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METHOD FOR MAKING BIOLOGICAL TISSUE TRANSPARENT AND REAGENT FOR SAME

Foreign code F190009778
File No. (S2017-0882-N0)
Posted date May 7, 2019
Country WIPO
International application number 2018JP025239
International publication number WO 2019009300
Date of international filing Jul 3, 2018
Date of international publication Jan 10, 2019
Priority data
  • P2017-132787 (Jul 6, 2017) JP
Title METHOD FOR MAKING BIOLOGICAL TISSUE TRANSPARENT AND REAGENT FOR SAME
Abstract Provided is a method for quickly making biological tissue transparent. A method for making biological tissue transparent, the method being characterized by including: a step for impregnating biological tissue with water-soluble ethylenic unsaturated monomers before, during, or after the fixing of the biological tissue by a fixing agent, the water-soluble ethylenic unsaturated monomers including at least a water-soluble ethylenic unsaturated monomer that has an ionic leaving group; a step for forming a hydrogel by polymerizing the water-soluble ethylenic unsaturated monomers inside the biological tissue post-fixing; and a step for removing lipids from the biological tissue post-fixing.
Outline of related art and contending technology BACKGROUND ART
Fluorescence imaging, diagnosis of diseases or in vivo biological phenomenon at the time of 1 as one of useful tools have been used, in the conventional technique, a tissue (for example the slice) as a two-dimensional image of the vicinity of the surface, it is difficult to obtain a three-dimensional image. In general, the biological tissue is a protein, lipid, nucleic acid or the like is configured, for each of these substances may have different refractive indexes, a refractive index thereof is in the living tissue and the non-uniform, and thus light do not pass through living tissue, living tissue can be observed from the inside of the surface is not possible.
Therefore, the refractive index of the entire living tissue, protein main component thereof is brought close to the scattering of light in the living tissue as a result suppress, a biological tissue has been developed a technique to be transparent. Chung et al. 'CLARITY' method, light scattering and refractive index of the non-uniform major lipid, protein polyacrylamide hydrogels were fixed are removed by the electrophoresis, liquid tissue, protein a solution close to that of the refractive index can be substituted by a, tissue in the art to be transparent (non-patent document 1, Patent Document 1).
CLARITY method, tissue destruction is likely to occur by electrophoresis for a problem in that, instead of the electrophoresis method of removing lipids gradually by shaking has been developed (Non-Patent Document 2, Patent Document 1, 2). However, in this method, tissue destruction hardly occurs on the other hand, the removal by shaking (2-3 weeks) for a long time was a problem that it takes.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 生体組織を固定剤で固定する前、若しくは固定している間、又は固定した後、該生体組織に水溶性エチレン性不飽和モノマーを浸潤させる工程であって、該水溶性エチレン性不飽和モノマーは、イオン性解離基を有する水溶性エチレン性不飽和モノマーを少なくとも含む工程、
 固定後の生体組織内で、前記水溶性エチレン性不飽和モノマーを重合させることによりヒドロゲルを形成する工程、及び
 前記固定後の生体組織から脂質を除去する工程
を含んでなることを特徴とする生体組織の透明化方法。

[請求項2]
前記イオン性解離基がアニオン性解離基である請求項1に記載の方法。

[請求項3]
前記イオン性解離基がスルホン酸基、カルボン酸又はリン酸基である請求項1又は2に記載の方法。

[請求項4]
前記イオン性解離基を有する水溶性エチレン性不飽和モノマーが(メタ)アクリル酸、イタコン酸、マレイン酸、フマル酸及びそれらの塩から選択される請求項1~3のいずれか1項に記載の方法。

[請求項5]
前記イオン性解離基を有する水溶性エチレン性不飽和モノマーがスチレンスルホン酸、(メタ)アクリロキシベンゼンスルホン酸、2-(メタ)アクリルアミド-2-メチルプロパンスルホン酸、2-(メタ)アクリルアミドプロパンスルホン酸、3-(メタ)アクリルアミド-2-ヒドロキシプロパンスルホン酸、3-(メタ)アクリロキシ-1-プロパンスルホン酸及びその塩から選択される請求項1~3のいずれか1項に記載の方法。

[請求項6]
前記水溶性エチレン性不飽和モノマー中の前記イオン性解離基を有する水溶性エチレン性不飽和モノマーの割合が少なくとも50モル%である請求項1~5のいずれか1項に記載の方法。

[請求項7]
前記水溶性エチレン性不飽和モノマーが(メタ)アクリルアミドベースのモノマーを含む請求項1~6のいずれか1項に記載の方法。

[請求項8]
脂質の除去が受動拡散により行われる請求項1~7のいずれか1項に記載の方法。

[請求項9]
生体組織に、請求項1~8のいずれか1項に記載の生体組織の透明化方法を適用することを特徴とする、透明化生体組織が包埋されたヒドロゲルの製造方法。

[請求項10]
生体組織透明化のため又は透明化生体組織が包埋されたヒドロゲルの製造ための、イオン性解離基を有する水溶性エチレン性不飽和モノマーの使用。

[請求項11]
イオン性解離基を有する水溶性エチレン性不飽和モノマーを含む生体組織透明化試薬又はキット。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • OSAKA PREFECTURE UNIVERSITY
  • Inventor
  • KOJIMA, Chie
  • MATSUMOTO, Akikazu
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG

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