JP2015198592A - Cell arrangement chip and method for arranging cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell arrangement chip with which a time required for accommodating a cell in a microchamber can be shortened.SOLUTION: A cell arrangement chip 100 includes a flow channel where fluid flows, a plurality of recess aligned on a bottom face of the flow channel, and one or two or more salients 133 aligned on a top surface of the flow channel. In a cross section of the flow channel which passes along a flow direction of fluid in the flow channel and depth direction of the recess and through the center of the recess, it is preferable that a bottom edge 134 of an upstream side's side face of the salient is located, in the flowing direction of fluid in the flow channel, between a point positioned at upstream side in half length of opening diameter of the recess from upstream side opening edge, and a point positioned at downstream side in half length of the opening diameter of the recess from the downstream side opening edge.

Description

  The present invention relates to a cell array chip and a cell array method using the cell array chip.

  Circulating tumor cells (CTC), circulating vascular endothelial cells (CEC), circulating vascular endothelial progenitor cells (CEP), various stem cells (hereinafter also referred to as “rare cells”) are present in the blood depending on the disease state. For example, detecting CTC, which is a cancer cell circulating in the blood, is very useful from the viewpoint of early detection of cancer metastasis. However, rare cells are very rare in blood and are extremely difficult to detect.

  Thus, in recent years, various rare cell detection methods have been proposed. For example, as a method for detecting rare cells, a cell suspension derived from a specimen (eg, blood) is provided to a cell array chip having a plurality of recesses (hereinafter also referred to as “microchamber”), and the cells are accommodated in the microchamber. After that, a method for analyzing whole cells has been proposed.

  For example, Patent Document 1 describes a cell detection method using a microarray chip having a microchamber for containing cells. In the cell detection method described in Patent Document 1, a cell suspension is provided to a microarray chip using a micropipette, and the cells are accommodated in a microchamber.

  On the other hand, Patent Document 2 describes a method of forming a cell tissue body using a cell tissue body microdevice having a microchamber and a flow path for introducing a cell suspension. In the method for forming a cell tissue described in Patent Document 2, a cell suspension is introduced into a flow path by a pump mechanism, and cells are accommodated in a microchamber.

International Publication No. 2010/027003 JP 2006-122012 A

  As described in Patent Document 1 and Patent Document 2, when cells are accommodated in a microchamber, the cells are generally precipitated by utilizing their own weight. Therefore, the movement of the cells into the microchamber becomes rate limiting, and the time required for accommodating the cells in the microchamber cannot be shortened.

  This invention is made | formed in view of this point, and it aims at providing the cell arrangement | sequence chip which can shorten the time required to accommodate a cell in a microchamber. Another object of the present invention is to provide a cell arrangement method using this cell arrangement chip.

  In the flow path in which a plurality of concave portions (microchambers) are formed on the bottom surface, the present inventors have formed the above-mentioned problem by forming a convex portion on the top surface for allowing the liquid flowing in the flow path to flow toward the concave portion. The present invention has been completed by finding out that the problem can be solved and further studying it.

  That is, the present invention relates to the following cell array chip.

[1] A cell array having a flow path through which liquid flows, a plurality of concave portions disposed on the bottom surface of the flow path, and one or more convex portions disposed on the top surface of the flow path. Chip.
[2] In the cross section of the flow path passing through the center of the recess along the flow direction of the liquid in the flow path and the depth direction of the recess, the lower end of the upstream side surface of the protrusion is the flow path In the direction of liquid flow at a point located upstream from the upstream opening edge of the concave portion by a half length of the opening diameter and from the downstream opening edge of the concave portion to a downstream length of half the opening diameter. The cell array chip according to [1], wherein the cell array chip is disposed between
[3] In the cross section, the lower end of the upstream side surface of the convex portion is disposed between the upstream opening edge of the concave portion and the downstream opening edge of the concave portion in the liquid flow direction in the flow path. The cell array chip according to [2].
[4] The convex portions are convex strips extending in a direction perpendicular to the liquid flow direction in the flow path on the concave portion and the depth direction of the concave portion, [1] to [3]. The cell array chip according to any one of the above.
[5] The bottom surface of the recess has cell adhesion, the opening diameter of the recess is 20 μm or more and 500 μm or less, and the depth of the recess is 10 μm or more and 250 μm or less. [4] The cell array chip according to any one of [4].
[6] In the cross section of the flow path along the flow direction of the liquid in the flow path and the depth direction of the concave portion, the lower end of the convex portion is more than the opening edge of the concave portion in the depth direction of the concave portion. The space between the top surface of the flow channel and the region where the convex portion of the top surface of the flow channel is not disposed, and the region where the concave portion of the bottom surface of the flow channel is not disposed, The cell array chip according to any one of [1] to [5], which is 20 μm or more and 1000 μm or less.

  The present invention also relates to the following cell sequencing method.

[7] A cell array having a flow path through which a liquid flows, a plurality of concave portions disposed on the bottom surface of the flow path, and one or more convex portions disposed on the top surface of the flow path. A cell arraying method comprising: a step of preparing a chip; and a step of introducing a liquid containing cells into the flow path and collecting the cells in the recess.
[8] The cell arrangement method according to [7], wherein the introduction of the liquid is performed intermittently.

  ADVANTAGE OF THE INVENTION According to this invention, the cell arrangement | sequence chip which can shorten the time required to accommodate a cell in a microchamber can be provided. Therefore, according to the present invention, for example, the time required for detecting rare cells using a cell array chip can be shortened.

1A is a plan view of the cell array chip according to the embodiment, and FIG. 1B is a cross-sectional view taken along line AA shown in FIG. 1A. 2A is a plan view of the microchamber chip, FIG. 2B is a plan view of the frame, and FIG. 2C is a bottom view of the top plate. FIG. 3A is a partially enlarged cross-sectional view of the cell array chip according to the embodiment, and FIG. 3B is a partially enlarged cross-sectional view of a conventional cell array chip that does not have a convex portion. FIG. 4 is a bottom view of a top plate according to a modification of the embodiment. FIG. 5 is a graph showing the results of fluid simulation.

  Embodiments of the present invention will be described in detail with reference to the drawings.

[Cell array chip]
FIG. 1 is a diagram showing a cell array chip 100 according to an embodiment of the present invention. 1A is a plan view of the cell array chip 100, and FIG. 1B is a cross-sectional view taken along line AA shown in FIG. 1A. As shown in FIGS. 1A and 1B, the cell array chip 100 includes a microchamber chip 110, a frame body 120, and a top plate 130.

  2A is a plan view of the microchamber chip 110, FIG. 2B is a plan view of the frame body 120, and FIG. 2C is a bottom view of the top plate 130. FIG. As shown in FIG. 1B, a microchannel chip 110, a frame body 120, and a top plate 130 are stacked in this order, thereby forming a flow path 121 through which a liquid flows. That is, the flow path 121 refers to a space through which liquid flows when liquid is introduced from an inlet 131 described later.

(Microchamber chip)
As shown in FIG. 1B and FIG. 2A, the microchamber chip 110 includes a substrate 111 and a plurality of microchambers 112 (concave portions) formed on one surface of the substrate 111. As shown in FIG. 1B, the surface of the substrate 111 on which the microchamber 112 is formed is the bottom surface of the flow path 121 through which a liquid (for example, a cell suspension, a washing solution, a staining solution, etc.) flows. Each microchamber 112 is open to the channel 121. The number and arrangement of the micro chambers 112 are not particularly limited. In the present embodiment, as shown in FIG. 2A, the microchambers 112 are arranged in a matrix at the center of the flow channel 121.

The material of the substrate 111 (microchamber chip 110) is not particularly limited. The material of the substrate 111 can be appropriately selected depending on the type of cells accommodated in the microchamber 112. For example, the material of the substrate 111 may be selected in consideration of the charged state, hydrophilicity, hydrophobicity and the like so as to impart suitable cell adhesiveness to the cells accommodated in the microchamber 112. Here, “cell adhesion” means the property of the surface of the substrate 111 such that the adhesive force of the cells to the substrate 111 is increased. “Cell adhesion” refers to the force with which half (50%) of cells peel from the plane when a force is applied to a plurality of cells attached to the plane in a direction substantially parallel to the plane. Value (unit: N). For example, when a liquid is caused to flow through the flow path 121 in which a plurality of cells are attached on the bottom surface (plane), the force F applied to the cells is calculated by the following equation (1).
F = 3πμVd (1)
Where F is the force applied to the cell (N), μ is the viscosity coefficient of the liquid (N / m ² · s), V is the flow velocity of the liquid (m / s), and d is the diameter of the cell (m). . Specifically, the cell adhesion force F is preferably more than 0.01 nN, more preferably 0.1 nN or more.

  Further, the substrate 111 may be surface-treated. Examples of the surface treatment method include plasma treatment, corona discharge treatment, blocking treatment with a blocking agent, and coating treatment with a hydrophilic polymer, protein, lipid and the like. These may be used alone or in combination of two or more. Examples of the material of the substrate 111 include resins such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane, acrylic resin, and cycloolefin polymer. In the present embodiment, the substrate 111 is made of a hydrophobic resin (eg, polystyrene, polypropylene, acrylic resin, polycarbonate, cycloolefin polymer, etc.), and the bottom surface of the microchamber 112 is hydrophobic. The thickness of the substrate 111 is not particularly limited as long as necessary strength can be ensured, and can be set as appropriate according to the depth of the microchamber 112 and the like.

  The shape of the microchamber 112 is not particularly limited as long as it can accommodate one cell or two or more cells. Examples of the shape of the microchamber 112 include a cylinder, a polygonal column, an inverted truncated cone, and an inverted polygonal truncated cone. The size of the microchamber 112 is not particularly limited, and can be set as appropriate according to the type of cells accommodated in the microchamber 112, the number of cells accommodated, and the like. Usually, the size of the microchamber 112 is preferably such that about 10 to 15 cells can adhere to the bottom surface of the microchamber 112. For example, the opening diameter of the microchamber 112 is in the range of 20 to 500 μm, and the depth of the microchamber 112 is in the range of 10 to 250 μm.

  A method for forming the microchamber 112 on the substrate 111 is not particularly limited. Examples of a method for forming the microchamber 112 on the substrate 111 include a mold forming method, a lithography method, a method using a focused ion beam (FIB), and the like. Further, a film in which the micro chamber 112 is formed may be attached to the substrate 111, or a commercially available substrate 111 (the micro chamber chip 110) in which the micro chamber 112 is already formed may be purchased.

  The kind of cell accommodated in the micro chamber 112 is not specifically limited. Examples of cells housed in the microchamber 112 include circulating tumor cells (CTC), circulating vascular endothelial cells (CEC), circulating vascular endothelial progenitor cells (CEP), circulating fetal cells, antigen-specific T cells, and various stem cells. Etc. are included.

(Frame)
As shown in FIG. 1B and FIG. 2B, the frame body 120 is a thin plate having a through-hole 122 disposed between the microchamber chip 110 and the top plate 130. The shape of the through-hole 122 is not particularly limited as long as the cell suspension can flow over the microchamber 112, and can be appropriately selected depending on the application. The thickness of the frame 120 is the distance between the region where the top surface convex portion 133 of the channel 121 is not disposed and the region where the microchamber 112 is not disposed on the bottom surface of the channel 121 (the depth of the channel). Is equivalent to The thickness of the frame 120 is not particularly limited, and is appropriately set according to the desired height (depth) of the flow path. For example, the thickness of the frame 120 is in the range of 20 to 1000 μm. By setting the thickness of the frame 120 within the range of 20 to 1000 μm, the cells attached to the surface outside the microchamber 112 in the flow channel 121 can be easily peeled off by the force of the liquid flowing in the flow channel 121. It becomes possible. In addition, it is possible to shorten the time until the cells in the cell suspension in the channel 121 settle in the microchamber 112 while preventing the clogging by the cells in the channel 121.

  The material of the frame 120 is not particularly limited, and may be the same material as a known microplate. Examples of the material of the frame 120 include resins such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane, polymethylmethacrylate, and cyclic olefin copolymer. The frame 120 may be a silicon sheet (double-sided seal) having adhesiveness on both sides. By using a double-sided seal as the frame body 120, the microchamber chip 110, the frame body 120, and the top plate 130 can be easily fixed.

  An inner surface of the through hole 122 of the frame body 120 is a side surface of the flow path 121. Therefore, the inner surface of the through hole 122 of the frame body 120 is in contact with the cell suspension when the cell array chip 100 is used. Therefore, the inner surface of the through hole 122 of the frame body 120 may be surface-treated as necessary. Examples of the surface treatment include plasma treatment, corona discharge treatment, coating treatment with hydrophilic polymer, protein, lipid and the like. For example, the inner surface of the through hole 122 of the frame body 120 may be subjected to a blocking process for preventing nonspecific binding of cells. Examples of the blocking agent include casein, skim milk, albumin (including bovine serum albumin), gelatin, high molecular compounds such as polyethylene glycol; phospholipid, low molecular weight compounds such as ethylenediamine and acetonitrile, and the like. These may be used alone or in combination of two or more. The frame body 120 may be integrated with the microchamber chip 110 or the top plate 130.

(Top board)
As shown in FIG. 1B and FIG. 2C, the top plate 130 is arranged on the frame body 120, the first through hole 131, the second through hole 132, and one or more on one surface. It is a thin plate which has the convex part 133 of. The surface on which the convex portion 133 of the top plate 130 is disposed becomes the top surface of the flow path 121 through which the liquid flows. The first through hole 131 serves as an inlet 131 for introducing a liquid into the channel 121 when the microchamber chip 110, the frame body 120, and the top plate 130 are stacked. The second through hole 132 serves as a discharge port 132 for discharging the liquid from the flow path 121. Usually, the inlet 131 communicates with one end of the flow path 121. The discharge port 132 communicates with the other end of the flow path 121. The shapes of the inlet 131 and the outlet 132 are not particularly limited. Moreover, the thickness of the top plate 130 is not particularly limited as long as necessary strength can be secured.

  As will be described later, the convex portion 133 changes the traveling direction of the liquid flowing through the flow path 121 and promotes the movement of cells into the microchamber 112. In the present embodiment, the top plate 130 has the same number of protrusions 133 as the microchambers 112 of the microchamber chip 110. Each convex portion 133 is disposed at a position corresponding to each microchamber 112.

  The components in the traveling direction of the liquid on the microchamber 112 are the flow direction of the liquid, the depth direction of the microchamber 112, and the direction perpendicular to the flow direction of the liquid and the depth direction of the microchamber 112 (hereinafter referred to as “channel”). 121 ”in the width direction). Here, the “flow direction of the liquid” refers to a direction in which the liquid flows on the microchamber 112 when the top plate 130 is viewed in plan. The liquid flow direction, the depth direction of the microchamber 112, and the width direction of the flow path 121 are orthogonal to each other.

  The shape of the convex portion 133 is not particularly limited as long as it can change the traveling direction of the liquid and promote the movement of cells into the microchamber 112. Examples of the shape of the convex portion 133 include a polygonal prism such as a triangular prism or a quadrangular prism; a polygonal pyramid such as a triangular pyramid or a quadrangular pyramid.

  The length of the convex portion 133 in the width direction of the flow path 121 is not particularly limited as long as it can change the traveling direction of the liquid and promote the movement of the cells into the micro chamber 112. The opening diameter of the micro chamber 112, It can be set as appropriate depending on the position of the convex 133 and the like.

  The length of the convex portion 133 in the liquid flow direction is not particularly limited as long as the convex portion 133 can secure a necessary strength against the liquid flow pressure, and depends on the interval between the convex portions 133 in the liquid flow direction. Can be set appropriately.

  The height of the projection 133 is not particularly limited as long as it can change the traveling direction of the liquid and promote the movement of cells into the microchamber 112, and is appropriately set depending on the thickness of the frame 120, the shape and position of the projection 133, and the like. Can be done. The lower end of the convex portion 133 may be disposed closer to the bottom surface side of the microchamber 112 than the opening edge of the microchamber 112. However, from the viewpoint of suppressing the movement of cells in the microchamber 112 to the outside of the microchamber 112, the lower end of the convex portion 133 is disposed on the top surface side of the flow channel 121 with respect to the opening edge of the microchamber 112. Preferably it is.

  The position of the convex portion 133 is not particularly limited as long as the moving direction of the liquid can be changed and the movement of the cells into the microchamber 112 can be promoted. From the viewpoint of promoting cell arrangement, the convex portion 133 is preferably disposed in the vicinity of the microchamber 112.

  3A is a partially enlarged cross-sectional view of the cell array chip 100 according to the present embodiment, and FIG. 3B is a partially enlarged cross-sectional view of a conventional cell array chip 100 ′ that does not have the convex portion 133. 3A and 3B are cross-sectional views along the liquid flow direction and the depth direction of the microchamber 112. The left side in the figure is upstream, and the right side is downstream.

  As shown in FIG. 3A, let R be the opening diameter of the microchamber 112 in the liquid flow direction. In the cross section of the flow path 121 passing through the center of the micro chamber 112 along the liquid flow direction and the depth direction of the micro chamber 112, the lower end 134 on the upstream side surface of the convex portion 133 is the flow of the liquid in the flow path 121. In the direction, half the length of the opening diameter (R / 2) from the upstream opening edge of the microchamber 112 (R / 2), and half the opening diameter from the downstream opening edge of the microchamber 112 (R / 2) It is preferably arranged between the points located on the downstream side (between the broken line A and the broken line B in FIG. 3A). Further, in the cross section, the lower end 134 of the upstream side surface of the convex portion 133 is between the upstream opening edge of the microchamber 112 and the downstream opening edge of the microchamber 112 in the liquid flow direction in the flow path 121. More preferably, they are arranged. Here, “the center of the microchamber 112” means the center of gravity of the opening surface of the microchamber 112.

  As shown in FIG. 3A, in the cell array chip 100 according to the present embodiment, the convex portion 133 changes the traveling direction of the liquid. Thereby, the component in the depth direction of the microchamber 112 among the components in the liquid traveling direction increases, and the moving speed of the cells 10 contained in the liquid to the microchamber 112 increases. Further, when the convex portion 133 is disposed in the vicinity of the microchamber 112, the liquid traveling direction can be changed in the vicinity of the microchamber 112 in which the convex portion 133 is disposed, and thus the region where the microchamber 112 is disposed. The proportion of cells 10 that settle to the surface increases. As a result, the cell array chip 100 according to the present embodiment can reduce the time required for accommodating the cells 10 in the microchamber 112.

  On the other hand, as shown in FIG. 3B, in the conventional cell array chip 100 ′ that does not have the convex portion 133, the cell 10 must move to the microchamber 112 only by its own weight. Therefore, the moving speed of the cell 10 to the microchamber 112 is slow. For this reason, in the cell array chip 100 ′ that does not have the convex 133, it takes a long time to accommodate the cells 10 in the microchamber 112.

  The method for forming the convex portion 133 on the top plate 130 is not particularly limited. Examples of the method for forming the convex portion 133 include mold forming processing, lithography, etching, and focused ion beam (FIB). Moreover, you may affix the film in which the convex part 133 was formed on a flat plate.

  The material of the top plate 130 is not particularly limited, but from the viewpoint of observing the inside of the channel 121 from the outside using a fluorescent microscope or the like, a material having light transmittance is preferable. Examples of the material of the top plate 130 include resins such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane, acrylic resin, and cycloolefin polymer. Similarly to the frame body 120, the surface (the surface on which the convex portion 133 is disposed) that becomes the top surface of the channel 121 of the top plate 130 may also be subjected to surface treatment (for example, blocking treatment).

  With the above configuration, the cell array chip 100 according to the present embodiment can be manufactured. For example, the cell array chip 100 can be manufactured by stacking the microchamber chip 110, the frame body 120, and the top plate 130 in this order and fixing them together. The method for fixing them is not particularly limited, but it is preferable that they are fixed detachably from the viewpoint of observation and maintenance. Examples of the fixing method include fixing by engagement, fixing using a screw, and fixing using an adhesive.

[Cell arrangement method]
Next, a method for using the cell array chip 100 according to the present embodiment (the cell array method according to the present embodiment) will be described.

  First, the cell array chip 100 according to the present embodiment is prepared.

  Next, the cells 10 are collected in the micro chamber 112 of the cell array chip 100. Specifically, a cell-containing liquid (cell suspension) is introduced into the channel 121 from the inlet 131, and the channel 121 is filled with the cell suspension. Next, the cell array chip 100 is allowed to stand for a certain time (for example, 1 to 15 minutes). Thereby, a part of the cells 10 contained in the cell suspension can be precipitated in the microchamber 112. However, the remaining cells 10 settle out of the microchamber 112. Therefore, in order to collect the cells 10 that have settled out of the microchamber 112 into the microchamber 112, it is preferable to perform intermittent liquid feeding. By performing intermittent liquid feeding, a moving force can be intermittently applied to the cells 10 outside the microchamber 112, and the cells 10 can be collected in the microchamber 112.

  The intermittent liquid feeding includes a one-shot liquid feeding process for moving the cells 10 outside the microchamber 112 in the liquid feeding direction, and a stationary process for sedimenting the moved cells 10. When a device that performs liquid feeding generates a predetermined liquid feeding pressure in the channel 121 for a predetermined time and moves the cells 10 outside the microchamber 112 in the liquid feeding direction, “one shot” "Liquid feeding". On the other hand, if this apparatus does not generate liquid feeding pressure, it will be “standing still”. Assuming one cycle from the start of the one-shot liquid feeding process to the end of the stationary process, the cells 10 outside the microchamber 112 move about 50 μm in the liquid feeding direction in one cycle, for example. When the cell 10 outside the microchamber 112 moves directly above the microchamber 112, the cell 10 settles in the microchamber 112. From the viewpoint of collecting the cells 10 outside the microchamber 112 into the microchamber 112, the number of cycles of intermittent liquid feeding is preferably 2 times or more, and more preferably 10 times or more.

  In intermittent liquid feeding, liquid feeding may be performed not only from the inlet 131 but also from the outlet 132. For example, intermittent liquid feeding may be performed from the inlet 131 for one cycle or two cycles or more, and then intermittent liquid feeding may be performed from the discharge port 132 for one cycle or two cycles or more. By performing intermittent liquid feeding alternately from the inlet 131 and the outlet 132, the cells 10 can be collected in the microchamber 112 more efficiently.

  The amount of liquid in the one-shot liquid feeding is appropriately set according to the size of the flow path 121 and the like. For example, the amount of liquid in one-shot liquid feeding is preferably about 1/100 to 1/2 of the volume of the cell suspension in the channel 121. In addition, it is preferable to adjust the liquid feeding amount per unit time so that the flow velocity in the flow channel 121 is 1 mm / second or less. The standing time is not particularly limited, but is, for example, 1 to 30 seconds.

  As described above, in the cell arrangement chip 100 according to the present embodiment, the moving speed of the cells 10 into the microchamber 112 is faster than the cell arrangement chip 100 ′ that does not have the convex portion 133. Therefore, in the cell arrangement chip 100 according to the present embodiment, the standing time can be shortened compared to the cell arrangement chip 100 ′ that does not have the convex portion 133.

  As described above, by using the cell array chip 100 of the present embodiment, when the cell suspension is introduced into the flow path 121, the cells 10 can be efficiently moved into the microchamber 112. it can. For this reason, by using the cell array chip 100 of the present embodiment, the time required to accommodate the cells 10 in the microchamber 112 can be shortened.

  In the above embodiment, the cell array chip 100 to which the plurality of micro chambers 112 of the micro chamber chip 110 and the plurality of convex portions 133 of the top plate 130 correspond respectively has been described. However, the number of the micro chambers 112 and the convex portions 133 may not be the same. For example, as shown in FIG. 4, the cell array chip 100 may have fewer protrusions 133 (projections) extending in the width direction of the channel 121 than the microchamber 112. In this case, the length in the width direction of the flow path 121 of the protrusion 133 (protrusion) is longer than the length in the width direction of the flow path 121 of the microchamber 112, and each protrusion 133 (protrusion) has a plurality of lengths. Corresponds to the microchamber 112. By doing in this way, the liquid which flows between the convex parts 133 adjacent to the width direction of the flow path 121 is lost, and the liquid which flows toward the micro chamber 112 can be increased. In the present specification, the “projection” means an elongated projection that extends in one direction.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail with reference to an Example, this invention is not limited by these Examples.

[Example 1]
1. Production of Cell Array Chip A micro-chamber chip made of polystyrene and having the same size as a slide glass was produced by molding. In the center region (longitudinal direction 50 mm, short side direction 20 mm) of the microchamber chip, 10000 holes of microchambers (opening diameter 100 μm, bottom surface diameter 80 μm, depth 50 μm) were formed at a pitch of 300 μm.

  A top plate (longitudinal direction 100 mm, short side direction 50 mm) made of acrylic resin and having two through-holes (introduction port and discharge port) was produced by molding. 10,000 convex portions (longitudinal direction 50 μm, short side direction 50 μm, height 50 μm) were formed at a pitch of 300 μm in the central region (longitudinal direction 50 mm, short side direction 20 mm) of the top plate.

  A double-sided seal (thickness: 100 μm) having a through hole as a frame was laminated on the upper surface of the microchamber chip. Further, a cell array chip A was produced by stacking a top plate on the frame. In the cell array chip A, each microchamber formed on the upper surface (bottom surface of the channel) of the microchamber chip and each convex portion formed on the lower surface of the top plate (top surface of the channel) face each other. . When the cell array chip A is viewed in plan, each convex portion is arranged so that the lower end of the upstream side surface of each convex portion overlaps the center of the opening of the corresponding microchamber.

  In addition, as a comparative example, instead of using a top plate having convex portions, a cell array chip B is formed by a procedure similar to that of the cell array chip A using a top plate (a flat acrylic resin substrate) having no convex portions. Produced.

2. Cell Arrangement on Cell Array Chip According to the following procedure, the cell suspension is introduced into the cell array chip A (Example) and the cell array chip B (Comparative Example), and the proportion of cells contained in the microchamber is determined. It was measured.

A liquid feeding tube was connected to each of the inlet and outlet of the cell array chip via a tube connector. The other end of the liquid feeding tube having one end connected to the inlet was placed in a 30% aqueous ethanol solution. Moreover, the other end of the liquid feeding tube having one end connected to the discharge port was connected to a syringe pump. In this state, the ethanol aqueous solution was sucked at a rate of 15 mL / min using a syringe pump to wet the entire surface of the flow path (including the bottom and side surfaces of the microchamber). Next, the other end of the liquid feeding tube having one end connected to the introduction port was moved into a phosphate buffer solution (PBS). In this state, the phosphate buffer was sucked at a rate of 15 mL / min using a syringe pump to wash the inside of the flow path. The other end of the liquid feeding tube having one end connected to the inlet was placed in a cell suspension containing 2 × 10 ⁵ white blood cells in 100 μL. Moreover, the other end of the liquid feeding tube having one end connected to the discharge port was connected to a syringe pump. In this state, 100 μL of the cell suspension was sucked at a predetermined rate of 0.0005 to 10 mL / min using a syringe pump, and the flow path was filled with the cell suspension. After allowing to stand for 5 minutes, the number of cells accommodated in the microchamber was counted using an optical microscope, and the recovery rate of the cells into the microchamber at each flow rate was calculated. The experimental results are shown in Table 1.

  As shown in Table 1, in the cell array chip A according to the example, cells could be recovered at a higher rate than the cell array chip B according to the comparative example at all the flow rates performed this time. In particular, when the flow rate is 0.01 mL / min, the cell array chip B according to the comparative example can recover only 30% of the cells, whereas the cell array chip A according to the example recovers as many as 80% of the cells. I was able to.

  From the above results, it can be seen that the cell array chip having one or more convex portions on the top surface of the flow path can shorten the time required to accommodate cells in the microchamber.

[Example 2]
With respect to a plurality of cell array chips each having a different position of the convex portion, a fluid flow simulation in the vicinity of the microchamber was performed using fluid analysis software (FLOW-3D version 10; Flow Science). The opening diameter of the microchamber was set to 100 μm, the depth of the microchamber was set to 50 μm, the length of the convex portion in the longitudinal direction was set to 100 μm, and the height of the convex portion was set to 50 μm. The following five conditions (variables) were set for the position of the lower end of the upstream side surface of the convex portion in the liquid flow direction when the cell array chip was viewed in plan.
Condition 1: When there is no protrusion (hereinafter referred to as “no protrusion”)
Condition 2: When overlapping with the center of the opening of the corresponding microchamber (hereinafter referred to as “convex 50”)
Condition 3: When overlapping with the upstream opening edge of the corresponding micro chamber (hereinafter referred to as “convex 100”)
Condition 4: When located further 25 μm upstream from the upstream opening edge of the corresponding microchamber (hereinafter referred to as “convex 125”)
Condition 5: When located 50 μm upstream from the upstream opening edge of the corresponding micro chamber (hereinafter referred to as “convex 150”)

  As an evaluation index, the depth of the microchamber with respect to the liquid flow velocity in the liquid flow direction at a position of 22 μm (corresponding to a general cell diameter) from the substrate surface (based on the area outside the microchamber) to the top plate direction. The ratio of liquid flow rate in the vertical direction was adopted. This index suggests that the larger the numerical value, the easier the cells move in the depth direction of the microchamber.

  The result of the simulation is shown in FIG. In the graph of FIG. 5, the horizontal axis (r) indicates the position (μm) of the lower end of the upstream side surface of the convex portion in the liquid flow direction when the center of the opening of the micro chamber is 0. The microchamber is opened at a position where r is −50 to 50 (μm). On the other hand, the vertical axis (C) is the above-described evaluation index (flow rate ratio).

  As shown in FIG. 5, when there are convex portions on the top surface of the flow path (convex 50, convex 100, convex 125 and convex 150), compared to the case where there is no convex portion on the top surface of the flow path (no convexity). ), The flow velocity of the liquid in the depth direction increases in the vicinity of the microchamber, and it can be seen that the cells easily move into the microchamber. In particular, when the lower end of the upstream side surface of the convex portion is located on the microchamber (convex 50 and convex 100), it can be seen that the flow rate of the liquid in the depth direction increases significantly.

  The cell array chip according to the present invention is useful, for example, for rapid examination of diseases.

10 cells 100, 100 'cell array chip 110 microchamber chip 111 substrate 112 microchamber (recess)
DESCRIPTION OF SYMBOLS 120 Frame 121 Flow path 122 Through-hole 130 Top plate 131 Inlet port 132 Outlet port 133 Convex part 134 The lower end of the upstream side surface of a convex part

Claims (8)

A flow path through which liquid flows;
A plurality of recesses disposed on the bottom surface of the flow path;
One or more protrusions disposed on the top surface of the flow path;
A cell array chip.   In the cross section of the flow path passing through the center of the recess along the flow direction of the liquid in the flow path and the depth direction of the recess, the lower end of the upstream side surface of the protrusion is the liquid in the flow path In the flow direction, the point located on the upstream side half the opening diameter from the upstream opening edge of the recess, the point located on the downstream side half the opening diameter from the downstream opening edge of the recess, The cell array chip according to claim 1, which is disposed between the two.   In the cross section, the lower end of the upstream side surface of the convex portion is disposed between the upstream opening edge of the concave portion and the downstream opening edge of the concave portion in the liquid flow direction in the flow path. The cell array chip according to claim 2.   The said convex part is a protruding item | line extended in the direction orthogonal to the flow direction of the liquid in the said flow path on the said recessed part, and the depth direction of the said recessed part. The cell array chip according to 1. The bottom surface of the recess has cell adhesion,
The opening diameter of the recess is 20 μm or more and 500 μm or less,
The depth of the recess is 10 μm or more and 250 μm or less.
The cell arrangement chip according to any one of claims 1 to 4. In the cross section of the flow path along the flow direction of the liquid in the flow path and the depth direction of the concave portion, the lower end of the convex portion is more than the opening edge of the concave portion in the depth direction of the concave portion. Is located on the top side of
The distance between the area where the convex part of the top surface of the flow path is not disposed and the area where the concave part of the bottom surface of the flow path is not disposed is 20 μm or more and 1000 μm or less.
The cell arrangement chip according to any one of claims 1 to 5. A cell array chip having a flow path through which a liquid flows, a plurality of concave portions disposed on the bottom surface of the flow path, and one or more convex portions disposed on the top surface of the flow path is prepared. And a process of
Introducing a liquid containing cells into the flow path, and collecting the cells in the recess;
A cell array method comprising:   The cell arrangement method according to claim 7, wherein the introduction of the liquid is performed intermittently.

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