JP2007228818A - Cell culture vessel, method for producing the same, and method for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture vessel capable of reproducing in vivo cell function through raising cell culture density, and to provide a method for producing the vessel. <P>SOLUTION: The cell culture vessel has multiple microvessels each 5-1,000μm in width or diameter and 5-1,000μm in depth and enables a culture density of 3-20 cells/1,000μm<SP>2</SP>to be attained. Thereby, the objective cell culture vessel optimum for reproducing in vivo cell function through raising cell culture density can be obtained. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell culture container, a production method thereof, and a cell culture method.

  Techniques for testing and examining cells isolated from tissues are indispensable in biotechnology-related fields. It is widely used for diagnosing diseases and pathological conditions, searching for new drugs and determining their efficacy, animal testing, plant testing, and testing for environmental pollutants. Therefore, the cells used in the biotechnology field have been extremely diversified.

  The isolated cells may be used immediately for testing, but in many cases, cell culture is performed in a culture dish or a test tube. Various tests are performed using the cultured cells. Cell culture strains used for cell culture tests are required to exhibit drug sensitivities and toxic reactions similar to in vivo tests, so-called in vivo tests. In addition, since cell culture strains used for cell culture tests are extremely expensive, it is desired to improve cell survival rate and growth rate.

In the cell culture test, the amount and concentration of the drug to be evaluated are varied under the same conditions, and the effect is measured. Therefore, the material, shape, etc. of the cell culture container must be the same. As the cell culture container, a plastic petri dish, a glass petri dish, a glass plate fixed in the container, a well plate, or the like is generally used. Well plates include 6-well, 12-well, 48-well, and 96-well plates or petri dishes. In general, the size of the whole plate is substantially the same, and the larger the number of wells, the smaller the size of one well. One well corresponds to one culture dish. Moreover, as shown in Patent Document 1, a 384 well plate including a large number of culture dishes having a smaller diameter has begun to be used due to the recent trend toward a small amount.
JP-A-11-169166

  However, when a conventional cell culture vessel is used for culturing tissue cells, there is a problem in that the cells are thinly stretched and have a non-directional form, and the cell function in vivo cannot be reproduced. This is considered to be the same as culture on a plate for cells having a size of several μm to several tens of μm even though the well size is small.

  The present invention has been made to solve such problems, and provides a cell culture container capable of increasing cell culture density and reproducing cell functions in vivo, and a method for producing the same. With the goal.

The cell culture container according to the present invention includes a plurality of micro containers having a width or a diameter of 5 μm to 1000 μm and a depth of 5 μm to 1000 μm on the surface on which the cells are cultured, and has a culture density of ³ cells / 1000 μm ^{2 to 20} cells / 1000 μm ^2. It can be achieved.

The cell culture method according to the present invention uses a cell culture vessel provided with a plurality of microcontainers having a width or a diameter of 5 μm to 1000 μm and a depth of 5 μm to 1000 μm on the surface on which the cells are cultured, and 3 cells / 1000 μm ^{2 to 20} cells / A culture density of 1000 μm ² can be achieved.

  The method for producing a cell culture container according to the present invention includes a step of forming a pattern on a substrate, a step of forming a metal structure by attaching a metal according to the pattern formed on the substrate or a transfer pattern thereof, and And transferring a pattern of the metal structure to form a cell culture vessel.

  ADVANTAGE OF THE INVENTION According to this invention, the cell culture container which can raise the culture density of a cell and can reproduce the cell function in the living body, and its manufacturing method can be provided.

  As a result of diligent research, the inventors of the present invention have increased the cell culture density by forming a micro-container having a desired size on the surface where cells are cultured in a cell culture container such as a plate or petri dish. The present inventors have found that an optimal cell culture vessel can be provided to reproduce the cell functions of Embodiments of the present invention will be described below.

  Usually, in vivo tests are performed using a laboratory animal such as a rat to determine toxicity to drugs. Tests using laboratory animals have high reliability of test results, but the costs and ethical problems associated with using laboratory animals have been pointed out. For this reason, in the in vitro test using a cell culture container, the cell culture form which can reproduce an in vivo test result is desired.

  Compared with the culture on a flat plate, by culturing in a cell culture container having a micro container, it is possible to prevent the cells from extending thinly and having a non-directional form. The cells arranged in the micro container proliferate three-dimensionally in the micro container, and if the container is deep, it becomes a multilayered state. In addition, the growth rate is increased compared to the culture on a flat plate.

Embodiment The configuration of a cell culture container according to an embodiment will be described with reference to FIGS. 1 (a) and 1 (b). Fig.1 (a) is a top view which shows the structure of the cell culture container concerning Embodiment. FIG.1 (b) is AA 'sectional drawing of Fig.1 (a).

  In the cell culture container, as shown in FIG. 1 (a), a concavo-convex pattern constituting a plurality of micro containers is formed. The concavo-convex pattern is formed in a two-step staircase shape as shown in FIG. That is, a two-step convex portion 4 including a first convex portion 2 formed in a lattice shape and a rectangular parallelepiped second convex portion 3 formed on the surface of the cell culture vessel on which cells are cultured. Is formed. A space formed by the two-stage convex portions 4 is the micro container 1. That is, the micro container 1 includes a concave portion completely surrounded by the first convex portion 2 and a concave portion formed by the second convex portion 3. The side wall 5 of the first convex part 2 and the side wall 6 of the second convex part 3 are formed substantially perpendicular to the bottom surface.

  The 1st convex part 2 is arrange | positioned at the grid | lattice form so that the four sides of the rectangular-shaped micro container 1 may be enclosed, as shown to Fig.1 (a). The 2nd convex part 3 is arrange | positioned on the 1st convex part 2 between the adjacent microcontainers 1 at island shape. The second protrusions 3 are provided on each of the four sides of the rectangular micro container 1. Therefore, the micro container 1 is not completely partitioned, and communicates with the adjacent micro container 1 at the four apex portions of the rectangular micro container 1. The concave / convex pattern may have two or more steps, or one step.

  The width or diameter of the micro container is preferably 5 to 1000 μm, and more preferably 10 μm to 600 μm. In the case of the embodiment, the width of the micro container 1 corresponds to the interval between the opposing second convex portions 3. The micro container is not limited to a rectangular shape, and may be a circular shape or a composite shape thereof. Further, the side wall may be inclined or may be stepped. When the side walls are inclined or stepped, the width of the micro container refers to the widest part of the interval between the opposing side walls. When the container width or diameter is smaller than 5 μm, the number of cells that can be arranged decreases, and it becomes difficult to obtain the cell density necessary for culture. When the thickness is larger than 1000 μm, the cells are thinly stretched and have a non-directional form as in the case of culturing on a flat plate, and it becomes difficult to obtain the cell density necessary for the culturing.

  The depth of the micro container is preferably 5 to 1000 μm. When the depth of the micro container is less than 5 μm, the cells cannot be multilayered, the culture density is lowered, and the culture speed is also lowered. When the depth of the micro container is deeper than 1000 μm, the multilayered culture cells progress, so that the inner cells far from the outermost cells are blocked from supplying the culture solution, and the wastes discharged from the cells are replaced. Result in cell death. The depth of the micro container can be said to be the height of the side wall with respect to the bottom surface. In the case of the embodiment, the depth of the micro container corresponds to the height of the two-step convex portion 4.

To achieve a higher culture density than flat, the culture cell density is preferably from ^{3cells / 1000μm 2 ~20cells / 1000μm 2} , more preferably ^{4cells / 1000μm 2 ~15cells / 1000μm 2} . If it becomes ³ cells / 1000 micrometers ² or less, it will become equivalent to the culture density on a flat plate, and a culture speed will also fall. When the cell density is 20 cells / 1000 μm ² or more, it becomes difficult to supply the culture solution to the inner cells due to the multilayered culture cells, and there is concern about the occurrence of cell death.

The density of cells to be spread for culturing is 8 cells / 1000 μm ² or less, preferably 6 cells / 1000 μm ² or less. Since cell culture strains are extremely expensive, the lower the density of cells to be spread, the better. On the other hand, when the density of the cells to be spread becomes low, the culture rate becomes slow. For this reason, it is desirable to set the density of cells to be spread according to the intended use such as observation of cell morphology and drug sensitivity test. Since the cell culture container according to the present invention has a micro container, the culture density can be increased even when the density of cells to be dispersed is relatively low.

  By increasing the cell density, in addition to the test in the cell culture container, the cells can be lysed or detached and used for a colorimetric test or the like. In addition, cell lines used for cell culture tests are extremely expensive. Compared with the culture on a normal flat plate, a culture density several times higher can be obtained, so that the cost of purchasing a cell line can be reduced and the experimental efficiency can be improved.

  The distance between the micro containers is preferably as close as possible to increase the number of cells. However, since the distance between the micro containers becomes narrow, industrial fine processing becomes difficult and the cost becomes high. It is desirable to select according to the required application.

  To make the resin plate have the same light transmittance as that of the glass plate in order to enable the transmitted light observation, the light transmittance at a wavelength of 300 nm to 800 nm including the ultraviolet region is set to 80% or more and the haze value is set to 10% or less. It is preferable. In order to satisfy the above requirements, it is necessary to use an acrylic resin that does not contain an ultraviolet absorber for the cell culture container, or select a material that does not have a ring structure in its chemical structure, such as PC (polycarbonate), polystyrene, or the like. In addition, additives such as an antioxidant, a viscosity improver, a heat stabilizer, and an anti-sticking agent need not contain an ultraviolet absorber.

  In the fluorescence observation method, the light (excitation light) for causing the fluorescent dye to shine cannot be discriminated from the fluorescence (fluorescence radiation light) generated unless it passes through the cell culture vessel. Therefore, high light transmittance is required. The transparency necessary for identifying the fluorescence (fluorescence emission light) needs to be 80% or more of the total light transmittance of visible light and within 10% of the haze value. In order to satisfy the above requirements, it is preferable to use a material having excellent optical properties such as polymethylmethacrylate for the cell culture container, and when using a polyolefin-based resin which is a crystalline resin, it should be used in an amorphous state. preferable.

  Autofluorescence means that after a polymer molecule absorbs ultraviolet / visible light, it emits light and emits fluorescence. While glass plates do not emit autofluorescence, many resin plates do autofluorescence, making it impossible to identify fluorescence (fluorescent radiation) generated from the sample, making it difficult to perform microanalysis, which is a feature of fluorescence analysis.

  In order not to be affected by the autofluorescence, it is required that the autofluorescence does not occur by irradiating light with a wavelength of 230 nm to 800 nm. Therefore, it is necessary to select a resin material that does not have a ring structure in the chemical structure such as PC (polycarbonate) or polystyrene for the cell culture container. Further, in order to eliminate the possibility of autofluorescence as much as possible, it is preferable that the additives such as antioxidants, viscosity improvers, heat stabilizers, and anti-sticking agents be as small as possible or not added.

  In order to observe with a polarizing microscope or a differential interference microscope without reducing the contrast of the differential interference observation method, a material having a small optical distortion is required. Therefore, it is necessary to select a resin material that does not have a ring structure in the chemical structure such as PC (polycarbonate) or polystyrene for the cell culture container.

  By coating the cell culture vessel with an organic film or an inorganic film, it can be made hydrophilic or hydrophobic. Thereby, the adhesion prevention of the bubble to a fine processus | protrusion and the adhesion degree of a cell are controllable. For example, there are a method using low-temperature plasma treatment, corona discharge treatment, ultraviolet irradiation, and the like, and a method of applying collagen, which is a protein that promotes cell adhesion. Further, by masking a part, only the other part can be covered with an organic film or an inorganic film. Thereby, the range of culture test conditions can be further widened.

Inorganic film formation methods such as sputtering and vapor deposition can also be applied to oil immersion lens observation methods. Normally, an organic solvent is blended in the oil used for the oil immersion lens in order to make the optical properties compatible with the glass plate for culturing cells and the optical lens. Organic solvents may not be applicable because they penetrate into resinous cell culture vessels and cause whitening and dissolution problems. Inorganic materials are resistant to organic solvents at the same time as the gas barrier effect, so by forming an inorganic film on the bottom of the cell culture vessel that comes in contact with the oil in the oil immersion lens, it can also be applied to oil immersion lens observation methods. It becomes possible, and the application range of the resin-made cell culture container can be expanded. When performing transmitted light observation, it is preferable that the film thickness is 400 nm or less, or a transparent inorganic material such as SiO ₂ is used.

  The manufacturing method of the resin-made cell culture container which has said several micro container is demonstrated. In this manufacturing method, a micro space structure corresponding to a micro container is formed on a substrate, a metal is attached according to a micro space structure pattern formed on the substrate or a transfer pattern thereof, and the structure pattern of the resin plate is Forming a metal structure having an opposite pattern; and transferring a pattern of the metal structure to form a resin plate.

Details are as follows.
(I) Formation of first resist layer on substrate (ii) Position alignment between substrate and mask A (iii) Exposure of first resist layer using mask A (iv) Heat treatment of first resist layer (v) Formation of second resist layer on first resist layer (vi) Alignment of substrate and mask B (vii) Exposure of second resist layer using mask B (viii) Heat treatment of second resist layer (ix) The resist layer is developed to form a desired resist pattern.
(X) Further, after conducting the conductive treatment on the formed resist pattern, a metal structure is deposited on the substrate by plating according to the formed resist pattern.
(Xi) A cell culture container is manufactured by forming a resin molded product using this metal structure as a mold.
The steps (v) to (viii) are optional and can be omitted. On the other hand, the steps (v) to (viii) can be repeated a plurality of times.

The resist pattern forming process will be described in more detail.
For example, when a structure having a depth of 30 μm and a depth of 100 μm is to be obtained on the substrate, a first resist layer (thickness 70 μm) and a second resist layer (thickness 30 μm) are formed in this order, and each layer is exposed. Alternatively, exposure and heat treatment are performed. In the development step, a pattern having a depth of 30 μm, which is the second resist layer, is first obtained, and then a pattern having a depth of 100 μm is obtained by combining the first resist layer and the second resist layer. When a pattern having a depth of 100 μm is obtained, it is necessary to control the solubility of each layer in the developer so as not to dissolve or deform the pattern of the second resist layer having a depth of 30 μm in the developer. Is done.

  One method of developing alkali resistance using a photolytic positive resist is to increase the baking time (solvent drying time) and cure the resist. Usually, the baking time is set according to the film thickness, solvent concentration such as thinner, and sensitivity. By increasing this time, alkali resistance can be provided. In addition, if the baking of the first resist layer proceeds too much, the resist is extremely hardened, and it becomes difficult to form a pattern by dissolving the portion irradiated with light in the subsequent development, so the baking time is shortened. It is preferable to select as appropriate. The apparatus used for baking is not particularly limited as long as the solvent can be dried, and examples thereof include an oven, a hot plate, and a hot air dryer. Compared with a photo-crosslinking negative resist, since the range of alkali resistance is limited, the resist thickness to be set is preferably within a range of 5 to 200 μm, and within a range of 10 to 100 μm, including each layer. More preferably.

  As a method of developing alkali resistance using a photo-crosslinking negative resist, optimization of the crosslinking density can be mentioned in addition to optimization of the baking time. Usually, the crosslinking density of the negative resist can be set by the exposure amount. In the case of a chemically amplified negative resist, the crosslinking density can be set by the exposure amount and the heat treatment time. By increasing the exposure amount or the heat treatment time, alkali resistance can be exhibited. In the case of a photo-crosslinking negative resist, the resist thickness to be set is preferably in the range of 5 to 500 μm, more preferably in the range of 10 to 300 μm, for each layer.

(I) The formation of the first resist layer 12 on the substrate 11 will be described.
FIG. 2A shows a state where the first resist layer 12 is formed on the substrate 11. The flatness of the resin-made cell container obtained in the molded product forming step is determined by the process of forming the first resist layer 12 on the substrate 11. That is, the flatness at the time when the first resist layer is formed on the substrate is reflected in the flatness of the metal structure, and thus the cell culture container.

  The method for forming the first resist layer 12 on the substrate 11 is not limited at all, and generally includes a spin coat method, a dipping method, a roll method, and a dry film resist bonding. Among these, the spin coating method is a method of applying a resist on a rotating glass substrate, and has an advantage of applying the resist to a glass substrate having a diameter of more than 300 mm with high flatness. Therefore, the spin coating method is preferable from the viewpoint of realizing high flatness.

  The resist used as the first resist layer 12 may be either a positive resist or a negative resist. In either case, the depth of focus of the resist varies depending on the sensitivity of the resist and the exposure conditions. Therefore, for example, when a UV exposure apparatus is used, it is preferable to select the exposure time and UV output value according to the resist thickness and sensitivity.

  When the resist used as the first resist layer 12 is a wet resist, for example, as a method for obtaining a predetermined resist thickness by a spin coating method, there is a method by changing the spin coating rotational speed or adjusting the viscosity. The method by changing the spin coat rotational speed is to obtain a desired resist thickness by appropriately setting the spin coater rotational speed. The viscosity adjustment method adjusts the viscosity according to the flatness required in actual use because there is a concern that the flatness may decrease when the resist thickness is large or the coating area is large. is there.

  For example, in the case of the spin coat method, the thickness of the resist layer applied at one time is preferably in the range of 10 to 50 μm, more preferably in the range of 20 to 50 μm in consideration of maintaining high flatness. preferable. In order to obtain a desired resist layer thickness while maintaining high flatness, the resist layer can be formed in a plurality of times.

  When a positive resist is used for the first resist layer 12, if the baking time (drying of the solvent) proceeds excessively, the resist is extremely hardened, and it becomes difficult to form a pattern in subsequent development. Therefore, when the resist thickness to be set is not 100 μm or more, it is preferable to select appropriately such as shortening the baking time.

(Ii) The alignment between the substrate 11 and the mask A13 will be described.
In order to make the positional relationship between the pattern of the first resist layer 12 and the pattern of the second resist layer 14 as desired, it is necessary to perform accurate alignment at the time of exposure using the mask A13. For alignment, a method of cutting and pinning the same position of the substrate and the mask A, a method of positioning using a laser interferometer, a position mark at the same position of the substrate 11 and the mask A13, and position with an optical microscope The method of combining them can be given. As a method of aligning with an optical microscope, for example, a position mark is formed on a substrate by a photolithographic method, and a position mark is drawn on a mask A by a laser drawing apparatus. Even manual operation using an optical microscope is effective in that accuracy within 5 μm can be easily obtained.

(Iii) The exposure of the first resist layer 12 using the mask A13 will be described.
The mask A13 used in the step shown in FIG. 2B is not limited in any way, and examples thereof include an emulsion mask and a chrome mask. In the resist pattern forming step, the size and accuracy depend on the mask A13 used. And the dimension and precision are reflected also in a resin-made cell culture container. Therefore, in order to make each dimension and accuracy of the resin-made cell culture container predetermined, it is necessary to define the size and accuracy of the mask A13. The method for increasing the accuracy of the mask A13 is not limited at all. For example, the laser light source used for pattern formation of the mask A13 can be changed to one having a shorter wavelength, but the equipment cost is high, and the mask A13 is manufactured. Since the cost is high, it is preferable to appropriately define the resin-made cell culture container according to the accuracy required for practical use.

  The material of the mask A13 is preferably quartz glass from the viewpoint of temperature expansion coefficient and UV transmission absorption performance, but is relatively expensive. Therefore, it is preferable to appropriately define the resin molded product according to the accuracy required for practical use. In order to obtain structures having different desired depths or heights as designed, or structures having different first and second resist patterns, they are used for exposure of the first resist layer 12 and the second resist layer 14. The mask pattern design (transmission / shading part) needs to be reliable, and simulation using CAE analysis software is one of the solutions.

  The light source used for the exposure is preferably ultraviolet light or laser light, which has a low equipment cost. Synchrotron radiation can be used when the equipment cost is high and the price of the resin plate is substantially high, but it is desired to obtain a deep exposure depth.

  Since exposure conditions such as exposure time and exposure intensity vary depending on the material, thickness, and the like of the first resist layer 12, it is preferable to adjust appropriately according to the pattern to be obtained. In particular, adjustment of exposure conditions is important because it affects the size and accuracy of the spatial structure pattern. Further, since the depth of focus varies depending on the type of resist, for example, when a UV exposure apparatus is used, it is preferable to select the exposure time and the UV output value according to the thickness and sensitivity of the resist.

(Iv) The heat treatment of the first resist layer 12 will be described.
As heat treatment after exposure, heat treatment called annealing is known in order to correct the shape of the resist pattern. Here, it is performed only when a chemically amplified negative resist is used for the purpose of chemical crosslinking. The chemically amplified negative resist is mainly composed of a two-component system or a three-component system, and, for example, an epoxy group at the end of a chemical structure is opened by light during exposure, and is subjected to a crosslinking reaction by heat treatment. For example, when the heat treatment time is 100 μm, the crosslinking reaction proceeds in several minutes under the condition of a set temperature of 100 ° C.

  If the heat treatment of the first resist layer 12 proceeds excessively, it becomes difficult to dissolve the uncrosslinked portion and form a pattern in subsequent development. Therefore, when the resist thickness to be set is not 100 μm or more, the heat treatment time is shortened. It is preferable to select appropriately, for example, or only heat treatment of the second resist layer 14 later.

(V) The formation of the second resist layer 14 on the first resist layer 12 will be described.
FIG. 2C shows a state where the second resist layer 14 is formed. The formation of the second resist layer 14 is performed by the same method as the formation of the first resist layer 12 described in (i) above. Moreover, when forming a resist layer using a positive resist by a spin coat system, alkali resistance can be expressed by making baking time about 1.5 to 2.0 times normal. Thereby, dissolution or deformation of the resist pattern of the second resist layer 14 can be prevented at the end of development of the first resist layer 12 and the second resist layer 14.

(Vi) The alignment between the substrate 1 and the mask B15 will be described. The alignment between the substrate 1 and the mask B15 is performed by the same method as the alignment between the substrate 1 and the mask A13 described in (ii) above.

(Vii) Exposure of the second resist layer 14 using the mask B15 will be described. The exposure of the second resist layer 14 using the mask B15 is performed in the same manner as the exposure of the first resist layer 12 using the mask A13 described in (iii) above. FIG. 2D shows how the second resist layer 14 is exposed.

(Viii) The heat treatment of the second resist layer 14 will be described. The heat treatment of the second resist layer 14 is performed by the same method as the heat treatment of the first resist layer 12 described in (iv) above. The heat treatment of the second resist layer 14 is performed so that the pattern of the second resist layer 14 is not dissolved or deformed when the pattern of the first resist layer 12 is obtained in the subsequent development. Chemical crosslinking proceeds by heat treatment, and alkali resistance is developed by increasing the crosslinking density. It is preferable that the heat treatment time for developing alkali resistance is appropriately selected according to the thickness of the resist from the usual 1.1 to 2.0 times range.

(Ix) The development of the resist layers 12 and 14 will be described. In the developing step shown in FIG. 2E, it is preferable to use a predetermined developer corresponding to the resist used. Development conditions such as development time, development temperature, and developer concentration are preferably adjusted as appropriate according to the resist thickness and pattern shape. For example, if the development time is too long in order to obtain the required depth, it becomes larger than a predetermined dimension, so it is preferable to set conditions appropriately. By this development process, a resist pattern 16 is formed.

  Examples of methods for improving the plane accuracy of the top surface of the cell culture container or the bottom of the fine pattern include a method of changing the resist type (negative type or positive type) used in resist coating, and a method of polishing the surface of the metal structure. can give.

  When forming a plurality of resist layers in order to obtain a desired molding depth, the resist layers are exposed and developed at the same time, or after forming and exposing one resist layer, a resist layer is further formed. The two resist layers can be developed at the same time.

(X) The metal structure forming step will be described in more detail.
The metal structure forming step is to deposit a metal along the resist pattern 16 obtained in the resist pattern forming step and form a micro space structure surface of the metal structure 18 along the resist pattern 16, thereby forming the metal structure. 18 is a step of obtaining 18.

  As shown in FIG. 2F, in this step, a conductive film 17 is formed along the resist pattern 16 in advance. A method for forming the conductive film 17 is not particularly limited, but preferably, a vacuum deposition method, a sputtering method, or the like is used. Examples of the conductive material used for the conductive film 17 include gold, silver, platinum, copper, and aluminum.

  As shown in FIG. 2G, after the conductive film 17 is formed, a metal structure 18 is formed by depositing metal along the resist pattern 16 by plating. The plating method is not particularly limited, and examples thereof include electrolytic plating and electroless plating. Although the metal used is not specifically limited, Nickel, nickel-cobalt alloy, copper, and gold can be mentioned, and nickel is preferably used from the viewpoint of economy and durability.

  The metal structure 18 may be polished according to the surface state. However, since there is a concern that dirt adheres to the modeled object, it is preferable to perform ultrasonic cleaning after polishing. The metal structure 18 may be surface-treated with a release agent or the like in order to improve the surface state. In addition, the inclination angle in the depth direction of the metal structure 18 is desirably 50 ° to 90 °, more desirably 60 ° to 87 °, from the shape of the resin molded product. The metal structure 18 deposited by plating is separated from the resist pattern 16.

(Xi) The molded product forming step will be described in detail.
The molded product forming step is a process of forming a resin molded product 19 using the metal structure 18 as a mold, as shown in FIG. The method for forming the resin molded product 19 is not particularly limited, and examples thereof include injection molding, press molding, monomer cast molding, solvent cast molding, roll transfer method by extrusion molding, and the like, from the viewpoint of productivity and mold transferability. Injection molding is preferably used. In the case where the resin molded product 19 is formed by injection molding using the metal structure 18 with a predetermined dimension selected as a mold, the shape of the metal structure 18 can be reproduced on the resin molded product 19 with a high transfer rate. As a method for confirming the transfer rate, there are methods using an optical microscope, a scanning electron microscope (SEM), a transmission electron microscope (TEM), and the like.

  The minimum value of the flatness of the resin molded product 19 is preferably 1 μm or more from the viewpoint of easy industrial reproduction. The maximum value of the flatness of the resin molded product 19 is preferably 200 μm or less from the viewpoint of preventing the molded product from warping and coming into contact with the optical system unit. The dimensional accuracy of the molded part of the resin molded product is preferably within a range of ± 0.5 to 10% from the viewpoint of easy industrial reproduction.

  Below, the Example and comparative example concerning this invention are shown. In these Examples and Comparative Examples, the dimensions of the microcontainer were varied, and the cell culture density and ALP (alkaline phosphatase) activity were evaluated. The total light transmittance and haze value, which are optical properties, were measured using a visible light transmittance meter (model: HA-TR) manufactured by Suga Test Instruments Co., Ltd. Specifically, the total light transmittance was measured twice by a method based on JIS K6714, and the average value was obtained. The thickness of the deposited film was measured by a stylus method using a surface shape measuring instrument (DEKTAK3030) manufactured by ULVAC, Inc.

The culture density measurement method is as follows. In the density by spraying a 0.4cells / 1000μm ² and 1.1cells / 1000μm ² rat incisor pulp derived pulp cells (RPC-C2A), were cultured for 48 hours and 96 hours, it is a cell fluorescent dye for staining Cell nuclei were stained with Propidium Iodide (PI). The number of stained cells at 1000 μm ² was counted using a confocal laser scanning microscope (model: LSM 510 META) manufactured by Carl Zeiss. Ten fields of view were observed per condition, and the average value was obtained.

ALP is a phosphate enzyme that is present in all organs and tissues in the body. It is known that pulp cells derived from rat incisor pulp (RPC-C2A), which are ALP positive cells, show a positive reaction in a high density state. Density pulp cells (RPC-C2A) derived from rat incisor pulp with a density of 0.4 cells / 1000 μm ² and 1.1 cells / 1000 μm ² were sprayed and cultured for 48 hours and 96 hours, and then ALP activity was evaluated. ALP activity levels were classified into grades 0 and 1+ to 4+ depending on the staining properties. The larger the numerical value of the grade, the more active it is. DAKO New Fuchsin Substrate System (code No. K0698) manufactured by DAKO was used for ALP staining. The ALP staining method is an example and does not limit the scope of application of the present invention.

[Comparative Example 1]
A commercially available polystyrene petri dish (φ90 mm—depth 20 mm) was used. The optical physical properties were a total light transmittance of 85% and a haze value of 3.7%.

[Comparative Example 2]
A resin plate having a width of 24 mm, a length of 74 mm, and a thickness of 1.0 mm was produced by an injection molding method using an acrylic resin (Parapet GH-S) manufactured by Kuraray Co., Ltd., and then sterilized. The optical physical properties were a total light transmittance of 90% and a haze value of 1.8%.

[Comparative Example 3]
Using an acrylic resin (Parapet GH-S) manufactured by Kuraray Co., Ltd., on a resin plate with a width of 24 mm, a length of 74 mm, and a thickness of 1.0 mm, the length and width shown in FIG. A plate having a plurality of microcontainers 1 having a thickness of 3 μm and a depth of 3 μm was produced. Fig.3 (a) is a top view which shows typically the structure of the cell culture container concerning the comparative example 3, and FIG.3 (b) is AA 'sectional drawing of Fig.3 (a). The resin plate was sterilized. Next, in order to prevent bubbles from being mixed into the micro container, a silicon oxide (SiO ₂ ) film having a thickness of 0.3 μm was formed using an evaporation apparatus manufactured by ULVAC, Inc. (model: UEP). The optical physical properties were a total light transmittance of 86% and a haze value of 9.1%.

[Comparative Example 4]
Using an acrylic resin (Parapet GH-S) manufactured by Kuraray Co., Ltd., on a resin plate having a width of 24 mm, a length of 74 mm, and a thickness of 1.0 mm by an injection molding method using a stamper, the shape as shown in FIG. A plate having a plurality of containers having a length and width of 5 mm and a depth of 2 mm was prepared. The resin plate was sterilized. Next, in order to prevent bubbles from entering the container, a 0.3 μm thick silicon oxide (SiO ₂ ) film was formed using a vapor deposition apparatus manufactured by ULVAC, Inc. (model: UEP). The optical properties were a total light transmittance of 89% and a haze value of 4.5%.

[Example 1]
Using an acrylic resin (Parapet GH-S) manufactured by Kuraray Co., Ltd., on a resin plate with a width of 24 mm, a length of 74 mm, and a thickness of 1.0 mm, the length and width shown in FIG. A plate having a plurality of microcontainers 1 having a thickness of 50 μm and a depth of 20 μm was produced. FIG. 3A is also a plan view schematically showing the configuration of the cell culture container according to Example 1, and FIG. 3B is a cross-sectional view taken along line AA ′ of FIG. The resin plate was sterilized. Next, a silicon oxide (SiO ₂ ) film having a thickness of 0.3 μm was formed using a vapor deposition apparatus manufactured by ULVAC, Inc. (model: UEP) in order to prevent air bubbles from entering the micro container 1. The optical properties were a total light transmittance of 85% and a haze value of 9.5%.

[Example 2]
Using an acrylic resin (Parapet GH-S) manufactured by Kuraray Co., Ltd., on a resin plate with a width of 24 mm, a length of 74 mm, and a thickness of 1.0 mm, the length and width shown in FIG. A plate having a plurality of microcontainers 1 having a thickness of 90 μm and a depth of 20 μm was produced. FIG. 4A is a plan view schematically showing the configuration of the cell culture container according to Example 2, and FIG. 4B is a cross-sectional view taken along line AA ′ of FIG. This is a configuration in which the first protrusion 2 shown in FIG. 1 is not provided and only the second protrusion 3 is provided. The dimensions of the second protrusion 3 are a width of 10 μm, a length of 60 μm, and a height of 20 μm. The resin plate was sterilized. Next, a silicon oxide (SiO ₂ ) film having a thickness of 0.3 μm was formed using a vapor deposition apparatus manufactured by ULVAC, Inc. (model: UEP) in order to prevent air bubbles from entering the micro container 1. The optical physical properties were a total light transmittance of 86% and a haze value of 8.8%.

[Example 3]
Using an acrylic resin (Parapet GH-S) manufactured by Kuraray Co., Ltd., on a resin plate with a width of 24 mm, a length of 74 mm, and a thickness of 1.0 mm, the length and width shown in FIG. A plate having a plurality of microcontainers 1 having a thickness of 100 μm and a depth of 50 μm was produced. FIG. 3A is also a plan view schematically showing the configuration of the cell culture container according to Example 3, and FIG. 3B is a cross-sectional view taken along line AA ′ of FIG. The resin plate was sterilized. Next, a silicon oxide (SiO ₂ ) film having a thickness of 0.3 μm was formed using a vapor deposition apparatus manufactured by ULVAC, Inc. (model: UEP) in order to prevent air bubbles from entering the micro container 1. The optical physical properties were a total light transmittance of 88% and a haze value of 7.3%.

[Example 4]
Using an acrylic resin (Parapet GH-S) manufactured by Kuraray Co., Ltd., on a resin plate with a width of 24 mm, a length of 74 mm, and a thickness of 1.0 mm, the length and width shown in FIG. A plate having a plurality of microcontainers 1 having a thickness of 500 μm and a depth of 50 μm was produced. FIG. 3A is also a plan view schematically showing the configuration of the cell culture container according to Example 4, and FIG. 3B is a cross-sectional view taken along line AA ′ of FIG. The resin plate was sterilized. Next, a silicon oxide (SiO ₂ ) film having a thickness of 0.3 μm was formed using a vapor deposition apparatus manufactured by ULVAC, Inc. (model: UEP) in order to prevent air bubbles from entering the micro container 1. The optical physical properties were a total light transmittance of 90% and a haze value of 5.1%.

Table 1 summarizes the culture results for the above comparative examples and examples when the density of the dispersed cells is 0.4 cells / 1000 μm ² .

In the case of a comparative example using a cell culture vessel in which a flat plate or a plurality of micro vessels are not in the range of 5 to 1000 μm in width and 5 to 1000 μm in depth, the culture density is 3 cells / 1000 μm ² or less even after 96 hours of culture. Also, the grade value of ALP activity is low. FIG. 5 shows a photograph of cells stained with ALP after culturing for 48 hours in Comparative Example 1. The grade value for this ALP activity is zero.

  In the examples, by setting the width of the plurality of microcontainers to 5 to 1000 μm and the depth to 5 to 1000 μm, the culture density and ALP activity grade values are higher than those of the comparative examples even after 48 hours of culture. After 96 hours of culture, the culture density and ALP activity grade values are even higher. Moreover, in the Examples, high transparency, culture density and ALP activity are achieved, and it is particularly suitable for cell observation using transmitted light. FIG. 6 (a) shows a photograph of cell nuclei stained after incubation for 48 hours in Example 2, and FIG. 6 (b) shows a photograph of cell nuclei stained after 96 hours of culture in Example 2. FIG. 7 shows a photograph of cells that were stained with ALP after 96 hours in Example 2. The grade value for this ALP activity is 4+.

Next, Table 2 summarizes the culture results for the comparative examples and examples when the density of the spread cells is 1.1 cells / 1000 μm ² .

In the case of the comparative example, even if the number of spread cells increases, the culture density and the grade value of ALP activity hardly change. Even after 96 hours of culture, the culture density is 3 cells / 1000 μm ² or less, and the grade value of ALP activity is low.

  In the examples, as the number of spread cells increased, the culture density markedly improved, and after 48 hours of culture, the ALP activity grade value was 4+ in all examples. That is, application of the present invention can be expected to improve accuracy and efficiency in various tests and studies using cell culture.

It is a figure which shows typically the structure of the cell culture container concerning embodiment of this invention. It is a figure which shows typically the manufacturing method of the cell culture container concerning embodiment of this invention. It is a top view which shows typically the cell culture container concerning the comparative example 3, Example 1, Example 3, and Example 4 of this invention. It is a top view which shows typically the cell culture container concerning Example 2 of this invention. In Comparative Example 1, it is a photograph of cells stained with ALP after culturing for 48 hours. In Example 2, it is the photograph of the cell nucleus dye | stained after culture | cultivating for 48 hours and 96 hours. In Example 2, it is the photograph of the cell which carried out the ALP dyeing | staining after 96-hour culture | cultivation.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Micro container 2 1st convex part 3 2nd convex part 4 Two steps convex part 5 Side wall 6 of 1st convex part Side wall 11 of 2nd convex part Substrate 12 1st resist layer 13 Mask A
14 Second resist layer 15 Mask B
16 Resist pattern 17 Conductive film 18 Metal structure 19 Resin molded product

Claims (11)

A cell culture vessel comprising a plurality of microcontainers having a width or diameter of 5 μm to 1000 μm and a depth of 5 μm to 1000 μm on a surface on which cells are cultured, and capable of achieving a culture density of 3 cells / 1000 μm ^{2 to 20} cells / 1000 μm ² . A cell culture vessel used for cell culture having a density of cells spread for culturing of 8 cells / 1000 μm ² or less, wherein a plurality of cells having a width or diameter of 5 μm to 1000 μm and a depth of 5 μm to 1000 μm A cell culture container equipped with a micro container.   The cell culture container according to claim 1 or 2, wherein the cells are dental pulp cells.   It has transparency, The cell culture container of any one of Claims 1-3 characterized by the above-mentioned.   The cell culture container according to any one of claims 1 to 4, further comprising a hydrophilic or hydrophobic organic film or inorganic film on the whole or a part of the outermost surface of the cell culture container. A step of spraying cells having a density of 8 cells / 1000 μm ² or less onto a cell culture vessel provided with a plurality of micro vessels having a width or diameter of 5 μm to 1000 μm and a depth of 5 μm to 1000 μm on the surface on which the cells are cultured; Culturing until the cell reaches ³ cells / 1000 μm ² to ²⁰ cells / 1000 μm ² .   The cell culture method according to claim 6, wherein the cells are dental pulp cells. Forming a pattern on the substrate;
Attaching a metal according to a pattern formed on the substrate or a transfer pattern thereof to form a metal structure;
And a step of forming a cell culture container by transferring the pattern of the metal structure.   In the step of forming a pattern on the substrate, the step of forming the resist pattern includes repeating the formation and exposure of the resist layer a plurality of times until the resist layer is formed into a structure having a desired height or depth. The method for producing a cell culture container according to claim 8, comprising:   The method for manufacturing a cell culture container according to claim 9, further comprising a step of aligning a mask position in order to align a pattern position in the step of repeating the formation and exposure of the resist layer a plurality of times.   The method for producing a cell culture container according to claim 9 or 10, wherein a mask having a different pattern is used in the step of repeating the formation and exposure of the resist layer a plurality of times.