Microdevices and methods using the same

Abstract

To provide a microdevice that achieves easy solution exchange.SOLUTION: Provided is a micro device comprising a solution exchange member 100 and a cell retention cassette 200 that can be attached to the solution exchange member, the cell retention cassette having a well 210 that retains a first solution containing cells, the solution exchange member having a suction capillary 110 that suctions the first solution retained in the well, and a reservoir 120 that retains a second solution to be flowed into the well, and the device being configured such that by attaching the cell retention cassette that retains a first solution containing cells in the well between the suction capillary of the solution exchange member and the reservoir that retains a second solution, solution exchange is initiated due to the suction of the first solution from the well into the suction capillary by capillary forces and the flow of a second solution from the reservoir into the well, and then, by stopping the suction of the solution from the well to the suction capillary while the cells and solution are retained in the well, the solution exchange is stopped.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a microdevice and a method using the same. [Background technology]

[0002] Non-patent document 1 describes a microfluidic device for culturing cells in a microchamber while continuously perfusing the culture medium using a syringe pump. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Paul J. Hung et al., “A novel high aspect ratio microfluidic design to provide a stable and uniform microenvironment for cell growth in a high throughput mammalian cell culture array”, Lab Chip, 2005, 5, 44-48 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] However, conventionally, when changing the solution in a well in which cells were cultured, the change had to be done manually by an operator using a pipette or other instrument, or by using an external pump such as a syringe pump, which inevitably made the operation and equipment complicated.

[0005] This invention has been made in view of the above-mentioned problems, and one of its objectives is to provide a microdevice and a method using the same that enables simple solution exchange. [Means for solving the problem]

[0006] A microdevice according to one embodiment of the present invention for solving the above problems includes a solution exchange member and a cell holding cassette that can be attached to the solution exchange member, wherein the cell holding cassette has a well for holding a first solution containing cells, and the solution exchange member has a suction capillary tube for drawing in the first solution held in the well by capillary force and a reservoir for holding a second solution to be introduced into the well, and by attaching the cell holding cassette, which holds the first solution containing cells in the well, between the suction capillary tube of the solution exchange member and the reservoir holding the second solution, solution exchange is initiated by drawing the first solution from the well to the suction capillary tube by capillary force and introducing the second solution from the reservoir to the well, and thereafter, with the cells and solution still held in the well, the solution exchange is stopped when the drawing of the solution from the well to the suction capillary tube by capillary force is stopped. According to the present invention, a microdevice that enables simple solution exchange is provided.

[0007] In the microdevice, the suction capillary tube has a volume smaller than the sum of the volume of the first solution held in the well of the cell holding cassette before it is attached to the solution exchange member and the volume of the second solution held in the reservoir of the solution exchange member before it is attached, and has a downstream end that opens into the gas phase, and the microdevice may be configured such that, after the start of the solution exchange, the suction of the solution from the well to the suction capillary tube by capillary force stops when the solution suctioned from the well by capillary force reaches the downstream end of the suction capillary tube.

[0008] In the microdevice described above, the cell holding cassette may be configured to be attachable to and detachable from the solution exchange member after attachment. In this case, the microdevice may include a plurality of the solution exchange members used for multiple solution exchanges of the cell holding cassette. The microdevice may also include a plurality of the cell holding cassettes and a plurality of the solution exchange members used for solution exchange of the plurality of cell holding cassettes. Furthermore, the microdevice may be used without being connected to an external pump.

[0009] A solution exchange method according to one embodiment of the present invention for solving the above problems is a solution exchange method using any of the above microdevices, comprising: a preparation step of preparing a cell holding cassette that holds a first solution containing cells in the well and is not attached to the solution exchange member, and a solution exchange member that holds a second solution in the reservoir and to which the cell holding cassette is not attached; and a solution exchange step after the preparation step, in which the cell holding cassette is attached between the suction capillary of the solution exchange member and the reservoir, thereby initiating solution exchange by suction of the first solution from the well to the suction capillary by capillary force and inflow of the second solution from the reservoir to the well, and thereafter stopping the solution exchange by stopping the suction of the solution from the well to the suction capillary by capillary force while the cells and solution are held in the well. According to the present invention, a simple solution exchange method is provided.

[0010] In the above method, the suction capillary tube has a volume smaller than the sum of the volume of the first solution held in the well during the preparation step and the volume of the second solution held in the reservoir, and has a downstream end that opens into the gas phase, and in the solution exchange step, the suction of the solution from the well to the suction capillary tube by capillary force is stopped when the solution suctioned from the well by capillary force reaches the downstream end of the suction capillary tube.

[0011] In the above method, the cell holding cassette is configured to be attachable to and detachable from the solution exchange member, and the method may further include removing the cell holding cassette, in which the cells and solution are held in the wells, from the solution exchange member after the solution exchange in the solution exchange step has been stopped. In this case, the microdevice includes a plurality of solution exchange members, including a first solution exchange member, a second solution exchange member, and a third solution exchange member, and the cell holding cassette, which is attachable to and detachable from the solution exchange member, and the method includes a first preparation step and a first solution exchange step using the first solution exchange member as the preparation step and the solution exchange step, and further includes a second preparation step in which, after the solution exchange in the first solution exchange step has been stopped, the cell holding cassette is removed from the first solution exchange member to prepare the cell holding cassette, which is not attached to the solution exchange member and holds the cells and the third solution after the solution exchange in the wells, and the second solution exchange member, which holds the fourth solution in the reservoir and does not have the cell holding cassette attached. The process may also include: a second solution exchange step in which, after the second preparation step, the cell holding cassette is attached between the suction capillary of the second solution exchange member and the reservoir, thereby initiating solution exchange by capillary force drawing the third solution from the well to the suction capillary and flowing the fourth solution from the reservoir to the well, and thereafter, with the cells and the fifth solution after the solution exchange held in the well, the suction of the solution from the well to the suction capillary by capillary force is stopped, thereby stopping the solution exchange; a third preparation step similar to the second preparation step except that the third solution exchange member is used instead of the second solution exchange member; and a third solution exchange step similar to the second solution exchange step except that the third solution exchange member is used instead of the second solution exchange member.

[0012] In the above method, the microdevice includes a plurality of solution exchange members and a plurality of cell holding cassettes, and the method may include the preparation step and the solution exchange step using the plurality of solution exchange members for each of the plurality of cell holding cassettes. Furthermore, in the above method, the microdevice may be used without being connected to an external pump.

[0013] A cell culture method according to one embodiment of the present invention for solving the above problems is a cell culture method using any of the above microdevices, wherein the cell holding cassette is configured to be attachable to the solution exchange member and detachable after attachment, and the method comprises a preparation step of preparing the cell holding cassette which holds a first solution containing cells in the well and is not attached to the solution exchange member, and the solution exchange member which holds a second solution in the reservoir and to which the cell holding cassette is not attached, and after the preparation step, attaching the cell holding cassette between the suction capillary of the solution exchange member and the reservoir The present invention provides a cell culture method by simple solution exchange, which includes: a solution exchange step in which a solution is started by aspirating the first solution from the well to the suction capillary tube by capillary force and flowing the second solution from the reservoir to the well, and thereafter the solution exchange is stopped when the aspiration of the solution from the well to the suction capillary tube by capillary force is stopped while the cells and solution are held in the well; and a culture step in which, after the solution exchange in the solution exchange step is stopped, the cell holding cassette in which the cells and solution are held in the well is removed from the solution exchange member and the cells are cultured in the well of the removed cell holding cassette.

[0014] A method for producing a gene-introduced cell according to an embodiment of the present invention for solving the above problems is a method for producing a gene-introduced cell using any of the above microdevices, including holding a first solution containing cells in the well and the cell holding cassette not attached to the solution exchange member, and preparing a second solution for gene introduction containing a nucleic acid to be introduced into the cells in the reservoir and the solution exchange member not attached to the cell holding cassette. After the preparation step, the cell holding cassette is attached between the suction capillary of the solution exchange member and the reservoir, so that suction of the first solution from the well to the suction capillary by capillary force and inflow of the second solution from the reservoir to the well start solution exchange. Then, after the solution exchange stops by stopping the suction of the solution from the well to the suction capillary by capillary force while the cells and the solution containing the nucleic acid are held in the well, a gene introduction step is performed by holding the cells in the solution containing the nucleic acid in the well of the cell holding cassette to obtain gene-introduced cells into which the nucleic acid has been introduced. According to the present invention, a method for producing gene-introduced cells by simple solution exchange is provided.

Effects of the Invention

[0015] According to the present invention, a microdevice that realizes simple solution exchange and a method using the same are provided.

Brief Description of the Drawings

[0016] [Figure 1] It is an explanatory diagram showing an example of the microdevice according to the present embodiment in plan view. [Figure 2] It is an explanatory diagram showing a configuration example of the microdevice according to the present embodiment in perspective view. [Figure 3] It is an explanatory diagram showing a specific production example of the microdevice in the example according to the present embodiment. [Figure 4] It is an explanatory diagram showing the main steps included in an example of the method according to the present embodiment. [Figure 5] It is an explanatory diagram showing the main steps included in another example of the method according to this embodiment. [Figure 6] It is an explanatory diagram showing the main steps included in still another example of the method according to this embodiment. [Figure 7] It is an explanatory diagram showing the result of evaluating the odorant responsiveness of sensor cells in the example according to this embodiment.

Mode for Carrying Out the Invention

[0017] Hereinafter, a microdevice according to an embodiment of the present invention (hereinafter referred to as "this device") and a method using the same (hereinafter referred to as "this method") will be described. Note that the present invention is not limited to this embodiment.

[0018] FIG. 1 shows an example of this device in a plan view. FIG. 2 shows a configuration example of this device in a perspective view. FIG. 3 shows a specific manufacturing example of this device in the examples described later. FIG. 4 shows the main steps included in an example of this method. FIG. 5 shows the main steps included in another example of this method. FIG. 6 shows the main steps included in still another example of this method.

[0019] In this embodiment, the direction indicated by the double-headed arrow Z shown in the figure is referred to as the "vertical direction", the direction indicated by the arrowhead U is referred to as the "upper direction", and the direction indicated by the arrowhead D is referred to as the "lower direction". The vertical direction may be the vertical direction. Also, the direction indicated by the arrow A shown in the figure is referred to as the "suction direction A", and the direction indicated by the arrow B is referred to as the "inflow direction B".

[0020] This device 1 is a microdevice including a solution exchange member 100 and a cell holding cassette 200 that can be attached to the solution exchange member 100. The cell holding cassette 200 has a well 210 for holding a first solution containing cells. The solution exchange member 100 has a suction capillary 110 that sucks the first solution held in the well 210 of the cell holding cassette 200 by capillary force, and a reservoir 120 for holding a second solution to be introduced into the well 210.

[0021] The device 1 is configured such that, by attaching a cell-holding cassette 200, which holds a first solution containing cells in a well 210, between the suction capillary tube 110 of the solution exchange member 100 and a reservoir 120 that holds a second solution, solution exchange is initiated by the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force and the inflow of the second solution from the reservoir 120 to the well 210. Subsequently, while the cells and solution are held in the well 210, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force is stopped, thereby stopping the solution exchange.

[0022] Furthermore, this method, as one aspect, includes a solution exchange method using the device 1. This solution exchange method includes a preparation step and a solution exchange step. In the preparation step, a cell holding cassette 200, which holds a first solution containing cells in a well 210 and is not attached to the solution exchange member 100, and a solution exchange member 100, which holds a second solution in a reservoir 120 and is not attached to the cell holding cassette 200, are prepared.

[0023] In the solution exchange process, after the preparation process, the cell holding cassette 200 is attached between the suction capillary tube 110 of the solution exchange member 100 and the reservoir 120, thereby initiating solution exchange through the suction of the first solution from the well 210 of the cell holding cassette 200 to the suction capillary tube 110 of the solution exchange member 100 by capillary force, and the inflow of the second solution from the reservoir 120 into the well 210.

[0024] Furthermore, in the solution exchange process, after the start of the solution exchange, the solution exchange is stopped when the suction from the wells 210 to the suction capillaries 110 of the solution exchange member 100 is stopped due to capillary force while the cells and solution are still held in the wells 210 of the cell holding cassette 200.

[0025] In other words, this device 1 is configured such that, with only a simple operation of attaching a cell holding cassette 200, which holds a first solution containing cells in a well 210, to a solution exchange member 100 having a reservoir 120 that holds a second solution and a suction capillary tube 110, the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force spontaneously begins, and the inflow of the second solution from the reservoir 120 to the well 210 also begins. Furthermore, while the cells and solution are then held in the well 210, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force spontaneously stops. Therefore, by using this device 1, the solution exchange in the well 210 of the cell holding cassette 200 can be easily achieved.

[0026] The cell holding cassette 200 included in this device 1 has a base 220 and a well 210 formed as a bottomed hole in the base 220. The material constituting the base 220 is not particularly limited as long as the effects of the present invention are obtained, and may be one or more selected from the group consisting of resin, glass, ceramics and metal, for example, but is preferably composed of resin. The shape of the base 220 is not particularly limited as long as the effects of the present invention are obtained, but is preferably substrate-shaped, for example. That is, the base 220 is preferably a resin member configured in the shape of a substrate.

[0027] The base 220 of the cell holding cassette 200 may be composed of a first substrate 221 and a second substrate 222, as shown in Figure 2. In the example shown in Figure 2, a through hole is formed in the first substrate 221, and the second substrate 222 is bonded to the first substrate 221 so as to block the area below the through hole. However, the base 220 is not limited to the example shown in Figure 2, as long as the effects of the present invention are obtained, it may be composed of, for example, a single substrate with a bottomed hole forming a well 210.

[0028] The well 210 has a bottom surface 211, an inner surface 212 extending upward from the outer circumference of the bottom surface 211, and a holding space 213 surrounded by the bottom surface 211 and the inner surface 212. In the example shown in Figure 2, the inner surface of the through hole in the first substrate 221 constitutes the inner surface 212 of the well 210, and the portion of the upper surface of the second substrate 222 that is exposed into the through hole (holding space 213) constitutes the bottom surface 211 of the well 210.

[0029] The bottom surface 211 and inner surface 212 of the well 210 are preferably hydrophilic. The hydrophilicity of the bottom surface 211 and inner surface 212 of the well 210 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the bottom surface 211 and inner surface 212 is preferably 85° or less, and more preferably 60° or less.

[0030] The bottom surface 211 of the well 210 is preferably adhesive to cells when it is intended to hold adhesive cells in the well 210. In this regard, for example, cell adhesion may be imparted to the bottom surface 211 by applying a cell adhesion component (for example, a peptide, protein, glycan, polysaccharide, or compound in which cell adhesion groups are artificially introduced) to the bottom surface 211, which is the surface of the material constituting the base 220 (for example, a resin surface).

[0031] On the other hand, when holding non-adherent cells in well 210, or when placing a culture carrier in well 210 and supporting cells (adherent cells and / or non-adherent cells) on the culture carrier, the bottom surface 211 of well 210 may or may not be adhesive to the cells.

[0032] The well 210 may have a top surface 214 that closes all or part of its upper opening. Specifically, for example, the solution exchange in the well 210 is performed with the entire upper opening of the well 210 closed by the top surface 214. That is, during solution exchange, the well 210 is sealed except for the openings that communicate with the suction capillary tube 110 and reservoir 120 of the solution exchange member 100.

[0033] On the other hand, for example, when culturing cells in a well 210, it is preferable to leave at least a portion of the upper opening of the well 210 open to the gas phase containing oxygen. That is, in this case, for example, as shown in the figure, cells are cultured in a well 210 having a top surface 214 on which ventilation holes 215 are formed. Because the well 210 is in communication with the gas phase through the opening, oxygen can be efficiently supplied from the gas phase to the cultured cells in the well 210 through the opening.

[0034] However, if, for example, the well 210 has a top surface 214 made of a material with high oxygen permeability, it is not necessary for a part of the top surface 214 to be open. Also, by culturing cells in a well 210 that does not have a top surface 214, oxygen can be efficiently supplied to the cells from the gas phase outside the well 210.

[0035] The top surface 214 of the well 210 may be composed of a sealing member 230, as shown in Figure 2. The sealing member 230 is bonded to the upper surface 220a of the base 220 so as to close all or part of the upper opening of the well 210. As a result, the portion of the lower surface of the sealing member 230 that is exposed into the holding space 213 constitutes the top surface 214 of the well 210.

[0036] Furthermore, when exchanging the solution in a well 210 after culturing cells in a well 210 having a top surface 214 with ventilation holes 215, the well 210 can be sealed by, for example, blocking the ventilation holes 215 with another material.

[0037] The material constituting the sealing member 230 is not particularly limited as long as the effects of the present invention are obtained, and may be one or more selected from the group consisting of resin, glass, ceramics and metal, but it is preferable that it be composed of resin. The shape of the sealing member 230 is not particularly limited, but it is preferable that it be in the shape of a sheet, for example. That is, it is preferable that the sealing member 230 is a member made of resin in the shape of a sheet.

[0038] Preferably, at least a portion of the sealing member 230 constituting the top surface 214 is detachably adhered to the base 220. In this case, for example, after culturing cells in the well 210 or after changing the solution, at least a portion of the sealing member 230 can be detached from the base 220 to open the top of the well 210, making it easy to perform operations such as retrieving the cells through the opening.

[0039] The bottom surface 211 and top surface 214 of the well 210 are preferably translucent, and particularly preferably transparent. The translucency or transparency of the bottom surface 211 and top surface 214 of the well 210 facilitates observation of the cells within the well 210.

[0040] The shape of the well 210 is not particularly limited as long as the effects of the present invention are obtained. That is, for example, the shape of the well 210 may be any shape such as a circle, ellipse, or polygon in plan view. Furthermore, it is preferable that the well 210 has an expanding portion 216 at the end of the solution exchange member 100 on the reservoir 120 side, where the cross-sectional area (area of ​​the cross section cut perpendicular to the inflow direction B) expands toward the inflow direction B, and / or a contracting portion 217 at the end of the solution exchange member 100 on the suction capillary tube 110 side, where the cross-sectional area (area of ​​the cross section cut perpendicular to the suction direction A) decreases toward the suction direction A. That is, as shown in Figure 1, the well 210 may have a tapered expanding portion 216 and a contracting portion 217 in plan view. By having an expanding portion 216 and / or a contracting portion 217 in the well 210, the suction of the solution from the well 210 to the suction capillary tube 110, and / or the inflow of the solution from the reservoir 120 to the well 210 can be effectively achieved.

[0041] The size of the well 210 is not particularly limited as long as the effects of the present invention are obtained, but for example, the area of ​​the bottom surface 211 of the well 210 is 1000 mm². 2 The following are also acceptable, and 300mm 2 Preferably, the following: 100 mm 2The following is more preferable. Also, the area of ​​the bottom surface 211 of the well 210 is, for example, 1 mm 2 It may be greater than or equal to 3mm 2 It is preferable that the amount be greater than or equal to 12 mm 2 It is more preferable that the values ​​be greater than or equal to the above. The area of ​​the bottom surface 211 of the well 210 may be determined by any combination of the lower limit and the upper limit.

[0042] Furthermore, the volume of well 210 may be, for example, 5000 μL or less, preferably 500 μL or less, and more preferably 200 μL or less. Also, the volume of well 210 may be, for example, 1 μL or more, preferably 10 μL or more, and more preferably 30 μL or more. The volume of well 210 may be determined by any combination of the above lower limit and the above upper limit.

[0043] The cell holding cassette 200 preferably has an upstream channel 240 that connects the well 210 to the reservoir 120 of the solution exchange member 100, and / or a downstream channel 250 that connects the well 210 to the suction capillary 110 of the solution exchange member 100, as shown in Figure 1. The upstream channel 240 and the downstream channel 250 are formed as groove-shaped microchannels in the base 220. The upstream end 241 of the upstream channel 240 (the end on the reservoir 120 side) opens to a part 220b of the outer surface of the base 220, and the downstream end 242 of the upstream channel 240 (the end on the well 210 side) opens into the well 210 (for example, the inner surface 212). The upstream end 251 of the downstream channel 250 (the end on the well 210 side) opens into the well 210 (for example, the inner surface 212), and the downstream end 252 of the downstream channel 250 (the end on the suction capillary 110 side) opens into a part 220c of the outer surface of the base 220.

[0044] However, the configuration of connecting the well 210 of the cell holding cassette 200 with the reservoir 120 and suction capillary 110 of the solution exchange member 100 is not limited to the example shown in Figure 1. That is, for example, the downstream end 241 of the upstream channel 240 and / or the upstream end 251 of the downstream channel 250 may each branch out and communicate with the well 210. Specifically, for example, the downstream end 241 of the upstream channel 240 may branch out toward the inflow direction B and connect to the well 210, and have two or more downstream branch channels (not shown) that communicate with it. Also, for example, the upstream end 251 of the downstream channel 250 may branch out toward the opposite direction to the suction direction A and connect to the well 210, and have two or more upstream branch channels (not shown) that communicate with it.

[0045] Since the upstream channel 240 and the downstream channel 250 are in communication with the well 210, when the solution is held in the well 210, the same solution is also held in the upstream channel 240 and the downstream channel 250.

[0046] In this regard, the inner surfaces of the upstream channel 240 and the downstream channel 250 are hydrophilic. The hydrophilicity of the inner surfaces of the upstream channel 240 and the downstream channel 250 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the inner surface is preferably 85° or less, and more preferably 60° or less.

[0047] On the other hand, the outer surface 220b of the base 220 where the upstream end 241 of the upstream channel 240 opens, and the outer surface 220c of the base 220 where the downstream end 252 of the downstream channel 250 opens, are hydrophobic. The hydrophobicity of the outer surfaces 220b and 220c of the base 220 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the outer surfaces 220b and 220c is preferably 90° or more, and more preferably 100° or more.

[0048] The sizes of the upstream flow path 240 and the downstream flow path 250 are not particularly limited as long as the effects of the present invention can be obtained. For example, the cross-sectional area of the upstream flow path 240 (the area of the cross-section cut by a plane perpendicular to the inflow direction B), and the cross-sectional area of the downstream flow path 250 (the area of the cross-section cut by a plane perpendicular to the suction direction A) can each independently be 2 10 mm or less, preferably 4 mm 2 or less, and more preferably 1 mm 2 or less. Also, the cross-sectional areas of the upstream flow path 240 and the downstream flow path 250 can each independently be, for example, 0.0001 mm 2 or more, preferably 0.01 mm 2 or more. The cross-sectional areas of the upstream flow path 240 and the downstream flow path 250 can each independently be specified by arbitrarily combining any of the above lower limit values and any of the above upper limit values.

[0049] The solution exchange member 100 included in the present device 1 has a base 130, a suction capillary 110 formed as a micro flow path in the base 130, and a reservoir 120 formed as a bottomed hole in the base 130.

[0050] The material constituting the base 130 is not particularly limited as long as the effects of the present invention can be obtained. For example, it may be one or more selected from the group consisting of resin, glass, ceramics, and metal, and is preferably made of resin. The shape of the base 130 is not particularly limited as long as the effects of the present invention can be obtained, but for example, it is preferably in the shape of a substrate. That is, the base 130 is preferably a resin member configured in the shape of a substrate.

[0051] The base portion 130 of the solution exchange member 100 may be configured to include a first substrate 131, a second substrate 132, and a sealing member 133, as shown in Figure 2. In the example shown in Figure 2, the first substrate 131 has grooves that constitute the suction capillary tube 110 and a recess that constitutes the lower part of the reservoir 120, and the second substrate 132 has a through hole that constitutes the upper part of the reservoir 120. The reservoir 120 is formed by bonding the second substrate 132 to the upper surface of the first substrate 131 at a position corresponding to the recess. The suction capillary tube 110 is formed by bonding the sealing member 133 to the upper surface of the first substrate 131 at a position that closes above the groove. However, the base portion 130 is not limited to the example shown in Figure 2, as long as the effects of the present invention are obtained, and may be configured to include, for example, one substrate having grooves that constitute the suction capillary tube 110 and a bottomed hole that constitutes the reservoir 120, and a sealing member bonded to the upper surface of the substrate at a position that closes above the groove.

[0052] The suction capillary tube 110 is a microchannel that draws in and holds the solution in the well 210 of the cell holding cassette 200 by capillary force. As shown in Figure 1, the upstream end 111 (the end on the well 210 side) of the suction capillary tube 110 opens to a part 130c of the outer surface of the base 130. The suction capillary tube 110 is formed so that when the solution comes into contact with the opening of the upstream end 111, which holds a gas (for example, air), the solution is drawn into the suction capillary tube 110 by capillary action.

[0053] Furthermore, the downstream end 112 of the suction capillary tube 110 (the end opposite to the upstream end 111) opens into a part 130a of the outer surface of the base 130. That is, the downstream end 112 of the suction capillary tube 110 communicates with the outside gas phase (e.g., the atmosphere) through an opening 113 formed in the outer surface 130a of the base 130. More specifically, in the example shown in Figure 2, the opening 113 of the downstream end 112 of the suction capillary tube 110 is formed on the outer surface 130a, which is the upper surface of the base 130 (the upper surface 133a of the sealing member 133). The suction capillary tube 110 is designed so that the suction by capillary force spontaneously stops when the solution drawn in by capillary force reaches the downstream end 112 which is open to the gas phase.

[0054] The inner surface of the suction capillary tube 110 is hydrophilic. The hydrophilicity of the inner surface of the suction capillary tube 110 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the inner surface is preferably 85° or less, and more preferably 60° or less.

[0055] On the other hand, the outer surface 130a of the base portion 130, where the downstream end 112 of the suction capillary tube 110 opens, is hydrophobic. The hydrophobicity of the outer surface 130a of the base portion 130 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the outer surface 130a is preferably 90° or more, and more preferably 100° or more.

[0056] The shape of the suction capillary 110 is not particularly limited as long as the effects of the present invention are obtained. That is, the suction capillary 110 may be formed to meander in a plan view, for example, as shown in the figure. Alternatively, the suction capillary 110 may be formed in a spiral shape in a plan view, or in a shape that branches into multiple parts.

[0057] The size of the suction capillary tube 110 is not particularly limited as long as the effects of the present invention are obtained, but for example, the flow path cross-sectional area of ​​the suction capillary tube 110 (the area of ​​the cross-section when cut by a plane perpendicular to the suction direction A) is 10 mm 2 The following is also acceptable: 4mm 2 Preferably, the following: 1 mm 2The following is more preferable. Furthermore, the flow path cross-sectional area of ​​the suction capillary tube 110 is, for example, 0.0001 mm². 2 It may be greater than or equal to 0.01 mm 2 It is preferable that the values ​​are as described above. The flow path cross-sectional area of ​​the suction capillary tube 110 may be determined by any combination of the lower limit and the upper limit mentioned above.

[0058] Furthermore, the volume of the suction capillary tube 110 may be, for example, 10,000 μL or less, and preferably 1,000 μL or less. Also, the volume of the suction capillary tube 110 may be, for example, 1 μL or more, and preferably 10 μL or more. The volume of the suction capillary tube 110 may be determined by any combination of the above lower limit and the above upper limit.

[0059] The reservoir 120 has a bottom surface 121, an inner surface 122 extending upward from the outer circumference of the bottom surface 121, and a holding space 123 surrounded by the bottom surface 121 and the inner surface 122. In the example shown in Figure 2, the recess in the first substrate 131 constitutes the lower part of the reservoir 120, including the bottom surface 121, and a part of the recess and the inner surface of the through hole in the second substrate 132 constitute the inner surface 122 of the reservoir 120. The top of the reservoir 120 is open.

[0060] The bottom surface 121 and inner surface 122 of the reservoir 120 are preferably hydrophilic. The hydrophilicity of the bottom surface 121 and inner surface 122 of the reservoir 120 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the bottom surface 121 and inner surface 122 is preferably 85° or less, and more preferably 60° or less.

[0061] The shape of the reservoir 120 is not particularly limited as long as the effects of the present invention are obtained, and may be any shape such as a circle, ellipse, or polygon in plan view.

[0062] The size of the reservoir 120 is not particularly limited as long as the effects of the present invention are obtained, but for example, the volume of the reservoir 120 may be 10,000 μL or less, and preferably 1,000 μL or less. Also, the volume of the reservoir 120 may be 1 μL or more, and preferably 10 μL or more. The volume of the reservoir 120 may be determined by any combination of the above lower limit and the above upper limit.

[0063] The solution exchange member 100 preferably has an outflow channel 140 that connects the reservoir 120 and the wells 210 of the cell holding cassette 200. The outflow channel 140 is formed as a groove-shaped microchannel in the base portion 130. As shown in Figure 1, the upstream end 141 of the outflow channel 140 (the end on the reservoir 120 side) opens into the reservoir 120 (for example, the inner surface 122), and the downstream end 142 of the outflow channel 140 (the end on the well 210 side) opens into a part 130b of the outer surface of the base portion 130.

[0064] Since the outflow channel 140 is in communication with the reservoir 120, the solution held in the reservoir 120 is also held in the outflow channel 140. In this respect, the inner surface of the outflow channel 140 is hydrophilic. The hydrophilicity of the inner surface of the outflow channel 140 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the inner surface is preferably 85° or less, and more preferably 60° or less.

[0065] On the other hand, the outer surface 130b of the base portion 130, where the downstream end 142 of the outflow channel 140 opens, is hydrophobic. The hydrophobicity of the outer surface 130b of the base portion 130 is not particularly limited as long as the effects of the present invention are obtained, but for example, the water contact angle of the outer surface 130b is preferably 90° or more, and more preferably 100° or more.

[0066] The size of the outflow channel 140 is not particularly limited as long as the effects of the present invention are obtained, but for example, the cross-sectional area of ​​the outflow channel 140 (the area of ​​the cross-section when cut by a plane perpendicular to the inflow direction B) is 10 mm². 2The following is also acceptable: 4mm 2 Preferably, the following: 1 mm 2 The following is more preferable. Furthermore, the cross-sectional area of ​​the outflow channel 140 is, for example, 0.0001 mm². 2 It may be greater than or equal to 0.01 mm 2 It is preferable that the above conditions are met. The cross-sectional area of ​​the outflow channel 140 may be determined independently by any combination of either of the lower limits and either of the upper limits.

[0067] The device 1 is configured so that the cell holding cassette 200 can be attached between the suction channel 110 of the solution exchange member 100 and the reservoir 120. The configuration for attaching the cell holding cassette 200 to the solution exchange member 100 is not particularly limited as long as the effects of the present invention are obtained, but for example, it is preferable that the cell holding cassette 200 and the solution holding member 100 are configured so that one engages with the other.

[0068] In other words, for example, the solution exchange member 100 may have a mounting recess 150 into which the cell holding cassette 200 is fitted. Specifically, in the example shown in Figures 1 and 2, a mounting recess 150 is formed in the base 130 of the solution exchange member 100 at a position between the suction capillary tube 110 and the reservoir 120, into which the base 220 of the cell holding cassette 200 is fitted.

[0069] Furthermore, the cell holding cassette 200 and the solution exchange member 100 may have a pair of engaging portions that can engage with each other. That is, the cell holding cassette 200 and the solution exchange member 100 may have a pair of engaging portions, for example, one of which has a convex shape and the other has a concave shape.

[0070] Specifically, in the example shown in Figure 1, the cell holding cassette 200 has a convex first engaging portion 260 formed protruding from the base portion 220, and the solution exchange member 100 has a concave second engaging portion 160 that engages with the protruding portion 260. More specifically, the cell holding cassette 200 has a first engaging portion 260 at both the reservoir 120 side end and the suction capillary tube 110 side end of the base portion 220, and the solution exchange member 100 has a pair of second engaging portions 160 that engage with the first engaging portion 260.

[0071] Furthermore, in the example shown in Figure 1, the upstream channel 240 and the downstream channel 250 of the cell holding cassette 200 open to the outer surface 220b and 220c of the first engaging portion 260 on the upstream side, respectively. Correspondingly, the outflow channel 140 and the suction capillary tube 110 of the solution holding member 100 open to the outer surface 130b and 130c of the second engaging portion 160 on the downstream side, respectively.

[0072] The device 1 is configured such that a cell holding cassette 200, which holds a first solution containing cells in a well 210, is installed between the suction capillary tube 110 of the solution exchange member 100 and the reservoir 120, thereby initiating the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force.

[0073] In other words, the device 1 is configured such that the cell holding cassette 200 is attached to the solution exchange member 100, thereby creating communication between the wells 210 of the cell holding cassette 200 and the suction capillaries 110 of the solution exchange member 100.

[0074] Specifically, when the cell holding cassette 200 is attached to the solution exchange member 100, the downstream channel 250 of the cell holding cassette 200 is connected to the suction capillary tube 110 of the solution exchange member 100, and as a result, the well 210 of the cell holding cassette 200 and the suction capillary tube 110 are in communication via the downstream channel 250.

[0075] More specifically, the outer surface 220c of the base 220 into which the downstream channel 250 of the cell holding cassette 200 opens, and the outer surface 130c of the base 130 into which the upstream end 111 of the suction capillary tube 110 of the solution exchange member 100 opens, are configured to be in close contact with each other.

[0076] Therefore, in the cell-holding cassette 200, the downstream channel 250 that holds the first solution together with the well 210 is connected to the upstream end 111 of the suction capillary tube 110 that holds gas (for example, air), and by this connection, the first solution begins to be drawn from the well 210 and the downstream channel 250 into the suction capillary tube 110 by capillary force. In other words, simply by attaching the cell-holding cassette 200 to the solution exchange unit 100, the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force begins spontaneously.

[0077] Furthermore, the device 1 is configured such that when the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force is initiated, the inflow of the second solution from the reservoir 120 to the well 210 is initiated.

[0078] In other words, the device 1 is configured such that the cell holding cassette 200 is attached to the solution exchange member 100, thereby enabling communication between the wells 210 of the cell holding cassette 200 and the reservoir 120 of the solution exchange member 100.

[0079] Specifically, when the cell holding cassette 200 is attached to the solution exchange member 100, the upstream channel 240 of the cell holding cassette 200 is connected to the outflow channel 140 of the solution exchange member 100, and as a result, the wells 210 of the cell holding cassette 200 and the reservoir 120 are in communication via the upstream channel 240 and the outflow channel 140.

[0080] More specifically, the outer surface 220b of the base 220 into which the upstream channel 240 of the cell holding cassette 200 opens, and the outer surface 130b of the base 130 into which the outflow channel 140 of the solution exchange member 100 opens are configured to be in close contact with each other.

[0081] Furthermore, the wells 210 of the cell holding cassette 200 are sealed except for the upstream channel 240 and the downstream channel 250, which communicate with the reservoir 120 and the suction capillary tube 110, respectively, by having a top surface 214 that closes the opening above them.

[0082] Therefore, with well 210 and reservoir 120 in communication, as described above, when the suction of the first solution from well 210 to the suction capillary tube 110 by capillary force is initiated, the inflow of the second solution from reservoir 120 to well 210 is also initiated. In other words, simply by attaching the cell holding cassette 200 to the solution exchange unit 100, in addition to the suction of the first solution from well 210 to the suction capillary tube 110 by capillary force, the inflow of the second solution from reservoir 120 to well 210 is also initiated spontaneously.

[0083] In this device 1, the exchange of solutions within the well 210 is initiated when the first solution is drawn from the well 210 to the suction capillary tube 110 by capillary force, and the second solution flows from the reservoir 120 into the well 210.

[0084] Furthermore, after the solution exchange has started as described above, the device 1 is configured such that, while the cells and solution are retained in the well 210, the suction from the well 210 to the suction capillary tube 110 by capillary force is stopped.

[0085] Here, the configuration for stopping the suction from the well 210 to the suction capillary tube 110 by capillary force while the cells and solution are held in the well 210 is not particularly limited as long as the effects of the present invention are obtained. For example, the suction capillary tube 110 may have a volume smaller than the sum of the volume of the first solution held in the well 210 of the cell holding cassette 200 before it is attached to the solution exchange unit 100 and the volume of the second solution held in the reservoir 120 of the solution exchange member 100 before it is attached (hereinafter referred to as "the total volume of the first and second solutions at the start of solution exchange"), and may also have a downstream end 112 that opens into the gas phase.

[0086] In this case, the suction by capillary force is stopped when a volume of solution smaller than the total volume of the first and second solutions at the start of solution exchange reaches the downstream end 112 of the suction capillary tube 110, which has been drawn from the well 210 into the suction capillary tube 110 by capillary force.

[0087] Specifically, at the start of solution exchange, the suction capillary tube 110 retains gas inside, and its downstream end 112 is open to the gas phase (i.e., the atmosphere outside the device 1 (e.g., air)). Therefore, the suction capillary tube 110 draws the solution from the well 210 by capillary force while releasing the gas from the opening at its downstream end 112.

[0088] Next, since the downstream end 112 of the suction capillary tube 110 is open into the gas phase, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force spontaneously stops when the aspirated solution reaches the downstream end 112.

[0089] Here, since the volume of the suction capillary tube 110 is smaller than the total volume of the first and second solutions at the start of solution exchange, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force is stopped while the solution is retained in the well 210.

[0090] Thus, the suction capillary tube 110 not only spontaneously begins aspirating the solution from the well 210 by capillary force, but also spontaneously stops the aspiration by capillary force while cells and solution are retained in the well 210, that is, before all of the solution in the well 210 containing the cells is aspirated. Furthermore, the suction capillary tube 110 retains the solution aspirated from the well 210 by capillary force without discharging it from the start to the stop of the solution exchange.

[0091] During solution exchange, the suction of the first solution from well 210 to the suction capillary tube 110 and the inflow of the second solution from reservoir 120 into well 210 occur in parallel. As a result, a considerable amount of mixing occurs within well 210 between the remaining first solution and the inflowing second solution. Therefore, the solution held in well 210 at the time solution exchange is stopped will be a third solution whose composition does not perfectly match that of the second solution. In this regard, for example, from a hydrodynamic standpoint, by designing the device 1 so that the conditions (e.g., flow rate) for the suction of the first solution from well 210 to the suction capillary tube 110 and the inflow of the second solution from reservoir 120 into well 210 are within an appropriate range, it is possible to adjust the composition of the third solution held in well 210 at the time solution exchange is stopped so that it is close to the composition of the second solution.

[0092] If the suction capillary tube 110 has a volume smaller than the total volume of the first and second solutions at the start of solution exchange, as described above, the volume of the suction capillary tube 110 may be, for example, 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.8 times or less, 0.7 times or less, or 0.6 times or less of the total volume of the first and second solutions at the start of solution exchange. Also, the volume of the suction capillary tube 110 may be, for example, 0.1 times or more, 0.2 times or more, 0.3 times or more, or 0.4 times or more of the total volume of the first and second solutions at the start of solution exchange. The ratio of the volume of the suction capillary tube 110 to the total volume of the first and second solutions at the start of solution exchange may be determined by any combination of the lower limit and the upper limit mentioned above.

[0093] Furthermore, if the suction capillary tube 110 has a volume smaller than the combined volume of the first and second solutions at the start of solution exchange, then naturally the volume of the suction capillary tube 110 will be smaller than the sum of the volume of the well 210 and the volume of the reservoir 120.

[0094] Specifically, the volume of the suction capillary tube 110 may be, for example, 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.8 times or less, 0.7 times or less, or 0.6 times or less, the sum of the volume of the well 210 and the volume of the reservoir 120. Also, the volume of the suction capillary tube 110 may be, for example, 0.1 times or more, 0.2 times or more, 0.3 times or more, or 0.4 times or more, the sum of the volume of the well 210 and the volume of the reservoir 120. The ratio of the volume of the suction capillary tube 110 to the sum of the volume of the well 210 and the volume of the reservoir 120 may be determined by any combination of the lower limit and the upper limit mentioned above.

[0095] Furthermore, the volume of the suction capillary tube 110 may be greater than or equal to the volume of the second solution held in the reservoir 120 of the solution exchange member 100 before the cell holding cassette 200 is attached (hereinafter referred to as "the volume of the second solution at the start of solution exchange"). In this case, all of the second solution held in the reservoir 120 at the start of solution exchange can be used for solution exchange.

[0096] Specifically, the volume of the suction capillary tube 110 may be, for example, 1 or more times the volume of the second solution at the start of suction, 1.1 or more times, or 1.2 or more times. Also, the volume of the suction capillary tube 110 may be, for example, 10 or less times the volume of the second solution at the start of suction, 8 or less times, 6 or less times, 4 or less times, or 2 or less times. The ratio of the volume of the suction capillary tube 110 to the volume of the second solution at the start of suction may be determined by any combination of the above lower limit and the above upper limit.

[0097] Furthermore, the volume of the suction capillary tube 110 may be greater than or equal to the volume of the first solution held in the well 210 of the cell holding cassette 200 before it is attached to the solution exchange member 100 (hereinafter referred to as "the volume of the first solution at the start of solution exchange"), or it may be less than or equal to the volume of the first solution at the start of solution exchange. In other words, during solution exchange, the entire amount of the first solution at the start of solution exchange may be exchanged, or only a portion of the first solution at the start of solution exchange may be exchanged.

[0098] Specifically, the volume of the suction capillary tube 110 may be, for example, 0.1 times or more, 0.2 times or more, 0.4 times or more, 0.6 times or more, 0.8 times or more, 1 time or more, 2 times or more, or 3 times or more, of the volume of the first solution at the start of solution exchange. Also, the volume of the suction capillary tube 110 may be, for example, 10 times or less, 8 times or less, 6 times or less, or 5 times or less, of the volume of the first solution at the start of solution exchange. The ratio of the volume of the suction capillary tube 110 to the volume of the first solution at the start of solution exchange may be determined by any combination of the lower limit and the upper limit mentioned above.

[0099] Next, the details of the solution exchange method using device 1 will be described. The solution exchange method includes a preparation step and a solution exchange step. In the preparation step, as shown in Figure 4(A), a first solution containing cells is held in the well 210, and a cell holding cassette 200 that is not attached to the solution exchange unit 100 is prepared.

[0100] Here, not limited to the first solution, the solution held in the device 1 in this method is preferably an aqueous solution capable of maintaining the viability of the cells held in well 210. For example, equilibrium salt solutions such as phosphate buffer or Hanks' solution, or culture media, or aqueous solutions prepared based on these solutions are preferably used.

[0101] The volume of the first solution held in well 210 during the preparation step is not particularly limited as long as the effects of the present invention are obtained, but for example it may be 5000 μL or less, preferably 500 μL or less, and more preferably 200 μL or less. Also, the volume of the first solution held in well 210 during the preparation step may be 1 μL or more, preferably 10 μL or more, and more preferably 30 μL or more. The volume of the first solution held in well 210 during the preparation step may be determined by arbitrarily combining any of the above lower limits and any of the above upper limits.

[0102] Furthermore, since the upstream channel 240 and downstream channel 250 of the cell holding cassette 200 are in communication with the well 210, by holding the first solution in the well 210, the first solution is also held in the upstream channel 240 and downstream channel 250.

[0103] The cells retained in well 210 are not particularly limited as long as they provide the effects according to the present invention, but for example, cells from mammals, reptiles, amphibians, fish, or insects are preferably used, and mammalian or insect cells are particularly preferably used. Furthermore, the cells retained in well 210 may be adherent cells or non-adherent cells, but adherent cells are preferably used.

[0104] In this method, solution exchange is performed while the cells remain in the well 210. For this reason, it is preferable to adhere the cells to the bottom surface 211 of the well 210. Alternatively, for example, a culture carrier may be held in the well 210 and the cells may be held on the culture carrier. Alternatively, for example, the bottom surface 211 of the well 210 may be formed as a recess that is lower than the downstream channel 250, and the cells may be settled and held on the recessed bottom surface 211.

[0105] The method for retaining the first solution containing the cells in well 210 is not particularly limited as long as the effects of the present invention are obtained. For example, the cells may be retained in well 210 by placing a cell suspension prepared by suspending the cells in the first solution into well 210.

[0106] In the preparation step, cells may be cultured in the well 210 beforehand. In this case, a cell holding cassette 200 containing pre-cultured cells in the well 210 is prepared. Cell culture in the well 210 is preferably carried out by placing the cell holding cassette 200 containing the cells in the well 210 into an incubator. Furthermore, it is preferable that the top of the well 210 is open during cell culture.

[0107] In addition, during the preparation process, as shown in Figure 4(A), a solution exchange member 100 is prepared, which holds the second solution in the reservoir 120 and does not have the cell holding cassette 200 attached.

[0108] In the preparation step, the second solution held in the reservoir 120 is preferably an aqueous solution having a different composition from the first solution. However, the compositions of the first and second solutions may be adjusted as appropriate depending on the purpose. That is, the second solution may be, for example, an aqueous solution prepared by adding the component to be acted upon the cells in the well 210 to an equilibrium salt solution or culture medium.

[0109] The volume of the second solution held in the reservoir 120 during the preparation step is not particularly limited as long as the effects of the present invention are obtained, but may be, for example, 10,000 μL or less, and preferably 1,000 μL or less. Also, the volume of the second solution held in the reservoir 120 during the preparation step may be 1 μL or more, and preferably 10 μL or more. The volume of the second solution held in the reservoir 120 during the preparation step may be determined by arbitrarily combining either of the above lower limits and either of the above upper limits.

[0110] Furthermore, since the outflow channel 140 of the solution exchange member 100 is in communication with the reservoir 120, by holding the second solution in the reservoir 120, the second solution is also held in the outflow channel 140.

[0111] The volume of the second solution held in the reservoir 120 during the preparation step (i.e., the volume of the second solution at the start of solution exchange) may be, for example, greater than or equal to the volume of the first solution held in the well 210 during the preparation step (i.e., the volume of the first solution at the start of solution exchange), or less than or equal to the volume of the first solution at the start of solution exchange.

[0112] Specifically, the volume of the second solution at the start of solution exchange may be, for example, 0.1 times or more, 0.2 times or more, 0.4 times or more, 0.6 times or more, 0.8 times or more, 1 time or more, 2 times or more, or 3 times or more, of the volume of the first solution at the start of solution exchange. Also, the volume of the second solution at the start of solution exchange may be, for example, 10 times or less, 8 times or less, 6 times or less, or 5 times or less, of the volume of the first solution at the start of solution exchange. The ratio of the volume of the second solution at the start of solution exchange to the volume of the first solution at the start of solution exchange may be determined by any combination of the lower limit and the upper limit mentioned above.

[0113] In addition, in the solution exchange member 100 in which the second solution prepared in the preparation step is held in the reservoir 120, the second solution is also held in the outflow channel 140 which is in communication with the reservoir 120.

[0114] In the solution exchange member 100 prepared in the preparation step, gas is held in the suction capillary tube 110. That is, in the preparation step, a solution exchange member 100 is prepared that does not have a cell holding cassette 200 attached, with the second solution held in the reservoir 120 and gas held in the suction capillary tube 110. Specifically, the suction capillary tube 110 may be filled with gas. Alternatively, the suction capillary tube 110 may not hold any liquid.

[0115] In the subsequent solution exchange step, first, as shown in Figure 4(B), the cell holding cassette 200 is attached between the suction capillary tube 110 of the solution exchange member 100 and the reservoir 120. That is, for example, if the cell holding cassette 200 and the solution holding member 100 are configured such that one engages with the other, the cell holding cassette 200 is attached to the solution holding member 100 by engaging the cell holding cassette 200 with the solution holding member 100.

[0116] Specifically, for example, as shown in Figures 1 and 2, if the solution exchange member 100 has a mounting recess 150, the cell holding cassette 200 is fitted into the mounting recess 150. Also, as shown in Figure 1, if the cell holding cassette 200 and the solution exchange member 100 each have a first engaging portion 260 and a second engaging portion 160, the first engaging portion 260 and the second engaging portion 160 are engaged.

[0117] Then, in the solution exchange process, the cell holding cassette 200, which holds the first solution containing cells in the well 210, is attached between the suction capillary tube 110 of the solution exchange member 100 and the reservoir 120, thereby initiating the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force.

[0118] In other words, by attaching the cell holding cassette 200 to the solution exchange member 100 and connecting the well 210 of the cell holding cassette 200 with the suction capillary tube 110 of the solution exchange member 100, the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force is initiated.

[0119] Specifically, when the downstream channel 250, which holds the first solution of the cell holding cassette 200, is connected to the suction capillary tube 110, which holds the gas of the solution exchange member 100, the first solution begins to be drawn into the suction capillary tube 110 by capillary force.

[0120] In other words, simply by attaching the cell holding cassette 200 to the solution exchange unit 100, the aspiration of the first solution from the well 210 to the suction capillary tube 110 by capillary force is spontaneously initiated.

[0121] Furthermore, during the solution exchange process, the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force is initiated, which in turn initiates the inflow of the second solution from the reservoir 120 into the well 210.

[0122] In other words, by attaching the cell holding cassette 200 to the solution exchange member 100, the well 210 of the cell holding cassette 200 and the reservoir 120 of the solution exchange member 100 are in communication, and as described above, the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force is initiated, which also initiates the inflow of the second solution from the reservoir 120 to the well 210.

[0123] Specifically, the upstream channel 240 in which the first solution of the cell holding cassette 200 is held and the outflow channel 140 in which the second solution of the solution exchange member 100 is held are connected, and the well 210 of the cell holding cassette 200 is sealed except that it is in communication with the reservoir 120 and the suction capillary tube 110 via the upstream channel 240 and the downstream channel 250, respectively. As described above, when the first solution is drawn from the well 210 to the suction capillary tube 110 by capillary force, the second solution begins to flow from the reservoir 120 into the well 210.

[0124] In other words, simply by attaching the cell holding cassette 200 to the solution exchange unit 100, in addition to the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force, the inflow of the second solution from the reservoir 120 into the well 210 also starts spontaneously.

[0125] In this solution exchange process, the first solution is drawn from the well 210 to the suction capillary tube 110 by capillary force, and the second solution is introduced from the reservoir 120 into the well 210, thereby initiating the exchange of the solution within the well 210.

[0126] During the solution exchange process, as long as the solution continues to be drawn from the well 210 to the suction capillary tube 110 by capillary force, the second solution will continue to flow from the reservoir 120 into the well 210.

[0127] Furthermore, in the solution exchange process, after the solution exchange is initiated as described above, as shown in Figure 4(C), the suction from the well 210 to the suction capillary tube 110 by capillary force is stopped while the cells and solution are held in the well 210.

[0128] In other words, for example, as described above, if the suction capillary tube 110 has a volume smaller than the total volume of the first and second solutions at the start of solution exchange and has a downstream end 112 that opens into the gas phase, then in the solution exchange process, the suction by capillary force is stopped when the volume of solution smaller than the total volume of the first and second solutions at the start of solution exchange, which has been drawn from the well 210 to the suction capillary tube 110 by capillary force, reaches the downstream end 112 of the suction capillary tube 110. Furthermore, when the suction of solution from the well 210 to the suction capillary tube 110 by capillary force is stopped, the inflow of the second solution from the reservoir 120 to the well 210 is also stopped.

[0129] Thus, in the solution exchange process, the solution exchange is spontaneously stopped before the solution in the well 210 containing the cells is completely aspirated. In other words, during the solution exchange process, the solution is retained in the well 210 containing the cells from the time the solution exchange is started until it is stopped, and ultimately, the well 210 containing the cells and the solution after the solution exchange is obtained.

[0130] As described above, if the suction capillary tube 110 has a volume smaller than the total volume of the first solution and the second solution at the start of solution exchange, the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process may be, for example, 0.99 times or less, 0.95 times or less, 0.9 times or less, 0.8 times or less, 0.7 times or less, or 0.6 times or less of the total volume of the first solution and the second solution at the start of solution exchange. Also, the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process may be, for example, 0.1 times or more, 0.2 times or more, 0.3 times or more, or 0.4 times or more of the total volume of the first solution and the second solution at the start of solution exchange. The ratio of the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process to the total volume of the first solution and the second solution at the start of solution exchange may be determined by any combination of the above lower limit and the above upper limit.

[0131] In the solution exchange process, the volume of solution drawn from the well 210 to the suction capillary 110 may be greater than or equal to the volume of the second solution at the start of the solution exchange. In this case, all of the second solution held in the reservoir 120 at the start of the solution exchange can be used for the solution exchange.

[0132] Specifically, the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process may be, for example, 1.1 times or more, or 1.2 times or more, the volume of the second solution at the start of aspiration. Also, the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process may be, for example, 10 times or less, 8 times or less, 6 times or less, 4 times or less, or 2 times or less, the volume of the second solution at the start of aspiration. The ratio of the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process to the volume of the second solution at the start of aspiration may be determined by any combination of the above lower limit and the above upper limit.

[0133] In the solution exchange process, the volume of solution drawn from the well 210 to the suction capillary 110 may be greater than or equal to the volume of the first solution at the start of the solution exchange, or it may be less than or equal to the volume of the first solution at the start of the solution exchange. In other words, in the solution exchange, the entire amount of the first solution at the start of the solution exchange may be exchanged, or only a portion of the first solution at the start of the solution exchange may be exchanged.

[0134] Specifically, the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process may be, for example, 0.1 times or more, 0.2 times or more, 0.4 times or more, 0.6 times or more, 0.8 times or more, 1 time or more, 2 times or more, or 3 times or more, of the volume of the first solution at the start of the solution exchange. Furthermore, the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process may be, for example, 10 times or less, 8 times or less, 6 times or less, or 5 times or less, of the volume of the first solution at the start of the solution exchange. The ratio of the volume of solution drawn from the well 210 to the suction capillary tube 110 during the solution exchange process to the volume of the first solution at the start of the solution exchange may be determined by any combination of the above lower limit and the above upper limit.

[0135] Furthermore, in the solution exchange process, it is preferable that the number of cells retained in well 210 when the solution exchange is stopped (hereinafter referred to as the "number of cells at the time of stopping the solution exchange") does not decrease significantly from the number of cells retained in well 210 before the start of the solution exchange (hereinafter referred to as the "number of cells at the start of the solution exchange"), and is particularly preferable that it be maintained at approximately the same level.

[0136] In other words, in the solution exchange process, the ratio of the number of cells at the end of the solution exchange to the number of cells at the start of the solution exchange may be, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 85% or more, and particularly preferably 90% or more.

[0137] Thus, with this device 1 and method, the solution exchange in the wells 210 holding cells in the cell-holding cassette 200 can be effectively achieved by simply attaching the cell-holding cassette 200 to the solution exchange member 200.

[0138] The cell holding cassette 200 of this device 1 may be configured to be attachable to the solution exchange member 100 and removable after attachment. The configuration for making the cell holding cassette 200 attachable to the solution exchange member 100 and removable after attachment is not particularly limited as long as the effects of the present invention are obtained. For example, the cell holding cassette 200 and the solution holding member 100 may be configured so that one can engage with the other and can be separated after engagement.

[0139] Specifically, for example, as shown in Figures 1 and 2, if the solution exchange member 100 has a mounting recess 150, the cell holding cassette 200 may be configured to be attachable by fitting it into the mounting recess 150 and then removable by removing it from the mounting recess 150. Also, the first engaging portion 260 of the cell holding cassette 200 and the second engaging portion 160 of the solution exchange member 100 shown in Figure 1 may be configured to be engageable with each other and detachable after engagement.

[0140] If the cell holding cassette 200 of the device 1 is configured to be attachable to the solution exchange member 100 and detachable after attachment, the method may further include removing the cell holding cassette 200, in which the cells and the third solution after solution exchange are held in the wells 210, from the solution exchange member 100 after the solution exchange in the solution exchange step has been stopped, as shown in Figure 4(D).

[0141] In this case, the method may further include, for example, a culture step in which cells are cultured in the wells 210 of the cell holding cassette 200, which has been removed from the solution exchange member 100 after the solution exchange in the solution exchange step has been stopped as described above. That is, as one aspect, the method also includes a cell culture method that includes a preparation step, a solution exchange step, and a culture step. Cell culture in the culture step is preferably carried out, for example, by holding the cell holding cassette 200, which holds the third solution containing cells in the wells 210, in an incubator. In the cell culture method, the first solution and the second solution are preferably culture media, and it is particularly preferable that at least the second solution is a culture media.

[0142] The device 1 may include a plurality of solution exchange members 100 and a cell holding cassette 200 that can be attached to the solution exchange members 100 and can be removed after attachment. In this case, the plurality of solution exchange members 100 may have suction capillaries 110 and reservoirs 120 of the same shape, or they may have suction capillaries 110 and / or reservoirs 120 of different shapes.

[0143] Furthermore, if the device 1 includes a plurality of solution exchange members 100, including a first solution exchange member 100(I) and a second solution exchange member 100(II), and a cell holding cassette 200 that can be attached to the solution exchange member 100 and can be removed after attachment, the method may be carried out as a first preparation step and a first solution exchange step using the first solution exchange member 100(I), respectively, and may also include a second preparation step and a second solution exchange step using the second solution exchange member 100(II).

[0144] In other words, in this case, as shown in Figure 5(A), first in the first preparation step, a cell holding cassette 200 is prepared that holds the first solution containing cells in the well 210 and is not attached to the solution exchange member 100, and a first solution exchange member 100(I) is prepared that holds the second solution in the reservoir 120 and is not attached to the cell holding cassette 200.

[0145] Next, after the first preparation step, in the first solution exchange step, as shown in Figure 5(B), the cell holding cassette 200 is attached between the suction capillary tube 110 of the first solution exchange member 100(I) and the reservoir 120, thereby initiating solution exchange by the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force and the inflow of the second solution from the reservoir 120 into the well 210. Subsequently, as shown in Figure 5(C), with the cells and the third solution after solution exchange held in the well 210, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force is stopped, thereby stopping the solution exchange.

[0146] Furthermore, in the subsequent second preparation step, as shown in Figure 5(D), after the solution exchange in the first solution exchange step is stopped, the cell holding cassette 200 is removed from the first solution exchange member 100(I), and as shown in Figure 5(E), the cell holding cassette 200 is prepared, holding the cells and the third solution in the well 210 and not attached to the solution exchange member 100. At the same time, the second solution exchange member 100(II) is prepared, holding the fourth solution in the reservoir 120 and not attached to the cell holding cassette 200.

[0147] Here, the fourth solution held in the reservoir 120 during the second preparation step is preferably an aqueous solution having a different composition from the third solution. However, the composition of the fourth solution may be adjusted as appropriate depending on the purpose. Furthermore, the volume of the fourth solution held in the reservoir 120 may be the same as or different from the volume of the second solution held in the reservoir 120 during the first preparation step.

[0148] Then, after the second preparation step, in the second solution exchange step, as shown in Figure 5(F), the cell holding cassette 200 is attached between the suction capillary tube 110 of the second solution exchange member 100(II) and the reservoir 120, thereby initiating solution exchange by the suction of the third solution from the well 210 to the suction capillary tube 110 by capillary force and the inflow of the fourth solution from the reservoir 120 to the well 210. Subsequently, as shown in Figure 5(G), with the cells and the fifth solution after solution exchange held in the well 210, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force is stopped, thereby stopping the solution exchange.

[0149] Furthermore, if the multiple solution exchange members 100 of the device 1 further include a third solution exchange member 100(III) (not shown), the method further includes a third preparation step and a third solution exchange step using the third solution exchange member 100(III) instead of the second solution exchange unit 100(II).

[0150] In other words, in the third preparation step following the second solution exchange step, after the solution exchange in the second solution exchange step is stopped, the cell holding cassette 200 is removed from the second solution exchange member 100(II), and the cells and fifth solution are held in the well 210, and the cell holding cassette 200 that is not attached to the solution exchange member 100 is prepared, and the sixth solution is held in the reservoir 120, and the third solution exchange member 100(III) that does not have the cell holding cassette 200 attached is prepared.

[0151] Here, the sixth solution held in the reservoir 120 in the third preparation step is preferably an aqueous solution having a different composition from the fifth solution. However, the composition of the sixth solution may be adjusted as appropriate depending on the purpose. Furthermore, the volume of the sixth solution held in the reservoir 120 may be the same as, or different from, the volume of the second solution held in the reservoir 120 in the first preparation step and / or the volume of the fourth solution held in the reservoir 120 in the second preparation step.

[0152] Then, after the third preparation step, in the third solution exchange step, the cell holding cassette 200 is attached between the suction capillary tube 110 of the third solution exchange member 100(III) and the reservoir 120, thereby initiating solution exchange by the suction of the fifth solution from the well 210 to the suction capillary tube 110 by capillary force and the inflow of the sixth solution from the reservoir 120 to the well 210. Subsequently, with the cells and the seventh solution after the solution exchange held in the well 210, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force is stopped, thereby stopping the solution exchange.

[0153] If the device 1 includes four or more solution exchange members 100, the same preparation steps as the second and third preparation steps described above, and the same solution exchange steps as the second and third solution exchange steps may be repeated using the fourth and subsequent solution exchange members 100. With this method, multiple solution exchanges can be easily performed for a single cell holding cassette 200 well 210 while retaining cells in the well 210.

[0154] The device 1 may include a plurality of cell-holding cassettes 200 and a plurality of solution exchange members 100 used for exchanging the solutions in the plurality of cell-holding cassettes 200. In this case, the method may include a preparation step and a solution exchange step using the plurality of solution exchange members 100 for each of the plurality of cell-holding cassettes 200. That is, for each of the plurality of cell-holding cassettes 200, the solution exchange using the above-described preparation step and solution exchange step may be performed only once, or two or more solution exchanges may be performed using the same steps as the second preparation step and second solution exchange step described above.

[0155] Furthermore, the multiple solution exchange members 100 may have suction capillaries 110 and reservoirs 120 of the same shape, or they may have suction capillaries 110 and / or reservoirs 120 of different shapes. Also, the multiple cell holding cassettes 200 may have wells 210 of the same shape, or they may have wells 210 of different shapes.

[0156] Specifically, for example, if the device 1 includes a plurality of solution exchange members 100, including a first solution exchange member 100(I) and a second solution exchange member 100(II), and a plurality of cell holding cassettes 200, including a first cell holding cassette 200(I) and a second cell holding cassette 200(II), then in the preparation step of this method, as shown in Figure 6(A), the first cell holding cassette 200(I) and the second cell holding cassette 200(II), which hold the first solution containing cells in the well 210 and are not attached to the solution exchange member 100, are prepared, as well as the first solution exchange member 100(I) and the second solution exchange member 100(II), which hold the second solution in the reservoir 120 and are not attached to the cell holding cassette 200.

[0157] Here, the first cell-holding cassette 200(I) and the second cell-holding cassette 200(II) may have the same or different types or numbers of cells held in the well 210, and / or the same or different compositions or amounts of the first solution held in the well 210. Also, the first solution exchange member 100(I) and the second solution exchange member 100(II) may have the same or different compositions or amounts of the second solution held in the reservoir 120.

[0158] Next, after the preparation step, in the solution exchange step, as shown in Figure 6(B), the first cell holding cassette 200(I) and the second cell holding cassette 200(II) are attached to the first solution exchange member 100(I) and the second solution exchange member 100(II), respectively, thereby initiating the solution exchange of each well 210. Subsequently, as shown in Figure 6(C), the solution exchange is stopped while the cells and solution are retained in the well 210.

[0159] If the device 1, which includes multiple cell-holding cassettes 200, includes a number of solution exchange members 100 greater than the number of cell-holding cassettes 200, then multiple solution exchanges can be easily performed for one or more of the cell-holding cassettes 200 as described above. With this method, solution exchange can be easily performed for each well 210 of the multiple cell-holding cassettes 200.

[0160] In the examples shown in Figures 5 and 6, multiple solution exchange members 100 are configured as separate components, and multiple cell holding cassettes 100 are configured as separate components. However, the invention is not limited to these configurations. For example, multiple solution exchange members 100 may be configured to have a common base 130 and multiple pairs of suction capillaries 110 and reservoirs 120 formed spaced apart on the base 130, and / or multiple cell holding cassettes 200 may be configured to have a common base 220 and multiple wells 210 formed spaced apart on the base 220.

[0161] Device 1 and the method can be used for a variety of applications. Specifically, Device 1 and the method can be used, for example, to evaluate the response of cells to a target substance or to screen for substances that cause changes in cells. Furthermore, Device 1 and the method can also be used, for example, to produce genetically modified cells.

[0162] In other words, this method, as one aspect, includes a method for producing gene-transformed cells using the device 1 (hereinafter referred to as "this manufacturing method"). This manufacturing method includes a gene introduction step in addition to the preparation step and solution exchange step described above.

[0163] In the preparation step of this manufacturing method, as shown in Figure 4(A), a cell holding cassette 200 that holds a first solution containing cells in a well 210 and is not attached to the solution exchange member 100, and a solution exchange member 100 that holds a second solution for gene transfer containing nucleic acids to be introduced into the cells in a reservoir 120 and is not attached to the cell holding cassette 200 are prepared.

[0164] Here, the cells targeted for gene transfer (transfection) are not particularly limited as long as they are cells that can be subjected to nucleic acid using the second solution. The second solution is not particularly limited as long as it is an aqueous solution for gene transfer that contains nucleic acid to be introduced into the cells in well 210 and has a composition that allows the nucleic acid to be introduced into the cells by holding the cells in the second solution. For example, a solution prepared using a commercially available gene transfer kit is preferably used. Specifically, the second solution for gene transfer preferably contains, for example, nucleic acid (DNA and / or RNA) to be introduced into the cells and a cationic lipid.

[0165] In the solution exchange step of this manufacturing method, after the preparation step, as shown in Figure 4(B), the cell holding cassette 200 is attached between the suction capillary tube 110 of the solution exchange member 100 and the reservoir 120, thereby initiating solution exchange by the suction of the first solution from the well 210 to the suction capillary tube 110 by capillary force and the inflow of the second solution containing nucleic acids from the reservoir 120 to the well 210. Subsequently, as shown in Figure 4(C), the solution exchange is stopped when the suction of the solution from the well to the suction capillary tube 110 by capillary force is stopped while the cells and the solution containing nucleic acids are held in the well 210.

[0166] Furthermore, the solution retained in the well 210 after the solution exchange in the solution exchange process contains nucleic acids to be introduced into the cells retained in the well 210 as a result of the aspiration of the first solution from the well 210 and the inflow of the second solution into the well 210, and has a composition that allows for the introduction of the nucleic acids into the cells by retaining the cells in the solution.

[0167] In the subsequent gene transfer step, after the solution exchange step, the cells are held in a solution containing nucleic acid in the well 210 of the cell holding cassette 200, thereby introducing the nucleic acid into the cells and obtaining gene-transferred cells into which the nucleic acid has been introduced.

[0168] Cell retention during the gene transfer process is carried out, for example, by placing the cell retention cassette 200 in an incubator. The time for which cells are retained in the nucleic acid-containing solution during the gene transfer process is not particularly limited, as long as it is the time necessary to introduce the nucleic acid into the cells.

[0169] The gene transfer process may be carried out by holding cells in the wells 210 of the cell holding cassette 200 while it remains attached to the solution exchange member 100. However, if the cell holding cassette 200 is configured to be attachable to the solution exchange member 100 and detachable after attachment, it is preferable to carry out the process by holding cells in the wells 210 of the cell holding cassette 200 after it has been removed from the solution exchange member 100.

[0170] In other words, in the gene transfer process, as shown in Figure 4(D), after the solution exchange, it is preferable to remove the cell holding cassette 200, in which the solution containing cells and nucleic acids for gene transfer is held in the wells 210, from the solution exchange member 100, and to carry out gene transfer by holding the cells in the solution containing the nucleic acids in the wells 210 of the removed cell holding cassette 200 that is not attached to the solution exchange member 100.

[0171] Furthermore, if the device 1 includes a plurality of solution exchange members 100, including a first solution exchange member 100(I) and a second solution exchange member 100(II), and a cell holding cassette 200 that can be attached to the solution exchange member 100 and can be removed after attachment, the manufacturing method may include, as the above-described preparation step, solution exchange step, and gene introduction step, a first preparation step, a first solution exchange step, and a gene introduction step using the first solution exchange member 100(I), and may further include a second preparation step and a second solution exchange step using the second solution exchange member 100(II).

[0172] In other words, in this case, in the second preparation step, after performing the gene transfer step as described above using the first solution exchange member 100(I), the third solution containing the gene-transferred cells is held in the well 210, and a cell holding cassette 200 is prepared that has been removed from the first solution exchange member 100(I) and is not attached to the solution exchange member 100.

[0173] In this case, if the gene transfer process is carried out by holding cells in the wells 210 of the cell holding cassette 200 which remains attached to the first solution exchange member 100, the cell holding cassette 200 is removed from the first solution exchange member 100(I) after the gene transfer process.

[0174] On the other hand, when the gene transfer process is carried out by holding cells in the wells 210 of a cell holding cassette 200 removed from the solution exchange member 100, the cell holding cassette 200 is removed from the first solution exchange member 100(I) before or during the gene transfer process. In other words, in this case, the preparation of the solution exchange member 100 in the second preparation step and the gene transfer process are carried out in parallel.

[0175] In the second preparation step, a second solution exchange member 100(II) is also prepared, which holds the fourth solution in the reservoir 120 and does not have the cell holding cassette 200 attached. The fourth solution held in the reservoir 120 in the second preparation step is preferably an aqueous solution having a different composition from the third solution. However, the composition of the fourth solution may be adjusted as appropriate depending on the purpose. Specifically, the fourth solution may not contain nucleic acids contained in the third solution (nucleic acids that remain in the solution after the gene transfer step without being introduced into the cells). Also, the volume of the fourth solution held in the reservoir 120 may be the same as or different from the volume of the second solution held in the reservoir 120 in the first preparation step.

[0176] Then, after the second preparation step, in the second solution exchange step, as shown in Figure 5(F), the cell holding cassette 200 is attached between the suction capillary tube 110 of the second solution exchange member 100(II) and the reservoir 120, thereby initiating solution exchange by the suction of the third solution from the well 210 to the suction capillary tube 110 by capillary force and the inflow of the fourth solution from the reservoir 120 to the well 210. Subsequently, as shown in Figure 5(G), with the gene-transformed cells and solution held in the well 210, the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force is stopped, thereby halting the solution exchange.

[0177] Thus, this manufacturing method allows for the simple production of genetically modified cells without the need to perform the complicated procedures required in conventional methods of producing genetically modified cells. Although not specifically described in this manufacturing method, various aspects of the solution exchange method described above can also be applied to this manufacturing method.

[0178] This manufacturing method can be used to produce any gene-modified cells, but is preferably used for the production of sensor cells, for example. Sensor cells are cells that express receptors (for example, insect olfactory receptors) through gene transfer, and are used as one type of stoichiometric sensor that utilizes receptors.

[0179] Device 1 has a simple structure. That is, as described above, by attaching the cell holding cassette 200 to the solution exchange member 100, the solution exchange starts spontaneously and then stops spontaneously, so there is no need to connect to an external pump for circulating the solution (for example, an electrically driven pump such as a syringe pump or peristaltic pump).

[0180] Therefore, this device 1 can be used without being connected to an external pump. Furthermore, in this method, this device 1 can be used without being connected to an external pump. Because this device 1 is a pumpless microdevice, solution exchange can be easily performed without using complex equipment.

[0181] Furthermore, since this device 1 is configured such that, for example, by attaching the cell holding cassette 200 to the solution exchange member 100, the wells 210 of the cell holding cassette 210 and the suction capillaries 110 and reservoir 120 of the solution exchange member 100 are connected and communicated as a series of flow paths, there is no need for tubes (for example, fluororesin tubes or silicone tubes) to connect each component and circulate the solution.

[0182] Therefore, the device 1 can be a microdevice without tubes. Furthermore, the device 1 without tubes can be used in this method. Because the device 1 is a tubeless microdevice, solution exchange can be easily performed without requiring a complex structure.

[0183] Furthermore, in this method, the solution exchange of the well 210 can be achieved using only the solution held in the well 210 of the cell holding cassette 200 and the solution held in the reservoir 120 of the solution exchange member 100 before the solution exchange begins.

[0184] Therefore, this method can be carried out, for example, without supplying a solution to the device 1 from an external source, from the start of solution exchange by attaching the cell holding cassette 200 to the solution exchange member 100 until the stop of said solution exchange.

[0185] Furthermore, this method can be performed, for example, from the start of solution exchange by attaching the cell holding cassette 200 to the solution exchange member 100 until the stop of said solution exchange, without discharging any solution from the device 1 to the outside.

[0186] Next, specific examples of this embodiment will be described. [Examples]

[0187] [Preparation of cell retention cassettes] The first substrate 221 was cut from a 3mm thick acrylic sheet (Acrylite, Mitsubishi Chemical) using an NC cutting machine (MM-100, Modia Systems). The second substrate 222 was then cut from a 1.0mm thick acrylic sheet using the same NC cutting machine. Finally, the first substrate 221 and the second substrate 222 were heat-pressed together using a heat press machine (TM-1955-10000, Totem).

[0188] Next, using a plasma etching apparatus (FA-1, Samco), the well bottom surface 211 (acrylic resin surface) of the cell holding cassette 200 was subjected to oxygen plasma treatment (output 25W, oxygen gas flow rate 20mL / min, 30 seconds) to make the well bottom surface 211 hydrophilic.

[0189] Furthermore, cell adhesion properties were imparted to the hydrophilic well bottom surface 211. Specifically, first, an aqueous solution containing 0.1 mg / mL of poly-D-lysine (Sigma-Aldrich) was uniformly dropped onto the well bottom surface 211. Then, after 5 minutes, the poly-D-lysine aqueous solution in the well 210 was aspirated and removed. After that, the well bottom surface 211 was thoroughly washed with ultrapure water and dried for 2 hours. In this way, a cell holding cassette 200 having a well 210 and an upstream channel 240 and a downstream channel 250 was obtained, as shown in Figure 3(A).

[0190] [Fabrication of solution exchange components] The first substrate 131 was cut from a 3mm thick acrylic sheet (Acrylite, Mitsubishi Chemical) using an NC cutting machine (MM-100, Modia Systems). The second substrate 132 was then cut from a 4.0mm thick acrylic sheet using the same NC cutting machine. Finally, the first substrate 131 and the second substrate 132 were heat-pressed together using a heat press machine (TM-1955-10000, Totem).

[0191] Next, using a plasma etching apparatus (FA-1, Samco), the inner surface (acrylic resin surface) of the grooves constituting the suction capillary tube 110 formed on the first substrate 131 was subjected to oxygen plasma treatment (output 25W, oxygen gas flow rate 20mL / min, 15 seconds) to make the inner surface hydrophilic.

[0192] On the other hand, the surface of the mounting recess 150 formed on the first substrate 131 (including the outer surface 130b where the downstream end of the outflow channel 140 opens, and the outer surface 130c where the upstream end of the suction capillary tube 110 opens) was treated to hydrophobicity by applying a water-repellent coating agent (SF Coat SFE-B002H:SFE Solvent=1:1, AGC Seimi Chemical).

[0193] Subsequently, a sealing member 133 made of polypropylene sealing tape (microplate sealing tape 9795, 3M) was applied to the first substrate 131 so as to cover the opening above the groove formed therein, and the suction capillary tube 110 was formed by pressing it tightly against the substrate. A hole with a diameter of 2 mm was formed in the portion of the sealing tape located above the downstream end 112 of the suction capillary tube 110, thereby opening the downstream end into the gas phase. In this way, a solution exchange member 100 having a reservoir 120 and a suction capillary tube 110 was obtained, as shown in Figure 3(B).

[0194] [Manufacturing of genetically modified cells] Gene-transformed cells were produced using the device 1 manufactured as described above. Specifically, HEK293 (Human Embryonic Kidney 293) cells were used as the cells into which the genes were introduced. By introducing genes into these HEK293 cells, sensor cells were produced that co-expressed the insect olfactory receptor OR8 (Olfactory receptor 8), the olfactory receptor co-receptor Orco (Olfactory receptor co-receptor), and the calcium-sensitive fluorescent protein GCaMP. The device 1 consisted of one cell-holding cassette 200 and two solution exchange members 100 (first solution exchange member 100(I) and second solution exchange member 100(II)).

[0195] First, place 0.5 × 10⁶ HEK293 cells into well 210 of cell retention cassette 200. 6 The HEK293 cells were seeded into well 210 by adding 72 μL of a cell suspension containing cells / mL at a density of 10 cells / mL. The seeded HEK293 cells settled and adhered to the bottom surface 211 of well 210. Culture medium was also retained in the upstream channel 240 and downstream channel 250 of the cell retention cassette 200.

[0196] After seeding HEK293 cells, a sealing member 230 made of polypropylene sealing tape (microplate sealing tape 9795, 3M) with 2 mm diameter ventilation holes 215 was attached to the upper surface 220a of the base 220 of the cell holding cassette 200, so as to cover the upper opening of the well 210.

[0197] Subsequently, to prevent drying of well 210, the cell retention cassette 200 was placed in a commercially available plastic cell culture dish along with a cleanroom wiper soaked in ultrapure water. The plastic dish was then covered and placed in an incubator at 37°C with a CO2 concentration of 5%, and static culture was performed for 24 hours.

[0198] Next, gene transfer (transfection) was performed. For gene transfer, a commercially available transfection reagent, Lipofectamine LTX Reagent with PLUS Reagent (Thermo Fisher Scientific), was used.

[0199] Specifically, the mixture was prepared by adding 0.5 μg and 1 μg of Aedes aegypti-derived OR8 DNA and Orco DNA, respectively, and 2.5 μg of GCaMP DNA to 10 μL of Lipofectamine LTX Reagent, 800 μL of Opti-MEM I Reduced Serum medium, and 8 μL of PLUS Reagent. Then, 60 μL of the resulting mixture was diluted with 220 μL of culture medium (DMEM high glucose) to prepare the transfection solution.

[0200] Then, 280 μL of transfection solution was placed in the reservoir 120 of the first solution exchange member 100(I). As a result, the transfection solution was retained in the reservoir 120 and the outflow channel 140 of the first solution exchange member 100(I). In addition, the suction capillary tube 110 of the first solution exchange member 100(I) was filled with air.

[0201] Meanwhile, in the cell holding cassette 200, the ventilation holes 215 formed in the sealing member 230 covering the top of the well 210 were sealed with another propylene sealing tape, thereby sealing the well 210. As a result, in the well 210, the HEK293 cells adhered to the bottom surface 211 were held in the solution, and a gas phase consisting of air was sealed between the liquid surface of the solution and the top surface 214.

[0202] The cell-holding cassette 200 was then attached to the first solution exchange member 100(I) at a position between the suction capillary tube 110 and the reservoir 120. By attaching the cell-holding cassette 200 to the first solution exchange member 100(I), the aspiration of culture medium from the well 210 to the suction capillary tube 110 by capillary force spontaneously began, and the inflow of transfection solution from the reservoir 120 into the well 210 also began.

[0203] Subsequently, the aspirated solution reaches the downstream end 112 of the suction capillary tube 110, and as the suction capillary tube 110 is filled with the solution, the aspiration of the solution from the well 210 to the suction capillary tube 110 by capillary force spontaneously stops, with the cells and solution being held in the well 210, and the inflow of the transfection solution from the reservoir 120 to the well 210 also stops.

[0204] Thus, by simply attaching the cell holding cassette 200 to the first solution exchange member 100(I), solution exchange within the well 210 containing the cells was achieved. At the point when the solution exchange was stopped, almost no transfection solution remained in the reservoir 210. Meanwhile, approximately the same volume of solution as at the start of the solution exchange was retained in the well 210.

[0205] In the solution exchange described above, from a hydrodynamic standpoint, the culture medium is aspirated from well 210 to suction capillary tube 110 by capillary force, and the transfection solution flows from reservoir 120 to well 210 at a flow rate of approximately 5 μL / second. The shape of device 1 and the volume of solution held are set so that approximately 93% of the culture medium held in well 210 before the start of the solution exchange is replaced with the transfection solution approximately 205 seconds after the start of the solution exchange, while maintaining the viability of the cells in well 210.

[0206] Subsequently, the cell retention cassette 200 was removed from the first solution exchange member 100(I), and the ventilation holes 215 of the sealing member 133 covering the well 210 were opened. Then, the cell retention cassette 200 was left to stand in the incubator again for 24 hours to perform gene transfer to the HEK293 cells in the well 210.

[0207] Subsequently, 280 μL of observation buffer solution was added to the reservoir 120 of the second solution exchange member 100(II). As a result, the observation buffer solution was retained in the reservoir 120 and the outflow channel 140 of the second solution exchange member 100(II). In addition, the suction capillary tube 110 of the second solution exchange member 100(II) was filled with air.

[0208] On the other hand, in the cell holding cassette 200, the ventilation holes 215 formed in the sealing member 133 covering the top of the well 210 were sealed with another propylene sealing tape, thereby sealing the well 210. As a result, in the well 210, the gene-transformed cells attached to the bottom surface 211 were held in the solution, and a gas phase consisting of air was sealed between the liquid surface of the solution and the top surface 214.

[0209] The cell-holding cassette 200 was then attached to the second solution exchange member 100(II) between the suction capillary tube 110 and the reservoir 120. By attaching the cell-holding cassette 200 to the second solution exchange member 100(II), the suction of the solution from the well 210 to the suction capillary tube 110 by capillary force spontaneously began, and the inflow of the observation buffer solution from the reservoir 120 into the well 210 also began.

[0210] Subsequently, the aspirated solution reaches the downstream end 112 of the suction capillary tube 110, and as the suction capillary tube 110 is filled with the solution, the aspiration of the solution from the well 210 to the suction capillary tube 110 by capillary force spontaneously stops, with the cells and solution being held in the well 210, and the inflow of the observation buffer solution from the reservoir 120 into the well 210 also stops.

[0211] Thus, by simply attaching the cell holding cassette 200 to the second solution exchange member 100(II), the solution exchange within the well 210 was achieved. At the point when the solution exchange was stopped, almost no observation buffer solution remained in the reservoir 120. Meanwhile, approximately the same volume of solution as at the start of the solution exchange was retained in the well 210.

[0212] Based on the above, it was possible to introduce genes into HEK293 cells and produce sensor cells through a simple operation using this device 1, which consists of one cell-holding cassette 200 and two solution exchange members 100.

[0213] [Evaluation of Odor Substance Responsiveness of Sensor Cells] The odor substance responsiveness of the sensor cells prepared as described above was evaluated. 1-octen-3-ol was used as the odor substance. Specifically, a 500 mM stock solution was prepared by dissolving 1-octen-3-ol in DMSO and stored at -20°C. For observation, this stock solution was diluted with Hanks' BSS (Balanced Salt Solution) to prepare an additive solution containing 500 μM 1-octen-3-ol, which was then used.

[0214] First, the cell holding cassette 200, which held the observation buffer solution in the well 210 by solution exchange using the second solution exchange member 100(II) described above, was incubated in an incubator for 30 minutes.

[0215] Subsequently, the cell-holding cassette 200, removed from the incubator, was placed on the stage of an inverted microscope (IX71N, Olympus), and an amount of additive solution was added to the observation buffer solution in well 210 to obtain a final concentration of odorant (1-octen-3-ol) of 100 μM.

[0216] Then, time-lapse photography was performed using a microscope to record the change in fluorescence intensity in the well over 2 minutes at 0.4-second intervals following the addition of the odorant. A NIBA filter with Blue excitation (excitation wavelength: 470-490 nm) and Green detection (515-550 nm) was used.

[0217] Furthermore, as Comparative Example 1, the time course of fluorescence intensity in the wells was recorded in the same manner except that only DMSO (0.2% solution) was added instead of the odor substance (1-octen-3-ol). Furthermore, as Comparative Example 2, the time course of fluorescence intensity in the wells after the addition of the target substance (1-octen-3-ol) was recorded in the same manner except that a commercially available glass-bottom dish was used instead of Device 1, and sensor cells prepared using a conventional pipetting method were used instead of solution exchange using Device 1.

[0218] Figure 7 shows the results of measuring the change in fluorescence intensity over time. As shown in Figure 7, the sensor cells produced by solution exchange using this device 1 (Example) showed an increase in fluorescence intensity after the addition of 1-octen-3-ol to the well, similar to the sensor cells produced by conventional solution exchange using a pipette (Comparative Example 2).

[0219] This result suggests that the added 1-octen-3-ol binds to the olfactory receptor AaOR8 expressed by the sensor cells, causing calcium ions from the culture medium to flow into the cytoplasm of the sensor cells. The binding of these calcium ions to the calcium-sensitive fluorescent protein GCaMP in the cytoplasm then increased the fluorescence intensity. On the other hand, in Comparative Example 1, where DMSO was added instead of 1-octen-3-ol, no increase in fluorescence intensity was observed after adding 1-octen-3-ol to the wells; only fading over time was observed. Therefore, it was confirmed that sensor cells responsive to odor substances could be produced by gene transfer using the simple procedure of this device 1.

Claims

1. A microdevice comprising a solution exchange member and a cell holding cassette that can be attached to the solution exchange member, The cell-holding cassette has a base and wells formed in the base for holding a first solution containing cells, The solution exchange member comprises a suction capillary tube for drawing in the first solution held in the well by capillary force, and a reservoir for holding the second solution to be flowed into the well. The cell-holding cassette is An upstream channel connecting the well and the reservoir, wherein the reservoir-side end opens to a part of the outer surface of the base and the well-side end opens to the inner surface of the well, and / or A downstream channel connecting the well and the suction capillary, wherein the end on the well side opens to the inner surface of the well, and the end on the suction capillary side opens to a part of the outer surface of the base, It has, The cell-holding cassette, which holds the first solution containing the cells in the well, is installed between the suction capillary of the solution exchange member and the reservoir holding the second solution. This initiates solution exchange through the suction of the first solution from the well to the suction capillary by capillary force and the inflow of the second solution from the reservoir to the well. Subsequently, the solution exchange is stopped when the suction of the solution from the well to the suction capillary by capillary force is stopped while the cells and solution are held in the well. Microdevices.

2. The suction capillary has a volume smaller than the sum of the volume of the first solution held in the well of the cell holding cassette before it is attached to the solution exchange member and the volume of the second solution held in the reservoir of the solution exchange member before it is attached to the cell holding cassette, and has a downstream end that opens into the gas phase. After the start of the solution exchange, the suction of the solution from the well to the suction capillary tube by capillary force is stopped when the solution drawn from the well by capillary force reaches the downstream end of the suction capillary tube. The microdevice according to claim 1.

3. The cell holding cassette is configured to be attachable to the solution exchange member and to be removable after attachment. The microdevice according to claim 1 or 2.

4. The cell retention cassette includes a plurality of solution exchange members used for multiple solution exchanges of the cell retention cassette, The microdevice according to claim 3.

5. The system includes a plurality of cell-holding cassettes and a plurality of solution exchange members used for exchanging the solutions of the plurality of cell-holding cassettes. A microdevice according to any one of claims 1 to 4.

6. Used without being connected to an external pump, A microdevice according to any one of claims 1 to 5.

7. A solution exchange method using a microdevice according to any one of claims 1 to 6, Preparation steps include preparing a cell holding cassette that holds a first solution containing cells in the well and is not attached to the solution exchange member, and a solution exchange member that holds a second solution in the reservoir and is not attached to the cell holding cassette, After the preparation step, the cell holding cassette is attached between the suction capillary tube and the reservoir of the solution exchange member, thereby initiating solution exchange by capillary force drawing the first solution from the well to the suction capillary tube and flowing the second solution from the reservoir to the well. Subsequently, while the cells and solution are held in the well, the solution exchange is stopped by ceasing the capillary force drawing the solution from the well to the suction capillary tube, thus ending the solution exchange process. A method of solution exchange, including the exchange of solutions.

8. The suction capillary has a volume smaller than the sum of the volume of the first solution held in the well during the preparation step and the volume of the second solution held in the reservoir, and has a downstream end that opens into the gas phase. In the solution exchange step, when the solution drawn from the well by capillary force reaches the downstream end of the suction capillary tube, the suction of the solution from the well to the suction capillary tube by capillary force is stopped. The solution exchange method according to claim 7.

9. The cell holding cassette is configured to be attachable to the solution exchange member and to be removable after attachment. The process further includes removing the cell holding cassette, in which the cells and solution are held in the wells, from the solution exchange member after the solution exchange in the solution exchange step has been stopped, The solution exchange method according to claim 7 or 8.

10. The microdevice includes a plurality of solution exchange members, including a first solution exchange member, a second solution exchange member, and a third solution exchange member, and a cell holding cassette that can be attached to the solution exchange member and is removable after attachment. The preparation step and the solution exchange step include a first preparation step and a first solution exchange step using the first solution exchange member, and further, After the cessation of the solution exchange in the first solution exchange step, a second preparation step is performed, in which the cell holding cassette is removed from the first solution exchange member, the cell holding cassette is prepared to hold the cells and the third solution after the solution exchange in the well and is not attached to the solution exchange member, and the second solution exchange member is prepared to hold the fourth solution in the reservoir and is not attached to the cell holding cassette. After the second preparation step, the cell holding cassette is attached between the suction capillary tube and the reservoir of the second solution exchange member, thereby initiating solution exchange by capillary force drawing the third solution from the well to the suction capillary tube and flowing the fourth solution from the reservoir to the well. Subsequently, with the cells and the fifth solution after the solution exchange held in the well, the suction of the solution from the well to the suction capillary tube by capillary force is stopped, thereby halting the solution exchange in the second solution exchange step. The third preparation step is the same as the second preparation step, except that the third solution exchange member is used instead of the second solution exchange member. The third solution exchange process is the same as the second solution exchange process, except that the third solution exchange member is used instead of the second solution exchange member. The solution exchange method according to claim 9, including the method described in claim 9.

11. The microdevice includes a plurality of the solution exchange members and a plurality of the cell holding cassettes, Each of the plurality of cell holding cassettes includes the preparation step and the solution exchange step using the plurality of solution exchange members, A method for exchanging a solution according to any one of claims 7 to 10.

12. The microdevice is used without connecting it to an external pump. A method for exchanging a solution according to any one of claims 7 to 11.

13. A cell culture method using a microdevice according to any one of claims 1 to 6, The cell holding cassette is configured to be attachable to the solution exchange member and to be removable after attachment. Preparation steps include preparing a cell holding cassette that holds a first solution containing cells in the well and is not attached to the solution exchange member, and a solution exchange member that holds a second solution in the reservoir and is not attached to the cell holding cassette, After the preparation step, the cell holding cassette is attached between the suction capillary tube and the reservoir of the solution exchange member, thereby initiating solution exchange by capillary force drawing the first solution from the well to the suction capillary tube and flowing the second solution from the reservoir to the well. Subsequently, while the cells and solution are held in the well, the solution exchange is stopped by ceasing the capillary force drawing the solution from the well to the suction capillary tube, thus ending the solution exchange process. After the solution exchange in the solution exchange step is stopped, the cell holding cassette in which the cells and solution are held in the wells is removed from the solution exchange member, and the cells are cultured in the wells of the removed cell holding cassette. A cell culture method, including the following.

14. A method for producing gene-transformed cells using a microdevice according to any one of claims 1 to 6, Preparation steps include preparing a cell holding cassette that holds a first solution containing cells in the well and is not attached to the solution exchange member, and a solution exchange member that holds a second solution for gene transfer containing nucleic acids to be introduced into the cells in the reservoir and is not attached to the cell holding cassette, After the preparation step, the cell holding cassette is attached between the suction capillary tube and the reservoir of the solution exchange member, thereby initiating solution exchange by capillary force drawing the first solution from the well to the suction capillary tube and flowing the second solution from the reservoir to the well. Subsequently, with the cells and the solution containing the nucleic acid held in the well, the solution exchange is stopped when the capillary force drawing the solution from the well to the suction capillary tube is stopped, thus ending the solution exchange process. After the solution exchange step, the gene introduction step involves introducing the nucleic acid into the cells by holding the cells in the wells of the cell holding cassette in a solution containing the nucleic acid, thereby obtaining gene-transformed cells into which the nucleic acid has been introduced. A method for producing genetically modified cells, including

Images