JP6035844B2 - Cell migration test apparatus and cell migration test method - Google Patents

Description

  The present invention relates to a cell migration test apparatus and a cell migration test method.

  Cell migration is involved in various stages such as immune response, post-fertilization embryo morphogenesis, 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. In particular, a cell migration test in the presence of a drug is used for screening a drug. Whether the drug acts as a promoter for the cell by analyzing whether it has positive or negative chemotaxis to the drug applied by the cell, and its migration rate, etc. Or knowledge as to whether it acts as an inhibitor. Therefore, methods for measuring cell migration in vitro have been developed for many years.

  For example, commercially available cell migration test apparatuses include the classic Boyden chamber, cell culture insert, FluoroBlock (registered trademark) (BD Biosciences), and Cell Mobility HitKit (registered trademark) (Cellomics). However, these devices have a problem that the cell migration region cannot be controlled.

  A substrate for observing cells by migrating to a specific region is disclosed in (Non-Patent Document 1). This substrate seeds cells in a well, cultures confluently, then creates a wound pattern by scratching the cells with a pin, and observes the process of cells migrating from the surroundings toward the wound pattern ( Scratch assay). 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 due to physical contact between the pins and the cells, and cell contents leak out. Furthermore, there is a problem that the area of the wound is large and there is a large variation in the part where cells are buried earlier and the part where cells are buried later, which makes quantitative assay difficult and requires observation over time.

  In contrast, 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. An apparatus for cell migration test in which a cell adhesion region and a cell adhesion inhibition region are adjacent to each other has been developed (Patent Document 1). By seeding cells in this cell migration test device, attaching the cells to the cell adhesion region, and 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. Therefore, in the apparatus described in Patent Document 1, the cell migration test can be performed quantitatively by controlling the arrangement of the cells before the cells migrate, the migration direction of the cells, and the timing of the migration start. However, in Patent Document 1, the shape of the apparatus and the pattern shape of the cell adhesion region and the cell adhesion inhibitory region that can cope with both positive chemotaxis and negative chemotaxis with respect to a drug are disclosed. Is not described.

  Patent Document 2 discloses that a plurality of wells capable of storing a liquid sample in a stationary state communicate with each other through a channel, that a channel is provided in the channel, and the well is a glass substrate. A barrier that forms one or more grooves having a width and / or depth according to the diameter of the cell or its deformability, or a flat surface. A cell migration test device is described, wherein the flat surface is provided to form a depth corresponding to the diameter of the cell or its deformability with the glass substrate. . According to Patent Document 2, according to the present apparatus, cells placed in a well can gather in the vicinity of the flow path before the start of chemotaxis and can be arranged in the cell traveling direction. It is described that the movement based on it can be measured accurately. However, in this apparatus, since the surface of the glass substrate has uniform cell adhesiveness, the cells supplied through the well may be scattered by the liquid flow generated by the pipette. In addition, it is difficult to control the random movement of cells with this apparatus, and the reliability and reproducibility of the analysis results may be inferior. Moreover, since the migration direction of the cell is limited to one direction, this device cannot cope with both positive chemotaxis and negative chemotaxis.

  Moreover, as a commercially available apparatus which can perform a cell migration test under a concentration gradient, there are [mu] -Slide Chemotaxis (ibidi) and EZ-TAXIScan (GE Healthcare Japan). However, because the former has a small number of wells, it cannot perform a migration test efficiently with high throughput, and the latter can only observe a decrease in migration speed for negative chemotaxis. Not too much. In addition, since the existing cell migration test apparatus as described above cannot strictly control the cell arrangement and cell migration area before migration, cell tracking software is necessary for quantitative migration analysis. Is required.

JP 2011-101638 A JP 2003-180336 A

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

  An object of the present invention is to provide a device for cell migration test that can cope with positive and negative chemotaxis and is excellent in reproducibility, quantitative performance, and efficiency, and a cell migration test method using the device. To do.

  The inventors of the present invention have provided a drug supply unit capable of forming a concentration gradient in a specific direction on the surface of the base material in the cell migration test apparatus, and the cells on the surface of the base material provided with a linear conductive region. Reproducibility of positive and negative chemotaxis by forming adhesive regions and cell adhesion inhibitory regions in specific pattern shapes and allowing cells to migrate to cell adhesion inhibitory regions that have been changed to cell adhesiveness by applying voltage The present inventors have found that a cell migration test with high quantitativeness and efficiency can be performed.

That is, the present invention includes the following inventions.
(1) A device for cell migration test,
A substrate having a linear conductive region on its surface,
In the linear conductive region, a region including both ends thereof is a first cell adhesion-inhibiting region, and a region between the first cell adhesion-inhibiting regions is a first cell adhesive region, and the linear conductive region Is a second cell adhesion-inhibiting region,
The linear conductive area is in contact with the drug supply,
The drug supply unit can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region,
The device described above, wherein the first cell adhesion-inhibiting region can be changed to the second cell adhesion region by applying a voltage to the linear conductive region.

(2) The drug supply unit includes a first drug supply unit and a second drug supply unit,
The linear conductive region is in contact with the first drug supply unit and the second drug supply unit;
The apparatus according to (1) above, wherein the first drug supply unit and the second drug supply unit are arranged to face each other across the first cell adhesive region on the linear conductive region.
(3) The device according to (1) or (2), wherein the plurality of linear conductive regions are comb-shaped together with a base connected to each linear conductive region.

(4) A cell migration test method,
(A) a step of seeding cells on the base material of the device for cell migration test according to any one of (1) to (3), and attaching the cells to the first cell adhesion region;
(B) modifying the first cell adhesion-inhibiting region to the second cell adhesion region by applying a voltage to the linear conductive region;
(C) supplying a drug to the drug supply unit to form a concentration gradient of the drug on the surface of the base material; and (d) in the second cell adhesive region of the cells adhered to the first cell adhesive region. The above method comprising the step of observing migration.
(5) The method according to (4), further comprising a step of measuring the migration distance of the cells in the second cell adhesive region after a predetermined time has elapsed.

  According to the present invention, it is possible to perform a cell migration test with high reproducibility, quantitativeness, and efficiency for positive and negative chemotaxis.

FIG. 1 is a diagram showing a first embodiment of the present invention. FIG. 2 is a diagram showing a second embodiment of the present invention. FIG. 3 is a diagram showing an embodiment of the present invention. FIG. 4 is a diagram showing an embodiment of the present invention. FIG. 5 is a diagram showing an embodiment of the present invention.

  Hereinafter, an embodiment of the apparatus for cell migration test of the present invention (hereinafter referred to as “the apparatus of the present invention”) will be described.

1st embodiment is equipped with the base material c which has the linear electroconductive area | region b on the surface, as shown in FIG.
In the linear conductive region b, the region including both ends thereof is the first cell adhesion-inhibiting region 11, and the space between the first cell adhesion-inhibiting regions 11 is the first cell adhesion region 13. The periphery of the conductive region b is the second cell adhesion inhibiting region 12;
The linear conductive region b is in contact with the drug supply unit 14,
The drug supply unit 14 can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region b,
The 1st cell adhesion inhibition area | region 11 is a form which can be changed into a 2nd cell adhesion area | region by the voltage application to the linear conductive area | region b.

2nd embodiment is equipped with the base material c which has the linear electroconductive area | region b on the surface, as shown in FIG.
In the linear conductive region b, the region including both ends thereof is the first cell adhesion-inhibiting region 21, and the space between the first cell adhesion-inhibiting regions 21 is the first cell adhesion region 23. The periphery of the conductive region b is the second cell adhesion inhibiting region 22;
The linear conductive region b is in contact with the first drug supply unit 24 and the second drug supply unit 24 ′,
The first drug supply unit 24 and the second drug supply unit 24 ′ can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region b.
The first drug supply unit 24 and the second drug supply unit 24 ′ are arranged to face each other across the first cell adhesive region 23 on the linear conductive region b,
The first cell adhesion-inhibiting region 21 has a form that can be changed to the second cell adhesion region by applying a voltage to the linear conductive region b.

(Cell adhesion region and cell adhesion inhibitory region)
In the present invention, the first cell adhesive region and the second cell adhesive region may be simply referred to as a cell adhesive region.

  In the present invention, the 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 the surface of a substrate on which a cell adhesion region and a cell adhesion inhibition region are formed, cells adhere to the cell adhesion region, but cells do not adhere to the cell adhesion inhibition region.

  Since cell adhesion may vary depending on the cells to be adhered, cell adhesion means cell adhesion to certain types of cells. Accordingly, on the cell migration test apparatus, there may be a plurality of cell adhesion regions for a plurality of types of cells, that is, there may be two or more levels of cell adhesion regions having different cell adhesion properties.

(Conductive region and substrate)
The apparatus of the present invention includes a substrate for forming a conductive region on the surface thereof.
The substrate used in the apparatus of the present invention is not particularly limited as long as it is formed of a material capable of forming a conductive region. The base material preferably includes an insulating material. Specifically, glass, quartz glass, borosilicate glass, alumina, sapphire, ceramics, forsterite, photosensitive glass, ceramic, silicon, elastomer, plastic (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon) , Acrylic materials, fluororesins, polycarbonate resins, polyurethane resins, methylpentene resins, phenol resins, melamine resins, epoxy resins, vinyl chloride resins). The base material preferably contains a transparent material. The shape is not limited, and examples thereof include a flat shape such as a flat plate, a flat membrane, a film, and a porous membrane, and a shape in which irregularities are formed on the surface. 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.

  When forming a conductive region on a substrate, 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 shaping technique such as engraving a fine groove. Specifically, a conductive material such as a metal film or a metal oxide film is formed on a base material such as a glass base material, and this is patterned by using a known technique such as a photolithography technique, thereby providing a conductive material. The sex region can be formed.

  Film formation of the conductive material on the substrate can be performed by a known method. For example, microwave plasma CVD (Chemical vapor deposition) method, ECRCVD (Electrical cyclotron resonance) method, ICP (Inductive coupled plasma) method, direct current sputtering method, ICP (Inductive coupled plasma method) Examples thereof include an arc type vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method. The film formation may be performed by coating. Spin coating and various printing methods can also be used.

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

  Although it does not restrict | limit especially as a film | membrane of an electroconductive material, It is preferable that it is a transparent film | membrane, for example, an ITO film | membrane, an IZO film | membrane, the polyethylenedioxythiophene film | membrane of a conductive polymer, etc. are mentioned. Further, it is preferable that the film is transparent even after voltage application. In the present invention, it is preferable to form the conductive region by forming an ITO film by sputtering and then patterning. A transparent membrane is advantageous in observing cells.

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

  The base material with which the apparatus of this invention is provided has a linear electroconductive area | region on the surface. A first cell adhesion inhibiting region and a first cell adhesion inhibiting region are formed in the linear conductive region, and a second cell adhesion inhibiting region is formed around the linear conductive region. ing. In the apparatus of the present invention, since the first cell adhesion-inhibiting region is changed to the second cell adhesion region by applying a voltage to the linear conductive region, the linear conductive region portion on the substrate surface is a cell. It becomes a runway. Therefore, when the conductive region is linear, the direction in which the cells adhered to the first cell adhesion-inhibiting region migrate can be controlled, and the cell migration test can be performed quantitatively.

  A plurality of linear conductive regions are preferably present, and the number thereof is not particularly limited, but is usually 2 to 1536, preferably 4 to 384, and more preferably 12 to 96. By using such a number, it is possible to evaluate the number of specimens equivalent to 96, 384, and 1536 microplates generally used in high-throughput screening, and it is easy to correlate data. The linear conductive regions are preferably linear and substantially parallel to each other from the viewpoint of quantitatively observing cell migration, but are not limited thereto and may be curved. The plurality of linear conductive regions are preferably electrically connected to form one conductive region. Thereby, a voltage can be applied to a plurality of linear conductive regions at once and simultaneously, and simple and accurate quantitative observation becomes possible.

  Specifically, the patterning of the conductive region as described above can be performed by performing resist coating, exposure through a photomask, development, and etching on the formed metal film or metal oxide film.

(Cell adhesion inhibitory area)
The cell adhesion-inhibiting region is preferably formed by 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. Further, the cell adhesion-inhibiting region may be formed of a resist portion that is non-cell-adhesive and has no hydrophilic film formed on the surface. As a material for such a resist portion, for example, SU-8 (MicroChem) can be cited. The method for forming the resist portion is not particularly limited, but can be formed by, for example, coating film formation by spin coating or various printing techniques. The thickness of the resist portion is not particularly limited, but is preferably 1 to 100 μm.

  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.

  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 to form a substrate or conductive material. A hydrophilic thin film is obtained by physically or chemically bonding to the region.

  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. , Polysaccharides, and the like. 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), copolymers of methacryloyloxyethylphosphorylcholine and acrylic monomers, dextran, and heparin, but are not limited thereto.

  Specific water-soluble oligomer materials and water-soluble low molecular weight 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 thereof, 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 Glycols, 2- [methoxy (polyethyleneoxy) -propyltrimethoxysilane, and triethylene glycol-terminated-thiols, but are not limited to these.

  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 cell adhesion is unlikely to be affected by a region not covered with the hydrophilic thin film on the surface of the substrate. Moreover, if the average thickness is 500 μm or less, coating is relatively easy.

  As a method for forming a hydrophilic film on the surface of the substrate or the conductive region, a method of directly adsorbing the hydrophilic organic compound to the substrate or the conductive region, or a method of directly coating the hydrophilic organic compound on the substrate or the conductive region A method, a method in which a hydrophilic organic compound is coated on a substrate or a conductive region, and then a crosslinking treatment is performed; a method in which a hydrophilic thin film is formed in a multi-stage manner in order to improve adhesion to the substrate or the conductive region; In order to improve adhesion to the material or the conductive region, a base layer is formed on the base material or the conductive region, and then a hydrophilic organic compound is coated. A polymerization initiation point is formed on the surface of the base material or the conductive region. And then a method of polymerizing the 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 an underlayer on the base material or the conductive region in order to improve adhesion to the base material or the conductive region. And then coating with a hydrophilic organic compound. This is because using these methods makes it easy to improve the adhesion of the hydrophilic organic compound to the substrate or the conductive region. In this specification, the term “bonding layer” is used. The binding layer means a layer existing between the outermost hydrophilic thin film layer and the base material in the case of forming a hydrophilic organic compound thin film in a multistage manner, and is formed on the surface of the base material or the conductive region. When a base layer is provided and a hydrophilic thin film layer is formed on the base layer, the base layer 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 may be a linker. In these methods, before coating with a hydrophilic material, a tie layer is formed from a material having a linker on a substrate or a conductive region. 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 substrate to which the epoxy silane is added is typically 45 ° or more, desirably 47 ° or more. If it exists, the base material which has sufficient cell adhesion inhibitory property can be made by adding an ethylene glycol type material etc. in the presence of an acid catalyst next. In the present invention, the water contact angle refers to a water contact angle measured at 23 ° C.

(Formation of first cell adhesion region by oxidation treatment and / or degradation treatment of hydrophilic membrane)
In the present invention, the first cell adhesion region is 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 oxidation treatment and / or degradation treatment. The

  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 treatment” is decomposition accompanied by oxidation (that is, oxidative decomposition), “decomposition treatment” and “oxidation treatment” indicate the same treatment.

  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 first cell adhesive region is formed on the conductive region, 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 first cell adhesive region may be formed of a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density. In this embodiment, both the first cell adhesion region and the cell adhesion inhibition 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 first 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.

  As a method of controlling the density of the hydrophilic organic compound, a bonding layer is provided between the hydrophilic organic compound thin film and the surface of the substrate or the conductive region, and the bonding force of the bonding layer with the hydrophilic organic compound is adjusted. The method of doing is mentioned. 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 aspect of the invention, the density of the material having a linker in the tie layer of the first cell adhesion region is low. The water contact angle of the surface of the bonding layer before forming the hydrophilic organic compound thin film in the first cell adhesion region is determined by using a silane coupling agent having an epoxy group at the end as a material having a linker. By way of example, it is typically 10 ° to 43 °, desirably 15 ° to 40 °. As a method for forming such a bonding layer, after forming a coating (bonding layer) of a material having a linker on the surface of the substrate or the conductive region, the surface of the bonding layer is oxidized and / or decomposed. Is mentioned. 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 surface 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 the various conditions, the same conditions as in the method of forming the first cell adhesive region by oxidizing and / or decomposing the hydrophilic membrane can be applied. A first cell adhesive region can be formed by forming a hydrophilic organic compound thin film on the thus formed binding layer.

  In this aspect of the invention, 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 is obtained by forming a coating of a material having a linker on the surface of the substrate or the conductive region. 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 proportion 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 elemental 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.

(Manufacturing method of the device of the present invention)
The apparatus of the present invention includes, for example, a step of forming a linear conductive region on the substrate surface, a step of forming a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond on the substrate surface, and a line. It can be produced by a method including a step of modifying the hydrophilic membrane to cell adhesion by oxidizing and / or decomposing so that the first cell adhesion region is formed on the conductive region.

(Formation of second cell adhesive region by applying voltage to conductive region)
In the present invention, the second cell adhesive region is formed by applying a voltage to the conductive region to decompose the hydrophilic film formed in the cell adhesion inhibitory region and peeling it to make it cell adhesive. The Here, decomposition refers to a change in which two or more organic compounds are generated from one organic compound by breaking the bond of the organic compound. In the present invention, by applying a voltage to the conductive region, for example, a bonding portion (linker) existing in the bonding layer is cut, and at least a part of the organic compound constituting the hydrophilic film is decomposed or removed. Conceivable.

  According to the apparatus of the present invention, since the timing of starting the migration of the cells adhered to the first cell adhesive region can be controlled by applying a voltage to the conductive region, the cell migration test can be performed quantitatively. Can do. In addition, a cell migration test with higher reproducibility can be performed as compared with a conventional scratch assay.

  The voltage applied to the conductive region is preferably a positive voltage. By applying a positive voltage, the cell adhesion-inhibiting region can be effectively changed to the cell adhesion region, and in particular, when the conductive region is made of an ITO film, it can be prevented from blackening, and cell migration Can be observed satisfactorily.

  The voltage to be applied can be appropriately determined by those skilled in the art, but is usually 1 to 10 V, preferably 2 to 5 V, and the application time is usually 0.5 to 60 minutes, preferably 1 to 10 minutes. It is.

  The voltage to be applied varies depending on the type of solvent in contact with the electrode, the electrode material, and the electrode shape, but it is usually higher than the voltage at which the cell adhesion-inhibiting region can be changed to the cell adhesive region. It is better to apply a voltage that is low enough not to adversely affect the cells.

  The voltage may be applied between the conductive regions in the substrate plane (for example, between ITO and ITO), or a counter electrode such as Pt is provided in the substrate plane between the conductive region and the counter electrode. (For example, between ITO and Pt) may be applied. In order to precisely control the voltage, a reference electrode such as Ag / AgCl may be provided in the substrate plane. The counter electrode and the reference electrode may not be in the plane of the base material (the electrode may be immersed in the culture solution).

  The second cell adhesion region is a detail of the first cell adhesion region obtained by subjecting a cell adhesion-inhibiting hydrophilic membrane containing an organic compound having a carbon-oxygen bond to an oxidation treatment and / or a degradation treatment. Surface properties may vary.

(Pattern shape and drug supply unit)
The substrate provided in the apparatus of the present invention has a linear conductive region on the surface thereof, and the first cell adhesion inhibitory region is formed in a region including both ends of the linear conductive region. A first cell adhesion region is formed between the first cell adhesion inhibition regions, and a second cell adhesion inhibition region is formed around the linear conductive region. The linear conductive region is in contact with the drug supply unit, and the drug supply unit can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region. Are arranged to be. Here, that the linear conductive region contacts the drug supply unit means that the drug in the drug supply unit can diffuse into the cell migration path formed on the linear conductive region after voltage application. By providing the pattern shape and the drug supply unit as described above, a concentration gradient of the drug is formed on the substrate surface so that a part of the drug reaches the cells adhered to the first cell adhesive region, and the first The cells adhered to the cell adhesion-inhibiting region can sense the concentration gradient of the drug and migrate in the positive or negative direction on the cell migration path (second cell adhesion region). Specifically, positive chemotaxis of the drug supplied to the drug supply unit 31 of the cell A adhered to the first cell adhesive region 32 as shown in FIG. 3, that is, in the direction of increasing the drug concentration. 4 and negative chemotaxis of the cells A adhered to the first cell adhesive region 42 as shown in FIG. 4 with respect to the drug supplied to the drug supply unit 41, that is, in the direction of decreasing the drug concentration. It is possible to test both for migration. Further, as shown in FIG. 5, when the first medicine supply unit 51 and the second medicine supply unit 51 ′ exist, one of the first medicine supply unit 51 and the second medicine supply unit 51 ′. A high concentration drug is introduced into the substrate, and a low concentration drug or water is supplied to the other to form a concentration gradient on the surface of the substrate, so that a test for positive and negative chemotaxis can be performed. By forming a concentration gradient in the entire cell migration path, it becomes possible to prevent the migration of cells in the reverse direction, which cannot be expected, so that improvement in reproducibility and quantification can be expected. In addition, by introducing different drugs into the first drug supply unit 51 and the second drug supply unit 51 ′, it is possible to determine the strength of the drug as an accelerator or inhibitor.

  When a cell migration test is performed using the apparatus of the present invention, a sample containing cells may be introduced from the drug supply unit.

(cell)
The cells to be seeded in the apparatus of the present invention may be floating cells such as blood cells and lymphoid cells, or may be adherent 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, and ovarian cancer. Cancer cells such as cells, liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermis angle Epidermal cells such as keratinocytes, tracheal epithelial 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, Examples include pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. 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 has ended. Any of cells may be sufficient. Moreover, the cell may culture | cultivate single seed | species and may cocultivate two or more types of cells.

  The culture sample containing the target cells is preliminarily subjected to a dispersion process in which the biological tissue is finely dispersed in a liquid, a separation process in which impurities other than the target cells in the biological tissue and other cell debris are removed. It is preferable to go.

  Prior to seeding of the cells in the apparatus of the present invention, it is preferable to preliminarily culture a culture sample containing the target cells by various culture methods to increase the target cells. For the preliminary culture, a normal culture method such as monolayer culture, coat dish culture, or on-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 culture sample and a culture solution in a culture container and maintaining the environment at a certain level, only specific living cells are adhered to the surface of a support such as a culture container. Multiply. The apparatus to be used, the treatment conditions, etc. are performed according to the usual 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, glass dishes, plastic dishes, slide glasses, covers 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 preliminary culture, the culture solution in the culture vessel is removed, so that unnecessary components such as clumps and fibrous impurities that do not adhere to the support surface in the culture sample are removed, and only living cells that adhere to the support surface are removed. Can be recovered. Means such as EDTA-trypsin treatment can be applied to recovering living cells adhered to the support surface.

The cells pre-cultured as described above are seeded on the apparatus of the present invention in the culture medium. 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 so that the cells adhere in a single layer in an amount sufficient to prevent the cells from growing on the cell migration test apparatus. Usually, it is preferable to seed so that cells are contained on the order of 10 ⁴ to 10 ⁶ per ml of culture medium.

  It is preferable to adhere the cells to the first cell adhesion region by culturing the cells seeded in the apparatus of the present invention in a culture solution. Any 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 cell 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 (such as fetal bovine serum), various growth factors, antibiotics, amino acids and the like may be added to the basal medium. A commercially available serum-free medium such as Gibco serum-free medium (Invitrogen) can also be used.

Although the time which culture | cultivates a cell is influenced by the presence or absence of the cell operation at the time of culture | cultivation, it is 6 to 96 hours normally, Preferably it is 12 to 72 hours. The temperature for culturing is usually 37 ° C. It is preferable to culture in a CO ₂ concentration atmosphere of about 5% using a CO ₂ cell culture apparatus or the like. After culturing, by washing the apparatus of the present invention, non-adherent cells are washed away, and the cells can be adhered only to the first cell adhesive region.

(Drug)
Using the apparatus for cell migration test of the present invention, cell migration to various drugs can be analyzed. The drug to be tested in the present invention is not particularly limited, and examples thereof include various peptides, proteins (including enzymes or antibodies), nucleic acids (polynucleotide (DNA or RNA), oligonucleotides (naturally or artificially synthesized). siRNA, shRNA, decoy oligo, etc.) or peptide nucleic acid (PNA)), organic compounds, inorganic compounds, low molecular compounds, and other high molecular compounds. Specific examples include fetal bovine serum (FBS), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), Latrunculin B, cytochalasin and the like.

  Examples of combinations of drugs and cells include mouse fibroblasts (SWISS-3T3) and fibroblast growth factor (FGF), bovine vascular endothelial cells and FBS, bovine vascular endothelial cells and Latrunculin B, canine kidney-derived epithelial cells (MDCK) and epidermal growth factor, HeLa cells and FBS, and the like.

(Cell migration test)
The cell migration test method of the present invention comprises:
(A) a step of seeding cells on a substrate of the apparatus of the present invention, and attaching the cells to the first cell adhesive region;
(B) modifying the first cell adhesion-inhibiting region to the second cell adhesion region by applying a voltage to the linear conductive region;
(C) supplying a drug to the drug supply unit to form a concentration gradient of the drug on the surface of the base material; and (d) in the second cell adhesive region of the cells adhered to the first cell adhesive region. Including the step of observing migration.

  In the step (a), as described above, the cells may be cultured after seeding the cells on the substrate. In that case, in particular, after the step (a), a separation treatment for removing impurities other than the target cell such as cells and cell debris can be performed. For example, it is preferable to wash the surface of the base material after applying a voltage to wash away cells other than the target cells and non-adhered cells, and leave only the target cells and the adhered cells on the base material. By such a separation treatment, a substance that can affect cell migration can be removed, and thus a cell migration analysis test can be performed more quantitatively.

  Any of the steps (b) and (c) may be performed first. That is, even if a voltage is applied after supplying the drug, the cell adhesion inhibitory region on the linear conductive region is changed to the cell adhesive region, and cell migration to the formed cell migration path is started. Alternatively, the drug may be supplied after a cell migration path is formed by applying a voltage. Preferably, drug supply is performed after voltage application. By supplying the drug after applying the voltage, it is possible to prevent the reproducibility of the concentration gradient due to the drug molecule adsorbing to the substrate surface when the drug is a charged molecule. In addition, it is possible to prevent the suspended cell adhesion inhibitor from reacting with the drug and degrading the performance of the drug. Therefore, it is desirable to add the drug after removing the medium in which the cell adhesion inhibitory substance is suspended after voltage application and exchanging the medium. On the other hand, when voltage application is performed after drug supply, it is advantageous in that the timing of the start of migration can be accurately controlled.

Regarding the step (d), observation of cell movement includes measurement of the speed of cell movement, and observation of the migration direction, the cell shape during migration, the connection between cells, and the like.
Hereinafter, embodiments of the present invention will be described with reference to the drawings.

First Embodiment FIG. 1 shows a first embodiment of the apparatus of the present invention.

The apparatus 10 according to the first embodiment includes a base material c having a linear conductive region b on its surface,
In the linear conductive region b, the region including both ends thereof is the first cell adhesion-inhibiting region 11, and the space between the first cell adhesion-inhibiting regions 11 is the first cell adhesion region 13. The periphery of the conductive region b is the second cell adhesion inhibiting region 12;
The linear conductive region b is in contact with the drug supply unit 14,
The drug supply unit 14 can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region b,
The first cell adhesion-inhibiting region 11 can be changed to the second cell adhesion region by applying a voltage to the linear conductive region b.

  Here, the first cell adhesion-inhibiting region 11 corresponds to the region where the hydrophilic film a on the linear conductive region b is formed, and the first cell adhesive region 13 has the conductive region b on the surface. The second cell adhesion inhibiting region 12 corresponds to a region where the hydrophilic film a on the substrate c is formed. The second cell adhesion region corresponds to the first cell adhesion inhibition region 11 before voltage application.

  In the present embodiment, the conductive region is preferably patterned in a comb shape including a plurality of linear conductive regions b (comb teeth) and a base portion b ′ that supports each linear conductive region b (see FIG. 1a). In that case, the width of the comb teeth formed by the conductive region (the width w on the side orthogonal to the direction in which the comb teeth extend from the base (the length direction of the comb teeth)) is preferably 0.1 to 500 μm. In order to observe cell migration, it is preferably 5 to 500 μm, more preferably 10 to 200 μm. If the width of the comb teeth portion is too narrow, the electric resistance in the length direction of the comb teeth portion will increase, and when a constant voltage is applied, a voltage drop tends to occur in the length direction of the comb teeth portion, and the potential control is possible. It becomes difficult. Since the degree of the voltage drop varies depending on the conductivity of the conductive material to be used, the lower limit of the width of the comb tooth portion is appropriately determined according to the conductivity of the conductive material, but is generally preferably 5 μm or more. When the width of the conductive region on the substrate is narrowed to 5 μm or less, a wide electrode is provided on the substrate, a part of the surface of the electrode is covered with an insulating film, and the desired width is 5 μm or less. By exposing only the region having a width of 2 and making the region a conductive region, the problem of a high resistance value and a voltage drop can be solved. In addition, when the width of the comb tooth portion is narrower than the size of the cell to be measured, it becomes difficult for the cells to adhere to the comb tooth portion. Accordingly, the width of the comb tooth portion when observing cell migration is appropriately determined depending on the size of the cell to be measured, but is generally preferably 10 μm or more. If the width of the comb-tooth portion is wide, the number of comb-tooth portions entering the field of view when observing with a microscope is reduced, which is disadvantageous for statistical processing. Generally, since the field of view of a microscope is several mm or less, in order for at least one comb tooth part to enter the field of view, the width of the comb tooth part is preferably 500 μm or less. In addition, when the width of the comb-tooth portion is moderately narrow, the migration direction of the cell to be measured is limited, so that the migration distance after a lapse of a certain time is increased, which has an advantageous effect on measurement. The interval between the comb teeth is preferably 10 to 1000 μm, more preferably 50 to 500 μm. If the spacing between the comb teeth is too narrow, the cells will overcome the cell adhesion inhibitory region on the insulating region between the comb teeth and the cells will easily migrate between adjacent comb teeth, so the cell migration direction It becomes difficult to limit. Therefore, it is generally preferable that the distance between the comb teeth is 10 μm or more, more preferably 50 μm or more. If the interval between the comb teeth is too wide, the number of comb teeth entering the field of view when observing with a microscope is reduced, which is disadvantageous for statistical processing. In general, since the field of view of a microscope is several mm or less, the width of the comb teeth is preferably 1000 μm or less. Two comb-shaped patterns in which the comb teeth and the comb teeth are engaged with each other are particularly preferred (FIG. 6a). Such a pattern is preferable because a current flows effectively when a voltage is applied in the cell migration test.

  Although the apparatus 10 of this embodiment may introduce the sample containing a cell from the chemical | medical agent supply part 14, it is preferable to provide the cell introduction part 15 for introduce | transducing the sample containing a cell. The cells introduced from the drug supply unit 14 or the cell introduction unit 15 do not adhere to the cell adhesion-inhibiting regions 11 and 12, but are guided to and adhere to the first cell adhesion region 13 after a certain period. This is advantageous in that the cells can be finally arranged in a predetermined pattern even if the cells are scattered in the cell adhesion-inhibiting regions 11 and 12 at the time of cell introduction. Therefore, the arrangement of the cell introduction part 15 with respect to the pattern shape of the cell adhesive region and the cell adhesion inhibitory region on the surface of the substrate is not particularly limited, but the cells are efficiently bonded to the first cell adhesive region 13 in a short time. In order to achieve this, it is preferable to provide the cell introduction part 15 on the upper part of the first cell adhesive region 13 (see FIG. 1b).

  In the present embodiment, the first cell adhesive region 13 is formed at the center of the linear conductive region b, and the drug supply unit 14 is disposed at a position near the end of the linear conductive region. Preferably it is. Thereby, in drug screening, when it is unknown whether the drug is an accelerator or an inhibitor, a cell migration test can be performed equally for positive and negative chemotaxis of cells.

  The device 10 of this embodiment may include a lid member d, and the drug supply unit 14 and the cell introduction unit 15 may be formed in the lid member d. In that case, the space in which cells formed between the lid member d and the substrate surface migrate (hereinafter referred to as the cell migration space 16), the drug supply unit 14, and the cell introduction unit 15 are integrated into a flow path. Forming.

  The material of the lid member d is not particularly limited as long as it is made of a material that can form a concentration gradient in the cell migration space 16, but is preferably transparent from the viewpoint of easy observation. As such a material, for example, resins such as glass, polystyrene (PS), polyethylene terephthalate (PET), cycloolefin polymer (COP), polydimethylsiloxane (PDMS), and PMMA are preferable. Those subjected to are particularly preferred.

  After a concentration gradient is formed in the cell migration space 16 by supplying a drug from the drug supply unit 14, the supply port of the drug supply unit 14 and the introduction port of the cell introduction unit 15 are used to maintain a constant concentration gradient for a long time. Preferably has a structure capable of sealing the flow path.

The cell migration space 16 is formed on the linear conductive region b which is a cell migration path, and may be integrally formed on the plurality of linear conductive regions b (FIG. 1c). A plurality of conductive regions b may be formed (FIG. 1d). When a plurality of cell migration spaces 16 are formed, each cell migration space 16 may form a flow path integrally with the drug supply unit 14 having one supply port, or the drug supply unit When 14 is provided with a plurality of supply ports (not shown), a plurality of flow paths may be formed integrally with each supply port of the medicine supply unit. In the former case, since the same concentration gradient can be formed in a plurality of cell migration spaces 16 by supplying a single drug, the migration test can be analyzed easily and more quantitatively. In the latter case, different drugs can be tested with a single device, particularly when the linear conductive region b is electrically connected as shown in FIG. So that the rate of cell migration can be compared in real time for different drugs.
Examples of the shape of the cell migration space 16 include a rectangular parallelepiped and a square.

  In the present embodiment, the dimensions of the base material c and the lid member d are not particularly limited. For example, the dimensions of the base material c can be 20 to 30 mm in the X direction in FIG. 1e and 25 to 75 mm in the Y direction. . The dimensions of the lid member d can be 18 to 28 mm in the X direction, 23 to 73 mm in the Y direction, and 10 to 30 mm in the Z direction. When the cell migration space 16 is integrally formed on a plurality of linear conductive regions b as shown in FIG. 1c, the X direction is 16 to 26 mm, the Y direction is 21 to 71 mm, and the Z direction is 0. 0.05 to 2 mm, and when formed in a plurality so as to include each linear conductive region b as shown in FIG. 1d, the X direction is 16 to 26 mm, and the Y direction is 1.2 μm to 0.2 mm ( The width in the Z direction may be 0.05 to 2 mm.

  In the present embodiment, the concentration gradient formed on the substrate surface is preferably a linear gradient along the linear conductive region b from the viewpoint of more quantitative analysis.

  The direction of the concentration gradient on the substrate surface is not particularly limited as long as it is provided on the substrate surface so that the concentration is different at both ends of the linear conductive region b, but from the viewpoint of simpler and quantitative analysis, It is preferable that there is no concentration gradient in a direction (Y direction in FIG. 1d) that is formed in parallel to the linear conductive region b (X direction in FIG. 1d) as a runway and that is perpendicular to the linear conductive region b (Y direction in FIG. 1d). .

  FIG. 2 shows a second embodiment of the device according to the invention. Various conditions such as the shape of the apparatus in this embodiment are the same as those described in the above embodiment unless otherwise specified in this section.

The device 20 according to the second embodiment includes a base material c having a linear conductive region b on its surface,
In the linear conductive region b, the region including both ends thereof is the first cell adhesion-inhibiting region 21, and the space between the first cell adhesion-inhibiting regions 21 is the first cell adhesion region 23. The periphery of the conductive region b is the second cell adhesion inhibiting region 22;
The linear conductive region b is in contact with the first drug supply unit 24 and the second drug supply unit 24 ′,
The first drug supply unit 24 and the second drug supply unit 24 ′ can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region b.
The first drug supply unit 24 and the second drug supply unit 24 ′ are arranged to face each other across the first first cell adhesive region 23 on the linear conductive region b,
The first cell adhesion-inhibiting region 21 can be changed to the second cell adhesion region by applying a voltage to the linear conductive region b.

According to this embodiment, a high concentration drug is introduced into one of the first drug supply unit 24 and the second drug supply unit 24 ′, and a low concentration drug or water is supplied to the other to supply the substrate surface. A concentration gradient can be formed and tested for positive and negative chemotaxis. Furthermore, according to the present embodiment, different drugs are supplied to the first drug supply unit 24 and the second drug supply unit 24 ′, and respective concentration gradients are formed on the surface of the base material, so that positive and negative chemotaxis is achieved. Can be tested. For example, when both drugs are promoters, if the drug supplied from the first drug supply unit 24 has a stronger action as a promoter, the cells are placed on the cell migration path by the first drug supply unit 24. When the drug supplied to the second drug supply unit 24 ′ has a stronger action as a promoter, the cells are arranged on the cell migration path with the second drug supply unit 24 ′. Run in the direction. In this way, it is possible to comparatively analyze the strength of the action of the drug as an accelerator or inhibitor under the condition in which the two drugs exist simultaneously.
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to the range of an Example.

[ITO film formation]
A glass substrate (manufactured by Corning) was prepared, and ITO (indium tin oxide) was formed on the glass substrate to a thickness of 1500 mm by sputtering. By photolithography, patterning was performed so that 90 runways having a width of 10 μm and a length of 5 mm were arranged at a pitch of 30 μm.

[PEG coating]
A pre-hydrolyzed epoxy silane (manufactured by GE Toshiba Silicone) is coated on a patterned ITO film-forming glass substrate by spin coating, heated at 80 ° C. for 15 minutes, and washed with pure water to obtain a silane compound. A film was formed. At this time, the contact angle of the ITO film-forming glass substrate was 40 to 55 °. PEG400 (Sigma-Aldrich) as a polyethylene glycol (PEG) with a catalytic amount of sulfuric acid added on the substrate is coated by dip coating, heated at 100 ° C for 1 hour, and washed with pure water. did. The contact angle of this substrate was about 27 to 35 °.

[Photolithography conditions]
A photomask in which a pattern having a line-shaped opening having a width of 1000 μm is formed on a substrate coated with PEG is arranged so that the line-shaped opening is perpendicular to the runway, and is subjected to proximity exposure. VUV (wavelength: 172 nm) was irradiated to decompose PEG corresponding to the line-shaped opening.

[Dishing condition]
The base material produced as described above and a 35 mmφ polystyrene dish having a 18 mmφ hole in the bottom were joined with an adhesive, and left for more than one day to release outgas and form a dish. Five dishes were produced as described above.

[Culture conditions]
Mouse fibroblasts were seeded at 2.0 × 10 ⁵ cells / cm ^{2 in} each of the prepared dishes and cultured in a DMEM 10% FBS solution for 24 hours.

[Voltage application conditions]
After confirming that the cells adhered to the line pattern, connect the conductive region to the positive electrode, connect the conductive region on the opposite side to the negative electrode, apply a voltage at 2.0 V for 1 minute, and remove the PEG. Released.

[Drug addition conditions]
After applying voltage, remove the medium of each dish, adjust the concentration of FBS as 0%, 0.1%, 1%, 10%, 20% in the medium as a drug, and replace each dish with a medium of each concentration. did.

[Running observation]
The cells were photographed once every 5 minutes with a CCD camera, and the migration direction of the cells on the migration path was observed and the travel distance was quantified. Images were taken 24 hours after voltage application.

a: hydrophilic membrane b: linear conductive region b ′ conductive base c: base material d: lid member e: counter electrode 10, 20: device for cell migration test 11, 21: first cell adhesion inhibitory region 12, 22: Second cell adhesion-inhibiting region 13, 23: First cell adhesive region 14: Drug supply unit 15, 25: Cell introduction unit 16, 26: Cell migration space 24: First drug supply unit 24 ′: second drug supply unit 31, 41: drug supply unit 32, 42, 52: cell adhesive region 51: first drug supply unit 51 ′: second drug supply unit A: cell

Claims (4)

An apparatus for cell migration test,
A substrate having a linear conductive region on its surface,
In the linear conductive region, two regions each including both ends thereof are first cell adhesion-inhibiting regions, and are sandwiched adjacent to the first cell adhesion-inhibiting region on the linear conductive region. The region is a first cell adhesion region, and the periphery of the linear conductive region is a second cell adhesion inhibitory region,
The linear conductive area is in contact with the drug supply,
The drug supply unit can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region,
The concentration gradient on the substrate surface is formed in parallel to the linear conductive region and is not in the direction perpendicular to the linear conductive region,
The first cell adhesion-inhibiting region can be changed to the second cell adhesion region by applying a voltage to the linear conductive region,
The drug supply unit includes a first drug supply unit and a second drug supply unit,
The linear conductive region is in contact with the first drug supply unit and the second drug supply unit;
The apparatus, wherein the first drug supply unit and the second drug supply unit are arranged to face each other across the first cell adhesive region on the linear conductive region. An apparatus for cell migration test,
A substrate having a linear conductive region on its surface,
In the linear conductive region, two regions each including both ends thereof are first cell adhesion-inhibiting regions, and are sandwiched adjacent to the first cell adhesion-inhibiting region on the linear conductive region. The region is a first cell adhesion region, and the periphery of the linear conductive region is a second cell adhesion inhibitory region,
The linear conductive area is in contact with the drug supply,
The drug supply unit can provide a concentration gradient of the drug on the substrate surface so that the concentration is different at both ends of the linear conductive region,
The first cell adhesion-inhibiting region can be changed to the second cell adhesion region by applying a voltage to the linear conductive region,
The drug supply unit includes a first drug supply unit and a second drug supply unit,
The linear conductive region is in contact with the first drug supply unit and the second drug supply unit;
The first drug supply unit and the second drug supply unit are arranged to face each other across the first cell adhesive region on the linear conductive region,
A plurality of linear conductive region is a comb with a base to connect to each linear conductive region, the device. A cell migration test method comprising:
(A) a step of seeding cells on a substrate of the apparatus for cell migration test according to claim 1 or 2 and allowing the cells to adhere to the first cell adhesion region;
(B) modifying the first cell adhesion-inhibiting region to the second cell adhesion region by applying a voltage to the linear conductive region;
(C) supplying a drug to the drug supply unit to form a concentration gradient of the drug on the surface of the base material; and (d) in the second cell adhesive region of the cells adhered to the first cell adhesive region. The above method comprising the step of observing migration.   The method according to claim 3, further comprising a step of measuring a migration distance of the cell in the second cell adhesive region after a predetermined time has elapsed.

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