JP2016013088A - Substrate for cell culture, and cell culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate for cell culture and a cell culture vessel comprising the substrate for cell culture in which, in a substrate for cell culture having a comb-tooth electrode structure of 1-layer type, an inoculated cell can easily spread over a whole area of a comb-tooth electrode structure in which a cell adhesive region 5 and a cell adhesion inhibitory region 6 are formed in a pattern.SOLUTION: There is provided a substrate 10 for cell culture having a comb-tooth electrode structure in which a cell adhesive region 5 and a cell adhesion inhibitory region 6 are formed in a pattern. The comb-tooth electrode structure is constituted so as to have: a first comb-shaped electrode 11 configured by a base part 7 extending in a longitudinal direction X and a comb part 8 extending in two directions Y which crosses the base part 7; and a second comb-shaped electrode 12a and a third comb-shaped electrode 12b which are severally engaged with the comb part 8 of the first comb-shaped electrode 11 from two directions.

Description

  The present invention relates to a cell culture substrate and a cell culture container, and more particularly to a cell culture substrate having a single-layer comb-shaped electrode and a cell culture container incorporating the same.

  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 is also known to have a very important role in the progression of diseases such as cancer, atherosclerosis, 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 for cell migration assays include the classic Boyden chamber, 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.

  As a technique for controlling the adhesion and non-adhesion of cells, a technique is known in which voltage is applied and controlled on a conductive substrate whose entire surface is not patterned. However, in the control technology using this substrate, the properties of the entire surface on the substrate change due to the application of voltage, so after attaching cells on the substrate, only a specific region is changed to cell adhesiveness. However, it was difficult to control and migrate cells only in that region.

  As another technique for controlling the adhesion and non-adhesion of cells, a substrate for observing cells by migrating to a specific region is also known. The control technology using this substrate is a process in which cells are seeded in a well, cultured confluently, a wound pattern is created by scratching the cells with a pin, and the cells migrate from around to the wound pattern. Observe (scratch assay). Since the scratch assay using such a substrate forms a wound pattern with pins, there is a physical limitation on the size (width) of the wound, and it can be reduced to the order of millimeters at most. In addition, there is a problem that the edge of the scratch is dirty and the variation of the scratch shape between the wells is large. In addition, when wounded, there is a drawback that cells float due to physical contact between the pins and the cells, and cell contents leak out. In addition, there is a problem that an expert needs to learn to form a beautiful flaw.

  With respect to such a problem, Patent Document 1 includes 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. A cell culture substrate in which a cell adhesion region and a cell adhesion inhibition region are adjacent to each other has been proposed. In this technique, cells are seeded on a cell culture substrate, the cells are adhered to the cell adhesive region, and a voltage is applied to the conductive region to change the cell adhesion inhibitory region on the conductive region to the cell adhesive property. By this, it is said that the migration of the cells adhering to the cell adhesive region to the region changed to the cell adhesive property can be observed. The cell culture substrate includes a conductive region patterned into a comb shape including a plurality of comb portions and a base portion supporting each comb portion. The width of the comb portion and the interval between the comb portions are considered to be related to ease of cell adhesion, ease of cell migration, ease of current flow, and the like.

JP 2011-101638 A

  Incidentally, for example, in the cell culture substrate 10C of the form shown in FIG. 5, a polyethylene glycol film is patterned on two opposing comb electrodes 41, 42 constituting the comb electrode structure, and one of the comb electrodes 41 is formed as a positive electrode. A voltage is applied as an electrode, and the polyethylene glycol film, which is a cell adhesion-inhibiting region patterned on the positive electrode, is peeled off to change the peeled portion into a cell adhesive region. After that, a voltage is applied using the other comb-shaped electrode 42 as a positive electrode, and the polyethylene glycol film, which is a cell adhesion-inhibiting region patterned on the positive electrode, is peeled off to make the peeled portion a cell adhesive region. Can be changed. A cell observing the migration of the cell 30 from the cell adhesion region to which the seeded cells 30 had previously adhered to the newly formed cell adhesion region due to the change from the cell adhesion inhibition region to the cell adhesion region. A migration test can be performed.

  In the cell culture substrate 10C having such a comb electrode structure, the cells 30 are seeded on the substrate 10C for culturing. At that time, the seeded cells 30 are likely to gather near the center of the cell culture substrate 10C as shown in FIG.

  However, when the electrode shape is a comb shape, the cells 30 gathered in the vicinity of the center are difficult to move due to the slit portion 13 between the comb electrodes and the step of the organic compound layer 4, and as shown in FIG. This causes a problem that it does not spread over the entire substrate 10C. Such a problem is that cells cannot be spread over the entire surface of the comb-tooth electrode structure of the cell culture substrate 10C, so that a cell migration test cannot be sufficiently performed in each part.

  The present invention has been made in order to solve the above-mentioned problems, and the object thereof is to provide a cell culture substrate having a single-layered comb electrode structure, in which the seeded cells are composed of cell adhesion regions and cells. An object of the present invention is to provide a cell culture substrate that can easily spread over the entire surface of a comb-shaped electrode structure in which an adhesion-inhibiting region is patterned. Another object is to provide a cell culture container provided with the cell culture substrate.

  (1) A cell culture substrate according to the present invention for solving the above problems is a cell culture substrate having a comb electrode structure in which a cell adhesive region and a cell adhesion inhibitory region are patterned, The comb electrode structure includes a first comb electrode composed of a base portion extending in a longitudinal direction and a comb portion extending in two directions intersecting the base portion, and a first comb electrode meshed with the comb portion of the first comb electrode from two directions. It has two comb electrodes and a third comb electrode.

  In the cell culture substrate according to the present invention, the width of the tip side of the comb portion of the first comb electrode may be larger than the width of the base portion side of the comb portion of the first comb electrode.

  In the cell culture substrate according to the present invention, the cell adhesion-inhibiting region can be changed to a cell adhesive region by applying a voltage to the second comb electrode and the third comb electrode.

  In the cell culture substrate according to the present invention, one end or both ends in the width direction of the tip portion of the comb portion of the first comb electrode may be configured to have a protrusion.

  (2) A cell culture container according to the present invention for solving the above problems includes a cell culture substrate having a comb electrode structure in which a cell adhesive region and a cell adhesion inhibitory region are patterned, and the cell culture. A cell culture container having a container body on which a substrate is mounted, wherein the comb electrode structure includes a base extending in a longitudinal direction and a comb extending in two directions intersecting the base. And a second comb electrode and a third comb electrode respectively engaged with the comb portion of the first comb electrode from two directions.

  In the cell culture container according to the present invention, the cell adhesion-inhibiting region can be changed to a cell adhesive region by applying a voltage to the second comb electrode and the third comb electrode.

  In the cell culture container according to the present invention, the container body can be configured to include an opening for arranging the cell culture substrate on the bottom surface.

  In the cell culture container according to the present invention, each of the first to third comb-shaped electrodes of the cell culture substrate may have an external connection terminal extending outside the container body.

  According to the present invention, in the substrate for cell culture having a single-layer type comb-tooth electrode structure, the seeded cells have the entire surface of the comb-tooth electrode structure in which the cell adhesion region and the cell adhesion-inhibiting region are patterned. It is possible to provide a cell culture substrate that can be easily spread, and a cell culture container including the cell culture substrate.

It is a schematic diagram which shows an example of the cell culture container which concerns on this invention, (A) is a top view, (B) is a left view. It is sectional drawing which shows an example of the laminated constitution of the substrate for cell cultures concerning this invention. It is a top view which shows the example of the comb electrode which comprises the substrate for cell cultures concerning this invention, (A) does not have a processus | protrusion in the front-end | tip part of a 1st comb electrode, (B) is a 1st comb electrode Has a protrusion at the tip. It is explanatory drawing which shows the movement state of a cell when a cell is seed | inoculated on the substrate for cell cultures based on this invention, (A) is a form immediately after seed | inoculating a cell, (B) is the seeded cell moving. It is the form after doing. It is explanatory drawing which shows the movement state of a cell when a cell is seed | inoculated on the substrate for cell cultures of the comb-tooth electrode structure where two comb-shaped electrodes oppose, (A) is a form immediately after seed | inoculating a cell, ( B) is the form after the seeded cells have migrated. It is a schematic diagram which shows the structural form (A) of a comb-shaped electrode, and current distribution (B) when a voltage is applied to the comb-shaped electrode. It is a schematic diagram which shows the preparation process of the substrate for cell cultures. It is explanatory drawing about the cell seeding to the substrate for cell culture, and a cell migration test.

  Hereinafter, a cell culture substrate and a cell culture container according to the present invention will be described with reference to the drawings. The technical scope of the present invention is not limited to the following embodiments, but includes the scope including the gist of the present invention.

[Cell culture substrate]
A cell culture substrate 10 according to the present invention has a one-layered comb electrode structure in which a cell adhesive region 5 and a cell adhesion inhibitory region 6 are patterned as shown in FIGS. ing. The comb electrode structure includes a first comb electrode 11 including a base portion 7 extending in the longitudinal direction X and a comb portion 8 extending in two directions Y intersecting the base portion 7, and a comb portion of the first comb electrode 11. 8 has a second comb-shaped electrode 12a and a third comb-shaped electrode 12b that are respectively meshed in two directions.

  As shown in FIG. 4, the cell culture substrate 10 is sandwiched between positions where the second comb electrode 12a and the third comb electrode 12b are engaged with each other from two directions, that is, between the second comb electrode 12a and the third comb electrode 12b. Since the first comb-shaped electrode 11 is arranged at the position, the cells 30 that are likely to gather near the center during cell seeding are arranged on the entire surface of the comb-shaped electrode structure in which the cell adhesion region 5 and the cell adhesion inhibition region 6 are patterned. Can be spread easily. According to this cell culture substrate 10, for example, like the comb electrode structure shown in FIG. 5, the cells 30 gathered near the center are difficult to move due to the slits 13 between the comb electrodes and the steps of the organic compound layer 4. The problem of not spreading over the entire surface of the comb electrode structure can be solved.

  The reason why the cells 30 gathered near the center at the time of cell seeding easily spread over the entire surface of the comb electrode structure is as follows. (1) The cells 30 are provided at positions sandwiched between the second comb electrode 12a and the third comb electrode 12b. In the one comb electrode 11, the cells 30 gathered near the center easily spread on the base 7 extending in the longitudinal direction X without exceeding the slit 13 having a particularly large step, and further intersect the base 7 2. Easily spread on the short comb portion 8 extending in the direction Y. (2) Similarly, in the second comb electrode 12a and the third comb electrode 12b meshed from the two directions, the cells gathered near the center. 30 spreads easily on the base 14 extending in the longitudinal direction X without exceeding the slit 13 having a particularly large step, and further easily spread on the short comb portion 15 intersecting the base 14. It is based Rukoto, to. On the other hand, in the comb electrode structure shown in FIG. 5, in order for cells 30 gathering near the center at the time of cell seeding to spread easily over the entire surface of the comb electrode structure, the slit portions 13 having particularly large steps must be passed one after another. It is not easy, and it is necessary to move on the comb portions 8 and 8 'having a long length, and the cells 30 are difficult to spread compared to the comb electrode structure according to the present invention. .

  Hereinafter, the configuration of the cell culture substrate will be described in detail.

(Cell adhesion region and cell adhesion inhibitory region)
The cell adhesion region 5 and the cell adhesion inhibition region 6 shown in FIG. 2 are patterned on the comb electrodes 11, 12a, 12b of the cell culture substrate 10, as shown in FIGS. . The comb electrode structure includes a first comb electrode 11, a second comb electrode 12a, and a third comb electrode 12b. Each of these comb-shaped electrodes is provided adjacent to each other with the slit portion 13 interposed therebetween. In the example of FIGS. 1 to 3, in the first comb electrode 11 provided at a position where the second comb electrode 12a and the third comb electrode 12b are engaged with each other from two directions, the second comb shape is formed from one direction (left side in plan view). The electrodes 12a are engaged with each other, and the third comb-shaped electrodes 12b are provided adjacently so as to be engaged with each other from the other direction (right side in plan view).

  The cell adhesion herein means that the cells 30 adhere or the cells 30 are easy to adhere, and the cell adhesion inhibitory means that the cells 30 are difficult to adhere or the cells 30 do not adhere. I mean. Therefore, when the cells 30 are seeded on the cell culture substrate 10 in which the cell adhesion region 5 and the cell adhesion inhibitory region 6 are formed in a predetermined pattern, as shown in FIGS. 7 and 8, the cell adhesion region The cell 30 adheres to 5, but the cell 30 does not adhere to the cell adhesion-inhibiting region 6. As a result, the cells 30 are arranged in the same pattern as the cell adhesive region 5 on the surface of the cell culture substrate 10. In addition, since cell adhesiveness may change with the cells 30 which are going to adhere | attach, cell adhesiveness means that it is cell adhesiveness with respect to a certain kind of cell 30. FIG. Accordingly, there may be a case where a plurality of cell adhesion regions 5 for a plurality of types of cells are present on the cell culture substrate 10 (that is, there are two or more cell adhesion regions 5 having different cell adhesion properties).

  Examples of detailed structures of the cell adhesion region 5 and the cell adhesion inhibition region 6 include the following two forms.

  In the first embodiment, a cell adhesion region 5 is obtained by applying a voltage to a cell adhesion-inhibiting organic compound film 4 (for example, polyethylene glycol film) containing an organic compound having a carbon-oxygen bond for decomposition and removal (peeling). It is a form. In this embodiment, the organic compound film 4 is formed on the entire surface of the substrate, and then the organic compound film 4 is patterned. Then, among the comb-shaped electrodes 11, 12a, and 12b having the patterned organic compound film 4, for example, the first By applying a positive voltage to one comb-shaped electrode 11 to decompose and remove (peel) the organic compound film pattern provided on the first comb-shaped electrode 11, a place where the cell adhesion inhibitory region 6 was initially (pattern formation) The formed organic compound film 4) is changed to the cell adhesion region 5.

  The second form is a form in which a pattern is formed with a hydrophilic film containing an organic compound having a carbon-oxygen bond at a low density. In this embodiment, an organic compound film pattern containing an organic compound having a carbon-oxygen bond at a high density becomes the cell adhesion-inhibiting region 6, whereas a hydrophilic film pattern containing the compound at a low density is used in the cell adhesion region 5. It is a thing using what becomes. A first region where the compound is likely to bind to the substrate surface and a second region where the compound is difficult to bind are patterned on the surface of the substrate, and a film of the compound is formed on the surface of the substrate. And the second region is the cell adhesive region 5.

(Base material)
As shown in FIG. 2, the base material 1 is not particularly limited as long as it is an insulating material on which the conductive film 2 can be formed. Moreover, it is preferable that it is formed with the material which can form the organic compound film | membrane 4 which has a carbon oxygen bond on the surface. Specifically, glass, quartz glass, borosilicate glass, photosensitive glass, aluminum oxide, sapphire, ceramics, forsterite, silicon, elastomer, plastic (for example, polyester resin, polyethylene resin, polypropylene resin, polystyrene resin, ABS resin) , Nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin) and the like.

  The shape of the base material 1 is not particularly limited as long as it is a flat plate shape that can be arranged on the cell culture container main body 21 as shown in FIG. For example, it may be a flat plate shape such as a plate, a sheet, a film, a membrane, or a porous membrane. Moreover, the surface of the base material 1 may be formed with unevenness. When the film is used as the substrate 1, its thickness is not particularly limited, but is usually in the range of 0.1 μm or more and 1000 μm or less, preferably in the range of 1 μm or more and 500 μm or less, more preferably 10 μm or more and 200 μm or less. Is within the range.

  In particular, cell adhesion on the cell adhesion region 5 and the cell adhesion inhibitory region 6 using the substrate 1 having fine irregularities in the range of 1 nm or more and 10 μm or less smaller than the size of the cell. In the case of the same uneven shape, the shape and behavior of the adhered cells can be controlled to effectively perform the test. For example, in the case of a line pattern, the fine unevenness is a depth of 1 nm or more and 10 μm or less, a line protrusion width of 1 nm or more and 10 μm or less, and a line recess width of 1 nm or more. It means within the range of 10 μm or less.

(Conductive film)
As shown in FIGS. 2 and 7, the conductive film 2 is provided on the substrate 1 with a comb electrode structure. As shown in FIGS. 1 and 3, the comb electrode structure includes a first comb electrode 11, a second comb electrode 12 a, and a third comb electrode 12 b, each having a comb tooth sandwiching a slit portion 13 between the comb electrodes. It is a structure arranged in a shape.

  Examples of the conductive film 2 include a metal film, a metal oxide film, a film in which metal fine particles and metal nanofibers are dispersed in an insulator, and a film made of a conductive organic material. Examples of the metal include gold 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. Examples of the metal nanofibers include carbon nanotubes, and examples of the conductive organic material include polyethylene dioxythiophene (PEDOT).

  The conductive film 2 is preferably a transparent film in observing the cells 30, and examples thereof include an ITO film, an IZO film, and a polyethylenedioxythiophene film of a conductive polymer. Further, it is preferable that the film is transparent even after voltage application. In the present invention, an ITO film is formed by a sputtering method and then patterned to form three comb electrodes (first comb electrode 11, second comb electrode 12a, and third comb electrode 12b through the slit portion 13). ), And the patterning pattern of the organic compound film 4 thereafter becomes the cell adhesion-inhibiting region 6, and the part where the organic compound film 4 is not provided becomes the cell adhesion region 5. The cells can be observed with an inverted microscope.

  The conductive film 2 can be formed by various methods. For example, a microwave plasma CVD (Chemical vapor deposition) method, an ECRCVD (Electrical cyclotron resonance) method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ECR method. Examples thereof include an arc type vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method. The film formation may be performed by coating, and spin coating and various printing methods can also be used.

  The thickness of the conductive film 2 is usually within the range of not less than the thickness of the monomolecular film and not more than 100 μm, preferably not less than 2 nm and not more than 1 μm, more preferably not less than 5 nm and not more than 500 nm. .

  The comb electrodes 11, 12a, and 12b can be formed using a patterning technique or a laser processing technique. Examples of the patterning technique include a gravure printing method, a screen printing method, an offset printing method, a flexographic printing method and a contact printing method, various printing methods, a method using various lithography methods, a method using an inkjet method, and other fine methods. A three-dimensional shaping method for engraving a deep groove can be used. Specifically, for example, a metal film or a metal oxide film is formed on a glass substrate, for example, and patterned using a known technique such as a photolithography technique, whereby the first comb electrode 11 and the second electrode A comb-shaped electrode 12a and a third comb-shaped electrode 12b can be formed. Further, as a laser processing technique, for example, a metal film or a metal oxide film is formed on a glass substrate, for example, and the film is cut by irradiating a laser on the film to thereby form the first comb electrode 11 and the first electrode. Two comb electrodes 12a and a third comb electrode 12b can be formed.

  The width of the slit 13 between the comb electrodes 11, 12a, 12b is not particularly limited, and can be in the range of 1 μm or more and 500 μm or more. For example, in the case of laser processing, the width of the slit 13 is usually about 100 μm to 150 μm, but the width varies depending on the spot diameter of the laser. For example, if the spot diameter is 100 μm, the slit width is also about 100 μm. become.

(Comb)
The first comb electrode 11 includes a base portion 7 extending in the longitudinal direction X and a comb portion 8 extending in two directions Y intersecting the base portion 7. On the other hand, the second comb electrode 12a and the third comb electrode 12b have comb portions 15 respectively engaged with the comb portion 8 of the first comb electrode 11 from two directions, and the second comb electrode 12a and the third comb electrode 12b are The first comb electrode 11 is disposed on both sides. That is, the first comb electrode 11 is sandwiched from both sides (two directions) by the second comb electrode 12a and the third comb electrode 12b, and the second comb electrode 12a is one side of the first comb electrode 11 The third comb electrode 12b is arranged on the other one-direction side of the first comb electrode 11 (right side in the plan view in FIG. 1).

  Each comb-shaped electrode is disposed at each of the left side, the middle side, and the right side through the slit portion 13 in plan view of FIG. 1 and the like, and the first comb-shaped electrode 11 is a second one disposed on the left side. Between the comb-shaped electrode 12a and the third comb-shaped electrode 12b disposed on the right side, the comb portion is disposed so as to be engaged. The first comb-shaped electrode 11 is arranged at a position on the inner side of the cell culture substrate 10, but may be provided at a strict center position or may be slightly shifted from the center position. Any region sandwiched between the second comb electrode 12a and the third comb electrode 12b may be used. In the example of FIGS. 1 to 3, “engaged from two directions” means that the second comb electrode 12 a is engaged from one direction (left side in plan view) of the first comb electrode 11 and the other direction (plan view). The third comb-shaped electrode 12b is meshed from the right).

  If it has such a comb-tooth electrode structure, the width | variety and length of each comb part 8 and 15 will not be specifically limited. The widths of the comb portions 8 and 15 are preferably within an average width of 10 mm or more and 100 mm or less. The width of the comb portion 8 is the width on the side orthogonal to the direction Y in which the comb portion 8 extends from the base portion 7 (W1 in FIG. 3 is the width on the tip side 16 and W2 is the width on the base side 17). Is the width on the side orthogonal to the direction Y in which the comb portion 15 extends from the base portion 14.

  In the comb electrode structure illustrated in FIGS. 1A and 3A, each part of the comb part 8 has the same width. In this comb electrode structure, when a positive voltage is applied to the comb electrode, for example, as shown in FIG. 6 (B), at the tip of the first comb electrode 11, a polyethylene glycol film is formed by current distribution during voltage application. A location B that cannot be sufficiently peeled may occur.

  The reason is that, for example, when the first comb electrode 11 is a positive electrode and the second comb electrode 12a and the third comb electrode 12b are negative electrodes, the organic pattern patterned on the first comb electrode 11 to which a positive voltage is applied. The compound film 4 was decomposed and peeled, but at that time, the current at the location B was small, and it was considered that the organic compound film 4 at the location B was sufficiently decomposed and could not be peeled off.

  Therefore, in the present invention, the structural form of the first comb-shaped electrode 11 is defined to have the base portion 7 and the comb portion 8 extending from the base portion 7, and the width W2 of the tip side 16 of the comb portion 8 is By making the width W1 larger than the width W1 on the base side 7, the difference in the current distribution is reduced, and the portion where the applied current is small is reduced. As shown in the portion A in FIG. In order to obtain such a width W 2, one end or both ends in the width direction of the front end side 16 of the comb portion 8 of the first comb electrode 11 can be configured to have a protrusion 19. The width W2 is preferably larger than the width W1 by about 5 mm or more and 20 mm or less. Such a width W2 can be easily realized by providing a protrusion 19 or the like on the distal end side 16 as shown in FIG. In addition, the protrusion shape is not specifically limited, The protrusion 19 may be provided in one of the width direction of a front-end | tip part, and may be provided in both. Such protrusions 19 can be formed together with patterning of the comb-shaped electrodes 11, 12a, 12b.

(Organic compound film)
As shown in FIGS. 2 and 7, the organic compound film 4 is patterned on each of the comb-shaped electrodes 11, 12 a, 12 b to act to constitute the cell adhesion-inhibiting region 6. Such an organic compound film 4 is a hydrophilic film formed of an organic compound having a carbon-oxygen bond. This organic compound film 4 is a film having water solubility and water swellability, and is a film made mainly of an organic compound having a carbon-oxygen bond. The organic compound film 4 before being oxidized or decomposed is not particularly limited as long as it has cell adhesion inhibitory properties and has cell adhesion properties after being oxidized and / or decomposed.

  Specifically, the organic compound film 4 has a high cell adhesion inhibitory property before being oxidized or decomposed by applying a positive voltage, and itself is cell-adhering after being oxidized or decomposed by applying a positive voltage. The conductive film 2 exposed by removing the organic compound film 4 may exhibit cell adhesion. For example, when a polyethylene glycol film is used as the organic compound film 4, by applying a positive voltage, the polyethylene glycol film is decomposed and peeled off, and a cell-adhesive conductive film 2 appears at that location, and cell adhesion Region 5 is entered.

  Here, 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.

  Examples of the main raw material constituting the organic compound film 4 include a water-soluble polymer, a water-soluble oligomer, a water-soluble organic compound, a surface active substance, an amphiphilic substance, and the like, and these are mutually physical or chemical. To form a hydrophilic organic compound film 4 by physically or chemically bonding to the substrate.

  Specific water-soluble polymer materials include polyalkylene glycol and derivatives thereof, polyacrylic acid and derivatives thereof, polymethacrylic acid and derivatives thereof, polyacrylamide and derivatives thereof, polyvinyl alcohol and derivatives thereof, and zwitterionic polymers. , Polysaccharides, and the like. Examples of the molecular shape include a straight chain, a branched one, and a dendrimer. More specifically, polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, such as Pluronic F108, Pluronic F127, poly (N-isopropylacrylamide), poly (N-vinyl-2-pyrrolidone), poly (2- Hydroxyethyl methacrylate), poly (methacryloyloxyethylphosphorylcholine), a copolymer of methacryloyloxyethylphosphorylcholine and an acrylic monomer, dextran, and heparin, 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) -propyltrimethoxysilane, and triethylene glycol-terminated-thiol.

  The average thickness of the organic compound film 4 is preferably in the range of 0.8 nm to 500 μm, and more preferably in the range of 0.8 nm to 100 μm. An average thickness of 0.8 nm or more is preferable because it is difficult to be affected by a region not covered with the organic compound film 4 on the substrate 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 the organic compound film 4, a method of directly adsorbing the organic compound to the conductive film 2, a method of directly coating the organic compound on the conductive film 2, and a crosslinking treatment after coating the organic compound on the conductive film 2 are performed. A method, a method of forming the organic compound film 4 in a multistage manner in order to improve the adhesion to the conductive film 2, a base film is formed on the conductive film 2 in order to improve the adhesion with the conductive film 2, and then organic Examples thereof include a method of coating a compound, a method of forming a polymerization initiation point on the surface of the conductive film 2, and then polymerizing a hydrophilic polymer brush.

  Among the above film forming methods, particularly preferable methods include a method of forming the organic compound film 4 in a multistage manner, and a base film is formed on the conductive film 2 in order to improve the adhesion to the conductive film 2, and then A method of coating an organic compound can be mentioned. By using these methods, the adhesion of the organic compound to the conductive film 2 can be increased.

  As shown in FIGS. 2 and 7, the organic compound film 4 formed on the conductive film 2 is patterned to have a predetermined pattern. The shape of the pattern is not particularly limited. For example, the pattern width can be about 300 μm, and the space width between adjacent patterns can be about 50 μm. In this case, for example, the pattern of the organic compound film 4 having a width of 300 μm is the cell adhesion inhibiting region 6, and for example, the space having a width of 50 μm is the cell adhesive region 5. Therefore, the seeded cells 30 are initially attached to the cell adhesion region 5 having a width of 50 μm, as illustrated in FIG. 8, for example. By changing to the region 5, the cell 30 attached to the cell adhesive region 5 can migrate to a location where the cell adhesive region 5 is newly changed.

  The organic compound film 4 can be patterned by various patterning means such as a general lithography method. For example, as shown in FIG. 7A, a conductive film 2 is formed on a base material 1, and thereafter, a base film 3 and an organic compound are formed on the conductive film 2 as shown in FIG. 7B. A film 4 is provided in order, and then, as shown in FIG. 7C, the organic compound film 4 having a predetermined pattern to be the cell adhesion-inhibiting region 6 is left and the organic compound film having a predetermined pattern to be the cell adhesion region 5 is left. Patterning is performed by lithography or the like so as to remove 4. Thus, the cell culture substrate 10 before the cells 30 are seeded can be formed.

(Undercoat)
The base film 3 is provided as necessary. As shown in FIGS. 2 and 7, the base film 3 is provided between the organic compound film 4 and the conductive film 2, and the organic compound film 4 is applied to the conductive film 2. Acts to improve adhesion. The base film 3 is preferably a film containing a material having a binding portion (linker). The combination of the linker and the functional group at the end of the material to be bonded to the linker includes epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde Etc. In each combination, any may be a linker. In these methods, the base film 3 is formed on the conductive film 2 with a material having a linker before the organic compound film 4 is formed. Preferably, the silane coupling agent (epoxy silane) which has an epoxy group at the terminal can be mentioned.

  The thickness of the base film 3 is not particularly limited, but can be in the range of 3 nm to 500 μm, for example. The base film 3 can be formed by adjusting a coating liquid having a base film forming material and applying the coating liquid.

(Cell migration experiment)
As shown in FIG. 7C, the cell culture substrate 10 has a cell adhesion inhibiting region 6 where the organic compound film 4 is patterned, and a cell adhesion region where the organic compound film 4 is not formed. It is the sex region 5. When the cells 30 are seeded on the cell culture substrate 10 thus formed, the cells 30 adhere to the cell adhesion region 5 and do not adhere to the cell adhesion inhibition region 6 as shown in FIG.

  Thereafter, as shown in FIG. 8B, when a positive voltage is applied to one of the comb electrodes, the organic compound film 4 (cell adhesion inhibiting region 6) patterned on the applied comb electrode is peeled off. As a result, the cell adhesion-inhibiting region 6 is changed to the cell adhesion region 5. Then, as shown in FIG. 8 (C), the cells 30 migrate from the cell adhesive region 5 to a region that has newly changed to the cell adhesive region 5 (peeled portion). Thus, cell migration experiments can be performed.

  The positive voltage to be applied can be appropriately determined, but is usually in the range of 1 V or more and 10 V or less, preferably in the range of 2 V or more and 5 V or less, and the application time is usually 0.5 minutes or more, 60 Within a range of minutes or less, preferably within a range of 1 minute or more and 10 minutes or less. The voltage to be applied varies depending on the type of solvent with which the electrode is in contact, the material of the electrode, and the shape of the electrode. Usually, the voltage that can change the cell adhesion-inhibiting region 6 to the cell adhesion region 5 is used. Thus, it is preferable to apply a voltage that is low enough not to adversely affect the cells.

  Observation of cell movement includes measurement of the speed of cell movement, and observation of the migration direction, cell morphology during migration, connection between surrounding cells, and the like. Measurement of the speed at which the cells move can be carried out by measuring the area and distance that the cells infiltrate in a pattern, for example, a linear pattern such as a comb portion.

[Cell culture vessel]
As shown in FIGS. 1 to 3, the cell culture container 20 according to the present invention includes the cell culture substrate 10 described above. Specifically, a cell including a cell culture substrate 10 having a comb electrode structure in which a cell adhesion region 5 and a cell adhesion inhibition region 6 are patterned, and a container body 21 on which the cell culture substrate 10 is mounted. It is a culture vessel 20. The comb electrode structure has a first comb-shaped electrode 11 composed of a base portion 7 extending in the longitudinal direction X and a comb portion 8 extending in the two directions Y intersecting the base portion 7 as described above, and the first The comb-shaped electrode 11 is characterized by having a second comb-shaped electrode 12a and a third comb-shaped electrode 12b that are respectively engaged with the comb portion 8 from two directions.

  The shape of the container main body 21 is not particularly limited, and may be a flask-type container illustrated in FIG. 1 or a dish-type container (not shown). The container body 21 includes an opening 23 that is disposed at a position where the cell culture substrate 10 is used as a bottom surface 24 for cell culture, and a culture solution injection port 22 for injecting a culture solution. In the example of the cell culture container 20 of FIG. 1, a lid 26 is attached to the culture solution inlet 22, and a cell culture substrate 10 is attached to the opening 23.

  The cell culture substrate 10 is bonded with an adhesive or the like so as to cover the opening 23 of the container body 21. The opening 23 is provided on the bottom surface 24 of the container main body 21 as shown in FIG. The cell culture substrate 10 is bonded so as to cover the opening 23. Therefore, the size of the opening 23 is designed according to the size and type of the cell culture substrate 10. In the example of FIG. 1, a rectangular opening 23 is provided on the bottom surface 24 of the rectangular parallelepiped container, and the rectangular cell culture substrate 10 is mounted in the opening 23.

  As shown in FIG. 1A, the cell culture substrate 10 has a first comb electrode 11, a second comb electrode 12a, and a third comb electrode 12b. The first to third comb electrodes 11, 12 a and 12 b have external connection terminals 11 ′, 12 a ′ and 12 b ′ extending outside the container body 21. The external connection terminals 11 ′, 12 a ′ and 12 b ′ are provided in a form extending from the cell culture substrate 10, for example, as shown in FIG. In the example of FIG. 1, the external connection terminals 11 ′, 12 a ′, and 12 b ′ are taken out from the side where the culture solution inlet 22 is provided, but may be taken out from other sides.

(cell)
The cells 30 are put into a container as cells to be seeded on the cell culture substrate 10. Such cells 30 may be floating cells such as blood cells and lymphoid cells, or may be adherent cells. In the present invention, the cell adhesive region 5 constituting the cell culture substrate 10 has adhesiveness. It is preferable that the cell has. Moreover, it uses suitably with respect to the cell which has the property to migrate.

  Examples of such cells include liver cancer cells, glioma cells, colon cancer cells, kidney cancer cells, pancreatic cancer cells, prostate cancer cells, colon cancer cells, breast cancer cells, lung cancer cells, and ovarian cancer. Cancer cells such as cells, liver cells that are liver parenchymal cells, Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermis angle Epidermal cells such as keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, corneal epithelial cells, etc. Examples include pancreatic islets of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells collected directly from tissues or organs, or may be passaged from one generation to another. Furthermore, these cells are undifferentiated embryonic stem cells, pluripotent stem cells such as pluripotent mesenchymal stem cells, unipotent stem cells such as vascular endothelial progenitor cells that have unipotency, and differentiation is completed. Any of cells may be sufficient. In addition, the cells may be cultivated as a single species, or two or more types of cells may be co-cultured.

  The culture sample containing the target cells is pre-dispersed by dispersing the biological tissue in a liquid and separating it to remove impurities such as cells other than the target cells and other cellular debris in the biological tissue. It is preferable to go. Prior to seeding of cells on a cell culture substrate, it is preferable to preliminarily culture a culture sample containing the target cells by various culture methods to increase the target cells.

  By using the cell culture container according to the present invention, such a cell 30 can be easily expanded in a comb electrode structure in which the cell adhesion region 5 and the cell adhesion inhibition region 6 are patterned. Specifically, as shown in FIG. 4, the first comb-shaped electrode 12a and the third comb-shaped electrode 12b are engaged with each other from two directions, that is, the first comb-shaped electrode is sandwiched between the second comb-shaped electrode 12a and the third comb-shaped electrode 12b. Since the electrode 11 is disposed, the cells 30 that are likely to gather near the center during cell seeding can be efficiently spread over the entire surface of the comb electrode structure. According to the cell culture substrate 10, for example, like the comb electrode structure shown in FIG. 5, the cells 30 gathered near the center are difficult to move due to the slits 13 between the comb electrodes and the step of the organic compound layer 4. The problem of not spreading over the entire surface of the comb electrode structure can be solved. As a result, a cell migration test in which cells migrate to the cell adhesive region can be performed satisfactorily.

DESCRIPTION OF SYMBOLS 1 Base material 2 Conductive film 3 Base film (silane coupling film)
4 Organic compound film (polyethylene glycol film)
5 Cell adhesion region 6 Cell adhesion inhibition region 7 Base (base of first comb electrode)
8 Comb (comb of the first comb electrode)
8 'Comb portion of comb electrode facing first comb electrode 10, 10A, 10B, 10C Cell culture substrate 11 First comb electrode 12a Second comb electrode 12b Third comb electrode 11', 12a ', 12b' External connection terminal 13 Slit between comb electrodes 14 Base (base of second and third comb electrodes)
15 Comb part (comb part of the second and third comb electrodes)
16 Tip side of the comb portion of the first comb electrode 17 Base portion side of the comb portion of the first comb electrode 19 Projection 20 Cell culture vessel 21 Container body 22 Culture fluid inlet 23 Opening portion (cell culture substrate mounting portion)
24 bottom surface 25 upper surface 26 lid 30 cell 41 first comb electrode 42 second comb electrode A, B specific location W1 width on the base side of the comb portion of the first comb electrode W2 width on the tip side of the comb portion of the first comb electrode

Claims (8)

A cell culture substrate having a comb electrode structure in which a cell adhesion region and a cell adhesion inhibition region are patterned,
The comb electrode structure includes a first comb electrode composed of a base portion extending in a longitudinal direction and a comb portion extending in two directions intersecting the base portion, and a first comb electrode meshed with the comb portion of the first comb electrode from two directions. A cell culture substrate comprising two comb electrodes and a third comb electrode.   2. The cell culture substrate according to claim 1, wherein the width of the tip side of the comb portion of the first comb electrode is larger than the width of the base side of the comb portion of the first comb electrode.   The cell culture substrate according to claim 1 or 2, wherein the cell adhesion-inhibiting region is changed to a cell adhesion region by applying a voltage to the second comb electrode and the third comb electrode.   The substrate for cell culture according to any one of claims 1 to 3, wherein one end or both ends in the width direction of the tip portion of the comb portion of the first comb-shaped electrode have protrusions. A cell culture vessel comprising a cell culture substrate having a comb electrode structure in which a cell adhesion region and a cell adhesion inhibitory region are patterned, and a container body equipped with the cell culture substrate,
The comb electrode structure includes a first comb electrode composed of a base portion extending in a longitudinal direction and a comb portion extending in two directions intersecting the base portion, and a first comb electrode meshed with the comb portion of the first comb electrode from two directions. A cell culture vessel comprising two comb electrodes and a third comb electrode.   The cell culture container according to claim 5, wherein the cell adhesion-inhibiting region is changed to a cell adhesion region by applying a voltage to the second comb electrode and the third comb electrode.   The cell culture container according to claim 5 or 6, wherein the container body includes an opening for arranging the cell culture substrate on a bottom surface.   The cell culture container according to any one of claims 5 to 7, wherein each of the first to third comb-shaped electrodes included in the cell culture substrate has an external connection terminal extending outside the container body. .

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