WO2019163657A1 - Embedding agent - Google Patents

Embedding agent Download PDF

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Publication number
WO2019163657A1
WO2019163657A1 PCT/JP2019/005491 JP2019005491W WO2019163657A1 WO 2019163657 A1 WO2019163657 A1 WO 2019163657A1 JP 2019005491 W JP2019005491 W JP 2019005491W WO 2019163657 A1 WO2019163657 A1 WO 2019163657A1
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Prior art keywords
frozen
embedding agent
embedding
synthetic collagen
specimen
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PCT/JP2019/005491
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French (fr)
Japanese (ja)
Inventor
かおり 牛田
直也 浅井
高橋　雅英
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国立大学法人名古屋大学
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Application filed by 国立大学法人名古屋大学 filed Critical 国立大学法人名古屋大学
Priority to JP2020501724A priority Critical patent/JPWO2019163657A1/en
Publication of WO2019163657A1 publication Critical patent/WO2019163657A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples

Definitions

  • This disclosure relates to embedding agents, and particularly to embedding agents containing synthetic collagen.
  • a frozen tissue specimen may be described as “frozen block” in which a collected biological tissue is embedded with a freezing embedding agent ⁇ freeze (hereinafter, a living tissue is embedded with a freezing embedding agent and frozen. ) ⁇ Thin slice ⁇ Dye.
  • frozen embedding agents those using polymers such as polyvinyl alcohol (PVA) and polyethylene glycol (PEG) are known, and are commercially available as frozen embedding agents.
  • the present inventors use gelatin as a sample preparation embedding agent that is in a liquid state at 15 ° C. to 25 ° C. and in a solid state at 4 ° C. when it is used as an aqueous solution, or the gelatin for a known frozen embedding agent. It has been found that the above-mentioned problems can be solved by adding, and a patent application has been filed (see Patent Document 1).
  • the embedding agent comprising gelatin described in Patent Document 1 or the embedding agent obtained by adding gelatin to the frozen embedding agent leaves gelatin remaining in the living tissue as a residue.
  • the embedding agent comprising gelatin described in Patent Document 1 or the embedding agent obtained by adding gelatin to the frozen embedding agent leaves gelatin remaining in the living tissue as a residue.
  • an object of the disclosure in the present specification is to provide an embedding agent that can improve the sliceability of a known frozen embedding agent and has a low background when a living tissue is stained.
  • the disclosure in this specification relates to the embedding agent shown below.
  • An embedding agent for preparing a frozen section comprising: With frozen embedding agents, Synthetic collagen, Including embedding agents.
  • [2] Carboxylic acid for improving the sliceability of frozen sections The embedding agent according to [1], further comprising: [3]
  • the synthetic collagen is represented by the following formula (1). -(Gly-XY) n- (1) The embedding agent according to the above [1] or [2].
  • the synthetic collagen is represented by the following formula (2). -(Pro-Z-Gly) n- (2) The embedding agent according to the above [1] or [2].
  • [5] Z in the formula (2) is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp). The embedding agent according to the above [4].
  • the embedding agent disclosed in this specification can improve the sliceability of a frozen block produced using the embedding agent by adding synthetic collagen.
  • the synthetic collagen can be washed away with water after the living tissue is adhered on the support, the background when dyed is reduced.
  • FIG. 1 is a drawing-substituting photograph
  • FIG. 1A is a photograph of a frozen block produced using the embedding agents of Examples 1 and 2 and Comparative Example 1.
  • FIG. 1B is a photograph of a frozen section obtained by slicing a frozen block.
  • FIG. 2 is a drawing-substituting photograph
  • FIG. 2A is a photograph of a frozen block produced using the embedding agents of Examples 3 and 4 and Comparative Example 2.
  • FIG. 2B is a photograph of a frozen section obtained by slicing a frozen block.
  • FIG. 3 is a drawing-substituting photograph
  • FIG. 3A is a photograph of the HE-stained specimen prepared in Example 5.
  • FIG. 4 is a photograph substituted for a drawing
  • FIG. 4A is a photograph of a frozen block prepared in Example 6
  • FIG. 4B is a photograph of a frozen section obtained by slicing a frozen block.
  • the embedding agent disclosed in this specification includes at least a frozen embedding agent and synthetic collagen.
  • synthetic collagen means non-animal-derived polymer collagen synthesized using only amino acids generated from fermentation or synthesis.
  • animal-derived collagen obtained by extraction from animals such as cows, pigs, and fish is very difficult to be non-brominated because it contains impurities such as odorous components. Therefore, the ratio of impurities and the like varies depending on the raw materials, and it is difficult to homogenize the quality between products.
  • synthetic collagen is produced by synthesis, impurities such as odor components are not mixed.
  • synthetic collagen having a desired molecular weight can be obtained by making the synthesis conditions constant.
  • the synthetic collagen is not particularly limited as long as it is a non-animal-derived polymer collagen synthesized using only amino acids generated from fermentation or synthesis.
  • a type 1 collagen molecule has a primary structure containing a repetition of three amino acid residues represented by formula (1).
  • This polypeptide is known to have a triple helix tertiary structure formed by gathering three of them in the same direction. -(Gly-XY) n- (1)
  • X and Y are each independently selected from the following amino acid residues.
  • Ala L-alanine residue
  • Arg L-arginine residue
  • Asn L-asparagine residue
  • Asp L-aspartic acid residue
  • Cys L-cysteine residue
  • Gln L-glutamine residue
  • Glu L-glutamic acid residue
  • His L-histidine residue
  • Hyp L-hydroxyproline residue
  • Ile L-isoleucine residue
  • Leu L-leucine residue
  • Lys L-lysine residue
  • Met L-methionine residue
  • Phe L-phenylalanine residue
  • Pro L-proline residue
  • Sar Sarcosine residue
  • Ser L-serine residue
  • Thr L-threonine residue
  • Trp L-tryptophan residue
  • Tyr L-tyrosine residue
  • Val L-valine residue
  • the synthetic collagen represented by the formula (1) may be produced by a known production method. For example, it can be synthesized by subjecting a peptide oligomer containing a repeating unit of-(Gly-XY)-to a condensation reaction in dimethyl sulfoxide or an aqueous solvent containing ethylenediamine. When the condensation reaction is performed in an aqueous solvent, the molecular weight can be adjusted by adjusting the phosphate ion concentration in the solvent (see Japanese Patent Application Laid-Open No. 2017-8094). Further, when the side chain of the synthetic collagen has an amino group, it may be succinylated, phthalated or acetylated by a known method as necessary. By eliminating the free amino group, the synthetic collagen is hard to solidify.
  • Z is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp). That is, the synthetic collagen represented by the above formula (2) is Consisting of (Pro-Pro-Gly) units, poly (Pro-Pro-Gly), Consisting of (Pro-Hyp-Gly) units, poly (Pro-Hyp-Gly), (Pro-Pro-Gly) units and poly (Pro-Pro or Hyp-Gly) in which (Pro-Hyp-Gly) units are condensed in any order, Embodiments are included.
  • the repeating number n of the formulas (1) and (2) can be appropriately set according to the target molecular weight and the coexisting sequence.
  • the lower limit of the number of repetitions n is 10 or more, 20 or more, 30 or more, 40 or more, 50 or more
  • the upper limit is 50000 or less, 5000 or less, 1000 or less, 750 or less, 500 or less, 250 or less. Etc.
  • Synthetic collagen may be produced using a known method, or commercially available one may be used.
  • Examples of commercially available synthetic collagen include hybrid collagen (manufactured by UNIX Corporation), pure colla (manufactured by JNC Corporation), and the like.
  • a known frozen embedding agent may be used.
  • O.C.T compound manufactured by Sakura Finetech
  • white tissue coat manufactured by UIC
  • FSC22 manufactured by Leica
  • cryomount low viscosity and high viscosity; manufactured by Muto Chemical Co., Ltd.
  • cryomatrix manufactured by Thermo Fisher Scientific
  • SCAM SECTION-LAB
  • Addition of synthetic collagen to the frozen embedding agent can improve the sliceability of the frozen block. Moreover, after producing a frozen section from a frozen block, the synthetic collagen in the frozen section can be easily washed with water. Therefore, the amount of synthetic collagen added to the frozen embedding agent is not particularly limited as long as the desired slicing performance can be obtained and the frozen embedding function of the frozen embedding agent is not lost. Although not particularly limited, for example, about 0.001 to 1.0% by weight of synthetic collagen may be added to 100 parts by weight of the frozen embedding agent.
  • a “curable substrate-penetrating specimen” and a “curable substrate non-penetrating specimen” can be produced.
  • "Curable substrate penetration specimen” is fixed with a known fixative such as formalin, paraformaldehyde, glutaraldehyde, etc., because the tissue on the specimen is permanently fixed so that it will not break even if stored for a long period of time.
  • a curable base material such as paraffin, epoxy resin, methacrylate, or alkaline polyester.
  • a typical example is a paraffin specimen.
  • the specimen may be prepared by a known method.
  • the “curable base material non-penetrating specimen” means a specimen prepared without using the above “curable base material”.
  • a typical example is a frozen specimen.
  • a slice obtained by slicing may be attached to a support, and then the biological tissue may be fixed with a fixing solution.
  • a fixing solution for example, formalin
  • the gelatin is also fixed, and the gelatin cannot be washed away after the fixing step.
  • synthetic collagen having no free amino group it is not fixed with an aldehyde-based fixing solution.
  • the synthetic collagen when using synthetic collagen that does not have a free amino group, even when a biological tissue is subjected to an immobilization step using an aldehyde-based fixing solution in a state containing synthetic collagen, the synthetic collagen can be washed away after the immobilization step. The background of staining can be lowered.
  • the free amino group may be succinylated, phthalated or acetylated as described above.
  • synthetic collagen of formula (2) may be used.
  • synthetic collagen having a free amino group may be used. Therefore, although synthetic collagen can be used regardless of the presence or absence of a free amino group, it is preferable to use synthetic collagen that does not have a free amino group from the viewpoint that it can be used for various specimen preparation methods.
  • the embedding agent disclosed in this specification may contain a water-soluble substance as necessary.
  • water-soluble substances selected from the group consisting of sugars, salts, extracts, vitamins, pH adjusters, pigments, and surfactants.
  • the water-soluble substance includes saccharides (eg, agarose, sucrose, sorbitol, maltitol, starch syrup, lactose, fructose, oligosaccharide, etc.); processed starches (eg, roasted dextrin, enzyme) Dextrins such as modified dextrins); salts (eg, sodium chloride, calcium chloride, magnesium chloride, sodium sulfate, etc.); extracts (eg, extracts from cattle, pigs, shellfish, vegetables, etc.); vitamins (eg, vitamin C) PH adjusters (eg, citric acid, sodium citrate, succinic acid, phthalic acid, hydrochloric acid, sulfuric acid, acetic acid, malic acid, tartaric acid, etc.); pigments (eg, Red No.
  • saccharides eg, agarose, sucrose, sorbitol, maltitol, starch syrup, lactose, fructose, oligos
  • the support for preparing the specimen is not particularly limited as long as a slice of a living tissue can be attached and observed with a microscope, and examples thereof include a slide glass and a light-transmitting resin film.
  • the pathological examination using the produced curable substrate non-penetrating specimen is as described above.
  • staining such as HE staining, PAS staining, and Alcian blue staining
  • immunostaining such as enzyme antibody method (DAB) and fluorescent antibody method It may be dyed by a known method such as a method and observed under a microscope.
  • the specimen preparation sample is an adipose tissue
  • carboxylic acid compounds include monocarboxylic acid compounds such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and caproic acid; dicarboxylic acid compounds such as oxalic acid, malonic acid, succinic acid, and glutaric acid; tricarboxylic acids such as citric acid Compounds; aromatic carboxylic acid compounds such as benzoic acid and phthalic acid; hydroxy acid compounds such as lactic acid and malic acid; carboxylic acid compounds substituted with halogen such as chloroacetic acid; and the like.
  • the addition amount of the carboxylic acid compound is not particularly limited as long as the sliceability is improved, and may be appropriately adjusted according to the type of the embedding agent and the carboxylic acid compound.
  • Example 1 As a frozen embedding agent, O.D. C. A T compound (manufactured by Sakura Finetech) was used, and 0.5% hybrid collagen (manufactured by UNIX) was used as synthetic collagen. And embedding agent was produced by mixing frozen embedding agent: synthetic collagen in the ratio of 9: 1. The amount of synthetic collagen contained in 0.5% hybrid collagen is 0.5% by weight. Therefore, O.D. C. The amount of synthetic collagen added to the T compound is 9: 0.005, and the amount of synthetic collagen added to 100 parts by weight of the frozen embedding agent is 0.056 weight.
  • Example 2 By adding 30 ⁇ l of acetic acid (manufactured by Wako) to 1 g of the embedding agent prepared in Example 1, an embedding agent to which acetic acid was added was prepared.
  • Example 3 O. C. An embedding agent was prepared in the same procedure as in Example 1 except that white tissue coat FL (manufactured by UI Kasei Co., Ltd.) was used instead of T compound (manufactured by Sakura Finetech Co., Ltd.).
  • Example 4 By adding 30 ⁇ l of acetic acid (manufactured by Wako) to 1 g of the embedding agent produced in Example 3, an embedding agent to which acetic acid was added was produced.
  • frozen sections were prepared by the following procedure.
  • a copper block (70 ⁇ 110 ⁇ 30 mm), a frozen section preparation metal chuck, and a metal weight are cooled at ⁇ 50 ° C. in an aluminum block ultra-low temperature layer Micro Cool MC-100 (manufactured by TOMY) in advance. did.
  • Human fat was used as a sample, and the prepared embedding agent was adjusted to the sample so as not to contain bubbles.
  • the bottom of the cryomold 3 (manufactured by Sakura Finetech Japan Co., Ltd.) was placed on a copper block, and the sample (2) and the embedding agent were placed in cryomold 3.
  • FIG. 1A is a photograph of a frozen block produced using the embedding agents of Examples 1 and 2 and Comparative Example 1.
  • FIG. 1B is a photograph of a frozen section obtained by slicing a frozen block.
  • FIG. 2A is a photograph of frozen blocks produced using the embedding agents of Examples 3 and 4 and Comparative Example 2.
  • FIG. 2B is a photograph of a frozen section obtained by slicing a frozen block.
  • the frozen sections prepared only with the frozen embedding agents of Comparative Examples 1 and 2 had many sample parts missing.
  • the number of missing samples was smaller than in Comparative Examples 1 and 2.
  • the synthetic collagen and acetic acid were added to the frozen embedding agent, the sample was less removed and the wrinkles of the frozen section were reduced.
  • Example 5 Using a mouse kidney as a sample and the embedding agent of Example 1 as an embedding agent, a frozen block was prepared with isopentane cooled with liquid nitrogen, and then sliced with a cryostat to obtain a frozen section. Next, dyeing was performed according to the following procedure.
  • a curable base material non-penetrating specimen was prepared by pressing and pasting the obtained frozen section against a slide glass.
  • the obtained curable substrate non-penetrating specimen was rinsed with tap water to wash away the synthetic collagen on the slide glass.
  • HE staining was performed using hematoxylin and eosin (Merck).
  • FIG. 3A is a photograph of a HE-stained specimen prepared in Example 5.
  • ⁇ Comparative Example 3> As an embedding agent, a fish-derived gelatin MAX-F (manufactured by Nippi Co., Ltd.) aqueous solution (concentration: 5% by weight) and O.D. C. A HE-stained curable substrate non-penetrating specimen was prepared in the same procedure as in ⁇ Example 5> except that a mixture of T compound at a ratio of 1: 1 was used.
  • FIG. 3B is a photograph of the HE-stained specimen prepared in Comparative Example 3.
  • the HE-stained specimen prepared using the embedding agent containing gelatin was stained with HE around the tissue. This is considered to be an effect of gelatin remaining without flowing with water when the curable substrate non-penetrated specimen was rinsed with tap water.
  • the background of HE staining was not confirmed around the tissue of the HE-stained specimen prepared using the embedding agent containing synthetic collagen.
  • synthetic collagen like gelatin, has the effect of improving the sliceability including the biological tissue part when preparing a frozen section from a frozen block by adding it to a frozen embedding agent. Became clear.
  • synthetic collagen when rinsing with tap water after a curable substrate non-penetrating specimen, synthetic collagen is much easier to flow than gelatin.
  • synthetic collagen when synthetic collagen is added to the frozen embedding agent, in addition to improving the sliceability of the frozen block, the background due to staining can be reduced.
  • Example 6 As a frozen embedding agent, 9.5 g of white tissue coat FL (manufactured by UI Chemical Co., Ltd.), 0.5 g of 0.5% hybrid collagen (manufactured by UNIX) as synthetic collagen, 10% oxalic acid (Katayama Chemical Co., Ltd.) The embedding agent was produced by mixing 300 ⁇ l of the product manufactured by the company. Next, a frozen section was prepared in the same procedure as the above [Preparation of frozen section] using human fat as a living tissue and the prepared embedding agent.
  • FIG. 4A is a photograph of a frozen block, and FIG.
  • FIG. 4B is a photograph of a frozen section obtained by slicing a frozen block. As is clear from FIG. 4B, even when oxalic acid was used, a frozen section with good sliceability of the biological sample portion was obtained. From the results of Examples 2, 4, and 6, it was found that even if the temperature of the frozen block was lowered to the temperature at which the adipose tissue was frozen by further adding a carboxylic acid compound in addition to the frozen embedding agent and synthetic collagen, It was confirmed that the slicing property of can be improved.
  • the embedding agent disclosed in this specification is used, the background when the prepared specimen is stained is lowered. Therefore, it is useful not only for rapid pathological diagnosis during surgery but also for preparing curable substrate non-penetrating specimens and curable substrate penetrating specimens in research institutions such as medical institutions and university medical departments, general hospitals, and the like.

Abstract

The present invention addresses the problem of providing an embedding agent whereby it becomes possible to reduce background in a specimen sectioned from a frozen block when the specimen is dyed. The problem can be solved by an embedding agent for frozen section production use, which comprises a freezing and embedding agent and synthetic collagen.

Description

包埋剤Embedding agent
　本開示は包埋剤に関し、特に、合成コラーゲンを含む包埋剤に関するものである。 This disclosure relates to embedding agents, and particularly to embedding agents containing synthetic collagen.
　近年、医療の現場では、手術中に生体組織を採取し、良性か悪性か、また、切除断端への浸潤・リンパ節転移の有無を迅速に検査することが求められている。手術中に当該検査を行うことで、手術中に術式を変更したり切除範囲を適切に決定することができ、手術の精度を上げることができる。 Recently, in the medical field, it is required to collect a living tissue during an operation and to quickly examine whether it is benign or malignant, and whether there is invasion or lymph node metastasis to the resected margin. By performing the examination during the operation, it is possible to change the operation method during the operation or to appropriately determine the resection range, and to improve the accuracy of the operation.
　手術中の生体組織の検査は、迅速性や抗原性の保持が優れていることから、一般的には、凍結組織標本を作製し、顕微鏡観察で行われている。凍結組織標本は、採取した生体組織を凍結包埋剤で包埋→凍結（以下、生体組織を凍結包埋剤で包埋して凍結したものを、「凍結ブロック」と記載することがある。）→薄切→染色することで作製できる。凍結包埋剤としては、ポリビニルアルコール（ＰＶＡ）やポリエチレングリコール（ＰＥＧ）等のポリマーを用いたものが知られており、凍結包埋剤として市販されている。 The examination of living tissue during surgery is generally performed by microscopic observation by preparing a frozen tissue specimen because of its excellent rapidity and retention of antigenicity. A frozen tissue specimen may be described as “frozen block” in which a collected biological tissue is embedded with a freezing embedding agent → freeze (hereinafter, a living tissue is embedded with a freezing embedding agent and frozen. ) → Thin slice → Dye. As frozen embedding agents, those using polymers such as polyvinyl alcohol (PVA) and polyethylene glycol (PEG) are known, and are commercially available as frozen embedding agents.
　しかしながら、市販されている凍結包埋剤を用いて薄切すると、シワ、破れ等が発生し易く、きれいな薄膜（切片）を得ることが困難な場合がある。特に、多くの病院・研究機関では、凍結包埋剤としてＯ．Ｃ．Ｔコンパウンドが使用されているが、生体組織がＯ．Ｃ．Ｔコンパウンドとなじみが悪い場合は、薄切が難しいという問題がある。更に、凍結組織標本は、薄切した生体組織の切片をスライドガラスに接着して作製するが、従来の凍結包埋剤を用いた場合は、スライドガラスから剥がれやすいという問題がある。 However, when sliced with a commercially available frozen embedding agent, wrinkles and tears are likely to occur, and it may be difficult to obtain a clean thin film (section). In particular, in many hospitals and research institutions, O.D. C. T compound is used, but living tissue is O.D. C. If you are not familiar with T compound, there is a problem that it is difficult to slice. Furthermore, a frozen tissue specimen is produced by bonding a slice of a living tissue to a slide glass. However, when a conventional frozen embedding agent is used, there is a problem that the frozen tissue specimen is easily peeled off from the slide glass.
　本発明者らは、水溶液とした際に１５℃～２５℃では液体状態で且つ４℃では固体状態となるゼラチンを標本作製用包埋剤として用いる、又は、公知の凍結包埋剤に前記ゼラチンを添加することで、上記問題を解決できることを見出し、特許出願をしている（特許文献１参照）。 The present inventors use gelatin as a sample preparation embedding agent that is in a liquid state at 15 ° C. to 25 ° C. and in a solid state at 4 ° C. when it is used as an aqueous solution, or the gelatin for a known frozen embedding agent. It has been found that the above-mentioned problems can be solved by adding, and a patent application has been filed (see Patent Document 1).
国際公開第２０１５／１９９１９５号International Publication No. 2015/199195
　ところで、ゼラチンは動物性蛋白質のため、標本を作製する生体組織に馴染みやすい。更に、ゼラチンは粘着性を有する。そのため、特許文献１に記載のゼラチンからなる包埋剤、或いは、凍結包埋剤にゼラチンを添加した包埋剤は、生体組織に入り込んだゼラチンが残渣として残ることから、硬化性基材非浸透標本を作製する際に、生体組織の支持体への密着性が向上するというメリットがある。しかしながら、硬化性基材非浸透標本をヘマトキシリン・エオジン（ＨＥ）等を用いて染色した場合、残渣として支持体上に残ったゼラチンも染色される。そのため、染色した標本を画像解析ソフトにより解析する場合、染色されたゼラチンも含めて解析されることから、解析精度が落ちるという問題がある。 By the way, since gelatin is an animal protein, it is easy to become familiar with the living tissue from which the specimen is prepared. Furthermore, gelatin is sticky. Therefore, the embedding agent comprising gelatin described in Patent Document 1 or the embedding agent obtained by adding gelatin to the frozen embedding agent leaves gelatin remaining in the living tissue as a residue. When preparing a specimen, there is a merit that adhesion of a living tissue to a support is improved. However, when a curable substrate non-penetrating specimen is stained with hematoxylin / eosin (HE) or the like, gelatin remaining on the support as a residue is also stained. Therefore, when a stained specimen is analyzed by image analysis software, the analysis includes the stained gelatin, which causes a problem that analysis accuracy is lowered.
　本明細書における開示は、上記従来の問題を解決するためになされたものであり、鋭意研究を行ったところ、（１）公知の凍結包埋剤への添加剤として合成コラーゲンを用いると、凍結ブロックの薄切性が向上すること、（２）合成コラーゲンは０℃～常温で液体であることから、標本作製時の水の温度が低くても洗い流しやすく、生体組織を染色した時のバックグラウンドを減少できること、を新たに見出した。 The disclosure in the present specification has been made in order to solve the above-described conventional problems, and as a result of extensive research, (1) when synthetic collagen is used as an additive to a known frozen embedding agent, (2) Synthetic collagen is liquid at 0 ° C to room temperature, so it can be easily washed out even when the temperature of water is low, and the background when a living tissue is stained It was found that it can be reduced.
　すなわち、本明細書における開示の目的は、公知の凍結包埋剤の薄切性を改善でき、生体組織を染色した時のバックグラウンドが少ない包埋剤を提供することである。 That is, an object of the disclosure in the present specification is to provide an embedding agent that can improve the sliceability of a known frozen embedding agent and has a low background when a living tissue is stained.
　本明細書における開示は、以下に示す包埋剤に関する。 The disclosure in this specification relates to the embedding agent shown below.
［１］凍結切片作製用の包埋剤であって、該包埋剤は、
　　凍結包埋剤と、
　　合成コラーゲンと、
を含む、包埋剤。
［２］凍結切片の薄切性を改善するためのカルボン酸、
を更に含む、上記［１］に記載の包埋剤。
［３］前記合成コラーゲンが、下記式（１）で表される、
　　－（Ｇｌｙ－Ｘ－Ｙ）ｎ－　（１）
上記［１］または［２］に記載の包埋剤。
［４］前記合成コラーゲンが、下記式（２）で表される、
　　－（Ｐｒｏ－Ｚ－Ｇｌｙ）ｎ－　（２）
上記［１］または［２］に記載の包埋剤。
［５］前記式（２）中のＺが、プロリン残基（Ｐｒｏ）又はヒドロキシプロリン残基（Ｈｙｐ）から選択される、
上記［４］に記載の包埋剤。 [1] An embedding agent for preparing a frozen section, the embedding agent comprising:
With frozen embedding agents,
Synthetic collagen,
Including embedding agents.
[2] Carboxylic acid for improving the sliceability of frozen sections,
The embedding agent according to [1], further comprising:
[3] The synthetic collagen is represented by the following formula (1).
-(Gly-XY) n- (1)
The embedding agent according to the above [1] or [2].
[4] The synthetic collagen is represented by the following formula (2).
-(Pro-Z-Gly) n- (2)
The embedding agent according to the above [1] or [2].
[5] Z in the formula (2) is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp).
The embedding agent according to the above [4].
　本明細書で開示する包埋剤は、合成コラーゲンの添加により、包埋剤を用いて作製した凍結ブロックの薄切性を向上できる。また、支持体上に生体組織を貼着した後、合成コラーゲンを水で洗い流すことができるので、染色した時のバックグランドが少なくなる。 The embedding agent disclosed in this specification can improve the sliceability of a frozen block produced using the embedding agent by adding synthetic collagen. In addition, since the synthetic collagen can be washed away with water after the living tissue is adhered on the support, the background when dyed is reduced.
図１は、図面代用写真で、図１Ａは、実施例１及び２、並びに、比較例１の包埋剤を用いて作製した凍結ブロックの写真である。また、図１Ｂは、凍結ブロックを薄切した凍結切片の写真である。FIG. 1 is a drawing-substituting photograph, and FIG. 1A is a photograph of a frozen block produced using the embedding agents of Examples 1 and 2 and Comparative Example 1. FIG. 1B is a photograph of a frozen section obtained by slicing a frozen block. 図２は、図面代用写真で、図２Ａは、実施例３及び４、並びに、比較例２の包埋剤を用いて作製した凍結ブロックの写真である。また、図２Ｂは、凍結ブロックを薄切した凍結切片の写真である。FIG. 2 is a drawing-substituting photograph, and FIG. 2A is a photograph of a frozen block produced using the embedding agents of Examples 3 and 4 and Comparative Example 2. FIG. 2B is a photograph of a frozen section obtained by slicing a frozen block. 図３は、図面代用写真で、図３Ａは、実施例５で作製したＨＥ染色標本の写真である。図３Ｂは、比較例３で作製したＨＥ染色標本の写真である。FIG. 3 is a drawing-substituting photograph, and FIG. 3A is a photograph of the HE-stained specimen prepared in Example 5. FIG. 3B is a photograph of the HE-stained specimen prepared in Comparative Example 3. 図４は、図面代用写真で、図４Ａは実施例６で作製した凍結ブロックの写真、図４Ｂは凍結ブロックを薄切りした凍結切片の写真である。FIG. 4 is a photograph substituted for a drawing, FIG. 4A is a photograph of a frozen block prepared in Example 6, and FIG. 4B is a photograph of a frozen section obtained by slicing a frozen block.
　以下に、包埋剤の実施形態について詳しく説明する。 Hereinafter, embodiments of the embedding agent will be described in detail.
　本明細書で開示する包埋剤は、凍結包埋剤及び合成コラーゲンを少なくとも含んでいる。なお、本明細書において「合成コラーゲン」とは、発酵や合成から生成したアミノ酸のみを使用して合成した非動物由来型の高分子コラーゲンを意味する。 The embedding agent disclosed in this specification includes at least a frozen embedding agent and synthetic collagen. In the present specification, “synthetic collagen” means non-animal-derived polymer collagen synthesized using only amino acids generated from fermentation or synthesis.
　牛、豚、魚等の動物から抽出して得られた動物由来コラーゲンは、臭い成分などの不純物が混入しているため、無臭化することは非常に困難であると言われている。そのため、原料の違いにより、不純物等の比率が異なり、製品間の品質を均質化することは難しい。一方、合成コラーゲンは合成により製造されるため、臭い成分等の不純物が混入することはない。また、合成条件を一定にすることで、所期の分子量の合成コラーゲンを得ることもできる。 It is said that animal-derived collagen obtained by extraction from animals such as cows, pigs, and fish is very difficult to be non-brominated because it contains impurities such as odorous components. Therefore, the ratio of impurities and the like varies depending on the raw materials, and it is difficult to homogenize the quality between products. On the other hand, since synthetic collagen is produced by synthesis, impurities such as odor components are not mixed. Moreover, synthetic collagen having a desired molecular weight can be obtained by making the synthesis conditions constant.
　更に、動物由来の材料には夾雑物が混入する恐れがある。そのため、動物由来の材料を含む包埋剤を用いて標本を作製すると、染色の際に夾雑物も染色されバックグラウンドとなる可能性がある。一方、合成コラーゲンには夾雑物が混入する恐れがないことから、染色の際に、包埋剤に起因するバックグラウンドの発生を抑えることができる。 Furthermore, there is a risk of contamination from animal-derived materials. Therefore, when a specimen is prepared using an embedding agent containing animal-derived material, impurities may be stained and become a background during staining. On the other hand, since there is no fear that contaminants are mixed in the synthetic collagen, it is possible to suppress the occurrence of background due to the embedding agent at the time of staining.
　合成コラーゲンは、上記の通り、発酵や合成から生成したアミノ酸のみを使用して合成した非動物由来型の高分子コラーゲンであれば、特に制限はない。例えば、一型コラーゲン分子は、式（１）で表す三アミノ酸残基の繰り返しを含む一次構造を有する。このポリペプチドは、同じ向きに３本寄り集って形成された三重らせんの三次構造をとることが知られている。
　　－（Ｇｌｙ－Ｘ－Ｙ）ｎ－　（１） As described above, the synthetic collagen is not particularly limited as long as it is a non-animal-derived polymer collagen synthesized using only amino acids generated from fermentation or synthesis. For example, a type 1 collagen molecule has a primary structure containing a repetition of three amino acid residues represented by formula (1). This polypeptide is known to have a triple helix tertiary structure formed by gathering three of them in the same direction.
-(Gly-XY) n- (1)
　上記式（１）中、Ｘ及びＹは、夫々独立して以下のアミノ酸残基から選択される。
Ａｌａ：Ｌ－アラニン残基、
Ａｒｇ：Ｌ－アルギニン残基
Ａｓｎ：Ｌ－アスパラギン残基
Ａｓｐ：Ｌ－アスパラギン酸残基
Ｃｙｓ：Ｌ－システイン残基
Ｇｌｎ：Ｌ－グルタミン残基
Ｇｌｕ：Ｌ－グルタミン酸残基
Ｈｉｓ：Ｌ－ヒスチジン残基
Ｈｙｐ：Ｌ－ヒドロキシプロリン残基
Ｉｌｅ：Ｌ－イソロイシン残基
Ｌｅｕ：Ｌ－ロイシン残基
Ｌｙｓ：Ｌ－リジン残基
Ｍｅｔ：Ｌ－メチオニン残基
Ｐｈｅ：Ｌ－フェニルアラニン残基
Ｐｒｏ：Ｌ－プロリン残基
Ｓａｒ：サルコシン残基
Ｓｅｒ：Ｌ－セリン残基
Ｔｈｒ：Ｌ－トレオニン残基
Ｔｒｐ：Ｌ－トリプトファン残基
Ｔｙｒ：Ｌ－チロシン残基
Ｖａｌ：Ｌ－バリン残基 In the above formula (1), X and Y are each independently selected from the following amino acid residues.
Ala: L-alanine residue,
Arg: L-arginine residue Asn: L-asparagine residue Asp: L-aspartic acid residue Cys: L-cysteine residue Gln: L-glutamine residue Glu: L-glutamic acid residue His: L-histidine residue Hyp: L-hydroxyproline residue Ile: L-isoleucine residue Leu: L-leucine residue Lys: L-lysine residue Met: L-methionine residue Phe: L-phenylalanine residue Pro: L-proline residue Sar: Sarcosine residue Ser: L-serine residue Thr: L-threonine residue Trp: L-tryptophan residue Tyr: L-tyrosine residue Val: L-valine residue
　上記の通り、Ｘ、Ｙは任意の組み合わせであるが、ＸがＰｒｏ、ＹがＨｙｐの場合が好ましい。式（１）で表される合成コラーゲンは、公知の製造方法により製造すればよい。例えば、－（Ｇｌｙ－Ｘ－Ｙ）－の繰り返し単位を含むペプチドオリゴマーを、ジメチルスルホキシド中やエチレンジアミンを含む水系溶媒等中で縮合反応させることで合成できる。なお、水系溶媒中で縮合反応を行う場合、溶媒中のリン酸イオン濃度を調節することで、分子量を調整できる（特開２０１７－８０９４号公報参照）。また、合成コラーゲンの側鎖がアミノ基を持つ場合は、必要に応じて、公知の方法によりサクシニル化、フタル化、アセチル化してもよい。遊離アミノ基を無くすことで、合成コラーゲンが固まり難くなる。 As described above, X and Y are arbitrary combinations, but it is preferable that X is Pro and Y is Hyp. The synthetic collagen represented by the formula (1) may be produced by a known production method. For example, it can be synthesized by subjecting a peptide oligomer containing a repeating unit of-(Gly-XY)-to a condensation reaction in dimethyl sulfoxide or an aqueous solvent containing ethylenediamine. When the condensation reaction is performed in an aqueous solvent, the molecular weight can be adjusted by adjusting the phosphate ion concentration in the solvent (see Japanese Patent Application Laid-Open No. 2017-8094). Further, when the side chain of the synthetic collagen has an amino group, it may be succinylated, phthalated or acetylated by a known method as necessary. By eliminating the free amino group, the synthetic collagen is hard to solidify.
　また、式（１）以外の合成コラーゲンとしては、以下の式（２）で表す三アミノ酸残基の繰り返しからなるものも知られている。
　　－（Ｐｒｏ－Ｚ－Ｇｌｙ）ｎ－　（２） In addition, as synthetic collagen other than the formula (1), one comprising a repetition of three amino acid residues represented by the following formula (2) is also known.
-(Pro-Z-Gly) n- (2)
　上記式（２）中、Ｚはプロリン残基（Ｐｒｏ）又はヒドロキシプロリン残基（Ｈｙｐ）から選択される。つまり、上記式（２）で表される合成コラーゲンは、
　　（Ｐｒｏ－Ｐｒｏ－Ｇｌｙ）単位からなる、ポリ（Ｐｒｏ－Ｐｒｏ－Ｇｌｙ）、
　　（Ｐｒｏ－Ｈｙｐ－Ｇｌｙ）単位からなる、ポリ（Ｐｒｏ－Ｈｙｐ－Ｇｌｙ）、
　　（Ｐｒｏ－Ｐｒｏ－Ｇｌｙ）単位、及び、（Ｐｒｏ－Ｈｙｐ－Ｇｌｙ）単位が、任意の順番で縮合したポリ（Ｐｒｏ－Ｐｒｏ又はＨｙｐ－Ｇｌｙ）、
の態様が含まれる。 In the above formula (2), Z is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp). That is, the synthetic collagen represented by the above formula (2) is
Consisting of (Pro-Pro-Gly) units, poly (Pro-Pro-Gly),
Consisting of (Pro-Hyp-Gly) units, poly (Pro-Hyp-Gly),
(Pro-Pro-Gly) units and poly (Pro-Pro or Hyp-Gly) in which (Pro-Hyp-Gly) units are condensed in any order,
Embodiments are included.
　式（１）及び（２）の繰り返し数ｎは、目的の分子量や、共存する配列に応じて適宜設定することができる。例えば、繰り返し数ｎの下限値は、１０以上、２０以上、３０以上、４０以上、５０以上等が挙げられ、上限値は、５００００以下、５０００以下、１０００以下、７５０以下、５００以下、２５０以下等が挙げられる。 The repeating number n of the formulas (1) and (2) can be appropriately set according to the target molecular weight and the coexisting sequence. For example, the lower limit of the number of repetitions n is 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, and the upper limit is 50000 or less, 5000 or less, 1000 or less, 750 or less, 500 or less, 250 or less. Etc.
　合成コラーゲンは、公知の方法を用いて製造してもよいし、市販されているものを用いてもよい。市販されている合成コラーゲンとしては、ハイブリッドコラーゲン（ユニクス株式会社製）、ピュアコラ（ＪＮＣ株式会社製）等が挙げられる。 Synthetic collagen may be produced using a known method, or commercially available one may be used. Examples of commercially available synthetic collagen include hybrid collagen (manufactured by UNIX Corporation), pure colla (manufactured by JNC Corporation), and the like.
　本明細書で開示する包埋剤に含まれる凍結包埋剤としては、公知の凍結包埋剤を用いればよい。例えば、Ｏ.Ｃ.Ｔコンパウンド（サクラファインテック社製）、ホワイトティシュコート（ユーアイ化成社製）、ＦＳＣ２２（ライカ社製）、クリオマウント（低粘度及び高粘度；武藤化学社製）、クリオマトリックス(サーモフィッシャーサイエンティフィック)、ＳＣＡＭ（ＳＥＣＴＩＯＮ－ＬＡＢ）等が挙げられる。 As a frozen embedding agent contained in the embedding agent disclosed in the present specification, a known frozen embedding agent may be used. For example, O.C.T compound (manufactured by Sakura Finetech), white tissue coat (manufactured by UIC), FSC22 (manufactured by Leica), cryomount (low viscosity and high viscosity; manufactured by Muto Chemical Co., Ltd.), cryomatrix (Thermo Fisher Scientific), SCAM (SECTION-LAB) and the like.
　合成コラーゲンを凍結包埋剤に添加することで、凍結ブロックの薄切性を向上することができる。また、凍結ブロックから凍結切片を作製した後は、凍結切片中の合成コラーゲンは、水で簡単に流すことができる。したがって、凍結包埋剤に対する合成コラーゲンの添加量は、所期の薄切性能が得られ、凍結包埋剤の凍結包埋機能を喪失しない範囲内であれば特に制限はない。特に制限はされないが、例えば、凍結包埋剤１００重量部に対し、合成コラーゲンは、０．００１～１．０重量％程度添加されていればよい。 Addition of synthetic collagen to the frozen embedding agent can improve the sliceability of the frozen block. Moreover, after producing a frozen section from a frozen block, the synthetic collagen in the frozen section can be easily washed with water. Therefore, the amount of synthetic collagen added to the frozen embedding agent is not particularly limited as long as the desired slicing performance can be obtained and the frozen embedding function of the frozen embedding agent is not lost. Although not particularly limited, for example, about 0.001 to 1.0% by weight of synthetic collagen may be added to 100 parts by weight of the frozen embedding agent.
　本明細書で開示する包埋剤を用いて生体組織を包埋し、凍結・薄切することで「硬化性基材浸透標本」及び「硬化性基材非浸透標本」を作製することができる。「硬化性基材浸透標本」とは、標本上の組織が長期間保存しても壊れないように永久的に固定するため、ホルマリン、パラフォルムアルデヒド、グルタルアルデヒド等の公知の固定液により固定化した生体組織を脱脂・脱水などの処理をした後に、パラフィン、エポキシ樹脂、メタクリレート、アルカリ系ポリエステル等の硬化性基材を、組織の内部まで浸透させて生体組織を硬化した標本を意味する。代表的なものとしてパラフィン標本が挙げられる。標本は公知の方法により作製すればよい。 By embedding a living tissue using the embedding agent disclosed in the present specification, and freezing and slicing it, a “curable substrate-penetrating specimen” and a “curable substrate non-penetrating specimen” can be produced. . "Curable substrate penetration specimen" is fixed with a known fixative such as formalin, paraformaldehyde, glutaraldehyde, etc., because the tissue on the specimen is permanently fixed so that it will not break even if stored for a long period of time. It means a specimen in which a living tissue is cured by degreasing and dehydrating the living tissue and then infiltrating a curable base material such as paraffin, epoxy resin, methacrylate, or alkaline polyester into the inside of the tissue. A typical example is a paraffin specimen. The specimen may be prepared by a known method.
　そして、「硬化性基材非浸透標本」とは、上記「硬化性基材」を使用せずに作製した標本を意味する。例えば、上記の固定液で固定化した生体組織、または、未固定生体組織を、包埋剤等を用いて硬度を持たせ、薄切して得られた切片を支持体に貼着した標本が挙げられる。代表的なものとして凍結標本が挙げられる。 The “curable base material non-penetrating specimen” means a specimen prepared without using the above “curable base material”. For example, a specimen in which a biological tissue fixed with the above-described fixing solution or an unfixed biological tissue is hardened using an embedding agent, and a slice obtained by slicing is attached to a support. Can be mentioned. A typical example is a frozen specimen.
　硬化性基材非浸透標本を用いて病理検査を行う例として、（１）水で硬化性基材非浸透標本を洗うことで、支持体上に付着している包埋剤を洗い流し、（２）ＨＥ染色、脂肪染色等の染色法、又は、酵素抗体法（ＤＡＢ）、蛍光抗体法等の免疫染色法等、公知の方法で染色し、（３）顕微鏡観察する、手順が挙げられる。当該手順（１）において、包埋剤（或いは薄切性改善剤）としてゼラチンを用いた場合、水で洗いきれずに残渣として付着したゼラチンが染色されるという問題があった。一方、合成コラーゲンは０℃～常温で液体である。そのため、水の温度が低くても、支持体上に残渣として付着する合成コラーゲンが少なくなり、染色時のバックグラウンドを低くできる。 As an example of performing pathological examination using a curable base material non-penetrable specimen, (1) washing the curable base material non-penetrated specimen with water, washing away the embedding agent adhering to the support, (2 ) Staining methods such as HE staining, fat staining, etc., or immunostaining methods such as enzyme antibody method (DAB), fluorescent antibody method, etc., and (3) Microscopic observation are included. In the procedure (1), when gelatin is used as an embedding agent (or sliceability improving agent), there is a problem that gelatin adhered as a residue is dyed without being washed with water. On the other hand, synthetic collagen is liquid at 0 ° C. to room temperature. Therefore, even if the temperature of water is low, the amount of synthetic collagen adhering as a residue on the support is reduced, and the background during staining can be lowered.
　なお、「硬化性基材非浸透標本」を作製する際には、薄切して得られた切片を支持体に貼着した後に、固定液で生体組織を固定する場合がある。その際に、固定液として遊離アミノ基を架橋するアルデヒド系固定液（例えば、ホルマリン）を用いるとゼラチンも固定され、固定化工程後にゼラチンを洗い流すことはできなかった。一方、遊離アミノ基を有しない合成コラーゲンを用いた場合は、アルデヒド系固定液で固定されない。したがって、遊離アミノ基を有しない合成コラーゲンを用いると、合成コラーゲンを含んだ状態で生体組織をアルデヒド系固定液による固定化工程を行った場合でも、固定化工程後に合成コラーゲンを洗い流すことができ、染色のバックグラウンドを低くできる。 When producing a “curable base material non-penetrable specimen”, a slice obtained by slicing may be attached to a support, and then the biological tissue may be fixed with a fixing solution. At that time, when an aldehyde-based fixing solution (for example, formalin) that crosslinks a free amino group is used as the fixing solution, the gelatin is also fixed, and the gelatin cannot be washed away after the fixing step. On the other hand, when synthetic collagen having no free amino group is used, it is not fixed with an aldehyde-based fixing solution. Therefore, when using synthetic collagen that does not have a free amino group, even when a biological tissue is subjected to an immobilization step using an aldehyde-based fixing solution in a state containing synthetic collagen, the synthetic collagen can be washed away after the immobilization step. The background of staining can be lowered.
　遊離アミノ基を有しない合成コラーゲンを得るためには、上記のとおり、遊離アミノ基をサクシニル化、フタル化、アセチル化すればよい。或いは、式（２）の合成コラーゲンを用いればよい。なお、アルコール固定等、固定液として遊離アミノ基を架橋しない固定液を用いる場合は、遊離アミノ基がある合成コラーゲンでもよい。したがって、合成コラーゲンとしては、遊離アミノ基の有無に関係なく用いることができるが、多様な標本作成方法に対応できるとの観点からは、遊離アミノ基を有しない合成コラーゲンを用いることが好ましい。 In order to obtain synthetic collagen having no free amino group, the free amino group may be succinylated, phthalated or acetylated as described above. Alternatively, synthetic collagen of formula (2) may be used. In the case of using a fixing solution that does not crosslink free amino groups as a fixing solution, such as alcohol fixation, synthetic collagen having a free amino group may be used. Therefore, although synthetic collagen can be used regardless of the presence or absence of a free amino group, it is preferable to use synthetic collagen that does not have a free amino group from the viewpoint that it can be used for various specimen preparation methods.
　本明細書で開示する包埋剤は、必要に応じて、水溶性物質を加えてもよい。例えば、糖類、塩類、エキス類、ビタミン類、ｐＨ調整剤類、色素類、界面活性剤類からなる群から選択される水溶性物質が挙げられる。 The embedding agent disclosed in this specification may contain a water-soluble substance as necessary. Examples thereof include water-soluble substances selected from the group consisting of sugars, salts, extracts, vitamins, pH adjusters, pigments, and surfactants.
　前記水溶性物質についてより具体的に記載すると、糖類（例えば、アガロース、スクロース、ソルビト－ル、マルチト－ル、水飴、乳糖、果糖、オリゴ糖等）；加工澱粉類（例えば、焙焼デキストリン、酵素変性デキストリン等のデキストリン）；塩類（例えば、塩化ナトリウム、塩化カルシウム、塩化マグネシウム、硫酸ナトリウム等）；エキス類（例えば、牛、豚、貝、野菜由来のエキス等）；ビタミン類（例えば、ビタミンＣ、ビタミンＣナトリウム、ビタミンＢ１塩酸塩等）；ｐＨ調整剤類（例えば、クエン酸、クエン酸ナトリウム、コハク酸、フタル酸、塩酸、硫酸、酢酸、リンゴ酸、酒石酸等）；色素類（例えば、赤色２号，３号，１０２号，１０５号，１０６号、黄色４号，５号、青色２号等）；界面活性剤類（例えば、蔗糖脂肪酸エステル、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、プロピレングリコ－ル脂肪酸エステル等）が挙げられ、それぞれ単独で用いても良いし２種以上併用しても良い。 More specifically, the water-soluble substance includes saccharides (eg, agarose, sucrose, sorbitol, maltitol, starch syrup, lactose, fructose, oligosaccharide, etc.); processed starches (eg, roasted dextrin, enzyme) Dextrins such as modified dextrins); salts (eg, sodium chloride, calcium chloride, magnesium chloride, sodium sulfate, etc.); extracts (eg, extracts from cattle, pigs, shellfish, vegetables, etc.); vitamins (eg, vitamin C) PH adjusters (eg, citric acid, sodium citrate, succinic acid, phthalic acid, hydrochloric acid, sulfuric acid, acetic acid, malic acid, tartaric acid, etc.); pigments (eg, Red No. 2, No. 3, No. 102, No. 105, No. 106, Yellow No. 4, No. 5, Blue No. 2, etc.); Fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, propylene glycol - Le fatty ester) are exemplified, each be used alone or in combination of two or more.
　標本を作製する際の支持体としては、薄切した生体組織の切片を貼着し顕微鏡観察できるものであれば特に制限は無く、スライドガラスや光透過性の樹脂フィルム等が挙げられる。 The support for preparing the specimen is not particularly limited as long as a slice of a living tissue can be attached and observed with a microscope, and examples thereof include a slide glass and a light-transmitting resin film.
　作製した硬化性基材非浸透標本を用いての病理検査は、上記のとおりである。また、作製した硬化性基材浸透標本を用いて病理検査する場合は、ＨＥ染色、ＰＡＳ染色、アルシアン青染色、等の染色法、又は、酵素抗体法（ＤＡＢ）、蛍光抗体法等の免疫染色法等、公知の方法で染色し、顕微鏡観察すればよい。 The pathological examination using the produced curable substrate non-penetrating specimen is as described above. In addition, when pathological examination is performed using the prepared curable substrate penetrating specimen, staining such as HE staining, PAS staining, and Alcian blue staining, or immunostaining such as enzyme antibody method (DAB) and fluorescent antibody method It may be dyed by a known method such as a method and observed under a microscope.
　また、標本作製用試料が脂肪組織の場合、脂肪組織が凍結する温度まで冷却しても凍結ブロックの良好な薄切性が得られる必要がある。その場合、必要に応じて、包埋剤にカルボン酸を添加してもよい。カルボン酸化合物としては、ギ酸、酢酸、プロピオン酸、酪酸、吉草酸、カプロン酸等のモノカルボン酸化合物；シュウ酸、マロン酸、コハク酸、グルタル酸等のジカルボン酸化合物；クエン酸等のトリカルボン酸化合物；安息香酸、フタル酸等の芳香族カルボン酸化合物；乳酸、リンゴ酸等のヒドロキシ酸化合物；クロロ酢酸等のハロゲンで置換されたカルボン酸化合物；等が挙げられる。カルボン酸化合物の添加量は、薄切性が改善する量であれば特に制限は無く、包埋剤やカルボン酸化合物の種類に応じて適宜調整すればよい。 In addition, when the specimen preparation sample is an adipose tissue, it is necessary to obtain a good slicing property of the frozen block even if it is cooled to a temperature at which the adipose tissue is frozen. In that case, you may add carboxylic acid to an embedding agent as needed. Examples of carboxylic acid compounds include monocarboxylic acid compounds such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, and caproic acid; dicarboxylic acid compounds such as oxalic acid, malonic acid, succinic acid, and glutaric acid; tricarboxylic acids such as citric acid Compounds; aromatic carboxylic acid compounds such as benzoic acid and phthalic acid; hydroxy acid compounds such as lactic acid and malic acid; carboxylic acid compounds substituted with halogen such as chloroacetic acid; and the like. The addition amount of the carboxylic acid compound is not particularly limited as long as the sliceability is improved, and may be appropriately adjusted according to the type of the embedding agent and the carboxylic acid compound.
　以下に実施例を掲げ、包埋剤の実施形態を具体的に説明するが、この実施例は単に実施形態の具体的な態様の参考のために提供されているものである。これらの例示は、発明の範囲を限定したり、あるいは制限することを表すものではない。 Hereinafter, embodiments of the embedding agent will be specifically described with reference to examples, but these examples are provided merely for reference of specific aspects of the embodiments. These illustrations are not meant to limit or limit the scope of the invention.
［包埋剤の作製］
＜実施例１＞
　凍結包埋剤として、Ｏ．Ｃ．Ｔコンパウンド（サクラファインテック社製）を用い、合成コラーゲンとして、０．５％ハイブリッドコラーゲン（ユニクス社製）を用いた。そして、凍結包埋剤：合成コラーゲンを、９：１の割合で混合することで包埋剤を作製した。なお、０．５％ハイブリッドコラーゲンに含まれる合成コラーゲンの量は０．５重量％である。したがって、Ｏ．Ｃ．Ｔコンパウンドに対する合成コラーゲンの添加量は、９：０．００５となり、凍結包埋剤１００重量部に対する合成コラーゲンの添加量は、０．０５６重量となる。 [Production of embedding agent]
<Example 1>
As a frozen embedding agent, O.D. C. A T compound (manufactured by Sakura Finetech) was used, and 0.5% hybrid collagen (manufactured by UNIX) was used as synthetic collagen. And embedding agent was produced by mixing frozen embedding agent: synthetic collagen in the ratio of 9: 1. The amount of synthetic collagen contained in 0.5% hybrid collagen is 0.5% by weight. Therefore, O.D. C. The amount of synthetic collagen added to the T compound is 9: 0.005, and the amount of synthetic collagen added to 100 parts by weight of the frozen embedding agent is 0.056 weight.
＜実施例２＞
　実施例１で作製した包埋剤１ｇに、酢酸（Ｗａｋｏ社製）３０μｌを添加することで、酢酸を添加した包埋剤を作製した。 <Example 2>
By adding 30 μl of acetic acid (manufactured by Wako) to 1 g of the embedding agent prepared in Example 1, an embedding agent to which acetic acid was added was prepared.
＜実施例３＞
　Ｏ．Ｃ．Ｔコンパウンド（サクラファインテック社製）に代え、ホワイトティシュコートＦＬ（ユーアイ化成社製）を用いた以外は、実施例１と同様の手順で包埋剤を作製した。 <Example 3>
O. C. An embedding agent was prepared in the same procedure as in Example 1 except that white tissue coat FL (manufactured by UI Kasei Co., Ltd.) was used instead of T compound (manufactured by Sakura Finetech Co., Ltd.).
＜実施例４＞
　実施例３で作製した包埋剤１ｇに、酢酸（Ｗａｋｏ社製）３０μｌを添加することで、酢酸を添加した包埋剤を作製した。 <Example 4>
By adding 30 μl of acetic acid (manufactured by Wako) to 1 g of the embedding agent produced in Example 3, an embedding agent to which acetic acid was added was produced.
＜比較例１＞
　合成コラーゲンを添加していないＯ．Ｃ．Ｔコンパウンドのみを、比較例１の包埋剤とした。 <Comparative Example 1>
O. with no synthetic collagen added. C. Only the T compound was used as the embedding agent of Comparative Example 1.
＜比較例２＞
　合成コラーゲンを添加していないホワイトティシュコートＦＬのみを、比較例２の包埋剤とした。 <Comparative Example 2>
Only the white tissue coat FL to which no synthetic collagen was added was used as the embedding agent of Comparative Example 2.
［凍結切片の作製］
　次に、実施例１～４及び比較例１～２の包埋剤を用いて、以下の手順で凍結切片を作製した。
（１）予め、アルミブロック超低温層Ｍｉｃｒｏ　Ｃｏｏｌ　ＭＣ－１００（ＴＯＭＹ社製）内に、銅ブロック（７０×１１０×３０ｍｍ）、凍結切片作製用金属チャック、及び、金属重りを、－５０℃で冷却した。
（２）試料としてヒト脂肪を用い、作製した包埋剤を気泡が入らないように試料に馴染ませた。
（３）クリオモルド３号（サクラファインテックジャパン社製）の底部をくり抜いたものを銅ブロック上に置き、上記（２）の試料と包埋剤をクリオモルド３号内に置いた。次に、凍結切片作製用金属チャックを被せ、さらに金属重りを載せて、Ｍｉｃｒｏ　Ｃｏｏｌの蓋をし、試料及び包埋剤の凍結ブロックを作製した。
（４）金属重り及びクリオモルドを外し、クライオスタットミクロトーム（ライカ社製ＣＭ３０５０）を用いて凍結ブロックの薄切を行い、厚さ約５μｍの凍結切片を得た。 [Preparation of frozen section]
Next, using the embedding agents of Examples 1 to 4 and Comparative Examples 1 and 2, frozen sections were prepared by the following procedure.
(1) A copper block (70 × 110 × 30 mm), a frozen section preparation metal chuck, and a metal weight are cooled at −50 ° C. in an aluminum block ultra-low temperature layer Micro Cool MC-100 (manufactured by TOMY) in advance. did.
(2) Human fat was used as a sample, and the prepared embedding agent was adjusted to the sample so as not to contain bubbles.
(3) The bottom of the cryomold 3 (manufactured by Sakura Finetech Japan Co., Ltd.) was placed on a copper block, and the sample (2) and the embedding agent were placed in cryomold 3. Next, it was covered with a metal chuck for preparing a frozen section, a metal weight was further placed thereon, a Micro Cool cover was applied, and a frozen block of a sample and an embedding agent was prepared.
(4) The metal weight and cryomold were removed, and the frozen block was sliced using a cryostat microtome (Leica CM3050) to obtain a frozen section having a thickness of about 5 μm.
　図１Ａは、実施例１及び２、並びに、比較例１の包埋剤を用いて作製した凍結ブロックの写真である。また、図１Ｂは、凍結ブロックを薄切りした凍結切片の写真である。 FIG. 1A is a photograph of a frozen block produced using the embedding agents of Examples 1 and 2 and Comparative Example 1. FIG. 1B is a photograph of a frozen section obtained by slicing a frozen block.
　図２Ａは、実施例３及び４、並びに、比較例２の包埋剤を用いて作製した凍結ブロックの写真である。また、図２Ｂは、凍結ブロックを薄切りした凍結切片の写真である。 FIG. 2A is a photograph of frozen blocks produced using the embedding agents of Examples 3 and 4 and Comparative Example 2. FIG. 2B is a photograph of a frozen section obtained by slicing a frozen block.
　図１Ｂ及び図２Ｂの写真から明らかなように、比較例１及び２の凍結包埋剤のみで作製した凍結切片は、試料部分の抜けが多かった。一方、合成コラーゲンを添加した実施例１～４の包埋剤を用いて作製した凍結切片は、比較例１及び２より、試料の抜けは少なかった。更に、凍結包埋剤に合成コラーゲン及び酢酸を添加した方が、試料の抜けが少なくなると共に、凍結切片のしわが少なくなった。以上の結果より、凍結包埋剤に合成コラーゲンを添加することで、凍結ブロックの薄切性、特に、試料部分の薄切性が向上し、更に酢酸を添加することで、試料部分の薄切性がより向上することが明らかとなった。 As is clear from the photographs of FIGS. 1B and 2B, the frozen sections prepared only with the frozen embedding agents of Comparative Examples 1 and 2 had many sample parts missing. On the other hand, in the frozen sections prepared using the embedding agents of Examples 1 to 4 to which synthetic collagen was added, the number of missing samples was smaller than in Comparative Examples 1 and 2. Furthermore, when the synthetic collagen and acetic acid were added to the frozen embedding agent, the sample was less removed and the wrinkles of the frozen section were reduced. Based on the above results, the addition of synthetic collagen to the frozen embedding improves the sliceability of the frozen block, especially the sliceability of the sample portion, and the addition of acetic acid further improves the sliceability of the sample portion. It became clear that the sex improved more.
［染色性の比較］
＜実施例５＞
　試料としてマウスの腎臓、包埋剤として実施例１の包埋剤を用い、液体窒素で冷却したイソペンタンにて凍結ブロックを作製後、クライオスタットで薄切をし、凍結切片を得た。次に、以下の手順で染色を行った。
（ａ）得られた凍結切片を、スライドガラスを押し当て貼着することで硬化性基材非浸透標本を作製した。
（ｂ）得られた硬化性基材非浸透標本を水道水で濯ぐことで、スライドガラス上の合成コラーゲンを洗い流した。次いでヘマトキシリン・エオジン（メルク社製）を用い、ＨＥ染色を行った。図３Ａは、実施例５で作製したＨＥ染色標本の写真である。 [Comparison of staining properties]
<Example 5>
Using a mouse kidney as a sample and the embedding agent of Example 1 as an embedding agent, a frozen block was prepared with isopentane cooled with liquid nitrogen, and then sliced with a cryostat to obtain a frozen section. Next, dyeing was performed according to the following procedure.
(A) A curable base material non-penetrating specimen was prepared by pressing and pasting the obtained frozen section against a slide glass.
(B) The obtained curable substrate non-penetrating specimen was rinsed with tap water to wash away the synthetic collagen on the slide glass. Next, HE staining was performed using hematoxylin and eosin (Merck). FIG. 3A is a photograph of a HE-stained specimen prepared in Example 5.
＜比較例３＞
　包埋剤として、魚由来のゼラチンであるＭＡＸ－Ｆ（株式会社ニッピ社製）水溶液（濃度は５重量％）とＯ．Ｃ．Ｔコンパウンドを１：１の割合で混合したものを用いた以外は、上記＜実施例５＞と同様の手順で、ＨＥ染色した硬化性基材非浸透標本を作製した。図３Ｂは、比較例３で作製したＨＥ染色標本の写真である。 <Comparative Example 3>
As an embedding agent, a fish-derived gelatin MAX-F (manufactured by Nippi Co., Ltd.) aqueous solution (concentration: 5% by weight) and O.D. C. A HE-stained curable substrate non-penetrating specimen was prepared in the same procedure as in <Example 5> except that a mixture of T compound at a ratio of 1: 1 was used. FIG. 3B is a photograph of the HE-stained specimen prepared in Comparative Example 3.
　図３Ｂから明らかなように、ゼラチンを含む包埋剤を用いて作製したＨＥ染色標本は、組織周辺がＨＥにより染色されていた。これは、硬化性基材非浸透標本を水道水で濯いだ際に、水で流れずに残ったゼラチンの影響と考えられる。一方、図３Ａから明らかなように、合成コラーゲンを含む包埋剤を用いて作製したＨＥ染色標本は、組織周辺にＨＥ染色のバックグラウンドは確認されなかった。 As is clear from FIG. 3B, the HE-stained specimen prepared using the embedding agent containing gelatin was stained with HE around the tissue. This is considered to be an effect of gelatin remaining without flowing with water when the curable substrate non-penetrated specimen was rinsed with tap water. On the other hand, as is clear from FIG. 3A, the background of HE staining was not confirmed around the tissue of the HE-stained specimen prepared using the embedding agent containing synthetic collagen.
　以上の結果より、合成コラーゲンはゼラチンと同様、凍結包埋剤に添加することで、凍結ブロックから凍結切片を作製する際に、生体組織部分も含めた薄切性を向上するという効果を有することが明らかとなった。一方、硬化性基材非浸透標本後に水道水で濯いだ際に、ゼラチンと比較して合成コラーゲンは非常に流れやすい。その結果、合成コラーゲンを凍結包埋剤に添加すると、凍結ブロックの薄切性の向上に加え、染色によるバックグラウンドを低減できるという顕著な効果を奏することが明らかとなった。 From the above results, synthetic collagen, like gelatin, has the effect of improving the sliceability including the biological tissue part when preparing a frozen section from a frozen block by adding it to a frozen embedding agent. Became clear. On the other hand, when rinsing with tap water after a curable substrate non-penetrating specimen, synthetic collagen is much easier to flow than gelatin. As a result, it has been clarified that when synthetic collagen is added to the frozen embedding agent, in addition to improving the sliceability of the frozen block, the background due to staining can be reduced.
［シュウ酸を用いた包埋剤］
＜実施例６＞
　凍結包埋剤として、ホワイトティシュコートＦＬ（ユーアイ化成社製）を９．５ｇ、合成コラーゲンとして、０．５％ハイブリッドコラーゲン（ユニクス社製）を０．５ｇ、１０％シュウ酸（片山化学工業株式会社製）を３００μｌ混合することで、包埋剤を作製した。次に、生体組織としてヒト脂肪を用い、作製した包埋剤を用いて、上記［凍結切片の作製］と同様の手順で、凍結切片を作製した。図４Ａは凍結ブロックの写真、図４Ｂは凍結ブロックを薄切りした凍結切片の写真である。図４Ｂから明らかなように、シュウ酸を用いた場合も、生体試料部分の薄切性が良好な凍結切片が得られた。実施例２、４、及び６の結果より、凍結包埋剤及び合成コラーゲンに加え、カルボン酸化合物を更に添加することで、脂肪組織が凍結する温度まで凍結ブロックの温度を下げても、凍結切片の薄切性を向上できることを確認した。 [Embedding agent using oxalic acid]
<Example 6>
As a frozen embedding agent, 9.5 g of white tissue coat FL (manufactured by UI Chemical Co., Ltd.), 0.5 g of 0.5% hybrid collagen (manufactured by UNIX) as synthetic collagen, 10% oxalic acid (Katayama Chemical Co., Ltd.) The embedding agent was produced by mixing 300 μl of the product manufactured by the company. Next, a frozen section was prepared in the same procedure as the above [Preparation of frozen section] using human fat as a living tissue and the prepared embedding agent. FIG. 4A is a photograph of a frozen block, and FIG. 4B is a photograph of a frozen section obtained by slicing a frozen block. As is clear from FIG. 4B, even when oxalic acid was used, a frozen section with good sliceability of the biological sample portion was obtained. From the results of Examples 2, 4, and 6, it was found that even if the temperature of the frozen block was lowered to the temperature at which the adipose tissue was frozen by further adding a carboxylic acid compound in addition to the frozen embedding agent and synthetic collagen, It was confirmed that the slicing property of can be improved.
　本明細書で開示する包埋剤を用いると、作製した標本を染色した際のバックグラウンドが低くなる。したがって、手術中の迅速病理診断のみならず、医療機関や大学医学部などの研究機関、一般病院等において、硬化性基材非浸透標本及び硬化性基材浸透標本の作製に有用である。 If the embedding agent disclosed in this specification is used, the background when the prepared specimen is stained is lowered. Therefore, it is useful not only for rapid pathological diagnosis during surgery but also for preparing curable substrate non-penetrating specimens and curable substrate penetrating specimens in research institutions such as medical institutions and university medical departments, general hospitals, and the like.

Claims (5)

  1. 　凍結切片作製用の包埋剤であって、該包埋剤は、
    　　凍結包埋剤と、
    　　合成コラーゲンと、
    を含む、包埋剤。     An embedding agent for preparing a frozen section, the embedding agent comprising:
    With frozen embedding agents,
    Synthetic collagen,
    Including embedding agents.
  2. 　凍結切片の薄切性を改善するためのカルボン酸、
    を更に含む、請求項１に記載の包埋剤。     Carboxylic acid to improve the sliceability of frozen sections,
    The embedding agent according to claim 1, further comprising:
  3. 　前記合成コラーゲンが、下記式（１）で表される、
    　　－（Ｇｌｙ－Ｘ－Ｙ）ｎ－　（１）
    請求項１または２に記載の包埋剤。     The synthetic collagen is represented by the following formula (1):
    -(Gly-XY) n- (1)
    The embedding agent according to claim 1 or 2.
  4. 　前記合成コラーゲンが、下記式（２）で表される、
    　　－（Ｐｒｏ－Ｚ－Ｇｌｙ）ｎ－　（２）
    請求項１または２に記載の包埋剤。     The synthetic collagen is represented by the following formula (2):
    -(Pro-Z-Gly) n- (2)
    The embedding agent according to claim 1 or 2.
  5. 　前記式（２）中のＺが、プロリン残基（Ｐｒｏ）又はヒドロキシプロリン残基（Ｈｙｐ）から選択される、請求項４に記載の包埋剤。
          The embedding agent according to claim 4, wherein Z in the formula (2) is selected from a proline residue (Pro) or a hydroxyproline residue (Hyp).
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JP2014009194A (en) * 2012-06-29 2014-01-20 Jnc Corp Method for manufacturing collagen-like polypeptides
WO2015199195A1 (en) * 2014-06-27 2015-12-30 国立大学法人名古屋大学 Embedding medium for specimen preparation, method for preparing curable base material non-penetrating specimen, method for preparing curable base material penetrating specimen, curable base material non-penetrating specimen, thin-slice-performance improver for frozen embedding medium, and frozen embedding medium

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JP2014009194A (en) * 2012-06-29 2014-01-20 Jnc Corp Method for manufacturing collagen-like polypeptides
WO2015199195A1 (en) * 2014-06-27 2015-12-30 国立大学法人名古屋大学 Embedding medium for specimen preparation, method for preparing curable base material non-penetrating specimen, method for preparing curable base material penetrating specimen, curable base material non-penetrating specimen, thin-slice-performance improver for frozen embedding medium, and frozen embedding medium

Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN112557159A (en) * 2020-11-26 2021-03-26 华中科技大学同济医学院附属协和医院 Specimen embedding device and frozen slicing method
CN112557159B (en) * 2020-11-26 2023-11-07 华中科技大学同济医学院附属协和医院 Specimen embedding device and frozen slicing method

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