Space information providing sheet, space fractionation device, and fraction piece recovery method

Abstract

To provide a space information providing sheet that can specify the position of each fraction piece in a tissue section before fractionation even if a plurality of fraction pieces are collectively recovered.SOLUTION: A space information providing sheet 1 is a sheet that is spatially partitioned and cut together with a tissue slice TS in a state of being overlapped with the tissue slice TS. The space information providing sheet 1 has a spatial information pattern that can identify its position in the entirety from a partial pattern. The spatial information pattern may be a random dot pattern formed of a plurality of microbeads randomly dispersed inside a sheet substrate.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a spatial information - imparting sheet, a spatial fractionating device, and a fraction fragment recovery method. More specifically, the present invention relates to a sheet for imparting spatial information to a tissue section, a device for spatially fractionating a tissue section, and a method for spatially fractionating a tissue section and recovering fragments.

Background Art

[0002] Omics analysis, which comprehensively analyzes the information of biomolecules contained in animals and plants, has become one of the main tools in life science research. Since omics analysis technology is a fundamental technology for understanding the living body at the cellular level, in addition to research on human diseases typified by cancer research, it is used in basic life sciences such as neuroscience, developmental science, and plant science.

[0003] However, there is a problem that it is not known in which position and in what form the cells analyzed by omics were present in the tissue. Tissues are composed of various cell types and states, and their spatial composition dominates interactions and functions. However, cell recovery from tissues causes loss of spatial information. Therefore, it is required to link the spatial information of the cells to the results of omics analysis of the cells collected from the tissue.

[0004] As a method applicable to spatial analysis, there is a method of directly separating cells from a tissue. As a device capable of separating cells with high resolution, a cell recovery device having a suction / discharge mechanism using a glass capillary is commercially available. However, this device is not assumed to recover all the cells of a tissue section, and comprehensive analysis cannot be performed.

[0005] The inventor of the present application has proposed a fine - structure blade for two - dimensionally partitioning and cutting cells (Patent Document 1). If the fine - structure blade is pressed against a tissue section, the tissue section can be spatially fractionated. Since a plurality of fraction fragments can be obtained by a single operation, comprehensive analysis becomes possible.

Prior Art Documents

Patent Documents

[0006] Patent Document 1 Japanese Patent Application Laid-Open No. 2015-31549 Summary of the Invention Problems to be Solved by the Invention

[0007] However, in order to collect each of a plurality of fractionated fragments obtained by cutting with a fine-structured blade while retaining the spatial information, it is necessary to collect the fractionated fragments one by one using a glass capillary or the like, and the throughput is extremely low. Considering the comprehensive collection of fractionated fragments from the tissue, this collection throughput is the biggest bottleneck.

[0008] In view of the above circumstances, an object of the present invention is to provide a spatial information-added sheet that can identify the position of each fractionated fragment in a tissue section before fractionation even when a plurality of fractionated fragments are collected in a batch. Alternatively, an object of the present invention is to provide a spatial fractionation device that can spatially fractionate a tissue section while retaining spatial information. Alternatively, an object of the present invention is to provide a fractionated fragment collection method that can collect a plurality of fractionated fragments with high throughput while associating them with spatial information. Means for Solving the Problems

[0009] The spatial information-added sheet according to the first aspect is a sheet that is spatially partitioned and cut together with the tissue section in a state of being superimposed on the tissue section, and has a spatial information pattern capable of specifying the position of the part in the whole from the partial pattern. The spatial information-added sheet according to the second aspect is, in the first aspect, a sheet base material having translucency, and a plurality of microbeads randomly dispersed inside the sheet base material, and the spatial information pattern is a random dot pattern formed by the plurality of microbeads. The spatial information-added sheet according to the third aspect is, in the second aspect, characterized in that the microbeads are fluorescent microbeads. In the fourth aspect, the spatial information - imparting sheet is characterized in that, in the second aspect, the plurality of micro - beads include micro - beads of a plurality of colors. In the fifth aspect, the spatial information - imparting sheet is characterized in that, in the second aspect, the average number of micro - beads per section is 9.2 or more. In the sixth aspect, the spatial information - imparting sheet is characterized in that, in the first aspect, the spatial information pattern is a concavo - convex pattern attached to the sheet surface. In the seventh aspect, the spatial information - imparting sheet is characterized in that, in the first aspect, the spatial information pattern is an array of one - dimensional codes or two - dimensional codes indicating the position of the part in the whole. The spatial fractionating device according to the eighth aspect includes any one of the spatial information - imparting sheets according to the first to seventh aspects, a fine - structure blade having a blade structure for spatially partitioning and cutting, and a pressing device for pressing the fine - structure blade against a laminate in which the spatial information - imparting sheet is superposed on the tissue section. The fraction fragment recovery method according to the ninth aspect includes a laminating step of superposing any one of the spatial information - imparting sheets according to the first to seventh aspects and the tissue section to obtain a laminate, a spatial fractionating step of spatially partitioning and cutting the laminate, a recovery step of collectively recovering a plurality of fraction fragments obtained by cutting the laminate, a separating step of individually separating the recovered plurality of fraction fragments, and a position - specifying step of specifying the position of each fraction fragment in the tissue section from the partial pattern possessed by each of the plurality of fraction fragments. In the tenth aspect, the fraction fragment recovery method is characterized in that, in the ninth aspect, in the laminating step, the laminate is attached to a substrate with an adhesive, and in the recovery step, the adhesive is dissolved to collectively recover the plurality of fraction fragments from the substrate.

Advantages of the Invention

[0010] According to the first aspect, since each of the plurality of fraction fragments obtained by spatial fractionation has a partial pattern, even if the plurality of fraction fragments are collectively recovered, the position of each fraction fragment in the tissue section before fractionation can be specified. According to the second aspect, spatial information can be imparted to a tissue section by a random dot pattern formed of a plurality of microbeads. According to the third aspect, since the fluorescent microbeads emit light in a dark place, they can be easily distinguished from foreign substances attached to the tissue section, and the random dot pattern can be easily recognized. According to the fourth aspect, since the color of the microbeads can be used as one of the feature amounts of the pattern, it is easy to specify the position from the partial pattern to the whole. According to the fifth invention, since the probability that one or more microbeads are included in one fraction fragment is high, the yield of identifying the position of the fraction fragment can be increased. According to the sixth aspect, spatial information can be imparted to a tissue section by an uneven pattern. According to the seventh aspect, by reading the code attached to the fraction fragment, the position of the fraction fragment in the tissue section can be specified. According to the eighth aspect, since spatial fractionation is performed in a state where the tissue section and the spatial information imparting sheet are overlapped, the tissue section can be spatially fractionated while maintaining the spatial information. According to the ninth aspect, after collecting a plurality of fraction fragments at once, the position of each fraction fragment in the tissue section is specified, so that the plurality of fraction fragments can be collected with high throughput while being associated with spatial information. According to the tenth aspect, by dissolving the adhesive after spatial fractionation, a plurality of fraction fragments attached to the substrate can be easily collected at once.

Brief Description of Drawings

[0011]

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Figure 9

Embodiments for Carrying out the Invention

[0012] Next, embodiments of the present invention will be described based on the drawings. (Spatial fractionation device) As shown in FIG. 1, a spatial fractionation device AA according to an embodiment of the present invention is a device that spatially partitions and cuts a tissue section TS in a two - dimensional space. The tissue section TS is a sample obtained by slicing an animal or plant tissue. The spatial fractionation device AA partitions and cuts the tissue section TS, for example, in a honeycomb shape.

[0013] Hereinafter, the process of spatially partitioning and cutting an object in a two - dimensional space is referred to as "spatial fractionation". The smallest unit area of the partitioning is referred to as a "compartment". Also, a fragment obtained by spatially fractionating an object is referred to as a "fraction fragment". The shapes of the compartments and fraction fragments can be selected from various shapes such as hexagons, squares, and rectangles. Also, the dimensions of the compartments and fraction fragments can be set to various dimensions according to the purpose.

[0014] The spatial partitioning device AA has a spatial information - attaching sheet 1 that is superimposed on the tissue section TS. The spatial information - attaching sheet 1 is a sheet that attaches spatial information to the tissue section TS. "Spatial information" means position information in a two - dimensional space. By attaching spatial information to the tissue section TS, the position of the tissue section TS before fractionation of the fractionated fragments can be specified. The spatial information - attaching sheet 1 will be described in detail later.

[0015] The thing obtained by superimposing the spatial information - attaching sheet 1 on the tissue section TS is called a laminate 2. The spatial partitioning device AA has a substrate 3 that supports the laminate 2. The material of the substrate 3 is not particularly limited, but if paper is used, it is possible to suppress the damage of the micro - structure blade 4 due to the Poisson effect.

[0016] The spatial partitioning device AA has a micro - structure blade 4 and a presser 5 that presses the micro - structure blade 4 against the laminate 2. By pressing the micro - structure blade 4 against the laminate 2, the laminate 2 can be spatially partitioned. The micro - structure blade 4 has a blade structure that two - dimensionally partitions and cuts an object.

[0017] In the examples shown in FIGS. 2(A) and 2(B), the micro - structure blade 4 has a honeycomb - like blade structure. That is, the micro - structure blade 4 has a structure in which unit blades in the shape of a regular - hexagonal cylinder in plan view are arranged in a plane without gaps. The blade structure of the micro - structure blade 4 is not limited to the honeycomb - like shape, and various shapes such as a lattice shape and a slit shape can be adopted according to the shape of the required fractionated fragments.

[0018] The partition width W (width of the unit blade) of the micro - structure blade 4 can be arbitrarily set according to the purpose. If the partition width W is set to be approximately the same as the size of one cell included in the tissue section TS, individual cells can be separated by spatially partitioning the tissue section TS. Thereby, single - cell spatial omics analysis becomes possible. In this case, depending on the target cells, the partition width W may be set to 10 - 30 μm.

[0019] The sectional area W may be made larger than the area occupied by one cell. For example, the sectional area W may be set to 30 to 200 μm. In this case, analysis can be performed on a state where several to several tens of cells are grouped together. Although the positional resolution is lowered, the number of samples can be suppressed while comprehensively analyzing a wide range of tissues.

[0020] The sectional area W may be made smaller than the area occupied by one cell. For example, the sectional area W may be set to 2 to 30 μm. In this case, minute regions inside the cell can be analyzed.

[0021] The microstructured blade 4 having such a blade structure can be produced, for example, using semiconductor processing techniques such as silicon etching.

[0022] The press 5 may be any device that can press the microstructured blade 4 against the laminate 2. As the press 5, a hand press, an automatic press, or the like can be used.

[0023] By using the spatial fractionation device AA of the present embodiment, spatial fractionation can be performed in a state where the tissue section TS and the spatial information - imparting sheet 1 are superposed. Therefore, the tissue section TS can be spatially fractionated while retaining the spatial information.

[0024] (Spatial information - imparting sheet) Next, the spatial information - imparting sheet 1 will be described. As described above, the spatial information - imparting sheet 1 is spatially fractionated together with the tissue section TS in a state of being superposed on the tissue section TS. The spatial information - imparting sheet 1 has a spatial information pattern. The "spatial information pattern" means a pattern that can specify the position of a part in the whole from a partial pattern (sub - pattern) of the whole pattern.

[0025] When the laminate 2 obtained by superimposing the spatial information - imparting sheet 1 on the tissue section TS is spatially fractionated, a plurality of fraction fragments are obtained. Each fraction fragment is a superposition of a fragment of the tissue section TS and a fragment of the spatial information - imparting sheet 1. Therefore, the fraction fragment has a pattern (partial pattern) of a part of the entire spatial information pattern. Since the position of that part in the whole can be specified from the partial pattern, the position of each fraction fragment in the tissue section TS before fractionation can be specified.

[0026] The spatial information - imparting sheet 1 is not particularly limited as long as it has a spatial information pattern, but three examples will be described below.

[0027] (A) Random dot pattern As shown in FIG. 3, the spatial information - imparting sheet 1A of the first example has a random dot pattern as the spatial information pattern. The spatial information - imparting sheet 1A has a sheet base material 10 and a plurality of micro - beads 11 randomly dispersed inside the sheet base material 10. The plurality of dispersed micro - beads 11 form a random dot pattern. If the spatial information - imparting sheet 1A is superimposed on the tissue section TS, spatial information can be imparted to the tissue section TS by the random dot pattern.

[0028] FIG. 4 shows an example of the fraction fragment 6 obtained by spatially fractionating the spatial information - imparting sheet 1A. This fraction fragment 6 contains four micro - beads 11. The arrangement of these micro - beads 11 corresponds to the partial pattern. The characteristic amount of the partial pattern can be extracted from the arrangement of the micro - beads 11. For example, let the lengths of the line segments between any one micro - bead 11 and the other three micro - beads 11 be L 1 、L 2 、L 3 respectively. Also, let the angles formed by the three line segments be θ 1 、θ 2 、θ 3 respectively. These lengths L 1 、L 2 、L 3 and the angles θ 1 、θ 2 、θ 3It can be used as a feature amount of the partial pattern. By searching for a portion that matches the feature amount of the partial pattern from the entire random dot pattern, the position of the fraction fragment 6 can be specified.

[0029] The sheet base material 10 has translucency to such an extent that the internal microbeads 11 can be recognized. From the viewpoint of recognizing the microbeads 11, it is more preferable that the transmittance of the sheet base material 10 in the visible light region is higher, and 70% or more is preferable. Further, the sheet base material 10 can be cut by the fine structure blade 4 and is required not to affect the analysis of cells. Examples of the material of such a sheet base material 10 include resins such as polydimethylsiloxane (PDMS), polystyrene, cycloolefin polymer (COC), polyvinyl chloride (PVC), and polyethylene naphthalate (PEN).

[0030] The microbeads 11 have a color. It is preferable to use fluorescent microbeads as the microbeads 11. Since the fluorescent microbeads emit light in a dark place, they are easily distinguishable from foreign substances attached to the tissue section TS and are easy to recognize the random dot pattern. A fluorescence microscope is preferably used for recognizing the random dot pattern formed by the fluorescent microbeads.

[0031] The spatial information - imparting sheet 1A may contain microbeads 11 of one color or may contain microbeads 11 of a plurality of colors. If a random dot pattern is formed with microbeads 11 of a plurality of colors, the color of the microbeads 11 can be used as one of the feature amounts of the pattern. Therefore, it is easy to specify the position from the partial pattern to the whole.

[0032] The particle diameter of the microbeads 11 is preferably sufficiently smaller than the section frame W. Specifically, the particle diameter of the microbeads 11 is preferably 0.01 - 5% of the section frame W. Alternatively, the particle diameter of the microbeads 11 is preferably 0.01 - 10 μm.

[0033] In order to enable identification of the position of the fraction fragment 6, it is preferable that one or more microbeads 11 are included in one section. As shown in the following formula, assuming that the probability that k events in which an average of λ microbeads 11 enter one section follows a Poisson distribution, in order to make the probability of k = 0 0.01% or less, λ>9.2 is sufficient. [Number]

[0034] Therefore, it is preferable that the average number of microbeads 11 contained in the spatial information-imparting sheet 1A is 9.2 or more per section. In this way, the probability that one fraction fragment 6 contains one or more microbeads 11 is high (99.99% or more), so the yield of identifying the position of the fraction fragment 6 can be increased.

[0035] The larger the number of microbeads 11 contained in the spatial information-imparting sheet 1A, the more feature amounts of the pattern, and thus the easier it is to identify the position of the fraction fragment 6. However, it is preferable that the microbeads 11 do not overlap and are in a separated state. Therefore, the upper limit of the average number of microbeads 11 depends on the particle size of the microbeads 11. Under general conditions, the average number of microbeads 11 per section may be set to 20 or less.

[0036] The spatial information-imparting sheet 1A can be obtained, for example, by mixing microbeads 11 in a liquid resin, thinning it, and curing the resin. Thereby, a spatial information-imparting sheet 1A with a thickness of 3 to 50 μm can be obtained.

[0037] (B) Concavo-convex pattern The spatial information-imparting sheet of the second example has a concavo-convex pattern provided on the sheet surface as a spatial information pattern. The spatial information-imparting sheet having a concavo-convex pattern can be formed by, for example, methods such as injection molding and photolithography. The concavo-convex pattern can be composed of any pattern, figure, character, symbol, etc. If this spatial information-imparting sheet is superimposed on the tissue section TS, spatial information can be imparted to the tissue section TS by the concavo-convex pattern.

[0038] (C) Code array As shown in FIG. 5(A), the spatial information-providing sheet 1C of the third example has a code array as a spatial information pattern. The code array is an array of one-dimensional codes or two-dimensional codes. Each code indicates the position of its part in the whole pattern. The array of codes may be a staggered array or a lattice array. However, it is preferable that the codes are arranged for each section at positions corresponding to the sections.

[0039] As the code, for example, the two-dimensional code shown in FIG. 5(B) may be adopted. This two-dimensional code has a direction mark 12 at the upper left. Further, the two-dimensional code has circular dots indicating row numbers in the upper right half region 13 and rectangular dots indicating column numbers in the lower left half region 14. The row number and the column number can be indicated by binary numbers according to the presence or absence of each dot. Note that the code can be formed in the same manner as the concavo-convex pattern.

[0040] By using the spatial information-providing sheet 1C, a code indicating the position can be attached to each of the fraction fragments. By reading the code attached to the fraction fragment, the position of the fraction fragment in the tissue section TS can be specified.

[0041] (Fraction fragment recovery method) Next, based on FIGS. 6 to 8, a fraction fragment recovery method will be described. Note that (1) to (6) in FIGS. 6 to 8 correspond to the numbers assigned to the steps described below, respectively.

[0042] (1) Laminating step First, the spatial information-providing sheet 1 and the tissue section TS are overlapped to obtain a laminate 2. When the tissue section TS is dried after being placed on the spatial information-providing sheet 1, the tissue section TS adheres to the spatial information-providing sheet 1. Thereby, spatial information can be imparted to the tissue section TS.

[0043] (2) Overall pattern acquisition step Next, photograph the laminate 2 with a microscope camera to obtain an overall image of the spatial information pattern. Note that the acquisition of the overall pattern may be performed before overlaying the spatial information - imparting sheet 1 on the tissue section TS. Also, when the spatial information pattern is known, such as when the spatial information - imparting sheet 1 has a concavo - convex pattern or a code array, this step can be omitted.

[0044] (3) Spatial fractionation step Next, press the fine - structure blade 4 against the laminate 2 to spatially fractionate the laminate 2. As a result, a plurality of fraction fragments 6 are obtained. Each fraction fragment 6 is a superposition of a fragment of the tissue section TS and a fragment of the spatial information - imparting sheet 1. Therefore, the fraction fragment 6 has a partial pattern.

[0045] (4) Recovery step Next, collectively recover the plurality of fraction fragments 6 obtained by cutting the laminate 2. For example, recover the plurality of fraction fragments 6 in a separated state into a liquid.

[0046] (5) Separation step Next, individually separate the plurality of fraction fragments 6 recovered in the liquid and transfer them, for example, to a well plate. This operation may be performed manually using a micropipette or automatically using a dispensing robot.

[0047] (6) Position identification step Next, photograph each fraction fragment 6 with a microscope camera to obtain an image of the partial pattern that the fraction fragment 6 has. Then, search for the part that matches the partial pattern in the spatial information pattern to identify the position of the fraction fragment 6. Repeat this to identify the position of each of the plurality of fraction fragments 6 in the tissue section TS of the fraction fragment 6 from the partial pattern that the fraction fragment 6 has.

[0048] Thus, since each of the plurality of fraction fragments 6 obtained by spatial fractionation has a partial pattern, even if the plurality of fraction fragments 6 are collected together, the position of each fraction fragment 6 in the tissue section TS before fractionation can be specified. Also, after collecting the plurality of fraction fragments 6 together, since the position of each fraction fragment 6 in the tissue section TS is specified, the plurality of fraction fragments 6 can be collected with high throughput while being associated with spatial information.

[0049] (7) Analysis step Finally, the fraction fragment 6 is subjected to analysis. For example, by introducing a surfactant or a digestive enzyme into each well or irradiating with ultrasonic waves, unnecessary structures in the cell membrane and inside the cell contained in the fraction fragment 6 are destroyed, and the target molecule to be analyzed is diffused into the solution. Next, a marker molecule that specifically binds to the target molecule is introduced to label the target molecule in a detectable state. Thereafter, the target molecule is detected with a sensor or the like.

[0050] In the above fraction fragment collection method, in the collection step, the plurality of fraction fragments 6 are collected together. Usually, after spatial fractionation, the fraction fragment 6 is held inside the unit blade of the fine structure blade 4. Therefore, for example, by applying a liquid flow to the fine structure blade 4 or applying a centrifugal force, the fraction fragment 6 is separated from the fine structure blade 4 and collected. Alternatively, if processed according to the following procedure, the collection of the fraction fragment 6 becomes easy.

[0051] As shown in FIG. 9(A), in the lamination step, the laminate 2 is attached to the substrate 3 with an adhesive. That is, the substrate 3, the adhesive layer 7, the spatial information-providing sheet 1, and the tissue section TS are laminated in this order. An adhesive that can be dissolved later is used. For example, polyvinyl alcohol (PVAL) is suitable because it is soluble in water. Also, gelatin, agar, or the like can be used as the adhesive.

[0052] As shown in FIG. 9(B), in the fractionation step, the tissue section TS, the spatial information-imparting sheet 1, and the adhesive layer 7 are cut by the microstructured blade 4. Since the fractionated fragment 6 is adhered to the substrate 3 by the adhesive layer 7, as shown in FIG. 9(C), even if the microstructured blade 4 is pulled up, the fractionated fragment 6 remains on the substrate 3 side. That is, no fractionated fragment 6 remains on the microstructured blade 4.

[0053] In the recovery step, when the substrate 3 with the fractionated fragments 6 attached is placed in warm water, the adhesive dissolves. As a result, a plurality of fractionated fragments 6 can be recovered from the substrate 3 all at once. In this way, by dissolving the adhesive after spatial fractionation, a plurality of fractionated fragments 6 attached to the substrate 3 can be easily recovered all at once.

Explanation of Reference Numerals

[0054] AA Spatial fractionation device TS Tissue section 1 Spatial information-imparting sheet 10 Sheet base material 11 Microbeads 2 Laminate 3 Substrate 4 Microstructured blade 5 Presser 6 Fractionated fragment

Claims

1. A sheet that is cut together with a tissue section in a state where the sheet is superimposed on the tissue section, and is spatially compartmentalized, A spatial information pattern that allows the position of a part in the whole to be identified from the partial pattern A spatial information imparting sheet.

2. A light-transmitting sheet base material; A plurality of microbeads randomly dispersed within the sheet substrate; The spatial information pattern is a random dot pattern formed by the plurality of microbeads.

2. The spatial information imparting sheet according to claim 1.

3. The microbeads are fluorescent microbeads.

3. The spatial information imparting sheet according to claim 2.

4. The plurality of microbeads includes microbeads of multiple colors.

3. The spatial information imparting sheet according to claim 2.

5. The average number of microbeads per compartment is 9.2 or more.

3. The spatial information imparting sheet according to claim 2.

6. The spatial information pattern is a concave-convex pattern applied to the surface of the sheet.

2. The spatial information imparting sheet according to claim 1.

7. The spatial information pattern is a one-dimensional or two-dimensional code sequence that indicates the location of the part in the whole.

2. The spatial information imparting sheet according to claim 1.

8. A spatial information imparting sheet according to any one of claims 1 to 7, A microstructured blade having a blade structure that cuts in a spatially compartmentalized manner; and a presser for pressing the fine structure blade against a laminate obtained by superposing the spatial information imparting sheet on the tissue slice. A spatial fractionation device comprising:

9. A lamination step of superposing the spatial information imparting sheet according to any one of claims 1 to 7 on the tissue section to obtain a laminate; A spatial partitioning step of spatially partitioning and cutting the laminate; a recovery step of collectively recovering a plurality of fraction pieces obtained by cutting the laminate; a separation step of separating the collected fraction fragments into individual fragments; and a position specifying step of specifying a position of each of the plurality of fraction pieces in the tissue section based on the partial pattern of the fraction piece. A method for recovering a fraction comprising the steps of:

10. In the lamination step, the laminate is attached to a substrate with an adhesive; In the recovering step, the adhesive is dissolved to recover the plurality of fractional pieces from the substrate at once. The method for recovering a fraction according to claim 9 .

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