WO2023228814A1 - Microarray chip and cell culture method using microarray chip - Google Patents

Microarray chip and cell culture method using microarray chip Download PDF

Info

Publication number
WO2023228814A1
WO2023228814A1 PCT/JP2023/018246 JP2023018246W WO2023228814A1 WO 2023228814 A1 WO2023228814 A1 WO 2023228814A1 JP 2023018246 W JP2023018246 W JP 2023018246W WO 2023228814 A1 WO2023228814 A1 WO 2023228814A1
Authority
WO
WIPO (PCT)
Prior art keywords
microarray chip
cells
wells
cell
culture
Prior art date
Application number
PCT/JP2023/018246
Other languages
French (fr)
Japanese (ja)
Inventor
昌平 山村
秀貴 上野
元 重藤
Original Assignee
国立研究開発法人産業技術総合研究所
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 国立研究開発法人産業技術総合研究所 filed Critical 国立研究開発法人産業技術総合研究所
Publication of WO2023228814A1 publication Critical patent/WO2023228814A1/en

Links

Images

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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms

Definitions

  • the present invention relates to a microarray chip and a cell culture method using the microarray chip.
  • the present invention relates to a microarray chip that can reliably separate large numbers of single cells and culture monoclonal cell colonies with high efficiency, and a cell culture method using the same.
  • the cells that make up cancer differ in their resistance to anticancer drugs, and in order to detect drug-resistant strains, it is necessary to separate many cells into single cells and collect the separated single cells. It is necessary to cultivate and evaluate. However, it is extremely difficult, for example, to detect and isolate a single target cancer cell from a large amount of cells in blood or tissue, and to culture and evaluate the isolated single cell. By culturing cell populations in petri dishes, it is difficult to efficiently detect and clone resistant strains at the single-cell level. Therefore, in order to trap and culture cells, microchannel devices of various shapes having microchannels with fine structures have been proposed.
  • a microchannel device for single cell separation comprising an upper layer of glass slide and a lower layer made of PDMS having microchannels and wells, and captures a single cell at the downstream end of each well.
  • a device in which a U-shaped trap is formed is known (for example, see Non-Patent Document 1).
  • each well also has a large surface area that provides space for the captured single cells to expand and grow.
  • a dual-well type microfluidic device which is configured to capture and culture single cells by arranging microwells of a minute size and microwells of a larger size to face each other vertically.
  • Non-Patent Document 1 and Non-Patent Document 2 mentioned above disclose a method of trapping and culturing cells using a microchannel device.
  • Due to the complexity of operations and the difficulty of recovering cells from microchannels there is still a need for devices and methods that can reliably separate many single cells and culture them with high efficiency.
  • the present invention was made in view of the above-mentioned circumstances, and its purpose is to provide a microarray chip and a microarray chip that can reliably separate a large number of single cells and culture monoclonal colonies with high efficiency.
  • An object of the present invention is to provide a cell culture method using the present invention.
  • a microarray chip in which a plurality of wells capable of accommodating cells are formed, each of the plurality of wells including a first part capable of accommodating a plurality of cells, and a first part capable of accommodating a plurality of cells. and a second portion capable of accommodating only cells, the first portion being formed as a recess recessed from the surface of the microarray chip, and the second portion being formed from the bottom surface of the first portion.
  • the present invention relates to a microarray chip formed as a concave recess.
  • the present invention is a cell culture method using the above-mentioned microarray chip, comprising: (a) applying a liquid containing a plurality of cells onto the microarray chip; (b) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in step (a); (c) culturing the single cells accommodated in the plurality of wells obtained in step (b).
  • the present invention is a method for evaluating a single cell using the above-mentioned microarray chip, comprising: (A) applying a liquid containing a plurality of cells onto the microarray chip; (B) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in the step (A); (C) culturing the single cells accommodated in the plurality of wells obtained in the step (B); (D) The present invention relates to a method including the step of evaluating the cell characteristics of a single cell based on the state after culturing in the step (C).
  • the present invention is a method for producing a monoclonal cell colony using the above-mentioned microarray chip, comprising: (a) applying a liquid containing a plurality of cells onto the microarray chip; (b) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in step (a); (c) culturing the single cells accommodated in the plurality of wells obtained in step (b).
  • the present invention it is possible to realize a microarray chip and a cell culture method using the microarray chip that can reliably separate a large number of single cells and obtain monoclonal colonies with high efficiency.
  • FIG. 1 is a top perspective view schematically showing the configuration of wells in a microarray chip according to a first embodiment of the present invention.
  • FIG. 2 is a sectional view taken along line AA in FIG.
  • FIG. 3 is a top view schematically showing a microarray chip.
  • 4A and 4B are diagrams showing modified examples of wells.
  • 5A to 5E are diagrams showing modified examples of wells.
  • FIGS. 6A to 6C are conceptual diagrams showing an example of cell behavior in a single well in a cell culture method using a microarray chip according to the second embodiment of the present invention.
  • FIGS. 7A and 7B are fluorescence images of wells 3 hours after seeding HeLa cells on the microarray chip (FIG.
  • FIG. 8 is a graph showing the single-cell accommodation rate of HeLa cells in each well when the well structure of the microarray chip is changed.
  • 9A and 9B are fluorescence images of the microarray chip immediately after seeding HeLa cells on the microarray chip (FIG. 9A) and 15 days after culture (FIG. 9B).
  • FIG. 10 is a graph showing the relationship between the pitch between wells and the single-cell accommodation rate of HeLa cells.
  • FIG. 11 is a graph showing the single-cell proliferation rate of HeLa cells in culture using microarray chips with different pitches between wells.
  • FIG. 12 is a fluorescence image of a microarray chip provided with wells with an inner diameter Y of 110 ⁇ m in the first portion 21 after 6 days of culturing HeLa cells.
  • FIG. 13 is a graph showing the single-cell proliferation rate of HeLa cells in culture using a microarray chip equipped with a well in which the first portion 21 has an inner diameter Y of 110 ⁇ m.
  • FIG. 14 is a fluorescence image of a microarray chip having a well with an inner diameter Y of 130 ⁇ m in the first portion 21 after 6 days of culturing HeLa cells.
  • 15 is a graph showing the single-cell proliferation rate of HeLa cells in culture using a microarray chip equipped with a well in which the first portion 21 has an inner diameter Y of 130 ⁇ m.
  • 16A and 16B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 16A) and on the 15th day of culture (FIG. 16B) in culture using the microarray chip of Comparative Example 1.
  • 17A and 17B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 17A) and on the 15th day of culture (FIG. 17B) in culture using the microarray chip of Comparative Example 2.
  • FIG. 18A and 18B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 18A) and on the 15th day of culture (FIG. 18B) in culture using the microarray chip of Comparative Example 3.
  • 19A and 19B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 19A) and on the 15th day of culture (FIG. 19B) in culture using the microarray chip of Comparative Example 4.
  • Figures 20A and 20B show fluorescence and bright field images of the microarray chip immediately after seeding of PC9 cells (Figure 20A) and on the third day of culture with 5 ⁇ M Gefitinib ( Figure 20B) in culture using the microarray chip of the present invention. It is.
  • FIG. 21 is a graph showing the single cell proliferation rate of PC9 cells.
  • FIG. 1 is a top perspective view schematically showing the configuration of wells in a microarray chip according to a first embodiment of the present invention
  • FIG. 2 is a sectional view taken along line AA in FIG.
  • the microarray chip 1 is formed with a plurality of wells 2 capable of accommodating cells, and each of the plurality of wells 2 has a first portion 21 capable of accommodating a plurality of cells. and a second portion 22 that can accommodate only a single cell.
  • the first portion 21 is formed as a recessed portion recessed from the surface 3 of the microarray chip 1 .
  • the second portion 22 is formed as a recessed portion recessed from the bottom surface 211 of the first portion 21 . That is, each well 2 is configured as a two-stage well having one first part 21 in the upper stage and one second part 22 in the lower stage.
  • the bottom surface 211 of the first portion 21 may be parallel to the surface 3 or may be inclined. When the bottom surface 211 has an inclined shape, it may be inclined downward toward the center of the bottom surface 211, for example.
  • the bottom surface 221 of the second portion 22 may be parallel to the surface 3 or may be slightly recessed.
  • the second portion 22 functions as a trap that separates and captures only a single cell.
  • the first part 21 functions as a chamber for culturing and proliferating the single cells captured in the second part 22. Therefore, the inner diameter X of the opening of the second portion 22 is set smaller than the inner diameter Y of the first portion 21 (X ⁇ Y).
  • the openings of the first portion 21 and the second portion 22 may have a circular shape, for example, as shown in FIG. 1.
  • the shape of the opening is not limited to this, and the shape of the opening may be a regular polygon as described later. Below, as an example, a case will be described in which the openings of the first portion 21 and the second portion 22 are each circular and concentric.
  • the inner diameter X of the second portion 22 is dimensioned to accommodate only a single cell. That is, within the second portion 22, two or more cells to be captured cannot be arranged side by side in the radial direction of the second portion 22. Therefore, the inner diameter X of the second portion 22 is set to a size that allows some margin for the size of a single cell to be captured. Note that the size of a single cell can be appropriately set by those skilled in the art depending on the acquisition source, characteristics, and size distribution of the cells to be captured. Preferably, the inner diameter X can be set to a dimension with a margin of about 0 to 50 ⁇ m relative to the size of a single cell, or a dimension of 100% to 300% relative to the cell size.
  • the inner diameter X can be several tens of ⁇ m, for example about 5 to 100 ⁇ m depending on the type of cell. However, it is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the inner diameter X according to the size of the cell to be captured.
  • the inner diameter Y of the first part 21 is dimensioned to allow the formation of a cell colony from a single cell captured in the second part 22. That is, within the first portion 21, the dimensions are set such that, for example, a plurality of cells can be arranged side by side in the radial direction. Note that the inner diameter Y of the first portion 21 is such that unless the cells largely protrude from the first portion 21 and cause cross-contamination with the adjacent well 2, the cells will not line up in the radial direction and the well 2 will be The dimensions may be such that a plurality of cells overlap when viewed from above. Based on these considerations, for example, the inner diameter Y can be several hundred ⁇ m, for example about 50 to 500 ⁇ m. However, it is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the inner diameter Y according to the size of the cells to be cultured.
  • the depth S of the second portion 22, i.e. the length from the bottom surface 211 of the first portion 21 to the bottom surface 221 of the second portion 22, is dimensioned to accommodate only a single cell. . That is, within the second portion 22, two or more cells to be captured cannot be arranged side by side in the depth direction of the second portion 22. Therefore, the depth S of the second portion 22 is set to be slightly larger (deeper) than the size of one cell, or slightly smaller (shallower) than the size of one cell, with some allowance for the size of one cell. Set to dimensions. Regarding the depth S, the dimension with a slight margin can be set in the same way as the design of the inner diameter On the other hand, the size can be set to 100 to 300%.
  • the size slightly smaller than the size of one cell can be set to 50 to 100% of the size of the cell.
  • the depth S can be several tens of ⁇ m, for example about 5 to 40 ⁇ m depending on the type of cell.
  • the depth S is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the depth S according to the size of the cell to be captured.
  • the depth T of the first portion 21, that is, the length from the surface 3 of the microarray chip 1 to the bottom surface 211 of the first portion 21, is set to a dimension that can limit cell proliferation.
  • the depth is such that multiple layers of cells are not formed in the depth direction of the first portion 21, that is, the depth is such that only a single layer of cells is formed. can do.
  • the depth T of the first portion 21 is set such that multiple layers can be formed in the depth direction as long as the cells do not protrude significantly from the first portion 21 and cause cross-contamination with the adjacent well 2. It may be of any size. Based on these considerations, for example, the depth T can be several tens of ⁇ m, for example about 1 to 50 ⁇ m. However, the depth T is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the depth T according to the size of the cells to be cultured.
  • the wall angle U of the first portion 21 and the second portion 22 is the angle between the inner wall surfaces of the first portion 21 and the second portion 22 and a line perpendicular to the surface 3 of the first microarray chip 1. be.
  • the wall angle U is determined depending on the conditions at the time of manufacturing the microarray chip 1, and can be, for example, approximately 0 to 45 degrees, and preferably 0 to 20 degrees.
  • the first portion 21 and the second portion 22 each have a cylindrical shape
  • the wall angle U is larger than 0°
  • the first portion 21 and the second portion 22 have a cylindrical shape.
  • each has a truncated cone shape. Note that the wall angle U of the first portion 21 and the wall angle U of the second portion 22 may have different values.
  • the pitch V between adjacent wells 2, that is, the distance between the center of the opening of the second part 22 of the well 2 and the center of the opening of the second part 22 of the adjacent well 2 is the first The distance may be sufficient to reduce cross-contamination when culturing cells within the portion 21 of the cell.
  • the pitch V depends on the cell type, the inner diameter Y of the first portion 21, the inner diameter X of the second portion 22, etc., but can be set to about 100 to 500 ⁇ m, for example.
  • the material of the microarray chip 1 is not particularly limited, but may include polymers such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), silicone, etc.
  • polymers such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), silicone, etc.
  • examples include metal, glass, quartz glass, and combinations of multiple materials such as polymer, glass, metal, etc. (for example, PDMS and glass).
  • Preferred are polystyrene, PMMA, glass, silicon, etc.
  • the transparency and permeability of the microarray chip 1 are not particularly limited, but preferably have transparency and permeability suitable for observing cells accommodated in the wells 2.
  • the wells 2 have translucency.
  • transparent means that the transmittance of a predetermined light for observation is 80% or more, and preferably 90% or more.
  • the microarray chip 1 can be produced by various known methods.
  • the microarray chip 1 can be produced, for example, by directly forming wells 2 on a substrate material, by attaching a film in which through holes or wells 2 are formed to a substrate material, by plastic molding, or the like.
  • plastic molding specifically, lithography technology (optical lithography, electron beam lithography, etc.) in the semiconductor technology field, cutting using a micro drill, laser processing, etc. may be used. I can do it.
  • a plastic master of the microarray chip 1 is produced by lithography and etching, and a mold is produced by electroforming the master. Thereafter, the microarray chip 1 is manufactured by injection molding using the manufactured mold.
  • injection molding using a mold as described above it is possible to produce a microarray chip 1 with a smooth surface.
  • the microarray chip 1 may be subjected to surface treatment if necessary.
  • the surface treatment method is not particularly limited, but plasma treatment, corona discharge treatment, etc. can be used.
  • a hydrophilic treatment such as plasma treatment, for example oxygen plasma treatment, can be performed.
  • a hydrophilic material for example, silicon, glass, etc.
  • surface treatment can be performed by coating the well 2 with protein, lipid, etc.
  • examples include surface treatment with collagen, fibronectin, Poly-D-Lysine, Poly-L-Lysine, Laminin, Vitronectin, Matrigel, etc.
  • collagen fibronectin
  • Poly-D-Lysine Poly-D-Lysine
  • Poly-L-Lysine Laminin
  • Vitronectin Matrigel, etc.
  • FIG. 3 shows a top view schematically showing the entire microarray chip 1.
  • the microarray chip 1 is formed as a rectangular plate-like member as a whole.
  • a plurality of wells 2 are regularly arranged on the surface 3 of the microarray chip 1.
  • the microarray chip 1 includes a plurality of rows 1 to 6 along the long sides 11 and 12 of the microarray chip 1 and a plurality of columns A to P along the short sides 13 and 14.
  • a plurality of blocks 4 are arranged in a matrix.
  • the plurality of blocks 4 are arranged with gaps from the long sides 11, 12 and short sides 13, 14 of the microarray chip 1.
  • the plurality of blocks 4 are arranged within a range of, for example, about 50 mm x about 15 to 18 mm on the microarray chip 1, although this is not particularly limited.
  • each block 4 the plurality of wells 2 described above are arranged in a matrix. For example, several thousand wells 2 can be placed in each block 4. For example, if each block 4 includes 1000 wells 2, 96,000 wells 2 are arranged in the entire microarray chip 1 shown in FIG. Note that the number of wells 2 arranged on the microarray chip 1 is not limited.
  • the microarray chip 1 can include at least two wells 2. Note that it is not essential that a plurality of blocks be formed on one microarray chip, and one microarray chip may include only one block.
  • the microarray chip according to this embodiment may be an open type microarray chip with no cover or lid above the wells.
  • a large number of cells can be applied to the microarray chip at the same time, and it is also easy to bring the culture medium and drugs into contact with the cells, and to collect the cells. becomes.
  • a removable cover or lid may be provided above the well for the purpose of preventing contamination during culture.
  • microarray chip 1 According to the microarray chip 1 according to the present embodiment described above, the following effects can be achieved.
  • the microarray chip 1 is formed with a plurality of wells 2 that can accommodate cells.
  • Each of the plurality of wells 2 has a first portion 21 that can accommodate a plurality of cells and a second portion 22 that can accommodate only a single cell.
  • the first portion 21 is formed as a recess recessed from the surface 3 of the microarray chip 1
  • the second portion 22 is formed as a recess recessed from the bottom surface 211 of the first portion 21.
  • the microarray chip 1 is configured such that each well 2 has a two-tiered structure of an upper first part 21 and a lower second part 22.
  • the lower second portion 22 is configured to accommodate only a single cell, and is therefore suitable for separating a large number of cells into single cells.
  • the upper first part 21 is configured to be able to accommodate a plurality of cells, it is suitable for culturing and proliferating a single cell accommodated in the second part 22. In this manner, the microarray chip 1 can achieve reliable separation of single cells and highly efficient culture of the separated cells using one well 2 with a two-tiered structure. This makes it possible to detect and clone drug-resistant strains using the microarray chip 1.
  • tumor tissue in brain tumors with extremely poor prognosis (e.g., malignant glioma, etc.), tumor tissue can be developed cell by cell on a chip, and anti-cancer strains can be developed. It becomes possible to conduct drug resistance and susceptibility tests.
  • prognosis e.g., malignant glioma, etc.
  • the opening of the first part 21 and the opening of the second part 22 have circular shapes, appropriate shapes can be selected based on various conditions such as the cell type and the method of manufacturing the microarray chip 1. I can do it.
  • FIGS. 4A and 4B show modified examples of the well 2.
  • FIG. 4A shows an example in which the openings of the first part 21 and the second part 22 are regular pentagons
  • FIG. 4B shows an example in which the openings of the first part 21 and the second part 22 are regular triangles. An example is shown.
  • the diameter of the circumscribed circle CC of the regular polygon is taken as the inner diameter of the opening, and the inner diameter Y of the first portion 21 and the inner diameter X of the second portion 22 are determined as described above. can be set.
  • the length of each side of the equilateral triangle is used as the diameter of the opening instead of the diameter of the circumscribed circle, and the inner diameter Y of the first portion 21 and the The inner diameter X of the portion 22 of No. 2 may be set.
  • the cell type and the method for manufacturing the microarray chip 1 can be changed as in the case of the circular shape described above.
  • An appropriate shape can be selected based on various conditions such as.
  • FIGS. 5B to 5E show modified examples of well 2.
  • 5A shows an example in which the openings of the first part 21 and the second part 22 are concentric circles
  • FIGS. 5B to 5E show that the openings of the first part 21 and the second part 22 are concentric. This shows an example where the center of the opening is off-center.
  • the wells 2 shown in FIGS. 5B to 5E are arranged in the microarray chip 1 shown in FIG. Let's make an assumption and explain.
  • the center of the opening of the second portion 22 is located closer to the long side 11 of the microarray chip 1 shown in FIG. 3 with respect to the center of the opening of the first portion 21.
  • the center of the opening of the second portion 22 is located closer to the long side 12 of the microarray chip 1 with respect to the center of the opening of the first portion 21.
  • the center of the opening of the second portion 22 is located closer to the short side 13 of the microarray chip 1 shown in FIG. 3 with respect to the center of the opening of the first portion 21.
  • the center of the opening of the second portion 22 is located closer to the short side 14 of the microarray chip 1 with respect to the center of the opening of the first portion 21.
  • the opening of the first portion 21 and the opening of the second portion 22 are arranged so as to be in contact with each other when viewed from above the well 2, but there is no limitation to this.
  • the opening of the first portion 21 and the opening of the second portion 22 may be arranged apart from each other.
  • the shape of the opening of the first portion 21 and the shape of the opening of the second portion 22 are made the same, but the present invention is not limited to this.
  • the shape of the opening of the second portion 22 and the shape of the opening of the second portion 22 may be formed to be different.
  • the opening of the first portion 21 may be circular and the opening of the second portion 22 may be a regular polygon, or the opening of the first portion 21 may be a regular polygon and the opening of the second portion 22 may be a regular polygon.
  • the opening of the portion 22 may also be circular.
  • the outer shape of the microarray chip 1 is rectangular has been described, but the outer shape is not limited to this, and the outer shape may be other than the rectangular shape.
  • the plurality of wells 2 are arranged in a matrix on the microarray chip 1, but the invention is not limited to this, and the plurality of wells 2 may be arranged alternately. Furthermore, the shapes and dimensions of the openings of the plurality of wells 2 arranged on the microarray chip 1 may be the same, or may be set differently for each block 4, for example.
  • the second embodiment relates to a cell culture method using a microarray chip.
  • the cell culture method according to the second embodiment can also be considered as a method for using a microarray chip or a method for producing a monoclonal cell colony.
  • the method includes the following steps. (a) applying a liquid containing a plurality of cells onto the microarray chip; (b) washing the surface of the microarray chip obtained in step (a) to remove the second portion of the plurality of wells; (c) A step of culturing the cells accommodated in the plurality of wells obtained in the step (b).
  • FIGS. 6A to 6C are diagrams illustrating the cell culture method using the microarray chip according to the first embodiment, and conceptually illustrate the state of cells in one well 2 on the microarray chip 1 illustrated in FIGS. 1 to 3.
  • FIG. 6A to 6C are diagrams illustrating the cell culture method using the microarray chip according to the first embodiment, and conceptually illustrate the state of cells in one well 2 on the microarray chip 1 illustrated in FIGS. 1 to 3.
  • a liquid containing a plurality of cells is applied onto the microarray chip 1. That is, cells are seeded onto the microarray chip 1.
  • the cell concentration in the liquid may be at least as high as to cover the entire surface 3 of the microarray chip 1 with a single layer of cells at the time of applying the cell-containing liquid, for example, 1.0 x 10 cells. It is preferable to set it as 6 cells/mL or more. Further, the cell concentration in the liquid is, for example, about 1.0 ⁇ 10 10 cells/mL or less, preferably about 1.0 ⁇ 10 9 cells/mL or less, or about 1.0 ⁇ 10 8 cells/mL or less. can do.
  • a medium may be used as a type of liquid for seeding, and there may be cases where the seeds are cultured in a seeded state.
  • the method of applying the liquid onto the microarray chip is not particularly limited.
  • liquid can be dropped onto the microarray chip with a pipette.
  • the amount of the droplet is not particularly limited because it varies depending on the area of the microarray chip. A person skilled in the art can appropriately determine the amount to be added. After dropping, it is preferable to allow sufficient standing time for the cells to settle to the bottom of the well before the next step.
  • FIG. 6A schematically shows the state of well 2 and cells H after performing step (a).
  • the second portion 22 of well 2 contains single cells.
  • a plurality of cells are accommodated in the first portion 21, and cells are also present on the surface 3 of the microarray chip 1 around the well 2.
  • step (b) the surface 3 of the microarray chip 1 obtained in the step (a) is washed. This removes excess cells. Washing can be carried out, for example, by washing away excess cells present on the surface 3 of the microarray chip 1 with a liquid such as a buffer solution or a culture solution using a pipette. As another example, a scraper can be moved along the surface 3 of the microarray chip 1 to remove excess cells present on the surface 3 of the microarray chip.
  • FIG. 6BI is a diagram conceptually showing how surplus cells are removed, and FIG. 6BII shows that as a result of the removal of surplus cells, a single cell is accommodated in the second portion 22 of one well 2.
  • FIG. This makes it possible to efficiently accommodate a single cell in one well 2.
  • step (b) after performing a washing operation, imaging is performed using a microscope to identify wells containing single cells. Through this operation, wells in which monoclonal cell colonies can be produced are identified.
  • steps (a) and (b) tools commonly used in cell experiments, such as pipettes and scrapers, may be used in addition to the microarray chip 1 according to the present invention. It can be handled under certain conditions. Furthermore, although it depends on the size of the microarray chip 1, steps (a) and (b) can be carried out in about 30 to 120 minutes, for example. For example, for a microarray chip 1 having the size of a general slide glass (76 x 26 mm), approximately 30,000 to 40,000 cells, and about 60,000 cells at most, can be separated one by one at the same time and accommodated in each well. The state can be set as follows. However, the required time and the number of cells that can be accommodated are not limited to the exemplified values.
  • a step of obtaining the single cell accommodation rate can be performed. This step can be performed by imaging using a microscope.
  • step (c) the cells accommodated in the wells 2 of the microarray chip 1 obtained in the step (b) are cultured under predetermined conditions. That is, the microarray chip 1 obtained in step (b) can be used as is for culture. Culture conditions vary depending on the cells accommodated and are not particularly limited.
  • it may include a step of adding a medium necessary for culturing the cells, or a step of immersing the microarray chip in the medium.
  • the microarray chip 1 is open and integrated and easy to handle, so it can be adapted to any culture conditions.
  • FIG. 6C shows well 2 in which cells have proliferated and a monoclonal cell colony C has been obtained after culturing for a predetermined period in step (c).
  • 6A to 6C illustrate the behavior of cells in one well, but cells proliferate similarly in other wells of the microarray chip containing one cell, and monoclonal cell colonies can be obtained. . Note that there may be wells that do not accommodate one cell, and there may also be wells that accommodate two or more cells, and there may be cases where the growth shown in the figure is not possible for all the wells. However, by using the microarray chip according to the first embodiment, it is possible to achieve a single cell accommodation rate of at least 30% as a whole.
  • microarray chip 1 By using the microarray chip 1 according to the present invention, cells can be cultured easily and without disadvantages such as cross-contamination through steps (a) to (c), and monoclonal cell colonies can be obtained. I can do it.
  • step (d) may include the step of recovering cell colonies from each well after completion of step (c).
  • a pipette or a manipulator can be used to collect cell colonies.
  • the cell colonies recovered in this manner can be subjected to any desired purpose, such as various analytical steps or further growth steps.
  • the third embodiment relates to a method for evaluating single cells.
  • the single cell evaluation method includes the following steps.
  • step (D) Based on the state after culturing in the step (C). , Assessing Cellular Properties of a Single Cell
  • step (C) may include culturing the cells in the presence of a candidate substance capable of influencing the properties of the cells.
  • the evaluation of a single cell according to the third embodiment of the present invention refers to individually evaluating the characteristics of each of a plurality of cells contained in the cell population applied to the microarray chip in step (A). shall be said.
  • the number of multiple cells is not limited in theory, but from the viewpoint of handling, it is possible to evaluate the individual characteristics of about 10 to 10,000 cells substantially simultaneously.
  • Steps (A) to (C) in the single cell evaluation method may be the same as steps (a) to (c) in the second embodiment.
  • Step (C) may include a step of culturing the cells in the presence of a candidate substance that can affect the characteristics of the cells in relation to the characteristics to be evaluated.
  • the candidate substance may be a low-molecular compound, a high-molecular compound, a biologically derived substance, or a combination thereof, which is a candidate drug for a disease, or may be an anticancer drug, an antibiotic, or the like.
  • step (C) ie after the production of monoclonal cell colonies, it may include the step of contacting the cell colony with a candidate substance that influences the property of the cells in relation to the property to be evaluated.
  • step (D) based on the state after culturing in the step (C), each single cell housed in each of the plurality of wells or a monoclonal cell colony generated by each single cell is grown. Assess cell characteristics. Evaluation of cell characteristics may be, for example, cell proliferation rate, cell life/death, specific physiological activity of cells, adhesion strength of cells to a chip, etc., and is not particularly limited. Thereby, in step (A), individual characteristics of each cell contained in the cell population applied to the microarray chip can be obtained.
  • the cells to be cultured in step (A) are cells derived from a malignant tumor.
  • Cells derived from a malignant tumor may be collected from peripheral blood of a human suffering from a malignant tumor.
  • circulating cancer cells (CTCs) can be collected from human peripheral blood.
  • cells derived from a malignant tumor can be collected from a tissue section of malignant glioma tumor tissue. Steps (A) and (B) are performed using these cells.
  • an anticancer drug is applied to each well of the microarray chip 1.
  • step (D) After culturing for a predetermined period, by observing and evaluating the state of the cells in step (D), for example, anticancer drug-resistant strains can be identified. Similarly, a step of confirming the sensitivity of cells to multiple types of anticancer drugs can also be carried out. This makes it possible to culture and evaluate single cancer cells from a specific patient, which can greatly contribute to cancer prognosis and treatment policy decisions.
  • the single cell evaluation method According to the single cell evaluation method according to the third embodiment, multiple monoclonal cell colonies can be evaluated on a microarray chip. Therefore, evaluation of single cells, which conventionally required general cloning, can be performed more easily and accurately.
  • microarray chip of the example shown in FIG. 3 was manufactured. Polystyrene was used as the material, and a mold was used to manufacture a microarray chip with wells of a specific shape, size, and pitch. A comparative microarray chip without a two-tiered structure was also manufactured in the same manner.
  • FIG. 7A is fluorescence images of wells 3 hours after seeding HeLa cells on the microarray chip (FIG. 7A) and 7 days after seeding (FIG. 7B). From FIG. 7A, it was confirmed that a single cell was accommodated in the second part of the well. Furthermore, from FIG. 7B, it was confirmed that monoclonal cell colonies were produced in the wells after 7 days of culture.
  • the well structure was arranged as follows. Column ABOP in FIG. 3 has the structure in FIG. 5E, column CDMN in FIG. 3 has the structure in FIG. 5D, column EF in FIG. 3 has the structure in FIG. 5C, column GH in FIG. 3 has the structure in FIG. 5B, and column IJKL in FIG. The structure was as shown in FIG. 5A.
  • the parameters X, Y, T, S, U, and V shown in FIG. 2 were set as follows.
  • the depth T of the first portion of the well was 20 ⁇ m
  • the depth S of the second portion was 28 ⁇ m.
  • the inner diameter X of the opening of the second portion 22 of one well and the inner diameter Y of the first portion 21 correspond to rows 1 to 6 and columns A to P of the block in FIG. Therefore, the following table was used.
  • the numerical values in the table represent "X ( ⁇ m)/Y ( ⁇ m)".
  • the pitch V between wells was 130 ⁇ m when Y was 110 ⁇ m, and 150 ⁇ m when Y was 130 ⁇ m.
  • the angle U of the well wall was 15°.
  • Figure 8 shows a graph of the single cell accommodation rate in each well.
  • a to J correspond to columns A to J of the block of FIG.
  • the diameter is the value of the inner diameter X of the opening of the second portion 22. From the results in Figure 8, it is possible to accommodate single cells when targeting HeLa cells if the inner diameter X is in the range of about 26 to 36 ⁇ m and the inner diameter Y is in the range of about 110 to 130 ⁇ m. was confirmed.
  • FIG. 10 is a graph showing the relationship between the pitch between wells and the single cell accommodation rate. From FIG. 10, it was confirmed that for HeLa cells, if the upper stage diameter was approximately 130 to 300 ⁇ m, the storage rate would be 20 to 30%.
  • FIG. 11 is a graph showing single cell proliferation rate. From FIG. 11, it was confirmed that when HeLa cells are targeted, proliferation is possible when the pitch V is approximately 300 ⁇ m or less.
  • FIG. 12 is a fluorescence image of a microarray chip cultured for 7 days using wells with a Y of 110 ⁇ m
  • FIG. 13 is a graph showing the single cell proliferation rate
  • FIG. 14 is a fluorescence image of a microarray chip using wells with Y of 130 ⁇ m after 7 days of culture
  • FIG. 15 is a graph showing the single cell proliferation rate.
  • the cell proliferation rate itself was equivalent when Y was 110 ⁇ m and when Y was 130 ⁇ m (data details not shown).
  • Y was 110 ⁇ m, cross-contamination might occur, so the single cell proliferation rate was low, but monoclonal colonies could be formed.
  • Y is a little smaller than 110 ⁇ m, for example, even if it is about 80 to 100 ⁇ m, it is considered that monoclonal colonies of HeLa cells can be formed.
  • 16A and 16B are fluorescence images of the microarray chip of Comparative Example 1 immediately after seeding (FIG. 16A) and on the 15th day of culture (FIG. 16B). Although one cell was accommodated, it was confirmed that the cells proliferated after 15 days of culture, causing cross-contamination.
  • 17A and 17B are fluorescence images of the microarray chip immediately after seeding (FIG. 17A) and on the 15th day of culture (FIG. 17B) in Comparative Example 2. Due to the influence of the seeded cell concentration, only a few wells contained a single cell, and almost no growth was observed during the 15 days of culture.
  • 18A and 18B are fluorescence images of the microarray chip immediately after seeding (FIG.
  • FIG. 18A is fluorescence images of the microarray chip of Comparative Example 4 immediately after seeding (FIG. 19A) and on the 15th day of culture (FIG. 19B). It was confirmed that a plurality of cells were accommodated in one well immediately after seeding, and that cells proliferated after 15 days of culture, causing cross-contamination.
  • the microarray chip was immersed in a medium containing an anticancer drug (Gefitinib (ZD1839)), and cultured for 7 days. Immediately after seeding and 3 days later, the state of the cells was observed using an inverted microscope (IX-73; Olympus) and a CCD camera (DP80; Olympus). The concentration of Gefitinib was varied at 0, 1, 5, 10, 100 ⁇ M.
  • FIGS. 20A and 20B are fluorescence and bright field images of the microarray chip immediately after seeding of PC9 cells (FIG. 20A) and on the third day of culture with 5 ⁇ M Gefitinib (FIG. 20B). From FIGS. 20A and 20B, it can be seen that some PC9 cells are proliferating within the chamber.
  • FIG. 21 is a graph showing the single cell proliferation rate of PC9 cells. From FIG. 21, it was found that the number of chambers in which one cell proliferated was inversely proportional to the anticancer drug concentration, and it was found that only drug-resistant cells could be cultured using this microchip.

Abstract

The present invention provides a microarray chip that makes it possible to reliably separate a large number of single cells and highly efficiently culture monoclonal colonies.　Provided is a microarray chip 1 having formed therein a plurality of wells 2 that can accommodate cells, wherein each of the plurality of wells has a first portion 21 that can accommodate a plurality of cells and a second portion 22 that can accommodate only a single cell, the first portion is formed as a recess that is recessed from the surface of the microarray chip, and the second portion is formed as a recess that is recessed from the bottom surface of the first portion.

Description

マイクロアレイチップおよびマイクロアレイチップを用いた細胞培養方法Microarray chip and cell culture method using microarray chip
　本発明は、マイクロアレイチップおよびマイクロアレイチップを用いた細胞培養方法に関する。とくに、多数の単一細胞を確実に分離し、モノクローンの細胞コロニーを高効率で培養可能なマイクロアレイチップ、及びこれを用いた細胞培養方法に関する。 The present invention relates to a microarray chip and a cell culture method using the microarray chip. In particular, the present invention relates to a microarray chip that can reliably separate large numbers of single cells and culture monoclonal cell colonies with high efficiency, and a cell culture method using the same.
　がんを構成する細胞は、一細胞ごとに抗がん剤への耐性が異なっており、薬剤耐性株の検出のためには多数の細胞を単一細胞に分離し、分離した単一細胞を培養して評価する必要がある。しかし、例えば、血液や組織中の大量の細胞の中から標的の単一がん細胞を検出し、分離すること、さらに分離した単一細胞を培養して評価することは非常に困難である。細胞集団をシャーレなどで培養する方法では、一細胞レベルでの効率的な耐性株の検出とそのクローニングは困難である。そこで、細胞のトラップと培養を行うために、微細な構造のマイクロ流路を有する種々の形状のマイクロ流路デバイスが提案されている。 The cells that make up cancer differ in their resistance to anticancer drugs, and in order to detect drug-resistant strains, it is necessary to separate many cells into single cells and collect the separated single cells. It is necessary to cultivate and evaluate. However, it is extremely difficult, for example, to detect and isolate a single target cancer cell from a large amount of cells in blood or tissue, and to culture and evaluate the isolated single cell. By culturing cell populations in petri dishes, it is difficult to efficiently detect and clone resistant strains at the single-cell level. Therefore, in order to trap and culture cells, microchannel devices of various shapes having microchannels with fine structures have been proposed.
　例えば、単一細胞分離のためのマイクロ流路デバイスであって、スライドグラスの上層と、マイクロ流路およびウェルを有するＰＤＭＳ製の下層とを備え、各ウェルの下流端に、単一細胞を捕捉して保持するＵ字形状のトラップが形成されたものが知られている（例えば、非特許文献１を参照）。各ウェルには、トラップに加えて、大きな表面エリアも形成され、捕捉された単一細胞を拡張、増殖させるための空間が提供されている。 For example, a microchannel device for single cell separation, comprising an upper layer of glass slide and a lower layer made of PDMS having microchannels and wells, and captures a single cell at the downstream end of each well. A device in which a U-shaped trap is formed is known (for example, see Non-Patent Document 1). In addition to the trap, each well also has a large surface area that provides space for the captured single cells to expand and grow.
　また、微小なサイズのマイクロウェルと、それよりも大きなマイクロウェルを上下に向かい合うように配置し、単一細胞の捕捉と培養とを行うように構成されたデュアルウェルタイプのマイクロ流路デバイスが知られている（例えば、非特許文献２を参照）。 In addition, a dual-well type microfluidic device is known, which is configured to capture and culture single cells by arranging microwells of a minute size and microwells of a larger size to face each other vertically. (For example, see Non-Patent Document 2).
　上述した非特許文献１および非特許文献２には、マイクロ流路デバイスを用いて細胞のトラップと培養を行う方法が開示されている。しかし、操作の煩雑性やマイクロ流路からの細胞回収の難しさから、依然として、多くの単一細胞を確実に分離し、高効率で培養することが可能なデバイスおよび方法が望まれている。 Non-Patent Document 1 and Non-Patent Document 2 mentioned above disclose a method of trapping and culturing cells using a microchannel device. However, due to the complexity of operations and the difficulty of recovering cells from microchannels, there is still a need for devices and methods that can reliably separate many single cells and culture them with high efficiency.
　本発明は、上記のような実状に鑑みてなされたものであって、その目的は、多数の単一細胞を確実に分離し、高い効率でモノクローンコロニーを培養可能なマイクロアレイチップおよびマイクロアレイチップを用いた細胞培養方法を提供することにある。 The present invention was made in view of the above-mentioned circumstances, and its purpose is to provide a microarray chip and a microarray chip that can reliably separate a large number of single cells and culture monoclonal colonies with high efficiency. An object of the present invention is to provide a cell culture method using the present invention.
　本発明は一実施形態によれば、細胞を収容可能な複数のウェルが形成されたマイクロアレイチップであって、複数のウェルのそれぞれは、複数の細胞を収容可能な第１の部分と、単一細胞のみを収容可能な第２の部分とを有し、前記第１の部分は、前記マイクロアレイチップの表面から凹んだ凹部として形成され、前記第２の部分は、前記第１の部分の底面から凹んだ凹部として形成されている、マイクロアレイチップに関する。 According to one embodiment of the present invention, there is provided a microarray chip in which a plurality of wells capable of accommodating cells are formed, each of the plurality of wells including a first part capable of accommodating a plurality of cells, and a first part capable of accommodating a plurality of cells. and a second portion capable of accommodating only cells, the first portion being formed as a recess recessed from the surface of the microarray chip, and the second portion being formed from the bottom surface of the first portion. The present invention relates to a microarray chip formed as a concave recess.
　本発明は別の実施形態によれば、前述のマイクロアレイチップを用いた、細胞培養方法であって、
　（ａ）前記マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程と、
　（ｂ）前記工程（ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程と、
　（ｃ）前記工程（ｂ）で得られた前記複数のウェルに収容された単一細胞を培養する工程と
を含む方法に関する。 According to another embodiment, the present invention is a cell culture method using the above-mentioned microarray chip, comprising:
(a) applying a liquid containing a plurality of cells onto the microarray chip;
(b) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in step (a);
(c) culturing the single cells accommodated in the plurality of wells obtained in step (b).
　本発明はまた別の実施形態によれば、前述のマイクロアレイチップを用いた、単一細胞の評価方法であって、
　（Ａ）前記マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程と、
　（Ｂ）前記工程（Ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程と、
　（Ｃ）前記工程（Ｂ）で得られた前記複数のウェルに収容された単一細胞を培養する工程と、
　（Ｄ）前記工程（Ｃ）の培養後の状態に基づき、単一細胞の細胞特性を評価する工程と
を含む方法に関する。 According to another embodiment, the present invention is a method for evaluating a single cell using the above-mentioned microarray chip, comprising:
(A) applying a liquid containing a plurality of cells onto the microarray chip;
(B) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in the step (A);
(C) culturing the single cells accommodated in the plurality of wells obtained in the step (B);
(D) The present invention relates to a method including the step of evaluating the cell characteristics of a single cell based on the state after culturing in the step (C).
　本発明はまた別の実施形態によれば、前述のマイクロアレイチップを用いた、モノクローンの細胞コロニーの製造方法であって、
　（ａ）前記マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程と、
　（ｂ）前記工程（ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程と、
　（ｃ）前記工程（ｂ）で得られた前記複数のウェルに収容された単一細胞を培養する工程と
を含む方法に関する。 According to another embodiment, the present invention is a method for producing a monoclonal cell colony using the above-mentioned microarray chip, comprising:
(a) applying a liquid containing a plurality of cells onto the microarray chip;
(b) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in step (a);
(c) culturing the single cells accommodated in the plurality of wells obtained in step (b).
　本発明によれば、多数の単一細胞を確実に分離し、高い効率でモノクローンコロニーを得ることが可能なマイクロアレイチップおよびマイクロアレイチップを用いた細胞培養方法を実現することができる。 According to the present invention, it is possible to realize a microarray chip and a cell culture method using the microarray chip that can reliably separate a large number of single cells and obtain monoclonal colonies with high efficiency.
図１は、本発明の第1実施形態に係るマイクロアレイチップにおけるウェルの構成を模式的に示す上面斜視図である。FIG. 1 is a top perspective view schematically showing the configuration of wells in a microarray chip according to a first embodiment of the present invention. 図２は、図１のＡ－Ａ断面図である。FIG. 2 is a sectional view taken along line AA in FIG. 図３は、マイクロアレイチップを模式的に示す上面図である。FIG. 3 is a top view schematically showing a microarray chip. 図４Ａ，４Ｂは、ウェルの変形例を示す図である。4A and 4B are diagrams showing modified examples of wells. 図５Ａ～５Ｅは、ウェルの変形例を示す図である。5A to 5E are diagrams showing modified examples of wells. 図６Ａ～Ｃは、本発明の第２実施形態に係るマイクロアレイチップを用いた細胞培養方法において、単一のウェルにおける細胞の挙動の一例を示す概念図である。FIGS. 6A to 6C are conceptual diagrams showing an example of cell behavior in a single well in a cell culture method using a microarray chip according to the second embodiment of the present invention. 図７Ａ，７Ｂは、マイクロアレイチップへのＨｅＬａ細胞の播種後３時間（図７Ａ）及び播種後７日（図７Ｂ）のウェルの蛍光イメージである。FIGS. 7A and 7B are fluorescence images of wells 3 hours after seeding HeLa cells on the microarray chip (FIG. 7A) and 7 days after seeding (FIG. 7B). 図８は、マイクロアレイチップのウェルの構造を変化させた場合の、各ウェルにおけるＨｅＬａ細胞の一細胞収容率を示すグラフである。FIG. 8 is a graph showing the single-cell accommodation rate of HeLa cells in each well when the well structure of the microarray chip is changed. 図９Ａ，９Ｂは、マイクロアレイチップへのＨｅＬａ細胞の播種直後（図９Ａ）及び培養１５日後（図９Ｂ）のマイクロアレイチップの蛍光イメージである。9A and 9B are fluorescence images of the microarray chip immediately after seeding HeLa cells on the microarray chip (FIG. 9A) and 15 days after culture (FIG. 9B). 図１０は、ウェル間のピッチと、ＨｅＬａ細胞の一細胞収容率との関係を示すグラフである。FIG. 10 is a graph showing the relationship between the pitch between wells and the single-cell accommodation rate of HeLa cells. 図１１は、ウェル間のピッチが異なるマイクロアレイチップを用いた培養における、ＨｅＬａ細胞の一細胞増殖率を示すグラフである。FIG. 11 is a graph showing the single-cell proliferation rate of HeLa cells in culture using microarray chips with different pitches between wells. 図１２は、第１の部分２１の内径Ｙが１１０μｍのウェルを備えるマイクロアレイチップの、ＨｅＬａ細胞の培養６日の蛍光イメージである。FIG. 12 is a fluorescence image of a microarray chip provided with wells with an inner diameter Y of 110 μm in the first portion 21 after 6 days of culturing HeLa cells. 図１３は、第１の部分２１の内径Ｙが１１０μｍのウェルを備えるマイクロアレイチップを用いた培養における、ＨｅＬａ細胞の一細胞増殖率を示すグラフである。FIG. 13 is a graph showing the single-cell proliferation rate of HeLa cells in culture using a microarray chip equipped with a well in which the first portion 21 has an inner diameter Y of 110 μm. 図１４は、第１の部分２１の内径Ｙが１３０μｍのウェルを備えるマイクロアレイチップの、ＨｅＬａ細胞の培養６日の蛍光イメージである。FIG. 14 is a fluorescence image of a microarray chip having a well with an inner diameter Y of 130 μm in the first portion 21 after 6 days of culturing HeLa cells. 図１５は、第１の部分２１の内径Ｙが１３０μｍのウェルを備えるマイクロアレイチップを用いた培養における、ＨｅＬａ細胞の一細胞増殖率を示すグラフである。FIG. 15 is a graph showing the single-cell proliferation rate of HeLa cells in culture using a microarray chip equipped with a well in which the first portion 21 has an inner diameter Y of 130 μm. 図１６Ａ，１６Ｂは、比較例１のマイクロアレイチップを用いた培養における、ＨｅＬａ細胞の播種直後（図１６Ａ）、及び培養１５日目（図１６Ｂ）のマイクロアレイチップの蛍光イメージである。16A and 16B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 16A) and on the 15th day of culture (FIG. 16B) in culture using the microarray chip of Comparative Example 1. 図１７Ａ，１７Ｂは、比較例２のマイクロアレイチップを用いた培養における、ＨｅＬａ細胞の播種直後（図１７Ａ）、及び培養１５日目（図１７Ｂ）のマイクロアレイチップの蛍光イメージである。17A and 17B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 17A) and on the 15th day of culture (FIG. 17B) in culture using the microarray chip of Comparative Example 2. 図１８Ａ，１８Ｂは、比較例３のマイクロアレイチップを用いた培養における、ＨｅＬａ細胞の播種直後（図１８Ａ）、及び培養１５日目（図１８Ｂ）のマイクロアレイチップの蛍光イメージである。18A and 18B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 18A) and on the 15th day of culture (FIG. 18B) in culture using the microarray chip of Comparative Example 3. 図１９Ａ，１９Ｂは、比較例４のマイクロアレイチップを用いた培養における、ＨｅＬａ細胞の播種直後（図１９Ａ）、及び培養１５日目（図１９Ｂ）のマイクロアレイチップの蛍光イメージである。19A and 19B are fluorescence images of the microarray chip immediately after seeding of HeLa cells (FIG. 19A) and on the 15th day of culture (FIG. 19B) in culture using the microarray chip of Comparative Example 4. 図２０Ａ，２０Ｂは、本発明のマイクロアレイチップを用いた培養における、ＰＣ９細胞の播種直後（図２０Ａ）、及び５μＭのGefitinibでの培養３日目（図２０Ｂ）のマイクロアレイチップの蛍光および明視野イメージである。Figures 20A and 20B show fluorescence and bright field images of the microarray chip immediately after seeding of PC9 cells (Figure 20A) and on the third day of culture with 5 μM Gefitinib (Figure 20B) in culture using the microarray chip of the present invention. It is. 図２１は、ＰＣ９細胞の一細胞増殖率を示すグラフである。FIG. 21 is a graph showing the single cell proliferation rate of PC9 cells.
［１．マイクロアレイチップ］
　以下、本発明の第１実施形態によるマイクロアレイチップについて、図面を参照して詳細に説明する。図１は、本発明の第１実施形態に係るマイクロアレイチップにおけるウェルの構成を模式的に示す上面斜視図であり、図２は、図１のＡ－Ａ断面図である。 [1. Microarray chip]
Hereinafter, a microarray chip according to a first embodiment of the present invention will be described in detail with reference to the drawings. FIG. 1 is a top perspective view schematically showing the configuration of wells in a microarray chip according to a first embodiment of the present invention, and FIG. 2 is a sectional view taken along line AA in FIG.
　図１および図２に示すように、マイクロアレイチップ１は、細胞を収容可能な複数のウェル２が形成されており、複数のウェル２のそれぞれは、複数の細胞を収容可能な第１の部分２１と、単一細胞のみを収容可能な第２の部分２２とを有している。第１の部分２１は、マイクロアレイチップ１の表面３から凹んだ凹部として形成されている。第２の部分２２は、第１の部分２１の底面２１１から凹んだ凹部として形成されている。すなわち、各ウェル２は、上段の１つの第１の部分２１と、下段の１つの第２の部分２２とを有する２段構造のウェルとして構成されている。なお、第１の部分２１の底面２１１は、表面３に対して平行であっても、傾斜していてもよい。底面２１１を傾斜した形状とする場合は、例えば底面２１１の中心に向かって下り傾斜とすることができる。また、第２の部分２２の底面２２１は、表面３に対して平行であっても、底面２２１が若干くぼんだ形状としてもよい。 As shown in FIGS. 1 and 2, the microarray chip 1 is formed with a plurality of wells 2 capable of accommodating cells, and each of the plurality of wells 2 has a first portion 21 capable of accommodating a plurality of cells. and a second portion 22 that can accommodate only a single cell. The first portion 21 is formed as a recessed portion recessed from the surface 3 of the microarray chip 1 . The second portion 22 is formed as a recessed portion recessed from the bottom surface 211 of the first portion 21 . That is, each well 2 is configured as a two-stage well having one first part 21 in the upper stage and one second part 22 in the lower stage. Note that the bottom surface 211 of the first portion 21 may be parallel to the surface 3 or may be inclined. When the bottom surface 211 has an inclined shape, it may be inclined downward toward the center of the bottom surface 211, for example. Furthermore, the bottom surface 221 of the second portion 22 may be parallel to the surface 3 or may be slightly recessed.
　第２の部分２２は、単一細胞のみを分離して捕捉するトラップとして機能する。一方、第１の部分２１は、第２の部分２２に捕捉された単一細胞を培養し、増殖させるためのチャンバとして機能する。そのため、第２の部分２２の開口部の内径Ｘは、第１の部分２１の内径Ｙよりも小さく設定されている（Ｘ＜Ｙ）。 The second portion 22 functions as a trap that separates and captures only a single cell. On the other hand, the first part 21 functions as a chamber for culturing and proliferating the single cells captured in the second part 22. Therefore, the inner diameter X of the opening of the second portion 22 is set smaller than the inner diameter Y of the first portion 21 (X<Y).
　第１の部分２１および第２の部分２２の開口部の形状は、例えば図１に示すように円形とすることができる。ただし、これには限定されず、後述するように開口部の形状を正多角形とすることもできる。以下では、一例として、第１の部分２１および第２の部分２２の開口部がそれぞれ円形、かつ同心円である場合を説明する。 The openings of the first portion 21 and the second portion 22 may have a circular shape, for example, as shown in FIG. 1. However, the shape of the opening is not limited to this, and the shape of the opening may be a regular polygon as described later. Below, as an example, a case will be described in which the openings of the first portion 21 and the second portion 22 are each circular and concentric.
　第２の部分２２の内径Ｘは、単一細胞のみを収納可能となるように寸法決めされる。すなわち、第２の部分２２内には、捕捉対象とする２つ以上の細胞を、第２の部分２２の径方向に並べて配置することはできない。そこで、第２の部分２２の内径Ｘは、捕捉対象とする単一細胞のサイズに若干の余裕を持たせた寸法に設定される。なお、単一細胞のサイズは、補足対象の細胞の取得源、特性や、サイズ分布に応じて、当業者が適宜設定することができる。好ましくは、内径Ｘは、単一細胞のサイズに対し、０から５０μｍ程度の余裕を持たせた寸法、または、細胞のサイズに対し、１００％から３００％の寸法に設定することができる。例えば、内径Ｘは、数十μｍとすることができ、例えば、細胞の種類に応じて約５～１００μｍとすることができる。ただし、これには限定されず、細胞種に応じて変更可能であり、捕捉対象の細胞のサイズに合わせて適切な寸法を内径Ｘとして設定することができる。 The inner diameter X of the second portion 22 is dimensioned to accommodate only a single cell. That is, within the second portion 22, two or more cells to be captured cannot be arranged side by side in the radial direction of the second portion 22. Therefore, the inner diameter X of the second portion 22 is set to a size that allows some margin for the size of a single cell to be captured. Note that the size of a single cell can be appropriately set by those skilled in the art depending on the acquisition source, characteristics, and size distribution of the cells to be captured. Preferably, the inner diameter X can be set to a dimension with a margin of about 0 to 50 μm relative to the size of a single cell, or a dimension of 100% to 300% relative to the cell size. For example, the inner diameter X can be several tens of μm, for example about 5 to 100 μm depending on the type of cell. However, it is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the inner diameter X according to the size of the cell to be captured.
　第１の部分２１の内径Ｙは、第２の部分２２に捕捉した単一細胞から、細胞コロニーを形成できるように寸法決めされる。すなわち、第１の部分２１内において、例えば複数の細胞を径方向に並んで配置することができるような寸法に設定される。なお、第１の部分２１の内径Ｙは、細胞が第１の部分２１から大きくはみ出して隣接するウェル２とクロスコンタミネーションを引き起こさなければ、複数の細胞が径方向に並ばずに、ウェル２の上方から見て複数の細胞が重なるような寸法としてもよい。これらを踏まえて、例えば、内径Ｙは、数百μｍとすることができ、例えば約５０～５００μｍとすることができる。ただし、これには限定されず、細胞種に応じて変更可能であり、培養対象の細胞のサイズに合わせて適切な寸法を内径Ｙとして設定することができる。 The inner diameter Y of the first part 21 is dimensioned to allow the formation of a cell colony from a single cell captured in the second part 22. That is, within the first portion 21, the dimensions are set such that, for example, a plurality of cells can be arranged side by side in the radial direction. Note that the inner diameter Y of the first portion 21 is such that unless the cells largely protrude from the first portion 21 and cause cross-contamination with the adjacent well 2, the cells will not line up in the radial direction and the well 2 will be The dimensions may be such that a plurality of cells overlap when viewed from above. Based on these considerations, for example, the inner diameter Y can be several hundred μm, for example about 50 to 500 μm. However, it is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the inner diameter Y according to the size of the cells to be cultured.
　第２の部分２２の深さＳ、すなわち、第１の部分２１の底面２１１から第２の部分２２の底面２２１までの長さは、単一細胞のみを収納可能となるように寸法決めされる。すなわち、第２の部分２２内には、捕捉対象とする２つ以上の細胞を、第２の部分２２の深さ方向に並べて配置することはできない。そこで、第２の部分２２の深さＳは、一細胞のサイズに若干の余裕を持たせ、一細胞のサイズよりも若干大きい（深い）寸法、あるいは一細胞のサイズよりも若干小さい（浅い）寸法に設定される。深さＳについて、若干の余裕を持たせた寸法は、内径Ｘの設計と同様に設定することができ、細胞のサイズより、例えば０～５０μｍ程度大きい（深い）寸法、または、細胞のサイズに対し、１００～３００％の寸法に設定することができる。一方、一細胞のサイズよりも若干小さい寸法は、細胞のサイズに対し、５０～１００％の寸法に設定することができる。例えば、深さＳは、数十μｍとすることができ、例えば細胞の種類に応じて約５～４０μｍとすることができる。ただし、これには限定されず、細胞種に応じて変更可能であり、捕捉対象の細胞のサイズに合わせて適切な寸法を深さＳとして設定することができる。 The depth S of the second portion 22, i.e. the length from the bottom surface 211 of the first portion 21 to the bottom surface 221 of the second portion 22, is dimensioned to accommodate only a single cell. . That is, within the second portion 22, two or more cells to be captured cannot be arranged side by side in the depth direction of the second portion 22. Therefore, the depth S of the second portion 22 is set to be slightly larger (deeper) than the size of one cell, or slightly smaller (shallower) than the size of one cell, with some allowance for the size of one cell. Set to dimensions. Regarding the depth S, the dimension with a slight margin can be set in the same way as the design of the inner diameter On the other hand, the size can be set to 100 to 300%. On the other hand, the size slightly smaller than the size of one cell can be set to 50 to 100% of the size of the cell. For example, the depth S can be several tens of μm, for example about 5 to 40 μm depending on the type of cell. However, the depth S is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the depth S according to the size of the cell to be captured.
　第１の部分２１の深さＴ、すなわち、マイクロアレイチップ１の表面３から第１の部分２１の底面２１１までの長さは、細胞の増殖を制限できる寸法に設定される。例えば、第１の部分２１内において、第１の部分２１の深さ方向に細胞の複数の層が形成されないような深さ、すなわち、細胞の単一の層のみが形成されるような深さとすることができる。なお、第１の部分２１の深さＴは、細胞が第１の部分２１から大きくはみ出して隣接するウェル２とクロスコンタミネーションを引き起こさなければ、深さ方向に複数の層が形成可能となるような寸法としてもよい。これらを踏まえて、例えば、深さＴは、数十μｍとすることができ、例えば約１～５０μｍとすることができる。ただし、これには限定されず、細胞種に応じて変更可能であり、培養対象の細胞のサイズに合わせて適切な寸法を深さＴとして設定することができる。 The depth T of the first portion 21, that is, the length from the surface 3 of the microarray chip 1 to the bottom surface 211 of the first portion 21, is set to a dimension that can limit cell proliferation. For example, within the first portion 21, the depth is such that multiple layers of cells are not formed in the depth direction of the first portion 21, that is, the depth is such that only a single layer of cells is formed. can do. The depth T of the first portion 21 is set such that multiple layers can be formed in the depth direction as long as the cells do not protrude significantly from the first portion 21 and cause cross-contamination with the adjacent well 2. It may be of any size. Based on these considerations, for example, the depth T can be several tens of μm, for example about 1 to 50 μm. However, the depth T is not limited to this, and can be changed depending on the cell type, and an appropriate dimension can be set as the depth T according to the size of the cells to be cultured.
　第１の部分２１および第２の部分２２の壁面角度Ｕは、第１の部分２１および第２の部分２２の内壁面と第１のマイクロアレイチップ１の表面３に直交する線とのなす角度である。壁面角度Ｕは、マイクロアレイチップ１の作製時の条件等によって決定されるが、例えば約０～４５°とすることができ、０～２０°とすることが好ましい。壁面角度Ｕが０°の場合、第１の部分２１および第２の部分２２は、それぞれ円筒形状となり、壁面角度Ｕが０°よりも大きい場合、第１の部分２１および第２の部分２２は、それぞれ円錐台形状となる。なお、第１の部分２１の壁面角度Ｕと第２の部分２２の壁面角度Ｕとを異なる値としてもよい。 The wall angle U of the first portion 21 and the second portion 22 is the angle between the inner wall surfaces of the first portion 21 and the second portion 22 and a line perpendicular to the surface 3 of the first microarray chip 1. be. The wall angle U is determined depending on the conditions at the time of manufacturing the microarray chip 1, and can be, for example, approximately 0 to 45 degrees, and preferably 0 to 20 degrees. When the wall angle U is 0°, the first portion 21 and the second portion 22 each have a cylindrical shape, and when the wall angle U is larger than 0°, the first portion 21 and the second portion 22 have a cylindrical shape. , each has a truncated cone shape. Note that the wall angle U of the first portion 21 and the wall angle U of the second portion 22 may have different values.
　隣接するウェル２間のピッチＶ、すなわち、ウェル２の第２の部分２２の開口部の中心と、隣接するウェル２の第２の部分２２の開口部の中心との間の距離は、第１の部分２１内で細胞を培養する際にクロスコンタミネーションを低減するのに十分な距離とすることができる。ピッチＶは、細胞種、第１の部分２１の内径Ｙおよび第２の部分２２の内径Ｘ等にも依存するが、例えば、約１００～５００μｍとすることができる。 The pitch V between adjacent wells 2, that is, the distance between the center of the opening of the second part 22 of the well 2 and the center of the opening of the second part 22 of the adjacent well 2 is the first The distance may be sufficient to reduce cross-contamination when culturing cells within the portion 21 of the cell. The pitch V depends on the cell type, the inner diameter Y of the first portion 21, the inner diameter X of the second portion 22, etc., but can be set to about 100 to 500 μm, for example.
　マイクロアレイチップ１の材料は、とくには制限されないが、ポリスチレン、ポリエチレン、ポリプロピレン、ポリアミド、ポリカーボネート、ポリジメチルシロキサン（ＰＤＭＳ）、ポリメチルメタクリレート（ＰＭＭＡ）、環状オレフィンコポリマー（ＣＯＣ）等のポリマー、シリコン等の金属、ガラス、石英ガラス、またポリマーとガラスや金属等を貼り合わせたような複数の素材を組み合わせたもの（例えばＰＤＭＳとガラス）等が例示される。好ましくは、ポリスチレン、ＰＭＭＡ、ガラス、シリコン等である。 The material of the microarray chip 1 is not particularly limited, but may include polymers such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), silicone, etc. Examples include metal, glass, quartz glass, and combinations of multiple materials such as polymer, glass, metal, etc. (for example, PDMS and glass). Preferred are polystyrene, PMMA, glass, silicon, etc.
　マイクロアレイチップ１の透明性および透過性は、とくには制限されないが、ウェル２に収容された細胞を観察するのに適した透明性および透過性を有することが好ましい。例えば、マイクロアレイチップ１を倒立顕微鏡で観察する場合には、ウェル２が透光性を有することが好ましい。ここで、透明とは、観察する所定の光の透過率が８０％以上であることをいい、９０％以上であることが好ましい。 The transparency and permeability of the microarray chip 1 are not particularly limited, but preferably have transparency and permeability suitable for observing cells accommodated in the wells 2. For example, when observing the microarray chip 1 with an inverted microscope, it is preferable that the wells 2 have translucency. Here, transparent means that the transmittance of a predetermined light for observation is 80% or more, and preferably 90% or more.
　マイクロアレイチップ１は、既知の種々の方法によって作製することが可能である。マイクロアレイチップ１は、例えば、基板材料にウェル２を直接加工する方法、貫通孔またはウェル２が形成されたフィルムを基板材料に貼り付ける方法、プラスチック成型による方法等によって作製することができる。例えば、プラスチック成型によってマイクロアレイチップ１を作製する場合、具体的には、半導体技術分野におけるリソグラフィ技術（光リソグラフィ、電子線リソグラフィ等）、マイクロドリル等を用いた切削加工、レーザ加工などを利用することができる。 The microarray chip 1 can be produced by various known methods. The microarray chip 1 can be produced, for example, by directly forming wells 2 on a substrate material, by attaching a film in which through holes or wells 2 are formed to a substrate material, by plastic molding, or the like. For example, when producing the microarray chip 1 by plastic molding, specifically, lithography technology (optical lithography, electron beam lithography, etc.) in the semiconductor technology field, cutting using a micro drill, laser processing, etc. may be used. I can do it.
　一例として、リソグラフィ技術を用いてプラスチック成型により作製する場合、具体的には、リソグラフィとエッチングによりマイクロアレイチップ１のプラスチック製のマスターを作製し、マスターに電気鋳造を施すことによって金型を作製する。その後、作製した金型を用いて射出成型によりマイクロアレイチップ１を作製する。このように金型を用いた射出成型を採用することにより、表面が滑らかなマイクロアレイチップ１を作製することができる。 As an example, when producing by plastic molding using lithography technology, specifically, a plastic master of the microarray chip 1 is produced by lithography and etching, and a mold is produced by electroforming the master. Thereafter, the microarray chip 1 is manufactured by injection molding using the manufactured mold. By employing injection molding using a mold as described above, it is possible to produce a microarray chip 1 with a smooth surface.
　マイクロアレイチップ１には、必要に応じて表面処理を施してもよい。表面処理方法は、とくには限定されないが、プラズマ処理、コロナ放電処理等を利用することができる。マイクロアレイチップ１の基板材料が疎水性の材料（例えば、ポリスチレン、ＰＭＭＡ等のポリマー）の場合、プラズマ処理、例えば酸素プラズマ処理等の親水化処理を行うことができる。マイクロアレイチップ１の基板材料が親水性の材料（例えば、シリコンやガラス等）の場合、親水化処理を行う必要はない。また、タンパク質や脂質等でウェル２をコーティングすることにより表面処理を行うことができ、コラーゲン、フィブロネクチン、Ｐｏｌｙ－Ｄ－Ｌｙｓｉｎｅ、Ｐｏｌｙ－Ｌ－Ｌｙｓｉｎｅ、Ｌａｍｉｎｉｎ、Ｖｉｔｒｏｎｅｃｔｉｎ、マトリゲル等による表面処理が挙げられるが、これらには限定されない。 The microarray chip 1 may be subjected to surface treatment if necessary. The surface treatment method is not particularly limited, but plasma treatment, corona discharge treatment, etc. can be used. When the substrate material of the microarray chip 1 is a hydrophobic material (for example, a polymer such as polystyrene or PMMA), a hydrophilic treatment such as plasma treatment, for example oxygen plasma treatment, can be performed. When the substrate material of the microarray chip 1 is a hydrophilic material (for example, silicon, glass, etc.), there is no need to perform a hydrophilic treatment. In addition, surface treatment can be performed by coating the well 2 with protein, lipid, etc., and examples include surface treatment with collagen, fibronectin, Poly-D-Lysine, Poly-L-Lysine, Laminin, Vitronectin, Matrigel, etc. However, it is not limited to these.
　図３に、マイクロアレイチップ１の全体を模式的に示す上面図を示す。図３に示すように、マイクロアレイチップ１は、全体として長方形の板状の部材として形成される。マイクロアレイチップ１の表面３には、複数のウェル２が規則的に配置されている。図３に示す一例においては、マイクロアレイチップ１には、マイクロアレイチップ１の長辺１１，１２に沿った複数の行１～６と、短辺１３，１４に沿った複数の列Ａ～Ｐとからなる行列の状態で複数のブロック４が配置されている。 FIG. 3 shows a top view schematically showing the entire microarray chip 1. As shown in FIG. 3, the microarray chip 1 is formed as a rectangular plate-like member as a whole. On the surface 3 of the microarray chip 1, a plurality of wells 2 are regularly arranged. In the example shown in FIG. 3, the microarray chip 1 includes a plurality of rows 1 to 6 along the long sides 11 and 12 of the microarray chip 1 and a plurality of columns A to P along the short sides 13 and 14. A plurality of blocks 4 are arranged in a matrix.
　複数のブロック４は、マイクロアレイチップ１の長辺１１，１２，および短辺１３，１４から隙間を空けて配置されている。複数のブロック４は、とくには限定されないが、マイクロアレイチップ１上において、例えば約５０ｍｍ×約１５～１８ｍｍの範囲内に配置される。 The plurality of blocks 4 are arranged with gaps from the long sides 11, 12 and short sides 13, 14 of the microarray chip 1. The plurality of blocks 4 are arranged within a range of, for example, about 50 mm x about 15 to 18 mm on the microarray chip 1, although this is not particularly limited.
　各ブロック４には、上述した複数のウェル２が行列状に配置されている。例えば、各ブロック４には数千個のウェル２を配置することができる。例えば、各ブロック４に１０００個のウェル２が含まれているとすると、図３に示すマイクロアレイチップ１全体には、９６，０００個のウェル２が配置されていることになる。なお、マイクロアレイチップ１に配置されるウェル２の数は限定されない。マイクロアレイチップ１は、少なくとも２個のウェル２を備えることができる。なお、１つのマイクロアレイチップに複数のブロックが形成されることは必須ではなく、１つのマイクロアレイチップに１つのブロックのみを備えていてもよい。 In each block 4, the plurality of wells 2 described above are arranged in a matrix. For example, several thousand wells 2 can be placed in each block 4. For example, if each block 4 includes 1000 wells 2, 96,000 wells 2 are arranged in the entire microarray chip 1 shown in FIG. Note that the number of wells 2 arranged on the microarray chip 1 is not limited. The microarray chip 1 can include at least two wells 2. Note that it is not essential that a plurality of blocks be formed on one microarray chip, and one microarray chip may include only one block.
　本実施の形態によるマイクロアレイチップは、ウェルの上方にカバーや蓋の存在しない開放型のマイクロアレイチップであってよい。これにより、後述する培養方法において、多数の細胞を実質的に同時にマイクロアレイチップに適用することができ、また、培養において用いる培地や薬剤を細胞と接触させる操作や、細胞を回収する操作等が容易となる。また同時に、培養中のコンタミネーションを防ぐ目的等によりウェルの上方に取り外し式のカバーや蓋などが存在してもよい。 The microarray chip according to this embodiment may be an open type microarray chip with no cover or lid above the wells. As a result, in the culture method described below, a large number of cells can be applied to the microarray chip at the same time, and it is also easy to bring the culture medium and drugs into contact with the cells, and to collect the cells. becomes. At the same time, a removable cover or lid may be provided above the well for the purpose of preventing contamination during culture.
　以上説明した本実施の形態によるマイクロアレイチップ１によると、以下のような作用効果を奏することができる。 According to the microarray chip 1 according to the present embodiment described above, the following effects can be achieved.
　マイクロアレイチップ１は、細胞を収容可能な複数のウェル２が形成されている。複数のウェル２のそれぞれは、複数の細胞を収容可能な第１の部分２１と、単一細胞のみを収容可能な第２の部分２２とを有している。第１の部分２１は、マイクロアレイチップ１の表面３から凹んだ凹部として形成され、第２の部分２２は、第１の部分２１の底面２１１から凹んだ凹部として形成されている。 The microarray chip 1 is formed with a plurality of wells 2 that can accommodate cells. Each of the plurality of wells 2 has a first portion 21 that can accommodate a plurality of cells and a second portion 22 that can accommodate only a single cell. The first portion 21 is formed as a recess recessed from the surface 3 of the microarray chip 1, and the second portion 22 is formed as a recess recessed from the bottom surface 211 of the first portion 21.
　マイクロアレイチップ１は、各ウェル２が上段の第１の部分２１と下段の第２の部分２２の２段構造を有するように構成されている。下段の第２の部分２２は単一細胞のみを収容可能に構成されているため、多数の細胞を単一細胞に分離するのに適している。一方、上段の第１の部分２１は複数の細胞を収容可能に構成されているため、第２の部分２２に収容された単一細胞を培養し、増殖させるのに適している。このように、マイクロアレイチップ１は、単一細胞の確実な分離と、分離された細胞の高い効率での培養とを、２段構造の１つのウェル２によって実現することができる。これにより、マイクロアレイチップ１を用いた薬剤耐性株の検出、クローニングが可能となり、例えば予後が極めて悪い脳腫瘍（例えば、悪性グリオーマ等）において、腫瘍組織を一細胞ごとにチップに展開し、抗がん剤耐性や感受性試験を行うことが可能となる。 The microarray chip 1 is configured such that each well 2 has a two-tiered structure of an upper first part 21 and a lower second part 22. The lower second portion 22 is configured to accommodate only a single cell, and is therefore suitable for separating a large number of cells into single cells. On the other hand, since the upper first part 21 is configured to be able to accommodate a plurality of cells, it is suitable for culturing and proliferating a single cell accommodated in the second part 22. In this manner, the microarray chip 1 can achieve reliable separation of single cells and highly efficient culture of the separated cells using one well 2 with a two-tiered structure. This makes it possible to detect and clone drug-resistant strains using the microarray chip 1. For example, in brain tumors with extremely poor prognosis (e.g., malignant glioma, etc.), tumor tissue can be developed cell by cell on a chip, and anti-cancer strains can be developed. It becomes possible to conduct drug resistance and susceptibility tests.
　第２の部分２２の内径Ｘは、第１の部分２１の内径Ｙよりも小さいので、単一細胞の確実な分離を実現できる。 Since the inner diameter X of the second portion 22 is smaller than the inner diameter Y of the first portion 21, reliable separation of single cells can be achieved.
　第１の部分２１の開口部および第２の部分２２の開口部は、円形の形状を有するので、細胞種、マイクロアレイチップ１の作製方法等の種々の条件に基づいて適切な形状を選択することができる。 Since the opening of the first part 21 and the opening of the second part 22 have circular shapes, appropriate shapes can be selected based on various conditions such as the cell type and the method of manufacturing the microarray chip 1. I can do it.
－変形例－
（１）上述した一実施の形態においては、２段構造のウェル２を構成する第１の部分２１および第２の部分２２の開口部がそれぞれ円形である例を説明したが、開口部の形状は、円形には限定されない。開口部の形状を真円ではなく、楕円形としてもよい。また、開口部を、三角形、四角形、六角形等の多角形、好ましくは正多角形とすることもできる。図４Ａ，４Ｂに、ウェル２の変形例を示す。図４Ａは、第１の部分２１および第２の部分２２の開口部が正五角形である例を示し、図４Ｂは、第１の部分２１および第２の部分２２の開口部が正三角形である例を示している。 -Modified example-
(1) In the embodiment described above, an example has been described in which the openings of the first portion 21 and the second portion 22 constituting the two-stage well 2 are each circular. is not limited to a circular shape. The shape of the opening may be oval instead of a perfect circle. Further, the opening may be formed into a polygon such as a triangle, a quadrangle, or a hexagon, preferably a regular polygon. FIGS. 4A and 4B show modified examples of the well 2. FIG. 4A shows an example in which the openings of the first part 21 and the second part 22 are regular pentagons, and FIG. 4B shows an example in which the openings of the first part 21 and the second part 22 are regular triangles. An example is shown.
　開口部の形状が正多角形である場合、正多角形の外接円ＣＣの直径を、開口部の内径として、上述したように第１の部分２１の内径Ｙおよび第２の部分２２の内径Ｘを設定することができる。なお、図４Ｂに示す正三角形の場合は、外接円の直径の代わりに、正三角形の各辺の長さを、開口部の直径として、上述したように第１の部分２１の内径Ｙおよび第２の部分２２の内径Ｘを設定してもよい。第１の部分２１の開口部および第２の部分２２の開口部を正多角形の形状を有するように構成することによって、上述した円形の場合と同様に、細胞種、マイクロアレイチップ１の作製方法等の種々の条件に基づいて適切な形状を選択することができる。 When the shape of the opening is a regular polygon, the diameter of the circumscribed circle CC of the regular polygon is taken as the inner diameter of the opening, and the inner diameter Y of the first portion 21 and the inner diameter X of the second portion 22 are determined as described above. can be set. In the case of the equilateral triangle shown in FIG. 4B, the length of each side of the equilateral triangle is used as the diameter of the opening instead of the diameter of the circumscribed circle, and the inner diameter Y of the first portion 21 and the The inner diameter X of the portion 22 of No. 2 may be set. By configuring the opening of the first part 21 and the opening of the second part 22 to have a regular polygonal shape, the cell type and the method for manufacturing the microarray chip 1 can be changed as in the case of the circular shape described above. An appropriate shape can be selected based on various conditions such as.
（２）上述した一実施の形態においては、２段構造のウェル２を構成する第１の部分２１および第２の部分２２の開口部が同心円である例を説明したが、第１の部分２１と第２の部分２２の位置関係は、これには限定されない。図５Ａ～５Ｅに、ウェル２の変形例を示す。図５Ａは、第１の部分２１および第２の部分２２の開口部が同心円である例を示し、図５Ｂ～５Ｅは、第１の部分２１の開口部の中心に対して第２の部分２２の開口部の中心がずれている例を示している。なお、ここでは、第１の部分２１と第２の部分２２の位置関係について、図５Ｂ～５Ｅに示すウェル２が、図示した向きのまま、図３に示すマイクロアレイチップ１に配置されていると仮定して説明する。 (2) In the embodiment described above, an example has been described in which the openings of the first portion 21 and the second portion 22 constituting the two-stage well 2 are concentric circles. The positional relationship between the second portion 22 and the second portion 22 is not limited to this. 5A to 5E show modified examples of well 2. 5A shows an example in which the openings of the first part 21 and the second part 22 are concentric circles, and FIGS. 5B to 5E show that the openings of the first part 21 and the second part 22 are concentric. This shows an example where the center of the opening is off-center. Here, regarding the positional relationship between the first portion 21 and the second portion 22, it is assumed that the wells 2 shown in FIGS. 5B to 5E are arranged in the microarray chip 1 shown in FIG. Let's make an assumption and explain.
　図５Ｂの例では、第１の部分２１の開口部の中心に対して、第２の部分２２の開口部の中心が、図３に示すマイクロアレイチップ１の長辺１１寄りに配置されている。図５Ｃの例では、第１の部分２１の開口部の中心に対して、第２の部分２２の開口部の中心が、マイクロアレイチップ１の長辺１２寄りに配置されている。図５Ｄの例では、第１の部分２１の開口部の中心に対して、第２の部分２２の開口部の中心が、図３に示すマイクロアレイチップ１の短辺１３寄りに配置されている。図５Ｅの例では、第１の部分２１の開口部の中心に対して、第２の部分２２の開口部の中心が、マイクロアレイチップ１の短辺１４寄りに配置されている。 In the example of FIG. 5B, the center of the opening of the second portion 22 is located closer to the long side 11 of the microarray chip 1 shown in FIG. 3 with respect to the center of the opening of the first portion 21. In the example of FIG. 5C, the center of the opening of the second portion 22 is located closer to the long side 12 of the microarray chip 1 with respect to the center of the opening of the first portion 21. In the example of FIG. 5D, the center of the opening of the second portion 22 is located closer to the short side 13 of the microarray chip 1 shown in FIG. 3 with respect to the center of the opening of the first portion 21. In the example of FIG. 5E, the center of the opening of the second portion 22 is located closer to the short side 14 of the microarray chip 1 with respect to the center of the opening of the first portion 21.
　なお、図５Ｂ～５Ｅにおいては、ウェル２の上方から見たときに第１の部分２１の開口部と第２の部分２２の開口部とが接するように配置されているが、これには限定されず、第１の部分２１の開口部と第２の部分２２の開口部とが互いに離れて配置されてもよい。 Note that in FIGS. 5B to 5E, the opening of the first portion 21 and the opening of the second portion 22 are arranged so as to be in contact with each other when viewed from above the well 2, but there is no limitation to this. Alternatively, the opening of the first portion 21 and the opening of the second portion 22 may be arranged apart from each other.
（３）上述した一実施の形態では、第１の部分２１の開口部の形状と第２の部分２２の開口部の形状を同一にしたが、これには限定されず、第１の部分２１の開口部の形状と第２の部分２２の開口部の形状を異なるように形成してもよい。例えば、第１の部分２１の開口部を円形とし、第２の部分２２の開口部を正多角形とすることができ、あるいは、第１の部分２１の開口部を正多角形とし、第２の部分２２の開口部を円形とすることもできる。 (3) In the embodiment described above, the shape of the opening of the first portion 21 and the shape of the opening of the second portion 22 are made the same, but the present invention is not limited to this. The shape of the opening of the second portion 22 and the shape of the opening of the second portion 22 may be formed to be different. For example, the opening of the first portion 21 may be circular and the opening of the second portion 22 may be a regular polygon, or the opening of the first portion 21 may be a regular polygon and the opening of the second portion 22 may be a regular polygon. The opening of the portion 22 may also be circular.
（４）上述した一実施の形態では、マイクロアレイチップ１の外形が長方形である例を説明したが、これには限定されず、長方形以外の形状とすることもできる。 (4) In the above-described embodiment, the example in which the outer shape of the microarray chip 1 is rectangular has been described, but the outer shape is not limited to this, and the outer shape may be other than the rectangular shape.
（５）上述した一実施の形態では、マイクロアレイチップ１上に複数のウェル２を行列状に配置したが、これには限定されず、複数のウェル２を互い違いに配置してもよい。また、マイクロアレイチップ１上に配置される複数のウェル２の開口部の形状および寸法は、それぞれ同一としてもよいし、例えばブロック４ごとに異なるように設定してもよい。 (5) In the embodiment described above, the plurality of wells 2 are arranged in a matrix on the microarray chip 1, but the invention is not limited to this, and the plurality of wells 2 may be arranged alternately. Furthermore, the shapes and dimensions of the openings of the plurality of wells 2 arranged on the microarray chip 1 may be the same, or may be set differently for each block 4, for example.
［２．マイクロアレイチップを用いた細胞の培養方法］
　次に、本発明の第２実施形態について説明する。第２実施形態は、マイクロアレイチップを用いた細胞培養方法に関する。第２実施形態による、細胞培養方法は、別の観点からは、マイクロアレイチップの使用方法、またはモノクローンの細胞コロニーの製造方法とも捉えることができる。当該方法は、以下の工程を含む。
　（ａ）マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程
　（ｂ）前記工程（ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程
　（ｃ）前記工程（ｂ）で得られた前記複数のウェルに収容された細胞を培養する工程 [2. Cell culture method using microarray chip]
Next, a second embodiment of the present invention will be described. The second embodiment relates to a cell culture method using a microarray chip. From another perspective, the cell culture method according to the second embodiment can also be considered as a method for using a microarray chip or a method for producing a monoclonal cell colony. The method includes the following steps.
(a) applying a liquid containing a plurality of cells onto the microarray chip; (b) washing the surface of the microarray chip obtained in step (a) to remove the second portion of the plurality of wells; (c) A step of culturing the cells accommodated in the plurality of wells obtained in the step (b).
　図６Ａ～６Ｃは、第１実施形態によるマイクロアレイチップを用いた細胞培養方法を説明する図であって、図１～３に例示するマイクロアレイチップ１上の１つのウェル２における細胞の様子を概念的に示す図である。 6A to 6C are diagrams illustrating the cell culture method using the microarray chip according to the first embodiment, and conceptually illustrate the state of cells in one well 2 on the microarray chip 1 illustrated in FIGS. 1 to 3. FIG.
　工程（ａ）では、マイクロアレイチップ１上に、複数の細胞を含む液体を適用する。すなわち、細胞をマイクロアレイチップ１に播種する。液体における細胞濃度は、細胞を含む液体を適用する時点で、マイクロアレイチップ１の表面３全体を、細胞の単一層で覆うことができる程度の濃度以上であればよく、例えば、１．０×１０６ｃｅｌｌｓ／ｍＬ以上とすることが好ましい。また、液体における細胞濃度は、例えば、１．０×１０１０ｃｅｌｌｓ／ｍＬ以下程度、好ましくは１．０×１０９ｃｅｌｌｓ／ｍＬ以下程度、あるいは、１．０×１０８ｃｅｌｌｓ／ｍＬ以下程度とすることができる。細胞濃度が低すぎると、十分な数の細胞がチャンバにトラップされない場合があり、細胞濃度が高すぎると、培地や細胞を浪費するおそれがある。播種に用いる液体の一種としては、培地もあってよく、播種した状態のまま培養する場合もありうる。 In step (a), a liquid containing a plurality of cells is applied onto the microarray chip 1. That is, cells are seeded onto the microarray chip 1. The cell concentration in the liquid may be at least as high as to cover the entire surface 3 of the microarray chip 1 with a single layer of cells at the time of applying the cell-containing liquid, for example, 1.0 x 10 cells. It is preferable to set it as 6 cells/mL or more. Further, the cell concentration in the liquid is, for example, about 1.0×10 10 cells/mL or less, preferably about 1.0×10 9 cells/mL or less, or about 1.0×10 8 cells/mL or less. can do. If the cell concentration is too low, not enough cells may be trapped in the chamber; if the cell concentration is too high, media and cells may be wasted. A medium may be used as a type of liquid for seeding, and there may be cases where the seeds are cultured in a seeded state.
　マイクロアレイチップ上への液体の適用方法は特には限定されない。例えば、ピペットにより液体をマイクロアレイチップ上に滴下することができる。滴下量は、マイクロアレイチップの面積により異なるため、特には限定されない。当業者が滴下量を適宜決定することができる。滴下後は、次の工程までの間に、細胞がウェルの下部にまで沈降するのに十分な静置時間を確保することが好ましい。 The method of applying the liquid onto the microarray chip is not particularly limited. For example, liquid can be dropped onto the microarray chip with a pipette. The amount of the droplet is not particularly limited because it varies depending on the area of the microarray chip. A person skilled in the art can appropriately determine the amount to be added. After dropping, it is preferable to allow sufficient standing time for the cells to settle to the bottom of the well before the next step.
　図６Ａは、工程（ａ）を実施した後のウェル２及び細胞Ｈの状態を模式的に示す。ウェル２の第２の部分２２には単一細胞が収容されている。これに加え、第１の部分２１にも複数の細胞が収容され、ウェル２の周囲のマイクロアレイチップ１の表面３にも細胞が存在する。 FIG. 6A schematically shows the state of well 2 and cells H after performing step (a). The second portion 22 of well 2 contains single cells. In addition, a plurality of cells are accommodated in the first portion 21, and cells are also present on the surface 3 of the microarray chip 1 around the well 2.
　続く工程（ｂ）では、前記工程（ａ）で得られたマイクロアレイチップ１の表面３を洗浄する。これにより、余剰細胞を除去する。洗浄は、例えば、マイクロアレイチップ１の表面３に存在する余剰の細胞を、ピペットを用いて緩衝液や培養液などの液体で流し取ることにより、実施することができる。別の例としては、スクレイパーを、マイクロアレイチップ１の表面３に沿って移動させ、マイクロアレイチップの表面３に存在する余剰の細胞を除去することができる。 In the following step (b), the surface 3 of the microarray chip 1 obtained in the step (a) is washed. This removes excess cells. Washing can be carried out, for example, by washing away excess cells present on the surface 3 of the microarray chip 1 with a liquid such as a buffer solution or a culture solution using a pipette. As another example, a scraper can be moved along the surface 3 of the microarray chip 1 to remove excess cells present on the surface 3 of the microarray chip.
　図６ＢＩは、余剰の細胞が除去される様子を概念的に示す図であり、図６ＢＩＩは、余剰の細胞が除去された結果、１つのウェル２の第２の部分２２に単一細胞が収容された様子を概念的に示す図である。これにより、１つのウェル２に、効率的に単一細胞を収容することが可能となる。 FIG. 6BI is a diagram conceptually showing how surplus cells are removed, and FIG. 6BII shows that as a result of the removal of surplus cells, a single cell is accommodated in the second portion 22 of one well 2. FIG. This makes it possible to efficiently accommodate a single cell in one well 2.
　工程（ｂ）では、洗浄の操作を行った後、顕微鏡を用いたイメージングを行い、単一細胞が収容されたウェルを特定する。この操作により、モノクローンの細胞コロニーを製造可能なウェルが特定される。 In step (b), after performing a washing operation, imaging is performed using a microscope to identify wells containing single cells. Through this operation, wells in which monoclonal cell colonies can be produced are identified.
　工程（ａ）及び（ｂ）の実施には、本発明によるマイクロアレイチップ１以外には、ピペットやスクレイパーなど、細胞実験において一般的に用いる道具を用いればよく、マイクロアレイチップ１は通常の実験室の条件にて取り扱うことができる。また、マイクロアレイチップ１のサイズにもよるが、工程（ａ）及び（ｂ）は、例えば、３０～１２０分程度で実施することができる。また、例えば、一般的なスライドガラスのサイズ（７６×２６ｍｍ）のマイクロアレイチップ１について、概ね３００００～４００００個、最大で６００００個程度の細胞を、同時に、１つずつ分離して、各ウェルに収容した状態とすることができる。しかし、所要時間及び収容可能な細胞の数は、例示された値に限定されるものではない。 To carry out steps (a) and (b), tools commonly used in cell experiments, such as pipettes and scrapers, may be used in addition to the microarray chip 1 according to the present invention. It can be handled under certain conditions. Furthermore, although it depends on the size of the microarray chip 1, steps (a) and (b) can be carried out in about 30 to 120 minutes, for example. For example, for a microarray chip 1 having the size of a general slide glass (76 x 26 mm), approximately 30,000 to 40,000 cells, and about 60,000 cells at most, can be separated one by one at the same time and accommodated in each well. The state can be set as follows. However, the required time and the number of cells that can be accommodated are not limited to the exemplified values.
　任意選択的に、工程（ａ）及び（ｂ）の終了後に、単一細胞の収容率を取得する工程を行うことができる。この工程は、顕微鏡を用いたイメージングにより実施することができる。 Optionally, after completing steps (a) and (b), a step of obtaining the single cell accommodation rate can be performed. This step can be performed by imaging using a microscope.
　次いで、工程（ｃ）では、前記工程（ｂ）で得られたマイクロアレイチップ１のウェル２に収容された細胞を、所定の条件下で培養する。すなわち、工程（ｂ）で得られたマイクロアレイチップ１をそのまま使用して、培養を行うことができる。培養条件は、収容された細胞によって異なり、特に限定されるものではない。任意選択的に、工程（ｂ）の後であって、工程（ｃ）の前に、細胞の培養に必要な培地を添加する工程、もしくは培地中にマイクロアレイチップを浸漬する工程を含んでもよい。マイクロアレイチップ１は、開放型であり、かつ一体型で取り扱いが容易であるため、あらゆる培養条件に適合させることができる。図６Ｃは、工程（ｃ）により所定の期間の培養を経て、細胞が増殖し、モノクローンの細胞コロニーＣが得られた状態のウェル２を示す。 Next, in step (c), the cells accommodated in the wells 2 of the microarray chip 1 obtained in the step (b) are cultured under predetermined conditions. That is, the microarray chip 1 obtained in step (b) can be used as is for culture. Culture conditions vary depending on the cells accommodated and are not particularly limited. Optionally, after step (b) and before step (c), it may include a step of adding a medium necessary for culturing the cells, or a step of immersing the microarray chip in the medium. The microarray chip 1 is open and integrated and easy to handle, so it can be adapted to any culture conditions. FIG. 6C shows well 2 in which cells have proliferated and a monoclonal cell colony C has been obtained after culturing for a predetermined period in step (c).
　図６Ａ～６Ｃでは、一つのウェルにおける細胞の挙動を例示したが、マイクロアレイチップの、一細胞が収容された他のウェルについても同様に細胞が増殖し、モノクローンの細胞コロニーを得ることができる。なお、一細胞が収容されないウェルが存在する場合もあり、二以上の細胞が収容されるウェルもあり、全てのウェルについて、図示したような増殖ができない場合もありうる。しかし、第１実施形態によるマイクロアレイチップを用いることで、全体として、少なくとも３割程度の一細胞収容率を達成することができる。 6A to 6C illustrate the behavior of cells in one well, but cells proliferate similarly in other wells of the microarray chip containing one cell, and monoclonal cell colonies can be obtained. . Note that there may be wells that do not accommodate one cell, and there may also be wells that accommodate two or more cells, and there may be cases where the growth shown in the figure is not possible for all the wells. However, by using the microarray chip according to the first embodiment, it is possible to achieve a single cell accommodation rate of at least 30% as a whole.
　本発明に係るマイクロアレイチップ１を用いることで、工程（ａ）～（ｃ）により、簡便にかつ、クロスコンタミネーションなどの不利益なく細胞を培養することができ、モノクローンの細胞コロニーを得ることができる。 By using the microarray chip 1 according to the present invention, cells can be cultured easily and without disadvantages such as cross-contamination through steps (a) to (c), and monoclonal cell colonies can be obtained. I can do it.
　任意選択的な工程（ｄ）として、工程（ｃ）の完了後、各ウェルから細胞コロニーを回収する工程を含んでもよい。細胞コロニーの回収には、ピペットを用いることもできるし、マニピュレータを用いることもできる。このようにして回収された細胞コロニーは、任意の用途、例えば、各種の分析工程や、さらなる増殖工程に供することができる。 Optionally, step (d) may include the step of recovering cell colonies from each well after completion of step (c). A pipette or a manipulator can be used to collect cell colonies. The cell colonies recovered in this manner can be subjected to any desired purpose, such as various analytical steps or further growth steps.
　［３．単一細胞の評価方法］
　次に、本発明の第３実施形態について説明する。第３実施形態は、単一細胞の評価方法に関する。
　単一細胞の評価方法は、以下の工程を含む。
　（Ａ）前記マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程
　（Ｂ）前記工程（Ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程
　（Ｃ）前記工程（Ｂ）で得られた前記複数のウェルに収容された細胞を培養する工程
　（Ｄ）前記工程（Ｃ）の培養後の状態に基づき、単一細胞の細胞特性を評価する工程
　任意選択的に、前記工程（Ｃ）が、細胞の特性に影響を与えうる候補物質の存在下で細胞を培養する工程を含んでいてもよい。 [3. Single cell evaluation method]
Next, a third embodiment of the present invention will be described. The third embodiment relates to a method for evaluating single cells.
The single cell evaluation method includes the following steps.
(A) Applying a liquid containing a plurality of cells onto the microarray chip. (B) Washing the surface of the microarray chip obtained in step (A) to remove the second cells in the plurality of wells. (C) A step of culturing the cells accommodated in the plurality of wells obtained in the step (B). (D) Based on the state after culturing in the step (C). , Assessing Cellular Properties of a Single Cell Optionally, step (C) may include culturing the cells in the presence of a candidate substance capable of influencing the properties of the cells.
　ここで、本発明の第３実施形態による単一細胞の評価とは、工程（Ａ）でマイクロアレイチップに適用した細胞集団に含有される複数の細胞のそれぞれについて、個別に特性を評価することをいうものとする。複数の細胞の個数は、理論上は限定されないが、取り扱い上の観点から、概ね１０～１００００個の細胞について、実質的に同時に、個別の特性を評価することができる。 Here, the evaluation of a single cell according to the third embodiment of the present invention refers to individually evaluating the characteristics of each of a plurality of cells contained in the cell population applied to the microarray chip in step (A). shall be said. The number of multiple cells is not limited in theory, but from the viewpoint of handling, it is possible to evaluate the individual characteristics of about 10 to 10,000 cells substantially simultaneously.
　単一細胞の評価方法における工程（Ａ）から工程（Ｃ）は、第２実施形態の工程（ａ）から工程（ｃ）と同様であってよい。工程（Ｃ）において、評価する特性と関連して、細胞の特性に影響を与えうる候補物質の存在下で細胞を培養する工程を含んでもよい。例えば、候補物質は、疾患に対する候補薬剤となる低分子化合物、高分子化合物、生体由来物質、またはそれらの組み合わせであってよく、抗がん剤、抗生物質等であってもよい。工程（Ｃ）の完了後、すなわちモノクローンの細胞コロニーの生成後に、評価する特性と関連して、細胞の特性に影響を与える候補物質と細胞コロニーを接触させる工程を含んでもよい。 Steps (A) to (C) in the single cell evaluation method may be the same as steps (a) to (c) in the second embodiment. Step (C) may include a step of culturing the cells in the presence of a candidate substance that can affect the characteristics of the cells in relation to the characteristics to be evaluated. For example, the candidate substance may be a low-molecular compound, a high-molecular compound, a biologically derived substance, or a combination thereof, which is a candidate drug for a disease, or may be an anticancer drug, an antibiotic, or the like. After the completion of step (C), ie after the production of monoclonal cell colonies, it may include the step of contacting the cell colony with a candidate substance that influences the property of the cells in relation to the property to be evaluated.
　工程（Ｄ）では、前記工程（Ｃ）の培養後の状態に基づき、複数のウェルのそれぞれに収容された各単一細胞について、または各単一細胞が増殖して生成したモノクローンの細胞コロニーについて、細胞特性を評価する。細胞特性の評価は、例えば、細胞の増殖率、細胞の生死、細胞の特定の生理活性、細胞のチップへの接着強度等であってよく、特には限定されない。これにより、工程（Ａ）で、マイクロアレイチップに適用した細胞集団中に含まれる各細胞の個別の特性を得ることができる。 In the step (D), based on the state after culturing in the step (C), each single cell housed in each of the plurality of wells or a monoclonal cell colony generated by each single cell is grown. Assess cell characteristics. Evaluation of cell characteristics may be, for example, cell proliferation rate, cell life/death, specific physiological activity of cells, adhesion strength of cells to a chip, etc., and is not particularly limited. Thereby, in step (A), individual characteristics of each cell contained in the cell population applied to the microarray chip can be obtained.
　より具体的な応用例として、工程（Ａ）において、培養の対象となる細胞が、悪性腫瘍由来の細胞である場合について説明する。悪性腫瘍由来の細胞は、悪性腫瘍に罹患したヒト末梢血から採取されたものであってよい。例えば、ヒト末梢血から、循環がん細胞（ＣＴＣ）を採取することができる。あるいは、悪性グリオーマ腫瘍組織の組織切片から、悪性腫瘍由来の細胞を採取することができる。これらの細胞を用いて工程（Ａ）、及び（Ｂ）を実施する。次いで、工程（Ｃ）における培養時に、マイクロアレイチップ１の各ウェルに、例えば、抗がん剤を適用する。所定の期間の培養後、工程（Ｄ）にて細胞の状態を観察し、評価することで、例えば、抗がん剤耐性株を同定することができる。同様にして、複数種の抗がん剤に対して細胞の感受性を確認する工程を実施することもできる。これにより、特定の患者の単一がん細胞の培養及び評価が可能となり、がんの予後診断や、治療方針の決定に大きく寄与することができる。 As a more specific application example, a case will be described in which the cells to be cultured in step (A) are cells derived from a malignant tumor. Cells derived from a malignant tumor may be collected from peripheral blood of a human suffering from a malignant tumor. For example, circulating cancer cells (CTCs) can be collected from human peripheral blood. Alternatively, cells derived from a malignant tumor can be collected from a tissue section of malignant glioma tumor tissue. Steps (A) and (B) are performed using these cells. Next, during culturing in step (C), for example, an anticancer drug is applied to each well of the microarray chip 1. After culturing for a predetermined period, by observing and evaluating the state of the cells in step (D), for example, anticancer drug-resistant strains can be identified. Similarly, a step of confirming the sensitivity of cells to multiple types of anticancer drugs can also be carried out. This makes it possible to culture and evaluate single cancer cells from a specific patient, which can greatly contribute to cancer prognosis and treatment policy decisions.
　第３実施形態による単一細胞の評価方法によれば、マイクロアレイチップ上で、複数のモノクローンの細胞コロニーを評価することができる。そのため、従来、一般的なクローニングを必須としていた単一細胞の評価を、より簡便、かつ正確に実施することができる。 According to the single cell evaluation method according to the third embodiment, multiple monoclonal cell colonies can be evaluated on a microarray chip. Therefore, evaluation of single cells, which conventionally required general cloning, can be performed more easily and accurately.
　以下に、実施例を参照して、本発明をより詳細に説明する。しかし、以下の実施例は、本発明を限定するものではない。 Hereinafter, the present invention will be explained in more detail with reference to Examples. However, the following examples are not intended to limit the invention.
　（１）マイクロアレイチップの製造
　図３に示す実施例のマイクロアレイチップを製造した。材料は、ポリスチレンを用い、金型を用いて、特定の形状、サイズ、ピッチを持つウェルを備えるマイクロアレイチップを製造した。２段構造を持たない比較例のマイクロアレイチップも同様にして製造した。 (1) Manufacture of microarray chip The microarray chip of the example shown in FIG. 3 was manufactured. Polystyrene was used as the material, and a mold was used to manufacture a microarray chip with wells of a specific shape, size, and pitch. A comparative microarray chip without a two-tiered structure was also manufactured in the same manner.
　（２）細胞培養
　ＧＦＰを発現するＨｅＬａ細胞を、Ｄｕｌｂｅｃｃｏ’ｓ　ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ’ｓ　ｍｅｄｉｕｍ（ＤＭＥＭ，１２８００－０１７；Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）に、１０ｖ／ｖ％　ｆｅｔａｌ　ｂｏｖｉｎｅ　ｓｅｒｕｍ（ＦＢＳ；Ｓｉｇｍａ－Ａｌｄｒｉｃｈ　Ｃｏ．ＬＬＣ）と、５ｖ／ｖ％　ｐｅｎｉｃｉｌｌｉｎ（Ｐ４３３３；Ｓｉｇｍａ－Ａｌｄｒｉｃｈ　Ｃｏ．ＬＬＣ）を加えた溶液に懸濁し、１．０×１０７ｃｅｌｌｓ／ｍＬの細胞懸濁液を調製した。この細胞懸濁液を、ピペットを用いてマイクロアレイチップに滴下し（播種）、約３０～６０分間静置して細胞をウェルに沈降させた。次いで、余剰細胞を、スクレイパーを用いて洗浄した。この操作により、ウェルの第２の部分に単一細胞を収容し、倒立顕微鏡（ＩＸ－７３；Ｏｌｙｍｐｕｓ）とＣＣＤ　ｃａｍｅｒａ（ＤＰ８０；Ｏｌｙｍｐｕｓ）により一細胞収容率を取得した。次いで、ＣＯ２　ｉｎｃｕｂａｔｏｒ（３７°Ｃ，５％　ＣＯ２）で、５～１４日間細胞の培養を行い、倒立顕微鏡（ＩＸ－７３；Ｏｌｙｍｐｕｓ）とＣＣＤ　ｃａｍｅｒａ（ＤＰ８０；　Ｏｌｙｍｐｕｓ）を用いて細胞状態を観察した。 (2) Cell culture HeLa cells expressing GFP were placed in Dulbecco's modified Eagle's medium (DMEM, 12800-017; Thermo Fisher Scientific) supplemented with 10v/v% fetal bovine serum ( FBS; Sigma-Aldrich Co.LLC ) and 5v/v% penicillin (P4333; Sigma-Aldrich Co. LLC) to prepare a cell suspension of 1.0 x 107 cells/mL. This cell suspension was dropped onto a microarray chip (seeding) using a pipette, and allowed to stand for about 30 to 60 minutes to allow the cells to settle into the wells. Excess cells were then washed away using a scraper. By this operation, a single cell was accommodated in the second part of the well, and the single cell accommodation rate was obtained using an inverted microscope (IX-73; Olympus) and a CCD camera (DP80; Olympus). Next, cells were cultured in a CO2 incubator (37°C, 5% CO2) for 5 to 14 days, and the state of the cells was observed using an inverted microscope (IX-73; Olympus) and a CCD camera (DP80; Olympus). .
　（３）結果
（ａ）モノクローンの細胞コロニーの観察
　図７Ａは、マイクロアレイチップへのＨｅＬａ細胞の播種後３時間（図７Ａ）及び播種後７日（図７Ｂ）のウェルの蛍光イメージである。図７Ａから、単一細胞が、ウェルの第２の部分に収容されていることが確認された。また、図７Ｂから、７日間の培養により、ウェルにおいてモノクローンの細胞コロニーが製造されたことが確認された。 (3) Results (a) Observation of monoclonal cell colonies FIG. 7A is fluorescence images of wells 3 hours after seeding HeLa cells on the microarray chip (FIG. 7A) and 7 days after seeding (FIG. 7B). From FIG. 7A, it was confirmed that a single cell was accommodated in the second part of the well. Furthermore, from FIG. 7B, it was confirmed that monoclonal cell colonies were produced in the wells after 7 days of culture.
（ｂ）ウェル構造の検討
　図３のマイクロアレイチップにおいて、ブロックごとに、ウェルのサイズ及び構造を変化させて、一細胞の収容率を評価した。 (b) Examination of well structure In the microarray chip shown in FIG. 3, the size and structure of the wells were varied for each block, and the accommodation rate of one cell was evaluated.
　ウェルの構造の配置は、以下のようにした。図３の列ＡＢＯＰは図５Ｅの構造、図３の列ＣＤＭＮは図５Ｄの構造、図３の列ＥＦは図５Ｃの構造、図３の列ＧＨは図５Ｂの構造、図３の列ＩＪＫＬは図５Ａの構造とした。 The well structure was arranged as follows. Column ABOP in FIG. 3 has the structure in FIG. 5E, column CDMN in FIG. 3 has the structure in FIG. 5D, column EF in FIG. 3 has the structure in FIG. 5C, column GH in FIG. 3 has the structure in FIG. 5B, and column IJKL in FIG. The structure was as shown in FIG. 5A.
　ウェルの詳細構造は、図２に示すパラメータＸ、Ｙ、Ｔ、Ｓ、Ｕ、Ｖを以下のように設定した。ウェルの第１の部分の深さＴは２０μｍ、第２の部分の深さＳは２８μｍとした。各ブロックにおける、１つのウェルの第２の部分２２の開口部の内径Ｘ、及び第１の部分２１の内径Ｙは、図３のブロックの行１から行６、列Ａから列Ｐに対応して、以下の表のとおりとした。表中の数値は、「Ｘ（μｍ）／Ｙ（μｍ）」を表す。ウェル間のピッチＶは、Ｙが１１０μｍの場合は１３０μｍ、Ｙが１３０μｍの場合は１５０μｍとした。
ウェル壁の角度Ｕは、１５°とした。 For the detailed structure of the well, the parameters X, Y, T, S, U, and V shown in FIG. 2 were set as follows. The depth T of the first portion of the well was 20 μm, and the depth S of the second portion was 28 μm. In each block, the inner diameter X of the opening of the second portion 22 of one well and the inner diameter Y of the first portion 21 correspond to rows 1 to 6 and columns A to P of the block in FIG. Therefore, the following table was used. The numerical values in the table represent "X (μm)/Y (μm)". The pitch V between wells was 130 μm when Y was 110 μm, and 150 μm when Y was 130 μm.
The angle U of the well wall was 15°.
Figure JPOXMLDOC01-appb-T000001
  
Figure JPOXMLDOC01-appb-T000001
  
　図８に、各ウェルにおける一細胞収容率のグラフを示す。図８のグラフにおいて、ＡからＪは、図３のブロックの列Ａから列Ｊに対応する。また、径は、第２の部分２２の開口部の内径Ｘの値である。図８の結果から、内径Ｘが約２６～３６μｍの範囲、かつ、内径Ｙが約１１０～１３０μｍの範囲であれば、ＨｅＬａ細胞を対象とする場合に、単一細胞の収容が可能であることが確認された。 Figure 8 shows a graph of the single cell accommodation rate in each well. In the graph of FIG. 8, A to J correspond to columns A to J of the block of FIG. Further, the diameter is the value of the inner diameter X of the opening of the second portion 22. From the results in Figure 8, it is possible to accommodate single cells when targeting HeLa cells if the inner diameter X is in the range of about 26 to 36 μm and the inner diameter Y is in the range of about 110 to 130 μm. was confirmed.
（ｃ）ウェル間ピッチの検討
　次に、Ｘを３４μｍ、Ｔを２０μｍ、Ｓを２８μｍに固定し、内径ＹとピッチＶの値を変えて、一細胞収容率及び一細胞増殖率を評価した。一細胞増殖率の評価は、一細胞が増殖したウェル数をカウントすることで行った。クロスコンタミネーションはカウントしなかった。変化させたＹとＶの値の組み合わせは以下の通りとした。単位はいずれもμｍである。
Figure JPOXMLDOC01-appb-T000002
   (c) Examination of pitch between wells Next, X was fixed at 34 μm, T was fixed at 20 μm, and S was fixed at 28 μm, and the values of inner diameter Y and pitch V were changed to evaluate the single cell accommodation rate and single cell growth rate. The single cell proliferation rate was evaluated by counting the number of wells in which a single cell had grown. Cross contamination was not counted. The combinations of the values of Y and V that were changed were as follows. The unit is μm.
Figure JPOXMLDOC01-appb-T000002
　図９Ａ，９Ｂは、播種直後（図９Ａ）、及び培養１５日後（図９Ｂ）の、Ｙ＝１００、Ｖ＝１３０としたウェルを備えるマイクロアレイチップの蛍光イメージである。第２の部分に単一細胞が収容されたウェルは、１４個存在し、図９Ａのイメージ中、これらを白色の矢印で示した。そのうち１５日間の培養で１０個のウェルがクロスコンタミなく増殖し、モノクローンのコロニーを形成した。図９Ｂ中、モノクローンのコロニーを形成した１０個のウェルを白色の矢印で示す。図示はしないが、マイクロアレイチップ全体では、割合は低いものの、２個以上の細胞が収納されたウェルが存在していた。 FIGS. 9A and 9B are fluorescence images of a microarray chip equipped with wells with Y=100 and V=130 immediately after seeding (FIG. 9A) and 15 days after culture (FIG. 9B). There were 14 wells containing single cells in the second part, and these were indicated by white arrows in the image of FIG. 9A. After 15 days of culture, 10 wells grew without cross contamination, forming monoclonal colonies. In FIG. 9B, 10 wells in which monoclonal colonies were formed are indicated by white arrows. Although not shown, in the entire microarray chip, there were wells containing two or more cells, although the percentage was low.
　図１０は、ウェル間のピッチと、一細胞収容率との関係を示すグラフである。図１０から、ＨｅＬａ細胞については、上段径が１３０から３００μｍ程度であれば２０～３０％の収納率となることが確認された。図１１は、一細胞増殖率を示すグラフである。図１１から、ＨｅＬａ細胞を対象とする場合には、ピッチＶが概ね３００μｍ以下の場合に、増殖可能であることが確認された。 FIG. 10 is a graph showing the relationship between the pitch between wells and the single cell accommodation rate. From FIG. 10, it was confirmed that for HeLa cells, if the upper stage diameter was approximately 130 to 300 μm, the storage rate would be 20 to 30%. FIG. 11 is a graph showing single cell proliferation rate. From FIG. 11, it was confirmed that when HeLa cells are targeted, proliferation is possible when the pitch V is approximately 300 μm or less.
　次に、Ｘを２６～３６μｍ、Ｔを２０μｍ、Ｓを２８μｍに固定し、ＹとＶの値を変えて、一細胞収容率及び一細胞増殖率を評価した。変化させたＹとＶの値（単位はμｍ）の組み合わせを以下の表３に示す。 Next, X was fixed at 26 to 36 μm, T was fixed at 20 μm, and S was fixed at 28 μm, and the values of Y and V were changed to evaluate the single cell accommodation rate and single cell growth rate. The combinations of the changed Y and V values (in μm) are shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000003
  
Figure JPOXMLDOC01-appb-T000003
  
　図１２は、Ｙが１１０μｍのウェルを用いた培養７日のマイクロアレイチップの蛍光イメージであり、図１３は、一細胞増殖率を示すグラフである。図１４は、Ｙが１３０μｍのウェルを用いた培養７日のマイクロアレイチップの蛍光イメージであり、図１５は、一細胞増殖率を示すグラフである。細胞増殖率自体は、Ｙが１１０μｍの場合と、Ｙが１３０μｍの場合とで同等であった（データ詳細は示さず）。Ｙが１１０μｍの場合はクロスコンタミネーションを生じる場合があるために一細胞増殖率は低くなったが、モノクローンのコロニーが形成可能であった。Ｙが１１０μｍよりも少し小さい場合、例えば、８０～１００μｍ程度であっても、ＨｅＬａ細胞のモノクローンのコロニーが形成可能であると考えられる。 FIG. 12 is a fluorescence image of a microarray chip cultured for 7 days using wells with a Y of 110 μm, and FIG. 13 is a graph showing the single cell proliferation rate. FIG. 14 is a fluorescence image of a microarray chip using wells with Y of 130 μm after 7 days of culture, and FIG. 15 is a graph showing the single cell proliferation rate. The cell proliferation rate itself was equivalent when Y was 110 μm and when Y was 130 μm (data details not shown). When Y was 110 μm, cross-contamination might occur, so the single cell proliferation rate was low, but monoclonal colonies could be formed. When Y is a little smaller than 110 μm, for example, even if it is about 80 to 100 μm, it is considered that monoclonal colonies of HeLa cells can be formed.
（ｄ）比較例
　ウェルを２段構造ではなく、１段構造とした比較例のマイクロアレイチップを製造し、一細胞の収容及び細胞増殖について評価した。比較例のマイクロアレイチップについて、ウェルの開口部径をＦ、ウェルの深さをＧ、ウェル間のピッチをＨとし、以下の４種のウェルを設計した。Ｆ、Ｇ、Ｈの値（単位はμｍ）と播種細胞濃度を以下の表４に示す。 (d) Comparative Example A microarray chip of a comparative example in which the wells had a single-layer structure instead of a two-layer structure was manufactured, and single-cell accommodation and cell proliferation were evaluated. Regarding the microarray chip of the comparative example, the following four types of wells were designed, with the well opening diameter being F, the well depth being G, and the pitch between the wells being H. The values of F, G, and H (in μm) and the seeded cell concentration are shown in Table 4 below.
Figure JPOXMLDOC01-appb-T000004
  
Figure JPOXMLDOC01-appb-T000004
  
　図１６Ａ，１６Ｂは、比較例１の播種直後（図１６Ａ）、及び培養１５日目（図１６Ｂ）のマイクロアレイチップの蛍光イメージである。一細胞は収容されるものの、１５日間の培養により細胞が増殖してクロスコンタミネーションが生じることが確認された。図１７Ａ，１７Ｂは、比較例２の播種直後（図１７Ａ）、及び培養１５日目（図１７Ｂ）のマイクロアレイチップの蛍光イメージである。播種細胞濃度の影響で一細胞が収容されたウェルは少なく、１５日間の培養による増殖が殆どみられなかった。図１８Ａ，１８Ｂは、比較例３の播種直後（図１８Ａ）、及び培養１５日目（図１８Ｂ）のマイクロアレイチップの蛍光イメージである。一細胞が収容されたウェルは存在するもの、１５日間の培養による増殖が確認できなかった。図１９Ａ，１９Ｂは、比較例４の播種直後（図１９Ａ）、及び培養１５日目（図１９Ｂ）のマイクロアレイチップの蛍光イメージである。播種直後から、１つのウェルに複数の細胞が収容され、１５日間の培養により細胞が増殖してクロスコンタミネーションが生じることが確認された。 16A and 16B are fluorescence images of the microarray chip of Comparative Example 1 immediately after seeding (FIG. 16A) and on the 15th day of culture (FIG. 16B). Although one cell was accommodated, it was confirmed that the cells proliferated after 15 days of culture, causing cross-contamination. 17A and 17B are fluorescence images of the microarray chip immediately after seeding (FIG. 17A) and on the 15th day of culture (FIG. 17B) in Comparative Example 2. Due to the influence of the seeded cell concentration, only a few wells contained a single cell, and almost no growth was observed during the 15 days of culture. 18A and 18B are fluorescence images of the microarray chip immediately after seeding (FIG. 18A) and on the 15th day of culture (FIG. 18B) in Comparative Example 3. Although there were wells containing single cells, no growth was observed during 15 days of culture. 19A and 19B are fluorescence images of the microarray chip of Comparative Example 4 immediately after seeding (FIG. 19A) and on the 15th day of culture (FIG. 19B). It was confirmed that a plurality of cells were accommodated in one well immediately after seeding, and that cells proliferated after 15 days of culture, causing cross-contamination.
　（４）薬剤耐性株の検出
　ＰＣ９細胞を、ＲＰＭＩ１６４０（３０２６４－５６，ナカライテスク）に１０ｖ／ｖ％　ｆｅｔａｌ　ｂｏｖｉｎｅ　ｓｅｒｕｍ（ＦＢＳ；Ｓｉｇｍａ－Ａｌｄｒｉｃｈ　Ｃｏ．　ＬＬＣ）と５ｖ／ｖ％　ｐｅｎｉｃｉｌｌｉｎ（Ｐ４３３３；Ｓｉｇｍａ－Ａｌｄｒｉｃｈ　Ｃｏ．ＬＬＣ）を加えた溶液に懸濁し、１．０×１０７ｃｅｌｌｓ／ｍＬの細胞懸濁液を調製した。この細胞懸濁液を、ピペットを用いてマイクロアレイチップに滴下し、３０～６０分間静置した。マイクロアレイチップのウェルの仕様は、Ｘを３４μｍ、Ｔを２０μｍ、Ｓを２８μｍ、Ｖを１１０～１５０μｍとし、Ｙは１１０μｍまたは１３０μｍとした。次いで、余剰細胞を、スクレイパーを用いて洗浄した。この操作により、ウェルの第２の部分に単一細胞を格納し、先の実施例と同様にして一細胞収容率を取得した。次いで、抗がん剤（Ｇｅｆｉｔｉｎｉｂ（ＺＤ１８３９））を含む培地中にマイクロアレイチップを浸漬し、７日間の培養を行った。播種直後及び３日後に倒立顕微鏡（ＩＸ－７３；Ｏｌｙｍｐｕｓ）とＣＣＤ　ｃａｍｅｒａ（ＤＰ８０；Ｏｌｙｍｐｕｓ）を用いて細胞状態を観察した。Ｇｅｆｉｔｉｎｉｂの濃度は、０、１、５、１０、１００μＭで変化させた。 (4) 10V / V % FETAL BOVINE SERUM (FBS; SIGMA -ALDRICH CO. LLC) and 5V / V % PENI on RPMI1640 (30264-56, Nakarai Tesk). CILLIN (P4333; SIGMA- Aldrich Co. LLC) was added to prepare a cell suspension of 1.0×10 7 cells/mL. This cell suspension was dropped onto the microarray chip using a pipette and left to stand for 30 to 60 minutes. The specifications of the wells of the microarray chip were as follows: Excess cells were then washed away using a scraper. By this operation, a single cell was stored in the second part of the well, and the single cell accommodation rate was obtained in the same manner as in the previous example. Next, the microarray chip was immersed in a medium containing an anticancer drug (Gefitinib (ZD1839)), and cultured for 7 days. Immediately after seeding and 3 days later, the state of the cells was observed using an inverted microscope (IX-73; Olympus) and a CCD camera (DP80; Olympus). The concentration of Gefitinib was varied at 0, 1, 5, 10, 100 μM.
　図２０Ａ，２０Ｂは、ＰＣ９細胞の播種直後（図２０Ａ）、及び５μＭのＧｅｆｉｔｉｎｉｂでの培養３日目（図２０Ｂ）のマイクロアレイチップの蛍光および明視野イメージである。図２０Ａ，２０Ｂから、一部のＰＣ９細胞はチャンバ内で増殖していることがわかる。図２１は、ＰＣ９細胞の一細胞増殖率を示すグラフである。図２１から、１細胞が増殖したチャンバ数が抗がん剤濃度に反比例することがわかり、本マイクロチップを用いて薬剤耐性を有する細胞のみを培養できることがわかった。 FIGS. 20A and 20B are fluorescence and bright field images of the microarray chip immediately after seeding of PC9 cells (FIG. 20A) and on the third day of culture with 5 μM Gefitinib (FIG. 20B). From FIGS. 20A and 20B, it can be seen that some PC9 cells are proliferating within the chamber. FIG. 21 is a graph showing the single cell proliferation rate of PC9 cells. From FIG. 21, it was found that the number of chambers in which one cell proliferated was inversely proportional to the anticancer drug concentration, and it was found that only drug-resistant cells could be cultured using this microchip.
　１　マイクロアレイチップ
　２　ウェル
　２１　第１の部分
　２１１　底面
　２２　第２の部分
　２２１　底面
　３　表面
  1 Microarray chip 2 Well 21 First portion 211 Bottom surface 22 Second portion 221 Bottom surface 3 Surface

Claims (7)

  1. 　細胞を収容可能な複数のウェルが形成されたマイクロアレイチップであって、
    　複数のウェルのそれぞれは、複数の細胞を収容可能な第１の部分と、単一細胞のみを収容可能な第２の部分とを有し、
    　前記第１の部分は、前記マイクロアレイチップの表面から凹んだ凹部として形成され、
    　前記第２の部分は、前記第１の部分の底面から凹んだ凹部として形成されている、マイクロアレイチップ。     A microarray chip in which a plurality of wells capable of accommodating cells are formed,
    Each of the plurality of wells has a first part capable of accommodating a plurality of cells and a second part capable of accommodating only a single cell,
    The first portion is formed as a recess recessed from the surface of the microarray chip,
    In the microarray chip, the second portion is formed as a concave portion recessed from the bottom surface of the first portion.
  2. 　前記第２の部分の内径は、前記第１の部分の内径よりも小さい、請求項１に記載のマイクロアレイチップ。 The microarray chip according to claim 1, wherein an inner diameter of the second portion is smaller than an inner diameter of the first portion.
  3. 　前記第１の部分の開口部および前記第２の部分の開口部は、円形または正多角形の形状を有する、請求項１または２に記載のマイクロアレイチップ。 The microarray chip according to claim 1 or 2, wherein the opening in the first portion and the opening in the second portion have a circular or regular polygonal shape.
  4. 　請求項１に記載のマイクロアレイチップを用いた、細胞培養方法であって、
    　（ａ）前記マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程と、
    　（ｂ）前記工程（ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程と、
    　（ｃ）前記工程（ｂ）で得られた前記複数のウェルに収容された単一細胞を培養する工程と
    を含む方法。     A cell culture method using the microarray chip according to claim 1, comprising:
    (a) applying a liquid containing a plurality of cells onto the microarray chip;
    (b) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in step (a);
    (c) A method comprising the step of culturing the single cells accommodated in the plurality of wells obtained in the step (b).
  5. 　請求項１に記載のマイクロアレイチップを用いた、単一細胞の評価方法であって、
    　（Ａ）前記マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程と、
    　（Ｂ）前記工程（Ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程と、
    　（Ｃ）前記工程（Ｂ）で得られた前記複数のウェルに収容された細胞を培養する工程と、
    　（Ｄ）前記工程（Ｃ）の培養後の状態に基づき、単一細胞の細胞特性を評価する工程を含む方法。     A method for evaluating a single cell using the microarray chip according to claim 1, comprising:
    (A) applying a liquid containing a plurality of cells onto the microarray chip;
    (B) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in the step (A);
    (C) culturing the cells accommodated in the plurality of wells obtained in the step (B);
    (D) A method comprising the step of evaluating the cell characteristics of a single cell based on the state after culturing in step (C).
  6. 　前記工程（Ｃ）が、細胞の特性に影響を与えうる候補物質の存在下で細胞を培養する工程を含む、請求項５に記載の方法。 The method according to claim 5, wherein the step (C) includes culturing the cells in the presence of a candidate substance that can affect the properties of the cells.
  7. 　請求項１に記載のマイクロアレイチップを用いた、モノクローンの細胞コロニーの製造方法であって、
    　（ａ）前記マイクロアレイチップ上に、複数の細胞を含む液体を適用する工程と、
    　（ｂ）前記工程（ａ）で得られたマイクロアレイチップの表面を洗浄することにより、前記複数のウェルの前記第２の部分に単一細胞のみを収容する工程と、
    　（ｃ）前記工程（ｂ）で得られた前記複数のウェルに収容された単一細胞を培養する工程と
    を含む方法。
          A method for producing a monoclonal cell colony using the microarray chip according to claim 1, comprising:
    (a) applying a liquid containing a plurality of cells onto the microarray chip;
    (b) accommodating only single cells in the second portions of the plurality of wells by washing the surface of the microarray chip obtained in step (a);
    (c) A method comprising the step of culturing the single cells accommodated in the plurality of wells obtained in the step (b).
PCT/JP2023/018246 2022-05-26 2023-05-16 Microarray chip and cell culture method using microarray chip WO2023228814A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2022-086270 2022-05-26
JP2022086270A JP2023173785A (en) 2022-05-26 2022-05-26 Microarray chip, and cell culture method using microarray chip

Publications (1)

Publication Number Publication Date
WO2023228814A1 true WO2023228814A1 (en) 2023-11-30

Family

ID=88919173

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/018246 WO2023228814A1 (en) 2022-05-26 2023-05-16 Microarray chip and cell culture method using microarray chip

Country Status (2)

Country Link
JP (1) JP2023173785A (en)
WO (1) WO2023228814A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008532539A (en) * 2005-03-16 2008-08-21 ロビオ システムズ リミテッド Maturation and transport system of cellular entities
JP2012529643A (en) * 2009-06-09 2012-11-22 オックスフォード・ジーン・テクノロジー・アイピー・リミテッド Picowell capture device for single cell or other particle analysis
JP2016007178A (en) * 2014-06-25 2016-01-18 大日本印刷株式会社 Cell culture vessel

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008532539A (en) * 2005-03-16 2008-08-21 ロビオ システムズ リミテッド Maturation and transport system of cellular entities
JP2012529643A (en) * 2009-06-09 2012-11-22 オックスフォード・ジーン・テクノロジー・アイピー・リミテッド Picowell capture device for single cell or other particle analysis
JP2016007178A (en) * 2014-06-25 2016-01-18 大日本印刷株式会社 Cell culture vessel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YAMAMURA, SHOHEI ET AL.: "Single cell separation, detection, and collection technology using cell chips,", BUNRI GIJUTSU [SEPARATION TECHNOLOGY], TECHNICAL COMMITTEE ON SEPARATION TECHNOLOGY, JAPAN, vol. 51, no. 5, 1 January 2021 (2021-01-01), Japan , pages 273 - 280, XP009550868, ISSN: 1343-7860 *

Also Published As

Publication number Publication date
JP2023173785A (en) 2023-12-07

Similar Documents

Publication Publication Date Title
CN107407691B (en) Device and system for molecular barcoding of nucleic acid targets in single cells
US9957472B2 (en) Deterministic high-density single-cell trap array
US10087408B2 (en) System and method for microfluidic cell culture
US7374906B2 (en) Biological assays using gradients formed in microfluidic systems
CN108026499B (en) Device for propagating micro-tissue
US20230086120A1 (en) Monolayer of pbmcs or bone-marrow cells and uses thereof
WO2017177839A1 (en) Super-hydrophobic micro-pit array chip, preparation method therefor and applications thereof
Wang et al. Trapping cells on a stretchable microwell array for single-cell analysis
JP6218185B2 (en) Cell deployment device and method for detecting rare cells
KR20180097726A (en) Flow cell with micro-retainer for discrete seeding microspots
ES2887105T3 (en) Integrated platform for single cell analysis
US20190054461A1 (en) Multi-use combined micro and nanowell plates
Pang et al. Microstructure-based techniques for single-cell manipulation and analysis
Chao et al. SpheroidChip: patterned agarose microwell compartments harboring HepG2 spheroids are compatible with genotoxicity testing
Chen et al. MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays
AU2003287170A1 (en) Biological assays using gradients formed in microfluidic systems
WO2023228814A1 (en) Microarray chip and cell culture method using microarray chip
WO2016140327A1 (en) Microchamber array plate
TWI588256B (en) Device and method for single cell isolation and cultivation
Li et al. High-throughput microfluidic single-cell trapping arrays for biomolecular and imaging analysis
JP6395712B2 (en) Microarray chip and cell analysis method using the chip
US20230235261A1 (en) Droplet trapping structure array, method for spheroid transfer and formation of spheroid array using the same
WO2020158178A1 (en) Particle capture device, particle capture method, and microscope system
Acevedo et al. Microtechnology applied to stem cells research and development
US20230221299A1 (en) A device for studying interactions of a first cell type with a second cell type and related method

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23811688

Country of ref document: EP

Kind code of ref document: A1