Sample collection method

Abstract

To provide a sampling method, a gel plate, and a sampling set which can perform sampling excellent in stability, reproductivity, and location accuracy.SOLUTION: In a sampling method, a gel for supporting samples to be collected is formed into a sheet shape on a film 2 so that a gel sheet 4 is provided. The gel sheet 4 on the film 2 is punctured by an injection needle 7B having a hollow shaped part 7C so as to be partially cut out. A cutout gel sheet portion 4E is stored in the hollow shaped part 7C. The gel sheet portion 4E in the hollow shaped part 7C is discharged from the inside of the hollow shaped part 7C, so that a portion of the gel sheet 4 is collected.SELECTED DRAWING: Figure 14

Description

【Technical Field】 【0001】 The present disclosure relates to a sample collection method, a gel plate, and a sample collection set. 【Background Art】 【0002】 In recent years, technologies for selecting target cells and cell populations by analyzing single cells have been rapidly developing. As technologies for selecting target cells, cell selection methods such as the limiting dilution method, cell sorter, microfluidics, and colony pickup method have attracted attention. 【0003】 In addition, as a technology for handling a small amount of cells, a printing technology for handling a small amount of droplets has attracted attention. The printing technology includes an inkjet method, a dispenser method, and a coating method. 【0004】 However, there are cases where it is preferable to collect only one cell. From the viewpoint of appropriately collecting only one arbitrary cell, a technology for precisely extracting a smaller amount of droplets with higher precision than the above-described printing technology is required. From this viewpoint, a method using a manipulator disclosed in Japanese Patent Application Laid-Open No. 2008-152044 (Patent Document 1), and a method for cutting and collecting a biological sample such as a cell using a sampling needle disclosed in International Publication No. 2017 / 061387 (Patent Document ②) have been developed. 【Prior Art Documents】 【Patent Documents】 【0005】 【Patent Document 1】 Japanese Patent Application Laid-Open No. 2008-152044 【Patent Document 2】 International Publication No. 2017 / 061387 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0006】 In Japanese Patent Publication No. 2008-152044, a solution containing the cells to be collected is drawn into the hollow part of a tube. Since the cells to be collected are contained in the solution, if the solution is discharged into a container, the cells will disperse inside the container. This presents a challenge in that it is difficult to collect samples with high stability and reproducibility. 【0007】 In International Publication No. 2017 / 061387, the gel comes into contact with the cell specimen. The cut cell specimen is temporarily placed inside the collection needle. Then, liquid is injected into the collection needle, and the pressure causes the cell specimen inside the needle to be expelled from the needle. However, the gel is not fixed and supported around the cells. Therefore, especially with cells that are not adherent to the gel, the cell specimen may break down and disperse when expelled from the collection needle. There is also a risk of damaging the cells by piercing them with the collection needle. The position of the cells is not fixed by the gel, making it difficult to collect cells with high precision and reproducibility. 【0008】 This disclosure has been made in view of the above-mentioned problems. The purpose of this disclosure is to provide a sample collection method that enables stable, reproducible, and highly positional sample collection, as well as a gel plate and sample collection set used in said method. [Means for solving the problem] 【0009】 In a sample collection method for collecting a sample that is fixed to be collected according to this disclosure, a gel sheet is formed on a film to support the sample to be collected. The gel sheet on the film is punctured with a needle having a hollow shape, thereby cutting off a portion of the gel sheet, and the cut-off portion of the gel sheet is housed in the hollow shape. The portion of the gel sheet is collected by discharging it from the hollow shape. 【0010】 A gel plate on which a sample to be collected is fixed, according to this disclosure, comprises a film and a gel sheet. The gel sheet is formed in a sheet shape on the film and contains a gel that supports the sample to be collected. 【0011】 A sample collection set for fixing a sample to be collected according to this disclosure comprises a gel material and a film. The gel material is capable of supporting the sample to be collected by forming a gel. The film is capable of supporting the gel. [Effects of the Invention] 【0012】 According to this disclosure, a sample collection method, gel plate, and sample collection set are available that enable stable, reproducible, and highly accurate sample collection. [Brief explanation of the drawing] 【0013】 [Figure 1] This is a schematic front view of the inside of the sample collection device according to this embodiment. [Figure 2] Figure 1 is a schematic diagram illustrating the sampling mechanism of the sample collection device shown. [Figure 3] This is a schematic diagram illustrating the configuration of the sampling needle holder in the sampling mechanism shown in Figure 2. [Figure 4] This is a schematic cross-sectional view showing the first step of the sample collection method according to this embodiment. [Figure 5] This is a schematic cross-sectional view showing the second step of the sample collection method according to this embodiment. [Figure 6] This is a schematic cross-sectional view showing the third step of the sample collection method according to this embodiment. [Figure 7] This is a schematic cross-sectional view showing the fourth step of the sample collection method according to this embodiment. [Figure 8] This is a schematic perspective view of a gel plate. [Figure 9] This is a schematic cross-sectional view showing the fifth step of the sample collection method according to this embodiment. [Figure 10] Figure 9 is a schematic enlarged perspective view showing the plate included in the figure and the gel plate on top of it. [Figure 11] This is a schematic cross-sectional view showing the sixth step of the sample collection method according to this embodiment. [Figure 12]It is a schematic enlarged cross-sectional view of the area XII surrounded by the dotted line in FIG. 11. [Figure 13] It is a schematic cross-sectional view showing the seventh step of the sample collection method according to the present embodiment. [Figure 14] It is a schematic enlarged cross-sectional view of the area XIV surrounded by the dotted line in FIG. 13. [Figure 15] It is a schematic cross-sectional view showing the eighth step of the sample collection method according to the present embodiment. [Figure 16] It is a schematic cross-sectional view showing the first modification of the present embodiment. [Figure 17] It is a schematic cross-sectional view showing the second modification of the present embodiment. [Figure 18] It is a schematic cross-sectional view showing the third modification of the present embodiment. [Figure 19] It is a schematic cross-sectional view showing the step of cutting out the gel obtained from the flowing sample in FIG. 18. [Figure 20] It is a schematic view showing a sample collection set for fixing the sample to be collected according to the present embodiment. [Figure 21] It is a schematic cross-sectional view showing an example of the step of dissolving the gel from the gel sheet portion and collecting only the cells. 【Embodiments for Carrying Out the Invention】 【0014】 Hereinafter, the present embodiment will be described with reference to the drawings. 【0015】 (Sample Collection Method) First, the features of the present embodiment will be briefly described. As shown in FIGS. 7 and 8, a gel sheet 4 on which a sample 4B to be collected is supported by a gel 4D is placed on a film 2. As shown in FIG. 12, a part of the gel sheet 4 on the film 2 is cut out by puncturing it with an injection needle 7B having a hollow-shaped portion 7C, and the cut gel sheet portion 4E is housed in the hollow-shaped portion 7C. As shown in FIG. 15, the gel sheet portion 4E in the hollow-shaped portion 7C is discharged from the hollow-shaped portion 7C, whereby the gel sheet portion 4E is collected. Hereinafter, the present embodiment will be described in detail. 【0016】 (Overall configuration of the sample collection device) Figure 1 is a schematic front view of the interior of the sample collection device according to Embodiment 1. For the sake of explanation, the X, Y, and Z directions are introduced. Referring to Figure 1, the sample collection device 100 is a device for collecting a sample from a gel plate 6, which will be described later. The sample collection device 100 mainly comprises a processing chamber, a sample XY stage 101 located inside the processing chamber, a container XY stage 102, and a collection mechanism 104. Although the sample collection device 100 in Figure 1 has only one collection mechanism 104, it may include multiple such mechanisms. 【0017】 The sample XY stage 101 (sample stage) is movable horizontally, i.e., along the XY direction (X direction and Y direction). Specifically, for example, a guide is installed on the lower surface of the sample XY stage 101. This guide is slidably connected to a guide rail installed on the bottom surface of the processing chamber. The upper surface of the sample XY stage 101 is a mounting surface on which the gel plate 6 can be fixed. At least a portion of the sample XY stage 101 has a stage penetration portion 101A that penetrates it. It is preferable that the gel plate 6 is fixed in a position that overlaps with the stage penetration portion 101A. 【0018】 Below the sample XY stage 101, a container XY stage 102 (container stage) is positioned. The container XY stage 102 is movable horizontally, i.e., along the XY direction (X direction and Y direction). Specifically, for example, a guide is installed on the underside of the container XY stage 102. This guide is slidably connected to a guide rail installed on the bottom surface of the processing chamber. The container XY stage 102 fixes a plate 8 on which a container 9A capable of housing a portion of the gel plate 6 is formed. As a result, in Figure 1, the sampling mechanism 104, the gel plate 6, and the plate 8 with the container 9A formed on it are arranged in the order from top to bottom in the Z direction. 【0019】 The sampling mechanism 104 and the observation optical system 106 are connected to a member that is movable in the Z direction, such as a Z-axis table. In other words, the sampling mechanism 104 and the observation optical system 106 are held within the sample collection device 100 so as to be movable in the Z direction. The observation optical system 106 observes and measures the position of the sample to be collected contained in the gel plate 6. The observation optical system 106 may be equipped with a CCD camera that converts the observed image into an electrical signal. Observation of the gel plate 6 by the observation optical system 106 may be performed using visible light. However, observation of the gel plate 6 is not limited to visible light; it may also be performed using infrared light, X-rays, ultrasound, etc., and depending on the material of the gel plate 6, it may be possible to observe the gel plate 6 using magnetism. The gel plate 6 observed by means other than visible light does not need to be transparent or translucent, and may be opaque. 【0020】 Although not shown in the diagram, a control unit is installed outside the processing chamber. The control unit includes a monitor, a control computer, and an operation panel. In the control unit, command values ​​for the operating speed of the needle for sampling, obtained from the operation panel, are input and stored in the control computer's storage device. For example, during sample collection, the command value for the speed is read from the storage device and sent to the control program of the sampling mechanism 104. Based on this command value, the control program of the sampling mechanism 104 determines the rotational speed of the servo motor 41 (described later) included in the sampling mechanism 104 and moves the sampling needle unit 24 up and down at a predetermined speed. This performs the sampling operation of the sampling mechanism 104. If the control computer is communicating with an upstream control system, the above command values ​​may be received from that control system. In addition, parameters corresponding to the material of the gel sheet from which the sample is collected may be stored in the storage device, and the command value for the sampling operation speed may be calculated according to the specified gel sheet material, thickness, and sampling method. 【0021】 (Configuration of the sampling mechanism and sampling needle holder) Figure 2 is a schematic diagram showing the sampling mechanism of the sample collection device shown in Figure 1. Figure 3 is a schematic diagram illustrating the configuration of the sampling needle holder in the sampling mechanism shown in Figure 2. The sampling mechanism 104 described above will be explained in more detail with reference to Figures 2 and 3. 【0022】 The sampling mechanism 104 in Figure 1 includes a sampling needle holder 7H containing an injection needle 7B, which is a sampling needle. The sampling mechanism 104 includes a first drive unit 40. The sampling needle holder 7H is detachably connected to the first drive unit 40. Any structure can be used for connecting the sampling needle holder 7H to the first drive unit 40. 【0023】 Any configuration can be adopted for the first drive unit 40. For example, as shown in Figure 2, the first drive unit 40 includes a servo motor 41, a cam 43, a bearing 44, a cam connecting plate 45, and a movable part 46. The servo motor 41 is installed such that its rotation axis extends in the direction along the Z direction as shown in Figure 1. The cam 43 is connected to the rotation axis of the servo motor 41. The cam 43 is rotatable about the rotation axis of the servo motor 41. 【0024】 The cam 43 includes a central part connected to the rotation axis of the servo motor 41 and a flange part connected to one end of the central part. The upper surface of the flange part (the surface on the servo motor 41 side) is the cam surface. This cam surface is formed in an annular shape along the outer circumference of the central part. Furthermore, as shown in Figure 2, the cam surface is formed in a slope shape so that the distance from the bottom surface of the flange part varies. For example, the cam surface includes an upper flat region where the distance from the bottom surface of the flange part is greatest, a lower flat region located at a distance from the upper flat region and where the distance from the bottom surface of the flange part is smallest, and a slope part connecting the upper flat region and the lower flat region. 【0025】 A bearing 44 is positioned so as to be in contact with the cam surface of the cam 43. As shown in Figure 2, the bearing 44 is positioned in a specific direction (to the right of the servo motor 41) when viewed from the cam 43. The bearing 44 maintains contact with the cam surface when the cam 43 rotates due to the rotation of the servo motor 41's rotation axis. A cam connecting plate 45 is connected to this bearing 44. In the cam connecting plate 45, one end connected to the bearing 44 and the other end opposite to it are fixed to a movable part 46. A collection needle holder housing is connected to the movable part 46. The collection needle holder 7H described above is housed in this collection needle holder housing. For example, a first connecting member such as a magnet may be placed on the surface of the collection needle holder 7H facing the collection needle holder housing. Alternatively, a second connecting member such as a magnet or magnetic material may be placed on the collection needle holder housing side to fix the first connecting member on the collection needle holder 7H side. 【0026】 A fixing pin is installed on the movable part 46. Another fixing pin is installed on the frame that holds the servo motor 41. A spring is installed connecting these two fixing pins. Due to this spring, the movable part 46 is subjected to a downward tensile force. This tensile force from the spring acts on the bearing 44 via the movable part 46 and the cam connecting plate 45. This tensile force from the spring maintains the bearing 44 in a state of being pressed against the cam surface of the cam 43. 【0027】 Furthermore, the movable part 46 and the collection needle holder housing are connected to a linear guide installed on the frame. The linear guide is positioned to extend in the Z direction. Therefore, the movable part 46 and the collection needle holder housing (including the injection needle 7B and collection needle holder 7H) are movable along the Z direction. 【0028】 Figure 3 is a schematic diagram illustrating the configuration of the sampling needle holder in the sampling mechanism shown in Figure 2. Referring to Figure 3, the sampling needle holder 7H is capable of gripping the central sampling equipment consisting of a syringe 7A and an injection needle 7B. The injection needle 7B has a hollow, or hollow, section 7C inside. The injection needle 7B can cut and penetrate the target object by descending. 【0029】 The injection needle 7B is fixed to the syringe 7A. In the collection needle holder 7H, the syringe 7A is fixed by the upper syringe fixing part 7D and the lower syringe fixing part 7E. A syringe cap 7CP is attached to the upper side of the syringe 7A. A tube 7G is connected to the upper side of the syringe cap 7CP. Gas or liquid is injected into the syringe 7A from the tube 7G through the syringe cap 7CP. As a result, a portion of the cut gel plate 6, which is housed in the hollow section 7C of the injection needle 7B, is pushed downward and discharged from the injection needle 7B. 【0030】 The collection needle holder 7H houses the above-mentioned components within the holder shell 7F. However, parts of the injection needle 7B and tube 7G may protrude outside the holder shell 7F. 【0031】 In the following explanation, the central collection device, consisting of syringe 7A and injection needle 7B as shown in Figure 3, will be referred to as syringe 7. 【0032】 (Details of sample collection method) Figure 4 is a schematic cross-sectional view showing the first step of the sample collection method according to this embodiment. Referring to Figure 4, for example, a film 2 is placed on a glass slide 1. For example, the glass slide 1 is a thin glass plate material with a thickness of about 1.2 mm. The film 2 is made thin from a soft material so that it can be easily torn by needle puncture. For example, the film 2 is polyvinylidene chloride with a thickness of about 10 μm. That is, it is preferable to use a thin film for food packaging as the film 2. A spacer 3 is placed on the film 2. The spacer 3 may be made from the same glass material as the glass material that forms the glass slide 1. The spacer 3 may have a thickness of 100 μm or more and 200 μm or less. For example, the thickness of the spacer 3 may be 100 μm or 200 μm. The spacer 3 has a through hole 3A formed in its center when viewed from above (plan view) in Figure 4. The through hole 3A penetrates the spacer 3. Therefore, the inside of the through hole 3A is hollow, and no material such as glass that constitutes the spacer 3 is placed inside. The planar shape of the through-hole 3A can be arbitrary, such as a rectangle. 【0033】 Next, a fluid sample 4C, which contains one or more samples 4B within a fluid gel raw material 4A capable of forming a gel, is supplied into the through-hole 3A. Sample 4B is cells to be collected. In Figure 4, multiple samples 4B are arranged one by one, spaced apart from each other. Gel raw material 4A is, for example, a 1.5% agarose gel, which is made by adding a fluorescent dye to 1.5 mL of gel precursor. However, gel raw material 4A is not limited to this; for example, it may be collagen, or a mixture of alginic acid and collagen. 【0034】 Gel raw material 4A is a material that supports cells, for example, by embedding them. Therefore, gel raw material 4A is required to be biocompatible. In this way, the final gel obtained will be a gel made from biocompatible gel raw material 4A. Here, biocompatibility means that even when biological tissues such as cells come into contact with the material, it does not exhibit toxicity such as inflammation and has low invasiveness to cells. This suppresses damage to cells (sample 4B) caused by the gel. The biocompatibility of a material can be confirmed, for example, by observing that the number of living cells does not decrease when cells are cultured on the material and stained to distinguish between living and dead cells, or when inflammatory markers expressed by cells or contents eluted from cells are measured. Alternatively, the biocompatibility of a material can be confirmed by observing that the expression of inflammatory markers does not increase, or that contents do not elute from cells. 【0035】 The gel material 4A is preferably transparent or translucent, from the viewpoint of allowing observation of the position of the embedded sample 4B. From the viewpoint of satisfying the above, the gel material 4A is selected from the group consisting of collagen, gelatin, fibrin, sodium alginate, gellan gum, agarose, hyaluronic acid, chitin, chitosan, polyethylene glycol, polyvinyl alcohol, and 2-methacryloyloxyethyl phosphorylcholine (MPC polymer). 【0036】 Figure 5 is a schematic cross-sectional view showing the second step of the sample collection method according to this embodiment. Referring to Figure 5, a film 2 is attached to the lower surface of the slide glass 5. The slide glass 5 may be of the same material and size as the slide glass 1 in Figure 4. The film 2 on the upper side of the fluid sample 4C in Figure 5 may be of the same material and size as the film 2 in Figure 4. 【0037】 Figure 6 is a schematic cross-sectional view showing the third step of the sampling method according to this embodiment. Referring to Figure 6, a glass slide 5 with film 2 attached is placed on a spacer 3. The film 2 attached to the glass slide 5 comes into contact with the fluid sample 4C and the spacer 3. The glass slide 5 and film 2 press down on the fluid sample 4C and the spacer 3. As a result, the fluid sample 4C and the spacer 3 are sandwiched between the glass slide 1 with film 2 attached below it and the glass slide 5 with film 2 attached above it. This results in the following stacking order from bottom to top: glass slide 1, film 2, spacer 3 and fluid sample 4C, film 2, and glass slide 5. 【0038】 Next, if, for example, the gel material 4A is a material that solidifies depending on temperature, the entire stacked structure is cooled to 4°C. Cooling causes the gel material 4A constituting the fluid sample 4C to solidify into gel 4D, and a gel sheet 4 is formed inside the through-hole 3A with the sample 4B supported within the gel 4D. Although the gel 4D is solidified, it is soft enough to be easily broken by needle puncture. The statement that the sample 4B is supported by the gel 4D means, for example, that the sample 4B is embedded so that it is covered and filled with the hard gel 4D, and the sample 4B does not flow. 【0039】 Figure 7 is a schematic cross-sectional view showing the fourth step of the sample collection method according to this embodiment. Figure 8 is a schematic perspective view of the gel plate. Referring to Figures 7 and 8, after the gel sheet 4 is provided, the film 2 and slide glass 5 placed on top of the gel sheet 4 are removed, and the slide glass 1 and spacer 3 are also removed. This forms a gel plate 6 comprising the film 2 and the gel sheet 4 placed on the film 2. The gel sheet 4 is a sample 4B to be collected, supported, for example, by embedding in a solidified gel 4D. The gel 4D is formed in a sheet shape on the film 2. In other words, the gel sheet 4 is formed in a sheet shape on the film 2 and includes the gel 4D that supports the sample 4B to be collected. In this embodiment, "providing the gel sheet 4" means that the process of obtaining the gel sheet 4 by supporting the sample 4B with the gel 4D by embedding or other means may be performed on the film 2, or a gel sheet 4 that has been formed in advance externally may be attached to the film 2 afterward. As a method for forming the gel sheet 4, it is conceivable to create it in advance on an arbitrary flat surface. In Figure 8, PBS (Phosphate-buffered saline) is added to gel plate 6 to prevent drying. 【0040】 Figure 9 is a schematic cross-sectional view showing the fifth step of the sample collection method according to this embodiment. Figure 10 is a schematic enlarged perspective view showing the plate included in Figure 9 and the gel plate on it. Referring to Figures 9 and 10, the formed gel plate 6 is punctured from above with a syringe 7. Specifically, the syringe 7 is, for example, a 1 mL syringe 7A fitted with a 20 G diameter needle 7B. The needle 7B has a hollow section 7C formed so as to penetrate it along its extending direction. The hollow section 7C is empty, and no material forming the needle 7B is placed inside. 【0041】 Meanwhile, the gel plate 6 is placed on top of the plate 8. The plate 8 is a culture plate for cell culture. Multiple containers 9A are formed on the plate 8 to serve as collection sections. The multiple containers 9A are recessed portions of the upper surface of the plate 8. Because of their recessed shape, a portion of the cut-out gel sheet 4 (gel sheet portion 4E) can be stored inside the containers 9A. The multiple containers 9A may be spaced apart from each other, for example, in 4 rows in the depth direction and 24 rows in the width direction as shown in Figure 10, for a total of 96 containers. The planar shape of the containers 9A can be arbitrary, such as a circle. In Figures 9 and 10, the film 2 that makes up the gel plate 6 is in contact with the upper surface of the plate 8 so as to block at least a portion of the multiple containers 9A. To achieve this, a portion of the plate 8 (the upper region) may be placed within the stage penetration portion 101A in Figure 1. 【0042】 A sheet fixing member 21 may be placed on top of the gel plate 6. The sheet fixing member 21 suppresses displacement of the gel plate 6 caused by the force applied to the gel 4D from above during the process of cutting the gel sheet 4. Instead of the sheet fixing member 21, an adhesive film tape may be used as a means to suppress displacement of the gel plate 6. 【0043】 The plate 8 on which the culture vessel container 9A is formed is made of one of the materials selected from the group consisting of polystyrene, polypropylene, polycarbonate, and polyethylene terephthalate. 【0044】 Figure 11 is a schematic cross-sectional view showing the sixth step of the sample collection method according to this embodiment. Figure 12 is a schematic enlarged cross-sectional view of region XII enclosed by the dotted line in Figure 11. Referring to Figures 11 and 12, the syringe 7 is lowered relative to the state in Figure 9. The position where the syringe 7 should be lowered is the position where the target sample 4B to be collected is located, as can be seen from the image acquired using the observation optical system (such as the observation optical system 106 in Figure 4). At this time, the gel sheet 4 of the gel plate 6 is punctured by the injection needle 7B. That is, the punctured portion of the gel sheet 4 is damaged by the passage of the sharp tip of the injection needle 7B. The injection needle 7B penetrates the gel sheet 4 while tearing it. The part of the gel sheet 4 that is cut off from the original gel sheet 4 by the injection needle 7B is the gel sheet portion 4E. Preferably, the cut-off gel sheet portion 4E consists of gel 4D and the sample 4B embedded inside it. The cut-out gel sheet portion 4E is guided into the hollow-shaped portion 7C and housed within the hollow-shaped portion 7C. 【0045】 Figure 13 is a schematic cross-sectional view showing the seventh step of the sample collection method according to this embodiment. Figure 14 is a schematic enlarged cross-sectional view of region XIV enclosed by the dotted line in Figure 13. However, the plate 8 is not shown in Figure 14. Referring to Figures 13 and 14, the injection needle 7B continues to descend from the state shown in Figures 11 and 12. The injection needle 7B penetrates the gel sheet portion 4E that is to be cut off and the portion of the film 2 located directly below the gel sheet portion 4E. As a result, the film 2 is damaged in the same way as the gel sheet 4, as shown in Figure 14. 【0046】 Figure 15 is a schematic cross-sectional view showing the eighth step of the sample collection method according to this embodiment. Referring to Figure 15, the injection needle 7B continues to descend from the state in which it has penetrated the gel sheet 4 and the film 2 as shown in Figures 13 and 14. At this time, the injection needle 7B discharges the gel sheet portion 4E from within the hollow-shaped portion 7C. The discharged gel sheet portion 4E is collected in the container 9A. 【0047】 The gel sheet portion 4E is discharged from the hollow section 7C as follows: When at least the tip of the injection needle 7B is lowered to a position where it reaches inside the container 9A, the piston located inside the syringe 7A is pushed downward. The resulting air pressure pressurizes the hollow section 7C of the injection needle 7B. As a result, the gel sheet portion 4E inside the hollow section 7C is pressed downward and pushed out of the hollow section 7C. At this time, the top of the container 9A is sealed by the gel plate 6, and the space inside the container 9A is a sealed chamber, which is preferably filled with PBS. 【0048】 As the injection needle 7B penetrates the gel plate 6, the injection needle 7B pierces both the gel sheet 4 and the film 2. However, as shown in Figure 14, it is preferable that only the gel sheet 4 is contained within the hollow portion 7C, and the film 2 is not contained within it. As a result, as shown in Figure 15, it is preferable that only the gel sheet portion 4E is recovered into the container 9A, and the film 2 is not recovered into the container 9A. 【0049】 Figure 16 is a schematic cross-sectional view showing a first modified example of this embodiment. Referring to Figure 16, the embodiments shown here are basically the same as those in Figure 15, so the same reference numerals are used for the same components as in Figure 15, and the descriptions are not repeated as long as the functions, etc., are the same. However, in Figure 16, a substrate 9B installed inside the container 9A is used as the recovery unit. The gel sheet portion 4E discharged from the hollow-shaped portion 7C is recovered on the upper surface of the substrate 9B. In this respect, Figure 16 differs in configuration from Figure 15, where the container 9A itself is the recovery unit. This embodiment is acceptable as long as the upper surface of the substrate 9B can receive the gel sheet portion 4E discharged from the hollow-shaped portion 7C. 【0050】 In the above, for example as shown in Figure 7, the gel sheet 4 contains multiple samples 4B. Thus, for example, before the gel sheet 4 is cut and partially stored by the injection needle 7B, the multiple samples 4B originally contained in the gel sheet 4 (in the gel raw material 4A) may be dispersed so that they are spaced apart from each other. As a result, as shown in Figure 14, the gel sheet portion 4E that is cut and stored in the hollow-shaped portion 7C may contain only one sample 4B. However, in this embodiment, the samples 4B may be arranged as in the following modified example. 【0051】 Figure 17 is a schematic cross-sectional view showing a second modified example of this embodiment. Referring to Figure 17, the embodiments shown herein are basically the same as those in Figure 14, so the same reference numerals are used for the same components as in Figure 14, and the descriptions are not repeated as long as the functions, etc., are the same. However, in Figure 17, multiple samples 4B are arranged one by one, spaced apart from each other, within the gel sheet portion 4E that is cut out and housed in the hollow-shaped portion 7C. In this respect, Figure 17 differs from the example in Figure 14 in which only one sample 4B is contained within the gel sheet portion 4E. In Figure 17, two samples 4B are contained within the gel sheet portion 4E. However, the number of samples 4B contained within a single gel sheet portion 4E is not limited to this, and may be, for example, three or more. 【0052】 In this embodiment, the gel sheet portion 4E, which is housed within the hollow-shaped portion 7C by the penetration of the injection needle 7B, contains an isolated sample 4B. Here, "isolated" means both the case where only one sample 4B is contained within the gel sheet portion 4E, as shown in Figures 14 to 16, and the case where multiple samples 4B are contained one by one, spaced apart from each other, as shown in Figure 17. 【0053】 Figure 18 is a schematic cross-sectional view showing a third modified example of this embodiment. Referring to Figure 18, the embodiments shown here are basically the same as those in Figure 4, so the same reference numerals are used for the same components as in Figure 4, and the explanations are not repeated as long as the functions, etc., are the same. However, in Figure 18, multiple samples 4B are gathered together as a single mass within the gel material 4A that constitutes the fluid sample 4C. This mass of multiple samples 4B is called an aggregate. In Figure 18, as an example, three samples 4B are gathered in the aggregate, but the aggregate is not limited to this, and there may be two, four or more samples 4B constituting the aggregate. 【0054】 Figure 19 is a schematic cross-sectional view showing the process of cutting the gel obtained from the fluid sample in Figure 18. Referring to Figure 19, the embodiments shown here are basically the same as those in Figures 14 and 17, so the same reference numerals are used for identical components, and the explanations are not repeated as long as the functions, etc., are the same. However, in Figure 19, the gel sheet portion 4E housed in the hollow-shaped portion 7C contains aggregates of multiple samples 4B. In this respect, Figure 19 differs in structure from the above figures. The aggregates must be small enough to pass through the hollow-shaped portion 7C. That is, the aggregates must be smaller than the width in the direction intersecting the direction in which the hollow-shaped portion 7C extends. 【0055】 Figure 20 is a schematic diagram showing a sample collection set for fixing a sample to be collected according to this embodiment. Referring to Figure 20, this embodiment may be implemented in the form of a sample collection set 11 comprising the components used above, namely a gel raw material 4A (medicine) capable of supporting the sample 4B to be collected by forming a gel, and a film 2 on which the gel can be placed. In other words, the gel raw material 4A and the film 2 are not integrated into the sample collection set 11 but are included as separate components. The sample collection set 11 allows the user to support the sample using the fluid gel raw material 4A before it is fixed. The sample collection set 11 allows the user to arbitrarily create a gel plate 6, etc., with a high degree of freedom. Using the sample collection set 11, the above sample collection method can be easily carried out by the user. 【0056】 (Effects and Benefits) This embodiment discloses a sample collection method for collecting a sample that is fixed in place. In this sample collection method, a gel 4D supporting the sample 4B to be collected is formed in a sheet shape on a film 2, and a gel sheet 2 is provided. The gel sheet 4 on the film 2 is punctured with a needle (injection needle 7B) having a hollow shape portion 7C, thereby cutting off a portion of the gel sheet 4, and the cut-off portion of the gel sheet (gel sheet portion 4E) is housed in the hollow shape portion 7C. The gel sheet portion 4E is collected by discharging it from the hollow shape portion 7C. 【0057】 Generally, when cutting and harvesting a gel sheet 4, specifically to contain only one cell, if the cell is adhering to the sheet material (gel), for example, the cell will be attached and fixed to the sheet material when cut out. However, if the cell is not adhering to the sheet material, it may not be possible to fix the cell to the sheet material, and the cell may break down or fall off the sheet material. In this case, it becomes difficult to accurately harvest the desired cell. 【0058】 Therefore, in this embodiment, the sample 4B is supported (for example, fixed by embedding) by the gel 4D within the gel sheet 4. The sample 4B does not flow relative to the gel sheet 4. This suppresses the collapse and dissipation of the sample 4B when the gel sheet portion 4E is placed in the hollow-shaped portion 7C and when the sample is collected. Since the sample 4B is supported by the gel 4D, the possibility of the sample 4B moving in an unintended direction and damaging the sample 4B during puncture is reduced. Thus, sample collection with high stability, reproducibility, and positional accuracy can be performed. If the gel 4D and film 2 are made of a soft and easily torn material, they will easily break when penetrated by the injection needle 7B. Therefore, the process of cutting out a part of the gel sheet 4 is not difficult. 【0059】 In the above sample collection method, the step of collecting the gel sheet portion 4E may involve the injection needle 7B cutting through the gel sheet portion 4E and the portion of the film 2 positioned directly beneath the gel sheet portion 4E. After the above penetration step, the gel sheet portion 4E may be collected in the collection section. The gel plate 6 uses film 2. Therefore, the injection needle 7B can cut through and penetrate the gel 4D, and also easily break the film 2 that is in contact with the underside, allowing the needle to penetrate the film 2. This enables sample collection with high stability, reproducibility, and positional accuracy. 【0060】 In the above sample collection method, the recovery unit is either the substrate 9B or the container 9A, and the recovery unit may be positioned directly below the injection needle 7B. This allows the cut-off gel sheet portion 4E to be easily recovered. 【0061】 In the above sample collection method, in the step where the gel sheet portion 4E is collected in the collection unit, only the gel sheet portion 4E may be collected in the collection unit. Generally, when cutting out a sheet member to which cells are attached, the sheet member is also cut out together with the cells. As a result, the material of the sheet member may be mixed with the collected cells, which can make accurate analysis of the cells difficult. However, according to this embodiment, unlike the sheet member, the film 2, which is a separate component from the gel sheet portion 4E, is not collected in the collection unit. Therefore, the possibility that the material of the film 2 may be mixed with the collected sample 4B and affect the accuracy of the analysis of sample 4B can be eliminated. 【0062】 Specifically, to prevent the torn film 2 from adhering to the gel sheet portion 4E as shown in Figure 14, and to prevent the film 2 from falling to the recovery section, the following conditions are preferable. For example, it is preferable that the contact angle of water with respect to the surface of the film 2 is 60° or more. In this way, the film 2 can be easily peeled off from the gel sheet 4. Alternatively, it is preferable that the thickness of the film 2 is 100 μm or less. 【0063】 In the above sample collection method, the gel sheet portion 4E may have a gel 4D and a sample 4B supported within the gel 4D. This ensures that the sample 4B is securely fixed to the gel 4D. 【0064】 In the above sample collection method, the gel sheet 4 may contain multiple samples 4B, and before the step of housing the gel sheet portion 4E within the hollow-shaped portion 7C, the method may further include a step of dispersing the multiple samples 4B within the gel sheet 4 so that they are spaced apart from each other. As a result, the gel sheet portion 4E that is housed within the hollow-shaped portion 7C in the housing step may contain isolated samples 4B. 【0065】 For example, if multiple samples 4B gather together in the gel sheet 4 to form an aggregate, there is a risk of damaging the samples 4B by puncturing them with the injection needle 7B, especially if the aggregate is large. Damage to the samples 4B may make it difficult to analyze, for example, a cross-section of the tissue. Therefore, it is preferable that the multiple samples 4B be dispersed so that they are far apart from each other, as described above. This way, damage to the samples 4B due to puncture can be suppressed. 【0066】 Specifically, in order to disperse multiple samples 4B within the gel sheet 4 so that they are spaced apart from each other, for example as shown in Figure 7, it is preferable that the gel solution be treated to ensure that the low-concentration sample 4B is sufficiently dispersed. Here, low concentration means, for example, 3.5 × 10⁻⁶ for a sample with a diameter of 10 μm. 4 This refers to a concentration of less than or equal to 1 / mL. 【0067】 However, in this embodiment, in the step of housing the gel sheet portion 4E within the hollow-shaped portion 7C, the gel sheet portion 4E housed within the hollow-shaped portion 7C may contain aggregates of samples 4B formed by the aggregation of multiple samples 4B. In particular, if the size of the aggregates is small, they can pass through the hollow-shaped portion 7C, and the possibility of the samples 4B being punctured by the injection needle 7B is small, then this configuration is acceptable. 【0068】 In the above, sample 4B is described as either a single (isolated) cell or an aggregate of multiple cells. However, sample 4B may be any microorganism other than a cell or a plant. Furthermore, sample 4B may be any non-biological material selected from the group consisting of metals, plastics, proteins, oils, and polysaccharides. 【0069】 Figure 21 is a schematic cross-sectional view showing an example of a process in which the gel is dissolved from the gel sheet portion and only the cells are recovered. Referring to Figure 21, it is also possible to recover only the cells (sample 4B) by dissolving the gel 4D that supports and fixes the sample 4B from the cut-out gel sheet portion 4E. Specifically, for example, if sample 4B is 5 × 10⁶ 5 A gel sheet portion 4E, in which cells / mL of fibroblasts are placed inside gel 4D, is housed inside the apparatus. A gel dissolving agent 10, such as a 1% EDTA (ethylenediaminetetraacetic acid) solution, is supplied inside the apparatus. In Figure 21, gel 4D consists of a mixture of alginic acid with a content of 1.0% and collagen with a content density of 1.2 mg / mL. The gel dissolving agent 10 dissolves gel 4D. As shown in the lower part of Figure 21, the gel dissolving agent 10 contains the dissolved gel 4D and accumulates in the substrate as gel dissolving agent 10A. Since this is liquid, only the sample 4B can be recovered by discarding it. 【0070】 EDTA, used as gel dissolving agent 10, chelates divalent ions such as calcium ions. Therefore, EDTA is considered to have low toxicity to cells. For alginate gel 4D, an enzyme that degrades gel 4D, such as alginate lyase, may be used as gel dissolving agent 10A. Even when an enzyme that degrades gel 4D, such as alginate lyase, is used, cells can be recovered with minimal invasiveness to the cells. 【0071】 Based on the above, the sample collection method according to this embodiment may further include a step of dissolving gel 4D from gel sheet portion 4E and a step of taking out the sample 4B remaining after the dissolution step. By using a mechanism to break the cross-linking structure of gel 4D and substances such as enzymes that selectively decompose gel 4D, it is possible to recover only cells from the recovered gel sheet portion 4E without damaging the cells. 【0072】 (modified version) The embodiment described above may have the following features. The features of each of the following components may be combined as appropriate. 【0073】 Firstly, it is preferable that the "film 2" in this embodiment is made of a material that is soft and brittle enough to be easily torn by a small pressure such as the downward movement of the injection needle 7B, allowing the injection needle 7B to penetrate it, but that the parts other than the puncture site do not tear significantly and maintain their pre-puncture state to the extent that they can support the gel sheet 4 even after penetration. From this viewpoint, the film 2 is not limited to a thin film for food packaging such as polyvinylidene chloride, but may be made of the following materials, for example. That is, the film 2 may be made of a plastic film such as polyethylene or polyvinyl chloride. Alternatively, the film 2 may be made of either polydimethylsiloxane or butyl rubber. Furthermore, from the viewpoint of making the puncture site even easier to tear by the downward movement of the injection needle 7B and preventing damage to parts other than the puncture site, it is preferable that the thickness of the film 2 is 10 μm or more and 100 μm or less. Also from the above viewpoint, it is preferable that the maximum elongation rate (ultimate elongation rate) of the film 2 is 100% or more and 1300% or less. Furthermore, in order to visually confirm the position of sample 4B through film 2, it is preferable that film 2 be transparent or translucent, suitable for observation by an optical system. 【0074】 Secondly, from the viewpoint of suppressing the problem of the shape of the gel sheet portion 4E, which is housed by the injection needle 7B upon puncture, collapsing, for example, in the hollow portion 7C, the inner wall surface of the injection needle 7B may have a tapered shape extending in a direction inclined with respect to the direction in which the injection needle 7B extends, for example, at its tip. The angle of the tapered shape with respect to the direction in which the injection needle 7B extends may be, for example, 90° or less. 【0075】 Thirdly, the needle used in this embodiment is not limited to the injection needle 7B, but can be any needle having a hollow portion. For example, it may be a pin in which the hollow portion is cylindrical (i.e., the entire needle is cylindrical). 【0076】 Fourthly, from the viewpoint of easily discharging the stored gel sheet portion 4E, it is preferable that the hollow-shaped portion 7C used in this embodiment is coated with a material that has low protein adsorption properties and low affinity with gel 4D. Specifically, the inner wall surface of the hollow-shaped portion 7C may be coated with, for example, a fluorine-based material. Here, gel 4D is, for example, MPC polymer (2-methacryloyloxyethyl phosphorylcholine). 【0077】 Furthermore, in this embodiment, gel 4D is not limited to commonly used materials such as the collagen gel described above, but may be formed from any of the following selected materials: tamarind seed gum, pectin, and carrageenan. 【0078】 The features of each example described in the embodiments above may be applied in appropriate combinations within a technically consistent range. 【0079】 The embodiments disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated by the claims rather than by the foregoing description, and all modifications within the meaning and scope equivalent to the claims are intended to be included. [Explanation of symbols] 【0080】 1,5 Slide glass, 2 Film, 3 Spacer, 3A Through hole, 4 Gel sheet, 4A Gel raw material, 4B Sample, 4C Flow sample, 4D Gel, 4E Gel sheet portion, 6 Gel plate, 7 Syringe, 7A Syringe, 7B Injection needle, 7C Hollow shape portion, 7CP Syringe cap, 7D Upper syringe fixing portion, 7E Lower syringe fixing portion, 7F Holder outer shell, 7G Tube, 7H Sampling needle holder, 8 Plate, 9A Container, 10,10A Gel dissolving agent, 11 Sample collection set, 21 Sheet fixing member, 40 First drive unit, 41 Servo motor, 43 Cam, 44 Bearing, 45 Cam connecting plate, 46 Movable part, 100 XY stage for container, 101 XY stage for sample, 101A Stage through portion, 102 XY stage for container, 104 sampling mechanism, 106 observation optical system.

Claims

[Claim 1] A sample collection method for collecting samples that have been fixed in place, A step of forming a gel sheet on a film to support the sample to be collected, and a step of providing the gel sheet, The process involves puncturing the gel sheet on the film with a needle having a hollow shape to cut off a portion of the gel sheet, and then storing the cut-off portion of the gel sheet inside the hollow shape, A method for collecting a sample, comprising the step of collecting a portion of the gel sheet by discharging the portion of the gel sheet from within the hollow-shaped portion. [Claim 2] The sampling step includes the step of penetrating the portion of the gel sheet cut by the needle and the portion of the film located directly below the portion of the gel sheet, The sample collection method according to claim 1, further comprising the step of collecting the portion of the gel sheet in a collection unit after the aforementioned penetrating step. [Claim 3] The aforementioned recovery unit is either a substrate or a container. The sample collection method according to claim 2, wherein the recovery unit is positioned directly below the needle. [Claim 4] The sample collection method according to claim 2 or 3, wherein in the recovery step, only the gel sheet portion is recovered in the recovery unit. [Claim 5] The sample collection method according to any one of claims 1 to 4, wherein the portion of the gel sheet comprises the gel and the sample supported within the gel. [Claim 6] The gel sheet contains a plurality of the samples, The sample collection method according to any one of claims 1 to 5, further comprising the step of dispersing the plurality of samples in the gel sheet so that they are arranged apart from each other, prior to the storage step. [Claim 7] The sample collection method according to claim 6, wherein the portion of the gel sheet stored in the hollow-shaped portion during the storage step contains the isolated sample. [Claim 8] The sample collection method according to any one of claims 1 to 5, wherein the portion of the gel sheet stored in the hollow-shaped part during the storage step includes aggregates of the samples formed by the collection of multiple samples. [Claim 9] The sample collection method according to any one of claims 1 to 8, wherein the gel is obtained by gelling a biocompatible gel raw material. [Claim 10] The process involves dissolving the gel from the gel sheet portion, A method for collecting a sample according to any one of claims 1 to 9, further comprising the step of taking out the sample remaining after the dissolution step.