WO2018155587A1

WO2018155587A1 - Reagent for enhancing transparency of biologically derived material

Info

Publication number: WO2018155587A1
Application number: PCT/JP2018/006564
Authority: WO
Inventors: 伸太郎麓; 茂川上; 孝洋西田
Original assignee: 国立大学法人長崎大学
Priority date: 2017-02-22
Filing date: 2018-02-22
Publication date: 2018-08-30
Also published as: JP7033795B2; JPWO2018155587A1

Abstract

The present invention provides: a reagent for enhancing transparency with which it is possible, in a short time and with a simple procedure, to enhance the transparency of a biologically derived material to an excellent degree of transparency while a lipid membrane is held, and to adjust pH within a specific range; a method for enhancing the transparency of a biologically derived material using said reagent; and a kit for enhancing the transparency of a biologically derived material, said kit including said reagent.

Description

Reagents for clearing biological materials

The present invention relates to a biological material-clearing reagent, a biological material-clearing method using the reagent, and a biological material-clearing kit containing the reagent.

In developing drug delivery systems and gene therapy methods, it is important to evaluate the pharmacokinetics and intracellular kinetics of the introduced carriers and genes in order to ensure their effectiveness and safety. After introducing a carrier or gene, the fixed tissue can be sliced and observed with a microscope to evaluate the distribution in the tissue. However, it is possible to obtain planar information. In recent years, after introducing a carrier or a gene, the tissue is made transparent and observed with a microscope, so that three-dimensional information can be obtained and the spatial distribution in the tissue can be evaluated.

The tissue is made transparent by electrophoresis or a method using a reagent. The electrophoresis method has high transparency efficiency, but has a drawback that a special apparatus is required and the procedure is complicated. Another problem is that small molecules are removed during membrane staining and electrophoresis because lipids are removed. The method using a reagent is transparent by infiltrating the tissue with the reagent, so that a special apparatus is not required and the procedure is simple. Several reagents for tissue clearing have been developed. Reagents containing formamide (Non-patent Document 1), reagents containing fructose (Non-patent Document 2), urea or urea derivatives, and surfactants and glycerol Reagents (Patent Document 1) and the like have been reported. In addition, as a method of clarifying, urea or urea at a concentration higher than the solution used in the step of infiltrating the biological material with a solution containing urea or a urea derivative at a predetermined concentration (first infiltration step) and the first infiltration step A method of infiltrating a solution containing a derivative into a biological material (second infiltration step) has been reported (Patent Document 2).

In addition, the brain, heart, lung, kidney, liver, pancreas, spleen, etc. of the mouse and the whole body of the mouse are cleared and imaged as 3D data using a clearing reagent containing urea, amino alcohol and surfactant. However, basic technologies for quantitative comparison between samples have been reported (Non-patent Documents 3 and 4).

WO2011 / 111876 WO2012 / 147965

When a tissue is made transparent using a conventional reagent, it takes time and the procedure is complicated because the reagent is infiltrated into the tissue for several days to several weeks and replaced with a reagent having a different composition in the middle. Further, when a tissue is clarified using a clarification reagent containing a surfactant, the transparency is high, but the lipid membrane is denatured or removed by the surfactant. On the other hand, when a reagent that does not contain a surfactant is used, the lipid membrane is retained, but the transparency is low and information on deeper tissues cannot be obtained. Furthermore, no reagent capable of adjusting the pH has been reported.

Therefore, the present invention provides a novel biological material-clearing reagent capable of at least partially solving the above-mentioned problems associated with conventional tissue-clearing reagents, and a method for clearing tissue of biological materials using the reagent And a kit for clarifying a biological material containing the reagent, and the like.

The inventors of the present invention have made extensive studies to solve the above-mentioned problems, and have found for the first time that polyethyleneimine has an effect of making the tissue transparent. By using polyethyleneimine for tissue transparency, transparency can be achieved in a short period of time with simple procedures, good transparency can be achieved while retaining the lipid membrane, and the pH of the reagent is within a specific range. The present inventors have found that it can be adjusted to The present inventors have further studied based on these findings and have completed the present invention.

The present invention provides the following.
[1] A reagent for clarifying a biological material, comprising polyethyleneimine.
[2] The reagent according to [1], wherein the polyethyleneimine contains a modified polyethyleneimine.
[3] The reagent according to [2], wherein the modified polyethyleneimine is at least one selected from the group consisting of propylene oxide modified polyethyleneimine, octadecyl isocyanate modified polyethyleneimine, and ethylene oxide modified polyethyleneimine.
[4] The reagent according to any one of [1] to [3], wherein the polyethyleneimine has a number average molecular weight of 300 to 70,000.
[5] The reagent according to any one of [1] to [3], wherein the polyethyleneimine has a number average molecular weight of 400 to 25,000.
[6] The reagent according to any one of [1] to [5], which does not substantially contain a surfactant.
[7] The reagent according to any one of [1] to [6], further comprising at least one compound selected from the group consisting of urea, formamide, lactamide, and derivatives thereof.
[8] The reagent according to any one of [1] to [7], further comprising at least one compound selected from the group consisting of glycerin, polyethylene glycol and propylene glycol.
[9] The reagent according to any one of [1] to [8], wherein the pH is 4 to 12.
[10] The reagent according to any one of [1] to [8], which has a pH of 7 to 11.
[11] The reagent according to any one of [1] to [10], wherein the biological material is a plant or animal-derived material.
[12] The animal-derived material is derived from at least one organ selected from the group consisting of brain, heart, liver, kidney, spleen, lung, stomach, small intestine, large intestine and muscle. reagent.
[13] The reagent according to [11], wherein the animal-derived material is derived from skin.
[14] A method for clarifying a biological material, comprising the step of infiltrating the biological material with the reagent according to any one of [1] to [13].
[15] The method according to [14], wherein the infiltration step is performed only once.
[16] The method according to [14] or [15], wherein the infiltration step is performed for 1 hour to 7 days.
[17] A kit for clarifying a biological material, comprising the reagent according to any one of [1] to [13].

According to the present invention, a reagent capable of clearing a biological material with good transparency while retaining a lipid membrane in a short period of time and a simple procedure, and a reagent capable of adjusting pH to a specific range are provided. . Moreover, according to this invention, the transparency method of the biological material using the said reagent is provided. Furthermore, according to the present invention, a kit for clarifying a biological material containing the reagent is provided.

FIG. 1 is a bright-field image of the liver and kidney of a mouse made transparent in Example 1. FIG. 2 is a bright-field image of the liver and kidney of the mouse clarified in Example 2. FIG. 3 is a bright field image of each tissue of the mouse made transparent in Example 3. FIG. 4 is a bright-field image of the liver and kidney of the mouse clarified in Example 4. FIG. 5 is a bright-field image of spinach leaves and stems clarified in Example 5. The upper row is leaves and the lower row is stems. FIG. 6 shows the blood vessels and gene expression of the mouse kidneys clarified in Example 6. FIG. 7 is a view showing blood vessels and parenchyma of the mouse brain cleared in Example 7. FIG. FIG. 8 is a diagram showing gene expression and generation of oxidative stress in the mouse liver cleared in Example 8. FIG. 9 is a graph showing the evaluation of the retention of the liposome membrane by the clearing reagent of the present invention and Scale A2 according to the FRET efficiency in Example 9. FIG. 10 is a bright-field image of the liver and kidney of the mouse clarified in Example 10. In the results of each reagent, the upper row is the kidney and the lower row is the liver. FIG. 11 is a bright field image of the liver of a mouse made transparent in Example 11. FIG. 12 is a view showing a bright-field image of a mouse kidney and various blood vessels from the glass surface, which were made transparent using various clarification reagents in Example 12. FIG. 13 is a bright-field image of the liver of a mouse made transparent in Example 13. FIG. 14 is a bright-field image of the liver and kidney of the mouse clarified in Example 14. FIG. 15 is a bright-field image of the liver and kidney of the mouse clarified in Example 15. FIG. 16 is a bright field image of each tissue of the mouse made transparent in Example 16. FIG. 17 is a bright-field image of the liver and kidney of the mouse clarified in Example 17. FIG. 18 is a diagram showing the spatial distribution of doxorubicin hydrochloride in the liver of a mouse clarified in Example 18.

1. Reagent for clearing biological material The present invention provides a reagent for transparentizing biological material (hereinafter also referred to as the reagent of the present invention). The reagent of the present invention is characterized by containing polyethyleneimine. The mechanism by which polyethyleneimine makes the biological material transparent is not clear, but is presumably due to the dehydrating action of polyethyleneimine.

The polyethyleneimine used in the reagent of the present invention is a polymer obtained by polymerizing ethyleneimine, and may be either linear or branched. The linear polyethyleneimine contains a secondary amine and one primary amine at the end, and has a linear structure. Examples of the branched type polyethyleneimine include polyethyleneimine having a branched structure containing primary, secondary and tertiary amines, and a completely branched dendrimer.

The number average molecular weight of polyethyleneimine is not particularly limited, but from the viewpoint of increasing the transparency of the biological material, for example, the lower limit is 300 or more, 400 or more, from the viewpoint of preventing deformation of the biological material, for example, the lower limit is 300 or more, 600 or more, 1400 or more, 1800 or more are mentioned, and the upper limit is 70,000 or less, 25,000 or less. Examples of the range of the number average molecular weight of polyethyleneimine include 300 to 70,000, 400 to 25,000, 500 to 12,000, and 600 to 10,000, for example, 10,000. Alternatively, the range of the number average molecular weight may be, for example, 1400 to 70,000, 1800 to 70,000. As the number average molecular weight, a molecular weight measured by a boiling point raising method can be used.

The polyethyleneimine may be a modified polyethyleneimine. Examples of the modified polyethyleneimine include derivatives of polyethyleneimine and isocyanate compounds, epoxy compounds, acrylic compounds, halogen compounds, isothiocyanate compounds, NHS (N-Hydroxysuccinimide) esters, and the like. Examples of the isocyanate compound include alkyl isocyanate, and examples of the alkyl isocyanate include tetradecyl isocyanate, pentadecyl isocyanate, hexadecyl isocyanate, heptadecyl isocyanate, octadecyl isocyanate, and nonadecyl isocyanate. Examples of the epoxy compound include alkylene oxide, and examples of the alkylene oxide include ethylene oxide, propylene oxide, butylene oxide, and the like. Examples of the acrylic compound include acrylonitrile. Examples of the halogen compound include alkyl halides. Examples of the isothiocyanate compound include pyridine isothiocyanate, iodophenyl isothiocyanate, ethoxyphenyl isothiocyanate, and chloroethyl isothiocyanate. Examples of NHS (N-Hydroxysuccinimide) ester include methyl polyethylene glycol-NHS. More specifically, as modified polyethyleneimine, for example, octadecyl isocyanate modified polyethyleneimine obtained by adding octadecyl isocyanate to polyethyleneimine, ethylene oxide modified polyethyleneimine obtained by adding ethylene oxide to polyethyleneimine, and propylene oxide added to polyethyleneimine Examples include propylene oxide-modified polyethyleneimine.

Polyethyleneimine may be used alone or in combination of two or more. In general, the higher the molecular weight of polyethyleneimine, the higher the transparency, but there is a tendency for tissue shrinkage to occur, so it may be effective to combine a high molecular weight polyethyleneimine with a low molecular weight polyethyleneimine. In this case, for example, a combination of polyethyleneimine having a molecular weight of 400 to 2,000 and polyethyleneimine having a molecular weight of 5,000 to 25,000 is used at a ratio of 1: 1 to 4: 1, and polyethyleneimine having a molecular weight of 600 and polyethyleneimine having a molecular weight of 10,000 is 1: 1. Examples include combinations used in a ratio of ~ 4: 1.

The content of polyethyleneimine in the reagent is not particularly limited, and usually ranges from 10 to 30% w / v%, 15 to 25% w / v%, for example, 20% w / v%.

Polyethyleneimine can be produced by a method known per se, or a commercially available polyethyleneimine may be used. Examples of commercially available polyethyleneimines include, for example, Wako Pure Chemical Industries, 163-17835, 166-17825, 169-17815, and Nippon Shokubai Co., Ltd., Epomin (registered trademark) SP-003, SP-006, SP-012. , SP-018, SP-200, P-1000, etc. Modified polyethyleneimines are also commercially available, and examples thereof include Nippon Shokubai Co., Ltd., Epomin (registered trademark) RP-20, PP-061 and the like.

The reagent of the present invention may further contain urea, formamide, lactamide, and / or a derivative thereof, preferably urea or formamide, lactamide, more preferably urea, or may not contain these. By including these amide compounds, it is possible to make clearing faster and with higher transparency. The mechanism of rapid and high transparency due to the inclusion of the amide compound is not clear, but it is presumed that the refractive index of the biological material is made uniform by the amide compound.

Derivatives include one, two, three, or four of the four hydrogen atoms in the urea formula, one, two, or three of the three hydrogen atoms in the formamide formula. Or one, two, three, four, five or six of the six hydrogen atoms in the formula of lactamide, independently of one another, a halogen atom (eg a fluorine atom, a chlorine atom, a bromine atom) , Iodine atom, etc.), or a hydrocarbon group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms (for example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group and the like).

The concentration of the above-mentioned amide compound in the reagent is not particularly limited, but usually ranges from 3 to 9M. When two or more amide compounds are used, the total concentration is meant. For example, when urea is used, 3M or more, 4M or more, 4.5M or more, 5M or more, 5.5M or more, 6M or more can be mentioned from the viewpoint of preventing the swelling of the biological material. From the viewpoint of preventing precipitation of urea, 9M or less, 8M or less, 7.5M or less, 7M or less, or 6.5M may be mentioned. Therefore, a preferable urea concentration is, for example, in the range of 4 to 9M, more preferably 4 to 8M, still more preferably 6 to 8M, and particularly preferably 7 to 8M.

The reagent of the present invention may further contain a polyhydric alcohol or may not contain these. Depending on the biological material, the transparency can be further increased by including a polyhydric alcohol. Although the mechanism for improving the transparency due to the inclusion of the polyhydric alcohol is not clear, it is presumed to be due to the dehydrating action of the polyhydric alcohol. The polyhydric alcohol is not particularly limited, and examples thereof include dihydric alcohols and trihydric alcohols. These polymers are also included in the dihydric alcohol and trihydric alcohol. Examples of the dihydric alcohol include ethylene glycol, propylene glycol, tetramethylene glycol, diethylene glycol, dipropylene glycol, and polyethylene glycol. Examples of the trivalent alcohol include glycerin, trimethylol ethane, trimethylol propane, diglycerin, and polyglycerin. Of these, propylene glycol, polyethylene glycol and glycerin are preferred. Polyethylene glycol having an average molecular weight of, for example, 200 to 20,000, preferably 400 to 10,000, more preferably 400 to 8,000 can be used. As the average molecular weight, an average molecular weight measured by a titration method can be used.

The solvent in the reagent of the present invention is not particularly limited as long as polyethyleneimine and other optional components are soluble, and examples thereof include water and a buffer solution. Examples of the buffer include PBS buffer, HEPES buffer, and Tris buffer.

The reagent of the present invention may contain further components such as a pH adjusting agent and an osmotic pressure adjusting agent.

The pH of the reagent of the present invention is not particularly limited, but from the viewpoint of increasing the transparency of the biological material, for example, pH 以上 4 or more, pH 以上 5 or more, pH 5.5 or more, pH 6 or more, pH 7 or more, pH 7.5 or more From the viewpoint of preventing discoloration of the fluorescent protein when observing the fluorescent protein in the transparent biological material, for example, the pH is 12 or less, the pH is 11 or less, and the pH is 10 or less. Therefore, the pH ranges are 4 to 12, pH 5 to 12, pH 5.5 to 12, pH 6 to 12, pH 7 to 12, pH 7.5 to 12, pH 8 to 12, pH 9 to 12, pH 4 -11, pH 5-11, pH 5.5-11, pH 6-11, pH 7-11, pH 7.5-11, pH 8-11, pH 9-11. Alternatively, the pH ranges are pH 5 to 10, pH 5.5 to 10, pH 6 to 10, pH 7 to 10, pH 7.5 to 10, pH 5 to 9, pH 5.5 to 9, pH 6 to 9, pH 7 ˜9, pH 、 7.5˜9.

The pH adjustment method is not particularly limited, and can be adjusted, for example, by adding an appropriate amount of a pH adjuster to the polyethyleneimine aqueous solution. Examples of pH adjusters include phosphoric acid, citric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, pyrophosphoric acid, sulfuric acid, nitric acid, acetic acid, glycolic acid, boric acid, lactic acid, silicic acid, phosphonic acid, and tartaric acid. Succinic acid, malic acid, sodium hydrogen carbonate, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, ammonia and the like.

The reagent of the present invention may be substantially free of surfactant. In the present specification, the term “substantially free of a surfactant” means that the surfactant is not included, or even when it is included, it is less than 0.1% w / v%, preferably less than 0.05% w / v%. More preferably, it is less than 0.025% w / v%.

The method for preparing the reagent of the present invention is not particularly limited, and for example, it can be prepared by dissolving polyethyleneimine in a solvent. Furthermore, arbitrary components as described above can be added. Moreover, it can adjust by adding a pH adjuster, adjusting to desired pH, adding an arbitrary component as needed, and melt | dissolving in a solvent.

The bio-derived material targeted by the reagent of the present invention to be clarified is not particularly limited, but a plant or animal-derived material is preferable. The biological material may be an individual (plant or animal) itself, or may be an organ, tissue, or cell. Animal organs are not particularly limited, for example, brain, heart, liver, kidney, pancreas, spleen, lung, stomach, small intestine, large intestine, skin, muscle, spinal cord, eyeball, gonad, thyroid, gallbladder, bone marrow, adrenal gland, digestion Examples include ducts, thymus, submandibular gland, prostate, testis, ovary, placenta, uterus and the like, preferably brain, heart, liver, kidney, spleen, lung, stomach, small intestine, large intestine, muscle, skin and the like. Plant organs are not particularly limited, and examples include leaves, petals, stems, roots, and seeds. The organs of animals and plants may be organs themselves, or organ homogenates and sliced sections of organs.

Animals are not particularly limited and include invertebrates and vertebrates, with vertebrates being preferred. Examples of vertebrates include fish, amphibians, reptiles, birds, and mammals, with mammals being preferred. Mammals include, for example, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), laboratory animals (eg, rabbits, etc.), livestock (eg, pigs, cows, goats, horses, sheep, etc.), pets ( Examples thereof include dogs and cats), primates (eg, humans, monkeys, orangutans, chimpanzees, etc.).

Plants are not particularly limited and include seed plants and spore plants. Seed plants include angiosperms (for example, dicotyledonous plants and monocotyledonous plants) and gymnosperms, and specific examples include spinach and ginkgo biloba. Examples of spore plants include bracken.

The biological material may be fixed or may not be fixed. In addition, the biological material may be a material expressing a fluorescent protein, or a material stained with a fluorescent chemical substance, a fat-soluble carbocyanine dye, or a coloring chemical substance.

Examples of fluorescent proteins include RFP, GFP, BFP, CFP, and YFP. Examples of the fluorescent chemical substance include DAPI® (4 ′, 6-diamidino-2-phenylindole), fluorescein and the like. Examples of the fat-soluble carbocyanine dye include DiI (1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine perchlorate), DiD (1,1'-dioctadecyl-3,3,3', 3 '-tetramethylindodicarbocyanineocyanperchlorate), DiO (3,3'-dioctadecyloxacarbo-cyanine perchlorate), DiR (1,1'-dioctadecyltetramethyl indotricarbocyanine iodide). Examples of chromogenic chemical substances include Evans Blue, which is a blue pigment, Indocyanine Green, which is a green pigment, Oil Red O, which is a red pigment, and X-gal (5-bromo-4-chloro), which is a chromogenic substrate. -3-indolyl-β-D-galactopyranoside), DAB (3,3'-diaminobenzidine), BCIP (5-bromo-4-chloro-3-indolyl-phosphate), NBT (Nitro Blue Tetrazolium Chloride) .

2. Method for clarifying biological material The present invention also provides a method for clarifying biological material (hereinafter also referred to as the method of the present invention). The method of the present invention comprises a step of infiltrating a biological material with the reagent of the present invention.

The method for infiltrating the biological material with the reagent of the present invention is not particularly limited, and examples thereof include a method of immersing the material in the reagent of the present invention. The period of infiltration is not particularly limited, but is preferably at least 1 hour from the viewpoint of sufficiently infiltrating the biological material with the reagent of the present invention. The period is preferably, for example, 1 hour to 7 days, more preferably 5 hours to 7 days, still more preferably 12 hours to 7 days, and even more preferably 1 day to 3 days. For example, in the case of organs such as the kidney and liver, good transparency can be achieved even for 1 hour to 3 days, or even 1 hour to 1 day. The infiltration temperature is not particularly limited as long as the reagent of the present invention is not significantly denatured. For example, when the reagent of the present invention contains urea, from the viewpoint of preventing precipitation of urea, for example, 25 ° C. to 45 ° C. is preferable, and 30 ° C. to 40 ° C. is more preferable.

The number of steps of the infiltration is not particularly limited, but with the reagent of the present invention, good transparency can be achieved without performing multiple infiltrations using a plurality of reagents with different concentrations. Can be infiltration.

3. Kit for clarification of biological material The present invention also provides a kit for clarification of biological material (hereinafter also referred to as the kit of the present invention). The kit of the present invention includes the reagent of the present invention. The kit of the present invention may further contain a tissue fixing solution, a washing buffer solution, and the like.

4. Clear biological material The reagent of the present invention or the biological material transparentized by the method of the present invention is stained with a fluorescent chemical substance, a fat-soluble carbocyanine dye, or a chromogenic chemical substance, and a stereomicroscope, It can be observed with a confocal laser microscope or a multiphoton excitation microscope. Further, the tissue homogenate can be clarified, and the concentration of the fluorescent chemical substance, fat-soluble carbocyanine dye, or chromogenic chemical substance in the tissue can be quantitatively measured using a fluorometer, an absorptiometer, or the like. Examples of the fluorescent chemical substance, the fat-soluble carbocyanine dye, and the color-forming chemical substance include those described above.

Since the reagent of the present invention of the embodiment which does not substantially contain a surfactant can make a biological material transparent while retaining the lipid membrane, the membrane can be formed using various fluorescent probes, fat-soluble carbocyanine dyes, and the like. Can be dyed. In addition, since the reagent of the present invention can be adjusted to a pH within a specific range, a reagent whose pH is adjusted according to various fluorescent probes can be used.

The transparent biological material can be directly observed with a stereo microscope, a confocal laser microscope, a multi-photon excitation microscope, or the like without slicing. Therefore, for example, evaluation of spatial distribution of DDS such as liposome preparations, evaluation of spatial distribution of gene expression in gene transfer methods, evaluation of localization of various proteins using fluorescent protein-expressing transgenic mice, blood vessels using lipid-soluble carbocyanine dyes And real visualization, cell death visualization, and oxidative stress visualization. As a spatial distribution evaluation of gene expression in the gene transfer method, for example, a gene encoding a fluorescent protein is targeted by gene transfer methods (for example, hydrodynamics method, tissue suction pressure method, method using bubble liposome and ultrasonic wave, etc.) After introduction into an animal, the organ is removed and the organ is clarified with the reagent of the present invention. After gene introduction as described above, a fat-soluble carbocyanine dye (for example, 1,1′- dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine perchlorate (DiI), 1,1'-dioctadecyl-3,3,3', 3'- tetramethylindodicarbocyanine perchlorate (DiD), 3,3'-dioctadecyloxacarbo-cyanine perchlorate (DiO), 1,1 Examples include a method of perfusing '-dioctadecyltetramethyl indotricarbocyanine diodide (DiR), etc.) to stain a blood vessel, and then removing the organ and clarifying the organ with the reagent of the present invention. In addition, as a method for visualizing blood vessels and parenchyma using a fat-soluble carbocyanine dye, the target animal is perfused with a fat-soluble carbocyanine dye, the blood vessel is stained, the organ is removed, and the reagent of the present invention is used. A method of clearing the organ, a method of immersing the organ in a fat-soluble carbocyanine dye and staining it, and then clarifying with the reagent of the present invention. After staining a blood vessel as described above, the organ is excised and a fat-soluble carbocyanine dye And the like, and a method of clarification with the reagent of the present invention. Furthermore, as a method for visualizing oxidative stress, for example, after exposing an animal to which an oxidative stress visualization reagent or a commercially available oxidative stress visualizing mouse is exposed to oxidative stress, the organ is removed and clarified with the reagent of the present invention. The method etc. are mentioned. Furthermore, these multiple events can be visualized simultaneously by multi-color deep imaging.

Also, the cleared biological material can be observed with a stereomicroscope, a confocal laser microscope, a multiphoton excitation microscope, etc. after the clearing reagent is washed once and then sliced. For example, microtubules can be observed with a confocal laser microscope by washing the clarified liver, slicing it, and staining the obtained sections with an anti-tubulin antibody and a fluorescently labeled secondary antibody. .

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

Example 1: Clarification of liver and kidney with polyethyleneimine
Preparation method of polyethyleneimine aqueous solution Adjust the pH of polyethyleneimine (number average molecular weight 600: 10,000 = 1: 1) dissolved in a small amount of water to pH 11 with 5M hydrochloric acid, add water, and add 20 w / v% polyethyleneimine An aqueous solution was prepared. Polyethyleneimine having a number average molecular weight of 600 was manufactured by Wako Pure Chemical Industries, 163-17835, and polyethyleneimine having a number average molecular weight of 10,000 was manufactured by Wako Pure Chemical Industries, 166-17825.
The mice under clear anesthesia of the liver and kidney were perfused with the fat-soluble carbocyanine dye 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindocarbocyanine perchlorate (DiI) transcardially. The tissue was then fixed by perfusing 4% paraformaldehyde. The liver and kidney were removed and immersed in a polyethyleneimine aqueous solution, and bright field images were obtained after 1 day and 3 days. The results are shown in FIG. Both the liver and kidney were clarified with a polyethyleneimine aqueous solution to the same extent as the 3-day immersion after 1-day immersion.

Example 2: Facilitation of liver and kidney transparency with urea
Preparation Method of Clarifying Reagent When preparing the polyethyleneimine aqueous solution of Example 1, urea was added to prepare an aqueous solution of 20 w / v% polyethyleneimine and 8M urea. In addition, 20 w / v% polyethyleneimine, 8M urea was obtained in the same manner as described above except that polyethyleneimine (number average molecular weight 600) or polyethyleneimine (number average molecular weight 10,000) was used and pH 9 was adjusted. An aqueous solution of was prepared. As urea, 210-01185 manufactured by Wako Pure Chemical Industries, Ltd. was used.
Mice under clear anesthesia of the liver and kidney were perfused with 4% paraformaldehyde and the tissue was fixed. The liver and kidney were removed and immersed in a clearing reagent, and a bright field image was obtained after 1 day. The results are shown in FIG. Each of the clarification reagents clarified the liver and kidney with high transparency by immersion for 1 day. When polyethyleneimine having a number average molecular weight of 10,000 was used, the transparency was highest. In addition, when polyethyleneimine having a number average molecular weight of 600 was used, the tissue did not shrink. When polyethyleneimine having a number average molecular weight of 600: 10,000 = 1: 1 was used, the tissue was not shrunk and clearing was promoted more than when a clearing reagent containing no urea was used (FIG. 1).

Example 3: Clarification of each tissue with a clarification reagent By the same method as in Example 2 except for pH, a clarification reagent (20 w / v% polyethyleneimine, molecular weight 600: 10,000 = concentration ratio 1: 1) Urea 8M, pH 11) was prepared.
In the same manner as in Example 1, DiI was perfused into the anesthetized mouse to fix the tissue. The brain, liver, kidney, lung, heart, spleen, stomach, small intestine, large intestine and muscle were removed and immersed in a clearing reagent, and bright field images of each tissue were obtained after 1 day and 3 days. The results are shown in FIG. All tissues were clarified with high transparency by a clarification reagent.

Example 4: Temporal change after immersing liver and kidney in clearing reagent A clearing reagent was prepared in the same manner as in Example 3.
In the same manner as in Example 1, DiI was perfused into the anesthetized mouse to fix the tissue. The liver and kidney were removed and immersed in a clearing reagent, and bright field images of each tissue were obtained 1 hour to 3 days later. The results are shown in FIG. Although the tissue shrinkage was observed after 1 hour, it was observed that the tissue became transparent. One day later, the tissue returned to its original size, and sufficient transparency was obtained.

Example 5: Clearance of spinach leaves and stems Spinach leaves and stems (unfixed) were immersed in a clearing reagent having the same composition as in Example 3, and bright field images were obtained after 1 day and 3 days later. . The results are shown in FIG. 5 (the upper row is leaves and the lower row is stems). Both leaves and stems were clarified with a clarification reagent after one day of immersion, and further clarified after three days of immersion.

Example 6: Simultaneous visualization of blood vessels and gene expression
Gene Transfer to Kidney Plasmid DNA encoding green fluorescent protein ZsGreen1 was administered into a mouse vein, and then suction was applied to the tissue using a suction device to perform gene transfer.
Twenty-four hours after introduction of the clearing gene of the kidney, DiI was perfused to the anesthetized mouse in the same manner as in Example 1 to fix the tissue. The kidney was removed, immersed in a clearing reagent (20 w / v% polyethyleneimine, molecular weight 1,800, urea 8M, pH 9), and 3 days later, the blood vessels stained with gene expression and DiI were observed with a confocal laser microscope did. The results are shown in FIG. The blood vessel and gene expression were visualized simultaneously by the clearing reagent, and it was confirmed that the plasmid administered intravenously leaked from the blood vessel and the gene was expressed outside the blood vessel.

Example 7: Visualization of cerebral blood vessels and parenchyma In the same manner as in Example 1, mice under anesthesia were perfused with DiI to fix the tissue. The brain was removed and roughly cut with a tissue slicer. After immersing the section in 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethylindodicarbocyanine perchlorate (DiD) solution and staining the substance, the clearing reagent (20 w / v% polyethyleneimine, molecular weight 600: It was immersed in 10,000 = concentration ratio 1: 1, urea 8M, pH 9), and blood vessels and parenchyma were observed with a confocal laser microscope one day later. The results are shown in FIG. Brain blood vessels and parenchyma were visualized simultaneously with the clearing reagent.

Example 8 Visualization of Oxidative Stress Occurrence When TdTomato Gene is Introduced After oxidative stress visualization reagent CellROX (registered trademark) deep red is administered intraperitoneally to mice, a plasmid DNA encoding red fluorescent protein tdTomato is hydrodynamically processed. It was introduced into the liver by (rapid intravenous administration of a large volume solution). Thereafter, the mice under anesthesia were fixed by perfusion with 4% paraformaldehyde. After the liver was removed, it was immersed in a clearing reagent prepared by the same method as in Example 7, and observed one day later using a confocal laser microscope. The results are shown in FIG. The generation of oxidative stress was visualized by the clearing reagent, and the positional relationship with the introduced gene expression could be analyzed.

Example 9: Measurement of membrane retention using FRET liposomes Liposomes labeled with NBD-DOPE (Avanti Polar Lipids, 810145) and Lissamine rhodamine B-DOPE (Avanti Polar Lipids, 810150) were treated with 0.1 w / surfactant. Mixing with clearing reagent Scale A2 (urea 4 M, glycerin 10 w / v%, Triton X-100 0.1 w / v%) containing v% or the clearing reagent of the present invention, the retention of liposome membrane is fluorescent. Evaluation was based on resonance energy (FRET) efficiency. The clearing reagent used was prepared in the same manner as in Example 7. The results are shown in FIG. The retention of the liposome membrane was 81.3% when the reagent of the present invention was used, and 8.27% when the clarifying reagent Scale A2 containing a surfactant was used, and the reagent of the present invention retained the lipid membrane. It was confirmed that the biological material was made transparent.

Example 10: Clarification of kidney and liver using reagents of each pH
Preparation of clearing reagent According to the method described in Example 3, clearing reagents of

pH

7, 9, and 11 were prepared. Using the prepared reagents and PBS as a control, the kidney and liver were clarified by the same method as in Example 2. The results are shown in FIG. 10 (in the results of each reagent, the upper row is the kidney and the lower row is the liver). Each of the clarification reagents clarified the liver and kidney with high transparency by immersion for 1 day. Moreover, the higher the pH, the higher the transparency.

Example 11: Improvement of transparency in the liver by adding polyhydric alcohol
Preparation of clearing reagent The urea concentration was set at 4M to avoid precipitation at low temperatures. Polyethyleneimine (molecular weight 1,800, manufactured by Wako Pure Chemical Industries, 169-17815) and urea are added with glycerin (Nacalai Tesque, 17018-25), 20 w / v% polyethyleneimine, 4M urea, 10 w An aqueous solution of / v% glycerin, pH 11 was prepared. In addition, it is made transparent by the same method as above except that polyethylene glycol (Nacalai Tesque, 28215-95, molecular weight 400) or propylene glycol (Nacalai Tesque, 29218-35) is used instead of glycerin. Reagents were prepared.
Clarification of liver DiI was perfused to anesthetized mice in the same manner as in Example 1, and the tissue was fixed. The liver was removed and immersed in each clearing reagent, and bright field images were obtained after 1 day and 3 days. The results are shown in FIG. When a clearing reagent containing glycerin, polyethylene glycol or propylene glycol was used, the transparency of the liver was improved.

Example 12: Clarification of kidney using various clarification reagents For comparison of lipid membrane retention and observation depth of blood vessel staining with DiI, as a clarification reagent, a clarification reagent Clear containing no surfactant ^T2 (Non-patent document 1), Scale S (non-patent document 5) containing a low concentration (0.2 w / v%) surfactant, CUBIC containing a high concentration (15 w / v%) surfactant (Non-patent Document 3) and pH 9 and pH 11 clearing reagents prepared by the same method as in Example 9 were used. PBS was used as a control. In the same manner as in Example 1, DiI was perfused into the anesthetized mouse to fix the tissue. The kidney was removed and immersed in various clearing reagents. About the reagent for clearing other than the reagent of this invention and PBS, it immersed according to the method as described in each literature. When using Clear ^T2 , the reagent of the present invention, and PBS, a bright field image is obtained after 1 day, with Scale S after 3 days, and with CUBIC after 9 days. Observed with. The results are shown in FIG. The reagent of the present invention clarified the kidney with high transparency by immersion for 1 day. When the reagent of the present invention was used, blood vessels with a depth of 302 μm and 705 μm could be observed from the glass surface after immersion for 1 day. When the reagent of the present invention having a pH of 11 was used, blood vessels with a depth of 1000 μm could be observed. On the other hand, even when the kidney was immersed in a reagent other than the reagent of the present invention for one day or more, a blood vessel having a depth of 302 μm could not be observed.

Example 13: Clarification of the liver with low and high molecular weight polyethyleneimines
Preparation method of clearing reagent A clearing reagent (20 w / v% polyethyleneimine, 8M urea, pH 11) was prepared in the same manner as in Example 2 except that polyethyleneimine (number average molecular weight 300 or 70,000) was used. Was prepared. Polyethyleneimine having a number average molecular weight of 300 was manufactured by Nippon Shokubai Co., Ltd., SP-003, and polyethyleneimine having a number average molecular weight of 70,000 was manufactured by Nippon Shokubai Co., Ltd., P-1000.
Liver was extracted from a mouse under clear anesthesia of the liver, and the tissue was fixed by immersion in 4% paraformaldehyde. Subsequently, it was immersed in a clearing reagent, and a bright field image was obtained after 1 day. The results are shown in FIG. The liver becomes transparent when polyethyleneimine of any molecular weight is used as a clearing reagent, but the liver swells with a clearing reagent prepared with a molecular weight of 300 of polyethyleneimine.

Example 14: Clarification of liver and kidney when using pH 5 clarification reagent
Preparation method of clearing reagent By the same method as in Example 2 except for pH and urea concentration, clearing reagent (20 w / v% polyethyleneimine, molecular weight 600: 10,000 = concentration ratio 1: 1, urea 6M, pH 5) was prepared.
The liver and kidney were removed from the mouse under clear anesthesia of the liver and kidney, and the tissue was fixed by immersion in 4% paraformaldehyde. Subsequently, it was immersed in a clearing reagent, and bright field images were obtained after 1 day and 3 days. The results are shown in FIG. Even at pH 5, the liver and kidney were cleared.

Example 15: Clarification of each tissue when using a modified polyethyleneimine aqueous solution
Preparation method of clearing reagent Add water to propylene oxide modified polyethyleneimine (molecular weight 1400), 10, 20, 50 w / v% propylene oxide modified polyethyleneimine aqueous solution (pH unadjusted (measured values are pH 11.2, 11.4, 11.9)) was prepared. As a propylene oxide-modified polyethyleneimine having a number average molecular weight of 1400, PP-061 manufactured by Nippon Shokubai Co., Ltd. was used.
The kidney and liver were removed from a mouse sacrificed by anesthesia with an excessive amount of clearing of the kidney and liver, and the tissue was fixed by immersion in 4% paraformaldehyde. Subsequently, it was immersed in a clearing reagent, and bright field images were obtained after 1 day and 3 days. The results are shown in FIG. The structure was also clarified by a propylene oxide-modified polyethyleneimine aqueous solution. Note that no significant shrinkage was observed even at a high concentration of 50%.

Example 16: Clarification of each tissue when using modified polyethyleneimine
Preparation method of clearing reagent According to the same method as in Example 2 except that propylene oxide modified polyethyleneimine was used instead of polyethyleneimine and pH, a clearing reagent (20 w / v% propylene oxide modified polyethyleneimine, A molecular weight of 1400, urea 8M, pH 5, 7.5 or 11) was prepared. As a propylene oxide-modified polyethyleneimine having a number average molecular weight of 1400, PP-061 manufactured by Nippon Shokubai Co., Ltd. was used.
Liver, kidney, lung, spleen, heart, muscle, small intestine, large intestine and skin were removed from mice sacrificed by anesthesia with a clear excess of each tissue, and the tissue was fixed by immersion in 4% paraformaldehyde. The skin was fixed after shaving with a clipper. Subsequently, it was immersed in a clearing reagent, and bright field images were obtained after 1 day and 3 days. The results are shown in FIG. It was shown that the structure can be made transparent even when propylene oxide-modified polyethyleneimine is used, like polyethyleneimine.

Example 17: Effect of urea concentration when using modified polyethyleneimine
Preparation method of clarification reagent Except for pH and urea concentration, a clarification reagent (20 w / v% propylene oxide-modified polyethyleneimine, molecular weight 1400, urea 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 M, pH 10) was prepared. As a propylene oxide-modified polyethyleneimine having a number average molecular weight of 1400, PP-061 manufactured by Nippon Shokubai Co., Ltd. was used.
The kidney and liver were removed from a mouse sacrificed by anesthesia with an excessive amount of clearing of the kidney and liver, and the tissue was fixed by immersion in 4% paraformaldehyde. Subsequently, it was immersed in a clearing reagent, and bright field images were obtained after 1 day and 3 days. The results are shown in FIG. Transparency increases with increasing urea concentration, but no significant difference was seen above 6 M.

Example 18: Visualization of spatial distribution of doxorubicin hydrochloride in the liver
Preparation method of clearing reagent A clearing reagent (20 w / v% propylene oxide-modified polyethyleneimine, molecular weight 1400, urea 8M, pH 5.5) was prepared in the same manner as in Example 16 except for pH. As a propylene oxide-modified polyethyleneimine having a number average molecular weight of 1400, PP-061 manufactured by Nippon Shokubai Co., Ltd. was used.
Liver- cleared doxorubicin hydrochloride (20 mg / kg) was intravenously administered to mice, and the liver was fixed by perfusion of PBS followed by 4% paraformaldehyde under anesthesia. After removing the liver, it was immersed in a clearing reagent and observed with a confocal laser microscope one day later. The results are shown in FIG. By adjusting the pH, leakage of doxorubicin hydrochloride from the liver when immersed in the clearing reagent was suppressed, and the spatial distribution of doxorubicin hydrochloride in the liver could be visualized.

This application is based on Japanese Patent Application No. 2017-030702 (filing date: February 22, 2017) filed in Japan, and all the contents thereof are included in this specification.

Claims

A reagent for clarification of bio-derived materials containing polyethyleneimine.
The reagent according to claim 1, wherein the polyethyleneimine contains a modified polyethyleneimine.
The reagent according to claim 2, wherein the modified polyethyleneimine is at least one selected from the group consisting of propylene oxide modified polyethyleneimine, octadecyl isocyanate modified polyethyleneimine and ethylene oxide modified polyethyleneimine.
The reagent according to any one of claims 1 to 3, wherein the number average molecular weight of polyethyleneimine is 300 to 70,000.
The reagent according to any one of claims 1 to 3, wherein the number average molecular weight of polyethyleneimine is 400 to 25,000.
The reagent according to any one of claims 1 to 5, which is substantially free of a surfactant.
The reagent according to any one of claims 1 to 6, further comprising at least one compound selected from the group consisting of urea, formamide, lactamide, and derivatives thereof.
The reagent according to any one of claims 1 to 7, further comprising at least one compound selected from the group consisting of glycerin, polyethylene glycol and propylene glycol.
The reagent according to any one of claims 1 to 8, wherein the pH is 4 to 12.
The reagent according to any one of claims 1 to 8, which has a pH of 7 to 11.
The reagent according to any one of claims 1 to 10, wherein the biological material is a plant or animal-derived material.
The reagent according to claim 11, wherein the animal-derived material is derived from at least one organ selected from the group consisting of brain, heart, liver, kidney, spleen, lung, stomach, small intestine, large intestine and muscle.
The reagent according to claim 11, wherein the animal-derived material is derived from skin.
A method for clarifying a biological material, comprising the step of infiltrating the biological material with the reagent according to any one of claims 1 to 13.
The method according to claim 14, wherein the infiltration step is performed only once.
The method according to claim 14 or 15, wherein the infiltration step is performed for 1 hour to 7 days.
A kit for clarifying a biological material, comprising the reagent according to any one of claims 1 to 13.