JP2014219261A - Manufacturing method of micro chamber chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of a micro chamber chip that allows easy manufacture of a micro chamber chip having a strong adhesive force at a cell in a bottom face of a micro chamber, and a weak adhesive force at a cell in a side face of the micro chamber and an outer face of the micro chamber.SOLUTION: A first substrate having a strong adhesive force at a cell in a surface thereof, and a second substrate having a weak adhesive force at a cell in a surface thereof are prepared. The second substrate includes one or more through holes. The first substrate and the second substrate are laminated and fixed to produce a micro chamber chip.

Description

  The present invention relates to a method for manufacturing a microchamber 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 very rarely present in blood depending on the pathological condition. . The detection of such rare cells is obviously difficult, but is extremely difficult. In recent years, various cell separation techniques have been applied to detect rare cells, and various detection devices have been commercialized. For example, when examining whether or not the target rare cells are present in a sample such as blood, it is proposed to analyze the expanded cells after spreading the cell suspension derived from the sample in a flat shape Has been.

  For example, Patent Document 1 discloses a microdevice having a plurality of fine concave portions (sample holding portions). The microdevice described in Patent Document 1 is manufactured by injection molding a thermoplastic resin. For this reason, the surface texture outside the recess is the same as the surface texture inside the recess. After the sample is held in each recess, the sample in each recess is irradiated with excitation light to detect fluorescence from the sample, whereby the sample can be analyzed using the microdevice described in Patent Document 1. .

  When rare cells are detected using the microdevice described in Patent Document 1, it is conceivable to provide a cell suspension individually for each recess as a method of retaining cells in each recess. However, in this method, it takes a long time to provide a cell suspension in each recess, and the analysis cannot be performed efficiently. Therefore, as another method, it is conceivable to widely provide a cell suspension on the surface of the microdevice where the recesses are formed. However, in this method, since cells adhere to an area outside the recess, only a part of the cells can be analyzed.

  As a means for preventing such adhesion of cells to the region outside the recess, it has been proposed to perform a surface treatment on the region outside the recess. For example, in Patent Document 2, a treatment for preventing non-specific adhesion of cells to the surface of a substrate (for example, ethylenediamine treatment) is performed, and then a plurality of fine recesses are formed on the substrate, so that a pre-biochip is formed. Is disclosed. The fine recess is formed using a focused ion beam. Thus, it is expected that the adhesion of cells outside the recess can be reduced by performing the surface treatment on the area outside the recess.

Japanese Patent Laid-Open No. 2004-212048 JP 2005-214889 A

  As described above, when a cell suspension is widely provided on one surface of the microdevice described in Patent Document 1, a large number of cells adhere to the outside of the recess. For this reason, when it is going to detect a rare cell using the microdevice of patent document 1, the rare cell contained in a test substance may be lost.

  On the other hand, in the pre-biochip described in Patent Document 2, a region outside the recess is surface-treated. For this reason, it is considered that the possibility that cells adhere to the outside of the recess is lower than that of the microdevice described in Patent Document 1. However, in the pre-biochip described in Patent Document 2, since the concave portion is formed after the surface treatment, the effect of the surface treatment may be impaired particularly at the periphery of the concave portion when the concave portion is formed. Therefore, even when trying to detect rare cells using the pre-biochip described in Patent Document 2, the rare cells contained in the specimen may be lost.

  Moreover, since the side surface of a recessed part is not surface-treated in the pre biochip of patent document 2, there also exists a problem that a cell will adhere to the side surface of a recessed part. If cells adhere to the side surfaces of the recesses in this way, some cells are hidden behind other cells, so that there is a possibility that rare cells contained in the specimen cannot be detected.

  The present invention has been made in view of the above points, and has a strong cell adhesion force to the bottom surface of the microchamber (concave portion) and a weak cell adhesion force to the side surface of the microchamber and the surface outside the microchamber. It is an object of the present invention to provide a method for manufacturing a microchamber chip capable of easily manufacturing a chip.

  The present inventors have found that the above problem can be solved by using a combination of two substrates having different cell adhesion to the surface, and have further studied and completed the present invention.

  That is, the present invention relates to the following microchamber chip manufacturing method.

[1] A method of manufacturing a microchamber chip having a microchamber for containing cells, the step of preparing a first substrate and a second substrate having one or more through-holes formed thereon, Laminating and fixing the first substrate and the second substrate, and the adhesive force of the cells on the surface of the first substrate facing the second substrate is the first substrate of the second substrate. A method of manufacturing a microchamber chip, which is stronger than the cell adhesive force on the surface not facing the surface and the inner surface of the through hole.
[2] The method for manufacturing a microchamber chip according to [1], wherein the maximum diameter of the through hole is in the range of 20 to 150 μm, and the thickness of the second thin plate is in the range of 20 to 100 μm.
[3] The cell adhesive force on the surface of the first substrate facing the second substrate is over 0.01 nN, and the surface of the second substrate not facing the first substrate and the inner surface of the through hole The method for producing a microchamber chip according to [1] or [2], wherein the cell adhesion is 0.01 nN or less.
[4] Cell adhesion force on the surface of the first substrate facing the second substrate is 0.1 nN or more, on the surface of the second substrate not facing the first substrate and on the inner surface of the through hole. The method for producing a microchamber chip according to [3], wherein the cell adhesion is 0.001 nN or less.
[5] The method for manufacturing a microchamber chip according to any one of [1] to [4], wherein the first substrate and the second substrate are resin substrates made of different materials.
[6] Any one of [1] to [5], wherein at least one of the first substrate and the second substrate is subjected to a surface treatment for adjusting cell adhesion before being laminated. The manufacturing method of the micro chamber chip | tip of description.
[7] The method of manufacturing a microchamber chip according to any one of [1] to [6], wherein the first substrate and the second substrate are fixed by heat welding, ultrasonic welding, or an adhesive.

  According to the present invention, a microchamber chip suitable for detection of rare cells can be easily produced. By using the microchamber chip manufactured according to the present invention, it becomes possible to detect rare cells with high accuracy.

FIG. 1A is a plan view of a cell deployment device, 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 plan view of the top plate. FIG. 3A is a partial enlarged cross-sectional view of the microchamber chip, and FIG. 3B is a partial enlarged cross-sectional view of the microchamber chip when the device for cell deployment is used. FIG. 4A is a plan view of the first substrate, and FIG. 4B is a plan view of the second substrate. 5A and 5B are schematic views for explaining the method of manufacturing the microchamber chip according to the present embodiment. 6A is a plan view of the frame according to the embodiment, FIG. 6B is a plan view of the top plate according to the embodiment, and FIG. 6C is a plan view of a state in which the double-sided seal and the top plate are overlaid. is there. FIG. 7A is a plan view of the first substrate according to the embodiment, FIG. 7B is a plan view of the second substrate according to the embodiment, and FIG. 7C is a side view of the device for cell deployment according to the embodiment. FIG. 7D is a plan view of the cell deployment device according to the example.

  Hereinafter, embodiments according to the present invention will be described in detail with reference to the drawings. The microchamber chip of the present embodiment can be used by being incorporated in, for example, a cell deployment device. Therefore, in the following description, a microchamber chip incorporated in a cell deployment device and a manufacturing method thereof will be described.

[Cell expansion device]
FIG. 1 is a diagram showing an example of a cell expansion device suitable for detection of rare cells. 1A is a plan view of the cell deployment device 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 deployment device 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 plan view of the top plate 130. FIG.

  The microchamber chip 110 has one or more microchambers 111 on one surface (see FIG. 2A). A microchamber chip 110 having a plurality of microchambers 111 is also referred to as a microchamber array (MCA). Here, the “microchamber” means a fine bottomed recess (microwell) for containing and holding one or more cells. Further, “holding cells” means that it is difficult for cells stored in the microchamber to come out of the microchamber when a liquid is flowed into the flow channel 121 described later.

  The surface of the microchamber chip 110 on which the microchamber 111 is formed is the bottom surface of the channel 121 of the cell deployment device 100. Each microchamber 111 is open to the channel 121. The arrangement position of the micro chamber 111 is not particularly limited. In the example shown in FIG. 2A, the plurality of microchambers 111 are arranged in a matrix at the center of the flow path. The configuration of the microchamber chip 110 will be described in detail separately.

  The frame body 120 is a thin plate having a through hole disposed between the microchamber chip 110 and the top plate 130 (see FIG. 2B). This through-hole becomes a flow path 121 for flowing a cell suspension derived from the specimen. The shape of the channel 121 (through hole) is not particularly limited as long as the cell suspension can flow over the microchamber 111, and can be appropriately selected according to the application. Moreover, the thickness of the frame 120 is not specifically limited, It sets suitably according to the height (depth) of a desired flow path. For example, the thickness of the frame 120 is in the range of 50 to 500 μm. By setting the height of the flow channel within the range of 50 to 500 μm, cells attached to the surface other than the micro chamber 111 of the micro chamber chip 110 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 flow channel 121 settle in the microchamber 111 while preventing clogging due to the cells in the flow 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, and the cell deployment device 100 can be manufactured.

  The inner surface of the through hole of the frame body 120 serves as a side surface of the flow path 121 and comes into contact with the cell suspension when the cell deployment device 100 is used. Therefore, the inner surface of the through hole 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 of the frame body 120 may be subjected to a blocking process for preventing nonspecific adhesion of cells. Examples of the blocking agent include casein, skim milk, albumin (including bovine serum albumin), hydrophilic polymers such as polyethylene glycol, phospholipids, low molecular compounds such as ethylenediamine and acetonitrile. These may be used alone or in combination of two or more.

  The top plate 130 is a thin plate having two through holes arranged on the frame body 120 (see FIG. 2C). These through-holes are respectively an inlet 131 for introducing a liquid (for example, a cell suspension, a washing liquid, a staining liquid, etc.) into the flow channel 121 and a discharge port for discharging the liquid from the flow channel 121. 132. Usually, the inlet 131 communicates with one end of the channel 121, and the outlet 132 communicates with the other end of the channel 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.

  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, polymethyl methacrylate, and cyclic olefin copolymer. Similarly to the frame body 120, the top surface of the flow path 121 of the top plate 130 may be surface-treated (for example, blocking treatment).

  The cell deployment device 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 from the viewpoint of observation and maintenance, it is preferable to fix them so as to be removable from each other. Examples of the fixing method include fixing by engagement, fixing using a screw, and fixing using an adhesive. Further, as described above, a double-sided seal may be used as the frame body 120.

  As shown in FIG. 1A and FIG. 1B, the flow path 121 formed in the cell deployment device 100 communicates with the outside through the inlet 131 and the outlet 132. By introducing a liquid (for example, a cell suspension) from the inlet 131, the liquid can be filled in the channel 121. At this time, the liquid flows in the flow path 121 from the inlet 131 toward the outlet 132. When the flow path 121 is filled with the cell suspension, the cells settle on the microchamber chip 110 and adhere to the bottom surface of the microchamber 111. That is, the cells are accommodated in the microchamber 111. Thereafter, washing and staining are performed, and various analyzes can be performed by observing cells housed in the microchamber 111 from the outside using a fluorescence microscope or the like. A method for detecting rare cells using the cell deployment device 100 will be described in detail again after the description of the microchamber chip.

[Microchamber chip]
Next, the microchamber chip 110 will be described in detail with reference to the drawings. 3A is a partially enlarged cross-sectional view of the microchamber chip 110, and FIG. 3B is a partially enlarged cross-sectional view of the microchamber chip 110 when the device 100 for cell deployment is used.

  As illustrated in FIG. 3A, the microchamber chip 110 includes a first substrate 112 and a second substrate 113 disposed on the first substrate 112. 4A is a plan view of the first substrate 112, and FIG. 4B is a plan view of the second substrate 113.

  As shown in FIG. 4B, one or two or more through holes 114 are formed in the second substrate 113. As shown in FIG. 3A, the bottomed microchamber 111 is formed by closing one opening of the through hole 114 with the first substrate 112. Therefore, a part of the surface of the first substrate 112 facing the second substrate 113 becomes the bottom surface of the microchamber 111. The inner surface of the through-hole 114 formed in the second substrate 113 is the side surface of the microchamber 111, and the surface of the second substrate 113 that does not face the first substrate 112 is the bottom surface of the channel 121 of the cell deployment device 100. It becomes.

  The materials of the first substrate 112 and the second substrate 113 are not particularly limited, but from the viewpoint of observing the inside of the flow path 121 from the outside using a fluorescent microscope or the like, it is a material having light transmittance like the top plate 130. Preferably there is. Examples of the material of the first substrate 112 and the second substrate 113 include resins such as polystyrene, polyethylene, polypropylene, polyamide, polycarbonate, polydimethylsiloxane, polymethylmethacrylate, and cyclic olefin copolymer. The thickness of the 1st board | substrate 112 will not be specifically limited if required intensity | strength can be ensured. On the other hand, the thickness of the second substrate 113 is appropriately selected according to the desired depth of the microchamber 111.

  The shape of the through hole 114 formed in the second substrate 113 is not particularly limited. Examples of the shape of the through hole 114 include a cylinder, a polygonal column, an inverted truncated cone, and an inverted polygonal truncated cone. The size of the through-hole 114 is not particularly limited, and can be appropriately set according to the type of cells to be accommodated, the number of cells to be accommodated in one microchamber, and the like. Usually, the size of the through-hole 114 is preferably such that about 10 to 15 cells can adhere to the bottom surface of the microchamber 111. For example, the maximum diameter of the through hole 114 is in the range of 20 to 150 μm, and the depth of the through hole 114 (the thickness of the second substrate 113) is in the range of 20 to 100 μm.

  The type of cell accommodated in the microchamber 111 is not particularly limited. Examples of cells accommodated in the microchamber 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. included.

  In order to accommodate all the cells contained in the cell suspension provided in the channel 121 in the microchamber 111 and to analyze them appropriately, only the bottom surface of the microchamber 111 is used as shown in FIG. 3B. It is preferable that the cell 10 adheres and the cell 10 does not adhere to the side surface of the microchamber 111 and the surface outside the microchamber 111 (the bottom surface of the channel 121). That is, the adhesion force of the cells 10 on the surface of the first substrate 112 facing the second substrate 113 (bottom surface of the microchamber 111) is the surface of the second substrate 113 not facing the first substrate 112 (bottom surface of the channel 121). It is preferable that the adhesive strength of the cells on the inner surface of the through hole 114 (side surface of the microchamber 111) is stronger.

In this specification, “cell adhesion” means that when a force is applied to a plurality of cells adhering to a plane in a direction substantially parallel to the plane, half (50%) of the cells are It means the value (unit: N) of the force that peels from the plane. For example, when a liquid is caused to flow through a flow path 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). .

When measuring “cell adhesion” in the present invention, at room temperature, a liquid is allowed to flow through a flow path in which a plurality of cells are attached at a cell density of 10 ² / mm ² on the bottom surface (plane), Apply force F to these cells for 1 minute. The force F is calculated by the above formula (1). About each of the upstream part of a flow path, a center part, and a downstream part, the peeling rate of the cell in the range of 200 micrometers x 200 micrometers is measured, and these average values are calculated. The force F when the obtained average value is 50% is defined as “cell adhesion”.

  Specifically, the cell adhesive force on the surface of the first substrate 112 facing the second substrate 113 is more than 0.01 nN, and the surface of the second substrate 113 not facing the first substrate 112 and the through hole 114. It is preferable that the cell adhesive force on the inner surface is 0.01 nN or less. In addition, the adhesive force of cells on the surface of the first substrate 112 facing the second substrate 113 is 0.1 nN or more, and the surface of the second substrate 113 that does not face the first substrate 112 and the inner surface of the through hole 114. The cell adhesion is more preferably 0.001 nN or less. By doing in this way, the cell 10 can be made to adhere only to the bottom face of the microchamber 111 more reliably. Regarding a method of changing the cell adhesive force on the surface of the first substrate 112 facing the second substrate 113 and the cell adhesive force on the surface of the second substrate 113 not facing the first substrate 112 and the inner surface of the through-hole 114. This will be described later when a method for manufacturing the microchamber chip 110 is described.

[Production method of micro chamber chip]
Next, a method for manufacturing the microchamber chip 110 will be described with reference to FIG. 5A and 5B are schematic views for explaining the method of manufacturing the microchamber chip according to the present embodiment. In these drawings, as in FIGS. 3A and 3B, only a part of the first substrate 112 and the second substrate 113 is shown. In addition, in order to show that the surface property of the first substrate 112 and the surface property of the second substrate 113 are different, the surface of the first substrate 112 is shown in white and the surface of the second substrate 113 is shown in black.

  The manufacturing method of the microchamber chip 110 according to the present embodiment includes (1) a first step of preparing the first substrate 112 and the second substrate 113, and (2) laminating the first substrate 112 and the second substrate 113. A second step. Hereinafter, each step will be described.

(1) First Step In the first step, as shown in FIG. 5A, a first substrate 112 and a second substrate 113 in which one or more through holes 114 are formed are prepared.

  As described above, the cell adhesive force on the surface of the first substrate 112 facing the second substrate 113 is greater than the cell adhesive force of the surface of the second substrate 113 not facing the first substrate 112 and the inner surface of the through hole 114. strong. Therefore, at least the surface properties of the surface of the first substrate 112 that is to face the second substrate 113 (shown in white in FIG. 5A), the surface of the second substrate 113 that is not to face the first substrate 112, and the through hole The surface properties of the inner surface of 114 (shown in black in FIG. 5A) are different from each other. 5A shows an example in which both surfaces of the first substrate 112 have the same surface property, and both surfaces of the second substrate 113 and the inner surface of the through hole 114 have the same surface property.

  The adhesion force of cells on the surface of the first substrate 112 that faces the second substrate 113 and the adhesion force of cells on the surface of the second substrate 113 that does not face the first substrate 112 and the inner surface of the through-hole 114. As a changing method, it is conceivable to use resin substrates made of different materials as the first substrate 112 and the second substrate 113. For example, as shown in the Examples, the adhesion of human leukemia cultured cells (CEM) on the surface of an untreated polystyrene substrate is 1N or more, but the adhesion of human leukemia cultured cells on the surface of an untreated polymethylmethacrylate substrate. The force is less than 0.01N. Therefore, an untreated polystyrene substrate may be used as the first substrate 112, and an untreated polymethyl methacrylate substrate may be used as the second substrate 113.

  Further, the cell adhesion force on the surface of the first substrate 112 that faces the second substrate 113, and the cell adhesion force on the surface of the second substrate 113 that does not face the first substrate 112 and the inner surface of the through hole 114. In order to adjust the adhesive force of cells before laminating at least one of the first substrate 112 or the second substrate 113 in which the through-holes 114 are formed in the second process, It is also conceivable to perform surface treatment. Examples of the surface treatment include plasma treatment, corona discharge treatment, coating treatment with hydrophilic polymer, protein, lipid and the like. These may be used alone or in combination of two or more. For example, plasma treatment or corona discharge may be used as a treatment for weakening the adhesion of cells to the surface of the second substrate 113 in which the through-hole 114 is formed (at least a surface not scheduled to face the first substrate 112 and the inner surface of the through-hole 114) Treatment, UV ozone treatment, bovine serum albumin (BSA) coating treatment, or polyvinyl alcohol (PVA) coating treatment may be applied. In addition, as a treatment for strengthening cell adhesion on the surface of the first substrate 112 (at least a surface that is to face the second substrate 113), an anti-EpCAM antibody immobilization treatment or poly-L-lysine (PLL) A coating treatment may be applied.

  A method for forming the through hole 114 in the second substrate 113 is not particularly limited. For example, the through holes 114 may be physically formed in the second substrate 113 by lithography, microneedles, or the like. Moreover, you may produce the 2nd board | substrate 113 which has the through-hole 114 by injection molding. Further, a porous film chemically synthesized by a polymer polymerization reaction may be used as the second substrate 113.

(2) Second Step In the second step, as shown in FIG. 5B, the first substrate 112 and the second substrate 113 are stacked and fixed. Thereby, the microchamber chip | tip 110 which has the bottomed microchamber 111 is produced.

  A method for fixing the first substrate 112 and the second substrate 113 is not particularly limited. For example, the second substrate 113 is fixed on one surface of the first substrate 112 by thermal welding, ultrasonic welding, or an adhesive.

  The microchamber chip 110 can be manufactured by the above procedure. According to the manufacturing method according to the present embodiment, the bottom surface of microchamber 111 is constituted by the surface of first substrate 112 (shown in white in FIG. 5B) having strong cell adhesion. On the other hand, the side surface of the microchamber 111 and the surface outside the microchamber 111 (the bottom surface of the flow path 121) are configured by the surface (shown in black in FIG. 5B) of the second substrate 113 with weak cell adhesion. Therefore, when a cell suspension is provided on the microchamber chip 110, almost all cells adhere to the bottom surface in the microchamber 111, as shown in FIG. 3B.

  As described above, the manufacturing method of the microchamber chip according to the present embodiment has a strong cell adhesion on the bottom surface of the microchamber suitable for the detection of rare cells, and the inner surface of the microchamber and the outside of the microchamber. A microchamber chip having weak cell adhesion on the surface can be easily manufactured.

[Rare cell detection method]
Finally, a method for detecting rare cells using the cell deployment device 100 will be described.

  For example, (A) a pretreatment step of wetting the bottom surface of the channel 121 of the cell deployment device 100, (B) an introduction step of introducing a cell suspension into the channel 121, and (C) cells in the channel 121 A cell expansion device by sequentially performing a recovery step of moving the cell and recovering the cells in the microchamber 111, and a detection step of (D) staining and detecting the rare cells recovered in the microchamber 111. 100 can be used to recover and detect rare cells from cell suspensions.

(A) Pretreatment step In the pretreatment step, an aqueous solution having a lower surface tension than water is introduced into the channel 121 of the cell deployment device 100, and the bottom surface of the channel 121 (in particular, the surface of the microchamber chip 110). ). By using an aqueous solution having a surface tension lower than that of water, not only the outside of the microchamber 111 but also the inside of the microchamber 111 can be wetted.

  The type of the aqueous solution introduced into the channel 121 is not particularly limited as long as the surface tension is lower than that of water. Examples of such aqueous solutions include aqueous solutions of alcohols such as ethanol, methanol, isopropyl alcohol, 0.01-1% of surfactants such as TWEEN 20 (trade name), Triton X (trade name), sodium dodecyl sulfate. (W / v) An aqueous solution is included. The surface tension of the aqueous solution at the temperature during use is preferably within the range of 10 to 60 mN / m, and more preferably within the range of 10 to 35 mN / m.

  After the pretreatment step, the flow path 121 is preferably filled with a phosphate buffer solution (PBS) or the like.

(B) Introduction Step In the introduction step, a cell suspension is introduced into the channel 121 from the introduction port 131, and the channel 121 is filled with the cell suspension. When the flow path 121 is filled with the phosphate buffer, the phosphate buffer is discharged from the outlet 132 at the same time as the cell suspension is introduced from the inlet 131.

  It is preferable to introduce the cell suspension so that the flow rate of the cell suspension in the channel 121 is constant within a predetermined range. By doing in this way, the cells contained in the cell suspension can be distributed substantially uniformly in the channel 121. The flow rate of the cell suspension in the channel 121 is appropriately set according to the shape of the channel 121. When the flow rate is too slow, the cells remain in the vicinity of the inlet 131, and the cell distribution in the longitudinal direction of the flow path 121 may be non-uniform. On the other hand, when the flow rate is too high, the cell distribution in the short direction of the channel 121 may be non-uniform.

  After introducing the cell suspension, it is preferable to let the cells contained in the cell suspension settle by allowing to stand for a certain time (for example, 1 to 15 minutes). At this time, some cells settle in the microchamber 111, but the remaining cells settle out of the microchamber 111. Since the adhesive force of the cells on the bottom surface of the microchamber 111 is strong, the cells that have settled in the microchamber 111 adhere firmly to the bottom surface of the microchamber 111. On the other hand, since the adhesive force of the cells outside the microchamber 111 is weak, the cells that have settled outside the microchamber 111 adhere weakly to the bottom surface of the channel 121 outside the microchamber 111.

(C) Recovery Step In the recovery step, cells outside the microchamber 111 are moved and the cells are recovered in the microchamber 111. Specifically, by performing intermittent liquid feeding, a moving force is intermittently applied to cells outside the microchamber 111.

  The intermittent liquid feeding includes a one-shot liquid feeding process for moving cells outside the microchamber 111 in the liquid feeding direction and a stationary process for sedimenting the moved cells. 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 outside the microchamber 111 in the liquid feeding direction, “one-shot liquid feeding” is performed. It becomes. 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, cells outside the microchamber 111 move, for example, about 50 μm in the liquid feeding direction in one cycle. When cells outside the microchamber 111 move directly above the microchamber 111, the cells settle in the microchamber 111 and are collected. From the viewpoint of collecting cells outside the microchamber 111 into the microchamber 111, the number of cycles of intermittent liquid feeding is preferably 2 times or more, and more preferably 10 times or more.

  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.

  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 cell recovery rate can be improved.

(D) Detection process In a detection process, the cell collect | recovered in the micro chamber 111 is dye | stained, and a rare cell is detected.

  Cell staining is performed by introducing a predetermined staining solution (for example, a solution of an antibody labeled with a fluorescent dye) into the channel 121. Thereafter, the cell and the inside of the channel 121 are washed by performing a process of introducing a washing solution (for example, phosphate buffer) into the channel 121 from the inlet 131 and discharging it from the outlet 132 at least once. . The type of staining solution is appropriately selected according to the type of cells to be detected.

  For example, circulating tumor cells (CTC) are detected as cells that are positive for nuclear staining and negative for CD45 in a blood-derived cell population. Circulating tumor cells (CTC) are also positive for cytokeratin (CK). Thus, for example, circulating tumor cells are detected by using a solution containing neutral red (nuclear staining) and a fluorescently labeled anti-CD45 antibody, or a solution further containing a fluorescently labeled anti-cytokeratin antibody in addition to these. be able to.

  Circulating vascular endothelial cells (CEC) are detected as cells positive for endothelial cell markers such as CD31 and CD51 / 61 in a blood-derived cell population. Therefore, for example, circulating vascular endothelial cells can be detected by using a solution containing a fluorescently labeled anti-CD31 antibody or anti-CD51 / 61 antibody. Whether or not the detected cell is a tumor cell may be confirmed by checking whether CD146 expressed when the endothelial cell becomes tumor is positive. In this case, a fluorescently labeled anti-CD146 antibody is also used.

  Circulating endothelial progenitor cells (CEP) are detected as cells with high ALDH activity in the blood-derived cell population. Therefore, for example, by using an ALDH activity measurement reagent (utilizing a fluorescence method), circulating vascular endothelial progenitor cells can be detected.

  The cell detection method is appropriately selected according to the staining method used. For example, when rare cells are stained with a dye, the rare cells are detected using an optical microscope or the like. When rare cells are fluorescently labeled, the rare cells are detected using a fluorescence microscope or the like. Thereafter, the target rare cells may be taken out using a micropipette or the like.

  Through the above steps, rare cells can be recovered from the cell suspension and detected using the cell deployment device 100.

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

1. Evaluation of cell adhesion to various substrates (1) Preparation of substrates The following 11 types of substrates were prepared as substrates to be evaluated. The thickness of each substrate is 1.5 mm.
(A) Untreated polymethylmethacrylate (PMMA) substrate (b) Untreated polypropylene (PP) substrate (c) Polystyrene (PS) substrate treated with UV ozone (d) Polystyrene treated with bovine serum albumin (BSA) Substrate (e) Plasma treated polystyrene substrate (f) Polystyrene alcohol (PVA) coated polystyrene substrate (g) Untreated polystyrene substrate (h) Untreated polycarbonate (PC) substrate (i) Untreated glass substrate (J) Polystyrene substrate coated with anti-human EpCAM antibody (k) Polystyrene substrate coated with poly-L-lysine (PLL)

  (C) The polystyrene substrate subjected to UV ozone treatment was prepared by irradiating ultraviolet rays with a UV ozone cleaner for 300 seconds on a polystyrene substrate having a thickness of 1.5 mm.

  (D) The polystyrene substrate treated with bovine serum albumin was prepared by immersing a polystyrene substrate having a thickness of 1.5 mm in a bovine serum albumin solution (concentration 1%) for 15 minutes and then drying the surface.

  (E) The polystyrene substrate subjected to plasma treatment was produced by subjecting the surface of a 1.5 mm thick polystyrene substrate to plasma treatment using a plasma surface treatment apparatus for 30 seconds.

  (F) Polystyrene alcohol coated polystyrene substrate is spin coated with a photocurable polyvinyl alcohol (BIOSURFINE-AWP-MRH; Toyo Gosei Co., Ltd.) solution (concentration 1%) on the surface of a 1.5 mm thick polystyrene substrate. It was produced by irradiating with UV rays and then curing.

  (J) The polystyrene substrate coated with the anti-human EpCAM antibody was prepared by immersing a 1.5 mm thick polystyrene substrate in an anti-human EpCAM antibody solution (concentration 1 μg / mL) for 15 minutes, and then drying the surface.

  (K) A polystyrene substrate coated with poly-L-lysine was coated with a poly-L-lysine solution (concentration: 0.1 mg / mL) on the surface of a polystyrene substrate having a thickness of 1.5 mm, and allowed to stand for 5 minutes. The solution was removed by suction filtration, and the surface was dried for 2 hours.

(2) Production of device for evaluation Evaluation was performed by attaching a double-sided seal (frame body) having a thickness of 100 μm and a polymethyl methacrylate substrate (top plate) having a thickness of 1.5 mm on one surface of a substrate to be evaluated. A device was prepared.

  6A is a plan view of the frame (double-sided seal) 120, FIG. 6B is a plan view of the top plate (polymethylmethacrylate substrate) 130, and FIG. 6C is a diagram in which the frame 120 and the top plate 130 are overlapped. FIG. As shown in FIG. 6A, the frame body 120 is formed with a hexagonal through-hole that becomes the flow path 121. The length of the channel 121 is 45 mm, and the width of the channel 121 is 15 mm. Further, as shown in FIG. 6B, the top plate 130 is formed with through holes having a diameter of 1 mm at positions corresponding to both ends of the channel 121. As shown in FIG. 6C, these through holes serve as an inlet 131 and an outlet 132 for the flow path, respectively.

(3) Evaluation of each substrate A liquid feeding tube was connected to each of the introduction port and the discharge port of the device for evaluation via a tube connector. The other end of the liquid feeding tube having one end connected to the introduction port was placed in a cell suspension containing 1 × 10 ⁵ human leukemia cultured cells (CEM) 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 rate of 1 mL / min using a syringe pump, and the flow path was filled with the cell suspension. After leaving still for 5 minutes, the number of cells present in the region of 200 μm × 200 μm was measured for each of the upstream portion, the central portion, and the downstream portion of the flow path using an optical microscope. Cell density in each area was either 10 a ^two / mm ^2.

  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 is sucked for 1 minute at a rate of 0.01, 0.1, 1, 10 mL / min using a syringe pump, and the inside of the channel is applied while applying force to the cells in the channel. Washed. The force F applied to each cell at this time is 0.001 nN (0.01 mL / min), 0.01 nN (0.1 mL / min), 0.1 nN (1 mL / min) or 1 nN (10 mL / min). Thereafter, the number of cells present in the same region (200 μm × 200 μm) in the flow channel was measured using an optical microscope, and the cell detachment rate (three locations) was determined from the change in the number of cells before and after the phosphate buffer solution was fed. Average value). In this experiment, “◯” was evaluated when the cell detachment rate was less than 50%, and “X” was evaluated when the cell detachment rate was 50% or more. Table 1 shows the evaluation results of each substrate.

  From the results shown in Table 1, the cell adhesive strength on the surface of the substrates (a) to (f) is 0.01 nN or less, and the cell adhesive strength on the surface of the substrates (d) and (f) is , 0.001 nN or less. Moreover, it turns out that the adhesive force of the cell in the surface of the board | substrate of (g)-(k) is more than 1 nN. In this example, (g) an untreated polystyrene substrate is used as the first substrate (cells are adhered) of the microchamber chip, and (a) untreated as the second substrate (cells are not adhered). The polymethylmethacrylate substrate or (d) BSA-coated polystyrene substrate was used.

2. Fabrication and evaluation of microchamber chip (1) Fabrication of microchamber chip and device for cell deployment according to examples An untreated polystyrene substrate (thickness 1.5 mm; first substrate) and a plurality of through-holes having a diameter of 100 μm are formed. Then, an untreated polymethylmethacrylate substrate or a BSA-coated polystyrene substrate (thickness 50 μm; second substrate) was laminated and bonded by a laser welding method to produce a microchamber chip according to the example. 7A is a plan view of the first substrate (polystyrene substrate) 112, and FIG. 7B is a second substrate (untreated polymethyl methacrylate substrate or polystyrene substrate with BSA coating) formed with a plurality of through holes 114. FIG. In FIG. 7B, the through holes 114 are schematically shown, and the size and number of the through holes are not accurate.

  The above-described frame body 120 (see FIG. 6A) and top plate 130 (see FIG. 6B) are further attached onto the second substrate 113 of the microchamber chip 110, thereby producing the cell deployment device according to the example. did. FIG. 7C is a side view of the device for cell deployment according to the example, and FIG. 7D is a plan view of the device for cell deployment according to the example. As shown in FIG. 7D, the plurality of through holes 114 formed in the second substrate 113 are all located in the flow path. Similarly to the above-described evaluation device, the two through holes formed in the top plate 130 serve as the inlet 131 and the outlet 132 for the flow path, respectively. In addition, in FIG. 7C, the thickness of each structural member is typically represented, and these thicknesses are not accurate.

(2) Production of microchamber chip and device for cell deployment according to comparative example A plurality of recesses having a diameter of 100 μm and a depth of 50 μm are formed on one surface of an untreated polystyrene substrate (thickness: 1.5 mm). The microchamber chip | tip which concerns on was produced. The position and shape of the recess are the same as those of the through hole formed in the second substrate described above (see FIG. 7B).

  The device for cell deployment according to the comparative example was manufactured by further attaching the above-described frame (see FIG. 6A) and the top plate (see FIG. 6B) on the surface of the microchamber chip where the recesses are formed. . For the device for cell deployment according to the comparative example, immediately before the evaluation experiment described below, a blocking solution (phosphate buffer solution containing 3% bovine serum albumin) is fed at a high flow rate (15 mL / min), The road inner surface was subjected to blocking treatment.

(3) Evaluation of each cell deployment device A liquid feeding tube was connected to each of the introduction port and the discharge port of the cell deployment device 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.

Next, the other end of the liquid feeding tube having one end connected to the inlet was moved into a cell suspension containing 1 × 10 ⁵ human leukemia cultured cells (CEM) in 100 μL. In this state, 100 μL of cell suspension is aspirated at a rate of 1 mL / min using a syringe pump, and the flow path is filled with the cell suspension, and then at a rate of 1 mL / min using a syringe pump. Aspiration of 0.1 μL of the cell suspension was repeated 20 times (stopped for 10 seconds after one aspiration), and the cells were collected in the microchamber.

  After the cells were collected, the number of cells existing outside the microchamber was counted using an optical microscope. As a result, in the cell deployment device according to the example, the proportion of cells existing outside the microchamber was 1% or less of the cells introduced into the flow path. In particular, in the cell deployment device using a polystyrene substrate coated with BSA as the second substrate, no cells were present outside the microchamber. On the other hand, in the device for cell deployment according to the comparative example, the proportion of cells existing outside the microchamber was 20% of the cells introduced into the flow path. The number of cells present in the microchamber was also counted using an optical microscope. As a result, in the cell deployment device according to the example, there were an average of 80 cells per microchamber and the variation in the number of cells per microchamber was small (CV = 10%). On the other hand, in the device for cell deployment according to the comparative example, the cells existed only in a part of the microchambers, and at most about 40 cells existed per one microchamber.

  From the above results, it can be seen that the microchamber chip manufactured by the manufacturing method according to the present invention can efficiently recover cells.

  The microchamber chip manufactured according to the present invention is useful, for example, for examination of diseases.

DESCRIPTION OF SYMBOLS 100 Cell expansion | deployment device 110 Microchamber chip | tip 111 Microchamber 112 1st board | substrate 113 2nd board | substrate 114 Through-hole 120 Frame body 121 Flow path 130 Top plate 131 Inlet port 132 Outlet port

Claims (7)

A method of manufacturing a microchamber chip having a microchamber for containing cells,
Preparing a first substrate and a second substrate in which one or more through holes are formed;
Laminating and fixing the first substrate and the second substrate,
The cell adhesive force on the surface of the first substrate facing the second substrate is stronger than the cell adhesive force on the surface of the second substrate not facing the first substrate and the inner surface of the through hole,
Manufacturing method of a micro chamber chip. The maximum diameter of the through hole is in the range of 20 to 150 μm,
The thickness of the second substrate is in the range of 20 to 100 μm.
The method for producing a microchamber chip according to claim 1. The cell adhesion force on the surface of the first substrate facing the second substrate is more than 0.01 nN,
The adhesive force of the cells on the surface of the second substrate that does not face the first substrate and the inner surface of the through-hole is 0.01 nN or less.
The manufacturing method of the micro chamber chip | tip of Claim 1 or Claim 2. The cell adhesive force on the surface of the first substrate facing the second substrate is 0.1 nN or more,
The adhesive force of the cells on the surface of the second substrate that does not face the first substrate and the inner surface of the through hole is 0.001 nN or less.
A method for manufacturing a microchamber chip according to claim 3.   The method of manufacturing a microchamber chip according to any one of claims 1 to 4, wherein the first substrate and the second substrate are resin substrates made of different materials.   6. The micro according to claim 1, wherein at least one of the first substrate and the second substrate is subjected to a surface treatment for adjusting cell adhesion before being laminated. A method for manufacturing a chamber chip.   The method for manufacturing a microchamber chip according to any one of claims 1 to 6, wherein the first substrate and the second substrate are fixed by thermal welding, ultrasonic welding, or an adhesive.

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