Cell manipulation method

Abstract

To provide a novel technique for detaching adherent cells from a solid phase.SOLUTION: A cell manipulation method includes: a step of culturing cells in a liquid; a step of disposing a flow path through which a gas is able to be introduced in the liquid; a step of forming an air bubble at an end portion of the flow path; and a step of attaching the cells to the air bubble.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present invention relates to an apparatus and a method for manipulating an organism. [Background technology]

[0002] Conventionally, in cell culture, protease such as trypsin or collagenase is used to detach cells adhered to a culture vessel such as a petri dish. Protease cuts adhesion factors such as integrins that attach cells to the inner wall surface of the culture vessel, and adhesion factors such as cadherins that adhere cells to each other, and can detach cells adhered to the culture vessel. In addition, a method and device for detaching cells by locally heating a scaffold material and irradiating it with shock waves has also been proposed (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2017-112923 A

[0004] However, when using proteolytic enzymes, all cells in the container are detached and selective detachment is not possible. In addition, the method of locally heating the scaffold material requires the use of a culture container that has been coated with a specific scaffold material in advance, and the method of applying shock waves requires a large-scale device. Summary of the Invention

[0005] In one embodiment, the adhesion manipulation method includes the steps of culturing cells in a liquid, placing a flow path into the liquid through which a gas can be introduced, forming an air bubble at the end of the flow path, and attaching the cells to the air bubble.

[0006] In one embodiment, a cell manipulation device is a cell manipulation device that detaches cells cultured in a liquid and attaches them to gas bubbles, and is equipped with a flow path that is placed in the liquid and is capable of introducing gas into the liquid, and is capable of forming the gas bubbles at the end of the flow path. [Brief description of the drawings]

[0007] [Figure 1] 1 is a photograph showing an example of a cell culture vessel. [Diagram 2] 1(a) to 1(c) are schematic diagrams showing the structure of a cell detachment device (cell manipulation device) according to one embodiment. [Diagram 3] 1(a) to 1(c) are schematic diagrams showing the structure of a cell detachment device according to one embodiment. [Figure 4] 1(a) to 1(c) are schematic diagrams showing the structure of a cell detachment device according to one embodiment. [Diagram 5] 1(a) to 1(c) are schematic diagrams illustrating an example of a method for detaching cells (a cell manipulation method). [Figure 6] 1(a) and (b) are schematic diagrams illustrating an example of a method for detaching cells. [Figure 7] 1(a) and (b) are schematic diagrams illustrating an example of a method for detaching cells. [Figure 8] 1(a) and (b) are schematic diagrams illustrating an example of a method for detaching cells. [Figure 9] 1(a) to 1(e) are micrographs taken of the flow paths of the cell culture vessel in Experimental Example 1. [Figure 10] 14(a) to 14(c) are micrographs taken during a cell detachment experiment in Experimental Example 2. [Figure 11] 14(a) to 14(c) are micrographs taken during a cell detachment experiment in Experimental Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings as needed. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and duplicate explanations will be omitted. In addition, the dimensional ratios in each drawing are exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[0009] [Method for manipulating cells (first embodiment)] In one embodiment, the present invention provides a method for manipulating cells, comprising the steps of culturing cells in a liquid, arranging a flow path capable of introducing a gas in the liquid, forming an air bubble at an end of the flow path, and attaching the cells to the air bubble. The method of this embodiment can also be said to be a method for detaching the adherent cells from a solid phase, comprising contacting an interface between a gas and a liquid with the adherent cells cultured on the surface of the solid phase, and moving the interface along the surface.

[0010] As described later in the Examples, the inventors have found that the method of this embodiment makes it possible to detach adherent cells from a solid phase without using protease or the like.

[0011] In the present specification, the term "adherent cells" refers to cells that are adhered to a solid phase. In the present specification, even if the cells are originally cultured in a suspended state, they are treated as adherent cells if they are cultured under conditions that allow them to adhere to a solid phase.

[0012] In the method of the present embodiment, the gas is not particularly limited, and examples thereof include air, nitrogen, and the like. The gas may be sterilized. The method of sterilizing the gas is not particularly limited, and examples thereof include passing the gas through a filter with a pore size of about 0.22 μm or less. In addition, the liquid may be a cell culture medium, a buffer solution, or the like. The interface between the gas and the liquid is not particularly limited as long as it is a surface where the gas and the liquid are in contact, and may be, for example, the periphery or surface of a bubble formed by the gas, the interface between the liquid and the gas when the liquid is flowed through the fluid device after the gas, or the interface between the liquid and the gas when the liquid is flowed through the fluid device after the gas.

[0013] (First embodiment) In the method for detaching cells according to the first embodiment, at least a part of the solid phase forms a flow path, and contacting the adherent cells with an interface between a gas and a liquid and moving the interface in a direction along the surface are performed by flowing air bubbles through the flow path.

[0014] Figure 1 is a photograph showing an example of a cell culture vessel. In the cell culture vessel shown in Figure 1, the solid phase forms a flow path, and adherent cells can be cultured on the surface of the solid phase by supplying a culture medium to the flow path. The flow path is provided with an inlet and an outlet. Culture medium, cells, gas, etc. can be introduced from the inlet and discharged from the outlet.

[0015] According to the method for detaching cells according to the first embodiment, by introducing gas from the inlet of the cell culture vessel illustrated in Fig. 1 and flowing it toward the outlet, the interface between the gas and the culture medium comes into contact with the adherent cells and they move along the surface of the flow channel, so that the adherent cells cultured on the surface of the flow channel of the cell culture vessel can be detached. It is necessary to introduce an amount of gas such that the interface between the gas and the liquid comes into contact with the cells on the surface of the flow channel.

[0016] As a method for moving the gas-liquid interface, gas may be continuously introduced from the inlet of the flow channel, or gas may be introduced from the inlet of the flow channel to form bubbles, and then liquid or gas may be introduced from the inlet of the flow channel to move the bubbles toward the outlet. The liquid may be a cell culture medium or a buffer solution. As a result, the gas-liquid interface comes into contact with the adherent cells, and the interface further moves in a direction along the surface of the flow channel, causing the cells to detach from the solid phase.

[0017] In the method for detaching cells according to the first embodiment, at least a part of the solid phase may be disposed in the flow channel. The surface of the solid phase refers to a surface on which adherent cells can be attached and cultured, and includes, but is not limited to, glass; resins such as polystyrene; metals; surfaces coated with one or more extracellular matrix components selected from collagen, fibronectin, laminin, polylysine, etc.; surfaces coated with various polymers (e.g., polymers whose hydrophilicity or adsorbability to cells can be controlled); and the like.

[0018] [Cell manipulation device] In one embodiment, the present invention provides a cell manipulation device that is disposed in a liquid, includes a flow path capable of introducing a gas into the liquid, is capable of forming bubbles at an end of the flow path, and detaches cells cultured in the liquid and causes the cells to adhere to the bubbles. The cell manipulation device of this embodiment can also be said to be a cell detachment device that includes a tubular portion capable of generating bubbles at an end when the end is disposed in a liquid. The cell detachment device of this embodiment is configured to allow the bubbles to come into contact with adherent cells cultured on the surface of a solid phase.

[0019] 2(a) to (c) are schematic diagrams showing the structure of a cell detachment device according to an embodiment. As shown in FIG. 2(a) and (c), the cell detachment device 200 has a cylindrical part 230 with a flow path 220 through which a gas 210 flows, and by introducing or discharging the gas 210 into or from the flow path 220, bubbles 211 can be generated at the end 231 of the cylindrical part 230 when the end 231 is placed in a liquid 240. The cell detachment device 200 shown in FIG. 2(a) is connected to a control unit 250 that manually or automatically controls the volume of the bubbles 211 by controlling the amount of the gas 210 introduced or discharged into the flow path 220, thereby constituting a cell detachment system. Therefore, the volume of the bubbles 211 can be controlled. In addition, the cell detachment device 200 can manually or automatically change the distance of the end 231 from a solid phase or a cell, and can move the end 231 along the solid phase surface.

[0020] The control unit 250 may be composed of, for example, a syringe, a pump, etc. In the cell detachment device 200, the flow channel 220 is the inner cavity of the tubular portion 230.

[0021] Fig. 2(b) is a cross-sectional view taken along line bb' of the cylindrical portion 230 shown in Fig. 2(a). As shown in Fig. 2(b), the cylindrical portion 230 of the cell detachment device 200 includes a flow channel 220 having a circular cross section in a plane perpendicular to the axial direction of the cylindrical portion.

[0022] The opening area at the end 231 of the channel 220 is not particularly limited as long as the cell can be peeled off, but for example, it can be an area larger than the adhesion area of ​​one cell. The shape of the tip of the channel 220 is not particularly limited, and the inner diameter of the channel 220 may be the same up to the tip, may gradually increase toward the tip, or may gradually decrease toward the tip. The end face may be a surface perpendicular to the axis of the channel 220, or may be a surface not perpendicular to the axis of the channel 220.

[0023] In the cell detachment device 200, the flow path 220 can be used to collect cells, but the tubular portion 230 may further include a second flow path for collecting the detached cells.

[0024] (Variation 1) 3(a) to (c) are schematic diagrams showing the structure of a cell detachment device according to one embodiment. As shown in FIG. 3(a), the tubular part 230 of the cell detachment device 300 has a double structure of an outer cylinder 221 and an inner cylinder 261. The space between the outer cylinder 221 and the inner cylinder 261 is a first flow path 220 through which the gas 210 flows, and the inside of the inner cylinder 261 is a second flow path 260 for collecting the detached cells. The cell detachment device 300 includes a control unit P that introduces or exhausts the gas 210 into the first flow path 220, and that controls the volume of the gas 211 at the end 231 of the tubular part 230 when the end 231 is placed in the liquid 240. 1 The control unit P may be connected to the 1As shown in FIG. 3(a), the cell detachment device 300 includes a control unit P 2 may be connected to

[0025] Fig. 3(b) is a cross-sectional view taken along line bb' of the cylindrical portion 230 shown in Fig. 3(a). As shown in Fig. 3(b), the cylindrical portion 230 of the cell detachment device 300 includes a first flow channel 220 having a doughnut-shaped cross section and a second flow channel 260 surrounded by the first flow channel 220.

[0026] In the above example, the gas 210 is introduced or discharged into the first flow path 220, and the detached cells are collected in the second flow path 260, but the gas 210 may be introduced or discharged into the second flow path 260, and the cells may be collected in the first flow path 220. The cell detachment device 300 is mainly different from the cell detachment device 200 in that the first flow path 220 through which the gas 210 flows to form bubbles and the second flow path 260 for collecting the cells are independent.

[0027] 3(c), the bubble 211 disposed at the end 231 of the tubular portion 230 of the cell detachment device 300 has a doughnut shape. According to the cell detachment device 300, the first flow path 220 and the second flow path 260 are independent of each other, so that the cells can be detached by the bubble 211 disposed at the end 231 of the first flow path 220, while the detached cells can be collected through the second flow path 260.

[0028] (Variation 2) 4(a) to (c) are schematic diagrams showing the structure of a cell detachment device according to one embodiment. As shown in FIG. 4(a), the cell detachment device 400 includes a cylindrical part 230 having a flow path 220 through which a gas 210 flows, and a control unit P that, when an end 231 of the cylindrical part 230 is placed in a liquid 240, places an air bubble 211 at the end 231 or controls the volume of the air bubble 211 by introducing or discharging the gas 210 into the flow path 220. 1 The control unit P may be connected to the 1may for example consist of a pump.

[0029] In the cell detachment device 400, the channel 220 also serves as a channel for recovering cells.

[0030] Fig. 4(b) is a cross-sectional view taken along line bb' of the cylindrical part 230 shown in Fig. 4(a). As shown in Fig. 4(b), the cylindrical part 230 of the cell detachment device 400 differs from the above-mentioned cell detachment device 200 mainly in the cross-sectional shape in a plane perpendicular to the axial direction of the cylindrical part 230.

[0031] Fig. 4(c) is a perspective view showing a state in which an air bubble 211 is disposed at the end 231 of the cylindrical portion 230 of the cell detachment device 400. As shown in Fig. 4(c), the air bubble 211 formed at the end 231 of the cylindrical portion 230 of the cell detachment device 400 is long in one direction. This makes it easy to detach adherent cells over a wider range. In particular, by moving the cylindrical portion 230 in a direction perpendicular to the longitudinal direction of the cylindrical portion 230, the adherent cells can be efficiently detached.

[0032] In the cell detachment device 400, the flow channel 220 also serves as a flow channel for collecting cells, but is not limited to this, and the flow channel 220 and the cell collection flow channel 260 may be independent. In this case, for example, the cell collection flow channel 260 may be disposed inside the flow channel 220, the flow channel 220 may be disposed inside the cell collection flow channel 260, or the flow channel 220 and the cell collection flow channel 260 may have the same shape and be disposed adjacent to each other.

[0033] [Method of manipulating cells (second to fourth embodiments)] Second embodiment The method for detaching cells according to the second embodiment includes contacting an interface between a gas and a liquid with adherent cells cultured on a surface of a solid phase, and moving the interface along the surface. The contacting of the interface between the gas and the liquid with the adherent cells is performed by contacting air bubbles generated at an end of a cylindrical portion of a cell detachment device having a cylindrical portion with the adherent cells. The moving of the interface along the surface is performed by moving the air bubbles along the surface of the solid phase.

[0034] 5(a) to (c) are schematic diagrams for explaining an example of the method of the second embodiment. Here, a case where cells are detached using the above-mentioned cell detachment device 200 will be explained. Fig. 5(a) shows a state where adherent cells 510 are cultured on the surface of a culture vessel 500. The adherent cells 510 are cultured in a medium (liquid) 240.

[0035] 5(a), gas 210 is introduced into the flow path 220 of the cell detachment device 200, and a gas bubble 211 is placed at the end 231 of the cylindrical portion 230. Then, the gas bubble 211 is brought into contact with the adherent cell 510. As a result, the interface 212 between the gas 210 and the liquid 240 (the periphery of the contact surface between the gas bubble 211 and the culture vessel 500) comes into contact with the adherent cell 510.

[0036] Next, the cell detachment device 200 is moved in a direction along the surface of the culture vessel (solid phase) while keeping the air bubble 211 in contact with the adherent cell 510. The moving direction of the cell detachment device 200 is indicated by an arrow in Fig. 5(a). The direction along the surface of the solid phase is not particularly limited as long as it is a direction parallel to the surface of the solid phase, and may be any direction.

[0037] As will be described later in the Examples section, it is unexpectedly possible to detach the adherent cells 510 from the culture vessel 500 by the above-mentioned operations.

[0038] Fig. 5(b) is a schematic diagram showing a state in which adherent cells 510 have been detached. By moving the cell detachment device 200 in a line-like manner, the adherent cells 510 can be detached in a line shape. Furthermore, by changing the size of the air bubbles 211, the adherent cells 510 can be detached in a line shape of a desired thickness. Furthermore, as shown in Fig. 5(b), the inventors have found that the detached adherent cells 510 attach to the surface of the air bubbles 211.

[0039] 5(c), the gas 210 in the flow channel 220 may be sucked to collect the cells 510 attached to the surface of the bubbles 211. According to this embodiment, the cells can be selectively detached and further collected by a device with a simple configuration.

[0040] 6(a) and (b) are schematic diagrams for explaining an example of the method of the second embodiment. Here, a case where cells are detached using the above-mentioned cell detachment device 400 will be explained. Fig. 6(a) is a top view showing a state where adherent cells 510 are cultured on the surface of a culture vessel 500. The adherent cells 510 are cultured in a medium (liquid) 240.

[0041] 6(a), gas 210 is introduced into the flow path 220 of the cell detachment device 400, and air bubbles 211 are placed at the end 231 of the tubular portion 230. Then, the air bubbles 211 are brought into contact with the adherent cells 510. As a result, the interface 212 between the gas 210 and the liquid 240 comes into contact with the adherent cells 510.

[0042] Next, the cell detachment device 400 is moved in a direction along the surface of the culture vessel (solid phase) while keeping the air bubble 211 in contact with the adherent cell 510. The moving direction of the cell detachment device 200 is indicated by an arrow in Fig. 6(a). The direction along the surface of the solid phase is not particularly limited as long as it is a direction parallel to the surface of the solid phase, and may be any direction.

[0043] 6(b) is a schematic diagram showing a state in which the adherent cells 510 have been detached. Thereafter, the detached adherent cells 510 may be collected through the channel 260.

[0044] The method of the second embodiment has been described above using the cell detachment devices 200 and 400 as an example. However, in the method of the second embodiment, a cell detachment device other than the cell detachment devices 200 and 400 may be used as the cell detachment device. Examples of cell detachment devices other than the cell detachment devices 200 and 400 include, but are not limited to, the cell detachment device 300 described above.

[0045] Third embodiment The method for detaching cells according to the third embodiment includes contacting an interface between a gas and a liquid with adherent cells cultured on a solid surface, and moving the interface in a direction along the surface. The contacting of the interface between the gas and the liquid with the adherent cells is performed by contacting the adherent cells with air bubbles of the above-mentioned cell detachment device, which has air bubbles disposed at the ends. The moving of the interface in a direction along the surface is performed by changing the volume of the air bubbles of the cell detachment device.

[0046] 7(a) and (b) are schematic diagrams for explaining an example of the method of the third embodiment. Here, a case where cells are detached using the above-mentioned cell detachment device 200 will be explained. Fig. 7(a) shows a state where adherent cells 510 are cultured on the surface of a culture vessel 500. The adherent cells 510 are cultured in a medium (liquid) 240.

[0047] 7(a), gas 210 is introduced into the flow path 220 of the cell detachment device 200, and air bubbles 211 are placed at the end 231 of the cylindrical portion 230. Then, the air bubbles 211 are brought into contact with the adherent cells 510. As a result, the interface 212 between the gas 210 and the liquid 240 comes into contact with the adherent cells 510.

[0048] Next, the volume of the air bubble 211 in the cell detachment device 200 is changed. Specifically, as shown in Fig. 7(b), a gas 210 is introduced into the flow channel 220 to increase the volume of the air bubble 211.

[0049] As a result, the gas-liquid interface 212 (the periphery of the contact surface between the gas bubble 211 and the culture vessel 500) moves in a direction along the surface of the culture vessel. That is, the surface of the gas bubble moves in all directions around the adherent cell 510 that was present directly below the flow channel 220.

[0050] As described later in the Examples, the above-mentioned operations also allow the adherent cells 510 to be detached from the culture vessel 500. Furthermore, the inventors have found that the detached adherent cells 510 attach to the surfaces of the air bubbles 211, as shown in FIG.

[0051] Here, the gas 210 in the flow channel 220 may be sucked to collect the cells 510 attached to the surface of the bubbles 211.

[0052] The method of the third embodiment has been described above by taking the case where the cell detachment device 200 is used as an example. However, in the method of the third embodiment, a cell detachment device other than the cell detachment device 200 may be used as the cell detachment device. Examples of cell detachment devices other than the cell detachment device 200 include, but are not limited to, the above-mentioned cell detachment device 300 and cell detachment device 400.

[0053] (Fourth embodiment) The method for detaching cells according to the fourth embodiment includes contacting an interface between a gas and a liquid with adherent cells cultured on a solid surface, and moving the interface in a direction along the surface. The contacting of the interface between the gas and the liquid with the adherent cells is performed by contacting the adherent cells with air bubbles of the cell detachment device described above, which has air bubbles disposed at its end. The moving of the interface in a direction along the surface is performed by changing the distance between the cell detachment device and the adherent cells.

[0054] 8(a) and (b) are schematic diagrams for explaining an example of the method of the fourth embodiment. Here, a case where cells are detached using the above-mentioned cell detachment device 200 will be explained. Fig. 8(a) shows a state where adherent cells 510 are cultured on the surface of a culture vessel 500. The adherent cells 510 are cultured in a medium (liquid) 240.

[0055] 8(a), gas 210 is introduced into the flow path 220 of the cell detachment device 200, and air bubbles 211 are placed at the end 231 of the tubular portion 230. Then, the air bubbles 211 are brought into contact with the adherent cells 510. As a result, the interface 212 between the gas 210 and the liquid 240 comes into contact with the adherent cells 510.

[0056] Next, the distance between the cell detachment device and the adherent cells is changed. Specifically, as shown in Fig. 8(b), the cylindrical part 230 is brought closer to the surface of the culture vessel. As a result, the air bubble 211 is deformed.

[0057] As a result, the gas-liquid interface 212 (the periphery of the contact surface between the gas bubble 211 and the culture vessel 500) moves in a direction along the surface of the culture vessel. That is, the surface of the gas bubble moves in all directions around the adherent cell 510 that was present directly below the flow channel 220.

[0058] By the above operation, the adherent cells 510 can also be detached from the culture container 500. Furthermore, the inventors discovered that the detached adherent cells 510 attach to the surfaces of the air bubbles 211, as shown in FIG.

[0059] Here, the gas 210 in the flow channel 220 may be sucked to collect the cells 510 attached to the surface of the bubbles 211.

[0060] The method of the fourth embodiment has been described above using the cell detachment device 200 as an example. However, in the method of the fourth embodiment, a cell detachment device other than the cell detachment device 200 may be used as the cell detachment device. Examples of cell detachment devices other than the cell detachment device 200 include, but are not limited to, the above-mentioned cell detachment device 300 and cell detachment device 400.

[0061] In the above embodiments, the technique for detaching adherent cells attached to a solid phase has been described, but the embodiments of the present invention are not limited thereto. For example, the detached adherent cells adhere to the surface of air bubbles by contacting the air bubbles, but the attachment of cells to the surface of air bubbles can also be applied to floating cells suspended in a culture solution.

[0062] [Cell manipulation system] In one embodiment, the present invention provides a cell detachment system comprising: (i) a cell detachment device having a tubular portion capable of generating gas bubbles at an end when the end is placed in a liquid; and (ii) a control unit that controls the generation of the gas bubbles by introducing or discharging the gas into the tubular portion of the cell detachment device.

[0063] The control unit is the same as that described above, and may be composed of, for example, a syringe, a pump, and a CPU for controlling them. The cell detachment system of this embodiment can efficiently carry out the cell detachment method described above. The detached cells can also be collected.

[0064] [Fluid device system] In one embodiment, the present invention provides a fluidic device system including a fluidic device having a solid phase capable of culturing adherent cells on a surface thereof disposed in a flow path, and the above-mentioned cell detachment system. The fluidic device system of this embodiment allows for culturing adherent cells and further for detaching the cells. The detached cells can also be collected.

[0065] As described above, the cell detachment device, cell detachment system, and fluid device system are simple devices that can selectively detach and recover cells. Therefore, for example, when target cells are cultured on feeder cells, they can be used to remove only unnecessary feeder cells, which can be applied to regenerative medicine. They are also useful in scratch assays to evaluate the cell migration speed to the area where the cells have been detached and recovered. EXAMPLES

[0066] The present embodiment will now be described with reference to examples, but the present invention is not limited to the following examples.

[0067] [Experimental Example 1] (Cell detachment in the flow channel) <<Preparation of culture vessel>> We produced a cell culture vessel, the photograph of which is shown in Figure 1. Specifically, we produced the cell culture vessel by laminating, in this order, a first polydimethylsiloxane (PDMS) sheet with a thickness of 0.1 mm, a first acrylic plate with a thickness of 2 mm, a second PDMS sheet with a thickness of 0.1 mm, and a second acrylic plate with a thickness of 2 mm on a glass substrate.

[0068] The first and second PDMS sheets and the first acrylic plate were laminated by cutting out the flow path pattern shown in Fig. 1 using a laser processing machine (model "VLS2.30", Yokohama Systems Co., Ltd.). The second acrylic plate was laminated with holes formed therein to serve as the inlet and outlet. The adapters shown in Fig. 1 were then attached to the inlet and outlet to obtain a cell culture vessel.

[0069] Cell Culture We cultured cells in the channels of the cell culture vessel. KatoIII cells, a human gastric cancer cell line, were used.

[0070] Cell detachment Approximately one day after the start of the culture, air was introduced into the flow channel of the cell culture vessel, followed by the medium, and the result was observed under a microscope. Figures 9(a) to (e) are micrographs of the flow channel of the cell culture vessel. The magnification is 4x. Figure 9(a) is a photograph of the flow channel before air was introduced. Figure 9(b) is a photograph of the flow channel in the middle of air flowing into the observation region of the flow channel. Figure 9(c) is a photograph of the flow channel observation region completely filled with air. Figure 9(d) is a photograph of the flow channel observation region in the middle of air flowing out of the observation region of the flow channel. Figure 9(e) is a photograph of the flow channel observation region filled with medium again.

[0071] As a result, it was confirmed that the cells in the channel detached and flowed out of the observation area (Figure 9(e)). This result demonstrated that the adherent cells cultured on the surface of the solid phase can be detached from the solid phase by contacting the gas-liquid interface with the cells and then moving the interface in a direction along the surface.

[0072] [Experimental Example 2] (Cell detachment using a cell detachment device 1) Adherent cells were detached using a cell detachment device having a configuration similar to that of the cell detachment device 200 described above.

[0073] First, cells were cultured using cell culture dishes. HeLa cells, a cell line derived from human cervical cancer, were used. After culturing the cells until they reached confluence, a detachment experiment was performed.

[0074] Figures 10(a) to (c) are micrographs taken during a cell detachment experiment. The magnification is 4x. First, as shown in Figure 10(a), the edge of the cell detachment device was brought close to the cells. The arrow indicates the edge of the cell detachment device.

[0075] Next, as shown in Fig. 10(b), an air bubble was formed at the edge of the cell detachment device to contact the cells, and the cell detachment device was moved in a direction parallel to the bottom surface of the cell culture dish. In Fig. 10(b), the arrow indicates the edge of the cell detachment device.

[0076] Figure 10(c) is a photograph taken after the cells had been detached. In Figure 10(c), the area surrounded by a dotted line indicates the area where the cells had been detached. It can be seen that the cells had detached in a line, just as the air bubbles had moved. This result further supports the idea that adherent cells cultured on the surface of a solid phase can be detached from the solid phase by contacting the gas-liquid interface with the cells and then moving the interface in a direction along the surface.

[0077] [Experimental Example 3] (Cell detachment using a cell detachment device 2) Adherent cells were detached using a cell detachment device having a configuration similar to that of the cell detachment device 200 described above.

[0078] First, cells were cultured using cell culture dishes. HeLa cells, a cell line derived from human cervical cancer, were used. After culturing the cells until they reached confluence, a detachment experiment was performed.

[0079] Figures 11(a) to (c) are micrographs taken during a cell detachment experiment. The magnification is 4x. First, as shown in Figure 11(a), the edge of the cell detachment device was brought close to the cells. In Figure 11(a), the arrow indicates the edge of the cell detachment device.

[0080] Next, as shown in Figure 11(b), air bubbles were formed at the end of the cell detachment device, and the amount of air introduced into the flow path of the cell detachment device was increased to increase the volume of the air bubbles. In Figure 11(b), the arrow indicates the end of the cell detachment device. The same operation was performed four times. The volume of the air bubbles was also changed each time.

[0081] FIG. 11(c) is a photograph of the state after the cells were detached. In FIG. 11(c), the area surrounded by a dotted line indicates the area from which the cells were detached. The cells were detached in an approximately circular area where the periphery of the air bubble had passed. It was revealed that the area from which the cells were detached could be controlled by changing the volume of the air bubble. This result further supports the idea that adherent cells cultured on the surface of a solid phase can be detached from the solid phase by contacting the gas-liquid interface with the cells and then moving the interface in a direction along the surface. [Explanation of symbols]

[0082] 200, 300, 400... cell detachment device (cell manipulation device), 210... gas, 211... air bubble, 212... interface, 220... flow path (first flow path), 221... outer cylinder, 230... cylindrical portion, 231... end portion, 240... liquid, 250, P 1 ,P 2 ...control section, 260...second flow path, 261...inner tube, 500...culture vessel, 510...adherent cells.

Claims

1. a flow channel having a tip portion disposed in a liquid containing cells disposed on a solid phase; a control unit that controls at least one of a volume of gas introduced into the flow channel or discharged from the flow channel and a distance between the cell and the flow channel; the cells are adherent cells, the control unit controls at least one of the volume of the gas and the distance between the cell and the flow channel to move a gas-liquid interface of the bubble formed and held at the tip portion along the solid phase while being in contact with the cell; The gas-liquid interface of the gas bubble formed and held at the tip is moved along the solid phase while being in contact with the cell, thereby detaching the cell from the solid phase. Biological body manipulation device.

2. The control unit moves an air-liquid interface of the air bubble formed and held at the tip portion along the solid phase while in contact with the cell, and then discharges the gas in the flow path to recover the cell through the flow path. The organism manipulation device of claim 1.

3. Placing a liquid and cells disposed on a solid phase in a container; placing a tip of a flow channel in the liquid; A control step of controlling at least one of a volume of gas introduced into the flow path or discharged from the flow path and a distance between the cell and the flow path, the cells are adherent cells, The control step controls at least one of the volume of the gas and the distance between the cell and the flow channel to move the gas-liquid interface of the bubble formed and held at the tip portion along the solid phase while being in contact with the cell; The control step causes an air-liquid interface of the air bubble formed and held at the tip portion to move along the solid phase while being in contact with the cell, thereby detaching the cell from the solid phase. Biological body manipulation method.

4. The control step includes controlling the gas discharged from the flow path, moving the gas-liquid interface of the bubble formed and held at the tip portion along the solid phase while in contact with the cell, and then discharging the gas from the flow path to recover the cell through the flow path. The method of claim 3 .

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