WO2007055171A1 - Cell culture container, cell culture method and method of regulating signal transduction in cultured cells - Google Patents

Cell culture container, cell culture method and method of regulating signal transduction in cultured cells Download PDF

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Publication number
WO2007055171A1
WO2007055171A1 PCT/JP2006/322096 JP2006322096W WO2007055171A1 WO 2007055171 A1 WO2007055171 A1 WO 2007055171A1 JP 2006322096 W JP2006322096 W JP 2006322096W WO 2007055171 A1 WO2007055171 A1 WO 2007055171A1
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WO
WIPO (PCT)
Prior art keywords
cell culture
groove
recesses
culture container
cells
Prior art date
Application number
PCT/JP2006/322096
Other languages
French (fr)
Japanese (ja)
Inventor
Taiji Nishi
Go Tazaki
Motohiro Fukuda
Original Assignee
Kuraray Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kuraray Co., Ltd. filed Critical Kuraray Co., Ltd.
Publication of WO2007055171A1 publication Critical patent/WO2007055171A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates

Definitions

  • the present invention relates to a cell culture container, a cell culture method, and a method for controlling signal transmission of cultured cells.
  • Apoptosis is a type of death of the cells that make up the body of a multicellular organism. It is a controlled and regulated cell that is actively awakened to keep an individual in better condition. It means suicide. In vivo, most cancerous cells (and other cells that have abnormalities inside) continue to be removed by apoptosis. This is known to prevent most tumor growth. In addition, in the process of development of organisms, cell death occurs at a fixed time and place (programmed cell death), which is the force that acts as a driving force for changes in the form of organisms. It happens by the mechanism of Apoto Isis.
  • cell culture dishes petri dishes
  • plates that are currently used for the purpose of culture tests and tissue culture in the medical and biotechnology fields, etc.
  • cell culture plates include 6-well, 12-well, 48-well, and 96-well plates (for example, Patent Document 1).
  • the signal transduction pathway in the cell tissue is configured. Construction can be said to be an extremely important parameter for reproducing living tissue.
  • the signal transduction pathway of a cultured tissue cultured in an existing cell culture dish (petri dish) or plate is a random network.
  • the left end cell and the right end network of the culture surface are connected to each other.
  • the current situation is to reproduce the form, which cannot occur in normal living tissues, such as short-circuiting and bonding.
  • Patent Document 1 JP-A-8-322593
  • the conventional cell culture container has a problem that a signal is transmitted through a route different from that of cells in a living tissue.
  • the problem to be solved by the present invention is to provide a cell culture container and a cell culture method capable of controlling signal transmission between cultured cells, and a method for controlling signal transmission of cultured cells.
  • the cell culture container according to the first aspect of the present invention includes a plurality of recesses and a groove portion provided between the plurality of recesses and communicating with the adjacent recesses, and communicates the recesses.
  • the length of the groove in the communication direction is 10 to 3000 / ⁇ ⁇ .
  • the cell culture container according to the second aspect of the present invention includes a plurality of recesses and a groove portion provided between the plurality of recesses and communicating the adjacent recesses, and the recesses communicate with each other.
  • a groove is provided so as to prevent a signal short circuit between cells in two of the plurality of recesses that are spaced apart from the adjacent recesses. Thereby, signal transduction between cultured cells can be controlled.
  • a cell culture container according to the third aspect of the present invention is the above-described cell culture container, wherein the depth of the groove is 1 to 50 m. Thereby, signal transmission between cultured cells can be controlled more reliably.
  • a cell culture container according to the fourth aspect of the present invention is the above-described cell culture container, wherein the groove has a width of 1 to 10 m. As a result, signal transmission between cultured cells can be controlled more reliably.
  • the cell culture container according to the fifth aspect of the present invention is the above-mentioned cell culture container, wherein the depth of the groove is 1 to 20 times the width of the groove. As a result, signal transmission between cultured cells can be controlled more reliably.
  • the cell culture container according to the sixth aspect of the present invention is the above-described cell culture container, wherein the depth of the recess is 5 to: LOOOm. Thereby, the signal transmission between the cultured cells can be controlled more reliably.
  • a cell culture container according to the seventh aspect of the present invention is the above-described cell culture container, wherein the length of the concave portion is 5 to 500 / ⁇ . Thereby, signal transmission between cultured cells can be controlled more reliably.
  • the cell culture container according to the eighth aspect of the present invention is the above-described cell culture container, wherein the depth of the concave portion is 1 to 20 times the length of the concave portion. Thereby, signal transmission between cultured cells can be controlled more reliably.
  • a method for controlling signal transmission of a cultured cell according to the ninth aspect of the present invention, the above-mentioned cell culture container is prepared, cells are injected into the recess of the cell culture container, and a culture solution is filled into the groove And culturing the cells, and between the cells contained in the adjacent recesses
  • a signal network for transmitting signals is formed. This makes it possible to easily control signal transduction between cultured cells.
  • a cell culture method according to the tenth aspect of the present invention provides the cell culture container described above, injects cells into the recess of the cell culture container, supplies a culture solution to the groove, Cells are cultured. Thereby, the cells can be cultured in a state in which signal transmission between the cultured cells is controlled.
  • a cell culture container and a cell culture method capable of controlling signal transmission between cultured cells, and a method for controlling signal transmission of cultured cells.
  • FIG. 1A is a plan view showing an example of the configuration of a cell culture container according to the present invention.
  • FIG. 1B is a cross-sectional view showing an example of the configuration of the cell culture container according to the present invention.
  • FIG. 2 is a diagram schematically showing a configuration of a cell culture container according to Example 1 of the present invention.
  • FIG. 3 is a diagram schematically showing a configuration of a cell culture container according to Example 2 of the present invention.
  • FIG. 4 is a diagram schematically showing a configuration of a cell culture container according to Example 3 of the present invention. Explanation of symbols
  • Cell culture dishes used for the purpose of culture tests and tissue culture in the medical and biotechnology fields, micro-space structures for culturing cells, and signal transduction in cell tissues
  • the micro-space structure for culturing cells has dimensions that allow about 1 to 5 cells to be arranged in the micro-space structure, as in living tissue.
  • Cell culture dish pre
  • collagen is appropriate for rat cardiomyocytes
  • polylysine is appropriate for nerve cells.
  • materials suitable for cell fixation are appropriately selected and coated, and the cells are arranged in the micro space structure.
  • the method for arranging the cells include a method in which the cell particle concentration is adjusted in advance and then sprayed together using a pipette, and a method in which the cells are arranged using an ink jet cell dispensing device.
  • Cells spread using a pipette or the like are initially placed outside the microspatial structure, but many cell types are microspatial structures with side walls that serve as scaffolds for easy differentiation and proliferation. Move in.
  • As a dimension that can arrange about 1 to 5 cells in the micro space structure it is preferable to select from the vertical and horizontal dimensions or a force of 5 to 500 111 in diameter and 5 to 1000 microns in depth.
  • the side walls arranged around the micro space structure have a role of preventing signal short-circuiting between cells in the long-distance micro space structure.
  • the particle size of a nerve cell is about 10 m
  • the length that can be developed from the neurite is about 30 to: LOOO / z m. Therefore, it is desirable that the length between the side walls dividing the micro space structure is selected from a force of 30 to: LOOO / zm.
  • the minute groove part for the cell in the micro space structure to form the signal network needs to have a width that allows only the network from the protrusion of the nerve cell to be formed, for example, so that the cell does not enter. . Therefore, it is desirable to select a force with a width of 1 to 10 ⁇ m and a depth of 1 to 50 ⁇ m.
  • FIG. 1A is a plan view showing a part of the configuration of a cell culture container that is useful in the embodiment of the present invention
  • FIG. 1B is a plan view showing a part of the configuration of a cell culture container that is useful in the embodiment of the present invention
  • the cell culture container 10 has a concavo-convex pattern.
  • a plurality of recesses 14 are formed by this uneven pattern.
  • Each of the recesses 14 has a microphone mouth space structure for injecting cells.
  • a large number of recesses 14 are arranged in a matrix.
  • the number of the concave portions 14 can be set to 3 ⁇ 3 or more, for example.
  • a first convex portion 13 is formed outside the plurality of concave portions 14.
  • the first convex portion 13 is formed so as to surround the outer periphery of the concave portion 14.
  • second protrusions 19 are formed.
  • the second convex portion 19 is lower than the first convex portion 13 in order to communicate the adjacent concave portion 14.
  • the surface of the cell culture vessel 10 has a two-step shape.
  • the height of the second convex portion 19 is lower than that of the first convex portion 13.
  • a groove 18 for communicating the recess 14 is provided on the second protrusion 19.
  • the side wall 19a of the second convex portion 19 can prevent a signal short circuit between cells of the long-distance microspace structure.
  • the planar shape of the recess 14 is rectangular, but the planar shape of the recess may be circular.
  • the size of the concave portion 14, that is, the length and width, or the diameter is preferably 5 to 500 / z m.
  • the depth of the recess 14 is preferably 5 to: LOOO m range force. That is, the height of the first convex portion 13 is preferably 5 to: LOOO m.
  • a suitable number of cells can be injected into the recess 14.
  • the ratio of the height and the length of the recess 14 is preferably 1: 1 to 1:20. That is, the length of the recess 14 is 1 to 20 times the depth of the recess 14.
  • the width of the second convex portion 19 is preferably 1 to: LO m. That is, it is preferable that the width of the second convex portion 19 provided between the adjacent concave portions 14 is 1 to: LO / z m. Therefore, the width of the groove 18 is also 1 to: LO m. In other words, a groove 18 having a width of 1 to 10 m is formed between adjacent recesses 14 so that the recesses 14 communicate with each other.
  • the width of the groove 18 is the length in the direction perpendicular to the paper surface of FIG. 1B. That is, the width of the groove 18 is the length in the direction perpendicular to the communication direction.
  • vertical to the communication direction of the part which the adjacent recessed part 14 communicates becomes 1-10 micrometers.
  • the depth of the groove 18 is preferably 1 to 50 ⁇ m. That is, it is preferable that the difference in height between the first convex portion 13 and the second convex portion 19 is 1 to 50 m. Therefore, a fine groove portion 18 having a cross-sectional force having a height of 1 to 50111 and a width of 1 to 10 / ⁇ ⁇ is formed on a plane perpendicular to the communication direction.
  • the depth of the groove 18 is less than the depth of the recess 14 / J.
  • the length of the groove 18 between the recesses 14 is preferably about 30 to: LOOO ⁇ m.
  • the length of the groove 18 is the distance in the communication direction between the adjacent recesses 14. In other words, the distance between the parts communicating with the adjacent recesses 14 is 30: LOOO m. Therefore, the adjacent recesses 14 are separated by a distance of 30 to: LOOO m.
  • the distance between the two opposite side walls 19a is 30 to 3000 / ⁇ ⁇ . Therefore, the distance in the direction in which the adjacent recesses 14 are communicated is 30 to: LOOO m.
  • the spacing force S30 between the opposing side walls 19a is: LOOO m.
  • the adjacent recesses 14 are arranged at a distance of 30 to: LOOO m, and communicate with each other by a groove portion 18 having a height of 1 to 50 111 and a width of 1 to: LO m.
  • a cross-section in a plane perpendicular to the communication direction has a height of 1 to 50 m, a width of 1 to: LO / z m, and a length of 30 to L000 m.
  • the ratio of the width and depth of the second convex portion 19 is preferably 1: 1 to 1:20.
  • the depth of the groove 18 is 1 to 20 times the groove of the groove 18.
  • cells in the micro-space structure form a signal network. For example, only a network of nerve cell protrusions is formed. Signals are transmitted between cells via this signal network. In this signal network, only adjacent microspace structures communicate.
  • a surface treatment on a region where the uneven pattern is provided it is preferable to perform a surface treatment on the upper surface and the side surface (side wall 19a) of the second convex portion 19 and the bottom surface of the concave portion 14.
  • a coating treatment of an organic material or an inorganic material can be performed.
  • the organic material or inorganic material a known material may be appropriately selected depending on the purpose.
  • the surface may be modified with an active energy ray such as an ultraviolet ray or an electron beam, or directly modified with other chemicals.
  • the concavo-convex pattern on the cell culture vessel can be formed by a semiconductor processing technique using silicon as a material, or a molding technique using a Ni stamper (master) using a photolithographic method.
  • a semiconductor processing technique using silicon as a material a resist is applied on a silicon substrate, and the resist is exposed and developed to pattern the resist. On this resist By etching the force silicon, the silicon substrate is patterned and an uneven pattern is formed. Then, by removing the resist, a silicon cell culture container having a desired uneven pattern is completed.
  • the substrate for the cell culture container is not limited to a silicon substrate, and may be a glass substrate or a resin substrate. As described above, by using the semiconductor processing technique, the uneven pattern can be formed with high accuracy.
  • a molding technique using a Ni stamper first, a first resist layer is applied onto a substrate and exposed, and then a second resist layer is applied and exposed. An uneven pattern can be formed on the substrate by batch original images of the first resist layer and the second resist. Then, a conductive film is deposited on the substrate on which the concave / convex pattern is formed by a vapor deposition method or a notching method. Then, the upper metal of the conductive thin film is deposited by plating to form a metal structure.
  • the stamper can be formed by peeling the metal structure from the substrate.
  • the cell culture container is formed of, for example, a resin material.
  • a structure for forming a metal structure may be formed using a silicon substrate, a glass substrate, or the like.
  • the method for producing the cell culture container is not limited to the above method.
  • the cell culture vessel 10 may be manufactured by mechanical cutting or an etching method for glass. This makes it possible to produce large-area 'cell culture strains.
  • cells can be easily observed by using a transparent material such as glass resin for the material of the cell culture container.
  • the cells can be cultured in a state in which signal transmission between the cells is controlled.
  • a cell culture container 10 having the above shape is prepared, and 1 to 5 cells, for example, are injected into the recess 14 of the cell culture container 10.
  • the culture medium is supplied to the groove 18 and the recess 14 to culture the cells.
  • signal transmission between the cultured cells is controlled by the side wall 19 a of the second convex portion 19.
  • the signal can be prevented from being transmitted to the cultured cells contained in the recesses 14 other than the adjacent recesses 14. Cultivation in an environment equivalent to that of living tissue becomes possible, and a signal transduction pathway that can reproduce living tissue is realized.
  • cells within the microspace structure form a signal network.
  • the protrusion of a nerve cell Only a power network is formed.
  • the specific dimensions of the cell culture container 10 described above are examples, and the present invention is not limited to the above range.
  • the width, depth, and length of the groove portion 18 are formed so as to prevent signal short-circuiting between cells in two of the plurality of recesses 14 that are separated from the adjacent recesses 14. do it.
  • the dimension of the groove 18 may be changed depending on the cultured cells.
  • FIG. 2 is a diagram schematically showing the shape of a cell culture vessel that is useful in this example.
  • a plurality of recesses 14 are formed on one substrate.
  • the recesses 14 are arranged side by side vertically and horizontally on the substrate surface.
  • a groove 18 is provided between the recesses 14.
  • the horizontal direction in FIG. 2 is defined as the width direction
  • the vertical direction perpendicular thereto is defined as the depth direction.
  • a cell culture vessel 10 is shown in which four recesses 14 are formed in the width direction and three in the depth direction. That is, twelve recesses 14 are formed in a matrix in the cell culture container 10.
  • the recesses 14 adjacent to each other in the width direction are communicated with each other by a single groove 18.
  • the recesses 14 adjacent to each other in the depth direction are communicated with each other by two groove portions 18. That is, the first convex portion 13 is provided in the groove portion 18 between the concave portions 14 adjacent in the depth direction.
  • the groove portion 18 between the concave portions 14 adjacent to each other in the depth direction is divided into two.
  • the size of the cell culture vessel 10 in the depth direction is A
  • the size of the cell culture vessel 10 in the width direction is B
  • the thickness (height) of the cell culture vessel 10 is C.
  • d be the pitch of adjacent recesses 14.
  • the plurality of recesses 14 are formed at substantially equal intervals.
  • the pitch in the depth direction of the groove 18 is also d.
  • the length of one recess 14 is e
  • the depth of the recess 14 is f.
  • the width of the groove 18 is g
  • the depth of the groove 18 is h
  • the length of the groove 18 is i.
  • Table 1 shows suitable numerical ranges of A, B, C, d, e, f, g, h, and i in the cell culture vessel 10 that is useful in this example.
  • the numerical values shown in parentheses indicate a more preferable numerical range for each dimension.
  • Table 1 lists dimensions suitable for cell culture tests and dimensions suitable for production of cultures and tissue culture.
  • fZe is assumed to be 1Z1 to 1Z20. That is, the length e of the recess 14 is set to 1 to 20 times the depth f of the recess 14.
  • gZh is assumed to be lZl to 20Zl. That is, the depth h of the groove 18 is set to 1 to 20 times the width g of the groove 18.
  • FIG. 3 is a diagram schematically showing the shape of a cell culture vessel that is useful in this example.
  • the cell culture container 10 is formed by bonding two substrates together. That is, the cell culture container 10 is formed by bonding the first substrate 21 and the second substrate 22 together.
  • a through hole 23 is formed in the first substrate 21 to form a recess 14.
  • the bonding surface of the second substrate 22 becomes the bottom surface of the recess 14.
  • a groove 18 is formed on the surface of the second substrate 22.
  • a fine recess 24 is formed for this purpose.
  • the first substrate 21 in which the through hole 23 is formed and the second substrate 22 in which the groove portion 18 is formed are brought into close contact with each other.
  • the surface on which the groove portion 18 of the second substrate 22 is formed becomes a bonding surface and is bonded.
  • the groove portion 18 of the second substrate 22 is provided in a predetermined shape at a position connecting adjacent through holes.
  • the through hole 23 has a circular planar shape.
  • the groove 18 extends to a position where the through hole 23 is provided. As a result, the adjacent recesses 14 communicate with the bottom surface.
  • a micro space structure for cell culture is formed by the through holes 23.
  • the side wall 19 a of the second convex portion 19 becomes the inner side surface of the through hole 23.
  • the through-hole 23 can be formed by, for example, a machining center, and the length of the tool is shortened if the size of the through-hole 23 is reduced.
  • the length that can be penetrated is about 0.5 mm.
  • the second substrate 22 having the fine groove 18 may be made of a soft and tacky material from the viewpoint of adhesion. In consideration of adhesion and bonding time, soft materials such as PDMS (polydimethylsiloxane) may be used. Further, a recess 24 for forming the groove 18 is formed in advance on the second substrate 22 and is brought into close contact with the first substrate 21. At this time, a recess 24 matching the positional relationship of the through hole 23 is formed on the second substrate 22 to form a groove 18. If position adjustment is necessary, it is possible to perform alignment by forming irregularities on the periphery of the substrate.
  • the cell-spreading side (through hole) and the network-forming side (groove 18) are different, the cells on the side wall do not cause a signal short circuit with the cells in the micro space structure. Therefore, a more precise network can be configured.
  • the lattice-shaped grooves 18 are formed on the first substrate 21.
  • the horizontal direction in FIG. 3 is defined as the width direction, and the direction (vertical direction) perpendicular thereto is defined as the depth direction.
  • a cell culture vessel 10 is shown in which four recesses 14 are formed in the width direction and three in the depth direction. That is, twelve recesses 14 are formed in a matrix in the cell culture container 10.
  • the recesses 14 adjacent to each other in the oblique direction are also communicated with each other by the groove portions 18. That is, the groove portion 18 has a width direction. And it is also provided in the diagonal direction between the depth direction. Therefore, a maximum of eight concave portions 14 provided around the concave portion 14 communicate with one concave portion 14.
  • the groove 18 provided in the oblique direction intersects with the groove 18 provided in the oblique direction at a different angle.
  • the groove part 18 provided in the diagonal direction becomes longer than the groove part 18 provided in the width direction and the depth direction.
  • the size of the cell culture vessel 10 in the depth direction is A
  • the size of the cell culture vessel 10 in the width direction is B
  • the thickness (height) of the cell culture vessel 10 is C.
  • the thickness of the cell culture vessel 10 is the total thickness of the first substrate 21 and the second substrate 22.
  • the pitch in the depth direction of the adjacent recesses 14 is d.
  • a plurality of recesses 14 are formed at substantially equal intervals in the width direction and the depth direction.
  • the pitch in the width direction and depth direction of the groove portion 18 is also d.
  • e is the length (diameter) of one recess 14
  • f is the depth of the recess 14.
  • the depth f of the recess 14 corresponds to the thickness of the first substrate 21.
  • the width of the groove 18 is g, the depth is h, and the length is i.
  • Table 2 shows suitable numerical ranges of A, B, C, d, e, f, g, h, and i in the cell culture container 10 that is useful in this example. In Table 2, the numerical values shown in parentheses indicate a more preferable numerical range for each dimension.
  • Table 2 lists dimensions suitable for cell culture tests and dimensions suitable for production of cultures and tissue culture.
  • the cell culture vessel 10 capable of controlling signal transmission can be realized.
  • fZe is assumed to be lZl to 20Zl. That is, the depth f of the recess 14 is set to 1 to 20 times the length e of the recess 14.
  • gZh is assumed to be lZl to 20Zl. That is, the width g of the groove 18 is set to 1 to 20 times the depth h of the groove 18.
  • FIG. 4 is a diagram schematically showing the shape of a cell culture vessel that is useful in this example.
  • a plurality of recesses 14 are formed on one substrate.
  • the recesses 14 are arranged side by side vertically and horizontally on the substrate surface.
  • a groove 18 is provided between the recesses 14.
  • the horizontal direction in FIG. 4 is defined as the width direction, and the direction perpendicular thereto is defined as the depth direction.
  • a cell culture container 10 is shown in which four recesses 14 are formed in the width direction and four in the depth direction. That is, the cell culture container 10 has 16 recesses 14 formed in a matrix.
  • the recesses 14 adjacent to each other in the oblique direction are also communicated by the groove portions 18. That is, the groove 18 is provided not only in the width direction and the depth direction but also in an oblique direction therebetween.
  • the diagonal groove portions 18 do not intersect each other. That is, the oblique grooves 18 are all provided in the same direction. Therefore, a maximum of six recesses 14 provided around the recess 14 communicate with one recess 14. Place these six recesses 14 in a regular hexagon.
  • the size of the cell culture vessel 10 in the depth direction is A
  • the size of the cell culture vessel 10 in the width direction is B
  • the thickness (height) of the cell culture vessel 10 is C.
  • d be the pitch of adjacent recesses 14.
  • the plurality of recesses 14 are formed at substantially equal intervals.
  • the pitch in the depth direction of the groove 18 is also d.
  • the length (diameter) of one recess 14 is e, and the depth of the recess 14 is defined.
  • the width of the groove 18 is g
  • the depth of the groove 18 is h
  • the length of the groove 18 is i.
  • Table 3 shows suitable numerical ranges of A, B, C, d, e, f, g, h, and i in the cell culture container 10 that is useful in this example.
  • the numbers shown in parentheses are The more preferable numerical range of each dimension is shown.
  • Table 3 lists dimensions suitable for cell culture tests and dimensions suitable for production of cultures and tissue culture.
  • the cell culture vessel 10 capable of controlling signal transmission can be realized.
  • fZe is assumed to be lZl to 20Zl. That is, the depth f of the recess 14 is set to 1 to 20 times the length e of the recess 14.
  • gZh is assumed to be lZl to 20Zl. That is, the width g of the groove 18 is set to 1 to 20 times the depth h of the groove 18.
  • the present invention is used, for example, in a cell culture vessel for culturing cells isolated from tissues and using them for testing and testing.

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Abstract

It is intended to provide a cell culture container by which intercellular signal transduction can be regulated, a cell culture method and a method of regulating signal transduction in cultured cells. A cell culture container (10) having a plural number of concaves (14) and a groove (18) which is located between the concaves (14) and connects concaves(14) adjacent to each other, wherein the length of the groove (18) in the connection direction is from 10 to 3000 μm. It is preferable to adjust the depth of the groove (18) to 1 to 50 μm. Owing to this constitution, intercellular signal transduction can be regulated.

Description

細胞培養容器、細胞培養方法及び培養細胞のシグナル伝達の制御方法 技術分野  Cell culture container, cell culture method, and control method for signal transmission of cultured cells
[0001] 本発明は、細胞培養容器、細胞培養方法及び培養細胞のシグナル伝達の制御方 法に関する。  The present invention relates to a cell culture container, a cell culture method, and a method for controlling signal transmission of cultured cells.
背景技術  Background art
[0002] 細胞死は、アポトーシス (apoptosis)と、ネクローシス (necrosis)に大別される。ァ ポトーシス（apoptosis)とは、多細胞生物の体を構成する細胞の死に方の一種で、 個体をより良 、状態に保っために積極的に弓 Iき起こされる、管理 ·調節された細胞の 自殺のことを意味する。生体内では、癌化した細胞 (そのほか内部に異常を起こした 細胞）のほとんどは、アポトーシスによって取り除かれ続けている。これにより、ほとん どの腫瘍の成長は未然に防がれていることが知られている。また、生物の発生過程 では、あら力じめ決まった時期、決まった場所で細胞死が起こり（プログラムされた細 胞死）、これが生物の形態変化などの原動力として働いている力 この細胞死もアポト 一シスの仕組みによって起こる。  Cell death is roughly classified into apoptosis and necrosis. Apoptosis is a type of death of the cells that make up the body of a multicellular organism. It is a controlled and regulated cell that is actively awakened to keep an individual in better condition. It means suicide. In vivo, most cancerous cells (and other cells that have abnormalities inside) continue to be removed by apoptosis. This is known to prevent most tumor growth. In addition, in the process of development of organisms, cell death occurs at a fixed time and place (programmed cell death), which is the force that acts as a driving force for changes in the form of organisms. It happens by the mechanism of Apoto Isis.
[0003] アポトーシスを開始させる細胞組織内のシグナル伝達経路は、例えば、線虫の遺 伝学的研究力 解明されてきている。そして、線虫や昆虫から哺乳類まで多細胞動 物のアポトーシス経路には共通点が多いことが知られている。また、細胞組織内のシ グナル伝達経路は、生体組織内の正常な機能を維持させるうえでも重要である。例 えば、心筋細胞の同期した拍動、神経細胞のネットワーク形成などにおいても整然と した形態が構築されて、正常な機能を発現することが可能となる。  [0003] Signal transduction pathways in cellular tissues that initiate apoptosis have been elucidated, for example, in nematode genetic research. It is known that there are many similarities in the apoptotic pathways of multicellular animals from nematodes and insects to mammals. The signal transmission pathway in the cell tissue is also important for maintaining normal functions in the living tissue. For example, an orderly form can be constructed in synchronized pulsation of cardiomyocytes, network formation of nerve cells, etc., and normal functions can be expressed.
[0004] ところで、現在、メディカル、バイオテクノロジー分野などで、培養試験、組織培養を 目的に使用されている細胞培養ディッシュ（シャーレ）、プレートなどは、その底面の 平板部分を培養面として使用されている。さらに、細胞培養プレートとしては、 6ゥェ ル、 12ゥエル、 48ゥエル、 96ゥエルの各プレートがある（例えば、特許文献 1)。  [0004] By the way, cell culture dishes (petri dishes) and plates that are currently used for the purpose of culture tests and tissue culture in the medical and biotechnology fields, etc., use the flat plate portion at the bottom as the culture surface. Yes. Furthermore, cell culture plates include 6-well, 12-well, 48-well, and 96-well plates (for example, Patent Document 1).
[0005] また、薬物の毒性判定などを目的としたアツセィ試験のための培養試験や、将来の 生体内移植を目的とした組織培養において、細胞組織内のシグナル伝達経路の構 築は、生体組織を再現させるうえで極めて重要なパラメーターであるといえる。 [0005] In addition, in the culture test for the assembly test for the purpose of determining the toxicity of the drug and the tissue culture for the purpose of future in vivo transplantation, the signal transduction pathway in the cell tissue is configured. Construction can be said to be an extremely important parameter for reproducing living tissue.
[0006] これに対し、既存の細胞培養ディッシュ (シャーレ）、プレートなどで培養された培養 組織のシグナル伝達経路は、ランダムネットワークであり、例えば、培養面の左端部 の細胞と右端部のネットワークが短絡して結合するなど、通常の生体組織内では起こ りえな 、形態を再現して 、るのが現状である。  [0006] On the other hand, the signal transduction pathway of a cultured tissue cultured in an existing cell culture dish (petri dish) or plate is a random network. For example, the left end cell and the right end network of the culture surface are connected to each other. The current situation is to reproduce the form, which cannot occur in normal living tissues, such as short-circuiting and bonding.
[0007] このため、細胞組織内の活性、活動電位を、電極上での電位変化やカルシウム濃 度変化による蛍光強度の測定にて評価する場合において、培養糸且織内における電 位差が生じな ヽ場合や、培養組織が一様な蛍光強度を発すると!ヽつた生体組織とは 異なる結果を生じて 、るのが現状である。  [0007] For this reason, when the activity and action potential in the cell tissue are evaluated by measuring the fluorescence intensity due to the potential change on the electrode and the calcium concentration change, a potential difference occurs in the cultured yarn and the tissue. In some cases, or when the cultured tissue emits uniform fluorescence intensity, it produces a result different from that of a living tissue.
[0008] 近年、シリコンを材料とした半導体カ卩ェ技術や、フォトリソグラフ法による Ni製スタン パー (原盤)を用いた成形技術の発達によって、ミリからマイクロ、更にはナノレベル の微細化が試みられている。しかしながら、これらの微細加工技術によって製造され た細胞培養多ゥエルプレートなどは、サンプルの微量化、集積ィ匕が可能になるものの 、培養細胞のシグナル伝達を制御することができず、生体組織を再現可能なシグナ ル伝達経路を実現するものではな 、。  [0008] In recent years, with the development of semiconductor molding technology using silicon as a material and molding technology using a Ni stamper (master) using photolithographic methods, attempts have been made to achieve micro- to micro- and even nano-level miniaturization. It has been. However, the cell culture multi-well plates produced by these microfabrication techniques can reduce the amount of samples and accumulate them, but cannot control the signal transmission of the cultured cells, so that the living tissue cannot be controlled. It is not a reproducible signal transmission path.
特許文献 1：特開平 8— 322593号公報  Patent Document 1: JP-A-8-322593
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] このように、従来の細胞培養容器では、生体組織内細胞と異なる経路でシグナルが 伝達されてしまうという問題点があった。 [0009] As described above, the conventional cell culture container has a problem that a signal is transmitted through a route different from that of cells in a living tissue.
本発明が解決しょうとする課題は、培養細胞間におけるシグナル伝達を制御可能 な細胞培養容器及び細胞培養方法並びに、培養細胞のシグナル伝達の制御方法 を提供することである。  The problem to be solved by the present invention is to provide a cell culture container and a cell culture method capable of controlling signal transmission between cultured cells, and a method for controlling signal transmission of cultured cells.
課題を解決するための手段  Means for solving the problem
[0010] 本発明の第 1の態様に力かる細胞培養容器は、複数の凹部と、前記複数の凹部の 間に設けられ、隣接する前記凹部を連通する溝部とを備え、前記凹部を連通する溝 部の連通方向の長さが 10〜3000 /ζ πιであるものである。これにより、培養細胞間に おけるシグナル伝達を制御することができる。 [0011] 本発明の第 2の態様に力かる細胞培養容器は、複数の凹部と、前記複数の凹部の 間に設けられ、隣接する前記凹部を連通する溝部とを備え、前記凹部を連通する溝 部が、前記複数の凹部のうち、前記隣接する凹部よりも離間した 2つの凹部内の細胞 間での信号短絡を防止することができるよう設けられているものである。これにより、培 養細胞間におけるシグナル伝達を制御することができる。 [0010] The cell culture container according to the first aspect of the present invention includes a plurality of recesses and a groove portion provided between the plurality of recesses and communicating with the adjacent recesses, and communicates the recesses. The length of the groove in the communication direction is 10 to 3000 / ζ πι. Thereby, signal transduction between cultured cells can be controlled. [0011] The cell culture container according to the second aspect of the present invention includes a plurality of recesses and a groove portion provided between the plurality of recesses and communicating the adjacent recesses, and the recesses communicate with each other. A groove is provided so as to prevent a signal short circuit between cells in two of the plurality of recesses that are spaced apart from the adjacent recesses. Thereby, signal transduction between cultured cells can be controlled.
[0012] 本発明の第 3の態様に力かる細胞培養容器は、上記の細胞培養容器において、前 記溝部の深さが 1〜50 mであるものである。これにより、培養細胞間におけるシグ ナル伝達をより確実に制御することができる。 [0012] A cell culture container according to the third aspect of the present invention is the above-described cell culture container, wherein the depth of the groove is 1 to 50 m. Thereby, signal transmission between cultured cells can be controlled more reliably.
[0013] 本発明の第 4の態様に力かる細胞培養容器は、上記の細胞培養容器にお!、て、前 記溝部の幅が 1〜10 mであるものである。これにより、培養細胞間におけるシグナ ル伝達をより確実に制御することができる。 [0013] A cell culture container according to the fourth aspect of the present invention is the above-described cell culture container, wherein the groove has a width of 1 to 10 m. As a result, signal transmission between cultured cells can be controlled more reliably.
[0014] 本発明の第 5の態様に力かる細胞培養容器は、上記の細胞培養容器において、前 記溝部の深さが前記溝部の幅の 1〜20倍であるものである。これにより、培養細胞間 におけるシグナル伝達をより確実に制御することができる。 [0014] The cell culture container according to the fifth aspect of the present invention is the above-mentioned cell culture container, wherein the depth of the groove is 1 to 20 times the width of the groove. As a result, signal transmission between cultured cells can be controlled more reliably.
[0015] 本発明の第 6の態様に力かる細胞培養容器は、上記の細胞培養容器において、前 記凹部の深さが 5〜： LOOO mであるものである。これにより、培養細胞間におけるシ グナル伝達をより確実に制御することができる。 [0015] The cell culture container according to the sixth aspect of the present invention is the above-described cell culture container, wherein the depth of the recess is 5 to: LOOOm. Thereby, the signal transmission between the cultured cells can be controlled more reliably.
[0016] 本発明の第 7の態様に力かる細胞培養容器は、上記の細胞培養容器において、前 記凹部の長さが 5〜500 /ζ πιであるものである。これにより、培養細胞間におけるシグ ナル伝達をより確実に制御することができる。 [0016] A cell culture container according to the seventh aspect of the present invention is the above-described cell culture container, wherein the length of the concave portion is 5 to 500 / ζπι. Thereby, signal transmission between cultured cells can be controlled more reliably.
[0017] 本発明の第 8の態様に力かる細胞培養容器は、上記の細胞培養容器において、前 記凹部の深さが前記凹部の長さの 1〜20倍であるものである。これにより、培養細胞 間におけるシグナル伝達をより確実に制御することができる。 [0017] The cell culture container according to the eighth aspect of the present invention is the above-described cell culture container, wherein the depth of the concave portion is 1 to 20 times the length of the concave portion. Thereby, signal transmission between cultured cells can be controlled more reliably.
[0018] 本発明の第 9の態様に力かる培養細胞のシグナル伝達の制御方法、上記の細胞 培養容器を用意し、前記細胞培養容器の前記凹部に細胞を注入し、前記溝部に培 養液を供給して前記細胞を培養し、前記隣接する凹部内に含まれる細胞間に対して[0018] A method for controlling signal transmission of a cultured cell according to the ninth aspect of the present invention, the above-mentioned cell culture container is prepared, cells are injected into the recess of the cell culture container, and a culture solution is filled into the groove And culturing the cells, and between the cells contained in the adjacent recesses
、信号が伝達するシグナルネットワークを形成させるものである。これにより、培養細 胞間におけるシグナル伝達を簡便に制御することができる。 [0019] 本発明の第 10の態様に力かる細胞培養方法は、上記の細胞培養容器を用意し、 前記細胞培養容器の前記凹部に細胞を注入し、前記溝部に培養液を供給して前記 細胞を培養するものである。これにより、培養細胞間におけるシグナル伝達が制御さ れた状態で細胞を培養することができる。 , A signal network for transmitting signals is formed. This makes it possible to easily control signal transduction between cultured cells. [0019] A cell culture method according to the tenth aspect of the present invention provides the cell culture container described above, injects cells into the recess of the cell culture container, supplies a culture solution to the groove, Cells are cultured. Thereby, the cells can be cultured in a state in which signal transmission between the cultured cells is controlled.
発明の効果  The invention's effect
[0020] 本発明によれば、培養細胞間におけるシグナル伝達を制御可能な細胞培養容器 及び細胞培養方法並びに、培養細胞のシグナル伝達の制御方法を提供することで ある。  [0020] According to the present invention, there are provided a cell culture container and a cell culture method capable of controlling signal transmission between cultured cells, and a method for controlling signal transmission of cultured cells.
図面の簡単な説明  Brief Description of Drawings
[0021] [図 1A]本発明にかかる細胞培養容器の構成の一例を示す平面図である。 FIG. 1A is a plan view showing an example of the configuration of a cell culture container according to the present invention.
[図 1B]本発明にかかる細胞培養容器の構成の一例を示す断面図である。  FIG. 1B is a cross-sectional view showing an example of the configuration of the cell culture container according to the present invention.
[図 2]本発明の実施例 1にかかる細胞培養容器の構成を模式的に示す図である。  FIG. 2 is a diagram schematically showing a configuration of a cell culture container according to Example 1 of the present invention.
[図 3]本発明の実施例 2にかかる細胞培養容器の構成を模式的に示す図である。  FIG. 3 is a diagram schematically showing a configuration of a cell culture container according to Example 2 of the present invention.
[図 4]本発明の実施例 3にかかる細胞培養容器の構成を模式的に示す図である。 符号の説明  FIG. 4 is a diagram schematically showing a configuration of a cell culture container according to Example 3 of the present invention. Explanation of symbols
[0022] 10 細胞培養容器、 13 第 1の凸部、 14 凹部、 18 溝部、 19 第 2の凸部  [0022] 10 cell culture container, 13 first convex part, 14 concave part, 18 groove part, 19 second convex part
19a 第 2の凸部の側壁、 21 第 1の基板、 22 第 2の基板、  19a side wall of the second convex portion, 21 first substrate, 22 second substrate,
23 貫通孔、 24 凹部  23 through holes, 24 recesses
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0023] メディカル、バイオテクノロジー分野などで、培養試験、組織培養を目的に使用する 細胞培養ディッシュ (シャーレ）、プレートにおいて、細胞を培養するためのマイクロ空 間構造、細胞組織内のシグナル伝達における遠距離マイクロ空間構造内の細胞どう しの信号短絡を防止するための側壁、マイクロ空間構造内の細胞がシグナルネットヮ ークを形成するための微細な溝を形成する。これにより、培養細胞間におけるシグナ ル伝達を制御可能な細胞培養容器を実現することができる。  [0023] Cell culture dishes (petri dishes) used for the purpose of culture tests and tissue culture in the medical and biotechnology fields, micro-space structures for culturing cells, and signal transduction in cell tissues A side wall for preventing signal short-circuiting between cells in the distance micro-space structure, and a minute groove for forming a signal network by the cells in the micro-space structure. As a result, a cell culture vessel capable of controlling signal transmission between cultured cells can be realized.
[0024] 細胞を培養するためのマイクロ空間構造は、生体組織と同様に、 1〜5個程度の細 胞がマイクロ空間構造に配置できる寸法とする。細胞培養ディッシュ（シャーレ）、プレ ート表面は、例えば、ラットの心筋細胞ではコラーゲン、神経細胞であればポリリジン など、あら力じめ細胞固定に適する材料を適宜選択、コートしたうえで細胞をマイクロ 空間構造内に配置する。細胞を配置する方法は、あらかじめ細胞粒子濃度を調整し た上でピペットを用いて一括して散布する方法、インクジェット式細胞分注装置を用 いて配置する方法などがあげられる。ピペットなどを用いて散布された細胞は、最初 、マイクロ空間構造の外にも配置されるが、細胞種の多くは、分化、増殖を容易に行 うための足場となる側壁を有するマイクロ空間構造内に移動する。 1〜5個程度の細 胞がマイクロ空間構造に配置できる寸法としては、縦'横、または直径5〜500 111、 深さ 5〜1000ミクロンのな力から選択することが好ましい。 [0024] The micro-space structure for culturing cells has dimensions that allow about 1 to 5 cells to be arranged in the micro-space structure, as in living tissue. Cell culture dish (petri dish), pre For example, collagen is appropriate for rat cardiomyocytes and polylysine is appropriate for nerve cells. For example, materials suitable for cell fixation are appropriately selected and coated, and the cells are arranged in the micro space structure. Examples of the method for arranging the cells include a method in which the cell particle concentration is adjusted in advance and then sprayed together using a pipette, and a method in which the cells are arranged using an ink jet cell dispensing device. Cells spread using a pipette or the like are initially placed outside the microspatial structure, but many cell types are microspatial structures with side walls that serve as scaffolds for easy differentiation and proliferation. Move in. As a dimension that can arrange about 1 to 5 cells in the micro space structure, it is preferable to select from the vertical and horizontal dimensions or a force of 5 to 500 111 in diameter and 5 to 1000 microns in depth.
[0025] マイクロ空間構造の周囲に配置される側壁は、遠距離マイクロ空間構造内の細胞 同士の信号短絡を防止する役割をもつ。例えば、神経細胞の粒子径は、約 10 m であり、その神経突起から進展可能な長さは、約 30〜： LOOO /z mであると推測する。 したがって、マイクロ空間構造どうしを分割する側壁間の長さは、 30〜： LOOO /z mのな 力から選択することが望まし 、。  [0025] The side walls arranged around the micro space structure have a role of preventing signal short-circuiting between cells in the long-distance micro space structure. For example, it is assumed that the particle size of a nerve cell is about 10 m, and the length that can be developed from the neurite is about 30 to: LOOO / z m. Therefore, it is desirable that the length between the side walls dividing the micro space structure is selected from a force of 30 to: LOOO / zm.
[0026] マイクロ空間構造内の細胞がシグナルネットワークを形成するための微細な溝部は 、細胞が入り込むことなぐ例えば、神経細胞の突起部からのネットワークのみが形成 される幅であることが必要となる。したがって、幅 1〜10 μ m、深さ 1〜50 μ mのなか 力も選択することが望ましい。  [0026] The minute groove part for the cell in the micro space structure to form the signal network needs to have a width that allows only the network from the protrusion of the nerve cell to be formed, for example, so that the cell does not enter. . Therefore, it is desirable to select a force with a width of 1 to 10 μm and a depth of 1 to 50 μm.
[0027] 以下に、本発明の実施の形態にカゝかる細胞培養容器の構成について図 1A及び 図 1Bを参照して説明する。図 1Aは、本発明の実施の形態に力かる細胞培養容器の 構成の一部を示す平面図であり、図 1Bは図 1 Aの A— A断面図である。  [0027] Hereinafter, the configuration of the cell culture container according to the embodiment of the present invention will be described with reference to FIGS. 1A and 1B. FIG. 1A is a plan view showing a part of the configuration of a cell culture container that is useful in the embodiment of the present invention, and FIG.
[0028] 細胞培養容器 10には、凹凸パターンが形成されている。この凹凸パターンによって 複数の凹部 14が形成される。この凹部 14のそれぞれが細胞を注入するためのマイク 口空間構造となる。なお、実際には多数の凹部 14がマトリクス状に配列される。この 凹部 14の数を、例えば、 3 X 3以上とすることが可能である。複数の凹部 14の外側に は、第 1の凸部 13が形成されている。第 1の凸部 13は、凹部 14の外周を囲むように 形成される。複数の凹部 14の間には、第 2の凸部 19が形成されている。ここで、第 2 の凸部 19は、隣接する凹部 14を連通するため、第 1の凸部 13よりも低くなつている。 すなわち、細胞培養容器 10の表面は 2段の階段状になっている。そして、第 2の凸 部 19の高さは、第 1の凸部 13よりも低くなつている。これにより、凹部 14を連通するた めの溝部 18が、第 2の凸部 19の上に設けられる。この第 2の凸部 19の側壁 19aによ つて、遠距離マイクロ空間構造の細胞間における信号短絡を防止することができる。 なお図 1において、凹部 14の平面形状が矩形であるが、凹部の平面形状は円形で あってもよい。 [0028] The cell culture container 10 has a concavo-convex pattern. A plurality of recesses 14 are formed by this uneven pattern. Each of the recesses 14 has a microphone mouth space structure for injecting cells. In practice, a large number of recesses 14 are arranged in a matrix. The number of the concave portions 14 can be set to 3 × 3 or more, for example. A first convex portion 13 is formed outside the plurality of concave portions 14. The first convex portion 13 is formed so as to surround the outer periphery of the concave portion 14. Between the plurality of recesses 14, second protrusions 19 are formed. Here, the second convex portion 19 is lower than the first convex portion 13 in order to communicate the adjacent concave portion 14. That is, the surface of the cell culture vessel 10 has a two-step shape. The height of the second convex portion 19 is lower than that of the first convex portion 13. As a result, a groove 18 for communicating the recess 14 is provided on the second protrusion 19. The side wall 19a of the second convex portion 19 can prevent a signal short circuit between cells of the long-distance microspace structure. In FIG. 1, the planar shape of the recess 14 is rectangular, but the planar shape of the recess may be circular.
[0029] 凹部 14の大きさ、すなわち縦 ·横の長さ、または直径は 5〜500 /z mであることが好 ましい。また、凹部 14の深さは 5〜： LOOO mの範囲力も選択することが好ましい。す なわち、第 1の凸部 13の高さを 5〜： LOOO mとすることが好ましい。これにより、凹部 14の中に、好適な数の細胞を注入することができる。さらに、凹部 14の高さと、長さ の比は、 1 : 1〜1 : 20とすることが好ましい。すなわち、凹部 14の長さを凹部 14の深 さの 1倍〜 20倍とする。  [0029] The size of the concave portion 14, that is, the length and width, or the diameter is preferably 5 to 500 / z m. The depth of the recess 14 is preferably 5 to: LOOO m range force. That is, the height of the first convex portion 13 is preferably 5 to: LOOO m. Thereby, a suitable number of cells can be injected into the recess 14. Further, the ratio of the height and the length of the recess 14 is preferably 1: 1 to 1:20. That is, the length of the recess 14 is 1 to 20 times the depth of the recess 14.
[0030] 第 2の凸部 19の幅は、 1〜： LO mであることが好ましい。すなわち、隣接する凹部 1 4の間に設けられた第 2の凸部 19の幅を 1〜： LO /z mとすることが好ましい。従って、 溝部 18の幅も 1〜： LO mとなる。換言すると、隣接する凹部 14の間に幅 1〜10 m の溝部 18を形成して、凹部 14間を連通する。なお、溝部 18の幅とは、図 1Bの紙面 に対して垂直な方向の長さとする。すなわち、溝部 18の幅とは、連通方向と垂直な方 向の長さとなる。これにより、隣接する凹部 14が連通する部分の連通方向と垂直な方 向の長さが 1〜10 μ mとなる。さらに、溝部 18の深さは 1〜50 μ mとすることが好まし い。すなわち、第 1の凸部 13と第 2の凸部 19の高さの差を 1〜50 mとすることが好 ましい。従って、連通方向と垂直な平面にぉぃて高さ1〜50 111、幅 1〜10 /ζ πιの断 面力もなる微細な溝部 18が形成される。もちろん、溝部 18の深さは、凹部 14の深さ よりち/ J、さくなる。  [0030] The width of the second convex portion 19 is preferably 1 to: LO m. That is, it is preferable that the width of the second convex portion 19 provided between the adjacent concave portions 14 is 1 to: LO / z m. Therefore, the width of the groove 18 is also 1 to: LO m. In other words, a groove 18 having a width of 1 to 10 m is formed between adjacent recesses 14 so that the recesses 14 communicate with each other. The width of the groove 18 is the length in the direction perpendicular to the paper surface of FIG. 1B. That is, the width of the groove 18 is the length in the direction perpendicular to the communication direction. Thereby, the length of the direction perpendicular | vertical to the communication direction of the part which the adjacent recessed part 14 communicates becomes 1-10 micrometers. Further, the depth of the groove 18 is preferably 1 to 50 μm. That is, it is preferable that the difference in height between the first convex portion 13 and the second convex portion 19 is 1 to 50 m. Therefore, a fine groove portion 18 having a cross-sectional force having a height of 1 to 50111 and a width of 1 to 10 / ζ πι is formed on a plane perpendicular to the communication direction. Of course, the depth of the groove 18 is less than the depth of the recess 14 / J.
[0031] 第 2の凸部 19の側壁 19aによって、遠距離マイクロ空間構造内の細胞同士の信号 短絡を防止することができる。すなわち、隣の凹部 14に注入された細胞力もの信号 は伝達される力 隣の凹部 14よりも遠い凹部 14に注入された細胞からの信号が伝達 されるのを防ぐことができる。このような構成とすることによって、隣同士の凹部 14内 の細胞間では、信号短絡が発生する。一方、複数の凹部 14のうち、隣接する凹部 14 以外の凹部 14内の細胞間での信号短絡を防止することができる。すなわち、 2っ以 上離れた凹部 14内の細胞同士で信号が伝達されるのを防ぐことができる。 [0031] By the side wall 19a of the second convex portion 19, signal short-circuit between cells in the long-distance microspace structure can be prevented. That is, the signal of the cell force injected into the adjacent recess 14 can be prevented from transmitting the signal transmitted from the cell injected into the recess 14 farther than the adjacent recess 14. By adopting such a configuration, a signal short circuit occurs between cells in the adjacent recesses 14. On the other hand, of the plurality of recesses 14, adjacent recesses 14 It is possible to prevent a signal short circuit between cells in the recess 14 except for. In other words, it is possible to prevent signals from being transmitted between cells in the recess 14 that are two or more apart.
[0032] ここで、凹部 14の間の溝部 18の長さを約 30〜： LOOO μ mとすることが好ましい。な お、溝部 18の長さとは、隣接する凹部 14の間の連通方向における距離となる。すな わち、隣接する凹部 14を連通する部分の距離が 30〜： LOOO mとなる。よって、隣 接する凹部 14は 30〜： LOOO mの距離で離間される。第 2の凸部 19において、対 向する 2つの側壁 19a間の距離が 30〜3000 /ζ πιとなる。よって、隣接する凹部 14を 連通する方向の距離が 30〜： LOOO mとなる。換言すると、対向する側壁 19a間の 間隔力 S30〜： LOOO mとなる。従って、隣接する凹部 14は 30〜： LOOO mの距離を 隔てて配置され、高さ1〜50 111、幅 1〜： LO mの断面を有する溝部 18で連通する 。換言すると、連通方向と垂直な平面における断面が高さ 1〜50 m、幅 1〜： LO /z m であり、長さが 30〜： L000 mの細長い溝部 18が形成される。  Here, the length of the groove 18 between the recesses 14 is preferably about 30 to: LOOO μm. The length of the groove 18 is the distance in the communication direction between the adjacent recesses 14. In other words, the distance between the parts communicating with the adjacent recesses 14 is 30: LOOO m. Therefore, the adjacent recesses 14 are separated by a distance of 30 to: LOOO m. In the second convex portion 19, the distance between the two opposite side walls 19a is 30 to 3000 / ζ πι. Therefore, the distance in the direction in which the adjacent recesses 14 are communicated is 30 to: LOOO m. In other words, the spacing force S30 between the opposing side walls 19a is: LOOO m. Accordingly, the adjacent recesses 14 are arranged at a distance of 30 to: LOOO m, and communicate with each other by a groove portion 18 having a height of 1 to 50 111 and a width of 1 to: LO m. In other words, a cross-section in a plane perpendicular to the communication direction has a height of 1 to 50 m, a width of 1 to: LO / z m, and a length of 30 to L000 m.
[0033] さらに、第 2の凸部 19の幅と、深さの比は、 1 : 1〜1 : 20とすることが好ましい。すな わち、溝部 18の深さを溝部 18の溝さの 1〜20倍とする。  [0033] Further, the ratio of the width and depth of the second convex portion 19 is preferably 1: 1 to 1:20. In other words, the depth of the groove 18 is 1 to 20 times the groove of the groove 18.
[0034] 上記の構成によって、マイクロ空間構造内の細胞がシグナルネットワークを形成す る。例えば、神経細胞の突起部力ものネットワークのみが形成される。このシグナルネ ットワークを介して細胞間を信号が伝達する。このシグナルネットワークでは、隣接す るマイクロ空間構造同士のみが連絡する。  [0034] With the above configuration, cells in the micro-space structure form a signal network. For example, only a network of nerve cell protrusions is formed. Signals are transmitted between cells via this signal network. In this signal network, only adjacent microspace structures communicate.
[0035] また、凹凸パターンが設けられた領域に表面処理を行うことが好ましい。すなわち、 第 2の凸部 19の上面及び側面 (側壁 19a)、並びに、凹部 14の底面に表面処理を行 うことが好ましい。表面処理としては、例えば、有機材料または無機材料のコーティン グ処理を行うことができる。有機材料または無機材料は目的によって適宜公知の材 料を選択すればよい。また、紫外線、電子線などの活性エネルギー線で処理したり、 その他薬品により直接表面を改質しても良い。  [0035] In addition, it is preferable to perform a surface treatment on a region where the uneven pattern is provided. That is, it is preferable to perform a surface treatment on the upper surface and the side surface (side wall 19a) of the second convex portion 19 and the bottom surface of the concave portion 14. As the surface treatment, for example, a coating treatment of an organic material or an inorganic material can be performed. As the organic material or inorganic material, a known material may be appropriately selected depending on the purpose. Further, the surface may be modified with an active energy ray such as an ultraviolet ray or an electron beam, or directly modified with other chemicals.
[0036] 細胞培養容器上の凹凸パターンは、シリコンを材料とした半導体加工技術や、フォ トリソグラフ法による Ni製スタンパー (原盤)を用いた成形技術によって形成することが できる。例えば、シリコンを材料とした半導体加工技術の場合、シリコン基板上にレジ ストを塗布し、レジストを露光、現像してレジストをパターユングする。このレジストの上 力 シリコンをエッチングすることによって、シリコン基板がパターユングされ、凹凸パ ターンが形成される。そして、レジストを剥離することにより、所望の凹凸パターンを有 するシリコン製細胞培養容器が完成する。もちろん、細胞培養容器用の基板はシリコ ン基板に限らず、ガラス基板ゃ榭脂基板であってもよい。このように半導体加工技術 を用いることによって、精度よく凹凸パターンを形成することができる。 The concavo-convex pattern on the cell culture vessel can be formed by a semiconductor processing technique using silicon as a material, or a molding technique using a Ni stamper (master) using a photolithographic method. For example, in the case of semiconductor processing technology using silicon as a material, a resist is applied on a silicon substrate, and the resist is exposed and developed to pattern the resist. On this resist By etching the force silicon, the silicon substrate is patterned and an uneven pattern is formed. Then, by removing the resist, a silicon cell culture container having a desired uneven pattern is completed. Of course, the substrate for the cell culture container is not limited to a silicon substrate, and may be a glass substrate or a resin substrate. As described above, by using the semiconductor processing technique, the uneven pattern can be formed with high accuracy.
[0037] また、 Niスタンパを用いた成形技術の場合、まず、基板上に第 1のレジスト層を塗 布、露光し、さらにその上力 第 2のレジスト層を塗布、露光する。この第 1のレジスト 層及び第 2のレジストを一括原像することによって基板上に凹凸パターンを形成する ことができる。そして、この凹凸パターンが形成された基板に導電性膜を蒸着法ゃス ノ ッタリング法によって付着する。そして、導電性薄膜の上力 金属をメツキにより堆 積して金属構造体を形成する。金属構造体を基板から剥離することによってスタンパ を形成することができる。このスタンパを用いて成形することによって、高精度の細胞 培養溶液を高い生産性で製造することができる。この場合、細胞培養容器は、例え ば、榭脂材料により形成される。もちろん、金属構造体を作成するための構造体をシ リコン基板やガラス基板などによって形成してもよい。さら〖こ、細胞培養容器の製造方 法は、上記の方法に限定されるものではない。具体的には、機械切削や、ガラスへの エッチング処理法などにより、細胞培養容器 10を製造してもよい。これによつて、大 面積 '細胞培養株製造も可能となる。また、細胞培養容器の素材をガラスゃ榭脂など の透明材料とすることによって、細胞の観察を容易に行うことができる。  [0037] In the case of a molding technique using a Ni stamper, first, a first resist layer is applied onto a substrate and exposed, and then a second resist layer is applied and exposed. An uneven pattern can be formed on the substrate by batch original images of the first resist layer and the second resist. Then, a conductive film is deposited on the substrate on which the concave / convex pattern is formed by a vapor deposition method or a notching method. Then, the upper metal of the conductive thin film is deposited by plating to form a metal structure. The stamper can be formed by peeling the metal structure from the substrate. By molding using this stamper, a highly accurate cell culture solution can be produced with high productivity. In this case, the cell culture container is formed of, for example, a resin material. Of course, a structure for forming a metal structure may be formed using a silicon substrate, a glass substrate, or the like. Furthermore, the method for producing the cell culture container is not limited to the above method. Specifically, the cell culture vessel 10 may be manufactured by mechanical cutting or an etching method for glass. This makes it possible to produce large-area 'cell culture strains. In addition, cells can be easily observed by using a transparent material such as glass resin for the material of the cell culture container.
[0038] 上記の細胞培養容器 10を用いて細胞を培養することにより、細胞間におけるシグ ナル伝達が制御された状態で細胞を培養することができる。具体的には、上記の形 状を有する細胞培養容器 10を用意し、細胞培養容器 10の凹部 14に例えば、 1〜5 個の細胞を注入する。そして、溝部 18及び凹部 14に培養液を供給して細胞を培養 する。このとき、培養細胞間におけるシグナルの伝達は、第 2の凸部 19の側壁 19aに よって制御される。具体的には、隣接する凹部 14以外の凹部 14に含まれる培養細 胞にシグナルが伝達されるのを防ぐことができる。生体組織と同等な環境での培養が 可能となり、生体組織を再現可能なシグナル伝達経路を実現する。従って、マイクロ 空間構造内の細胞がシグナルネットワークを形成する。例えば、神経細胞の突起部 力 のネットワークのみが形成される。なお、上記した細胞培養容器 10の具体的な寸 法は例示であり、本発明は、上記の範囲に限定されるものではない。例えば、溝部 1 8の幅、深さ及び長さを、複数の凹部 14のうち、隣接する凹部 14よりも離間した 2つ の凹部内の細胞間での信号短絡を防止することができるよう形成すればよい。さらに 、溝部 18の寸法は、培養細胞によって変化させてもよい。 [0038] By culturing cells using the cell culture vessel 10 described above, the cells can be cultured in a state in which signal transmission between the cells is controlled. Specifically, a cell culture container 10 having the above shape is prepared, and 1 to 5 cells, for example, are injected into the recess 14 of the cell culture container 10. Then, the culture medium is supplied to the groove 18 and the recess 14 to culture the cells. At this time, signal transmission between the cultured cells is controlled by the side wall 19 a of the second convex portion 19. Specifically, the signal can be prevented from being transmitted to the cultured cells contained in the recesses 14 other than the adjacent recesses 14. Cultivation in an environment equivalent to that of living tissue becomes possible, and a signal transduction pathway that can reproduce living tissue is realized. Thus, cells within the microspace structure form a signal network. For example, the protrusion of a nerve cell Only a power network is formed. The specific dimensions of the cell culture container 10 described above are examples, and the present invention is not limited to the above range. For example, the width, depth, and length of the groove portion 18 are formed so as to prevent signal short-circuiting between cells in two of the plurality of recesses 14 that are separated from the adjacent recesses 14. do it. Furthermore, the dimension of the groove 18 may be changed depending on the cultured cells.
[0039] 次に本発明にかかる細胞培養容器の実施例について図 2〜図 4を用いて説明する Next, an example of the cell culture container according to the present invention will be described with reference to FIGS.
[0040] [実施例 1] [0040] [Example 1]
実施例 1にかかる細胞培養容器の形状について図 2を用いて説明する。図 2は、本 実施例に力かる細胞培養容器の形状を模式的に示す図である。図 2では、複数の凹 部 14が 1枚の基板上に形成されている。凹部 14は基板面上に縦、横に並んで配列 されている。この凹部 14の間には、溝部 18が設けられている。  The shape of the cell culture container according to Example 1 will be described with reference to FIG. FIG. 2 is a diagram schematically showing the shape of a cell culture vessel that is useful in this example. In FIG. 2, a plurality of recesses 14 are formed on one substrate. The recesses 14 are arranged side by side vertically and horizontally on the substrate surface. A groove 18 is provided between the recesses 14.
[0041] ここで、細胞培養容器 10の表面上において、図 2中の横方向を幅方向とし、それと 直交する縦方向を奥行き方向とする。本実施例では、幅方向に 4つ、奥行き方向に 3 つの凹部 14が形成された細胞培養容器 10を示している。すなわち、細胞培養容器 10には、 12個の凹部 14がマトリクス状に形成されている。ここでは、幅方向に隣接す る凹部 14は、 1つの溝部 18で連通されている。一方、奥行き方向に隣接する凹部 14 は、 2つの溝部 18で連通されている。すなわち、奥行き方向に隣接する凹部 14の間 の溝部 18には、第 1の凸部 13が設けられている。この第 1の凸部 13によって、奥行 き方向に隣接する凹部 14の間の溝部 18が、 2つに区分けされて 、る。  [0041] Here, on the surface of the cell culture vessel 10, the horizontal direction in FIG. 2 is defined as the width direction, and the vertical direction perpendicular thereto is defined as the depth direction. In this embodiment, a cell culture vessel 10 is shown in which four recesses 14 are formed in the width direction and three in the depth direction. That is, twelve recesses 14 are formed in a matrix in the cell culture container 10. Here, the recesses 14 adjacent to each other in the width direction are communicated with each other by a single groove 18. On the other hand, the recesses 14 adjacent to each other in the depth direction are communicated with each other by two groove portions 18. That is, the first convex portion 13 is provided in the groove portion 18 between the concave portions 14 adjacent in the depth direction. By the first convex portion 13, the groove portion 18 between the concave portions 14 adjacent to each other in the depth direction is divided into two.
[0042] 図 2に示すように、細胞培養容器 10の奥行き方向の大きさを A、細胞培養容器 10 の幅方向の大きさを B、細胞培養容器 10の厚み（高さ）を Cとする。また、隣接する凹 部 14のピッチを dとする。ここでは、複数の凹部 14をほぼ等間隔で形成している。さら に、溝部 18の奥行き方向のピッチも dとなる。 1つの凹部 14の長さを eとし、凹部 14の 深さを fとする。また、溝部 18の幅を g、溝部 18の深さを h、溝部 18の長さを iとする。こ こで、本実施例に力かる細胞培養容器 10における A、 B、 C、 d、 e、 f、 g、 h及び iの好 適な数値範囲を表 1に示す。なお、表 1において、括弧内に示された数値は、各寸法 のより好適な数値範囲を示す。 [0043] [表 1] [0042] As shown in FIG. 2, the size of the cell culture vessel 10 in the depth direction is A, the size of the cell culture vessel 10 in the width direction is B, and the thickness (height) of the cell culture vessel 10 is C. . Also, let d be the pitch of adjacent recesses 14. Here, the plurality of recesses 14 are formed at substantially equal intervals. Furthermore, the pitch in the depth direction of the groove 18 is also d. The length of one recess 14 is e, and the depth of the recess 14 is f. Further, the width of the groove 18 is g, the depth of the groove 18 is h, and the length of the groove 18 is i. Here, Table 1 shows suitable numerical ranges of A, B, C, d, e, f, g, h, and i in the cell culture vessel 10 that is useful in this example. In Table 1, the numerical values shown in parentheses indicate a more preferable numerical range for each dimension. [0043] [Table 1]
Figure imgf000012_0001
Figure imgf000012_0001
[0044] 表 1には、細胞の培養試験に適した寸法と、培養株の製造及び組織培養に適した 寸法とが記載されている。表 1に示す寸法の範囲に凹部 14、溝部 18及び細胞培養 容器 10の大きさを設定することにより、シグナル伝達を制御可能な細胞培養容器を 実現することができる。ここで、 fZeが 1Z1〜1Z20とする。すなわち、凹部 14の長 さ eを凹部 14の深さ fの 1倍〜 20倍とする。さらに、 gZhが lZl〜20Zlとする。すな わち、溝部 18の深さ hを溝部 18の幅 gの 1〜 20倍とする。 [0044] Table 1 lists dimensions suitable for cell culture tests and dimensions suitable for production of cultures and tissue culture. By setting the size of the concave portion 14, the groove portion 18 and the cell culture vessel 10 within the dimensions shown in Table 1, a cell culture vessel capable of controlling signal transmission can be realized. Here, fZe is assumed to be 1Z1 to 1Z20. That is, the length e of the recess 14 is set to 1 to 20 times the depth f of the recess 14. Furthermore, gZh is assumed to be lZl to 20Zl. That is, the depth h of the groove 18 is set to 1 to 20 times the width g of the groove 18.
[0045] [実施例 2]  [0045] [Example 2]
実施例 2にかかる細胞培養容器の形状について図 3を用いて説明する。図 3は、本 実施例に力かる細胞培養容器の形状を模式的に示す図である。図 3では、 2枚の基 板を貼り合わせることによって細胞培養容器 10を形成している。すなわち、第 1の基 板 21と第 2の基板 22とを貼り合わせることによって細胞培養容器 10が形成される。こ こで、第 1の基板 21に貫通孔 23を形成して凹部 14としている。この場合、第 2の基板 22の接合面が、凹部 14の底面となる。第 2の基板 22の表面には、溝部 18を形成す るための微細な凹部 24が形成される。そして、貫通孔 23が形成された第 1の基板 21 と溝部 18が形成された第 2の基板 22とを密着させる。このとき、第 2の基板 22の溝部 18が形成されている面が接合面となって、貼り合わせられる。第 2の基板 22の溝部 1 8は、隣接する貫通孔を結ぶ位置に所定の形状で設けられている。また、貫通孔 23 は、平面形状が円形になっている。溝部 18は貫通孔 23が設けられている位置まで 延在される。これにより、隣接する凹部 14が底面側連通する。 The shape of the cell culture container according to Example 2 will be described with reference to FIG. FIG. 3 is a diagram schematically showing the shape of a cell culture vessel that is useful in this example. In FIG. 3, the cell culture container 10 is formed by bonding two substrates together. That is, the cell culture container 10 is formed by bonding the first substrate 21 and the second substrate 22 together. Here, a through hole 23 is formed in the first substrate 21 to form a recess 14. In this case, the bonding surface of the second substrate 22 becomes the bottom surface of the recess 14. A groove 18 is formed on the surface of the second substrate 22. A fine recess 24 is formed for this purpose. Then, the first substrate 21 in which the through hole 23 is formed and the second substrate 22 in which the groove portion 18 is formed are brought into close contact with each other. At this time, the surface on which the groove portion 18 of the second substrate 22 is formed becomes a bonding surface and is bonded. The groove portion 18 of the second substrate 22 is provided in a predetermined shape at a position connecting adjacent through holes. The through hole 23 has a circular planar shape. The groove 18 extends to a position where the through hole 23 is provided. As a result, the adjacent recesses 14 communicate with the bottom surface.
[0046] このような構成では、貫通孔 23によって細胞培養のためのマイクロ空間構造が形成 される。このとき、第 2の凸部 19の側壁 19aは貫通孔 23の内側面となる。本実施例に おいて、貫通孔 23の形成は、例えばマシユングセンター等が考えられ、その刀具の 長さは貫通孔 23のサイズが微細になれば短くなる。例えば、 φ 200 mの貫通孔 23 の場合、貫通可能な長さは 0. 5mm程度となる。この場合、第 1の基板 21の厚さを薄 くすることが好ましい。また、第 1の基板 21の厚さは、凹部 14が所定の深さになるよう に、選択される。 In such a configuration, a micro space structure for cell culture is formed by the through holes 23. At this time, the side wall 19 a of the second convex portion 19 becomes the inner side surface of the through hole 23. In this embodiment, the through-hole 23 can be formed by, for example, a machining center, and the length of the tool is shortened if the size of the through-hole 23 is reduced. For example, in the case of a through hole 23 of φ200 m, the length that can be penetrated is about 0.5 mm. In this case, it is preferable to reduce the thickness of the first substrate 21. Further, the thickness of the first substrate 21 is selected so that the concave portion 14 has a predetermined depth.
[0047] また、微細の溝部 18を有する第 2の基板 22は、密着性の観点から、軟質、タック感 を有する材料を用いることができる。密着性及び接合時間を考慮し、 PDMS (ポリジメ チルシロキサン)などの軟質材料を用いてもよい。また、溝部 18を形成するための凹 部 24を予め第 2の基板 22に形成して、第 1の基板 21と密着させる。このとき、貫通孔 23の位置関係に合った凹部 24を第 2の基板 22上に形成して、溝部 18とする。なお 、位置調整が必要な場合、基板周辺部に凹凸を形成して、位置合わせを行なうこと が可能である。本構成は、細胞を散布する側（貫通孔）と、ネットワークを構成する側（ 溝部 18)が異なるため、側壁上の細胞が、マイクロ空間構造内の細胞と信号短絡を 起こすことがなくなる。よって、より精密なネットワークを構成することが可能となる。な お、 2枚の基板を使用しない場合は、第 1の基板 21に格子状の溝部 18を形成する。  [0047] The second substrate 22 having the fine groove 18 may be made of a soft and tacky material from the viewpoint of adhesion. In consideration of adhesion and bonding time, soft materials such as PDMS (polydimethylsiloxane) may be used. Further, a recess 24 for forming the groove 18 is formed in advance on the second substrate 22 and is brought into close contact with the first substrate 21. At this time, a recess 24 matching the positional relationship of the through hole 23 is formed on the second substrate 22 to form a groove 18. If position adjustment is necessary, it is possible to perform alignment by forming irregularities on the periphery of the substrate. In this configuration, since the cell-spreading side (through hole) and the network-forming side (groove 18) are different, the cells on the side wall do not cause a signal short circuit with the cells in the micro space structure. Therefore, a more precise network can be configured. When two substrates are not used, the lattice-shaped grooves 18 are formed on the first substrate 21.
[0048] 細胞培養容器 10の表面上において、図 3中の横方向を幅方向とし、それと直交す る方向（縦方向）を奥行き方向とする。本実施例では、幅方向に 4つ、奥行き方向に 3 つの凹部 14が形成された細胞培養容器 10を示している。すなわち、細胞培養容器 10には、 12個の凹部 14がマトリクス状に形成されている。本実施例では、斜め方向 に隣接する凹部 14も溝部 18によって連通されている。すなわち、溝部 18は、幅方向 及び奥行き方向だけでなぐその間の斜め方向にも設けられている。従って、 1つの 凹部 14に対して、その周囲に設けられた最大 8個の凹部 14が連通される。さらに、 斜め方向に設けられた溝部 18は、異なる角度で斜め方向に設けられた溝部 18と交 差する。また、斜め方向に設けられた溝部 18は、幅方向及び奥行き方向に設けられ た溝部 18よりも長くなる。 [0048] On the surface of the cell culture vessel 10, the horizontal direction in FIG. 3 is defined as the width direction, and the direction (vertical direction) perpendicular thereto is defined as the depth direction. In this embodiment, a cell culture vessel 10 is shown in which four recesses 14 are formed in the width direction and three in the depth direction. That is, twelve recesses 14 are formed in a matrix in the cell culture container 10. In this embodiment, the recesses 14 adjacent to each other in the oblique direction are also communicated with each other by the groove portions 18. That is, the groove portion 18 has a width direction. And it is also provided in the diagonal direction between the depth direction. Therefore, a maximum of eight concave portions 14 provided around the concave portion 14 communicate with one concave portion 14. Furthermore, the groove 18 provided in the oblique direction intersects with the groove 18 provided in the oblique direction at a different angle. Moreover, the groove part 18 provided in the diagonal direction becomes longer than the groove part 18 provided in the width direction and the depth direction.
[0049] 図 3に示すように、細胞培養容器 10の奥行き方向の大きさを A、細胞培養容器 10 の幅方向の大きさを B、細胞培養容器 10の厚み（高さ）を Cとする。ここで、細胞培養 容器 10の厚みは、第 1の基板 21と第 2の基板 22との合計の厚さとなる。また、隣接す る凹部 14の奥行き方向のピッチを dとする。ここでは、幅方向及び奥行き方向に複数 の凹部 14をほぼ等間隔で形成している。また、溝部 18の幅方向及び奥行き方向の ピッチも dとなる。さらに、 1つの凹部 14の長さ（直径)を eとし、凹部 14の深さを fとする 。ここで、凹部 14の深さ fは、第 1の基板 21の厚さに対応する。また、溝部 18の幅を g 、深さを h、長さを iとする。ここで、本実施例に力かる細胞培養容器 10における A、 B 、 C、 d、 e、 f、 g、 h及び iの好適な数値範囲を表 2に示す。なお、表 2において、括弧 内に示された数値は、各寸法のより好適な数値範囲を示す。  [0049] As shown in FIG. 3, the size of the cell culture vessel 10 in the depth direction is A, the size of the cell culture vessel 10 in the width direction is B, and the thickness (height) of the cell culture vessel 10 is C. . Here, the thickness of the cell culture vessel 10 is the total thickness of the first substrate 21 and the second substrate 22. Further, the pitch in the depth direction of the adjacent recesses 14 is d. Here, a plurality of recesses 14 are formed at substantially equal intervals in the width direction and the depth direction. Further, the pitch in the width direction and depth direction of the groove portion 18 is also d. Further, e is the length (diameter) of one recess 14 and f is the depth of the recess 14. Here, the depth f of the recess 14 corresponds to the thickness of the first substrate 21. The width of the groove 18 is g, the depth is h, and the length is i. Here, Table 2 shows suitable numerical ranges of A, B, C, d, e, f, g, h, and i in the cell culture container 10 that is useful in this example. In Table 2, the numerical values shown in parentheses indicate a more preferable numerical range for each dimension.
[0050] [表 2] 寸法（ m)  [0050] [Table 2] Dimensions (m)
i口 、"央 培養株製造、組織培養  i Mouth, “Oh, culture production, tissue culture
A 3，000〜 150,000 150,000〜2，000，000  A 3,000 to 150,000 150,000 to 2,000,000
B 3，000〜 150,000 150,000〜2貝 000  B 3,000〜150,000 150,000〜2 shellfish 000
C 100〜 100,000 100〜200,000 d 100(200)〜2，000(1000) 100(200)〜2，000( 1000) e 5(15)〜500(200) 5(15)〜500(200) f 5(7)〜 1,000(300) 5(7)〜 1,000(300) g 1(2)〜10(7) 1(2)〜10(7)  C 100 to 100,000 100 to 200,000 d 100 (200) to 2,000 (1000) 100 (200) to 2,000 (1000) e 5 (15) to 500 (200) 5 (15) to 500 (200) f 5 (7) to 1,000 (300) 5 (7) to 1,000 (300) g 1 (2) to 10 (7) 1 (2) to 10 (7)
h 1(2)〜50(30) 1(2)〜50(30)  h 1 (2) to 50 (30) 1 (2) to 50 (30)
20(30)〜2,000(1000) 20(30)〜2,000(1000) f/e 1/1-1/20 1/1〜1/20  20 (30) to 2,000 (1000) 20 (30) to 2,000 (1000) f / e 1 / 1-1 / 20 1/1 to 1/20
g/h 1/·！〜 1/20 1/1〜1/20 [0051] 表 2には、細胞の培養試験に適した寸法と、培養株の製造及び組織培養に適した 寸法とが記載されている。表 2に示す寸法の範囲に凹部 14、溝部 18及び細胞培養 容器 10の大きさを設定することにより、シグナル伝達を制御可能な細胞培養容器 10 を実現することができる。ここで、 fZeが lZl〜20Zlとする。すなわち、凹部 14の 深さ fを凹部 14の長さ eの 1倍〜 20倍とする。さらに、 gZhが lZl〜20Zlとする。 すなわち、溝部 18の幅 gを溝部 18の深さ hの 1〜20倍とする。 g / h 1 / ·! ~ 1/20 1/1 ~ 1/20 [0051] Table 2 lists dimensions suitable for cell culture tests and dimensions suitable for production of cultures and tissue culture. By setting the size of the concave portion 14, the groove portion 18 and the cell culture vessel 10 within the range of dimensions shown in Table 2, the cell culture vessel 10 capable of controlling signal transmission can be realized. Here, fZe is assumed to be lZl to 20Zl. That is, the depth f of the recess 14 is set to 1 to 20 times the length e of the recess 14. Furthermore, gZh is assumed to be lZl to 20Zl. That is, the width g of the groove 18 is set to 1 to 20 times the depth h of the groove 18.
[0052] [実施例 3]  [0052] [Example 3]
実施例 3にかかる細胞培養容器の形状について図 4を用いて説明する。図 4は、本 実施例に力かる細胞培養容器の形状を模式的に示す図である。図 4では、複数の凹 部 14が 1枚の基板上に形成されている。凹部 14は基板面上に縦、横に並んで配列 されている。この凹部 14の間には、溝部 18が設けられている。  The shape of the cell culture container according to Example 3 will be described with reference to FIG. FIG. 4 is a diagram schematically showing the shape of a cell culture vessel that is useful in this example. In FIG. 4, a plurality of recesses 14 are formed on one substrate. The recesses 14 are arranged side by side vertically and horizontally on the substrate surface. A groove 18 is provided between the recesses 14.
[0053] ここで、細胞培養容器 10の表面上において、図 4中の横方向を幅方向とし、それと 直交する方向を奥行き方向とする。本実施例では、幅方向に 4つ、奥行き方向に 4つ の凹部 14が形成された細胞培養容器 10を示している。すなわち、細胞培養容器 10 には、 16個の凹部 14がマトリクス状に形成されている。本実施例では、斜め方向に 隣接する凹部 14も溝部 18によって連通されている。すなわち、溝部 18は、幅方向及 び奥行き方向だけでなぐその間の斜め方向にも設けられている。また、本実施例で は、実施例 2と異なり、斜め方向の溝部 18同士が交差していない。すなわち、斜め方 向の溝部 18は全て同じ方向に設けられている。よって、 1つの凹部 14に対して、その 周囲に設けられた最大 6個の凹部 14が連通される。この 6個の凹部 14を、正六角形 となるよう酉己置してちょい。  [0053] Here, on the surface of the cell culture vessel 10, the horizontal direction in FIG. 4 is defined as the width direction, and the direction perpendicular thereto is defined as the depth direction. In this embodiment, a cell culture container 10 is shown in which four recesses 14 are formed in the width direction and four in the depth direction. That is, the cell culture container 10 has 16 recesses 14 formed in a matrix. In the present embodiment, the recesses 14 adjacent to each other in the oblique direction are also communicated by the groove portions 18. That is, the groove 18 is provided not only in the width direction and the depth direction but also in an oblique direction therebetween. Further, in the present embodiment, unlike the second embodiment, the diagonal groove portions 18 do not intersect each other. That is, the oblique grooves 18 are all provided in the same direction. Therefore, a maximum of six recesses 14 provided around the recess 14 communicate with one recess 14. Place these six recesses 14 in a regular hexagon.
[0054] 図 4に示すように、細胞培養容器 10の奥行き方向の大きさを A、細胞培養容器 10 の幅方向の大きさを B、細胞培養容器 10の厚み（高さ）を Cとする。また、隣接する凹 部 14のピッチを dとする。ここでは、複数の凹部 14をほぼ等間隔で形成している。さら に、溝部 18の奥行き方向のピッチも dとなる。 1つの凹部 14の長さ（直径)を eとし、凹 部 14の深さを する。また、溝部 18の幅を g、溝部 18の深さを h、溝部 18の長さを iと する。ここで、本実施例に力かる細胞培養容器 10における A、 B、 C、 d、 e、 f、 g、 h及 び iの好適な数値範囲を表 3に示す。なお、表 3において、括弧内に示された数値は 、各寸法のより好適な数値範囲を示す。 As shown in FIG. 4, the size of the cell culture vessel 10 in the depth direction is A, the size of the cell culture vessel 10 in the width direction is B, and the thickness (height) of the cell culture vessel 10 is C. . Also, let d be the pitch of adjacent recesses 14. Here, the plurality of recesses 14 are formed at substantially equal intervals. Furthermore, the pitch in the depth direction of the groove 18 is also d. The length (diameter) of one recess 14 is e, and the depth of the recess 14 is defined. The width of the groove 18 is g, the depth of the groove 18 is h, and the length of the groove 18 is i. Here, Table 3 shows suitable numerical ranges of A, B, C, d, e, f, g, h, and i in the cell culture container 10 that is useful in this example. In Table 3, the numbers shown in parentheses are The more preferable numerical range of each dimension is shown.
[0055] [表 3] [0055] [Table 3]
Figure imgf000016_0001
Figure imgf000016_0001
[0056] 表 3には、細胞の培養試験に適した寸法と、培養株の製造及び組織培養に適した 寸法とが記載されている。表 3に示す寸法の範囲に凹部 14、溝部 18及び細胞培養 容器 10の大きさを設定することにより、シグナル伝達を制御可能な細胞培養容器 10 を実現することができる。ここで、 fZeが lZl〜20Zlとする。すなわち、凹部 14の 深さ fを凹部 14の長さ eの 1倍〜 20倍とする。さらに、 gZhが lZl〜20Zlとする。 すなわち、溝部 18の幅 gを溝部 18の深さ hの 1〜20倍とする。 [0056] Table 3 lists dimensions suitable for cell culture tests and dimensions suitable for production of cultures and tissue culture. By setting the size of the concave portion 14, the groove portion 18 and the cell culture vessel 10 within the dimensions shown in Table 3, the cell culture vessel 10 capable of controlling signal transmission can be realized. Here, fZe is assumed to be lZl to 20Zl. That is, the depth f of the recess 14 is set to 1 to 20 times the length e of the recess 14. Furthermore, gZh is assumed to be lZl to 20Zl. That is, the width g of the groove 18 is set to 1 to 20 times the depth h of the groove 18.
産業上の利用可能性  Industrial applicability
[0057] 本発明は、例えば、組織から単離した細胞を培養し、試験、検査に用いるための細 胞培養容器に利用される。 [0057] The present invention is used, for example, in a cell culture vessel for culturing cells isolated from tissues and using them for testing and testing.

Claims

請求の範囲 The scope of the claims
[1] 複数の凹部と、  [1] a plurality of recesses;
前記複数の凹部の間に設けられ、隣接する前記凹部を連通する溝部とを備え、 前記凹部を連通する溝部の連通方向の長さが 10〜3000 μ mである細胞培養容  A cell culture container, comprising: a groove portion provided between the plurality of concave portions and communicating with the adjacent concave portions, wherein the length of the groove portion communicating with the concave portion is 10 to 3000 μm.
[2] 複数の凹部と [2] With multiple recesses
前記複数の凹部の間に設けられ、隣接する前記凹部を連通する溝部とを備え、 前記凹部を連通する溝部が、前記複数の凹部のうち、前記隣接する凹部よりも離 間した 2つの凹部内の細胞間での信号短絡を防止することができるよう設けられてい る細胞培養容器。  A groove portion that is provided between the plurality of recesses and communicates with the adjacent recesses, and the groove portion that communicates with the recesses is in two recesses separated from the adjacent recesses among the plurality of recesses. A cell culture vessel provided to prevent signal short-circuiting between cells.
[3] 前記溝部の深さが 1〜50 μ mである請求項 1又は 2に記載の細胞培養容器。  [3] The cell culture container according to claim 1 or 2, wherein the groove has a depth of 1 to 50 μm.
[4] 前記溝部の幅が 1〜： LO μ mである請求項 1、 2又は 3に記載の細胞培養容器。 [4] The cell culture container according to [1], [2] or [3], wherein the groove has a width of 1 to: LO μm.
[5] 前記溝部の深さが前記溝部の幅の 1〜20倍である請求項 1乃至 4のいずれかに記 載の細胞培養容器。 [5] The cell culture container according to any one of [1] to [4], wherein the depth of the groove is 1 to 20 times the width of the groove.
[6] 前記凹部の深さが 5〜： LOOO μ mである請求項 1乃至 5のいずれかに記載の細胞培 養容器。  [6] The cell culture container according to any one of [1] to [5], wherein the recess has a depth of 5 to: LOOO μm.
[7] 前記凹部の長さが 5〜500 mである請求項 1乃至 6のいずれかに記載の細胞培 養容器。  [7] The cell culture container according to any one of [1] to [6], wherein the recess has a length of 5 to 500 m.
[8] 前記凹部の深さが前記凹部の長さの 1〜20倍である請求項 1乃至 7のいずれかに 記載の細胞培養容器。  [8] The cell culture container according to any one of [1] to [7], wherein the depth of the recess is 1 to 20 times the length of the recess.
[9] 請求項 1乃至 8のいずれかに記載の細胞培養容器を用意し、 [9] A cell culture container according to any one of claims 1 to 8 is prepared,
前記細胞培養容器の前記凹部に細胞を注入し、  Injecting cells into the recess of the cell culture container,
前記溝部に培養液を供給して前記細胞を培養して、  Supplying a culture solution to the groove and culturing the cells;
前記隣接する凹部内に含まれる培養細胞間に対して、信号が伝達するシグナルネ ットワークを形成させる培養細胞のシグナル伝達の制御方法。  A method for controlling signal transmission of cultured cells, wherein a signal network for transmitting signals is formed between the cultured cells contained in the adjacent recesses.
[10] 請求項 1乃至 8のいずれかに記載の細胞培養容器を用意し、 [10] A cell culture vessel according to any one of claims 1 to 8 is prepared,
前記細胞培養容器の前記凹部に細胞を注入し、  Injecting cells into the recess of the cell culture container,
前記溝部に培養液を供給して前記細胞を培養する細胞培養方法。  A cell culture method for culturing the cells by supplying a culture solution to the groove.
PCT/JP2006/322096 2005-11-08 2006-11-06 Cell culture container, cell culture method and method of regulating signal transduction in cultured cells WO2007055171A1 (en)

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