JP2012120453A - Container for cell test and method for testing cell using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multiwell container for a cell test provided with a cell-adhesive region and a cell adhesion inhibiting region in each well, and capable of effectively testing such as an observation of cell migration by using change of the cell adhesion inhibiting region to be cell-adhesive by applying a voltage to the cell adhesion inhibiting region.SOLUTION: The container for a cell test includes a substrate having a plurality of divided recessed parts and an electrode arranged in the plurality of recessed parts. On the electrode, the cell-adhesive region and the cell adhesion inhibiting region which is adjacent to the cell adhesive region and changes to be cell-adhesive by applying a voltage to the electrode are arranged.

Description

  The present invention relates to a cell test container and a cell test method using the container.

  Currently, various animal and plant cell cultures are being performed, and new cell culture methods have been developed. Cell culture techniques are used for the purpose of elucidating the biochemical phenomena and properties of cells and producing useful substances. In addition, attempts have been made to examine the physiological activity and toxicity of artificially synthesized drugs using cultured cells.

  Some cells (particularly many animal cells) have an adhesion dependency that grows by adhering to something, and cannot survive for a long time in a floating state in vitro. In order to culture such cells having adhesion dependency, a carrier for cell adhesion is required, and generally, a plastic culture in which cell adhesion proteins such as collagen and fibronectin are uniformly applied. A dish is used. These cell adhesion proteins are known to act on cultured cells, facilitate cell adhesion, and affect cell morphology.

  On the other hand, cell migration is involved in various stages such as immune response, embryo morphogenesis after fertilization, tissue repair and regeneration. It also plays an extremely important role in the progression of diseases such as cancer, atherosclerosis and arthritis. Specifically, cell migration through the vascular endothelium is an important phenomenon in the pathophysiology of conditions such as inflammation, atherosclerosis, and cancer metastasis. Therefore, methods for measuring cell migration in vitro have been developed for many years.

  Devices already on the market for cell migration assays include classic Boyden chambers, cell culture inserts, FluoroBlock® (BD Biosciences), Cell Mobility HitKit® (Cellomics). However, with such a device, it is difficult to quantitatively assay cell migration by controlling the migration direction of attached cells.

  (Non-Patent Document 1) describes a technique for controlling cell adhesion and non-adhesion by applying a voltage on a conductive substrate whose entire surface is not patterned. However, with this substrate, the properties of the entire surface of the substrate change due to the application of voltage, so after attaching cells on the substrate, only a specific region is changed to cell adhesion, and only in that region. Cells cannot be controlled to migrate.

  (Non-Patent Document 2) discloses that a cell adhesion-inhibiting film is formed by forming a cell adhesion-inhibiting film on a substrate having a conductive region and an insulating region, and applying a voltage to the specific conductive region. Describes a method of culturing cells by modifying them to cell adhesiveness and attaching cells only to the region. However, the cell adhesive region and the cell adhesion inhibitory region are not adjacent to each other on the conductive region of the substrate of (Non-Patent Document 2). Therefore, even if a cell is adhered to an area modified to cell adhesion and then applied to another conductive area by applying voltage to another cell, the already adhered cells are not adjacent to each other. It cannot be assayed by migrating to the adherent area.

  On the other hand, the present inventors include a base material having a conductive region and an insulating region, and a cell adhesive region and a cell adhesion inhibitory region formed on the conductive region, and the conductive region In the above, a cell culture substrate in which a cell adhesion region and a cell adhesion inhibitory region are adjacent to each other has been developed, and a patent application has already been filed (Japanese Patent Application No. 2010-232954). By seeding cells on this cell culture substrate, adhering cells to the cell adhesion region, applying a voltage to the conductive region to change the cell adhesion inhibitory region on the conductive region to cell adhesion, The migration of the cells adhering to the cell adhesion region to the cell adhesion inhibition region changed to cell adhesion can be observed. However, since this cell culture substrate is not a so-called multi-well type substrate that has a plurality of sites for seeding and observing cells, it is necessary to use the multi-well substrate for efficient tests such as cell migration. Technology development was desired.

  A substrate for observing cell migration in such a multiwell is disclosed in (Non-Patent Document 3). This substrate has a plurality of wells (for example, 96 wells), and after seeding cells in each well and culturing confluently, a wound pattern is created by scratching the cells with a pin, and surroundings toward the wound pattern The cell migration process is observed (scratch assay). When scratching with a pin, a sword-shaped part in which a large number of pins are erected on a plate-like member is used, and cells in a plurality of wells (for example, 96 wells) on the substrate are scratched together to form a wound. Since the scratch assay using such a substrate forms a wound pattern with pins, there is a physical limitation on the size (width) of the wound, and it can be reduced to the order of millimeters at most. In addition, there is a problem that the edge of the scratch is dirty and the variation of the scratch shape between the wells is large. In addition, when wounded, there is a drawback that cells float or cell contents leak due to physical contact between pins and cells. Furthermore, depending on the total number of wells, there are problems that the scratching operation with the sword-shaped member needs to be performed a plurality of times, and that it is necessary for the experimenter to learn to form beautiful scratches.

  Another multi-well substrate for observing cell migration is disclosed in (Non-Patent Document 4). This substrate is fitted with a silicone resin insert member so as to be in contact with a part of the bottom surface of each well, seeded with cells in that state, and when the cells are confluent, the insert member is removed, and the removed portion It forms a wound pattern. Compared to the technique of the above (Non-Patent Document 3), the wound pattern corresponds to the shape of the insert member, so the uniformity of the pattern between the wells is improved. There is a problem that a beautiful edge is still not obtained by entering the side. Similarly to the above (Non-Patent Document 3), the size of the flaw depends on the size of the insert member, so it is at most on the order of millimeters. When the insert member is removed, the cells float or crush due to physical contact. Cell contents may leak out. In addition, the presence of the insert member obstructs the operation and observation of the cells, and it is necessary to repeat the attachment of the insert member for each well, so that the experiment becomes complicated.

Jiang X, Ferrigno R, Mrksich M, Whitesides GM. 2003. "Electrochemical desorption of self-assembled monolayers noninvasively releases patterned cells from geometrical confinements." J. Am. Chem. Soc. 125: 2366-7. Sunny S. Shah, Ji Youn Lee, Stanislav Verkhoturov, Nazgul Tuleuova, Emile A. Schweikert, Erlan Ramanculov, and Alexander Revzin. 2008. "Exercising Spatiotemporal Control of Cell Attachment with Optically Transparent Microelectrodes." Langmuir 24: 6837-6844. Justic C Yarrow, et al., A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods, "BMC Biotechnology 2004, 4: 21. CELL BIOLABS, INC., Product Manual: CytoSelectTM 24-Well Wound Healing Assay, 2008-2010.

  In view of this, the present invention provides a cell adhesion region and a cell adhesion inhibitory region in each well, and observes cell migration by utilizing the fact that the cell adhesion is changed by applying a voltage to the cell adhesion inhibitory region. An object of the present invention is to provide a multi-well cell test container capable of efficiently performing such tests, and a cell test method using the same.

The gist of the present invention for solving the above problems is as follows.
(1) a base material having a plurality of partitioned recesses;
An electrode disposed in the plurality of recesses,
A cell test container in which a cell adhesion region and a cell adhesion inhibitory region that changes to cell adhesion by applying a voltage to the electrode adjacent to the cell adhesion region are disposed on the electrode.
(2) The plurality of recesses include a first recess and a second recess, and the first electrode disposed in the first recess and the second electrode disposed in the second recess are electrically independent, The cell test container according to (1), wherein voltage application to the first electrode and the second electrode can be controlled for each electrode.
(3) The plurality of concave portions include a first concave portion and a second concave portion, and the sizes and / or shapes of the cell adhesion-inhibiting regions of the first concave portion and the second concave portion are different from each other as described in (1) or (2) Cell test container.
(4) A plurality of comb-like electrodes combined with each other are disposed in the plurality of recesses, and a voltage is applied between the plurality of comb-like electrodes, thereby inhibiting cell adhesion along the comb-like electrodes. The cell test container according to (1), wherein the region changes to cell adhesiveness.
(5) A method for testing cells for cell migration observation, cell differentiation control, cell proliferation control or cell culture,
(I) a step of seeding a cell in the cell test container according to any one of (1) to (4) above and allowing the cell to adhere to a cell adhesive region;
(Ii) applying a voltage to the cell adhesion-inhibiting region to change the cell adhesion-inhibiting region to cell adhesion;
Including said method.

  With the cell test container of the present invention, cell migration in a multiwell can be observed. For example, it is possible to conduct an efficient drug discovery screening assay by administering a drug to each well and observing cell behavior. In addition, compared to conventional techniques such as a scratch assay, a wound pattern (cell adhesion region) is formed by applying a voltage, so there is no physical contact with the cells, and problems such as leakage of cell contents do not occur. .

It is a figure which illustrates typically a 1st embodiment of a container for cell examination of the present invention. It is a perspective view which shows 1st Embodiment of the container for a cell test of this invention. It is a figure for demonstrating the process of observing cell migration using the container for a cell test of this invention. It is a figure for demonstrating the process of observing cell migration using the container for a cell test of this invention. It is a figure for demonstrating the process of observing cell migration using the container for a cell test of this invention. It is a figure which shows 2nd Embodiment of the container for cell tests of this invention. It is a figure which shows the state after changing the cell adhesion inhibition area | region into cell adhesiveness in FIG. 4a. It is a figure which shows 3rd Embodiment of the container for cell tests of this invention. It is a figure which shows 3rd Embodiment of the container for cell tests of this invention. It is a figure which shows 4th Embodiment of the container for cell tests of this invention.

Hereinafter, the present invention will be described in detail.
First, a first embodiment of the cell test container of the present invention will be described with reference to FIGS. 1 and 2. As shown in FIG. 1, the cell test container 1 includes a base material 20 having a plurality of partitioned recesses 10 and electrodes 21 arranged in the plurality of recesses 10. An adhesive region 22 and a cell adhesion inhibitory region 23 that is adjacent to the cell adhesive region 22 and changes to cell adhesiveness by applying a voltage to the electrode 21 are arranged.

  In the present invention, cell adhesion means that cells adhere or cells adhere easily. Cell adhesion inhibitory means that cells are difficult to adhere or cells do not adhere. Therefore, when cells are seeded on an electrode in which a cell adhesion region and a cell adhesion inhibition region are arranged in a pattern, cells adhere to the cell adhesion region, but cells adhere to the cell adhesion inhibition region. Since it does not adhere, cells are arranged in a pattern on the electrode.

  Since the degree of cell adhesion may vary depending on the cells to be adhered, the cell adhesion means that the cell adhesion is to a certain kind of cells. Therefore, the cell test container may have a plurality of cell adhesion regions for a plurality of types of cells, that is, there may be two or more cell adhesion regions having different cell adhesion properties.

  Examples of the structure of the cell adhesion region and the cell adhesion inhibition region in the cell test container of the present invention include the following two forms.

  The first form is a form in which the cell adhesion region is a cell adhesion region obtained by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment. is there. In this embodiment, a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond is formed on the electrode, and then an oxidation treatment and / or a decomposition treatment are performed on a region where cell adhesion is desired. Thus, cell adhesion is imparted to the region to modify the region. The part not subjected to the treatment is a cell adhesion-inhibiting region.

  In the second form, the cell adhesive region is formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density. This form utilizes the fact that a hydrophilic film containing an organic compound having a carbon-oxygen bond at a high density has cell adhesion inhibitory properties, whereas a hydrophilic film containing the compound at a low density has a cell adhesion property. It is a thing. When a first region where the compound is likely to bind to the electrode and a second region where the compound is difficult to bind are provided on the electrode and a film of the compound is formed on the electrode, the first region becomes a cell adhesion inhibitory region, It becomes a cell adhesion region.

  Furthermore, in the cell test container of the present invention, the cell adhesion-inhibiting region on the electrode changes to cell adhesion by application of a voltage, preferably application of a positive voltage to the electrode disposed in the recess. The region changed to cell adhesion by application of voltage is obtained by subjecting the cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to oxidation treatment and / or degradation treatment as described above. The detailed surface texture may differ from the region.

(Base material)
The base material 20 has a plurality of partitioned concave portions 10 (wells). In FIG. 1, for the sake of convenience, a state in which a plurality of recesses 10 are arranged in a line is depicted, but in practice, a large number of recesses 10 arranged two-dimensionally can be provided as shown in FIG. The number of recesses 10 is not particularly limited. For example, the number of recesses is the same as that of various general multiwell plates such as 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, 1536 wells, and the like. 10 can be provided. The cell test container 1 shown in FIG. 2 includes, for example, a side wall member in which a through hole is provided in a base material 20 on which an electrode, a cell adhesion region, and a cell adhesion inhibition region (not shown) are arranged. By integrating 11 by means such as adhesion, a plurality of recesses 10 having the base material 20 as the bottom surface can be formed. In FIG. 1, in order to easily understand the structure of the container, the side wall member 11 between the plurality of recesses 10 is omitted and only the recesses 10 are shown. As shown in FIG. 1, a counter electrode 30 for the electrode 21 is provided in each recess 10. For example, when the cell culture solution is injected into the recess 10, the counter electrode 30 is held at a position where it can be applied to the electrode 21 by being immersed in the culture solution. FIG. 2 shows a state before the counter electrode 30 is arranged in the recess 10.

  The shape, size, depth, and other dimensions of the recess 10 are not particularly limited as long as cells can be seeded on the electrode 21 disposed in the recess 10. As the shape of the recess 10, a round bottom, a conical shape, or the like can be appropriately selected in addition to the flat bottom as shown in FIG. 1. Alternatively, the plurality of recesses may be connected to each other. In addition, the depth of the recess 10 is preferably set to such a depth that the counter electrode 30 can be sufficiently immersed in the culture solution when a culture solution containing cells is introduced into the recess 10 in order to seed cells. .

  In addition to combining the base material 20 and the side wall member 11 by bonding or the like as shown in FIG. 2, when the base material 20 is molded, it is integrally molded so that a plurality of concave portions are defined, and An electrode or the like may be provided on the bottom surface.

  The base material 20 used for the cell test container is not particularly limited as long as it is made of a material capable of forming the electrode 21. It is preferable to form the electrode 21 on a base material made of an insulating material. Specifically, glass, quartz glass, borosilicate glass, alumina, sapphire, forsterite, photosensitive glass, ceramic, silicon, elastomer, plastic (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic) Resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). The shape is not limited, for example, a flat shape such as a flat plate, a flat membrane, a film, a porous membrane, a three-dimensional shape such as a cylinder, a stamp, a multiwell plate, a microchannel, and irregularities are formed on the surface. Shape. When using a film, the thickness in particular is not restrict | limited, However, Usually, it is 0.1-1000 micrometers, Preferably it is 1-500 micrometers, More preferably, it is 10-200 micrometers.

  In particular, when a substrate having fine irregularities of about 1 nm to 10 μm smaller than the cell size is added to the surface and the cell adhesive region on the electrode has the same shape, It is possible to control the behavior and perform the test effectively. For example, in the case of a line pattern, the fine irregularities indicate irregularities having a depth of 1 nm to 10 μm, a line convex portion width of 1 nm to 10 μm, and a line concave portion width of 1 nm to 10 μm.

  When an electrode is formed on a base material made of an insulating material, a known patterning technique can be used. Known patterning techniques include, for example, gravure printing methods, screen printing methods, offset printing methods, flexographic printing methods, contact printing methods and other printing methods, various lithography methods, ink jet methods, and the like. For example, a three-dimensional molding method for engraving a fine groove can be used. Specifically, an electrode material such as a metal film or a metal oxide film is formed on a base material made of an insulating material, such as a glass base material, and is patterned using a known technique such as a photolithography technique. Thus, an electrode having a desired pattern can be formed.

  The electrode material can be deposited on the substrate by a known method. For example, microwave plasma CVD (Chemical vapor deposit) method, ECRCVD (Electric cyclotron resonance chemical vapor deposit) method, ICP (Inductive coupled plasma) method, DC sputtering method, ECR (Electric cyclotron resonance) method, sputtering method, ionization deposition method, Examples thereof include arc type vapor deposition, laser vapor deposition, EB (Electron beam) vapor deposition, and resistance heating vapor deposition. The film formation may be performed by coating. Spin coating and various printing methods can also be used.

  Examples of the material constituting the electrode include a metal or metal oxide, a film in which metal fine particles and conductive nanofibers are dispersed in an insulator, and a conductive organic material. Examples of the metal include silver, gold, copper, and platinum. Examples of the metal oxide include ITO (indium tin oxide) and IZO (indium zinc oxide). Examples of the metal fine particles include silver, gold, and copper. Fine particles such as platinum, conductive nanofibers include carbon nanotubes, and conductive organic materials include polyethylene dioxythiophene (PEDOT).

  The electrode is not particularly limited, but is preferably a transparent electrode, and examples thereof include an electrode made of ITO, IZO, polyethylenedioxythiophene which is a conductive polymer, or the like. Moreover, it is preferable that it is transparent even after voltage application. In the present invention, it is preferable to form an electrode by forming ITO on a substrate by sputtering and then patterning. Transparent electrodes are advantageous in observing cells.

  The thickness of the electrode is usually about a monomolecular film to about 100 μm, preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.

  In the case of patterning the electrodes as described above, specifically, the formed metal or metal oxide can be applied by resist application, exposure through a photomask, development and etching.

(Cell adhesion inhibitory area)
The cell adhesion-inhibiting region is preferably composed of a hydrophilic film formed of an organic compound having a carbon-oxygen bond. The hydrophilic film is a thin film mainly composed of an organic compound having a carbon-oxygen bond, which has water solubility and water swellability, and has a cell adhesion inhibitory property before being oxidized and is oxidized and / or decomposed. There is no particular limitation as long as it has cell adhesion.

  In the present invention, the carbon-oxygen bond means a bond formed between carbon and oxygen, and is not limited to a single bond but may be a double bond. Examples of the carbon-oxygen bond include a C—O bond, a C (═O) —O bond, and a C═O bond.

  The main raw materials include water-soluble polymers, water-soluble oligomers, water-soluble organic compounds, surfactants, amphiphiles, etc., which are physically or chemically cross-linked with each other, It becomes a hydrophilic thin film by chemically bonding.

  Specific water-soluble polymer materials include polyalkylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polymethacrylic acid and derivatives thereof, polyacrylamide and derivatives thereof, polyvinyl alcohol and derivatives thereof, and zwitterionic polymers. And polysaccharides. Examples of the molecular shape include a straight chain, a branched one, and a dendrimer. More specifically, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, such as Pluronic F108, Pluronic F127, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2- Hydroxyethyl methacrylate), poly (methacryloyloxyethylphosphorylcholine), a copolymer of methacryloyloxyethylphosphorylcholine and an acrylic monomer, dextran, heparin, and the like, but are not limited thereto.

  Specific water-soluble oligomer materials and water-soluble low-molecular compounds include alkylene glycol oligomers and derivatives thereof, acrylic acid oligomers and derivatives thereof, methacrylic acid oligomers and derivatives thereof, acrylamide oligomers and derivatives thereof, saponified vinyl acetate oligomers and Derivatives, oligomers composed of zwitterionic monomers and derivatives thereof, acrylic acid and derivatives thereof, methacrylic acid and derivatives thereof, acrylamide and derivatives thereof, zwitterionic compounds, water-soluble silane coupling agents, water-soluble thiol compounds, etc. be able to. More specifically, ethylene glycol oligomer, (N-isopropylacrylamide) oligomer, methacryloyloxyethylphosphorylcholine oligomer, low molecular weight dextran, low molecular weight heparin, oligoethylene glycol thiol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene Examples include, but are not limited to, glycol, 2- [methoxy (polyethyleneoxy) propyl] trimethoxysilane, and triethylene glycol-terminated-thiol.

  It is desirable that the hydrophilic film has a high cell adhesion inhibitory property before treatment and exhibits cell adhesion after oxidation treatment and / or degradation treatment.

  The average thickness of the hydrophilic film is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. An average thickness of 0.8 nm or more is preferable because it is difficult to be affected by the region not covered with the hydrophilic thin film on the substrate or electrode surface in protein adsorption or cell adhesion. Moreover, if the average thickness is 500 μm or less, coating is relatively easy.

  As a method for forming a hydrophilic film on the electrode, a method of directly adsorbing a hydrophilic organic compound to the electrode, a method of directly coating a hydrophilic organic compound on the electrode, or a crosslinking treatment after coating the hydrophilic organic compound on the electrode Applying method, forming multi-step hydrophilic thin film to improve adhesion to electrode, forming base layer on electrode to improve adhesion to electrode, then coating with hydrophilic organic compound And a method of forming a polymerization initiation point on the surface of the electrode and then polymerizing a hydrophilic polymer brush.

  Among the above film forming methods, particularly preferable methods include a method of forming a hydrophilic thin film in a multistage manner, and a base layer is formed on the electrode in order to improve adhesion to the electrode, and then a hydrophilic organic compound The method of coating can be mentioned. This is because it is easy to improve the adhesion of the hydrophilic organic compound to the electrode by using these methods. In this specification, the term “bonding layer” is used. The bonding layer means a layer existing between the outermost hydrophilic thin film layer and the electrode when a thin film of hydrophilic organic compound is formed in a multistage manner, and a base layer is provided on the surface of the electrode. When a hydrophilic thin film layer is formed on an underlayer, the underlayer is meant. The binding layer is preferably a layer containing a material having a binding portion (linker). Examples of combinations of the linker and the functional group at the end of the material to be bonded to the linker include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. Is mentioned. In each combination, any of them may be a linker. In these methods, before coating with a hydrophilic material, a bonding layer is formed from a material having a linker on the electrode. The density of the material in the bonding layer is an important factor that defines the bonding force. The density can be easily evaluated using the contact angle of water on the surface of the bonding layer as an index. For example, taking a silane coupling agent having an epoxy group at the end (epoxy silane) as an example, the water contact angle of the surface of the electrode to which epoxy silane is added is typically 45 ° or more, preferably 47 ° or more. For example, a region having a sufficient cell adhesion inhibitory property can be formed by adding an ethylene glycol material or the like in the presence of an acid catalyst. In the present invention, the water contact angle refers to a water contact angle measured at 23 ° C.

(Formation of cell adhesion region by oxidation and / or degradation of hydrophilic membrane)
In the present invention, the cell adhesion region may be formed by a region that is made cell adhesive by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to an oxidation treatment and / or a decomposition treatment. preferable.

  In the present invention, “oxidation” has a narrow meaning, and means a reaction in which an organic compound reacts with oxygen and the content of oxygen is higher than before the reaction.

  In the present invention, “decomposition” refers to a change in which a bond of an organic compound is broken to produce two or more organic compounds from one organic compound. “Decomposition treatment” typically includes, but is not limited to, decomposition by oxidation treatment, decomposition by ultraviolet irradiation, and the like. When the “decomposition process” is a decomposition accompanied by oxidation (that is, oxidative decomposition), the “decomposition process” and the “oxidation process” indicate the same process.

  Decomposition by ultraviolet irradiation means that an organic compound absorbs ultraviolet rays and decomposes through an excited state. In addition, when an ultraviolet ray is irradiated into a system in which an organic compound is present together with a molecular species containing oxygen (oxygen, water, etc.), the molecular species are activated in addition to the absorption of the ultraviolet ray by the compound and the decomposition. May react with organic compounds. The latter reaction can be classified as “oxidation”. A reaction in which an organic compound is decomposed by oxidation by an activated molecular species can be classified as “decomposition by oxidation” rather than “decomposition by ultraviolet irradiation”.

  As described above, “oxidation treatment” and “decomposition treatment” may overlap as operations, and the two cannot be clearly distinguished. Therefore, in this specification, the term “oxidation treatment and / or decomposition treatment” is used.

  Examples of the oxidation treatment and / or decomposition treatment include a method of subjecting the hydrophilic film to ultraviolet irradiation treatment, a method of photocatalytic treatment, and a method of treatment with an oxidizing agent. In the present invention, since the cell adhesive region and the cell adhesion inhibitory region are respectively formed on the electrode, the hydrophilic film is partially oxidized and / or decomposed in a pattern. In the case of partial oxidation treatment and / or decomposition treatment, a mask such as a photomask or a stencil mask or a stamp may be used. Further, the oxidation treatment and / or the decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser.

  In the case of the ultraviolet irradiation treatment, it is preferable to use a lamp that emits ultraviolet rays in the UV-C region from the VUV region, such as a mercury lamp that emits ultraviolet rays with a wavelength of 185 nm or 254 nm or an excimer lamp that emits ultraviolet rays with a wavelength of 172 nm. When the photocatalytic treatment is performed, it is preferable to use a light source that emits ultraviolet light having a wavelength of 365 nm or less, and it is more preferable to use a light source that emits ultraviolet light having a wavelength of 254 nm or less. As the photocatalyst, it is preferable to use a titanium oxide photocatalyst, a titanium oxide photocatalyst activated with a metal ion or a metal colloid. As the oxidizing agent, an organic acid or an inorganic acid can be used without any particular limitation. However, since a high concentration acid is difficult to handle, it is preferable to dilute it to a concentration of 10% or less. The optimum ultraviolet treatment time, photocatalyst treatment time, and oxidant treatment time can be appropriately determined according to various conditions such as the ultraviolet intensity of the light source used, the activity of the photocatalyst, the oxidizing power and concentration of the oxidant.

  The cell adhesion region may be formed by a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density. In this embodiment, both the cell adhesive region and the cell adhesion inhibitory region are formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond. The two regions differ in the density of the organic compound. The higher the density, the more difficult the cells adhere. In the cell adhesion region, the density of the organic compound is low enough to allow cells to adhere. On the other hand, in the cell adhesion-inhibiting region, the density of the organic compound is so high that cells cannot adhere.

  Examples of the method for controlling the density of the hydrophilic organic compound include a method in which a bonding layer is provided between the hydrophilic organic compound thin film and the electrode surface, and the bonding strength of the bonding layer with the hydrophilic organic compound is adjusted. . Here, the “binding layer” is as defined above, and may be composed of the preferred materials described above. The bonding force of the bonding layer increases as the density of the material having the linker in the bonding layer increases, and decreases as the density decreases. As described above, the density of the material having a linker in the bonding layer can be easily evaluated using the water contact angle on the surface of the bonding layer as an index.

  In this embodiment, the density of the material with the linker in the tie layer of the cell adhesion region is low. In the cell adhesion region, the water contact angle of the surface of the bonding layer before forming the hydrophilic organic compound thin film is taken as an example in the case of using a silane coupling agent having an epoxy group as a terminal as a material having a linker. And typically 10 ° to 43 °, desirably 15 ° to 40 °. Examples of a method for forming such a bonding layer include a method of forming a coating (bonding layer) of a material having a linker on the electrode surface and then oxidizing and / or decomposing the surface of the bonding layer. Examples of the method for oxidizing and / or decomposing the bonding layer surface include a method of treating the bonding layer surface with ultraviolet irradiation, a method of treating with a photocatalyst, and a method of treating with an oxidizing agent. The entire surface of the bonding layer may be oxidized and / or decomposed or partially processed. The partial processing can be performed by using a mask such as a photomask or a stencil mask, or by using a stamp. Further, the oxidation treatment and / or the decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser. As for various conditions, the same conditions as in the method of forming a cell adhesive region by oxidizing and / or decomposing a hydrophilic film can be applied. A cell adhesion region can be formed by forming a thin film of a hydrophilic organic compound on the binding layer thus formed.

  In this embodiment, the density of the material having a linker in the binding layer of the cell adhesion-inhibiting region is high. The water contact angle on the surface of the bonding layer before forming a thin film of hydrophilic organic compound in the cell adhesion inhibitory region is an example of using a silane coupling agent having an epoxy group at the end as a material having a linker. When it is taken, it is typically 45 ° or more, desirably 47 ° or more. Such a bonding layer can be obtained by forming a coating of a material having a linker on the electrode surface. When the bonding layer surface is partially oxidized and / or decomposed, the remaining portion not subjected to the treatment becomes the bonding layer having the water contact angle. A cell adhesion-inhibiting region can be formed by forming a hydrophilic organic compound thin film on the binding layer thus formed.

(Comparison between cell adhesion region and cell adhesion inhibitory region)
The amount of carbon in the cell adhesion region (including the bonding layer when a bonding layer is present) is lower than the amount of carbon in the cell adhesion inhibiting region (including the bonding layer when the bonding layer is present). It is preferable. Specifically, the amount of carbon in the cell adhesion region is preferably 20 to 99% with respect to the amount of carbon in the cell adhesion inhibition region. Corresponding to this range is particularly suitable when the thickness of the hydrophilic film (the total of the thickness of the bonding layer and the hydrophilic film when a bonding layer is present) is 10 μm or less. “Carbon content (atomic concentration,%)” is as defined below.

  In addition, the value of the ratio (%) of the carbon bonded to oxygen in the cell adhesive region (including the bonded layer when the bonded layer is present) is the cell adhesion inhibitory region (the bonded layer is It is preferable that the value be smaller than the value of the ratio (%) of carbon bonded to oxygen among the carbon in the carbon (including the bonded layer if present). Specifically, the value of the ratio (%) of carbon bonded to oxygen in the cell adhesion region is equal to the ratio of carbon bonded to oxygen among the carbon in the cell adhesion inhibitory region ( %) Is preferably 35 to 99%. Corresponding to this range is particularly suitable when the thickness of the hydrophilic film (the total of the thickness of the bonding layer and the hydrophilic film when a bonding layer is present) is 10 μm or less. “The ratio of carbon bonded to oxygen (atomic concentration,%)” is as defined below.

(Evaluation method of hydrophilic thin film)
Evaluation methods for the hydrophilic thin film of the present invention (including a bonding layer when a bonding layer is present) include contact angle measurement, ellipsometry, atomic force microscope observation, electron microscope observation, Auger electron spectroscopy measurement, X-ray measurement Photoelectron spectroscopy and various mass spectrometry methods can be used. Among these methods, X-ray photoelectron spectroscopy (XPS / ESCA) is most excellent in quantification. What is obtained by this measurement method is a relative quantitative value, and is generally calculated by an element concentration (atomic concentration,%). Hereinafter, the X-ray photoelectron spectroscopic analysis method in the present invention will be described in detail.

  In the present invention, the “carbon amount” of the hydrophilic thin film is defined as “the carbon amount obtained from the analytical value of the C1s peak obtained using an X-ray photoelectron spectrometer”. In the present invention, the “ratio of carbon bonded to oxygen” of the hydrophilic thin film is “the ratio of carbon bonded to oxygen determined from the analytical value of the C1s peak obtained using an X-ray photoelectron spectrometer. Is defined. Specific measurement can be performed as described in JP-A-2007-312736.

(Pattern shape)
In the cell test container of the present invention, it is preferable that the cell adhesion region and the cell adhesion inhibition region are arranged in a pattern. The shape of the pattern is not particularly limited as long as it is a two-dimensional pattern, and can be selected depending on the type of cell, the tissue to be formed, and the like. For example, a line shape, a tree shape (dendritic shape), a mesh shape, a lattice shape, a circular shape, a quadrangular pattern, a pattern in which the inside of a figure such as a circular shape and a quadrangular shape is a cell adhesive region or a cell adhesion inhibitory region Can be formed.

  The pattern of the cell adhesion region and the cell adhesion inhibition region is formed so that the cell adhesion region and the cell adhesion inhibition region are adjacent to each other on the electrode of the substrate. Since the cell adhesion region and the cell adhesion inhibitory region are adjacent to each other on the electrode, the cells are first adhered to the cell adhesion region, and then the cell adhesion is applied by applying a voltage (preferably positive voltage) to the electrode. When the inhibitory region changes to cell adhesion, cells can migrate to the region that has changed to cell adhesion. Therefore, a region that changes to cell adhesion can be determined in advance by patterning, and the region and direction in which cells migrate in a test such as cell migration observation can be controlled. Or in the case of the test etc. which cocultivate a cell, the magnitude | size and shape of the area | region of the other cell cocultured can be controlled.

  The cell test container 1 as shown in FIGS. 1 and 2 can be manufactured, for example, as follows. That is, a step of forming the electrode 21 on the substrate 20, a step of forming a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond on the electrode 21, a cell adhesive region and a cell on the electrode 21 The hydrophilic member is oxidized and / or decomposed into a pattern so as to be adjacent to the adhesion-inhibiting region, and the side wall member 11 having a through hole is integrated with the base material 20 and the base material 20. It can manufacture by the method including the process to do.

(cell)
The cells to be seeded in the cell test container may be floating cells such as blood cells and lymphoid cells, and may be adhesive cells, but the present invention is preferably used for cells having adhesiveness. It is also preferably used for cells having the property of migrating. Examples of such cells include liver cancer cells, glioma cells, colon cancer cells, kidney cancer cells, pancreatic cancer cells, prostate cancer cells, colon cancer cells, breast cancer cells, lung cancer cells, ovarian cancer cells and other cancer cells, liver Hepatocytes, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells such as epidermal keratinocytes, tracheal epithelium Cells, gastrointestinal epithelial cells, cervical epithelial cells, epithelial cells such as corneal epithelial cells, mammary cells, pericytes, muscle cells such as smooth muscle cells and cardiomyocytes, kidney cells, pancreatic Langerhans islet cells, peripheral neurons and Examples thereof include nerve cells such as optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells collected directly from tissues or organs, or may be obtained by substituting them for several generations. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation is completed. Any of the cells obtained may be used. The cells may be cultivated as a single species, or two or more cells may be co-cultured.

  Samples containing the target cells are preliminarily subjected to a dispersion process in which the biological tissue is finely dispersed in the liquid and a separation process to remove impurities other than the target cells in the biological tissue such as cells and cell debris. It is preferable to keep it.

  Prior to seeding of cells in a cell test container, it is preferable to pre-culture a sample containing the target cells in advance by various culture methods to increase the target cells. For the preculture, a normal culture method such as monolayer culture, coat dish culture, or gel culture can be employed. In the preculture, one of the methods of culturing with cells attached to the surface of the support is already known as a so-called monolayer culture method. Specifically, for example, by storing a sample containing a target cell and a culture solution in a culture container and maintaining the environment at a certain environmental condition, only specific living cells are supported on the surface of the support such as the culture container. It grows in a state where it adheres. The apparatus to be used, the treatment conditions, etc. are performed according to the normal monolayer culture method. As materials for the surface of the support on which cells adhere and grow, materials such as polylysine, polyethyleneimine, collagen, and gelatin that can adhere and proliferate well can be selected. A so-called cell adhesion factor, which is a chemical substance that favors cell adhesion and proliferation, is also applied to the surface of a support such as glass, plastic sheet, and plastic film.

  After pre-culture, removing the culture medium in the culture vessel removes unnecessary components such as clumps and fibrous impurities that do not adhere to the support surface in the sample, and removes only living cells that adhere to the support surface. Can be recovered. Means such as EDTA-trypsin treatment can be applied to recovering living cells adhered to the support surface.

Cells pre-cultured as described above are seeded on a cell test container. The seeding method and the seeding amount of the cell are not particularly limited, and for example, the method described in “A tissue culture technique (1999)” published by Asakura Shoten, pages 266 to 270 can be used. It is preferable to seed the cells in a sufficient amount so that the cells do not need to be grown on the cell test container so that the cells adhere in a single layer. Usually, it is preferable to seed so that cells are contained in the order of 10 ⁴ to 10 ⁶ per ml of culture medium, and seeding so that cells are contained in the order of 10 ⁴ to 10 ⁶ per 1 cm ^{2 of} electrode. Is preferred. Specifically, seeding is performed at about 2 × 10 ⁵ per 400 mm ² .

  The seeded cells are allowed to adhere to the cell adhesion region. Any cell culture medium can be used without particular limitation as long as it is a cell culture medium usually used in the art. For example, depending on the type of cells used, MEM medium, BME medium, DME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. A basal medium as described in "Tissue culture technology third edition" edited by the Japanese Society for Tissue Culture, page 581 can be used. Furthermore, serum (fetal bovine serum etc.), various growth factors, antibiotics, amino acids, etc. may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used.

(Cell test)
As described above, the cells are seeded on the cell test container of the present invention, preferably cultured, and after the cells are adhered to the cell adhesive region, a voltage is applied between the electrode and the counter electrode, Various tests such as observation of cell migration can be performed by changing the cell adhesion-inhibiting region on the electrode to cell adhesion. That is, the cell test method of the present invention comprises:
(I) seeding cells in a cell test container and attaching the cells to the cell adhesion region;
(Ii) applying a voltage to the cell adhesion-inhibiting region to change the cell adhesion-inhibiting region to cell adhesion;
It is characterized by including.

  Hereinafter, as one of the cell testing methods of the present invention, the case of observing cell migration will be described with reference to FIGS. 3a to 3c.

  First, as shown in FIG. 3 a, after seeding cells A in the respective recesses 10, cell culture is preferably performed, and the cells A are adhered to the cell adhesive regions 22. Furthermore, it is preferable to wash away the cell A that has not adhered by washing the cell test container 1 so that the cell A adheres only to the cell adhesive region 22.

  Subsequently, as shown in FIG. 3 b, the counter electrode 30 is installed at a predetermined position with respect to each recess 10. Then, by applying a voltage between the electrode 21 and the counter electrode 30, the cell adhesion-inhibiting region 23 is changed to the cell adhesion region 24. Here, the voltage applied to the electrode 21 is preferably a positive voltage. By applying a positive voltage, the cell adhesion-inhibiting region 23 can be effectively changed to the cell adhesion region 24, and in particular, when the electrode 21 is made of an ITO film, it is possible to prevent blackening. Observation of cell migration can be carried out satisfactorily. In FIG. 3b, the cell adhesion-inhibiting region 23 (for example, a hydrophilic film) is shown to have disappeared in order to clearly express a change caused by applying a voltage. It is presumed that decomposition products remain. In addition, in the cell adhesive region 22 in FIG. 3a, it is presumed that degradation products and the like generated by oxidation treatment and / or degradation treatment of the hydrophilic film remain.

  The applied voltage varies depending on the type of solvent in contact with the electrode, the material of the electrode, and the shape of the electrode, but it is usually higher than the voltage that can change the cell adhesion-inhibiting region to cell adhesion. Therefore, it is preferable to apply a low voltage that does not adversely affect the cells. Specifically, it is usually 1 to 10 V, preferably 2 to 5 V, and the application time is usually 1 second to 60 minutes, preferably 10 seconds to 10 minutes, but is not limited thereto.

  Then, as shown in FIG. 3c, since the cells A adhered to the cell adhesive region 22 migrate toward the cell adhesive region 24, this state can be observed with a microscope or the like. Observation of cell behavior includes, for example, measurement of the rate at which cells migrate, and observation of migration direction, cell morphology during migration, cell division frequency, and connections between surrounding cells. The speed at which the cells migrate can be measured by measuring the area and distance in which the cells fill the cell adhesion-inhibiting region that has been changed to cell adhesion, or by tracking individual cells. Since the present invention is a multi-well test container having a plurality of recesses, for example, a drug is administered to each recess and a voltage is applied to change the cell adhesion-inhibiting region together to cell adhesiveness, thereby creating a wound. A drug discovery screening assay or the like can be efficiently performed by forming a pattern and observing the migration behavior of cells toward the wound pattern.

  The cell test container of the present invention can be used for various cell tests such as cell differentiation control, cell growth control, cell culture, and co-culture of different cells in addition to the observation of cell migration. For example, regarding cell differentiation control and cell proliferation control, cells having differentiation ability are known to change their differentiation state due to the influence of mechanical stress received from the scaffold. Cells are seeded in a cell-adhesive region of size and / or differentiated. After the cells have differentiated, the cells are allowed to migrate and proliferate by changing the cell adhesion-inhibiting region adjacent to the cell adhesion region to cell adhesion. That is, since differentiated cells can migrate and proliferate, a large number of differentiated cells can be obtained by repeating such culture. In the case where different cells are co-cultured, for example, first, the first cells are seeded and cultured in the recesses where the cell adhesion region and the cell adhesion inhibitory region are adjacent to each other. After the cells are sufficiently adhered to the cell adhesion region, a voltage is applied to change the cell adhesion inhibitory region to cell adhesion. Then, immediately after (or immediately before) the voltage application, the second cells are seeded. As a result, before the first cell migrates / proliferates to the area changed to cell adhesion and covers this area, the second cell adheres first to the area changed to cell adhesion. Co-culture of different cells in the converted area is possible.

  Next, a second embodiment of the cell test container of the present invention will be described with reference to FIGS. 4a and 4b. 4a and 4b show a state in which a plurality of recesses in which an electrode, a cell adhesive region and a cell adhesion inhibitory region are arranged are viewed from above. In this embodiment, as shown in FIG. 4a, the plurality of recesses include the first recess 10A and the second recess B, and are disposed in the first electrode 21A disposed in the first recess 10A and the second recess 10B. The second electrode 21B is electrically independent from each other. Such a configuration can be produced, for example, by patterning the electrodes when forming the electrodes on the base material and forming a plurality of electrode regions (21A, 21B) that are not electrically connected to each other. The electrode pattern is not limited to the case of dividing corresponding to each row of recesses as shown in FIG. 4a (three rows in FIG. 4a), for example, the case of dividing corresponding to each of a plurality of rows of recesses, It can be set as appropriate, for example, in the case of dividing in correspondence with each group of island-shaped recesses instead of the shape.

  In the cell test container shown in FIG. 4a, voltage application to the first electrode 21A and the second electrode 21B can be controlled. Here, in the case of controlling the voltage application, the case where the magnitude of the voltage is changed for each electrode, the case where the timing for applying the voltage is changed for each electrode, and the like are included. By controlling the voltage application for each electrode, as shown in FIG. 4b, for example, by applying a voltage only to the first electrode 21A, only the cell adhesion-inhibiting region 23 on the first electrode 21A is made cell-adhesive. The area 24 can be changed. Accordingly, since the behavior of cells can be controlled for each recess in one cell test container, more efficient and diverse tests can be performed.

  Moreover, 3rd Embodiment of the container for cell tests of this invention is shown in FIG.5 and FIG.6. In this embodiment, it is comprised so that the magnitude | size and / or shape of each cell adhesion inhibition area | region 23 may differ about arbitrary 1st recessed parts 10A and 2nd recessed parts 10B among several recessed parts. FIG. 5 is an example in which the width of the cell adhesion-inhibiting region 23 is formed to be different for each column. In FIG. 6, the cell adhesion-inhibiting regions 23 are arranged in various shapes such as a vertical stripe shape, a horizontal stripe shape, a circle shape, or a square shape. Of course, the size and / or shape of the cell adhesion-inhibiting region 23 is not limited to that shown in FIGS. 5 and 6 and can be set as appropriate in consideration of, for example, the direction and distance of cell migration.

  Further, a fourth embodiment of the cell test container of the present invention will be described with reference to FIG. In this embodiment, a plurality of comb-like electrodes 21 </ b> C are arranged in the plurality of recesses 10. Similarly, comb-like counter electrodes 30C are combined with each other and arranged on the same plane. By applying a voltage between the comb-shaped electrode 21C and the comb-shaped counter electrode 30C, for example, when a positive voltage is applied to the comb-shaped electrode 21C side, cell adhesion inhibition on the comb-shaped electrode 21C is inhibited. The cell region A is changed to cell adhesiveness, so that the cells A adhered to the cell adhesive region 22 are aligned with the cell adhesion inhibitory region 23 (that is, the comb-like electrode 21C) changed to cell adhesiveness. And will run. In the present invention, since the pattern of the cell adhesion-inhibiting region and the cell adhesion-inhibiting region can be formed by a photolithography technique, the width of the cell adhesion-inhibiting region can be reduced to several μm. Therefore, it is possible to observe, for example, a process in which one cell migrates by forming the comb-like electrode 21C and the cell adhesion-inhibiting region 23 on the comb-like electrode 21C in an extremely small amount.

DESCRIPTION OF SYMBOLS 1 Cell test container 10 Recess 10A 1st recessed part 10B 2nd recessed part 11 Side wall member 20 Base material 21 Electrode 21A 1st electrode 21B 2nd electrode 21C Comb-like electrode 22 Cell adhesive region 23 Cell adhesion inhibitory region 24 Cell adhesion Region 30 counter electrode 30C counter electrode A cell

Claims (5)

A substrate having a plurality of partitioned recesses;
An electrode disposed in the plurality of recesses,
A cell test container in which a cell adhesion region and a cell adhesion inhibitory region that changes to cell adhesion by applying a voltage to the electrode adjacent to the cell adhesion region are disposed on the electrode.   The plurality of recesses include a first recess and a second recess, and the first electrode disposed in the first recess and the second electrode disposed in the second recess are electrically independent, The cell test container according to claim 1, wherein voltage application to the second electrode can be controlled for each electrode.   The cell test container according to claim 1 or 2, wherein the plurality of recesses include a first recess and a second recess, and the sizes and / or shapes of the cell adhesion-inhibiting regions of the first recess and the second recess are different.   A plurality of comb-like electrodes combined with each other are arranged in a plurality of recesses, and a voltage is applied between the plurality of comb-like electrodes, so that a cell adhesion-inhibiting region is a cell along the comb-like electrodes. The cell test container according to claim 1, which changes to adhesiveness. A method for testing cells for cell migration observation, cell differentiation control, cell proliferation control or cell culture comprising:
(I) seeding the cells in the cell test container according to any one of claims 1 to 4 and attaching the cells to the cell adhesion region;
(Ii) applying a voltage to the cell adhesion-inhibiting region to change the cell adhesion-inhibiting region to cell adhesion;
Including said method.

Images