JP5760410B2 - Cell test container and cell test method using the same - Google Patents

Description

  The present invention relates to a cell test container and a cell test method performed 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 test container 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 the cells in this cell test container, attaching the cells to the cell adhesive region, applying a voltage to the conductive region to change the cell adhesion inhibitory region on the conductive region to cell adhesiveness, 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, in the above technique, the conductive region functioning as the electrode and the counter electrode is formed on the same surface of the substrate, or the counter electrode is immersed in the culture solution applied on the cell test container (the counter electrode). The voltage was applied while being held by hand so that the electrode was inserted. Therefore, in the former case, since the counter electrode is arranged in the plane, there is a possibility that the effective area where voltage can be applied is limited, and the patterning of each electrode, cell adhesion region and cell adhesion inhibition region may be restricted. There is. In the latter case, it is difficult to keep the experimental conditions constant because the counter electrode is manually held during the experiment, and it may cause unevenness in voltage application. Therefore, there is still room for improvement in the configuration of the counter electrode.

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.

  An object of the present invention is to provide a cell test container capable of applying a voltage without causing unevenness in a cell adhesion-inhibiting region under a certain experimental condition, and a cell test method using the same. .

  The present inventors have a cell test container comprising a cell adhesion region and a cell adhesion inhibition region adjacent to the cell adhesion region, and changing the cell adhesion inhibition region to cell adhesion by applying a voltage. The inventors have found that the above-mentioned problems can be solved by using a lid member and fixing the counter electrode to the lid member, thereby completing the present invention.

That is, the present invention includes the following inventions.
(1) A cell test container comprising a base material having at least one recess, and a lid member arranged to face the recess,
Having an electrode on the bottom of the recess,
A cell adhesion region and a cell adhesion inhibitory region adjacent to the cell adhesion region are disposed on the electrode,
A counter electrode is fixed to the lid member,
The cell test container, wherein the cell adhesion-inhibiting region changes to cell adhesion by applying a voltage between the electrode and the counter electrode.
(2) The cell according to (1), wherein the substrate has a plurality of recesses, the lid member is disposed to face the plurality of recesses, and a plurality of counter electrodes are fixed corresponding to each recess. Test container.

(3) The cell test container according to (1) or (2), wherein the counter electrode is a mesh electrode.
(4) The cell test container according to (1) or (2), wherein the counter electrode is a flat electrode.
(5) The cell test container according to (1) or (2), wherein the counter electrode protrudes from the lid member toward the concave portion of the base material.

(6) A method for testing cells for cell migration observation, cell differentiation control, cell proliferation control or cell culture,
(I) a step of seeding cells in the cell test container according to any one of (1) to (5) above and allowing the cells to adhere to the cell adhesion region;
(Ii) a step of disposing a lid member facing the recess;
(Iii) changing the cell adhesion-inhibiting region to cell adhesion by applying a voltage between the electrode on the bottom surface of the recess and the counter electrode;
Including said method.

  According to the cell test container of the present invention, since the counter electrode is fixed at a predetermined position when the lid member is opposed to the recess, the experiment can be performed under constant conditions. In addition, since the voltage applied in the cell adhesion-inhibiting region is not uneven, the cell test can be performed with high accuracy and good reproducibility.

It is a cross-sectional view which shows one Embodiment of the container for a cell test of this invention. It is a top view which shows one Embodiment of the container for a cell test of this invention. It is a figure which shows typically the cross section of the container for cell tests before applying a voltage. It is a figure which shows typically the state which applied the voltage and changed the cell-adhesion inhibition area | region into cell-adhesiveness. It is a figure which shows typically the state which the cell migrated to the area | region changed to cell adhesiveness. It is a side view which shows another embodiment of the container for a cell test of this invention. It is a top view which shows another embodiment of the container for a cell test of this invention. It is a perspective view which shows another embodiment of the container for a cell test of this invention.

Hereinafter, the present invention will be described in detail.
One embodiment of the cell test container of the present invention will be described based on FIGS. 1a to 1b and FIGS. 2a to 2c. As shown in the cross-sectional view of FIG. 1 a, the cell test container 1 includes a base material 2 having at least one recess 20 and a lid member 3 disposed to face the recess 20. An electrode 21 is provided on the bottom surface, and a cell adhesive region and a cell adhesion inhibitory region adjacent to the cell adhesive region are disposed on the electrode 21, the counter electrode 30 is fixed to the lid member 3, and the cell The adhesion-inhibiting region is characterized by changing to cell adhesion by applying a voltage between the electrode 21 and the counter electrode 30.

  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 applying a voltage, preferably by applying a positive voltage to the electrode on the bottom of 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 substrate 2 has at least one recess 20 (one recess in the example of FIG. 1a). In this embodiment, the member of the concave portion 20 in which the through hole 22 is formed is integrated with the base material 2 having the electrode 21 formed on the surface by means such as adhesion, whereby the through hole 22 on the bottom surface of the concave portion 20 is formed. The electrode 21 is exposed at the position. In addition, this invention is not limited to the example of FIG. 1a, For example, the base material 2 and the member of the recessed part 20 are shape | molded integrally instead of separate, and the base material in the state in which the recessed part is formed is shape | molded by shaping | molding. An electrode may be provided on the bottom surface of the recess.

  The dimensions, such as the shape, size, and depth, of the recess 20 are not particularly limited as long as cells can be seeded on the electrode 21 on the bottom of the recess. As the shape of the recess 20, a round bottom, a conical shape, or the like can be appropriately selected in addition to a flat bottom as shown in FIG. The depth of the recess 20 is such that the counter electrode 30 is placed in the culture solution when the lid member 3 is disposed facing the recess 20 and a culture solution containing cells is introduced into the recess 20 in order to seed cells. It is preferable that the depth be sufficiently immersed in the substrate.

  The substrate 2 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.

(Cover member)
And the container for cell tests of this invention is provided with the cover member 3 arrange | positioned facing the recessed part 20, as shown to FIG. The lid member 3 is normally configured to be detachable, but may be fixed to a substrate having a recess when the cells can be seeded, observed or the like while attached. . Further, in the attached state, the lid member 3 is preferably held at a certain relative position with respect to the concave portion 20 by fitting into the concave portion 20 as shown in FIG. You may provide fixing means, such as a clip for fixing the cover member 3 and the recessed part 20, or the base material 2 as needed.

  Depending on the case, the upper surface of the lid member 3 may be open or transparent. When the upper surface is transparent, cells can be observed with the lid member disposed, that is, with the counter electrode disposed, which is advantageous.

  Further, a counter electrode 30 with respect to the electrode 21 is fixed to the lid member 3, and for example, power can be supplied through a lead 33. In this embodiment, a wire-like counter electrode 30 is stretched inside the lid member 3 so as to form a rhombus when viewed from above (FIG. 1b). The counter electrode 30 is preferably stretched so as to be parallel to the surface of the electrode 21 as shown in FIG. 1a. However, in some cases, the counter electrode 30 is inclined such as a shape that descends from the periphery toward the center of the electrode 21. It can also be provided. Further, when the net-like counter electrode 30 is provided, the density of the net can be arbitrarily set such as not only a rhombus as shown in FIG. If the counter electrode 30 is a mesh electrode, it is advantageous that cells can be observed through the counter electrode in a state where the counter electrode is arranged. The material of the counter electrode 30 is the same as that of the electrode 21 described above, but gold, platinum, etc. are particularly preferably used.

(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 to cocultivate can be controlled.

  The cell test container 1 as shown in FIG. 1a can be manufactured, for example, as follows. That is, a step of integrating the recess 20 having the through-hole 22 into the base material 2 having the electrode 21, a step of forming a cell adhesion-inhibiting hydrophilic film containing an organic compound having a carbon-oxygen bond on the electrode 21, In addition, it can be produced by a method including a step of modifying the hydrophilic film into a pattern by oxidizing and / or decomposing it so that the cell adhesion region and the cell adhesion inhibitory region are adjacent to each other on the electrode 21. .

(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, glass petri dishes, plastic petri dishes, glass slides, 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 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) a step of disposing a lid member facing the recess;
(Iii) a step of changing the cell adhesion-inhibiting region to cell adhesion by applying a voltage between the electrode on the bottom surface of the recess and the counter electrode;
It is characterized by including.

  Hereinafter, as one of the cell test methods of the present invention, the case of observing cell migration will be described with reference to FIGS. 2a to 2c.

  First, as shown in FIG. 2 a, after cell A is seeded, cell culture is preferably performed, and the cell A is adhered to the cell adhesive region 23. Furthermore, it is preferable to wash away the cell A which has not adhered by washing the cell test container 1 so that the cell A adheres only to the cell adhesive region 23.

  Subsequently, as shown in FIG. 2b, the counter electrode 30 is fixed at a predetermined position by disposing a lid member so as to face the recess. Then, by applying a voltage between the electrode 21 and the counter electrode 30, the cell adhesion-inhibiting region 24 is changed to the cell adhesion region 25. Here, the voltage applied to the electrode 21 is preferably a positive voltage. By applying a positive voltage, the cell adhesion-inhibiting region 24 can be effectively changed to the cell adhesion region 25, and particularly when the electrode 21 is made of an ITO film, it can be prevented from being blackened, Observation of cell migration can be carried out satisfactorily. In FIG. 2b, the cell adhesion-inhibiting region 24 (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, it is presumed that degradation products and the like generated by oxidation treatment and / or degradation treatment of the hydrophilic film remain in the cell adhesive region 23 in FIG. 2a.

  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. 2c, since the cells A adhered to the cell adhesive region 23 migrate toward the cell adhesive region 25, 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.

  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, another embodiment of the cell test container of the present invention is shown in FIGS. 3a and 3b. In this embodiment, the counter electrode 31 is a flat electrode. A flat plate is preferable because a uniform voltage can be applied to the electrode 21. Further, like the net-like counter electrode of FIG. 1a, this flat electrode is preferably parallel to the electrode 21, but is not limited thereto.

  Furthermore, FIG. 4 shows another embodiment of the cell test container of the present invention. In the embodiment of FIG. 4, the base material 2 has a plurality of recesses 20 (multiwell). In FIG. 4, for the sake of convenience, the number of recesses (wells) is small, but in practice, a large number of recesses such as 96, 384, and 1152 can be provided.

  And in this embodiment, the cover member 3 to which many counter electrodes 32 were fixed is provided. Each counter electrode 32 of the lid member 3 corresponds to each of the opposing recesses 20. By disposing the lid member 3 on the base material 2, the counter electrode is disposed at a predetermined position in each recess 20, and the cell adhesion-inhibiting region of each recess is changed to cell adhesiveness at a time by applying a voltage. Therefore, efficient cell testing can be performed. Alternatively, instead of changing the cell adhesion-inhibiting regions in the plurality of recesses 20 to cell adhesiveness at a time, a voltage can be applied to any recesses at any timing.

  In the example of FIG. 4, the counter electrode 32 is formed in a pin or needle shape, but is not limited thereto. For example, it may be a shape that protrudes from the lid member toward the concave portion of the substrate, such as a wedge shape, a cone shape, or a dome shape. Of course, you may provide the counter electrode which becomes parallel to the electrode of a base material like the above-mentioned embodiment.

DESCRIPTION OF SYMBOLS 1 Cell test container 2 Base material 3 Lid member 20 Recess 21 Electrode 22 Through-hole 23 Cell adhesion region 24 Cell adhesion inhibition region 25 Cell adhesion region 30 Counter electrode 31 Counter electrode 32 Counter electrode 33 Lead A cell

Claims (4)

A cell test container comprising a base material having at least one recess, and a lid member arranged to face the recess,
Having an electrode on the bottom of the recess,
A cell adhesion region and a cell adhesion inhibitory region adjacent to the cell adhesion region are disposed on the electrode,
A wire-like counter electrode stretched in a net shape so as to be parallel to the electrode is fixed to the lid member,
The cell test container, wherein the cell adhesion-inhibiting region changes to cell adhesion by applying a voltage between the electrode and the counter electrode. The cell test container according to claim 1, wherein the wire-like counter electrode stretched in a net shape is a grid-like electrode. Cell migration observation, a method of testing the cells for the cells growth control or cell culture,
(I) seeding the cells in the cell test container according to claim 1 or 2, and attaching the cells to the cell adhesion region;
(Ii) a step of disposing a lid member facing the recess;
(Iii) changing the cell adhesion-inhibiting region to cell adhesion by applying a voltage between the electrode on the bottom surface of the recess and the counter electrode;
Including said method. A method for testing cells for cell differentiation control comprising:
(I) seeding the cells in the cell test container according to claim 1 or 2, and attaching the cells to the cell adhesion region;
(Ii) a step of disposing a lid member facing the recess;
(Iii) changing the cell adhesion-inhibiting region to cell adhesion by applying a voltage between the electrode on the bottom surface of the recess and the counter electrode;
Including said method.

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