JP2017142250A - Ultrahigh density tissue array - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means which achieves a tissue array usable for a comprehensive assay.SOLUTION: A method for manufacturing an ultrahigh density tissue array includes: (1) a H-segment preparation step of hollowing out and collecting a part a tissue piece formed by slicing a tissue block with a hollow needle; (2) a binder piece preparation step of hollowing out and collecting a sheet of a binder with a hollow needle holding H-segment in a hollow; (3) a lamination step of repeating the steps (1) and (2) to laminate the H-segment and a binder piece in a longitudinal direction in the hollow needle; (4) a H-block preparation step of taking out, heating and fusing the laminate from the hollow needle; (5) an embedding step of directing the H-block in a lateral direction; (6) a V-segment preparation step of slicing the embedded H-block in a horizontal direction with microtome; (7) a V-block preparation step of laminating the V-segment while aligning one side of a long side of the V-segment; and (8) an ultrahigh density tissue array sheet production step of embedding the V-block in a direction at which all of the tissues are exposed to the upper surface, and slicing the V-block in a lamination direction of the V-segment.SELECTED DRAWING: None

Description

  The present invention relates to an ultra-high-density tissue array, and relates to a method for manufacturing a tissue array chip used for examination and analysis of a living tissue.

  Conventionally, a tissue array chip in which tissue pieces are arranged on a substrate has been used for examination and analysis of a living tissue. The tissue array chip is used for examination of the presence or absence of a diseased tissue, analysis of genes and proteins, screening, and the like by applying a staining solution that specifically stains a test substance.

  This tissue array chip is manufactured as follows. FIG. 11 is an explanatory diagram for explaining a conventional tissue array chip manufacturing method. (A) A core is collected by punching a tissue block obtained by blocking a living tissue. (B) The collected cores are inserted into holes provided in an array on the base block. (C) The surface of the base block in which the core is inserted into each hole is sliced with a thickness of several μm to produce a tissue array sheet in which tissue pieces are arranged. (D) A tissue array chip is fabricated by mounting the tissue array sheet on a substrate. Further, (c) and (d) are repeated to produce a plurality of tissue array chips.

  The tissue array block used for manufacturing the tissue array chip is manufactured by an apparatus having a punching mechanism. Patent Documents 1 and 2 include a punch for a base block and a punch for a tissue block. A hole is formed in the base block with the punch for the base block, and the tissue block is punched with the punch for the tissue block. An apparatus is disclosed in which a core is collected and the collected core is inserted into a hole in a base block.

Patent Documents 1 and 2 are methods for producing a tissue array in which a hole is formed in a tissue block, a core is extracted, and the core is transplanted to a recipient block. The following cases that cannot be produced by these coring methods are often assumed. (1) The tissue cannot be observed again in the coring site. For this reason, it may not be approved by the Ethics Committee. (2) There are facilities that prohibit the taking out of tissue blocks to the outside, and in such facilities, arrays cannot be produced by multi-institutional joint research. (3) Tissue blocks of interesting cases and valuable cases are important, and it is extremely difficult to make holes. (4) The extraction of the core is limited to a tissue block having a certain thickness. (5) When a target lesion such as a tumor is flying away, it is difficult to cover the entire lesion with a normal core. (6) Since a tissue block having a different thickness is used, a core chip occurs.
As means for solving the above problem, the present inventors have developed a technique (spiral tissue array) for producing a tissue array block and a tissue array sheet from a sliced tissue piece formed in a roll shape (Patent Document 3). In addition, the present inventors have developed a technique (pile-up tissue array) for producing a tissue array block and a tissue array sheet from sliced tissue pieces sliced from the tissue block (Patent Document 4).
In addition, a technique for producing a tissue array having a maximum of about 10,000 tissues per slide glass is also known (Patent Document 5, Non-Patent Document 1).

JP 2004-215667 A JP 2006-158394 A Japanese Patent No. 4625909 Japanese Patent No. 5629939 US2006 / 0257908A1

Nature Methods, Vol. 2 (7), 511-513, 2005

  The spiral tissue array is an excellent technique that substantially clears the drawbacks of conventional tissue arrays produced by punching. However, in the spiral tissue array, since the sliced tissue pieces are formed in a roll shape, the core diameter increases due to winding. On the other hand, since the tissue array chip is placed on a slide glass (2.5 cm × 7.5 cm) for observation, it is usually 2 cm × 3 cm in size. Therefore, in the spiral tissue array, the number of tissues placed on one sheet is limited to about several tens. The pile-up tissue array can produce one sheet with the same number of tissues as the number of tissues on the tissue array chip produced by conventional punching, but it is a comprehensive and high-throughput assay by collecting more tissues. There is a need for tissue arrays that can be used for In view of such circumstances, the present invention has an object to propose a new tissue array block manufacturing method and tissue array chip.

  As a result of intensive studies on the means for mounting the tissue on the slide glass at an extremely high density, the present inventors made an H-block in which the tissue was arrayed in the horizontal direction, and obtained a V-segment supplied from the H-block. By producing V-blocks that are stacked in an array in the vertical direction, the present inventors have succeeded in efficiently integrating tissues in three dimensions, thereby completing the present invention.

The present invention provides the following.
[1] (1) A step of cutting out a portion from a tissue piece sliced from a tissue block with a hollow needle and preparing an H-segment;
(2) The hollow needle holding the H-segment in the hollow, the sheet comprising the H-segment binder is cut out and prepared, and a binder section is prepared;
(3) A step of repeating steps (1) and (2) to alternately stack H-segments and binder sections in the vertical direction in the hollow needle,
(4) taking the laminate from the hollow needle, heating and fusing it, and producing an H-block;
(5) the step of embedding the H-block in the lateral direction;
(6) The embedded H-block is sliced horizontally with a microtome to produce a V-segment;
(7) A step of laminating the V-segment with one long side aligned to produce a V-block;
(8) embedding the V-block in the direction in which all tissues are exposed on the upper surface, and slicing in the V-segment lamination direction to produce an ultra-high-density tissue array sheet.
A method for producing an ultra-high density tissue array sheet.
[2] (1) A step of cutting out a part from a tissue piece sliced from a tissue block with a hollow needle and preparing an H-segment;
(2) The hollow needle holding the H-segment in the hollow, the sheet comprising the H-segment binder is cut out and prepared, and a binder section is prepared;
(3) A step of repeating steps (1) and (2) to alternately stack H-segments and binder sections in the vertical direction in the hollow needle,
(4) taking the laminate from the hollow needle, heating and fusing it, and producing an H-block;
(5) the step of embedding the H-block in the lateral direction;
(6) The embedded H-block is sliced horizontally with a microtome to produce a V-segment;
(7) A step of laminating the V-segment with one long side aligned to produce a V-block;
(8) a step of embedding the V-block in a direction in which all tissues are exposed on the upper surface, and slicing in a V-segment stacking direction to produce an ultra-high-density tissue array sheet;
(9) including a step of arranging the sheet on the substrate,
Manufacturing method of ultra-high density tissue array chip.
[3] The step (9) includes an operation of arranging the plurality of sheets obtained in the step (8) on a substrate made of paraffin, and after the step (9), the plurality of sheets and the substrate. The manufacturing method as described in [2] including the process of heating and uniting these.
[4] The method according to any one of [1] to [3], wherein the H-segment has a thickness of 50 μm or more and less than 400 μm.
[5] The production method according to any one of [1] to [4], wherein the binder section has a thickness of 40 μm to 400 μm.
[6] The production method according to any one of [1] to [5], wherein the binder is paraffin.
[7] The production method according to any one of [1] to [6], wherein the hollow needle has a circular, square, or polygonal hollow shape.
[8] The hollow needle has a circular shape having an inner diameter of 0.5 to 5.0 mm, or a rectangular or polygonal hollow shape having a long side and a short side of 0.5 to 5.0 mm, [1] to [7 ] The manufacturing method in any one of.

The tissue array sheet manufacturing method and tissue array chip of the present invention have the following advantages of the spiral tissue array:
(1) A tissue array can be produced without making a hole in the tissue block. (2) It is possible to select only the lesioned part, and the target lesion site (site of interest) is comprehensively taken into the array. (3) Since coring can be performed while observing the inhomogeneity of the tissue, the heterogeneity can also be comprehensively captured. In addition to the fact that joint research of
(5) An ultra-high density tissue array mounted on a substrate can accumulate tissues of around 24,000 specimens.
According to the present invention, it is possible to provide an ultra-high density tissue array chip that covers major tumors throughout the body. Using this chip, it is possible to comprehensively analyze the expression of candidate molecules and candidate signals for molecular target drugs. Further, according to the present invention, theoretically more than 1 million sections can be collected from the same tissue, and an extremely high banking function can be exhibited. Expression in a tissue of candidate molecules discovered by NGS (next-generation sequencing) or the like can be examined across the body and exhaustively on the same chip. According to the present invention, it is possible to realize an ultra-high-density array not only from a tissue but also from a cell line and the like, and it is also possible to acquire expression information of a target molecule in about 24000 cell lines at once.

It is a conceptual diagram of the manufacturing method of the ultra-high-density tissue array of this invention. An embodiment of the H-segment production process is shown. An embodiment of a process for producing an H-block from an H-segment is shown. An embodiment of a process for producing a V-segment from an H-block is shown. An embodiment of a process for producing a V-block from a V-segment is shown. FIG. 6 shows one embodiment of a process for producing a section from a V-block and attaching the section to a substrate. It is a hematoxylin-eosin dyeing | staining figure of the tissue array chip | tip mounted with 24000 specimens obtained by the manufacturing method 2 of this invention. It is the immuno-staining figure of the tissue array chip | tip mounted with 24000 specimens obtained by the manufacturing method 2 of this invention, and the one part enlarged view. It is the hematoxylin-eosin dyeing | staining figure of the tissue array chip | tip mounted with 24000 specimens obtained by the manufacturing method 2 of this invention, and the one part enlarged view. It is a figure which shows the ultra-high-density structure | tissue array chip | tip produced without (a) of heating and adhesion | attachment operation, and (b). It is explanatory drawing explaining the conventional tissue block, tissue array block, tissue array sheet, and tissue array chip.

Definitions In the present invention, “tissue block” refers to a tissue derived from a living body to be subjected to an ultrahigh density tissue array. The tissue block is typically a paraffin-embedded tissue block, but may be a frozen tissue block. The tissue block may be a cell cluster (also referred to as a cell block) obtained by culturing a cell line in two dimensions or three dimensions. In the case of a cell block, the cells to be used may be fixed by a conventional method and embedded in paraffin, or may be frozen by a conventional method.

  In the present invention, the “ultra high density tissue array block” refers to a block of tissue prepared from the tissue block according to steps (1) to (7) of the production method of the present invention and arranged in high density. -Also referred to as a block.

  In the present invention, the “ultra high density tissue array sheet” means a sheet obtained by slicing the “ultra high density tissue array block (V-block)”, and is also simply referred to as a section.

  In the present invention, the “ultra high density tissue array chip” refers to a substrate in which one or more of the “ultra high density tissue array sheets” are mounted on a substrate.

  In the present invention, the “substrate” refers to a material that forms the basis of the “ultra high density tissue array chip”. A typical example of the substrate is a slide glass, but it may be a plastic plate or a paper material. As will be described later, it is also preferable to use a substrate made of paraffin material. In this case, the assembly of tissue pieces can be performed on paraffin.

  In the present invention, “ultra high density tissue array” and “ultra high density tissue microarray” are used interchangeably.

  The concept of the manufacturing method of the ultra-high density tissue array chip of the present invention is shown in FIG.

Manufacturing method of ultra-high density tissue array sheet (Manufacturing method 1)
The manufacturing method of the ultra-high-density tissue array sheet of the present invention includes the following steps (1) to (8).
(1) Step of preparing a H-segment by extracting a portion from a tissue piece sliced from a tissue block with a hollow needle and (2) With the hollow needle holding the H-segment in the hollow, Cutting out a sheet of binder and preparing a binder section; and
(3) Repeat steps (1) and (2) to stack the H-segments and binder sections alternately in the vertical direction in the hollow needle (4) Take out the laminate from the hollow needle and heat (5) Step of embedding the H-block in the horizontal direction (6) Slicing the embedded H-block horizontally with a microtome Step of creating segment (7) Laminating the V-segment with the long sides aligned, and step of creating V-block (8) Orienting the V-block in the direction in which all the structures are exposed to the upper surface Embedding and slicing in the V-segment lamination direction to produce an ultra-high density tissue array sheet (section)

  Manufacturing method 1 will be described with reference to FIGS. 2-6 is an example of a manufacturing method. In the drawings and in the description of the following processes, paraffin is used as a binder for bonding between the segments, partitioning, and protecting the surface of the segments. By paraffin is meant paraffin containing paraffin alone or synthetic polymer additives such as polyethylene. The binder in the present invention is not limited to paraffin, and examples thereof include cyanoacrylate, carboxymethyl cellulose, polyethylene glycol, and polyvinyl alcohol.

(1) Step of preparing H-segment (Figure 2)
A tissue piece 1 is produced from a paraffin-embedded tissue block A of a case (specimen) used for producing a tissue microarray (TMA) using a microtome. The thickness of the tissue piece is usually 50 μm-400 μm, preferably 50 μm-250 μm, more preferably 150 μm-200 μm. Here, when slicing at about 200μm, keep the tissue block A in a 35-40 ℃ oven in advance or hold it in a hot tub of the same temperature for about 30 seconds to keep the section clean without cracking. Can be sliced.
The section to be mounted on the TMA in the tissue piece is determined in advance by microscopic observation or the like, and a part of the tissue piece 1 (1 section in the figure) is cut out and collected using a tissue arrayer equipped with a hollow needle. , H-segment 2 is produced. The operation of hollowing out the H-segment 2 from the tissue piece 1 with a hollow needle is performed while heating the tissue piece 1. The heating temperature is desirably a temperature that does not exceed the melting point of the paraffin used in the tissue block A.
The hollow of the hollow needle defines the shape of the H-segment, and examples of the shape include a circle, a square, a pentagon, and a hexagon. A circular or square hollow needle is preferable, and a square hollow needle is more preferable. The hollow needle is usually made of metal, but is not limited to metal as long as it is resistant to the heating conditions in the step (4). The length of the hollow needle is usually 1-5 cm. In FIG. 2, a hollow needle having a circular hollow is used.
When the hollow of the hollow needle is circular, it preferably has an inner diameter of 0.5 to 5.0 mm. When the hollow of the hollow needle is a quadrangle, it is preferable to have a long side and a short side of 0.5 to 5.0 mm. Here, the long side and the short side may have the same length. In the case where the hollow is a polygon such as a pentagon or a hexagon, the size may be appropriately set according to the circular inner diameter.
One H-segment has a thickness (height) of usually 50 μm-400 μm, preferably 50 μm-250 μm, more preferably 150 μm-200 μm, and its shape has a diameter of 0.5-5. It is a 0 mm cylinder or a rectangular column having a long side and a short side of 0.5 to 5.0 mm, and preferably a square column having a bottom of 0.5 to 5.0 mm on one side.

(2) A step of cutting out and collecting a sheet made of the H-segment binder with the hollow needle holding the H-segment in the hollow to prepare a binder section (FIG. 2)
Paraffin and other materials used as binders are made into sheets. In this case, when a sliced sheet made of a binder can be used, it may be used for this step as it is. When the binder can be used as a block, a sheet made of the binder is prepared using a microtome according to the method for producing a tissue piece from the tissue block in step (1).
When paraffin is used as the binder, the paraffin block B is prepared, and the paraffin sheet 3 is prepared using a microtome.
The sheet made of paraffin or other binder is set to a thickness of 0.2 to 2.0 times, preferably 0.5 times the thickness of the tissue piece. For example, for a 200 μm tissue piece, the thickness of the paraffin sheet is 40-400 μm, preferably 150-250 μm.
Using the tissue arrayer provided with the hollow needle for holding the H-segment in the hollow used in the step (1), the sheet 3 made of the binder is cut out and collected to produce the binder section 4. When paraffin is used as the binder, the operation of hollowing out the binder section 4 from the sheet 3 with a hollow needle is performed while heating the sheet 3. The heating temperature is preferably a temperature that does not exceed the melting point of the paraffin used as the binder.

(3) Repeating steps (1) and (2) to stack H-segments and binder sections alternately in the vertical direction in the hollow needle (FIG. 3)
In this step, a plurality of H-segments 2 and binder slices 4 are alternately stacked in the vertical direction. For this purpose, the steps (1) and (2) are repeated. Since the binder slice 4 also has an effect of protecting the tissue surface of the H-segment 2, the process (2) → the process (1) → the process (2) → the process (1)... → the process (2) In addition, it is desirable that the repeating step starts from step (2) and ends at step (2).
In order to achieve an ultra-high-density array, 5-50 H-segments 2 are usually stacked, preferably 10-30. The binder slices 4 may be the same as the number of H-segments, but it is desirable to stack a number one more than the number of H-segments.

(4) Step of removing the laminate from the hollow needle and heating and fusing it to make an H-block (Figure 3)
In step (3), a desired number of H-segments 2 are laminated in the vertical direction, and a laminate (columnar body) in which the binder slice 4 is sandwiched between the segments is obtained. One or both of the upper part and the lower part of the laminate are composed of the binder section 4.
The laminate is taken out from the hollow needle, the periphery of the laminate is heated at a temperature equal to or lower than the melting point of the material used as the binder, and then the laminate is fused by cooling. Thereby, the H-block 5 is produced. For example, an H-block made of 20 tissue pieces 2 (thickness 200 μm) and 20 paraffin sections 4 (thickness 100 μm) using a hollow needle with a diameter of 3 mm is a cylinder with a diameter of 3 mm and a height of 6 mm. Become. The heating temperature and heating time of the laminate taken out from the hollow needle can be appropriately set according to the common general technical knowledge in the technical field. If paraffin is used as the binder, heat around the laminate at 40-60 ° C. The heating time can be appropriately set according to the heating temperature, but is usually 5-120 minutes, preferably 10-90 minutes.

(5) Embedding the H-block in the horizontal direction (Fig. 3)
H-block 5 is embedded laterally with paraffin. Embedding is carried out in the same manner as the paraffin embedding method for tissues usually used in the art.

(6) Process of producing V-segment by slicing the embedded H-block horizontally with a microtome (FIG. 4)
Next, the H-block 5 embedded in paraffin is sliced with a microtome in the horizontal direction. It is desirable that the cylindrical H-blocks have the same size by chamfering (C1) before being sliced. Further, the double-sided tape 6 is cut in accordance with the size of the H-block 5.
A double-sided tape 6 is affixed on the surface of the H-block 5 ′ that is surfaced if necessary, and is sliced with a microtome at a lower position (C 2) parallel to the affixed surface to produce a V-segment 7. The thickness of the V-segment 7 is set equal to the thickness of the H-segment 2. Preferred V-segment thickness is 50 μm-400 μm, preferably 50 μm-250 μm, more preferably 150 μm-200 μm.
Another double-sided tape 6 is affixed on the remaining H-block 5 ′ after V-segment excision, and sliced with a microtome at a lower position (C2) parallel to the affixed surface, and the next V-segment 7 is produced. This operation is repeated to obtain a predetermined number of V-segments.
Or you may perform this process, without using a double-sided tape. In that case, the mutual adhesion of the V-segment 7 surface in the step (7) can be performed, for example, by adhesion with heating as described later.

(7) Step of fabricating the V-block by stacking the V-segments with the long side aligned on one side (FIG. 5)
The number of V-segments obtained can be determined from the size of the H-block and the thickness of the V-segment. Among them, the number of sheets used for producing the V-block is set according to the number of mounted ultra-high-density tissue arrays as the final target. For example, when supplying a section to be mounted on a substrate from a V-block consisting of 20 H-segments and 100 V-segments, 2000 specimens are aligned in one section, and a maximum of 12 sections are 1 sheet. Because it can be mounted on the same substrate, a tissue microarray of 24,000 specimens is completed. 24000 specimens are the goal of the ultra-dense tissue array of the present invention, and in order to achieve the purpose of the present invention, the segment of the segment is such that the product of the number of H-segments and the number of V-segments is approximately 2000. It is sufficient to set the number of. The product may be less than 2000 or more than 2000 in light of the intended use of the tissue microarray.
The obtained V-segments 7 are bonded to each other with a double-sided tape attached on the tissue. Or the mutual adhesion | attachment of the V-segment 7 surface can also be performed by the method with a heating. That is, the periphery of the laminate of V-segments 7 is heated to at least partially melt the binder (eg, paraffin) contained in the laminate. Conditions such as the heating temperature and the heating time may be the same as those for manufacturing the H-block 5 (step (4)). The V-segments 7 in which the binder is at least partially melted by heat treatment are brought into contact with each other (preferably pressed), and in this state, the binder is solidified again to adhere the surfaces of the V-segments 7 to each other. By performing the heating / bonding operation in this manner, it is possible to maintain the bonding or pressure bonding force between the binders.
The above work is performed while confirming with a stereomicroscope. When V-segments are made from cylindrical H-blocks, etc., when the lengths of the short sides of the segments are not aligned, the V-segments are stacked so that at least one of the long sides of the V-segments matches. I will do it. In this way, a V-block 8 in which a predetermined number of V-segments 7 are laminated can be produced.

(8) Embedding the V-block in the direction in which all tissues are exposed on the upper surface and slicing in the V-segment stacking direction to produce an ultra-high density tissue array sheet (section) (FIG. 6). )
By embedding the V-block 8 with the surface matched on one side of the long side of the V-segment facing upward, all the tissue appears on the upper surface. Embedding is carried out in the same manner as the paraffin embedding method for tissues usually used in the art. A tape 9 having a size covering the upper surface is affixed, and is sliced with a microtome at a predetermined position (C3) below and parallel to the affixed surface, and a section 10 is produced. Therefore, the cutting direction of the V-block is the stacking direction of the V-segments.
Examples of the tape used here include a protective seal, a tissue protector S for slide glass (Pasology Laboratories, Inc.), a transfer seal for tissue banks (BioBank KIT, Pasology Laboratories, Inc.), and the like. Similar sections can be made without the use of tape if the technology is capable of slicing with a uniform thickness without using tape.
The thickness of the section can be set to a thickness suitable for microscopic observation. Usually, it is 1 μm-20 μm, preferably 1 μm-10 μm, more preferably 4 μm-6 μm.
By repeating the above operation, a desired number of sections can be obtained.

Manufacturing method of ultra-high density tissue array chip (Manufacturing method 2)
The production method 2 of the present invention includes the following step (9) in addition to the steps (1) to (8).

(9) Step of arranging the ultra-high density tissue array sheet (section) obtained in step (8) on the substrate (FIG. 6)
The section 10 to which the tape is attached obtained in the step (8) is arranged on the substrate, and the tissue is transferred from the tape to the substrate.
The glass slide 11 is preferably used as the substrate. A typical glass slide is 2.5 cm × 7.5 cm in shape, and the sections are usually arranged in a 2 cm × 6 cm section. As a method for transferring the tissue from the tape to the substrate, there is a method in which the section is immersed in water and wetted, and lightly pressed onto the substrate to attach the tissue.
In this way, an ultra-high density tissue array chip can be produced.

Additionally or alternatively, step (9) can also be performed by a method involving heating as described below.
The plurality of ultra-high density tissue array sheets (sections) obtained in step (8) are heated and bonded together. That is, for example, the plurality of sections 10 arranged on the substrate are heated, and the binder (for example, paraffin) included in each section 10 is at least partially melted. Conditions such as the heating temperature and the heating time may be the same as those for manufacturing the H-block 5 (step (4)). The sections 10 in which the binder is at least partially melted by the heat treatment are brought into contact with each other (preferably pressed) in the horizontal direction, and the sections 10 are adhered to each other by solidifying the binder again in this state. By performing such a heating step and an adhesion step, it is possible to control the adhesive force between the binders, so in the produced array chip, it is possible to prevent the occurrence of gaps between the cores (sections), In addition, a plurality of cores can be easily mounted on the same block.
In a preferred embodiment, the plurality of sections 10 are preliminarily arranged in a predetermined arrangement on a paraffin substrate and then subjected to the heating and bonding treatment described above, thereby fusing the sections 10 and fusing the section 10 and the paraffin substrate. I do. As a result, assembly can be performed on paraffin, and the tissue can be easily transferred onto the paraffin substrate. As the paraffin substrate, a substrate having the same shape and size as those of the substrate described above with respect to the step (8) can be used. In this way, a high density tissue array chip on paraffin can be produced.
The above heating / bonding operations can be performed, for example, on a stereomicroscope equipped with a hot plate stage.

  The ultra-high density tissue array chip of the present invention can be subjected to a tissue cytochemistry method well known in the art. In that case, deparaffinization is performed, but if necessary, the tape can be removed by leaving the chip in xylene used for deparaffinization. The tissue arranged on the chip can be observed with a microscope. An example of a staining photograph is shown in Fig. 7-9.

The arrays in FIGS. 8 and 9 are arrays on which 24,000 specimens are mounted, and are made from specimens for 24,000 people. For example, specimens for 4000 cases are used for 6 fields per case. This makes it possible to produce an array with 24,000 specimens. In this case, multiple visual fields can be confirmed per case.
The ultra-high-density tissue array has a relatively small area per specimen and a narrow field of view in microscopic observation, but it can cover a narrow field of view by mounting multiple specimens per case. Can do. Furthermore, there is also an advantage that it is easy to compare non-uniformities in tissues by mounting a large number of distant parts of the same tissue.

  FIG. 10 shows the difference between the ultra-high density tissue array chips produced with or without the above heating and bonding operations. FIG. 10A shows an array chip on a slide glass manufactured without heating and bonding operations. In the figure, paraffin was stained in blue, a gap was generated as indicated by the black part (arrow), and a plurality of rows could not be mounted on the same block. On the other hand, FIG. 10B shows an array chip on paraffin produced by heating and bonding operations. By performing this operation, it can be seen that the gaps between the cores were eliminated and a plurality of cores could be mounted on the same block. In this example, 20,000 tissue pieces could be placed on paraffin. This made it easy to perform many 10-20 types of immunostaining simultaneously in almost the same visual field. In addition, since it is no longer necessary to assemble on a slide glass, it is possible to assemble on paraffin, so that it can be sampled as a tissue section by slicing into thin sections other than the final process. .

  In the present embodiment, the tissue block is described as an example of a paraffin-embedded tissue block. However, the present invention is not limited to this, and for example, a frozen tissue block in which a tissue is frozen may be used. In this case, an embedding agent called a compound is used instead of paraffin. It is preferable to freeze the tissue in a state where it is embedded in the compound and to carry out the above steps in a cooling environment in which the frozen state can be maintained. In this case, heating is not performed, or the temperature and the heating time are adjusted so as to be in a moderately flexible state. Other modifications can be made as appropriate within the scope of the object of the present invention.

  The present invention can greatly contribute to the field of cancer diagnosis and treatment. For example, according to the present invention, it is possible to produce an ultra-high-density tissue array chip that covers major tumors throughout the body, and to comprehensively analyze the expression of candidate molecules and candidate signals for molecular target drugs. In the present invention, theoretically more than 1 million sections can be collected from the same tumor, and it has an extremely high banking function. Not only tissues but also cell lines can be treated in the same way, and expression of 24,000 cell lines can be acquired at once.

  Although several specific embodiments of the present invention have been described in detail, those skilled in the art will recognize that various modifications may be made to the specific embodiments shown without departing from the teachings and advantages of the invention. It is possible to make changes. Accordingly, all such modifications and changes are intended to be included within the spirit and scope of the present invention as claimed in the following claims.

A Tissue block B Binder (paraffin) blocks C1, C2, C3 Cut surface 1 Tissue piece 2 H-segment 3 Binder (paraffin) sheet 4 Binder (paraffin) section 5 H-block 5 ′ surfaced H-block 6 Double-sided tape 7 V-segment 8 V-block 9 Tape 10 Section 11 Slide glass

Claims (8)

(1) A step of cutting out a portion from a tissue piece sliced from a tissue block with a hollow needle and preparing an H-segment;
(2) The hollow needle holding the H-segment in the hollow, the sheet comprising the H-segment binder is cut out and prepared, and a binder section is prepared;
(3) A step of repeating steps (1) and (2) to alternately stack H-segments and binder sections in the vertical direction in the hollow needle,
(4) taking the laminate from the hollow needle, heating and fusing it, and producing an H-block;
(5) the step of embedding the H-block in the lateral direction;
(6) The embedded H-block is sliced horizontally with a microtome to produce a V-segment;
(7) A step of laminating the V-segment with one long side aligned to produce a V-block;
And (8) embedding the V-block in a direction in which all tissues are exposed on the upper surface, and slicing the V-segment in the stacking direction of the V-segment to produce an ultra-high-density tissue array sheet. Manufacturing method of density tissue array sheet. (1) A step of cutting out a portion from a tissue piece sliced from a tissue block with a hollow needle and preparing an H-segment;
(2) The hollow needle holding the H-segment in the hollow, the sheet comprising the H-segment binder is cut out and prepared, and a binder section is prepared;
(3) A step of repeating steps (1) and (2) to alternately stack H-segments and binder sections in the vertical direction in the hollow needle,
(4) taking the laminate from the hollow needle, heating and fusing it, and producing an H-block;
(5) the step of embedding the H-block in the lateral direction;
(6) The embedded H-block is sliced horizontally with a microtome to produce a V-segment;
(7) A step of laminating the V-segment with one long side aligned to produce a V-block;
(8) a step of embedding the V-block in a direction in which all tissues are exposed on the upper surface, and slicing in a V-segment stacking direction to produce an ultra-high-density tissue array sheet;
(9) A method for producing an ultra-high-density tissue array chip, comprising the step of arranging the sheet on a substrate.   Step (9) includes an operation of arranging the plurality of sheets obtained in Step (8) on a substrate made of paraffin, and after the step (9), heating the plurality of sheets and the substrate. The manufacturing method of Claim 2 including the process of uniting these.   The manufacturing method of any one of Claims 1-3 whose thickness of H-segment is 50 micrometers or more and less than 400 micrometers.   The manufacturing method according to any one of claims 1 to 4, wherein the binder section has a thickness of 40 µm to 400 µm.   The manufacturing method of any one of Claims 1-5 whose binder is paraffin.   The manufacturing method according to claim 1, wherein the hollow needle has a circular, square, or polygonal hollow shape.   The hollow needle has a circular shape having an inner diameter of 0.5 to 5.0 mm or a rectangular or polygonal hollow shape having a long side and a short side of 0.5 to 5.0 mm. The production method according to item.