Microarray chip and cell culture method using microarray chip

Abstract

To provide a microarray chip that securely separates a large number of single cells, and is capable of culturing a monoclonal colony at high efficiency.SOLUTION: Provided is a microchip array 1 on which a plurality of wells 2 capable of housing cells are formed, where each of a plurality of wells has a first portion 21 capable of housing a plurality of cells and a second portion 22 capable of housing only single cells, the first portion is formed as a recess recessed from the surface of the microchip array, and the second portion is formed as a recess recessed from the bottom face of the first portion.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] 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 capable of reliably separating a large number of single cells and culturing monoclonal cell colonies with high efficiency, and a cell culture method using the same.

Background Art

[0002] The cells constituting cancer differ in resistance to anticancer drugs for each cell. In order to detect drug-resistant strains, it is necessary to separate a large number of cells into single cells, culture the separated single cells, and evaluate them. However, for example, it is very difficult to detect and separate target single cancer cells from a large number of cells in blood or tissues, and further culture and evaluate the separated single cells. In the method of culturing a cell population in a petri dish or the like, it is difficult to efficiently detect and clone drug-resistant strains at the single-cell level. Therefore, various microchannel devices having microchannels with fine structures have been proposed for trapping and culturing cells.

[0003] For example, a microchannel device for single cell separation is known, which includes an upper layer of a slide glass and a lower layer made of PDMS having microchannels and wells, and a U-shaped trap for capturing and holding a single cell is formed at the downstream end of each well (see, for example, Non-Patent Document 1). In addition to the trap, each well also forms a large surface area, providing a space for expanding and growing the captured single cells.

[0004] Also known is a dual-well type microchannel device configured to arrange a micro-well of a minute size and a larger micro-well facing each other vertically to perform capture and culture of single cells (see, for example, Non-Patent Document 2).

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] H. Chen et al., High-throughput, deterministic single cell trapping and long-term clonal cell culture in microfluidic devices, Lab Chip, 15, 1072-1083, 2015 [Non-Patent Document 2] C. Lin et al., A microfluidic dual-well device for high-throughput single-cell capture and culture, Lab Chip, 15, 2928-2938, 2015 [Overview of the project] [Problems that the invention aims to solve]

[0006] Non-patent documents 1 and 2 mentioned above disclose methods for trapping and culturing cells using microfluidic devices. However, due to the complexity of the operation and the difficulty of retrieving cells from the microfluidic channels, there is still a need for devices and methods that can reliably isolate many single cells and culture them with high efficiency.

[0007] The present invention has been made in view of the above-described circumstances, and its purpose is to provide a microarray chip and a cell culture method using the microarray chip that can reliably isolate a large number of single cells and culture monoclonal colonies with high efficiency. [Means for solving the problem]

[0008] According to one embodiment of the present invention, the present invention relates to a microchip chip having a plurality of wells capable of accommodating cells, wherein each of the plurality of wells has a first portion capable of accommodating a plurality of cells and a second portion capable of accommodating only a single cell, the first portion being formed as a recessed area from the surface of the microchip array, and the second portion being formed as a recessed area from the bottom surface of the first portion.

[0009] According to another embodiment of the present invention, the present invention relates to a cell culture method using the aforementioned microarray chip, (a) A step of applying a liquid containing multiple cells onto the microarray chip, (b) A step of cleaning the surface of the microarray chip obtained in step (a) to contain only single cells in the second portion of the plurality of wells, (c) A step of culturing the single cells contained in the plurality of wells obtained in step (b) above. Regarding methods including

[0010] According to another embodiment of the present invention, a method for evaluating single cells using the aforementioned microarray chip, (A) A step of applying a liquid containing multiple cells onto the microarray chip, (B) A step of cleaning the surface of the microarray chip obtained in step (A) to contain only single cells in the second portion of the plurality of wells, (C) A step of culturing the single cells contained in the plurality of wells obtained in step (B), (D) A step of evaluating the cellular characteristics of a single cell based on the state after culturing in step (C) above. Regarding methods including

[0011] According to another embodiment of the present invention, a method for producing monoclonal cell colonies using the aforementioned microarray chip, (a) A step of applying a liquid containing multiple cells onto the microarray chip, (b) By washing the surface of the microarray chip obtained in the step (a), a step of accommodating only single cells in the second portion of the plurality of wells; (c) A step of culturing the single cells accommodated in the plurality of wells obtained in the step (b); It relates to a method including.

Effect of the Invention

[0012] According to the present invention, it is possible to realize a microarray chip capable of surely separating a large number of single cells and obtaining monoclonal colonies with high efficiency, and a cell culture method using the microarray chip.

Brief Description of the Drawings

[0013] [Figure 1] 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. [Figure 2] FIG. 2 is a cross-sectional view taken along the line A-A of FIG. 1. [Figure 3] FIG. 3 is a top view schematically showing a microarray chip. [Figure 4] FIG. 4 is a diagram showing a modified example of a well. [Figure 5] FIG. 5 is a diagram showing a modified example of a well. [Figure 6] FIG. 6 is a conceptual diagram showing an example of the behavior of cells in a single well in a cell culture method using a microarray chip according to a second embodiment of the present invention. [Figure 7] FIG. 7 is a fluorescence image of wells 3 hours (a) and 7 days (b) after seeding HeLa cells on a microarray chip. [Figure 8] FIG. 8 is a graph showing the single-cell accommodation rate of HeLa cells in each well when the structure of the wells of the microarray chip is changed. [Figure 9] FIG. 9 is a fluorescence image of a microarray chip immediately after seeding HeLa cells (a) and 15 days after culturing (b). [Figure 10] Figure 10 is a graph showing the relationship between the pitch between wells and the single-cell accommodation rate of HeLa cells. [Figure 11] Figure 11 is a graph showing the single-cell growth rate of HeLa cells in culture using microarray chips with different pitches between wells. [Figure 12] Figure 12 is a fluorescence image of HeLa cells cultured for 6 days on a microarray chip having wells with an inner diameter Y of 110 μm in the first portion 21. [Figure 13] Figure 13 is a graph showing the single-cell growth rate of HeLa cells in culture using a microarray chip having wells with an inner diameter Y of 110 μm in the first portion 21. [Figure 14] Figure 14 is a fluorescence image of HeLa cells cultured for 6 days on a microarray chip having wells with an inner diameter Y of 130 μm in the first portion 21. [Figure 15] Figure 15 is a graph showing the single-cell growth rate of HeLa cells in culture using a microarray chip having wells with an inner diameter Y of 130 μm in the first portion 21. [Figure 16] Figure 16 is a fluorescence image of a microarray chip of Comparative Example 1 at immediately after seeding (a) and on the 15th day of culture (b) of HeLa cells in culture. [Figure 17] Figure 17 is a fluorescence image of a microarray chip of Comparative Example 2 at immediately after seeding (a) and on the 15th day of culture (b) of HeLa cells in culture. [Figure 18] Figure 18 is a fluorescence image of a microarray chip of Comparative Example 3 at immediately after seeding (a) and on the 15th day of culture (b) of HeLa cells in culture. [Figure 19] Figure 19 is a fluorescence image of a microarray chip of Comparative Example 4 at immediately after seeding (a) and on the 15th day of culture (b) of HeLa cells in culture. [Figure 20]Figure 20 shows fluorescence and bright-field images of the microarray chip immediately after seeding of PC9 cells (a) and on day 3 of culture with 5 μM Gefitinib (b) in a culture using the microarray chip of the present invention. [Figure 21] Figure 21 is a graph showing the single-cell proliferation rate of PC9 cells. [Modes for carrying out the invention]

[0014] [1. Microarray chip] Hereinafter, a microarray chip according to the first embodiment of the present invention will be described in detail with reference to the drawings. Figure 1 is a schematic top perspective view showing the well configuration in the microarray chip according to the first embodiment of the present invention, and Figure 2 is a cross-sectional view AA of Figure 1.

[0015] As shown in Figures 1 and 2, the microarray chip 1 has a plurality of wells 2 capable of containing cells, and each of the plurality of wells 2 has a first portion 21 capable of containing multiple cells and a second portion 22 capable of containing only a single cell. The first portion 21 is formed as a recessed area from the surface 3 of the microarray chip 1. The second portion 22 is formed as a recessed area from the bottom surface 211 of the first portion 21. That is, each well 2 is configured as a two-tiered well having one upper first portion 21 and one lower second portion 22. The bottom surface 211 of the first portion 21 may be parallel to the surface 3 or it may be inclined. If the bottom surface 211 is inclined, for example it can be sloped downwards toward the center of the bottom surface 211. The bottom surface 221 of the first portion 22 may be parallel to the surface 3 or it may be slightly recessed.

[0016] The second section 22 functions as a trap that isolates and captures only single cells. Meanwhile, the first section 21 functions as a chamber for culturing and growing the single cells captured in the second section 22. Therefore, the inner diameter X of the opening of the second section 22 is set to be smaller than the inner diameter Y of the first section 21 (X <Y)。

[0017] The openings of the first section 21 and the second section 22 can be circular, for example, as shown in Figure 1. However, they are not limited to this, and the openings can also be regular polygons, as will be described later. Below, as an example, we will describe the case where the openings of the first section 21 and the second section 22 are both circular and concentric.

[0018] The inner diameter X of the second section 22 is sized to accommodate only a single cell. That is, it is not possible to arrange two or more cells to be captured side by side in the radial direction of the second section 22. Therefore, the inner diameter X of the second section 22 is set to a size that allows for some leeway over the size of the single cell to be captured. The size of the single cell can be appropriately set by a person skilled in the art depending on the source, characteristics, and size distribution of the cells to be captured. Preferably, the inner diameter X can be set to a size that allows for a leeway of about 0 to 50 μm over the size of the single cell, or to a size that is 100% to 300% of the cell size. For example, the inner diameter X can be several tens of μm, and can be 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 size can be set as the inner diameter X according to the size of the cell to be captured.

[0019] The inner diameter Y of the first section 21 is sized to allow cell colonies to form from a single cell captured in the second section 22. That is, the dimensions are set so that, for example, multiple cells can be arranged radially within the first section 21. However, the inner diameter Y of the first section 21 may be set so that multiple cells overlap when viewed from above the well 2, rather than being arranged radially, as long as the cells do not protrude significantly from the first section 21 and cause cross-contamination with the adjacent well 2. Based on these considerations, 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.

[0020] The depth S of the second section 22, that is, the length from the bottom surface 211 of the first section 21 to the bottom surface 221 of the second section 22, is sized to accommodate only a single cell. In other words, it is not possible to place two or more cells to be captured side by side in the depth direction of the second section 22. Therefore, the depth S of the second section 22 is set to allow for some leeway in the size of a single cell, being slightly larger (deeper) or slightly smaller (shallower) than the size of a single cell. The depth S with some leeway can be set in the same way as the design of the inner diameter X, and can be set to a dimension that is, for example, 0 to 50 μm larger (deeper) than the size of the cell, or to a dimension that is 100 to 300% of the size of the cell. On the other hand, a dimension that is slightly smaller than the size of a single cell can be set to a dimension that is 50 to 100% of the size of the cell. For example, the depth S can be several tens of μm, and can be, for example, about 5 to 40 μm depending on the type of cell. However, this is not the only option; it can be modified depending on the cell type, and an appropriate dimension can be set as the depth S according to the size of the cells to be captured.

[0021] The depth T of the first section 21, that is, the length from the surface of the microarray chip 1 to the bottom surface 211 of the first section 21, is set to a dimension that can restrict cell proliferation. For example, the depth can be such that multiple layers of cells are not formed in the depth direction of the first section 21, that is, only a single layer of cells is formed. However, the depth T of the first section 21 may be set to a dimension that allows for the formation of multiple layers in the depth direction, provided that the cells do not protrude significantly from the first section 21 and cause cross-contamination with adjacent wells 2. Based on these considerations, for example, the depth T can be several tens of micrometers, for example, about 1 to 50 micrometers. 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 depth T according to the size of the cells to be cultured.

[0022] 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 of the first microarray chip 1. The wall angle U is determined by the conditions during the manufacturing of the microarray chip 1, but can be, for example, about 0 to 45°, and is preferably 0 to 20°. When the wall angle U is 0°, the first portion 21 and the second portion 22 are cylindrical in shape, and when the wall angle U is greater than 0°, the first portion 21 and the second portion 22 are frustoconical in shape. Note that the wall angle U of the first portion 21 and the wall angle U of the second portion 22 may be different values.

[0023] The pitch V between adjacent wells 2, i.e., the distance between the center of the opening of the second portion 22 of well 2 and the center of the opening of the second portion 22 of the adjacent well 2, can be a distance sufficient to reduce cross-contamination when culturing cells in the first portion 21. The pitch V depends on the cell type, the inner diameter Y of the first portion 21 and the inner diameter X of the second portion 22, etc., but can be, for example, about 100 to 500 μm.

[0024] The material of the microarray chip 1 is not particularly limited, but examples include polymers such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and cyclic olefin copolymer (COC), metals such as silicon, glass, quartz glass, and combinations of multiple materials such as polymers bonded to glass or metal (for example, PDMS and glass). Preferably, the material is polystyrene, PMMA, glass, silicon, etc.

[0025] The transparency and transmittance of the microarray chip 1 are not particularly limited, but it is preferable that they have transparency and transmittance suitable for observing the cells contained in well 2. For example, when observing the microarray chip 1 with an inverted microscope, it is preferable that well 2 is light-transmitting. Here, transparency means that the transmittance of the predetermined light to be observed is 80% or more, and preferably 90% or more.

[0026] The microarray chip 1 can be fabricated by various known methods. For example, it can be fabricated by directly processing the wells 2 into a substrate material, by attaching a film with through holes or wells 2 formed thereon to a substrate material, or by plastic molding. For example, when fabricating the microarray chip 1 by plastic molding, specific methods such as lithography (photolithography, electron beam lithography, etc.) in the semiconductor technology field, cutting using a micro-drill, or laser processing can be used.

[0027] For example, when manufacturing using lithography technology and plastic molding, a plastic master of the microarray chip 1 is created by lithography and etching, and a mold is created by electroforming the master. Then, the microarray chip 1 is manufactured by injection molding using the manufactured mold. By employing injection molding using a mold in this way, it is possible to manufacture a microarray chip 1 with a smooth surface.

[0028] The microarray chip 1 may be surface-treated as needed. The surface treatment method is not particularly limited, but plasma treatment, corona discharge treatment, etc., can be used. If the substrate material of the microarray chip 1 is a hydrophobic material (e.g., polymers such as polystyrene and PMMA), hydrophilization treatment such as plasma treatment, for example, oxygen plasma treatment, can be performed. If the substrate material of the microarray chip 1 is a hydrophilic material (e.g., silicon or glass), hydrophilization treatment is not necessary. Furthermore, surface treatment can be performed by coating the wells 2 with proteins or lipids, and examples of surface treatments using collagen, fibronectin, poly-D-lysine, poly-L-lysine, laminin, vitronectin, Matrigel, etc., are listed, but are not limited to these.

[0029] Figure 3 shows a schematic top view of the entire microarray chip 1. As shown in Figure 3, the microarray chip 1 is formed as a rectangular plate-shaped member as a whole. Multiple wells 2 are regularly arranged on the surface 3 of the microarray chip 1. In the example shown in Figure 3, multiple blocks 4 are arranged on the microarray chip 1 in a matrix state consisting of multiple rows 1 to 6 along the long sides 11 and 12 of the microarray chip 1 and multiple columns A to P along the short sides 13 and 14.

[0030] Multiple blocks 4 are arranged with gaps between them and the long sides 11, 12 and short sides 13, 14 of the microarray chip 1. The multiple blocks 4 are arranged on the microarray chip 1, for example, within an area of ​​approximately 50 mm x approximately 15-18 mm, although this is not particularly limited.

[0031] Each block 4 has multiple wells 2 arranged in a matrix. For example, each block 4 can contain several thousand wells 2. If each block 4 contains 1,000 wells 2, then the entire microarray chip 1 shown in Figure 3 will contain 96,000 wells 2. Note that the number of wells 2 arranged in the microarray chip 1 is not limited. The microarray chip 1 can have at least two wells 2. Note that it is not necessary for multiple blocks to be formed in a single microarray chip; a single microarray chip may contain only one block.

[0032] The microarray chip according to this embodiment may be an open-type microarray chip without a cover or lid above the wells. This allows a large number of cells to be applied to the microarray chip substantially simultaneously in the culture method described later, and also facilitates operations such as bringing the culture medium and drugs used in the culture into contact with the cells, and harvesting the cells.

[0033] The microarray chip 1 according to this embodiment, as described above, can achieve the following effects.

[0034] The microchip array 1 has 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 multiple cells and a second portion 22 capable of accommodating only a single cell, the first portion 21 is formed as a recessed area from the surface 3 of the microchip array 1, and the second portion 22 is formed as a recessed area from the bottom surface 211 of the first portion 21.

[0035] The microarray chip 1 is configured such that each well 2 has a two-tiered structure consisting of an upper first section 21 and a lower second section 22. The lower second section 22 is configured to accommodate only single cells, making it suitable for separating a large number of cells into single cells. On the other hand, the upper first section 21 is configured to accommodate multiple cells, making it suitable for culturing and growing the single cells contained in the second section 22. In this way, the microarray chip 1 can achieve reliable single-cell isolation and highly efficient culture of the isolated cells with just one well 2 in its two-tiered structure. This makes it possible to detect and clone drug-resistant strains using the microarray chip 1, and for example, in brain tumors with a very poor prognosis (e.g., malignant gliomas), it becomes possible to spread tumor tissue cell by cell onto the chip and perform anticancer drug resistance and sensitivity tests.

[0036] Since the inner diameter X of the second section 22 is smaller than the inner diameter Y of the first section 21, reliable separation of single cells can be achieved.

[0037] Since the openings of the first portion 21 and the second portion 22 have a circular shape, an appropriate shape can be selected based on various conditions such as the cell type and the method of manufacturing the microarray chip 1.

[0038] -Variations- (1) In the embodiment described above, an example was described in which the openings of the first part 21 and the second part 22 constituting the two-tiered well 2 are circular, but the shape of the opening is not limited to a circle. The shape of the opening may be an ellipse instead of a perfect circle. The opening can also be a polygon such as a triangle, quadrilateral, or hexagon, preferably a regular polygon. Figure 4 shows a modified example of well 2. Figure 4(a) shows an example in which the openings of the first part 21 and the second part 22 are regular pentagons, and Figure 4(b) shows an example in which the openings of the first part 21 and the second part 22 are equilateral triangles.

[0039] If the shape of the opening is a regular polygon, the diameter of the circumscribed circle CC of the regular polygon can be used as the inner diameter of the opening, and the inner diameter Y of the first part 21 and the inner diameter X of the second part 22 can be set as described above. In the case of an equilateral triangle shown in Figure 4(b), instead of the diameter of the circumscribed circle, the lengths of each side of the equilateral triangle can be used as the diameter of the opening, and the inner diameter Y of the first part 21 and the inner diameter X of the second part 22 can be set as described above. By configuring the openings of the first part 21 and the second part 22 to have the shape of a regular polygon, an appropriate shape can be selected based on various conditions such as the cell type and the method of manufacturing the microarray chip 1, similar to the case of a circle described above.

[0040] (2) In the embodiment described above, an example was described in which the openings of the first portion 21 and the second portion 22 constituting the two-tiered well 2 are concentric circles. However, the positional relationship between the first portion 21 and the second portion 22 is not limited to this. Figure 5 shows a modified example of well 2. Figure 5(a) shows an example in which the openings of the first portion 21 and the second portion 22 are concentric circles, and Figures 5(b) to (e) show examples in which the center of the opening of the second portion 22 is offset from the center of the opening of the first portion 21. Here, the positional relationship between the first portion 21 and the second portion 22 will be explained assuming that the well 2 shown in Figures 5(b) to (e) is arranged on the microarray chip 1 shown in Figure 3 in the orientation shown.

[0041] In the example shown in Figure 5(b), the center of the opening of the second portion 22 is positioned closer to the long side 11 of the microarray chip 1 shown in Figure 3, relative to the center of the opening of the first portion 21. In the example shown in Figure 5(c), the center of the opening of the second portion 22 is positioned closer to the long side 12 of the microarray chip 1, relative to the center of the opening of the first portion 21. In the example shown in Figure 5(d), the center of the opening of the second portion 22 is positioned closer to the short side 13 of the microarray chip 1 shown in Figure 3, relative to the center of the opening of the first portion 21. In the example shown in Figure 5(e), the center of the opening of the second portion 22 is positioned closer to the short side 14 of the microarray chip 1, relative to the center of the opening of the first portion 21.

[0042] In Figures 5(b) to 5(e), the opening of the first portion 21 and the opening of the second portion 22 are arranged to be in contact when viewed from above the well 2, but the design is not limited to this, and the openings of the first portion 21 and the second portion 22 may be arranged to be separated from each other.

[0043] (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 the same, but the invention is not limited to this, and the shape of the opening of the first portion 21 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 circular.

[0044] (4) In the embodiment described above, an example was given in which the outer shape of the microarray chip 1 is rectangular, but it is not limited to this, and it can also be in a shape other than rectangular.

[0045] (5) In the embodiment described above, multiple wells 2 are arranged in a matrix on the microarray chip 1, but the invention is not limited to this, and multiple wells 2 may be arranged alternately. Furthermore, the shape and dimensions of the openings of the multiple wells 2 arranged on the microarray chip 1 may be the same, or they may be set to be different for each block 4, for example.

[0046] [2. Cell culture method using microarray chips] 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 viewpoint, 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 monoclonal cell colonies. The method includes the following steps. (a) The process of applying a liquid containing multiple cells onto a microarray chip. (b) A step of cleaning the surface of the microarray chip obtained in step (a) to contain only single cells in the second portion of the plurality of wells. (c) A step of culturing the cells contained in the plurality of wells obtained in step (b) above.

[0047] Figure 6 is a diagram illustrating a cell culture method using a microarray chip according to the first embodiment, and conceptually shows the state of cells in one well 2 on the microarray chip 1 illustrated in Figures 1 to 3.

[0048] In step (a), a liquid containing multiple cells is applied to the microarray chip 1. That is, cells are seeded onto the microarray chip 1. The cell concentration in the liquid should be at least sufficient to cover the entire surface 3 of the microarray chip 1 with a single layer of cells at the time the liquid containing cells is applied, for example, 1.0 × 10⁻⁶ 6 It is preferable to have a concentration of cells / mL or higher. For example, 1.0 × 10 10 cells / mL or less, preferably 1.0 × 10⁶ 9 cells / mL or less, or 1.0 × 10 8 The cell concentration can be kept below approximately cells / mL. If the cell concentration is too low, a sufficient number of cells may not be trapped in the chamber, and if the cell concentration is too high, there is a risk of wasting culture medium and cells. Culture medium may also be used as one of the liquids for seeding, and it is also possible to culture the cells in the seeded state.

[0049] The method of applying liquid to the microarray tip is not particularly limited. For example, the liquid can be dropped onto the microarray tip using a pipette. The amount dropped is not particularly limited, as it will vary depending on the area of ​​the microarray tip. A person skilled in the art can determine the amount dropped as appropriate. 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.

[0050] Figure 6(a) schematically shows the state of well 2 and cell H after performing step (a). A single cell is contained in the second portion 22 of well 2. In addition, multiple cells are contained in the first portion 21, and cells are also present on the surface 3 of the microarray chip 1 surrounding well 2.

[0051] In the subsequent step (b), the surface 3 of the microarray chip 1 obtained in step (a) is washed. This removes excess cells. Washing can be performed, for example, by using a pipette to rinse off the excess cells present on the surface 3 of the microarray chip 1 with a liquid such as buffer or culture medium. Alternatively, a scraper can be moved along the surface 3 of the microarray chip 1 to remove the excess cells present on the surface 3 of the microarray chip.

[0052] Figure 6(bI) conceptually illustrates how excess cells are removed, and (bII) conceptually illustrates how, as a result of the removal of excess cells, single cells are contained in the second portion 22 of one well 2. This makes it possible to efficiently contain single cells in one well 2.

[0053] In step (b), after the washing procedure, imaging using a microscope is performed to identify wells containing single cells. This procedure identifies wells from which monoclonal cell colonies can be produced.

[0054] For steps (a) and (b), in addition to the microarray chip 1 according to the present invention, tools commonly used in cell experiments, such as pipettes and scrapers, may be used, and the microarray chip 1 can be handled under normal laboratory conditions. Furthermore, depending on the size of the microarray chip 1, steps (a) and (b) can be performed in approximately 30 to 120 minutes. For example, with a microarray chip 1 of a typical slide glass size (76 x 26 mm), approximately 30,000 to 40,000 cells, and up to a maximum of about 60,000 cells, can be simultaneously separated one by one and placed in each well. However, the required time and the number of cells that can be placed are not limited to the exemplified values.

[0055] Optionally, after the completion of steps (a) and (b), a step of obtaining the single-cell occupancy rate may be performed. This step can be carried out by imaging using a microscope.

[0056] Next, in step (c), the cells contained in well 2 of the microarray chip 1 obtained in step (b) are cultured under predetermined conditions. That is, the microarray chip 1 obtained in step (b) can be used as is for culturing. The culture conditions vary depending on the contained cells and are not particularly limited. Optionally, a step of adding the culture medium necessary for cell culture, or a step of immersing the microarray chip in the culture medium, may be included after step (b) and before step (c). Since the microarray chip 1 is open and integrated and easy to handle, it can be adapted to a wide range of culture conditions. Figure 6(c) shows well 2 after culturing for a predetermined period in step (c), where the cells have proliferated and monoclonal cell colonies C have been obtained.

[0057] Figure 6 illustrates the behavior of cells in one well, but cells will similarly proliferate in other wells of the microarray chip that contain a single cell, allowing for the creation of monoclonal cell colonies. It should be noted that some wells may not contain a single cell, and some wells may contain two or more cells; therefore, proliferation as illustrated may not occur in all wells. However, by using the microarray chip according to the first embodiment, a single-cell occupancy rate of at least 30% can be achieved overall.

[0058] By using the microarray chip 1 according to the present invention, cells can be cultured easily and without disadvantages such as cross-contamination by steps (a) to (c), and monoclonal cell colonies can be obtained.

[0059] An optional step (d) may be included after the completion of step (c) for collecting cell colonies from each well. A pipette or a manipulator may be used to collect the cell colonies. The collected cell colonies can then be used for any purpose, such as various analytical steps or further proliferation steps.

[0060] [3. Evaluation methods for single cells] Next, a third embodiment of the present invention will be described. The third embodiment relates to a method for evaluating single cells. The method for evaluating single cells includes the following steps: (A) A step of applying a liquid containing multiple cells onto the microarray chip. (B) A step of cleaning the surface of the microarray chip obtained in step (A) to contain only single cells in the second portion of the plurality of wells. (C) A step of culturing the cells contained in the plurality of wells obtained in step (B) above. (D) A step of evaluating the cellular characteristics of a single cell based on the state after culturing in step (C) above. Optionally, step (C) may include a step of culturing cells in the presence of a candidate substance that may affect the characteristics of the cells.

[0061] 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 the multiple cells contained in the cell population applied to the microarray chip in step (A). The number of multiple cells is not theoretically limited, but from a handling standpoint, it is possible to evaluate the individual characteristics of approximately 10 to 10,000 cells substantially simultaneously.

[0062] 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 culturing the cells in the presence of a candidate substance that may affect the cell properties in relation to the properties to be evaluated. For example, the candidate substance may be a small molecule compound, a large molecule compound, a bio-derived substance, or a combination thereof that is a candidate drug for a disease, and may be an anticancer drug, an antibiotic, etc. After the completion of step (C), i.e., after the generation of a monoclonal cell colony, the step may include contacting the cell colony with a candidate substance that may affect the cell properties in relation to the properties to be evaluated.

[0063] In step (D), based on the state after culturing in step (C), the cellular characteristics are evaluated for each single cell contained in each of the multiple wells, or for the monoclonal cell colonies generated by the proliferation of each single cell. The evaluation of cellular characteristics may include, but is not limited to, the cell proliferation rate, cell viability, specific physiological activity of the cell, or the adhesion strength of the cell to the chip. This makes it possible to obtain the individual characteristics of each cell contained in the cell population applied to the microarray chip in step (A).

[0064] As a more specific application example, we will describe the case where the cells to be cultured in step (A) are cells derived from malignant tumors. Cells derived from malignant tumors may be collected from the peripheral blood of a person affected by a malignant tumor. For example, circulating cancer cells (CTCs) can be collected from human peripheral blood. Alternatively, cells derived from malignant tumors can be collected from tissue sections of malignant glioma tumor tissue. Steps (A) and (B) are performed using these cells. Next, during the culture in step (C), an anticancer drug, for example, is applied to each well of the microarray chip 1. After a predetermined period of culture, the state of the cells is observed and evaluated in step (D), which allows for the identification of, for example, anticancer drug-resistant strains. Similarly, a step can be performed to confirm the sensitivity of cells to multiple types of anticancer drugs. This makes it possible to culture and evaluate single cancer cells from a specific patient, which can greatly contribute to prognosis diagnosis and treatment strategy determination for cancer.

[0065] According to the single-cell evaluation method of the third embodiment, multiple monoclonal cell colonies can be evaluated on a microarray chip. Therefore, the evaluation of single cells, which previously required general cloning, can be performed more simply and accurately. [Examples]

[0066] The present invention will be described in more detail below with reference to examples. However, the following examples are not intended to limit the present invention.

[0067] (1) Manufacturing of microarray chips A microarray chip of the embodiment shown in Figure 3 was manufactured. Polystyrene was used as the material, and a mold was used to produce a microarray chip with wells of a specific shape, size, and pitch. A comparative example microarray chip without a two-stage structure was manufactured in the same manner.

[0068] (2)Cell culture HeLa cells expressing GFP were suspended in a solution of Dulbecco's modified Eagle's medium (DMEM, 12800-017; Thermo Fisher Scientific) with 10 v / v% fetal bovine serum (FBS; Sigma-Aldrich Co. LLC) and 5 v / v% penicillin (P4333; Sigma-Aldrich Co. LLC) added, and measured in 1.0 × 10⁶ units. 7 A cell suspension at a concentration of cells / mL was prepared. This cell suspension was dropped onto a microarray tip using a pipette (seeding), and allowed to stand for approximately 30-60 minutes to allow the cells to settle in the wells. The excess cells were then washed using a scraper. This procedure resulted in single cells being contained in the second portion of the wells, and the single-cell occupancy rate was obtained using an inverted microscope (IX-73; Olympus) and a CCD camera (DP80; Olympus). The cells were then cultured in a CO2 incubator (37°C, 5% CO2) for 5-14 days, and the cell state was observed using an inverted microscope (IX-73; Olympus) and a CCD camera (DP80; Olympus).

[0069] (3) Results (a) Observation of monoclonal cell colonies Figure 7 shows fluorescence images of the wells of HeLa cells on a microarray chip 3 hours (a) and 7 days after seeding (b). From Figure 7(a), it was confirmed that a single cell was contained in the second portion of the well. Furthermore, from Figure 7(b), it was confirmed that monoclonal cell colonies were produced in the well after 7 days of culture.

[0070] (b) Examination of well structure In the microarray chip shown in Figure 3, the single-cell occupancy rate was evaluated by varying the well size and structure for each block.

[0071] The well structure was arranged as follows: Columns ABOP in Figure 3 correspond to structure (e) in Figure 5, columns CDMN in Figure 3 correspond to structure (d) in Figure 5, columns EF in Figure 3 correspond to structure (c) in Figure 5, columns GH in Figure 3 correspond to structure (b) in Figure 5, and columns IJKL in Figure 3 correspond to structure (a) in Figure 5.

[0072] The detailed structure of the well was determined by setting the parameters X, Y, T, S, U, and V shown in Figure 2 as follows: The depth T of the first part of the well was 20 μm, and the depth S of the second part was 28 μm. For each block, the inner diameter X of the opening of the second part 22 of one well, and the inner diameter Y of the first part 21, correspond to rows 1 to 6 and columns A to P of the block in Figure 3, as shown in the table below. The 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 well wall angle U was 15°.

[0073] [Table 1]

[0074] Figure 8 shows a graph of the single-cell occupancy rate in each well. In the graph in Figure 8, A to J correspond to columns A to J of the block in Figure 3. The diameter is the value of the inner diameter X of the opening of the second section 22. From the results in Figure 8, it was confirmed that single cells can be accommodated when HeLa cells are the target cells if the inner diameter X is in the range of approximately 26 to 36 μm and the inner diameter Y is in the range of approximately 110 to 130 μm.

[0075] (c) Examination of well pitch Next, with X fixed at 34 μm, T at 20 μm, and S at 28 μm, the single-cell occupancy rate and single-cell proliferation rate were evaluated by varying the values ​​of the inner diameter Y and pitch V. The single-cell proliferation rate was evaluated by counting the number of wells in which single cells proliferated. Cross-contamination was not counted. The combinations of Y and V values ​​that were varied are as follows. All units are in μm. [Table 2]

[0076] Figure 9 shows fluorescence images of a microarray chip with wells set to Y=100 and V=130, immediately after seeding (a) and after 15 days of culture (b). There were 14 wells containing single cells in the second section, which are indicated by white arrows in the image in Figure 9(a). Of these, 10 wells proliferated without cross-contamination after 15 days of culture and formed monoclonal colonies. In Figure 9(b), the 10 wells that formed monoclonal colonies are indicated by white arrows. Although not shown in the illustration, there were wells containing two or more cells throughout the entire microarray chip, albeit at a low percentage.

[0077] Figure 10 is a graph showing the relationship between the pitch between wells and the single-cell occupancy rate. From Figure 10, it was confirmed that for HeLa cells, a occupancy rate of 20-30% is achieved when the upper row diameter is approximately 130 to 300 μm. Figure 11 is a graph showing the single-cell proliferation rate. From Figure 11, it was confirmed that, for HeLa cells, proliferation is possible when the pitch V is approximately 300 μm or less.

[0078] Next, X was fixed at 26-36 μm, T at 20 μm, and S at 28 μm, and the single-cell occupancy rate and single-cell proliferation rate were evaluated by varying the values ​​of Y and V. The combinations of varied Y and V values ​​(in μm) are shown in Table 3 below.

[0079] [Table 3]

[0080] Figure 12 shows a fluorescence image of a microarray chip cultured for 7 days using wells with a Y of 110 μm, and Figure 13 is a graph showing the single-cell proliferation rate. Figure 14 shows a fluorescence image of a microarray chip cultured for 7 days using wells with a Y of 130 μm, and Figure 15 is a graph showing the single-cell proliferation rate. The cell proliferation rate itself was similar for both the 110 μm and 130 μm Y cases (data details are not shown). In the case of 110 μm Y, the single-cell proliferation rate was lower due to the possibility of cross-contamination, but monoclonal colonies could still be formed. It is thought that monoclonal colonies of HeLa cells can also be formed when Y is slightly smaller than 110 μm, for example, around 80-100 μm.

[0081] (d) Comparative example A comparative microarray chip with a single-stage well structure instead of a two-stage structure was fabricated, and its single-cell containment and cell proliferation were evaluated. For the comparative microarray chip, the well opening diameter was defined as F, the well depth as G, and the well pitch as H, and the following four types of wells were designed. The values ​​of F, G, and H (in μm) and the seeded cell concentration are shown in Table 4 below.

[0082] [Table 4]

[0083] Figure 16 shows fluorescence images of the microarray chip immediately after seeding (a) and after 15 days of culture (b) for Comparative Example 1. Although a single cell was contained, it was confirmed that the cells proliferated and cross-contamination occurred after 15 days of culture. Figure 17 shows fluorescence images of the microarray chip immediately after seeding (a) and after 15 days of culture (b) for Comparative Example 2. Due to the influence of the seeded cell concentration, few wells contained a single cell, and almost no proliferation was observed after 15 days of culture. Figure 18 shows fluorescence images of the microarray chip immediately after seeding (a) and after 15 days of culture (b) for Comparative Example 3. Although some wells contained a single cell, no proliferation was confirmed after 15 days of culture. Figure 19 shows fluorescence images of the microarray chip immediately after seeding (a) and after 15 days of culture (b) for Comparative Example 4. From immediately after seeding, multiple cells were contained in a single well, and it was confirmed that the cells proliferated and cross-contamination occurred after 15 days of culture.

[0084] (4) Detection of drug-resistant strains PC9 cells were suspended in a solution of RPMI1640 (30264-56, Nacalai Tesque) with 10 v / v% fetal bovine serum (FBS; Sigma-Aldrich Co. LLC) and 5 v / v% penicillin (P4333; Sigma-Aldrich Co. LLC) added, and measured in 1.0 × 10⁶ units. 7A cell suspension at a concentration of cells / mL was prepared. This cell suspension was added to a microarray tip using a pipette and allowed to stand for 30-60 minutes. The wells of the microarray tip were configured as follows: X = 34 μm, T = 20 μm, S = 28 μm, V = 110-150 μm, and Y = 110 μm or 130 μm. The excess cells were then washed using a scraper. This procedure allowed single cells to be placed in the second portion of the well, and the single-cell occupancy rate was obtained in the same manner as in the previous example. The microarray tip was then immersed in a culture medium containing the anticancer drug (Gefitinib (ZD1839)) and cultured for 7 days. Cellular conditions were observed immediately after seeding and 3 days later using an inverted microscope (IX-73; Olympus) and a CCD camera (DP80; Olympus). The concentration of Gefitinib was varied to 0, 1, 5, 10, and 100 μM.

[0085] Figure 20 shows fluorescence and bright-field images of the microarray chip immediately after seeding of PC9 cells (a) and on day 3 of culture with 5 μM Gefitinib (b). From Figure 20, it can be seen that some PC9 cells are proliferating within the chambers. Figure 21 is a graph showing the single-cell proliferation rate of PC9 cells. From Figure 21, it can be seen that the number of chambers in which a single cell proliferated is inversely proportional to the anticancer drug concentration, indicating that only drug-resistant cells can be cultured using this microchip. [Explanation of symbols]

[0086] 1 Microarray Chip 2 wells 21 Part 1 211 Bottom 22 Part 2 221 Bottom 3 surface

Claims

1. An open microchip array having multiple wells capable of containing cells, Each of the multiple wells has a first portion capable of accommodating multiple cells and a second portion capable of accommodating only a single cell. The first portion has an opening and is formed as a recessed area from the surface of the microchip array, the inner diameter of the opening of the first portion is 50 to 500 μm, and the depth of the first portion is 1 to 50 μm. The second portion has an opening and is formed as a recessed area from the bottom surface of the first portion, the inner diameter of the opening in the second portion is 5 to 100 μm, and the inner diameter of the opening in the second portion is smaller than the inner diameter of the opening in the first portion. An open microarray chip for culturing monoclonal cell colonies, with a pitch of 100–300 μm between adjacent wells.

2. The microarray chip according to claim 1, wherein the openings of the first portion and the second portion have a circular or regular polygonal shape.

3. A cell culture method using the microarray chip described in claim 1, (a) A step of applying a liquid containing multiple cells onto the microarray chip, (b) A step of cleaning the surface of the microarray chip obtained in step (a) to contain only single cells in the second portion of the plurality of wells, (c) A step of growing cells by culturing the single cells contained in the plurality of wells obtained in step (b) above. A method that includes this.

4. A method for evaluating a single cell using the microarray chip described in claim 1, (A) A step of applying a liquid containing multiple cells onto the microarray chip, (B) A step of cleaning the surface of the microarray chip obtained in step (A) to contain only single cells in the second portion of the plurality of wells, (C) A step of growing cells by culturing the cells contained in the plurality of wells obtained in step (B), (D) A step of evaluating the cellular characteristics of a single cell based on the state after culturing in step (C) above. A method that includes this.

5. The method according to claim 4, wherein step (C) includes culturing cells in the presence of a candidate substance that may affect the characteristics of the cells.

6. A method for producing monoclonal cell colonies using the microarray chip described in claim 1, (a) A step of applying a liquid containing multiple cells onto the microarray chip, (b) A step of cleaning the surface of the microarray chip obtained in step (a) to contain only single cells in the second portion of the plurality of wells, (c) A step of growing cells by culturing the single cells contained in the plurality of wells obtained in step (b) above. A method that includes this.

7. The method according to any one of claims 3, 4, or 6, wherein cleaning the surface includes washing away excess cells with a liquid.

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