Method for producing formalin-fixed paraffin-embedded thin-layer sections, and method for producing slide specimens.

Abstract

The present invention provides a method for producing small formalin-fixed paraffin-embedded thin-layer sections and a method for producing slide specimens, both of which can be used to prepare small formalin-fixed paraffin-embedded thin-layer sections. [Solution] A thin-layer section obtained from formalin-fixed paraffin-embedded tissue is spread out, the thin-layer section is solidified, and then the solidified thin-layer section is cut to the desired size to obtain a formalin-fixed paraffin-embedded thin-layer section.

Description

[Technical Field]

[0001] The present invention relates to a method for producing formalin-fixed paraffin-embedded thin-layer sections and a method for producing slide specimens. [Background technology]

[0002] Methods for preserving biological tissue include flash freezing using dry ice or liquid nitrogen, and freezing the tissue after fixing it with a fixative. However, tissue samples prepared by these methods must be stored at temperatures below -80°C, and are therefore not commonly used in routine pathological diagnosis.

[0003] On the other hand, formalin-fixed paraffin-embedded (FFPE) samples can be stored for long periods at 4°C, and are therefore widely used not only in routine pathological diagnosis but also in basic research (see, for example, Patent Documents 1-3).

[0004] Incidentally, one technique that has become increasingly popular in recent years is spatial transcriptomics. Spatial transcriptomics makes it possible to obtain transcriptome information of cells within a living tissue while preserving the positional information of the tissue. For this reason, spatial transcriptomics has become increasingly popular in recent years to understand the characteristics of pathological and normal tissues.

[0005] Spatial transcriptome analysis is a technique that was selected as "Method of the Year" by Nature Methods in 2020. Therefore, while the demand for spatial transcriptome analysis is expected to increase significantly in the future, the cost of such analysis remains high.

[0006] When performing spatial transcriptome analysis on FFPE samples, it is necessary to thin the FFPE sample to prepare FFPE thin-layer sections and then analyze these FFPE thin-layer sections. Slide specimens in which FFPE thin-layer sections are attached to a glass slide are prepared, for example, by (i) obtaining FFPE thin-layer sections from an FFPE sample, (ii) spreading the FFPE thin-layer sections using 40°C water, and (iii) directly attaching the FFPE thin-layer sections, which have been suspended in water, to a glass slide. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2022 / 211084 [Patent Document 2] Japanese Patent Publication No. 2019-207201 [Patent Document 3] Japanese Patent Publication No. 2018-148904 [Overview of the project] [Problems that the invention aims to solve]

[0008] However, with the conventional techniques described above, the number of FFPE thin-layer sections that can be analyzed by spatial transcriptome analysis is limited to one sample. This is because spatial transcriptome analysis requires the FFPE thin-layer section to be attached to a narrow area (6.5 mm × 6.5 mm) on a glass slide. However, the minimum size of an FFPE thin-layer section that can be prepared by conventional methods is approximately 4.0 mm × 4.0 mm, so the number of FFPE thin-layer sections that can be attached to a narrow area on a glass slide is limited to one sample.

[0009] One aspect of the present invention aims to provide a method for producing formalin-fixed paraffin-embedded thin-layer sections and a method for producing slide specimens, which can produce small formalin-fixed paraffin-embedded thin-layer sections. [Means for solving the problem]

[0010] As a result of diligent research to solve the above problems, the inventors of the present invention have discovered that small thin-layer sections can be easily obtained by solidifying an extended thin-layer section and then cutting the said thin-layer section, and have completed the present invention. The present invention encompasses the following inventions.

[0011] [1] A method for producing formalin-fixed paraffin-embedded thin-layer sections from formalin-fixed paraffin-embedded tissue, comprising: a thinning step of obtaining thin-layer sections from the formalin-fixed paraffin-embedded tissue; an stretching step of stretching the thin-layer sections obtained in the thinning step; a solidification step of solidifying the thin-layer sections stretched in the stretching step; and a cutting step of cutting the thin-layer sections solidified in the solidification step to a desired size to obtain formalin-fixed paraffin-embedded thin-layer sections.

[0012] [2] The method for producing a formalin-fixed paraffin-embedded thin-layer section according to [1], wherein in the spreading step, the thin-layer section is spread under conditions of 35°C or higher and 60°C or lower, and in the solidification step, the thin-layer section is solidified under conditions of 0°C or higher and less than 35°C.

[0013] [3] In the cutting process, the thickness of the formalin-fixed paraffin-embedded thin-layer section is 0.01 μm or more and 3000 μm or less, and the area of ​​the main surface is 0.2 mm². 2 Above 16mm 2 A method for producing a formalin-fixed paraffin-embedded thin-layer section according to [1] or [2], comprising cutting the solidified thin-layer section so that it is less than [amount].

[0014] A method for manufacturing slide specimens, comprising a step of attaching a formalin-fixed paraffin-embedded thin-layer section obtained by the manufacturing method described in any one of [4] [1] to [3] onto a slide glass. [Effects of the Invention]

[0015] According to one aspect of the present invention, a method for producing a small formalin-fixed paraffin-embedded thin section and a method for producing a slide specimen can be provided.

Brief Description of Drawings

[0016] [Figure 1] It is a diagram showing the steps of producing a slide specimen in an embodiment of the present invention. [Figure 2] It is a diagram showing the steps of producing a slide specimen in a comparative example of the present invention.

Mode for Carrying Out the Invention

[0017] Hereinafter, an example of an embodiment of the present invention will be described in detail, but the present invention is not limited thereto. The present invention can be variously modified within the scope shown in the claims. In addition, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, by combining the technical means disclosed in each embodiment, new technical features can be formed. All academic documents and patent documents described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, "X to Y" representing a numerical range is intended to mean "X or more and Y or less".

[0018] 〔1. Method for Producing Formalin-Fixed Paraffin-Embedded Thin Section〕 A method for producing formalin-fixed paraffin-embedded thin-layer sections according to one embodiment of the present invention is a method for producing formalin-fixed paraffin-embedded thin-layer sections from formalin-fixed paraffin-embedded tissue, comprising: a thinning step of obtaining thin-layer sections from the formalin-fixed paraffin-embedded tissue; an stretching step of stretching the thin-layer sections obtained in the thinning step; a solidification step of solidifying the thin-layer sections stretched in the stretching step; and a cutting step of cutting the thin-layer sections solidified in the solidification step to a desired size to obtain formalin-fixed paraffin-embedded thin-layer sections.

[0019] The specific composition of the formalin-fixed paraffin-embedded tissue is not limited. The formalin-fixed paraffin-embedded tissue may, for example, be (i) a living tissue (e.g., normal tissue or pathological tissue) fixed with formalin and embedded in paraffin, or (ii) an artificially manufactured tissue-like structure or organ-like structure fixed with formalin and embedded in paraffin.

[0020] The organisms from which the aforementioned biological tissue originates are not limited to humans and non-human animals (e.g., monkeys, chimpanzees, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats).

[0021] The normal tissues mentioned above are not limited to, but include, for example, skin, sweat gland tissue, hair follicle tissue, stomach, liver, kidney, lung, pancreas, heart, brain, breast, mammary gland, uterus, esophagus, pharynx, larynx, tongue, and thyroid gland.

[0022] The aforementioned pathological tissues are not limited to, but include, for example, inflamed tissues (e.g., skin), inflamed tissues (e.g., skin), skin cancer, oral cancer, pharyngeal cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, rectal cancer, lung cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, lymphoma, prostate cancer, bladder cancer, breast cancer, ovarian cancer, endometrial cancer, cervical cancer, sarcoma, osteosarcoma, melanoma, brain tumor, meningioma, and biliary tract cancer.

[0023] The aforementioned tissue-like structures and organ-like structures are not limited to, but include, for example, spheroids, organoids, and cell sheets. Furthermore, the types of cells constituting these tissue-like structures and organ-like structures are not limited.

[0024] The method for preparing the formalin-fixed paraffin-embedded tissue is not limited, and the formalin-fixed paraffin-embedded tissue can be prepared according to known methods. The method for preparing the formalin-fixed paraffin-embedded tissue may, for example, include an immobilization step, a dehydration step, an intermediate agent treatment step, and an embedding step.

[0025] The aforementioned immobilization step is, for example, the immobilization of the target tissue (e.g., biological tissue, or artificially manufactured tissue-like or organ-like structure) with formalin. In this immobilization step, for example, the target tissue can be immobilized by contacting it with a solution containing formalin (e.g., approximately 10% neutral buffered formalin, approximately 3.7% formaldehyde) for a desired time (e.g., 1 to 24 hours).

[0026] The dehydration step is a step of removing water from the immobilized target tissue. In this dehydration step, for example, water can be removed from the immobilized target tissue by bringing it into contact with alcohol (e.g., 70% to 100% ethanol) for a desired time (e.g., 1 hour to 24 hours).

[0027] The aforementioned intermediate treatment step is a step of removing unwanted substances such as alcohol from the target tissue from which water has been removed. In this intermediate treatment step, for example, unwanted substances can be removed from the target tissue by contacting the target tissue from which water has been removed with an intermediate agent (for example, xylene or chloroform) for a desired time (for example, 1 to 24 hours).

[0028] The aforementioned embedding step is a step of embedding the target tissue, from which unwanted substances have been removed, in paraffin. In this embedding step, for example, the target tissue from which unwanted substances have been removed is placed in dissolved paraffin, and then the paraffin is cooled and solidified to obtain formalin-fixed paraffin-embedded tissue in which the target tissue is embedded.

[0029] Formalin-fixed paraffin-embedded thin-layer sections can be produced from formalin-fixed paraffin-embedded tissue through the aforementioned thinning process, stretching process, solidification process, and cutting process. The thinning process, stretching process, solidification process, and cutting process will be described below in detail.

[0030] [1-1. Thinning process] The aforementioned thinning process is a process of obtaining thin-layer sections from formalin-fixed paraffin-embedded tissue.

[0031] The thinning step may be, for example, a step of cutting out thin sections from formalin-fixed paraffin-embedded tissue using a microtome (e.g., a rotary microtome or a sliding microtome).

[0032] The thickness of the thin-layer section is not limited and can be set appropriately depending on the microtome used. The thickness of the thin-layer section may be, for example, 0.01 μm to 3000 μm, 0.01 μm to 1000 μm, 0.01 μm to 100 μm, or 0.1 μm to 10 μm.

[0033] The size (area) of the main surface of the thin-layer section is not limited and can be set appropriately depending on the microtome used. Furthermore, since the thin-layer section is cut to a desired size (area) in the cutting process described later, the size (area) of the main surface of the thin-layer section obtained in the thin-layer preparation process is not limited. For example, the size (area) of the main surface of the thin-layer section may be 16 mm. 2 ~100mm 2 , 16mm 2 ~50mm 2 , or 16mm 2 ~25mm 2It is possible.

[0034] In this specification, "main surface" refers to the surface within a slide specimen that is the subject of observation when a formalin-fixed, paraffin-embedded thin-layer section is mounted on a glass slide to prepare the slide specimen.

[0035] [1-2. Extension process] The aforementioned stretching step is a step of stretching the thin-layer section obtained in the thin-layer preparation step.

[0036] The stretching step may be, for example, a step of stretching the thin-layer section obtained in the thin-layer preparation step by placing it in an environment at a predetermined temperature.

[0037] More specifically, the stretching step may be, for example, a step of stretching the thin-layer section obtained in the thin-layer preparation step by floating the section in hot water.

[0038] According to the above configuration, wrinkles in the thin-layer section can be easily removed.

[0039] The temperature of the stretching process (for example, the temperature of the environment in which the stretching process is performed, or the temperature of the hot water) is not limited to any temperature that can soften and stretch the thin-layer section. The temperature may be, for example, 35°C to 60°C, 35°C to 50°C, 38°C to 45°C, or 42°C. At such a temperature, wrinkles in the thin-layer section can be easily removed without excessive loss of paraffin.

[0040] [1-3. Solidification process] The solidification step is a process of solidifying the thin-layer sections that were stretched in the stretching step.

[0041] The solidification step may be, for example, (a1) a step of solidifying the thin-layer section that was stretched in the stretching step by moving it to another environment at a lower temperature than the environment used in the stretching step, or (b1) a step of solidifying the thin-layer section that was stretched in the stretching step by lowering the temperature of the environment used in the stretching step.

[0042] More specifically, the solidification step may be, for example, (a2) a step of solidifying the thin-layer section that has been stretched in the stretching step by transferring it to another room-temperature water at a lower temperature than the water used in the stretching step, or (b2) a step of solidifying the thin-layer section floating in the room-temperature water by lowering the temperature of the water used in the stretching step to make it room-temperature water.

[0043] According to the above configuration, the hardness of the thin-layer section after the stretching process is increased, thereby facilitating the cutting of the thin-layer section in the cutting process described later.

[0044] If the thin-layer section is solidified in a short time, the thin-layer section can be solidified without excessive loss of paraffin. Therefore, the solidification step is preferably the step described in (a1) or (a2) above.

[0045] The temperature of the solidification step (for example, the temperature of the environment in which the solidification step is performed, or the temperature of the room temperature water) is not limited to any temperature that can solidify the paraffin-containing thin-layer section. The temperature may be, for example, 0°C or higher and less than 35°C, 10°C to 32°C, 20°C to 30°C, or 28°C.

[0046] In this specification, "hot water" refers to a liquid with a temperature higher than "room temperature water." In this specification, the components of "hot water" and "room temperature water" are not limited. "Hot water" and "room temperature water" may be, for example, a liquid consisting of water, a liquid containing water and components other than water, or a liquid that does not contain water.

[0047] [1-4. Cutting process] The cutting step is a step of cutting the thin layer section solidified in the solidification step into a desired size to obtain the formalin-fixed paraffin-embedded thin layer section. In this cutting step, since the thin layer section is solidified and its hardness is increased, the thin layer section can be easily cut into a desired size.

[0048] The cutting step only needs to be a step capable of cutting the thin layer section solidified in the solidification step, and the specific configuration is not limited. For example, the cutting step can be a step of cutting a thin layer section floating in normal temperature water.

[0049] The method for cutting the thin layer section is not limited. For example, scissors and tweezers can be used to cut the thin layer section into a desired size.

[0050] The thickness of the formalin-fixed paraffin-embedded thin layer section obtained by the cutting step is not limited. For example, it can be 0.01 μm to 3000 μm, 0.01 μm to 1000 μm, 0.01 μm to 100 μm, or 0.1 μm to 10 μm.

[0051] The size (area) of the main surface of the formalin-fixed paraffin-embedded thin layer section obtained by the cutting step is not limited. For example, it can be less than 0.1 mm 2 to 16 mm 2 less than, 0.2 mm 2 to 16 mm 2 less than, or 1 mm 2 to 16 mm 2 less than.

[0052] [2. Method for manufacturing a slide specimen] The method for manufacturing a slide specimen according to an embodiment of the present invention is a method for manufacturing a slide specimen including an attaching step of attaching the formalin-fixed paraffin-embedded thin layer section obtained by the method for manufacturing a formalin-fixed paraffin-embedded thin layer section according to an embodiment of the present invention onto a slide glass.

[0053] The aforementioned attachment step is not limited to any specific configuration, as it is a step that allows the formalin-fixed paraffin-embedded thin-layer section to be attached to a glass slide. The attachment step may be, for example, (i) a step of placing a desired number of formalin-fixed paraffin-embedded thin-layer sections obtained by the cutting step onto a glass slide with room temperature water, and then removing the room temperature water, or (ii) a step of repeating step (i) multiple times.

[0054] The number of formalin-fixed paraffin-embedded thin-layer sections attached to the glass slide may be one or more, and is not limited to that number. The lower limit of the number of formalin-fixed paraffin-embedded thin-layer sections attached to the glass slide may be, for example, one or more, two or more, three or more, five or more, seven or more, ten or more, or fifteen or more. The upper limit of the number of formalin-fixed paraffin-embedded thin-layer sections attached to the glass slide may be, for example, 50 or less, 40 or less, or 30 or less.

[0055] The size (area) of the area on the glass slide to which the formalin-fixed paraffin-embedded thin-layer section is attached is not limited and can be an area appropriate to the instrument used for analysis. For example, it may be an area of ​​30 mm × 30 mm, 10 mm × 10 mm, or 6.5 mm × 6.5 mm.

[0056] The purpose of using the aforementioned slide specimen is not limited. The slide specimen may be, for example, a slide specimen used in spatial transcriptome analysis, immunohistofluorescence staining, or immunohistochemistry. With this configuration, multiple samples can be analyzed using a single slide specimen. Therefore, this configuration may offer advantages such as reduced analysis costs, improved analysis efficiency, reduced reagent usage, shorter acquisition time, and reduced image size.

[0057] This invention may also contribute to achieving Goal 9 of the United Nations' Sustainable Development Goals (SDGs), "Build resilient infrastructure, promote inclusive and sustainable industrialization and foster innovation." [Examples]

[0058] <Preparation of formalin-fixed, paraffin-embedded thin-layer sections (slide specimens)> <1. Examples> <1-1. Preparation of slide specimens> (Process 1) Formalin-fixed paraffin-embedded tissue was prepared. Specifically, excised skin was fixed by immersing it in 10% neutral buffered formalin (3.7% formaldehyde) and leaving it at 4°C for 24 hours. Formalin-fixed paraffin-embedded tissue was then prepared by dehydration, intermediate agent treatment, and paraffin embedding treatment of the fixed tissue. The dehydration, degreasing, and paraffin embedding treatments were carried out according to known methods.

[0059] (Process 2) The formalin-fixed paraffin-embedded tissue was placed in a microtome (Leica), and thin-layer sections (3 μm thick) were cut from the formalin-fixed paraffin-embedded tissue. The size of the tissue in these thin-layer sections, which contained both paraffin and tissue, was 10 mm × 5 mm.

[0060] (Step 3) The thin-layer sections were stretched by floating them in hot water (42°C). This removed any wrinkles present within the thin-layer sections.

[0061] (Step 4) The thin-layer sections were solidified by floating them in room temperature water (28°C). This increased the hardness of the thin-layer sections (see 101 in Figure 1).

[0062] (Step 5) Solidified thin-layer sections floating on room temperature water (28°C) were trimmed using scissors and tweezers. The size of the trimmed thin-layer sections (formalin-fixed paraffin-embedded thin-layer sections) was "1 mm × 1 mm" (see 102 and 103 in Figure 1).

[0063] (Step 6) After trimming the thin-layer sections, they were drawn up using a straw and placed within a designated area on a microscope slide (see 104 in Figure 1). Then, the water on the microscope slide was removed and the thin-layer sections were attached to the microscope slide.

[0064] (Step 7) By performing steps 4 to 6 described above multiple times, a slide specimen was prepared in which 25 formalin-fixed paraffin-embedded thin-layer sections were attached to a predetermined area (6.5 mm × 6.5 mm) on a glass slide (see 105 in Figure 1).

[0065] <1-2. Test Results> As shown in Figure 1, 105, 25 formalin-fixed paraffin-embedded thin-layer sections could be attached within a 6.5 mm × 6.5 mm area on a single glass slide.

[0066] <2. Comparative Examples> <2-1. Preparation of slide specimens> (Step 1') Formalin-fixed paraffin-embedded tissue was prepared. Specifically, excised skin was fixed by immersing it in 10% neutral buffered formalin (3.7% formaldehyde) and leaving it at 4°C for 24 hours. Formalin-fixed paraffin-embedded tissue was then prepared by dehydration, intermediate agent treatment, and paraffin embedding treatment of the fixed tissue. The dehydration, degreasing, and paraffin embedding treatments were carried out according to known methods.

[0067] (Step 2') The formalin-fixed paraffin-embedded tissue was placed in a microtome (Leica), and a thin-layer section (3 μm thick) was cut from the formalin-fixed paraffin-embedded tissue (see 201 in Figure 2). The size of the tissue in the thin-layer section, which contained paraffin and tissue, was "10 mm × 5 mm".

[0068] (Step 3') The thin-layer sections were stretched by floating them in hot water (42°C). This removed wrinkles present in the thin-layer sections (see 202 in Figure 2). The layers obtained in step 3' were subjected to steps 4' and 5', or steps 4'' and 5'', described later.

[0069] (Step 4') While the aforementioned thin-layer section was floating in hot water (42°C), the section was further spread using a brush (see 203 in Figure 2).

[0070] (Step 5') Slide specimens were prepared by attaching the aforementioned thin-layer sections to a glass slide using a brush (see 204 in Figure 2).

[0071] (Step 4'') The thin-layer sections floating on the hot water (42°C) were trimmed using scissors and tweezers (see 205 in Figure 2).

[0072] (Step 5'') We attempted to mount the trimmed thin-layer sections onto a microscope slide (see 206 in Figure 2).

[0073] <2-2. Test Results> As shown in Figure 2, 204, in the method that did not use steps 4 and 5, but did use steps 4' and 5', only one formalin-fixed paraffin-embedded thin-layer section could be attached to a 30 mm × 30 mm area on a single glass slide.

[0074] As shown in Figures 2, 205 and 206, when steps 4 and 5 were not used, and when steps 4'' and 5'' were used, operational problems occurred, such as the paraffin-containing thin-layer sections sticking to the instrument (e.g., tweezers), making it impossible to obtain trimmed thin-layer sections. [Industrial applicability]

[0075] This invention can be used in the medical field (e.g., pathological diagnosis) and the research field (e.g., various analyses using spatial transcriptome methods).

Claims

1. A method for preparing formalin-fixed paraffin-embedded thin-layer sections from formalin-fixed paraffin-embedded tissue, A thinning process to obtain thin-layer sections from the formalin-fixed paraffin-embedded tissue, A stretching step in which the thin-layer section obtained in the thin-layer preparation step is stretched, A solidification step in which the thin-layer section stretched in the stretching step is solidified, and A method for producing a formalin-fixed paraffin-embedded thin-layer section, comprising a cutting step of cutting the thin-layer section solidified in the solidification step into a desired size to obtain the formalin-fixed paraffin-embedded thin-layer section.

2. In the stretching process, the thin-layer section is stretched under conditions of 35°C or higher and 60°C or lower. The method for producing formalin-fixed paraffin-embedded thin-layer sections according to claim 1, wherein the thin-layer sections are solidified under conditions of 0°C or higher and less than 35°C in the solidification step.

3. In the cutting process, the thickness of the formalin-fixed paraffin-embedded thin-layer section is 0.01 μm or more and 3000 μm or less, and the area of ​​the main surface is 0.2 mm². ２ Above 16mm ２ A method for producing a formalin-fixed paraffin-embedded thin-layer section according to claim 1, comprising cutting the solidified thin-layer section so that it is less than [amount missing].

4. A method for manufacturing a slide specimen, comprising the step of attaching a formalin-fixed paraffin-embedded thin-layer section obtained by the manufacturing method described in claim 1 onto a slide glass.

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