JP2019095197A - Lid of tube for life science, tube set for life science, and selection method of cell - Google Patents

Abstract

To provide a lid of a tube for life science, a tube set, and a selection method of a cell allowing an efficient operation.SOLUTION: When a lid 32 is attached, a recess 34 is disposed on a surface forming the side of the tube for a life science, a bottom surface 34a of the recess 34 is flat, and is circular or is a polygon of four or more sides, and a side surface 34b is formed of an inclined surface having the direction in which the side surface extends from the bottom surface 34a to an opening. Furthermore, a tube set including a tube 50 used with the lid 32, and a selection method of a cell using the lid 32 are provided.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a lid for a life science tube, a tube set for life science, and a method for sorting cells, in particular, a lid for a life science tube suitable for image analysis of cells, a tube set for life science and a tube for life science The present invention relates to a method of sorting cells using a lid.

  As a method of obtaining target cells from a plurality of cells, sorting of target cells by flow cytometry is performed. Flow cytometry disperses fine cells in a fluid, finely flows the fluid, optically analyzes individual cells, and determines the cells to be obtained based on the analysis results, and performs sorting. It is a thing.

  In addition, a plurality of cells are collectively dropped onto a well slide, dropped into small wells, photographed, and an image analyzed to identify a target cell. Thereafter, operations for aspirating the specified target cells with a capillary and transferring them to a PCR (polymerase chain reaction) plate or tube are performed.

  As a container used for a gene analysis device, for example, Patent Document 1 below describes a reaction container having a removable lid on a tube used for PCR application.

JP, 2011-072263, A

  The lid of the reaction vessel used in Patent Document 1 is only described to be used for sealing in order to prevent the reaction liquid contained therein from evaporating and disappearing. Therefore, with regard to the reaction container described in Patent Document 1, after the target cells are determined using another apparatus and method, it is necessary to separate them into the reaction container, and the operation flow is complicated and time-consuming. There was a problem that the equipment and instruments used for the operation were expensive.

  In addition, in flow cytometry, there are cases in which cells other than the target are mixed among the sorted cells, and the ratio of the target cells is about several percent. Therefore, it was inefficient to perform analysis or pretreatment for analysis on all cells sorted by flow cytometry. In addition, since the bottom of the PCR plate or PCR tube was not flat, it was not focused, and the cells could not be observed on the PCR plate or in the PCR tube.

  The present invention has been made in view of such circumstances, and by conducting image analysis using a lid, a tube for life sciences that can be efficiently subjected to subsequent analysis or pretreatment of analysis. Lid, tube set for life science, and method of sorting cells.

  In order to achieve the above object, the present invention is a lid for a tube for life sciences, which has a recess on the side of the tube for life sciences, and the bottom surface of the recess is flat and is a polygon of circle or square or more. Provided is a lid for a life science tube, the side surface comprising an inclined surface in a direction extending from the bottom to the opening.

  According to the lid of the tube for life sciences of the present invention, the concave portion is provided on the side of the lid of the tube for life sciences on which the tube for life science is attached, and the bottom of the recess is made flat. An image of the sample can be taken. By making the bottom of the recess of the lid flat, it is possible to focus on the sample in the recess and to capture a good image.

  In addition, by making the side surface of the recess the inclined surface in the direction that spreads from the bottom toward the opening, the aperture can be made wider, and the sample can be easily entered into the recess. Further, by narrowing the bottom toward the bottom, the size of the bottom can be reduced, and the size of the bottom with respect to the sample can be set to a predetermined range. Therefore, in the image analysis of the specimen, the entire bottom surface can be imaged by imaging with one field of view, and imaging and analysis of the specimen can be efficiently performed.

  In addition, by making the side surface an inclined surface in the direction of spreading toward the opening, bubbles in the culture solution can be easily extracted, and light can be prevented from being refracted by the bubbles, and a good image can be obtained. It can be imaged.

  In the present specification, "for life science" refers to handling cells and biomolecules, and more specifically, includes culturing, detection, separation, purification, analysis and evaluation of cells, and biomolecules. It refers to those used for compound synthesis, detection, separation, purification, analysis and evaluation, etc. “Life science tube” refers to a container used for life science applications.

  The “Life Science Tube Side” is the side on which the Life Science Tube is mounted, and when used with the Life Science Tube, forms a space between the lid of the Life Science Tube and the Tube. It says the face side.

  In another aspect of the present invention, the side surface preferably has two or more steps of inclined surfaces having different angles with respect to the bottom surface.

  According to this aspect, the design flexibility of the side surface of the recess can be increased by forming the side surface of the recess of the lid with two or more inclined surfaces having different angles with respect to the bottom surface. For example, by increasing the angle on the opening side of the recess, the sample can be more easily introduced into the recess. In addition, by reducing the angle on the bottom surface side of the side surface, the bottom surface can be narrowed, and the bottom surface can be imaged with one image even if the magnification at the time of imaging is increased. By increasing the angle on the bottom surface side of the side surface, it is possible to prevent the specimen introduced into the recess from staying on the side surface and to drop it to the bottom surface.

  In another aspect of the present invention, the size of the bottom surface is preferably 0.05 mmφ or more and 1 mmφ or less when approximated to a circumscribed circle.

  By setting the size of the bottom surface in the above range, the entire bottom surface is imaged with a single image by imaging using an objective lens with a magnification (5 or more and 63 or less) used for imaging of a normal image. can do. Therefore, imaging and image analysis of the sample can be efficiently performed.

  In another aspect of the present invention, the transmittance for light with a wavelength of 350 nm or more and 800 nm or less is preferably 60% or more.

  According to this aspect, by setting the transmittance of the material used for the lid of the tube for life science to the light of the above-mentioned wavelength to 60% or more, a good image can be taken.

  In another aspect of the present invention, it is preferably made of acrylic resin, polypropylene or polystyrene.

  In this embodiment, the material used for the lid of the tube for life sciences is limited, and by using acrylic resin, polypropylene or polystyrene, the transparency of the lid can be obtained, and a good image is imaged. be able to. In addition, since heat resistance can be secured by using polypropylene or polystyrene, the process is performed using the lid as it is, even in the case of the process of applying temperature, for example, the PCR process and the like in the next step. It can be carried out.

  In another aspect of the present invention, it is preferable that the surface of the recess is subjected to a low cell adhesion treatment.

  According to this aspect, by performing the cell adhesion treatment on the surface of the recess, it is possible to prevent the cell from adhering to the recess of the lid when transferring the cells from the lid to the life science tube. Therefore, the sample can be reliably moved from the lid to the life science tube.

  In order to achieve the above-mentioned object, the present invention provides a tube set for life sciences provided with the above-mentioned lid for life sciences tubes and a tube for life sciences.

  According to the tube set for life sciences of the present invention, a sample subjected to image analysis using the lid for the life sciences tube described above is transferred to the life sciences tube used in the set, and the subsequent treatment is performed. Can. Therefore, the image analysis of the sample and the subsequent processing can be performed with one life science tube set having a lid, and the operation can be performed efficiently.

  In another aspect of the present invention, the life science tube is preferably a PCR tube or a protein low adsorption tube.

  This embodiment describes a specific example of a life science tube, and a PCR tube and a low-adsorption protein tube can be used as the life science tube.

  In order to achieve the above object, the present invention is based on the steps of separating cells into a lid of a tube for life sciences, imaging the separated cells and acquiring a cell image, and based on a cell image, And sorting the cells of interest.

  According to the cell sorting method of the present invention, the target cells can be sorted with high accuracy by sorting the target cells by cell images using the lid of the tube for life sciences, and for life sciences The target cells can be easily transferred to the tube.

  In another aspect of the present invention, a step of attaching a tube for life science to a lid of a tube for life sciences after a step of selecting a target cell, a step of transferring the cell of interest to a tube for life science, Processing the target cells in the tube.

  According to this aspect, image analysis of cells is performed using the lid of the tube for life science, the target cell is moved to the tube for life science, and the treatment of the target cell is performed in the tube for life science. Image analysis and subsequent processing can be performed with one life science tube provided.

  In another aspect of the present invention, the step of treating the target cell is preferably a PCR treatment.

  In this embodiment, the step of treating the target cell is limited, and PCR treatment can be performed.

  In another aspect of the present invention, the lid of the tube for life sciences has a recess on the side of the tube for life sciences, the bottom surface of the recess is a circle or a polygon of square or more, and the side faces from the bottom toward the opening It is preferable that it consists of an inclined surface in the direction of spreading.

  According to this aspect, the lid for the life science tube has a recess on the side to which the life science tube of the lid is attached, and the bottom surface of the recess is made flat to capture an image of cells in the recess. it can. By flattening the bottom of the recess of the lid, cells in the recess can be focused and a good image can be imaged.

  In addition, by setting the side surface of the recess to be an inclined surface in a direction that spreads from the bottom toward the opening, the opening can be made wider, and cells can easily enter the recess. In addition, by narrowing toward the bottom, the size of the bottom can be reduced, and the size of the bottom with respect to cells can be set to a predetermined range. Therefore, in the image analysis of the sample, the entire bottom surface can be imaged with a single image, and imaging and analysis of the sample can be efficiently performed.

  In addition, by making the side surface an inclined surface in the direction of spreading toward the opening, bubbles in the culture solution can be easily extracted, and light can be prevented from being refracted by the bubbles, and a good image can be obtained. It can be imaged.

  In another aspect of the present invention, the size of the bottom surface is preferably 0.05 mmφ or more and 1 mmφ or less when approximated to a circumscribed circle.

  By setting the size of the bottom surface in the above range, it is possible to image the entire bottom surface in shooting with one field of view. Therefore, imaging and image analysis can be performed efficiently.

  In another aspect of the present invention, in the step of acquiring a cell image, the magnification of the objective lens for photographing is 5 times or more and 63 times or less, and the size of the bottom is approximated to a circumscribed circle in the image photographing area It is preferable that the diameter at the time is shorter than the length of the short side of the two orthogonal sides of the image capturing area and is 1/2 or more of the length of the short side.

  According to this aspect, in the image capturing area where the magnification of the objective lens for capturing is 5 times or more and 63 times or less, the size of the bottom (diameter when approximated to a circumscribed circle) By setting the length shorter than the length of the short side and 1/2 or more of the length of the short side, it is possible to obtain an optimum size for observing the entire bottom surface and cells in the image pickup area.

According to the lid of the tube for life sciences of the present invention, it is possible to introduce an analyte (cell) to be observed into the recess and perform image analysis. And, by using it in combination with the tube for life science, image analysis of the sample and subsequent processing can be performed with one life science tube set having a lid, and efficient operation can be performed. In addition, according to the cell sorting method, the target cells can be sorted with high accuracy by sorting out the target cells by image analysis with the lid of the life sciences tube, and the purpose of the life sciences tube Cell migration can be easily performed.

It is a schematic block diagram which shows the structure of the apparatus which images a cell. FIG. 7 illustrates another aspect of the tray. It is sectional drawing which shows the shape of the lid | cover of the tube for life sciences. It is the figure which showed the relationship between the image imaged and the size of the bottom face of a recessed part. It is sectional drawing which shows the shape of the lid | cover of the tube for life sciences of other embodiment. It is sectional drawing which shows the shape of the lid | cover of the tube for life sciences of other embodiment. It is process drawing which shows the process of the selection direction of a cell. It is process drawing which shows the process of the selection direction of a cell. It is process drawing which shows the process of the selection direction of a cell. It is a figure explaining the process of attaching the tube for life sciences. It is a figure explaining the process of moving the object cell to the tube for life sciences.

  Hereinafter, a lid for a life science tube, a tube set for life science, and a cell sorting method according to the present invention will be described with reference to the attached drawings. In addition, in this specification, "-" is used in the meaning which includes the numerical value described before and after that as a lower limit and an upper limit. In the following, cells will be described as an example, but the lid of the tube for life science of the present invention and the tube set for life science are not limited to cells, and can be used for compounds containing biomolecules.

  First, an analysis apparatus provided with an imaging device for capturing an image by applying a lid (hereinafter, also simply referred to as a “lid”) of the tube for life sciences of the present embodiment will be described.

  FIG. 1 is a schematic configuration view showing a configuration of an apparatus for imaging target cells sorted by a lid of a tube for life sciences and acquiring optical information from the cells in the step of sorting. As a preferred embodiment, it is an analysis apparatus capable of acquiring information of fluorescence emission from a fluorescent dye labeled to cells fractionated by an antigen-antibody reaction or the like, and acquiring a light transmission image of the cell by visible light.

  The analyzer 10 shown in FIG. 1 is a fluorescent excitation light source 12 that emits light for measuring fluorescence emitted from target cells, and a light that emits light (visible light) for measuring transmitted light of cells. A light source device 14 for visual field, a tray 19 comprising a lid 32 and a plate 18 of a tube for life sciences containing cells 16 to be imaged, a lens 20, an excitation filter 22, a dichroic mirror 24 and a fluorescence filter 26 are held. A filter group (filter cube) 28 and an imaging device 30 for imaging fluorescence and transmitted light from the cells 16 are provided.

  The fluorescence excitation light source device 12 can use a high pressure mercury lamp, a high pressure xenon lamp, an LED (light emitting diode), a light amplification by stimulated emission of radiation (LASER), or the like. By using these light sources, by narrowing the wavelength region of the irradiation light to be applied to the cells 16, it becomes possible to reliably carry out analysis with high accuracy. In addition, as the fluorescence excitation light source device 12, a tungsten lamp, a halogen lamp, a white LED or the like can be used. Even in the case of using these light sources, the cell 16 can be irradiated with light of the target wavelength by transmitting only the target wavelength by the excitation filter 22. Also as the light source device 14 for bright field, the same light source as the excitation light source device 12 for fluorescence can be used.

  The tray 19 comprises a plate 18 and a lid 32 for containing the cells 16 collected. The lid 32 has a recess on the side to which a life science tube (hereinafter, also simply referred to as a "tube") is attached, and the collected cells are accommodated in the recess. In the present specification, the side to which the tube for life sciences of the lid 32 of the tube for life sciences is attached is referred to as the surface of the lid for the tube for life sciences, and the opposite side is referred to as the back. The plate 18 is a sample table for holding the lid 32. Since the cells 16 are imaged from the back side of the lid 32, the plate 18 has a through hole at the position of the plate 18 corresponding to the bottom of the recess of the lid 32. Alternatively, it is formed of a transparent material capable of transmitting light.

  FIG. 2 shows another aspect of the tray. The tray 319 shown in FIG. 2 differs from the tray 19 described in FIG. 1 in that the plate 318 and the lid 332 are integrally formed. As a tray used for the analysis apparatus 10, as shown in FIG. 2, the tray with which the plate 318 and the lid 332 were united can be used. By integrally forming the plate 318 and the lid 332, it can be manufactured at once by injection molding, and the lid 332 can be arranged with high accuracy. Also, the tray can be manufactured at low cost.

  Returning to FIG. 1, the lens 20 emits the fluorescence emitted from the cell 16 by the light output from the fluorescence excitation light source device 12 and the transmitted light from the light output from the bright field light source device 14 transmitted through the cell 16 Expanding. The lens 20 can be a lens generally used for optical applications.

  The filter group 28 includes an excitation filter 22, a dichroic mirror 24, and a fluorescence filter 26. As a specific example of such a filter group 28, it is preferable to use a filter cube, and for example, Zeiss Filter Set 49 (DAPI) can be used. The light emitted from the fluorescence excitation light source device 12 transmits only the light of the target wavelength region by the excitation filter 22. The light transmitted through the excitation filter 22 is reflected by the dichroic mirror 24 in the direction of the tray 19. The fluorescence emission from the cell 16 generated by the excitation light emitted from the fluorescence excitation light source device 12 passes through the lens 20, the dichroic mirror 24, and the fluorescence filter 26, and is imaged by the imaging device 30. The fluorescence emitted by the excitation light exists on the longer wavelength side of the wavelength region of the excitation light than the wavelength region of the excitation light. Therefore, by using the dichroic mirror 24, only the fluorescence emission can be transmitted. Further, by using the fluorescence filter 26 that transmits only the fluorescence without transmitting the excitation light, it is possible to capture an image with information of only the fluorescence emission from the cell 16 by the imaging device 30. Therefore, by transmitting only the fluorescence by the fluorescence filter 26, the image captured by the imaging device 30 can be acquired without being affected by the excitation light, and the accuracy of the inspection is improved by the fluorescence emission information. It can be done.

  Fluorescence imaging with light emitted from the fluorescence excitation light source device 12 is usually immunostained with a plurality of types of dyes in order to obtain a plurality of pieces of information for one cell in accordance with the examination purpose of the cells. In this case, with respect to the fluorescence from the respective dyes of the immunostained cells, by imaging using a filter group having transmission characteristics or reflection characteristics suitable for the fluorescence wavelengths of the respective dyes, optical wavelengths of different wavelengths are obtained. You can get information. In addition, when imaging the transmitted light of the cell 16 by the light source device 14 for bright field, imaging is performed with the filter group 28 removed. Thereby, the transmitted light can be imaged by the imaging device 30.

  The imaging device 30 is not particularly limited as long as the fluorescence or transmitted light of the cells 16 in the lid 32 on the tray 19 can be imaged. For example, a charge-coupled device (CCD) camera can be used.

«Leg for life science tubes»
FIG. 3 is a cross-sectional view showing the shape of the lid 32 of the tube for life sciences used in the present embodiment. In the analysis apparatus 10 shown in FIG. 1, excitation light is irradiated from the bottom side of the lid 32, and light including information from cells such as fluorescence from cells emitted by the excitation light transmitted through the lid 32 is received. The material of the lid 32 preferably has conditions such as being transparent to the light, not causing autofluorescence, and not scattering. In addition, in order to image the cells 16, the bottom surface 34a of the recess 34 of the lid 32 is flat. By making the bottom surface 34 a of the recess 34 flat, it is possible to focus on the cells 16, and image analysis of the cells 16 present on the bottom surface 34 a can be performed with high accuracy.

  Further, the shape of the bottom surface 34a is a circle or a polygon of quadrilateral or more. Further, when the size of the bottom surface 34a approximates a circle circumscribed to the bottom surface 34a, the diameter L of the circle is preferably 0.05 mmφ or more and 1 mmφ or less, more preferably 0.1 mmφ or more and 0.5 mmφ or less More preferably, they are 0.2 mmφ or more and 0.4 mmφ or less. In addition, in FIG. 3, the bottom surface 34a is described as circular. Taking the shape and size of the bottom surface 34a into the above shape and size, and using an objective lens with a magnification of 5 to 63 times, with a preferred cell image size, and taking a picture with one field of view (one shot The entire bottom surface 34 a can be imaged by imaging). In order to obtain multiple information for one cell depending on the examination purpose of the cells, immunostaining is usually performed using multiple types of dyes. Therefore, imaging of the cell 16 can be performed by analyzing fluorescence information from each dye and bright field imaging, superimposing each image after imaging if necessary, and analyzing the cell.

  FIG. 4 is a diagram showing the relationship between the image pickup area 40 imaged by the microscope and the size of the bottom surface 34 a of the recess 34. The size L of the bottom surface 34a is such that the diameter L of the bottom surface 34a is shorter than the length of the short side d and the length of the short side d among two long sides e and short sides d of the image capturing area 40. It is preferable to set it as 1/2 or more. By setting the length L of the bottom surface to d> L> d / 2, the flat bottom surface 34 a of the lid in which the cells are stored has a preferred cell image size in the image capturing area 40, and It enables imaging of cells by visual field. The size of the image capturing area 40 is determined by the magnification of the objective lens of the microscope and the capturing camera. By using a commonly used camera, for example, with a 20 × objective lens, it is possible to image one bottom surface 34 a in the image capturing area 40 by setting the diameter L of the bottom surface to 0.4 mmφ. . The diameter L of the bottom surface is 0.2 mmφ for a 40 × objective lens, 0.1 mm φ for an 63 × objective lens, and 1 mm φ for a 10 × objective lens. The bottom surface 34a can be imaged with one image. In order to observe cells, a high magnification is preferable, but if the magnification becomes high, the accuracy of the image analysis starts to be affected by the unevenness on the back side of the lid 32, etc. Therefore, the magnification of the objective lens is about 20 times Is preferred.

  The side surface 34 b of the recess 34 is formed obliquely in the direction extending from the bottom surface 34 a toward the opening, and has a tapered shape. By forming the side surface 34 b obliquely, air bubbles in the culture solution can be easily extracted, and light can be prevented from being refracted by the air bubbles. In addition, since a wide opening can be secured, the cells 16 can be easily stored in the recess 34, and the area of the bottom can be further reduced.

  The thickness t of the bottom surface 34a of the lid 32 is preferably 0.2 mm or more and 1 mm or less. As shown in FIG. 1, since imaging of the cell 16 is performed from the back surface side (bottom surface side) of the lid 32, when the thickness of the bottom surface 34a is 1 mm or less, the lens 20 is made to approach the cell 16. This is preferable because it enables imaging of cells at high magnification. In addition, it is possible to set the depth of focus in a narrow range if it is 0.2 mm or more, and even if there is a slight scratch or the like on the back surface side of the lid 32, the focus of the scratch may be shifted. It is preferable because the image of the flaw is not projected and only the image of the cell can be taken. The thickness t of the bottom surface 34a is more preferably 0.3 mm or more and 0.5 mm or less, and still more preferably 0.4 mm.

  The material of the lid 32 is preferably a material that easily transmits light when capturing an image. Specifically, a material selected from acrylic resin, polypropylene, or polystyrene can be used. The lid 32 made of these materials preferably has a transmittance of 60% or more, more preferably 70% or more, and further preferably 80% or more for light of a wavelength of 350 nm or more and 800 nm or less. preferable. From the viewpoint of light permeability, it is preferable to use an acrylic resin or polypropylene. In addition, when the treatment after selection of the target cells is PCR treatment, it is preferable to have heat resistance, and by using polypropylene or polystyrene, deterioration of the material in the PCR treatment can be prevented. In the present invention, “transmittance” is a value obtained by dividing transmitted light by incident light (transmittance = transmitted light / incident light), and, for example, the luminous flux transmitted when a luminous flux of 100 is incident is 60 The transmittance is calculated to be 60%.

  Moreover, it is preferable that the cell low attachment process is given to the surface (bottom 34a and side 34b) of the recessed part 34. FIG. The low cell adhesion treatment is a treatment for preventing cells, ie, proteins from adhering to the bottom surface 34a and the side surface 34b in the recess 34, and is performed by coating the surface with a material having a property to prevent protein adsorption. . The reason for the adsorption of the protein into the recess 34 is mainly due to the hydrophobic interaction in which the hydrophobic group on the resin surface, which is the material of the lid 32, and the hydrophobic group in the protein are bonded. By coating a material having the following, it is possible to obtain a surface subjected to a low cell adhesion treatment.

  As a material to be used for low cell adhesion treatment, a polymer containing a phosphocholine group (for example, Lipidur (registered trademark) (alias: MPC (2-methacryloyl ethylphosphorylcholine) polymer) (manufactured by NOF Corporation), polyvinyl pyrrolidone, polyethylene glycol, PVA (polyvinyl alcohol) hydrogel, BSA (Bovine serum albumin) etc. can be used. As a coating method, it is possible to perform coating by dipping after immersion in a dispersion in which the above-described material is dispersed in a solvent, and then drying. As a solvent, for example, in the case of lipidumer, ethanol can be used, and the lipidumeur is mixed and dispersed in ethanol in a proportion of 0.5 wt% to prepare a dispersion.

  By subjecting the surface of the recess 34 to a low cell adhesion treatment, it is possible to prevent the attachment to the side surface 34 b before reaching the bottom surface 34 a when the cells are sorted by flow cytometry etc. in the recess 34. . In addition, after image analysis, when moving cells to the tube, they can be easily moved to the tube without adhering to the bottom surface 34a.

  The low cell adhesion treatment can also be carried out by stabilizing an enzyme that degrades the attached protein.

FIG. 5 is a cross-sectional view showing the shape of the lid 132 of the life science tube of another embodiment. Side face of the recess 134 of the lid 132 shown in FIG. 5 is a side has an angle theta _B relative to the bottom surface 134a 134b, and, and a side surface 134c having an angle theta _C with respect to the bottom surface 134a. As shown in FIG. 5, the side surfaces can be formed by side surfaces 134b and 134c which are bent in multiple stages and are two inclined surfaces having different angles θ _B and θ _C with respect to the bottom surface 134a. By forming the side surface with the two-step inclined surface, the design freedom of the side surface in the recess 134 can be increased. By increasing the angle θ _C on the opening side of the recess 134, cells can be more easily introduced into the recess 134. In addition, the bottom surface 134a can be narrowed by reducing the angle θ _B on the bottom surface 134a side, and when capturing an image, the bottom surface 134a can be captured with one image even when capturing at a high magnification. it can. The relationship between the angles θ _B and θ _C is not particularly limited. In FIG. 5, the angle theta _C have been described at an angle theta _B larger view, the angle theta _B may be larger than the angle theta _C. By increasing the angle theta _B, cells remain in a side 134b, it is possible to prevent the cells do not reach the bottom surface 134a. In addition, in FIG. 5, although the side surface is made into the inclined surface of two steps, the number of steps is not specifically limited, It can also be set as an inclined surface of three or more steps.

  FIG. 6 is a cross-sectional view showing the shape of a lid 232 of a life science tube according to still another embodiment. The lid 232 shown in FIG. 6 is different from the other embodiments in that a spacer 236 is provided in the recess. By making the shape of the recess 234 of the spacer 236 into the shape of the recess 34, 134 of the lid 32, 132 described above and fitting it into the inside of the lid 232, the same effect as that of the above embodiment can be obtained.

<Tube Set for Life Science>
The lid of the above-mentioned life science tube is used as a life science tube set together with the life science tube. As tubes for life sciences, use tubes for applications such as cell culture, detection, separation, purification, analysis and evaluation, and synthesis, detection, separation, purification, analysis and evaluation of compounds containing biomolecules Can. Specifically, a protein low adsorption tube that reduces sample adhesion to the container can be used in protein and peptide analysis and the like. Alternatively, a PCR tube for performing PCR may be used. When used in combination with the lid of the tube for life science of the present embodiment, image analysis and subsequent processing, analysis, and the like can be performed with a single tube set for life science.

<Method of sorting cells>
Next, a method of sorting cells using the above-described lid for life science tubes and the tube set for life sciences will be described.

  7 to 9 are process diagrams showing the process of cell sorting direction. Further, FIG. 10 is a view for explaining a process of attaching a life science tube, and FIG. 11 is a view for explaining a process of moving a target cell to the life science tube. According to the cell sorting method of the present embodiment, based on the step of sorting the cells into the lid of the tube for life sciences, the step of imaging the sorted cells to obtain a cell image, and the cell image, And selecting the target cells. In addition, after the step of selecting the target cells, a step of attaching the life science tube to the lid of the life science tube, a step of moving the target cell to the life science tube, and a target cell in the life science tube And the processing step. Each step will be described below.

<< process to fractionate >>
First, as shown in FIG. 7, the culture solution 38 is dropped into the recess 34 of the lid 32. Next, as shown in FIG. 8, cells 16 are separated into the recess 34 having the culture solution 38. The step of separating target cells from a plurality of cells can be performed, for example, by flow cytometry. In flow cytometry, a sample liquid containing a substance to be measured, such as cells to be measured, is caused to flow toward the center of a laminar flow of sheath liquid in a flow cell, and laser light is irradiated to the substance to be measured in an optical detection unit. The size, structure, etc. of the substance to be measured are measured by measuring the scattered light and fluorescence produced. As parameters of measurement in the optical detection unit, there are forward scattered light, side scattered light and fluorescence, but in forward scattered light, the size of the object to be measured and the structure of the substance to be measured by side scattered light and fluorescence. Etc can be measured.

  Then, using the measurement parameters in the optical detection unit, the sorting system separates target cells on the lid on the tray. In flow cytometry, the sample / sheath solution is designed to flow from top to bottom, and as it flows in a laminar flow, it pops up and falls from a nozzle at the tip of the flow cell. A transducer (vibrator) is used to apply vertical vibration to the entire flow cell or the inside of the flow cell so that the sample / sheath liquid that has flowed out of the flow cell becomes droplets from the middle. Based on the separation conditions using the measurement parameters in the optical detection unit, it is determined whether it is a cell to be separated or not, and the entire sample / sheath liquid is charged with + or-just before it becomes a droplet. After that, the droplets fall between the two deflection plates, the positively charged droplets are drawn to the -plate side, and the negatively charged droplets are pulled to the + plate side, on the tray in the cell collection section. The cells can be sorted into each lid.

  In addition, although the said method was described as an example as a method of flow cytometry, it is not limited to said method, It can carry out by the method currently generally performed. Moreover, as a process to fractionate, it is not limited to flow cytometry, It can carry out also with another method.

<< Step of acquiring cell image >>
Next, the cells sorted by the lid 32 are imaged to obtain a cell image. FIG. 9 is a diagram showing a process of acquiring a cell image. As a device for capturing a cell image, the device shown in FIG. 1 can be used.

  As described above, the lid 32 of the tube for life sciences used in the present embodiment has a flat bottom surface 34a of the recess 34 and is a circle or a polygon having a square or more, and the side surface 34b extends from the bottom surface 34a to the opening It is formed obliquely in the direction in which it spreads. Therefore, it is suitable for imaging the image of the cell 16 present on the bottom surface 34a, and a good cell image can be efficiently obtained using the lid 32 of the tube for life sciences of the present embodiment.

<< Step of selecting target cells >>
Next, based on the cell image obtained in the step of acquiring a cell image, confirmation and sorting of the target cell are performed. Confirmation of the target cell by cell imaging includes, for example, presence or absence of nucleus, size of nucleus, shape of nucleus (ratio of area of nucleus area to area of cytoplasm, degree of circularity of nucleus), shape of cell (continuous or jagged) Peak value, average value, brightness distribution (whether the cell membrane is uniformly fluorescent or locally strongly fluorescent), absorption degree to transmitted light of a specific wavelength (hemoglobin or white blood cell) Discrimination), spectral characteristics attributable to differences in oxygen affinity of hemoglobin (the absorption coefficients of reduced hemoglobin [Hb] and oxygenated hemoglobin [HbO ₂ ] with respect to the wavelength), and the like.

«Process for attaching a tube for life sciences»
Next, the life science tube 50 is attached to the lid 32 of the life science tube after the step of selecting the target cells. FIG. 10 is a view for explaining the process of attaching the life science tube. The tube 50 may attach the tube 50 only to the lid 32 from which the target cell and the cells 16 sorted have been separated in the step of sorting the cells of interest, or may attach the tube 50 to all the lids 32. . The step of attaching the life science tube can be performed using a dedicated device.

  When the tube 50 is attached only to the lid 32 from which the target cells and the sorted cells 16 have been collected, only the target cells to which the tube 50 is attached are subjected to the next step. Therefore, it is preferable to link information on the tube 50 and the cells 16 in the tube 50. As a method of correlating the information of the cells 16 with the lid 32 or the tube 50, there is a method of marking the alignment information of the lid 32 arranged on the tray 19 on the lid 32 with characters. As a description example of the sequence information, if it is "C4", it means C row 4 column of the plate, and if it is "D 4" it means column 4 of D row. Further, instead of the sequence information of the tray 19, cell information may be printed by QR code (registered trademark). In addition, by providing a RFID (radio frequency identifier) tag storing the array information of the tray on the lid 32, it is possible to clarify the cell information.

<< Process of moving cells to tubes for life sciences >>
After attaching the tube 50 to the lid 32, the cells 16 in the lid 32 are moved to the tube 50. FIG. 11 is a view for explaining the process of moving a target cell to a life science tube. The cells 16 can be transferred from the lid 32 to the tube 50 by, for example, inverting the tube 50 after attaching the tube 50 to the lid 32, and performing centrifugation to move the cells 16 together with the culture fluid 38 into the tube 50. it can. The step of transferring the cells to the life science tube can be performed using a dedicated device.

<< Process of processing target cells >>
After moving the target cells to the tube 50, the target cells are processed. As a process of processing a target cell, PCR processing can be mentioned, for example. The PCR process is a method of amplifying a specific region of a DNA (deoxyribonucleic acid) molecule, and can be performed, for example, by the following method. However, the PCR process is not limited to the following method. Moreover, the process of processing the target cell is not limited to the PCR process, and other processes can also be performed.

[PCR processing]
(Step 1)
The reaction solution is heated to about 94 ° C., maintained at temperature for 30 seconds to 1 minute, and the double stranded DNA is separated into single strands.

(Step 2)
The reaction solution is rapidly cooled to about 60 ° C., the single stranded DNA and the primer are heated (annealed) to a predetermined temperature, and the single stranded DNA and the primer are heated.

(Step 3)
The DNA polymerase is reacted with the primer, and separation of the single-stranded DNA and the primer does not occur, and heating is performed to a temperature (about 60 to 72 ° C.) suitable for DNA polymerase activity. This condition is continued for the time required for DNA synthesis (usually 1 to 2 minutes depending on the length of amplification).

(Step 4)
A specific DNA fragment can be amplified by repeating steps 1 to 3 as one cycle, and repeating the steps 1 to 3. In general, n cycles of PCR treatment allow amplification of a target portion 2 n times from one double stranded DNA. Although a long DNA strand will be left until the end, by performing about 20 cycles, the amount of DNA can be reduced to a negligible amount compared to the specific DNA fragment required.

  As described above, according to the cell sorting method of the present embodiment, after the imaging of the cell image and the determination of the target cell are performed using the lid, the cell is moved to the tube and the target cell is processed. it can. Therefore, it is possible to carry out from image analysis to processing of cells with one life science tube set having a lid. In addition, movement of cells from the lid for image analysis to the tube for processing can be easily performed without requiring a difficult operation such as using a capillary.

DESCRIPTION OF SYMBOLS 10 Analysis apparatus 12 Excitation light source device 14 for fluorescence 14 Light source device 16 for bright fields Cells 18, 318 Plate 19, 319 Tray 20 Lens 22 Excitation filter 24 Dichroic mirror 26 Fluorescence filter 28 Filter group (filter cube)
30 Imager 32. 132, 232, 332 Tube science lid for life sciences
34, 134, 234 Recess 34a, 134a Bottom 34b, 134b, 134c Side 38 Culture solution 40 Image taking area 50 Life science tube (tube)
236 Spacer

Claims (14)

A lid for life science tubes,
The life science tube side has a recess,
A lid for a tube for life sciences, wherein a bottom surface of the recess is flat and is a circle or a polygon having a square or more, and a side surface is an inclined surface extending in a direction from the bottom toward the opening.   The lid for a tube for life science according to claim 1, wherein the side surface has two or more steps of inclined surfaces having different angles with respect to the bottom surface.   The lid for a tube for life sciences according to claim 1 or 2, wherein the size of the bottom surface is a diameter of 0.05 mm or more and 1 mm or less when approximated to a circumscribed circle.   The lid for a tube for life sciences according to any one of claims 1 to 3, which has a transmittance of 60% or more to light of a wavelength of 350 nm or more and 800 nm or less.   The lid for the tube for life sciences according to any one of claims 1 to 4, which is made of acrylic resin, polypropylene or polystyrene.   The lid for the tube for life sciences according to any one of claims 1 to 5, wherein a low cell adhesion treatment is applied to the surface of the recess.   A tube set for life science comprising the lid for the life science tube according to any one of claims 1 to 6 and a life science tube.   The tube set for life science according to claim 7, wherein the life science tube is a PCR tube or a protein low adsorption tube. Separating the cells into a lid of a life science tube;
Imaging the sorted cells to obtain a cell image;
Sorting the target cells based on the cell image. Attaching the life science tube to a lid of the life science tube after the step of selecting the target cells;
Transferring the target cell to the life science tube;
And D. processing the target cells in the life science tube.   The cell sorting method according to claim 10, wherein the step of processing the target cell is a PCR process.   The lid of the tube for life sciences has a recess on the side of the tube for life sciences, and the bottom surface of the recess is a circle or a polygon of quadrilateral or more, and an inclined surface in the direction in which the side surface extends from the bottom toward the opening The method for sorting cells according to claim 10 or 11, comprising the steps of   The cell sorting method according to claim 12, wherein the diameter of the bottom surface is approximately 0.05 mmφ or more and 1 mmφ or less when approximated to a circumscribed circle. In the step of obtaining the cell image, the magnification of the objective lens for imaging is 5 times or more and 63 times or less,
The size of the bottom surface is such that the diameter when approximated to a circumscribed circle in the image capturing area is shorter than the length of the short side of the two orthogonal sides of the image capturing area, and 1/1 of the length of the short side. The cell sorting method according to claim 13, which is two or more.

Images