JP2005027659A - Cell microchip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell microchip simply carrying out various measurements such as action, etc., of medicine in a short time by utilizing a biological reaction using a small amount of cells and to provide a measuring method using the same. <P>SOLUTION: The invention relates to the cell microchip comprising a micropassage 32 formed on a plastic substrate 31, a measuring method using a cell characterized by the use of the cell microchip, and a biological inspection method characterized by the use of the cell microchip. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a microchannel, particularly a cell microchip on which a microchannel having a branch path is formed, and a measurement method using the cell microchip.

  Conventionally, as a method of examining the action of drugs using cells, a method of determining the viability of cells by adding a drug-containing solution after planting and culturing a considerable number of cells in a dish-shaped incubator made of glass or plastic Was done. In the above method, the number of cells, the amount of medium and the amount of reagents that must be prepared in advance are considerable, and the method also occupies considerable space in the incubator for cell culture. In particular, when using rare cells and reagents, the measurement itself was difficult. Moreover, when observing or measuring all cells, it takes a considerable amount of time for measurement and determination.

On the other hand, many products have been developed in recent years for so-called DNA microchips and protein microchips in which a large number of nucleic acids such as DNA and proteins such as antibodies are aligned on a substrate to perform reaction and analysis in minute amounts (for example, patents) (See Literatures 1 and 2), which are commercially available, but a technique for analyzing cells themselves on a microchip is not known.
JP-T 6-504997 JP-T-2002-542487

  The present invention provides a cell microchip capable of easily performing various measurements such as the action of a drug in a short period of time using a biological reaction using a small amount of cells, and a measurement method using the same. Objective.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the above object can be achieved by using the following cell microchip, and have completed the present invention.

  That is, the present invention relates to a cell microchip formed by forming a microchannel on a plastic substrate. The cross section of the microchannel is preferably a rectangle, a triangle, a circle, an ellipse, or a cut shape thereof. The long side and short side or diameter of the cross section of the microchannel is preferably 2 to 200 μm.

  In the cell microchip of the present invention, the microchannel preferably has one or more branch points. The branch point is preferably a trifurcated branch point constituted by a flow path (A), a flow path (B), and a flow path (C).

  In the cell microchip of the present invention, it is preferable that the microchannel constitutes a set of measurement systems using cells, and a plurality of the measurement systems are formed on the single plastic substrate.

  Furthermore, in the cell microchip of the present invention, it is preferable that the plastic substrate is formed of an epoxy resin. The cell microchip of the present invention preferably has a haze value of 10% or less and a total light transmittance of 88% or more.

  The present invention relates to a measurement method using cells, characterized by using the cell microchip of the present invention.

  The present invention relates to a biological test method using cells, characterized by using the cell microchip of the present invention.

  The present invention includes a flow path (A), a flow path (B), a flow path (C) in which the flow path (A) and the flow path (B) merge on a plastic substrate, and the three flow paths. A method for examining the action of a drug on a cell using a cell microchip in which a microchannel having a trifurcated branch point is formed, wherein the culture medium is one of the channel (A) and the channel (B). And the cells are allowed to grow or survive, and a drug-containing solution is injected from the other side to bring the cells into contact with the drug. The channel (C) examines the path of the medium or solution or the action of the drug. It is related with the inspection method characterized by being an area | region for.

  In the method for inspecting a drug action, the contact between the cell and the drug is preferably performed at the three-way branch point. In the method for examining drug action, cells are grown or survived in a single layer linearly starting from one or more cells in the channel (A) or channel (B), which is a channel for growing or surviving cells. It is preferable.

  The method for inspecting a drug action preferably uses a cell microchip in which a plurality of the microchannels are formed on the plastic substrate, and inspects the action of the drug on the cells a plurality of times.

  According to the cell microchip of the present invention, various measurements such as the action of a drug can be easily performed in a short period of time using a biological reaction using a small amount of cells in a microchannel formed on a plastic substrate. Can be done. According to the present invention, it is possible to provide a cell microchip having microchannels having various shapes and sizes according to purposes. When the long side and short side or diameter of the cross section of the microchannel is set to 2 to 200 μm depending on the size of the cell to be used, the cell can be linearly grown or survived. Biological response can be measured. In addition, when the microchannel has one or a plurality of branch points, the reaction and measurement points can be set near the branch points. Thereby, the labor and time required for measurement or determination of the measurement result are reduced. Such an effect is more effective when the branch point is a three-way branch point constituted by the flow path (A), the flow path (B), and the flow path (C).

  In the cell microchip of the present invention, when the microchannel constitutes a set of measurement systems using cells, and a plurality of the measurement systems are formed on the single plastic substrate, Numerous biological reactions and measurements can be performed simultaneously on the microchip, further reducing the time and effort required to measure and determine the measurement results. In addition, the space for culturing cells can be saved, and the incubator can be used effectively in a small space.

  Furthermore, in the cell microchip of the present invention, when the plastic substrate is formed of an epoxy resin, the affinity with the cell is particularly excellent, and the cell can be proliferated or survived well in a minute space. It is particularly advantageous for the measurement utilizing the biological reaction of Moreover, the board | substrate which consists of an epoxy-type resin is excellent in the microscopic observation of a cell from the characteristic that transparency is favorable and a phase difference is small. Further, the cell microchip of the present invention has excellent transparency when the haze value is 10% or less and / or when the total light transmittance is 88% or more. Is suitable.

  According to the measurement method or biological test method using cells of the present invention, by using the cell microchip, it is possible to provide various measurement methods or test methods in a short period of time, in a simple manner and in a small space. it can.

  According to the method of inspecting the action of a drug on cells according to the present invention, the action of the drug can be controlled at the branch point or the test flow path by using a cell microchip on which a micro flow path having a three-way branch point is formed. Thus, it is possible to provide an inspection method that can be investigated in a short period of time and is simple and space-saving. When a cell microchip in which a plurality of the microchannels are formed on the plastic substrate is used, a large number and / or many types of tests can be performed at one time. When the test method of the present invention is used for drug screening or the like, a high-throughput assay using cells is possible, and development of a pharmaceutical product can be promoted.

  The cell microchip of the present invention is formed by forming a microchannel on a plastic substrate.

  The microchannel in the present invention is a groove formed on a plastic substrate and having a micrometer-scale width and depth, and is a path through which cells exist and liquid passes or is retained. Further, the present microchannel includes not only one microchannel but also an aggregate of individual microchannels (also simply referred to as channels).

  The shape of the microchannel is not particularly limited as long as it has at least one cell and allows liquid to pass or be retained. The cross section of the microchannel is preferably a rectangle, a triangle, a circle, an ellipse, or a cut shape thereof from the viewpoint of easy processing of a plastic substrate and a shape suitable for cell survival. Specifically, a quadrangle, a V shape, a semicircle, a U shape, and the like can be given.

  The size of the cross section of the microchannel can be appropriately set according to the object of the present invention, particularly the size of the cell to be used and the shape of the cross section. When a cross section is a rectangle, a triangle, or its cut form, a long side and a short side are 1-1000 micrometers normally, It is preferable that it is 2-200 micrometers, 10-100 micrometers is more preferable, 10-50 micrometers is further more preferable. When the cross section is a circle, an ellipse, or a cut shape thereof, the diameter is usually 1-1000 μm, preferably 2-200 μm, more preferably 10-100 μm, and even more preferably 10-50 μm. The diameter is calculated assuming that the shape is a circle or an ellipse. In the case of an ellipse, the major axis and the minor axis are assumed.

  The length of each of the microchannels can be arbitrarily set according to the object of the present invention and the size of the plastic substrate to be formed, and is not particularly limited. It is about 01 mm to 10 cm. Within such a range, handling is easy and it can be suitably used for various measurements.

  The starting point and / or the ending point of the flow path is any point on the tip of the plastic substrate or the substrate, and is not particularly limited. When the start point or end point of the flow path is at the tip of the substrate, the start point or end point may be open at the leading edge, or it is closed and positioned inside the substrate at an appropriate length from the leading edge of the substrate. Also good. When the start point or end point of the flow path is inside the substrate, a container for trapping the liquid may be provided at the start point or end point.

  Since the microchannel is also an assembly of a plurality of channels, it is preferable to have one or a plurality of branch points where the channels intersect to connect the individual channels. In order to be suitable for the measurement method of the present invention described below, the number of branch points is not particularly limited as long as it is 1 or 2 or more, but it is 2 to 100, preferably 2 in one microchannel assembly. -50, more preferably 2-10 are exemplified. Further, the number of flow paths branching off from the branch point is also exemplified by 2 to 1000, preferably 2 to 500, more preferably 2 to 100 in accordance with the object of the present invention. The branch points may be distributed in the microchannel assembly in any manner, and the distribution state thereof is a state in which the branching points are regularly or irregularly distributed at an appropriate interval on a single channel. There is no particular limitation on the state of regular or irregular distribution in the flow path.

  The microchannel may be a well (hole) provided in a large number to hold one cell (one cell) on a plastic substrate. Such a cell microchip can perform many kinds of assays using one cell at a time or many times at the same time.

  For example, FIGS. 1 and 2 show examples of cell microchips suitably used in the following inspection method of the present invention. In FIG. 1, a cell microchip 10 has a microchannel 2 formed on a plastic substrate 1. The micro-channel 2 is composed of a channel (A) 3, a channel (B) 4, and a channel (C) 5, and has a three-way branch point 6 where these three channels intersect. . The start point or end point of the flow path can be arbitrarily set, but in FIG. 1, the start points of the flow paths (A) and (B) are at the tip of the plastic substrate. With such a configuration, a sample containing cells and a solution containing a drug can be injected directly from the cell microchip 10.

  In FIG. 1, a microchannel 2 including a channel (A) 3, a channel (B) 4, a channel (C) 5, and a trifurcated branch point 6 includes cells in the channel (B) 4, for example. A set of measurement systems is configured by injecting a sample. It is preferable that a plurality of the measurement systems are formed on one plastic substrate in order to efficiently perform a large number of measurements simultaneously. The number of the measurement systems can be appropriately set depending on the size of the cell microchip and the processing technology of the microchannel. An example of such a cell microchip is shown in FIG.

  In FIG. 2, the microchannels are formed in columns. In addition, a part of the measurement system has a further branch point 7 and 8. With such a configuration, a plurality of drugs can be separately injected into the measurement system at different times, and the interaction of the drugs can be examined.

  Next, the manufacturing method of the cell microchip of this invention is demonstrated.

  As the resin for forming the plastic substrate constituting the cell microchip of the present invention, it is preferable to use a thermoplastic resin or a thermosetting resin. Examples of the thermoplastic resin include polycarbonate, polyacrylate, polyethersulfone, polysulfone, polyester, polymethyl methacrylate, polyetherimide, and polyamide. Thermosetting resins include epoxy resins, unsaturated polyesters, and polydiallyl phthalate. And polyisobornyl methacrylate. These resins can be used singly or in combination of two or more, and can be used as a copolymer or a mixture with other components.

  In the present invention, an epoxy resin is particularly preferably used because it has good transparency and a small phase difference, and is excellent in heat resistance and chemical resistance. In the present invention, since the state of cells present on the cell microchip is directly observed with a microscope or the like, the plastic substrate is required to have good transparency and a small phase difference. Since the cell microchip of the present invention may be heated during cell culture or during various measurements, heat resistance is required within that limit. In addition, since various liquids are allowed to pass through the microchannel, chemical resistance is required within that limit. Furthermore, the epoxy resin is excellent in affinity with not only cells but also proteins or nucleic acids, and is particularly preferable as a material for cell microchips.

  Examples of the epoxy resin include bisphenol A type, bisphenol F type, bisphenol S type and bisphenol type such as water addition thereof, novolak type such as phenol novolak type and cresol novolak type, triglycidyl isocyanurate type and hydantoin type. Such as nitrogen-containing ring type, alicyclic type, aliphatic type, aromatic type such as naphthalene type, low water absorption type such as glycidyl ether type, biphenyl type, dicyclo type, ester type and ether ester type, and their modifications Etc. These may be used alone or in combination. Among the various epoxy resins, it is preferable to use a bisphenol A type epoxy resin, an alicyclic epoxy resin, or a triglycidyl isocyanurate type from the viewpoint of discoloration prevention.

  As such an epoxy resin, generally an epoxy resin having an epoxy equivalent of 100 to 1000 and a softening point of 120 ° C. or less is preferably used in view of physical properties such as flexibility and strength of the obtained resin substrate. Furthermore, from the point of obtaining an epoxy resin-containing liquid that is excellent in coating property, developability to a sheet shape, etc., a two-component mixed type that exhibits a liquid state at a temperature equal to or lower than the coating temperature, particularly at room temperature, can be preferably used.

  Epoxy resins include curing agents, curing accelerators, and anti-aging agents, denaturing agents, surfactants, dyes, pigments, anti-discoloring agents, ultraviolet absorbers, organic fillers, and the like that are conventionally used as necessary. Various conventionally known additives can be appropriately blended.

  There is no limitation in particular about the said hardening | curing agent, The 1 type (s) or 2 or more types of suitable hardening | curing agents according to an epoxy resin can be used. Examples include tetrahydrophthalic acid and methyltetrahydrophthalic acid, organic acid compounds such as hexahydrophthalic acid and methylhexahydrophthalic acid, ethylenediamine and propylenediamine, diethylenetriamine and triethylenetetramine, their amine adducts and Examples include amine compounds such as phenylenediamine, diaminodiphenylmethane, and diaminodiphenylsulfone.

  Also amide compounds such as dicyandiamide and polyamide, hydrazide compounds such as dihydragit, methylimidazole, 2-ethyl-4-methylimidazole, ethylimidazole, isopropylimidazole, 2,4-dimethylimidazole, phenylimidazole, undecylimidazole. Examples of the curing agent include imidazole compounds such as heptadecyl imidazole and 2-phenyl-4-methylimidazole.

  Furthermore, imidazolines such as methyl imidazoline, 2-ethyl-4-methyl imidazoline, ethyl imidazoline, isopropyl imidazoline, 2,4-dimethyl imidazoline, phenyl imidazoline, undecyl imidazoline, heptadecyl imidazoline, 2-phenyl-4-methyl imidazoline. Examples of the curing agent include compounds, other phenolic compounds, urea compounds, and polysulfide compounds.

  In addition, acid anhydride compounds and the like are also mentioned as examples of the curing agent, and such an acid anhydride curing agent can be preferably used from the viewpoint of discoloration prevention. Examples include phthalic anhydride, maleic anhydride, trimellitic anhydride, pyromellitic anhydride, nadic anhydride, glutaric anhydride, tetrahydrophthalic anhydride, methyltetrahydrophthalic anhydride, hexahydrophthalic anhydride, Examples include methylhexahydrophthalic acid anhydride, methylnadic acid anhydride, dodecenyl succinic acid anhydride, dichlorosuccinic acid anhydride, benzophenone tetracarboxylic acid anhydride, and chlorendic acid anhydride.

  Particularly, an acid anhydride curing agent having a molecular weight of about 140 to about 200 and having a colorless or light yellow color such as phthalic anhydride, tetrahydrophthalic anhydride, hexahydrophthalic anhydride, or methylhexahydrophthalic anhydride. Can be preferably used.

  The mixing ratio of the epoxy resin and the curing agent is such that when an acid anhydride curing agent is used as the curing agent, the acid anhydride equivalent is 0.5 to 1.5 equivalents per 1 equivalent of the epoxy group of the epoxy resin. It is preferable to mix | blend so that it may become, More preferably, 0.7-1.2 equivalent is good. When the acid anhydride is less than 0.5 equivalent, the hue after curing is poor, and when it exceeds 1.5 equivalent, the moisture resistance tends to decrease. In addition, also when using another hardening | curing agent individually or in combination of 2 or more types, the usage-amount can be based on said equivalent ratio.

  Examples of the curing accelerator include tertiary amines, imidazoles, quaternary ammonium salts, organometallic salts, phosphorus compounds, urea compounds, etc., particularly tertiary amines and imidazoles. It is preferable to use it. These can be used alone or in combination.

  It is preferable that the compounding quantity of the said hardening accelerator is 0.05-7.0 weight part with respect to 100 weight part of epoxy resins, More preferably, 0.2-3.0 weight part is good. When the blending amount of the curing accelerator is less than 0.05 parts by weight, a sufficient curing accelerating effect cannot be obtained, and when it exceeds 7.0 parts by weight, the cured body may be discolored.

  Examples of the anti-aging agent include conventionally known compounds such as phenol compounds, amine compounds, organic sulfur compounds, and phosphine compounds.

  Examples of the modifying agent include conventionally known ones such as glycols, silicones and alcohols.

  The surfactant is added to smooth the surface of the sheet when the epoxy resin sheet is molded by casting the epoxy resin while being exposed to air. Examples of the surfactant include silicones, acrylics, and fluorines, and silicones are particularly preferable.

  Further, the plastic substrate may be formed of a plurality of layers, and a hard coat layer made of urethane resin, acrylic resin, silicone resin, or the like may be formed on the epoxy resin sheet. Among the hard coat layer forming resins, urethane resins are preferable, and urethane acrylates are particularly preferably used.

  The plastic substrate in the present invention can be formed by a conventionally known method such as a casting method or a casting method.

  In the casting method, a hard coat layer forming resin solution is applied to a flat plate mold and then cured by ultraviolet irradiation or heating. Thereafter, another flat plate mold subjected to the mold release treatment is disposed so as to face the mold release treatment surface and the hard coat layer, and the thickness of the two molds is fixed with a spacer or the like. Thereafter, a plastic substrate forming resin liquid is poured into the gap between the two flat plate molds and cured by heating or the like. Thereafter, the plastic substrate on which the hard coat layer is formed can be obtained by opening the two molds.

  In the casting method, a support composed of an endless belt is run at a constant speed of, for example, 0.1 to 50 m / min, preferably 0.2 to 5 m / min, via a driving drum and a driven drum, and on the support drum. After the hard coat layer forming resin liquid is applied by a die, it is cured by ultraviolet irradiation or heating, and the plastic substrate forming resin liquid is applied thereon by a die and then cured by heating or the like. Then, the plastic substrate in which the hard-coat layer was formed can be obtained by peeling from an endless belt.

  When the plastic substrate has a two-layer structure of an epoxy resin sheet and a hard coat layer, the microchannel may be formed on the epoxy resin sheet or the hard coat layer. Can be selected according to the cell to be treated.

  When the plastic substrate has a two-layer structure of an epoxy resin sheet and a hard coat layer, the surface roughness is preferably 0.1 to 5 nm on the epoxy resin sheet surface, more preferably 0.1 to 3 nm. Preferably, it is 0.1-2 nm. The surface roughness on the hard coat layer is preferably 5 to 100 nm, preferably 7 to 80 nm, and more preferably 10 to 50 nm.

  The thickness of the cell microchip of the present invention is preferably 100 μm to 800 μm, more preferably 200 μm to 600 μm, and still more preferably 200 μm to 400 μm. Moreover, when the hard coat layer is formed, the thickness of the hard coat layer is preferably 1 μm to 10 μm. If the thickness of the hard coat layer is less than 1 μm, a sufficient hard coat function cannot be imparted, and if it exceeds 10 μm, cracks tend to occur in the hard coat layer.

  The cell microchip of the present invention preferably has a haze value of 10% or less, more preferably 3% or less, particularly preferably 0 to 2%, from the viewpoint of ease of cell observation with a microscope or the like. is there. The haze value is a value measured according to JIS K 7136, and can be measured using a haze meter such as HM-150 manufactured by Murakami Color Co., Ltd.

  The cell microchip of the present invention preferably has a total light transmittance of 88% or more, more preferably 90% or more, and particularly preferably 92, from the viewpoint of easy observation of cells with a microscope or the like. ~ 100%. The total light transmittance is a value measured according to JIS K 7361-1, and is determined by measuring the total transmitted light at 550 nm using a high-speed spectrophotometer, for example, DOC-3C manufactured by Murakami Color Co., Ltd. be able to.

  Next, a microchannel is formed on the plastic substrate. Examples of a method for forming the microchannel include a processing method using the following laser.

1. Direct processing method (1) Direct etching processing by laser ablation This is a method of forming grooves by direct etching processing by ablation processing by laser beam beam scanning or imaging processing. It is preferable to use ablation processing by ultraviolet light absorption, which is non-thermal processing that does not go through a thermal processing process, because deterioration of edges and wall surfaces, deterioration of accuracy, and deterioration of appearance due to thermal damage during heating can be prevented.

As lasers used, Er-YAG laser, Nd-YAG laser, Nd-YVO ₄ laser, Nd-YLF laser, carbon dioxide laser, excimer laser, semiconductor laser, titanium-sapphire laser, copper vapor laser, free electron laser , Ar ion laser, Kr ion laser, ultrashort pulse laser using infrared vibration excitation, etc., but laser capable of ablation by ultraviolet absorption of 400 nm or less, for example, ArF excimer laser with oscillation wavelength of 193 nm, 248 nm KrF Excimer laser, 308 nm XeCl excimer laser, third harmonic of 355 nm Nd-YAG laser, fourth harmonic of 266 nm, third, fourth harmonic of Nd-YVO ₄ laser, or wavelength of 400 nm or more Holding Even a single laser can absorb light in the ultraviolet region via a multiphoton absorption process, and a pulse width represented by a titanium sapphire laser having a wavelength of about 750 to 850 nm is less than 1 × 10 ⁻¹¹ seconds or less. A short pulse laser or the like is particularly suitable.

As a specific processing method, for example, the following can be performed.
(I) Ablation processing is performed by irradiating a workpiece with laser light that has been shaped according to the width of a groove formed by mask imaging or a beam homogenizer optical system or a diffractive optical element, and the laser irradiation position is predetermined. Groove machining is performed by moving along the machining line.
(Ii) The entire image is directly ablated by a mask imaging method on the workpiece through a mask designed in accordance with the processing shape.

(2) Dry etching by vacuum process A resist layer or a metal thin film layer is formed on a workpiece, and a processing pattern is formed on the resist layer or the metal thin film layer by a normal photolithography process. Using this patterned thin film layer as a mask, etching is performed by a vacuum apparatus such as vacuum plasma, atmospheric pressure plasma, or reactive ion etching to form a groove.

(3) Method Using Wet Process A resist layer or a metal thin film layer is formed on a workpiece, and a processing pattern is formed on the resist layer or the metal thin film layer by a normal photolithography process. Using this pattern forming layer as a mask, etching is performed using an alkali, an acid, an organic solvent, or the like that can dissolve the workpiece, thereby forming a groove. Further, when a photosensitive resin is used as a processed product, a groove can be formed by a known photolithography process.

2. Transfer method (1), (2), and (3) are used to produce a desired shape, and this is used as a mother mold to produce a mold by electroforming.

  In electroforming, a metal thin film layer is formed on the surface of a mother die by vapor deposition or electroless plating, and then metal such as Ni or Cu is applied to thicken the metal, and then peeled off from the mother die. Using the metal mold thus formed, the shape is transferred to a plastic material by a method such as pressing, stamping, injection molding, or extrusion molding.

  In this method, the first matrix is the above 1. A material that can be easily processed by any one of the methods (1), (2), and (3) may be selected. The transfer method has the advantage that once a mold is made, it is easy to mass-produce. On the other hand, the material of the product needs to be a material that can be stamped or injection molded.

  As mentioned above, when processing a micro channel using a laser, the width and length of the channel can be set by designing a desired shape on the mask. The depth of the channel and the shape of the cross section can be set by appropriately adjusting the amount of laser to be irradiated.

  When processing the microchannel, the absorption of the laser can be enhanced by adding a UV absorber or an organic filler to the resin constituting the plastic substrate.

  In order to protect the resin surface of the processed cell microchip, it is preferable to attach a surface protective sheet until just before use.

  As the surface protective sheet, those described in Japanese Patent No. 3511386, Japanese Patent No. 3290261, Japanese Patent No. 2934398, and Japanese Patent No. 2983449 can be used, but the present invention is not limited thereto.

  The cell microchip of the present invention can be used by covering the top surface of the chip with a plastic lid in order to prevent evaporation of the liquid in the microchannel. Such a lid has a shape and an area covering at least the entire microchannel, and its thickness may be about 30 to 400 μm. The plastic material is the same as that of the substrate. Alternatively, when two or more cell microchips are used in an overlapping manner, the bottom of the microchip positioned directly above can be used as an alternative to the microchip lid positioned directly below.

  The cell microchip of the present invention thus produced can be used in a measurement method using cells, and the present invention provides a measurement method using the cell microchip.

  The present invention also provides a biological test method using cells, characterized by using the cell microchip.

  The cell used in the present invention is not particularly limited. Examples of the cells include bacteria, fungi (including yeast), plant cells, and animal cells, and protozoa (protozoa) are included as long as they match the size of the microchannel. Among these, animal cells widely used for various measuring methods are preferably used. Examples of animal cells include cells isolated from various animal tissues, passage cells, cell lines, germ cells (egg or sperm), embryonic stem cells, somatic stem cells, and nerve cells. For use in a measurement method useful for diagnosis, prevention or treatment of human diseases, human-derived cells are preferable, and examples include cells derived from pathological tissues of patients suffering from cancer and other diseases. When cells derived from a patient's pathological tissue are used, cells derived from normal tissues of the same person or cells derived from normal tissues of a healthy person are preferably used as controls. These cells may be cells that proliferate or survive by attaching to the bottom of the microchannel, may be cells that proliferate or survive in a floating state, may be undifferentiated cells, Cell may be used. Ordinary cells for gene transfer can be obtained from a public depository and are preferred. Examples of such cells include mouse or rat-derived cells such as COP, L, C127, Sp2 / 0, NS-1, NIH3T3 and ST2, hamster-derived cells such as BHK and CHO, COS1, COS3, COS7, CV1, and Vero. And monkey-derived cells such as HeLa and 293, and insect-derived cells such as Sf9, Sf21, and High Five. In the cell microchip of the present invention, not only cells but also proteins and nucleic acids may be present.

  The cells can be cultured in a cell microchip in a medium and environment suitable for the cells. In particular, when the plastic substrate is an epoxy resin, the affinity with cells is high, which is convenient. By culturing the cells, the cells can be grown in a single layer along the microchannel.

For example, a method of culturing mammalian cells in a cell microchip is performed by culturing in a commercially available medium supplemented with serum in a cell culture incubator (37 ° C., in a humidified state and in the presence of 5% CO ₂ ). be able to. In this case, in order to introduce cells into the microchannel, the cell-containing medium may be injected from directly above or next to the starting point of the channel using, for example, a micropipette or a microsyringe. During culture, the medium can be replaced by draining part of the medium in the microchannel using a micropipette or microsyringe, and then supplementing with fresh medium, or by using a micropump or other medium during the culture period. The method of flowing. Alternatively, in the case of an immersion method in which a culture dish of a size that can store a microchip is filled with a medium and the entire microchip is immersed in the culture, it is not necessary to replace the medium.

  The medium in the present invention includes not only a normal medium used for culturing cells as described above, but also a serum-free medium, physiological saline used for temporary survival of cells, phosphate buffer, etc. Solutions are also included.

  Hereinafter, a method for examining the action of a drug on cells using the cell microchip shown in FIG. 1 will be described.

  Consists of a flow path (A) 3, a flow path (B) 4, a flow path (A) 3 and a flow path (C) 5 where the flow path (B) 4 merges, and the three flow paths on the plastic substrate 1. In the method of inspecting the action of the drug on the cells using the cell microchip on which the microchannel 2 having the three-way branch point 6 is formed, either the channel (A) 3 or the channel (B) 4 is used. On the other hand, the medium is filled to allow the cells to grow or survive, and from the other side, a drug-containing solution is injected to bring the cells into contact with the drug.

  Cell culture in the channel (A) 3 or the channel (B) 4 is performed as described above. The cells to be used are preferably cells that start from one or more cells and grow or survive in a monolayer linearly in the flow path. The cells are brought into contact with a drug-containing solution injected from the other channel. Examples of the method of injecting the chemical-containing solution include a method of injecting from directly above or next to the starting point of the flow path using a micropipette or a microsyringe, or a method of flowing the solution at a low speed with a micropump or the like. In this case, the drug-containing solution is preferably a cell culture medium containing a predetermined concentration of drug. The contact is preferably a state-of-the-art single cell contact with a solution, and the contact point is preferably a three-way junction 6. The medium and the chemical-containing solution flow in the direction of the flow path (C) 5 and are finally discharged from the measurement system.

  In addition, when a cell microchip as shown in FIG. 2 in which a plurality of microchannels are formed is used, it is possible to inspect the action of a drug on cells a plurality of times using a single chip, which is preferable. In this case, the number of specimens (nk number) can be increased by using the same type of cells, and the action of the drug can be simultaneously examined for many types and concentrations of drugs using the same type of cells. A comparison of the effect of a single drug on cells can also be examined simultaneously. Moreover, when using a heterogeneous cell, it is preferable to use a negative control cell and / or a positive control cell as a control.

  For example, when the chemical resistance of a cell is inspected, the growth of a cell having no chemical resistance is stopped near the branch point 6. The cells having chemical resistance continue to grow using the flow path (C) 5 as a growth path.

  When cell growth stops, cell viability is determined by observation with a microscope or the like. In this case, since only one cell in contact with the chemical-containing solution in the vicinity of the branch point between the flow path (A) 3 and the flow path (B) 4 has to be observed, the time required for determination is shortened.

  It is a feature of the present invention that the drug concentration of the drug-containing solution can be changed even after the drug-containing solution is brought into contact with the cells. Specifically, when a drug-containing solution is injected from the flow path (A) 3, the tributary flowing through the flow path (A) 3 is processed, and a high concentration chemical solution is injected from the tributary to contact the cells. The chemical concentration of the chemical-containing solution can be increased. The concentration of the drug in contact with the cell can be determined by preparing a calibration curve in advance by the laser colorimetric method and measuring the drug-containing solution near the branch point 6 by the laser colorimetric method.

  In this way, since the drug concentration of the drug-containing solution can be continuously increased even after contact with the cells, the cells are grown again using the three-way microchannel, and the drug-containing solution with a high concentration is again added. It is efficient without the need to inject. Thereby, thresholds, such as chemical resistance of the measurement object cell, can be easily determined. As described above, the drug concentration of the drug-containing solution can be appropriately changed, so that the drug-containing solutions having different concentrations can be brought into contact with cells of the same target in a set of measurement systems and can be examined in time series. When a drug-containing solution with a different concentration is brought into contact with cells in a set of measurement systems, starting from a low-concentration drug-containing solution and gradually increasing the concentration, the concentration accuracy of the drug-containing solution and cell physiology It is preferable from the viewpoint.

  The micro flow path used in the inspection method of the present invention is not limited to a micro flow path having a trifurcated branch point, but has a branch point where three or more flow paths merge into a single flow path. You may have. In this case, it is preferable that the width of each flow path is the same. For example, when the shape is such that the flow paths (A), (B), and (C) merge to form the flow path (D), different types of flow paths (A), (B), and (C) When three types of cells are brought into contact with each other at the intersections of the flow paths (A), (B), and (C), signals generated by the interaction between these cells can be detected. Such signals are not particularly limited, and examples thereof include ligands, cytokines, growth factors, and changes in potential on the cell membrane.

[Example]
EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1
100 parts of 3,4-epoxycyclohexylmethyl-3,4-epoxycyclohexanecarboxylate represented by the chemical formula of (Chemical Formula 1) (parts by weight, the same shall apply hereinafter), and methylhexahydrophthalic anhydride represented by the chemical formula of Chemical Formula 2 125 parts of a product, 3.75 parts of tetra-n-butylphosphonium o, o-diethyl phosphorodithioate represented by the chemical formula of (Chemical Formula 3), 2.25 parts of glycerin and 0.07 part of a silicone surfactant are stirred. Mixing was performed to prepare an epoxy resin solution.

まず、（化４）の化学式で示されるウレタンアクリレートの１７重量％のトルエン溶液を、ステンレス製エンドレスベルトに走行速度０．３ｍ／分で流延塗布し、風乾してトルエンを揮発させた後、ＵＶ硬化装置を用いて硬化し、膜厚２μｍのハードコート層を形成した。続いて、エポキシ系樹脂液をダイより０．３ｍ／分で流延塗布し、加熱装置を用いて硬化させ、膜厚４００μｍのエポキシ系樹脂層を形成した。

First, a 17 wt% toluene solution of urethane acrylate represented by the chemical formula of (Chemical Formula 4) was cast and applied to a stainless steel endless belt at a running speed of 0.3 m / min, and air-dried to volatilize toluene, It hardened | cured using UV curing apparatus and the hard-coat layer with a film thickness of 2 micrometers was formed. Subsequently, an epoxy resin solution was cast and applied from a die at a rate of 0.3 m / min, and cured using a heating device to form an epoxy resin layer having a thickness of 400 μm.

前記ハードコート層およびエポキシ系樹脂層からなる積層体をエンドレスベルトから剥離し、窒素置換により酸素濃度が０．５％の雰囲気下でガラス板上に１８０℃×１時間放置し、アフターキュアを行った。キュア後の積層体を、縦２５ｍｍ、横７５ｍｍの寸法に切断し、細胞マイクロチップ用エポキシ基板を得た。

The laminate composed of the hard coat layer and the epoxy resin layer is peeled off from the endless belt, and is left on a glass plate at 180 ° C. for 1 hour in an atmosphere having an oxygen concentration of 0.5% by nitrogen substitution to perform after cure It was. The cured laminate was cut into dimensions of 25 mm length and 75 mm width to obtain an epoxy substrate for cell microchip.

  A chart of the total light transmittance measured from the epoxy resin layer surface of the epoxy substrate is shown in FIG. The surface roughness (Ra) defined in JIS B0601 was 10 nm for the hard coat layer and 0.5 nm for the epoxy resin layer surface. The retardation of the epoxy substrate was such that the in-plane retardation (Δnd) was 0.5 nm and the thickness direction retardation (Rth) was 20 nm.

Next, using a KrF excimer laser (manufactured by Lambda Physic) with a wavelength of 248 nm, the laser light is focused on the epoxy resin layer surface so that it becomes 50 μm square and the intensity is 1.28 J / cm ^2, and the scanning speed is 120 μm / sec. A single scan was performed to process the microchannel shown in FIG. 4 on the surface of the epoxy resin layer to obtain a cell microchip. In this case, the depth of the microchannel was 30 μm, and the cross section was trapezoidal. The haze value of the cell microchip was 0.5% for both the microchannel portion and the substrate portion.

Example 2
In the stage of processing the micro flow path, using a KrF excimer laser with a wavelength of 248 nm (manufactured by Lambda Physic), the laser beam is squeezed on the surface of the epoxy resin layer so as to be 50 μm square and the intensity is 1.28 J / cm ² , A microchannel illustrated in FIG. 4 was processed in the same manner as in Example 1 except that scanning was performed twice at a scanning speed of 120 μm / sec to obtain a cell microchip. In this case, the depth of the microchannel was 60 μm, and the cross section was trapezoidal. The haze value of the cell microchip was 0.5% for both the microchannel portion and the substrate portion.

Example 3
Using a KrF excimer laser (Lambda Physic) with a wavelength of 248 nm, the hard coat layer is focused to a 50 μm square with an intensity of 1.28 J / cm ² and scanned once at a scanning speed of 120 μm / sec. Except that, the microchannel was processed in the same manner as in Example 1 to obtain a cell microchip. In this case, the depth of the microchannel was 30 μm, and the cross section was trapezoidal. The haze value of the cell microchip was 0.5% for both the microchannel portion and the substrate portion.

Example 4
Using the cell microchip produced in Example 1, according to the method described in Journal of Japanese Society for Laser Medicine Vol. 17, No. 1, pp. 1-10 (1996), δ− against MTF7 which is a breast cancer cell of a rat is used. The effect of aminolevulinic acid (5ALA) was examined.

MTF7 is a Texas State University, M.C. D. Used from Dr. Stephan P. Tomasovic of Anderson Cancer Institute. MTF7 grows in a cell culture incubator (37 ° C., in the presence of 5% CO ₂ under humidification) in a medium supplemented with 10% fetal calf serum (FCS) in α-MEM medium (manufactured by Sigma). I let you.

  The cell microchip (FIG. 4) prepared in Example 1 was placed in a 90 mmφ petri dish, and the flow path (A) 33, the flow path (B) 34, and the flow path (C) 35 were filled with the above medium. Next, MTF7 was planted in the liquid reservoir 37 of the channel (A) 33 using a micropipette, and then the medium was added to the petri dish until the entire cell microchip was covered with the medium. Within the incubator, MTF7 cells were grown linearly along the flow path (A) 33 and continued to culture until the trifurcation branch point 36 was reached.

  Next, the cell microchip on which the MTF7 cells have grown is removed from the petri dish, and the medium is injected from the reservoir 37 of the flow path (A) 33 using a micropump, and various concentrations (25, 50, 100, The medium containing 200 A, 400 μg / ml) 5ALA (manufactured by Sigma) was injected from the liquid reservoir 37 of the channel (B) 34 and observed under a fluorescence microscope with an incubator. When the Ar ion laser of 488 nm was irradiated to the trifurcation branch point 36 (condition: 200 μW), it was observed that fluorescence was emitted by observation under a fluorescence microscope. Cells that were not injected with 5ALA containing media did not fluoresce.

  Furthermore, when the cells after laser irradiation were continuously cultured and observed under a microscope over time, the cells that emitted fluorescence stopped growing and eventually died, but MTF7 cells grown in a medium that did not contain 5ALA Continued to grow along channel (C) 35.

Schematic diagram showing one embodiment of the cell microchip of the present invention Schematic diagram showing another embodiment of the cell microchip of the present invention Schematic diagram showing the total light transmittance of the cell microchip of the present invention Schematic diagram showing another embodiment of the cell microchip of the present invention

Explanation of symbols

1 Plastic substrate 2 Micro flow path 3 Flow path (A)
4 Channel (B)
5 Channel (C)
6 Triangular branch point 7 Triangular branch point 8 Triangular branch point 10 Cell microchip 20 Cell microchip 30 Cell microchip 31 Plastic substrate 32 Microchannel 33 Channel (A)
34 Channel (B)
35 Channel (C)
36 Three-way junction point 37 Liquid reservoir

Claims (15)

A cell microchip formed by forming a microchannel on a plastic substrate. The cell microchip according to claim 1, wherein a cross section of the microchannel is a rectangle, a triangle, a circle, an ellipse, or a cut shape thereof. The cell microchip according to claim 2, wherein a long side and a short side or a diameter of the cross section of the microchannel are 2 to 200 µm. The cell microchip according to claim 1, wherein the microchannel has one or a plurality of branch points. The cell microchip according to claim 4, wherein the branch point is a trifurcated branch point constituted by a flow path (A), a flow path (B), and a flow path (C). The cell microchip according to any one of claims 1 to 5, wherein the microchannel constitutes a set of measurement systems using cells, and a plurality of the measurement systems are formed on the single plastic substrate. The cell microchip according to any one of claims 1 to 6, wherein the plastic substrate is formed of an epoxy resin. The cell microchip according to any one of claims 1 to 7, wherein a haze value is 10% or less. The cell microchip according to any one of claims 1 to 8, wherein the total light transmittance is 88% or more. A measurement method using cells, wherein the cell microchip according to claim 1 is used. A biological test method using cells, characterized by using the cell microchip according to claim 1. A flow path (A), a flow path (B), a flow path (C) where the flow path (A) and the flow path (B) merge on a plastic substrate, and a three-way branch point formed by the three flow paths A method for inspecting the action of a drug on a cell using a cell microchip having a microchannel with a cell filled with a medium in either the channel (A) or the channel (B) The cell is in contact with the drug by injecting a drug-containing solution from the other side, and the flow path (C) is a path for the medium or solution or an area for inspecting the action of the drug. An inspection method characterized by being. The method for inspecting a drug action according to claim 12, wherein the contact between the cell and the drug is performed at the three-way branch point. Starting from one or more cells in the flow path (A) or the flow path (B), which is a flow path for proliferating or surviving cells, the cells are linearly grown or live in a single layer. The method for inspecting a chemical action according to claim 12 or 13. 15. The method for inspecting a drug action according to claim 12, wherein the action of the drug on the cell is inspected a plurality of times using a cell microchip in which a plurality of the microchannels are formed on the plastic substrate. .

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