Method for manufacturing resin-embedded samples, method for manufacturing thin sections, thin sections and captured images

Abstract

This invention provides a method for manufacturing embedded samples that suppresses the curvature of sheet-like samples and the peeling and curling of membranes in sheet-like samples laminated on a film, making it suitable for calculating the thickness of sheet-like samples and preparing horizontally sliced ​​sections. [Solution] A method for producing a resin-embedded sample, comprising the steps of: (a) placing a membrane on which cell samples are stacked in a container; (b) adding a gel material in a sol state to the container and gelling it to obtain a gel-embedded sample in which the cell samples are embedded in the gel; and (c) removing the gel-embedded sample from the container and embedding it in resin to obtain a resin-embedded sample.

Description

Technical Field

[0001] The present invention relates to a method for producing an embedded resin sample, a method for producing a thin section, the thin section, and a captured image.

Background Art

[0002] Conventionally, in order to store a sample of biological tissue for a long time and use it for observation, a technique for producing an embedded resin sample in which the sample is embedded in resin is known. A thin section obtained by thinly slicing the embedded resin sample is stained and enlarged for observation using a microscope.

[0003] For example, Patent Document 1 describes a method for preparing a specimen for the purpose of examining tissue during surgery. The method of Patent Document 1 includes a step of embedding tissue with an embedding agent for specimen preparation, a step of preparing a block in which the tissue embedded with the embedding agent for specimen preparation is cooled and solidified, a step of preparing an immobilized block by immersing the block in a fixing solution, a step of preparing a curable substrate block in which the immobilized block is embedded with a curable substrate, and a step of thinly slicing the curable substrate block to obtain a support on which the thinly sliced tissue adheres.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors have found that when a thinly sliced embedded resin sample of a cell sample is prepared, the cell sample may be curved. Further, when the cell sample is in the form of a sheet laminated on a membrane, it has been found that when the embedded resin sample is thinly sliced, peeling or curling of the membrane may occur.

[0006] The present invention aims to provide a technique for suppressing the curvature of cell samples, membrane peeling, and curling that occur when resin-embedded cell samples are thinly sliced. [Means for solving the problem]

[0007] The present invention encompasses the following embodiments. [1] A method for producing a resin-embedded sample, comprising the steps of: (a) placing a membrane on which cell samples are stacked in a container; (b) adding a gel material in a sol state to the container and gelling it to obtain a gel-embedded sample in which the cell samples are embedded in the gel; and (c) removing the gel-embedded sample from the container and embedding it in resin to obtain a resin-embedded sample. [2] The method for producing a resin-embedded sample according to [1], wherein step (a) is a step of placing a membrane on which the cell sample is laminated on the bottom surface of the container. [3] A method for producing a resin-embedded sample according to [1] or [2], wherein step (a) includes the steps of: placing a sheet-like gel on the bottom surface of the container (a1); and placing a membrane on which the cell sample is laminated on the sheet-like gel (a2). [4] A method for producing a resin-embedded sample according to any one of [1] to [3], wherein the gel material comprises agarose. [5] The method for producing a resin-embedded sample according to [4], wherein the gel material further comprises gelatin. [6] The method for producing a resin-embedded sample according to [5], wherein the mass of the gelin contained in the gel material is less than the mass of the agarose. [7] A method for producing a resin-embedded sample according to any one of [1] to [6], wherein the cell sample is a three-dimensional cell tissue comprising cells, a cationic substance, extracellular matrix components, and a polyelectrolyte. A method for producing thin sections of a cell sample, comprising the step of thinly slicing a resin-embedded sample obtained by any of the manufacturing methods described in [8][1] to [7] in a direction perpendicular to the membrane or horizontal to the membrane. [9] A thin section of a cell sample, comprising a membrane and a cell sample laminated on the membrane, wherein the membrane and the cell sample are not curved and the membrane is not detached.

[10] The thin section according to [9], wherein the cell sample is a three-dimensional cell tissue comprising cells, a cationic substance, extracellular matrix components, and polyelectrolytes. A thin section according to [9] or

[10] , obtained by thinly slicing a resin-embedded sample manufactured by any of the manufacturing methods described in

[11] , [1], to the membrane in a direction perpendicular to the membrane. Image of a thin section as described in any of

[12] [9]~

[11] .

[13] 5mm 2 A thin section of a cell sample having the above area.

[14] The thin section according to

[13] , wherein the cell sample is a three-dimensional cell tissue comprising cells, a cationic substance, extracellular matrix components, and polyelectrolytes. A thin section according to

[13] or

[14] , obtained by thinly slicing a resin-embedded sample manufactured by any of the manufacturing methods described in

[15] , [1], to the membrane in a horizontal direction. Image of a thin section as described in any of

[16] ,

[13] , or

[15] . [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a technique for suppressing the curvature of cell samples and the peeling or curling of membranes when resin-embedded cell samples are sliced. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a schematic diagram illustrating one embodiment of a method for manufacturing a resin-embedded sample. [Figure 2] Figure 2 is a schematic diagram illustrating one embodiment of a method for manufacturing a resin-embedded sample. [Figure 3] Figure 3 shows optical microscope images of thin sections from Experimental Example 1. [Figure 4]Figure 4 is an optical microscope image of a thin section in Experimental Example 2. [Figure 5] Figure 5 is an optical microscope image of a thin section in Experimental Example 3.

Mode for Carrying Out the Invention

[0010] Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate. The dimensional ratios in the drawings may not necessarily match the actual dimensional ratios.

[0011] [Method for Producing Resin-Embedded Sample] 1 In one embodiment, the present invention provides a method for producing a resin-embedded sample, which includes: step (a) of disposing a membrane on which cell samples are stacked in a container; step (b) of putting a sol-state gel material into the container to cause gelation, and obtaining a gel-embedded sample in which the cell samples are embedded in the gel; and step (c) of taking out the gel-embedded sample from the container and embedding it in resin to obtain a resin-embedded sample.

[0012] The membrane on which cell samples are stacked may be, for example, the membrane of a cell culture insert on which cell culture has been performed. Cells are stacked on the membrane.

[0013] The membrane may be one commonly used for cell culture inserts. Examples of the material of the membrane include, but are not limited to, polyethylene terephthalate, polycarbonate, and the like.

[0014] The cells in the cell sample are not particularly limited, and cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats can be used. The origin site of the cells is not particularly limited, and somatic cells derived from bone, muscle, internal organs, nerves, brain, skin, or blood, etc. may be used, or germ cells may be used, or cancer cells may be used.

[0015] Examples of somatic cells derived from blood include immune cells such as lymphocytes, neutrophils, macrophages, and dendritic cells. Examples of cancer cells include those of gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, and liver cancer.

[0016] Furthermore, the cells may be pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells), or they may be tissue stem cells.

[0017] Furthermore, the cells may be primary cells, or cultured cells such as subcultured cells or cell lines. Additionally, one type of cell may be used alone, or a mixture of multiple types may be used.

[0018] The cell sample may be an artificially created three-dimensional cell tissue. More specifically, the cell sample may be a three-dimensional cell tissue containing cells, cationic substances, extracellular matrix components, and polyelectrolytes.

[0019] In recent years, in drug assay systems that require an environment close to that of a living organism, the superiority of using three-dimensionally organized cell tissues over cells grown on flat plates has been demonstrated.

[0020] Three-dimensional cell tissue can be produced by a method comprising the steps of: mixing cells with a cationic substance, extracellular matrix components, and polyelectrolytes to obtain a mixture; and culturing the cells in the obtained mixture to obtain three-dimensional cell tissue.

[0021] As cationic substances, any positively charged substance can be used, as long as it does not adversely affect cell growth and the formation of cell aggregates described later. Examples of cationic substances include, but are not limited to, cationic buffers such as tris-hydrochloric acid, tris-maleic acid, bis-tris, and HEPES, as well as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, and polyarginine. Among these, cationic buffers are preferred, and tris-hydrochloric acid is more preferred.

[0022] The concentration of the cationic substance is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. The concentration of the cationic substance used in this embodiment is preferably 10 to 100 mM relative to the total volume of the mixture, and may be, for example, 20 to 90 mM, 30 to 80 mM, 40 to 70 mM, or 45 to 60 mM.

[0023] When a cationic buffer is used as the cationic substance, the pH of the cationic buffer is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. The pH of the cationic buffer used in this embodiment is preferably 6.0 to 8.0. For example, the pH of the cationic buffer used in this embodiment may be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The pH of the cationic buffer used in this embodiment is more preferably 7.2 to 7.6, and even more preferably about 7.4.

[0024] Any component constituting the extracellular matrix (also known as the extracellular matrix, or ECM) can be used as the extracellular matrix component, as long as it does not adversely affect cell growth and the formation of cell aggregates described later. Examples of extracellular matrix components include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, enterin, fibrin, proteoglycans, and combinations thereof. The extracellular matrix component may also be a modified or variant of those mentioned above. The extracellular matrix component may be used alone or in combination of two or more types.

[0025] Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, and dermatan sulfate proteoglycan. Among the extracellular matrix components, collagen, laminin, and fibronectin are preferred, with collagen being particularly preferred.

[0026] The total content of extracellular matrix components is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. The total content of extracellular matrix components relative to the total volume of the mixture may be 0.005 mg / mL or more and 1.5 mg / mL or less, 0.005 mg / mL or more and 1.0 mg / mL or less, 0.01 mg / mL or more and 1.0 mg / mL or less, 0.025 mg / mL or more and 1.0 mg / mL or less, or 0.025 mg / mL or more and 0.1 mg / mL or less. The extracellular matrix components can be used dissolved in a suitable solvent. Examples of solvents include, but are not limited to, water, buffer solutions, and acetic acid. Among these, buffer solutions or acetic acid are preferred.

[0027] In this specification, a polyelectrolyte means a polymer having dissociable functional groups in its polymer chain. Any polyelectrolyte can be used as the polyelectrolyte in this embodiment, as long as it does not adversely affect cell growth and cell aggregate formation. Examples of polyelectrolytes include, but are not limited to, heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate and chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, glycosaminoglycans such as hyaluronic acid, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, polyacrylic acid, and combinations thereof. The polyelectrolyte may also be a derivative of those described above. These polyelectrolytes may be used individually or in combination of two or more.

[0028] The polyelectrolyte is preferably a glycosaminoglycan. Among these, heparin, chondroitin sulfate, and dermatan sulfate are preferred, with heparin being particularly preferred.

[0029] The concentration of the polyelectrolyte is not particularly limited, as long as it does not adversely affect cell growth and cell aggregate formation. Unlike extracellular matrix components, polyelectrolytes are effective at any concentration as long as it is below the solubility limit, and they do not inhibit the effects of extracellular matrix components. The concentration of the polyelectrolyte is preferably 0.005 mg / mL or more relative to the total volume of the mixture, but may be 0.005 mg / mL to 1.0 mg / mL, 0.01 mg / mL to 1.0 mg / mL, 0.025 mg / mL to 1.0 mg / mL, or 0.025 mg / mL to 0.1 mg / mL.

[0030] The polymer electrolyte can be used after being dissolved in a suitable solvent. Examples of solvents include, but are not limited to, water and buffer solutions. When a cationic buffer solution is used as the cationic substance mentioned above, the polymer electrolyte may also be used after being dissolved in the cationic buffer solution.

[0031] The mixing ratio (final concentration ratio) of the polyelectrolyte and the extracellular matrix component is preferably 1:2 to 2:1, but may also be 1:1.5 to 1.5:1, or 1:1.

[0032] In the manufacturing method of this embodiment, first, in step (a), a membrane on which cell samples are layered is placed in a container. As the membrane on which cell samples are layered, for example, the membrane of a cell culture insert in which cell culture has been performed can be removed and used.

[0033] The container used in step (a) is not particularly limited as long as it can hold the gel material in a sol state, gel it, and obtain a gel-embedded sample. Specifically, for example, the cap of a 1.5 mL tube or the wells of a well plate can be used as containers.

[0034] Step (a) may be a step of placing a membrane on which the cell sample is stacked on the bottom surface of the container. Hereinafter, this method may be referred to as the bottom-placement method.

[0035] Alternatively, step (a) may include the steps of placing a sheet-like gel on the bottom surface of the container (a1) and placing a membrane on which the cell sample is layered on top of the sheet-like gel (a2). Hereinafter, this method may be referred to as the pedestal-mounting method.

[0036] Next, in step (b), a gel material in sol form is placed in a container and gelled to obtain a gel-embedded sample in which the cell sample is embedded in the gel.

[0037] Figure 1 is a schematic diagram illustrating the process of preparing a gel-embedded sample using the bottom-placement method. In the bottom-placement method, a membrane containing the cell sample is placed on the bottom of a container, and then a gel material in a sol state is added to the container to gel, thereby obtaining a gel-embedded sample in which the cell sample is embedded in the gel. In a gel-embedded sample prepared using the bottom-placement method, the membrane containing the cell sample is exposed on the surface of the gel-embedded sample.

[0038] Figure 2 is a schematic diagram illustrating the process of preparing a gel-embedded sample using the pedestal-mounting method. In the pedestal-mounting method, first, in step (a1), a sheet of gel is placed on the bottom surface of the container. Next, in step (a2), a membrane containing the cell sample is placed on top of the sheet of gel. Subsequently, the gel material in a sol state is placed in the container and gelled to obtain a gel-embedded sample in which the cell sample is embedded in the gel. In a gel-embedded sample prepared using the pedestal-mounting method, the membrane containing the cell sample is located inside the gel-embedded sample and is not exposed on the surface.

[0039] As will be described later in the examples, agarose can be suitably used as the gel material in step (b). Furthermore, as will be described later in the examples, it is preferable that the gel material further contains gelatin. When the gel material contains agarose and gelatin, the curvature of the cell sample, peeling and lifting of the membrane when the resin-embedded sample is sliced ​​tend to be further suppressed.

[0040] As will be described later in the examples, when the gel material contains agarose and gelatin, it is preferable that the mass of gelatin contained in the gel material is less than the mass of agarose.

[0041] The agarose concentration in the gel material is preferably 0.4% to 2.0% by mass, and may be, for example, 0.7% to 1.8% by mass, 0.8% to 1.5% by mass, or 0.9% to 1.1% by mass.

[0042] Examples of gelatin include gelatin derived from cows or pigs (melting point around 30°C), and gelatin derived from fish (melting point below 20°C). Gelatin with a melting point below 20°C is preferably used. When fish-derived gelatin is dissolved in water, it is in a liquid state at 15°C to 25°C and in a solid state at 4°C.

[0043] When the gel material contains agarose and gelatin, the mass of gelatin may be 25% to 250% of the mass of agarose, for example, 30% to 60%, or for example, 45% to 55%. The mass of gelatin may be greater than the mass of agarose, but it is preferable that the mass of gelatin is less than or equal to the mass of agarose.

[0044] The method for gelling a gel material in a sol state should be one that is appropriate for the gel material being used. For example, when using agarose, a mixture of agarose and gelatin as the gel material, one method is to leave the container containing the gel material in a sol state undisturbed in a low-temperature environment such as a refrigerator.

[0045] Next, in step (c), the gel-embedded sample is removed from the container and embedded in resin to obtain a resin-embedded sample.

[0046] As the resin, any resin commonly used for resin-embedded specimens can be used, such as paraffin or OCT compound (Sakura FineTech Japan). Resin-embedded specimens may also be prepared using an automated embedding device.

[0047] [Method for producing thin sections of cell samples] In one embodiment, the present invention provides a method for producing thin sections of a cell sample, comprising the step of thinly slicing a resin-embedded sample obtained by the above-described manufacturing method in a direction perpendicular to the membrane or horizontal to the membrane.

[0048] As will be described later in the examples, the manufacturing method of this embodiment makes it possible to obtain thin sections in which the curvature of the cell sample and the peeling and curling of the membrane are suppressed.

[0049] Furthermore, as will be described later in the examples, it is difficult to slice conventional resin-embedded samples horizontally to the membrane, but according to the manufacturing method of this embodiment, it is possible to obtain thin sections sliced ​​horizontally to the membrane.

[0050] [Thin section (vertical thin section)] In one embodiment, the present invention provides a thin section of a cell sample comprising a membrane and a cell sample laminated on the membrane, wherein the membrane and the cell sample are not curved and the membrane is not peeled off.

[0051] The thin sections of this embodiment can be obtained by thinly slicing the resin-embedded sample produced by the manufacturing method described above, perpendicular to the membrane. Hereinafter, the thin sections of this embodiment may be referred to as perpendicular sections. The thickness of the perpendicular sections may be, for example, 10 μm or less, 7.5 μm or less, 6 μm or less, or 5 μm or less.

[0052] In the vertical thin section of this embodiment, the cell sample may be a three-dimensional cellular tissue containing cells, cationic substances, extracellular matrix components, and polyelectrolytes. The three-dimensional cellular tissue is as described above.

[0053] [Image of a vertical thin section] In one embodiment, the present invention provides captured images of the vertical thin sections described above. As will be described later in the examples, in the captured images of this embodiment, the membrane and cell sample are not curved, and membrane peeling and curling are suppressed. Therefore, the thickness of the cell sample and other parameters can be accurately analyzed.

[0054] [Thin section (horizontal thin section)] In one embodiment, the present invention relates to 5 mm 2 This provides thin sections of a cell sample having the above area.

[0055] The thin sections of this embodiment can be obtained by thinly slicing the resin-embedded sample produced by the manufacturing method described above in a direction horizontal to the membrane. Hereinafter, the thin sections of this embodiment may be referred to as horizontal thin sections. The thickness of the horizontal thin sections may be, for example, 10 μm or less, for example, 7.5 μm or less, for example, 6 μm or less, or for example, 5 μm or less. As described above, it is difficult to section conventional resin-embedded samples horizontally relative to the membrane, but the manufacturing method described above makes it possible to obtain horizontal sections with a larger area than conventional horizontal sections. The horizontal sections of this embodiment are, for example, 5 mm 2 It may have an area greater than or equal to 10 mm 2 It may have an area greater than or equal to the above, for example, 14 mm 2 It may have the above area.

[0056] In the horizontal thin section of this embodiment, the cell sample may be a three-dimensional cellular tissue containing cells, cationic substances, extracellular matrix components, and polyelectrolytes. The three-dimensional cellular tissue is as described above.

[0057] [Image of horizontal thin section] In one embodiment, the present invention provides imaging images of the horizontal thin sections described above. As described above, it is difficult to thinly section conventional resin-embedded samples in a direction horizontal to the membrane, but horizontal thin sections can be obtained by the manufacturing method described above. It is expected that new insights can be obtained by analyzing the imaging images of this embodiment.

[0058] The vertical or horizontal sections described above can be stained and analyzed using commonly practiced methods. The images of the vertical or horizontal sections may be used for image recognition or image analysis using software. [Examples]

[0059] The present invention will be described in detail below with reference to examples. The present invention is not limited to the following examples.

[0060] [Experimental Example 1] (Consideration of gel type) Resin-embedded cell samples were prepared using different types of gels. Subsequently, the resin-embedded samples were sectioned, and the curvature of the cell samples and the delamination and peeling of the membrane were evaluated.

[0061] For the gels used, agarose gel and gelatin-mixed agarose gel were investigated. For the cell samples, three-dimensional cell tissue cultured in a cell culture insert was used. Paraffin was used as the resin.

[0062] (Preparation of cell samples) 0.9 × 10⁶ normal human dermal fibroblasts (NHDF) per tissue sample were suspended in an equal mixture of 0.1 mg / mL heparin / 100 mM Tris-HCl buffer (pH 7.4) and 0.1 mg / mL collagen / 5 mM acetic acid solution (pH 3.7). The resulting mixture was centrifuged at 1,000 × g for 2 minutes at room temperature to obtain a viscous substance. The obtained viscous substance was suspended in DMEM containing 10% fetal bovine serum (FBS). Subsequently, the entire resulting suspension was seeded into a 96-well cell culture insert (Corning, catalog number 7369, membrane material polyester) and centrifuged at 400 × g for 1 minute at room temperature. This formed cell aggregates on the cell culture insert. The cells were then cultured for 24 hours at 37°C with a carbon dioxide concentration of 5% to obtain three-dimensional cell tissue. The cell sample had a diameter of 4.3 mm (corresponding to the diameter of the 96-well cell culture insert) and a thickness of approximately 100 μm. Next, the edge of the membrane of the cell culture insert was grasped with tweezers and peeled off from the adhesive surface to the well body, obtaining the membrane on which the cell sample was layered.

[0063] (Preparation of gel material) Gel materials were prepared. For the agarose gel, agarose (Agarose L03, Takara Bio) was mixed with water to a concentration of 1% by mass, and then heated in a microwave oven to dissolve it. For the gelatin-mixed agarose gel, gelatin (fish gelatin type A, Nitta Gelatin) and agarose (Agarose L03, Takara Bio) were mixed with water to a concentration of 0.4% by mass for gelatin and 1% by mass for agarose, and then heated in a microwave oven to dissolve them.

[0064] (Preparation of gel-embedded samples) Cell samples were gel-embedded using agarose gel or gelatin-mixed agarose gel, employing both a bottom-placement method and a pedestal-placement method.

[0065] In the bottom-placement gel embedding method, a lid of a 1.5 mL tube (131-8155CS-N, WATSON) was used as a container. The membrane containing the layered cell sample was placed at the bottom of the container, 80 μL of gel material was added, and the sample was left to harden in a refrigerator for 30 minutes to obtain the gel-embedded sample.

[0066] In the pedestal-based gel embedding method, 80 μL of gel material was placed in the lid of a 1.5 mL tube (131-8155CS-N, manufactured by Watson), left to harden in a refrigerator for 30 minutes, and then removed to prepare the pedestal. Next, using a 24-well plate as the container, the pedestal was placed at the bottom of the container, a membrane with layered cell samples was placed on top of the pedestal, 650 μL of gel material was added, and left to harden in a refrigerator for 30 minutes to obtain the gel-embedded sample.

[0067] (Preparation of paraffin-embedded samples) Gel-embedded samples were removed from their containers and embedded in paraffin to prepare paraffin-embedded samples. Paraffin embedding was performed using an automated embedding device (CT-PRO20, Genostaff).

[0068] (Preparation of thin sections) Paraffin-embedded samples were sectioned perpendicular to the membrane to prepare thin sections. A microtome (REM-710, Yamato Koki Kogyo) was used for sectioning the paraffin-embedded samples. The thickness of the sections was set to 5 μm. The sections were mounted on glass slides (CREST, Matsunami Glass Kogyo) and dried on a hot plate. Subsequently, the sections were stained with hematoxylin and eosin (HE) and observed under a light microscope.

[0069] Figure 3 shows optical microscope images of the thin sections. When using agarose gel, thin sections were obtained in which the curvature of the cell sample, membrane detachment, and curling were suppressed, regardless of whether the gel was placed on the bottom or on a pedestal.

[0070] When using gelatin-mixed agarose gel, gel-embedded samples placed on the bottom of the container were too soft and crumbled when removed from the container, making it impossible to prepare paraffin-embedded samples. Similarly, gel-embedded samples placed on a pedestal also crumbled when removed from the container, but because the gel was larger than that of the bottom-placed method, the cell samples were not damaged, and paraffin-embedded samples could be prepared. With the pedestal-placement method, thin sections were obtained with suppressed curvature of the cell samples, membrane detachment, and curling.

[0071] From these results, it was found that both agarose gel and gelatin-mixed agarose gel can be used as gel materials. Furthermore, it was considered more preferable to prepare the gel-embedded samples using the pedestal-mounting method.

[0072] [Experimental Example 2] (Investigation of the composition of gelatin-mixed agarose gel) Using gel materials with varying concentrations of agarose and gelatin, gel-embedded samples were prepared using a pedestal-mounting method, similar to that in Experimental Example 1. Subsequently, paraffin-embedded samples were prepared, similar to that in Experimental Example 1. Then, thin sections of the paraffin-embedded samples were prepared, and the curvature of the cell samples and the delamination and peeling of the membrane were evaluated.

[0073] The same agarose and gelatin as in Experimental Example 1 were used, but their concentrations were varied as shown in Table 1 below.

[0074] [Table 1]

[0075] Figure 4 shows optical microscope images of the thin sections. As a result, under all conditions, thin sections were obtained in which the curvature of the cell sample, membrane detachment, and curling were suppressed. When the gel material contained gelatin in addition to agarose, the curvature of the cell sample, membrane detachment, and curling tended to be suppressed even more.

[0076] When the gel material contained agarose and gelatin, thin sections were obtained in which the curvature of the cell sample, membrane detachment, and peeling were suppressed. Furthermore, it was observed that curvature of the cell sample, membrane detachment, and peeling tended to be suppressed more effectively when the mass of gelatin was less than the mass of agarose.

[0077] [Experimental Example 3] (Preparation of thin horizontal sections) The paraffin-embedded sample, prepared in the same manner as in Experimental Example 1, was thinly sectioned horizontally to the membrane to produce thin sections.

[0078] In paraffin-embedded cell samples that were not gel-embedded, it was difficult to obtain thin sections (hereinafter sometimes referred to as "horizontal sections") that were sliced ​​horizontally relative to the membrane. This was thought to be partly due to the curvature of the cell samples in the paraffin-embedded samples, making horizontal cutting difficult.

[0079] A gelin-mixed agarose gel containing 1% by mass of agarose and 0.5% by mass of gelatin was used as the gel material. Cell samples were embedded in the gel using a pedestal-mounting method to obtain gel-embedded samples. At this time, hematoxylin was added to 2% by mass to improve visibility when preparing horizontal thin sections.

[0080] Next, the gel-embedded samples were embedded in paraffin using an automated embedding device to obtain paraffin-embedded samples. Subsequently, the paraffin-embedded samples were sectioned horizontally relative to the membrane to obtain horizontal sections. The thickness of the sections was set to 5 μm.

[0081] Horizontal thin sections were mounted on glass slides (CREST, Matsunami Glass Industry Co., Ltd.), dried overnight on a hot plate, then stained with HE, and observed under a light microscope.

[0082] Figure 5 shows optical microscope images of horizontal thin sections. The horizontal thin sections in Figure 4 are serial sections, and the numbers in Figure 5 indicate the order in which they were sectioned. The thickness of each horizontal thin section was 5 μm. The area of ​​each horizontal thin section was 14.4 mm².2 As shown in Figure 5, it was confirmed that horizontal thin sections could be easily produced. [Industrial applicability]

[0083] According to the present invention, it is possible to provide a technique for suppressing the curvature of cell samples and the peeling or curling of membranes when resin-embedded cell samples are sliced.

Claims

1. (a) A step of placing a membrane on which cell samples are stacked into a container, (b) A step in which a gel material in a sol state is placed in the container and gelled, thereby obtaining a gel-embedded sample in which the cell sample is embedded in the gel, (c) A step in which the gel-embedded sample is removed from the container and embedded in resin to obtain a resin-embedded sample, A method for producing resin-embedded samples, including [the specified component].

2. The above step (a) is, The method for producing a resin-embedded sample according to claim 1, comprising the step of placing a membrane on which the cell sample is laminated on the bottom surface of the container.

3. The above step (a) is, The steps include (a1) placing a sheet-like gel on the bottom surface of the container, (a2) A step of placing the membrane on which the cell sample is laminated on the sheet-like gel, A method for producing a resin-embedded sample according to claim 1, including the method described in claim 1.

4. A method for producing a resin-embedded sample according to any one of claims 1 to 3, wherein the gel material includes agarose.

5. The method for producing a resin-embedded sample according to claim 4, wherein the gel material further comprises gelatin.

6. The method for producing a resin-embedded sample according to claim 5, wherein the mass of the gelatin contained in the gel material is less than the mass of the agarose.

7. A method for producing a resin-embedded sample according to any one of claims 1 to 3, wherein the cell sample is a three-dimensional cell tissue comprising cells, a cationic substance, extracellular matrix components, and a polymer electrolyte.

8. A method for producing thin sections of a cell sample, comprising the step of thinly slicing a resin-embedded sample obtained by the manufacturing method described in any one of claims 1 to 3 in a direction perpendicular to the membrane or horizontal to the membrane.

9. A thin section of a cell sample, The membrane comprises a cell sample stacked on the membrane, A thin section in which the membrane and the cell sample are not curved and the membrane is not detached.

10. The thin section according to claim 9, wherein the cell sample is a three-dimensional cell tissue comprising cells, a cationic substance, extracellular matrix components, and a polyelectrolyte.

11. A thin section according to claim 9, obtained by thinly slicing a resin-embedded sample manufactured by the manufacturing method described in any one of claims 1 to 3 in a direction perpendicular to the membrane.

12. Image of a thin section according to claim 9 or 10.

13. 5mm ２ A thin section of a cell sample having the above area.

14. The thin section according to claim 13, wherein the cell sample is a three-dimensional cell tissue comprising cells, a cationic substance, extracellular matrix components, and a polyelectrolyte.

15. A thin section according to claim 13, obtained by thinly slicing a resin-embedded sample manufactured by the manufacturing method described in any one of claims 1 to 3 in a direction horizontal to the membrane.

16. Image of a thin section according to claim 13 or 14.

Images