment. A 50 μg / mL collagen I solution (Cultrex Bovine Collagen I (registered trademark), solvent: 0.02 M acetic acid aqueous solution) was coated on the tip of the projection assembly 101 to be thinner than the height of the projection assembly 101. A smooth stamp made of PDMS with surface hydrophilization was brought into close contact, and the collagen I solution was adhered only to the tip of the projection assembly 101. After removing the smooth stamp, it is kept at room temperature for 1 hour and washed with PBS (phosphate buffer), so that only the tip of the projection assembly 101 is modified with collagen, and a surface treatment suitable for cell culture. Went.

  In this example, polystyrene was used for the cell culture vessel 100. However, not only polystyrene but also an organic substance such as polycarbonate, an inorganic substance such as glass, or a laminated structure thereof, as long as it has an affinity for the surface treatment to be applied. But you can. Moreover, although the size of the cell culture vessel 100 is 35 mm in diameter and the formation area of the projection aggregate 101 is 30 mm in diameter, this can be changed according to the size of the cells to be cultured. Further, the projection aggregate 101 is preferably arranged with a gap 103, and in this embodiment, the gap 103 is provided in a cross shape as shown in FIG. By forming the gap 103 in this manner, the culture solution can easily flow and nutrients can be efficiently supplied to the cells. Moreover, the waste product of the cell at the time of cell culture can be discharged | emitted efficiently.

In this embodiment, the modification with the collagen, which is a kind of protein, is performed after the formation of the protrusion aggregate 101. However, the present invention is not limited to this method, and the type of cell to be cultured, the cell culture container 100, and the protrusion aggregate. Oxygen plasma treatment (for example, 100 W, depending on the material of the body 101
30s), surface treatment such as UV irradiation, hydrophilization treatment by immersion in hydrogen peroxide water or ozone water, introduction of functional groups represented by amino groups, carboxyl groups, methyl groups, CF ₃ groups, etc. You can also. Further, by applying this surface treatment to only a part of the projection aggregate 101 by the method shown in FIGS. 5 and 6, the shape of the cells to be cultured can be controlled.

  Hereinafter, the example which cultured the cell using the cell culture container 100 of this invention is shown. FIG. 7 is a schematic diagram showing a state in which cells are cultured using the cell culture container 100 produced in the second embodiment.

The cell culture vessel 100 was immersed in a culture solution, and normal human epidermal keratinocytes were cultured in the cell culture vessel 100 by a conventional method (used medium: HuMedia-KB2 (Kurabo Co., Ltd., 37 ° C.). under 5% CO _2). as a result, epidermal keratinocytes in cell culture vessel 100 is normally attached, and grown in a sheet shape.

Fourteen days after the start of culture, a 2 cm diameter polyvinylidene difluoride (PVDF) membrane was placed on the cultured cells, and the medium was aspirated, so that sheet-like epidermal keratinocytes grown on the cell culture vessel 100 were obtained. Was peeled from the cell culture vessel 100 together with the PVDF membrane. The sheet-like epidermal keratinocytes could be easily peeled off from the PVDF membrane. The damage to the sheet-shaped epidermal keratinocytes due to peeling from the cell culture vessel 100 could be significantly reduced as compared with the case of using a normal glass petri dish or the like.

  Hereinafter, an example in which cells are cultured using the cell culture container 100 of the present invention and cell detachability is improved will be described.

  FIG. 11 shows an example of the shape of the cell culture vessel 100 produced in this example using the method of Example 1. FIG. 12 is an enlarged view of the columnar microprojections 801 constituting the projection aggregate 101 on the cell culture container 100. In this embodiment, in order to investigate the relationship between the peelability of cultured cells and the shape of the columnar microprojections 801 constituting the projection aggregate 101, the diameter R of the columnar microprojections 801 is 180 nm, 240 nm, 500 nm, and 2 μm. Four types of four cell culture vessels 100 were formed. The heights H of the formed columnar microprojections 801 are all 3 μm in the four cell culture containers 100. The arrangement of the columnar microprojections 801 is a two-dimensional square lattice as shown in FIGS. 11 and 12, and the interval D between adjacent columnar microprojections 801 is twice the diameter R.

For evaluation of exfoliation, human mesenchymal stem cells (CAMBREX, Cyro hMSC No.2051) were cultured on each cell culture vessel 100 (CAMBREX, bullet kit MSCGM (medium for stem cell proliferation)) The seeds were sown and cultured in an incubator at 37 ° C. and 5% CO ₂ for 5 days. For comparison, human mesenchymal stem cells were cultured under the same conditions in an animal cell culture dish (Corning) generally used for comparison.

  The detachability was evaluated by detaching human mesenchymal stem cells on the cell culture vessel 100 after culturing for 5 days by a water flow generated in the medium. For the water flow, attach a pipette tip with a capacity of 200 μl to an electric pipette for 250 μl (EDP plusEP-250), and discharge the medium used for culture at an angle of 70 degrees with respect to the bottom from the bottom of the cell culture vessel 100. Generated by.

  Table 1 shows the results of observing the detachment of the cells on the cell culture container 100 by discharging the medium once under the microscope observation. In any cell culture vessel 100 formed in the present invention, it was confirmed that the peelability of human mesenchymal stem cells was increased as compared to the animal cell culture dish used as a comparative example. It was shown that the peelability of human mesenchymal stem cells from the cell culture vessel 100 depends on the diameter R of the columnar microprojections 801, and that the peelability is greatest when the diameter is large R = 2 μm. From this, it was shown that the adhesive force of human mesenchymal stem cells, which are adhesive cells, is reduced on the columnar microprojections 801 and is excellent in peelability.

The schematic diagram which shows an example of the cell culture container by this invention. The schematic diagram which shows another example of the cell culture container by this invention. The schematic diagram which shows the 3rd example of the cell culture container by this invention. The schematic diagram which shows the method of forming a processus | protrusion group on the cell culture container surface. The schematic diagram which shows the surface modification method only to the front-end | tip of a processus | protrusion group. The schematic diagram which shows the modification method which forms distribution of the process to a processus | protrusion group front-end | tip part. The schematic diagram which shows the usage method of the cell culture container in this invention. The schematic diagram of the scanning electron micrograph of the protrusion assembly produced in the first example. The schematic diagram which shows the scanning electron micrograph which expanded the protrusion aggregate | assembly. The schematic diagram which shows the manufacturing process of a columnar microprotrusion. The schematic diagram which shows an example of the cell culture container produced in the 4th Example. The schematic diagram which shows the enlarged view of the columnar microprotrusion on the cell culture container produced in the 4th Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 ... Cell culture container, 101 ... Protrusion assembly, 102,501 ... Surface treatment, 401 ... Container, 402 ... Mold, 403 ... Mold shape, 502 ... Stamp, 503 ... Surface treatment agent,
701 ... Culture medium, 702 ... Cells, 801 ... Columnar microprojections, 802 ... Base.

Claims (15)

  A cell culture container, comprising a group of protrusions formed on a bottom surface of the cell culture container and having an equivalent diameter of 10 nm to 10 μm and a height of 10 nm to 1 mm.   2. The cell culture container according to claim 1, wherein an equivalent diameter of the protrusion group formed on the bottom surface of the cell culture container is smaller than a diameter of cells cultured in the cell culture container. .   2. The cell culture container according to claim 1, wherein an equivalent diameter of the protrusion group formed on the bottom surface of the cell culture container is 1/5 or less of a diameter of cells cultured in the cell culture container. A cell culture container.   2. The cell culture container according to claim 1, wherein the projection group formed on the bottom surface of the cell culture container includes one having a distance smaller than the diameter of the cells cultured in the cell culture container. A cell culture container.   2. The cell culture container according to claim 1, wherein the cell culture container and the projection group are made of the same material and are integrated.   The cell culture container according to claim 1, wherein the whole or a part of the cell culture container is subjected to at least one treatment of hydrophilic treatment, hydrophobization treatment, formation of a metal layer, or coating of an organic substance. A cell culture vessel characterized by being applied.   The cell culture container according to claim 1, wherein one or more treatments of hydrophilic treatment, hydrophobization treatment, formation of a metal layer, or coating of an organic substance are selectively applied to a specific region. A cell culture container characterized by the above.   The cell culture container according to claim 1, wherein one or more of a hydrophilic treatment, a hydrophobization treatment, formation of a metal layer, or coating of an organic substance is selectively applied to individual protrusions constituting the protrusion group. A cell culture vessel characterized by being treated.   2. The cell culture container according to claim 1, wherein only the tip of the projection group is subjected to hydrophilic treatment, hydrophobization treatment, formation of a metal layer, or coating with an organic substance.   2. The cell culture container according to claim 1, wherein the protrusion group has a row of vacant regions through which a liquid can pass through the surface of the cell culture container.   A cell culture container having a container for containing a culture solution and a projection group formed on a bottom surface of the container, wherein the container and the projection group are made of the same material.   The cell culture container according to claim 11, wherein an equivalent diameter of the protrusion group formed on the bottom surface of the cell culture container is smaller than a diameter of a cell cultured in the cell culture container.   12. The cell culture container according to claim 11, wherein an equivalent diameter of the projection group formed on the bottom surface of the cell culture container is not more than one fifth of a diameter of a cell cultured in the cell culture container. Cell culture container.   12. The cell culture container according to claim 11, wherein the projection group formed on the bottom surface of the cell culture container includes a projection whose spacing is smaller than the diameter of the cells cultured in the cell culture container. Cell culture container. A cultured cell characterized by being cultured in the culture container according to claim 1.

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