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WO2022102623A1 - Procédé et kit pour rendre transparent un matériau biologique - Google Patents

Procédé et kit pour rendre transparent un matériau biologique Download PDF

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Publication number
WO2022102623A1
WO2022102623A1 PCT/JP2021/041219 JP2021041219W WO2022102623A1 WO 2022102623 A1 WO2022102623 A1 WO 2022102623A1 JP 2021041219 W JP2021041219 W JP 2021041219W WO 2022102623 A1 WO2022102623 A1 WO 2022102623A1
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Prior art keywords
reagent
reagents
biological material
sphere
located inside
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PCT/JP2021/041219
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English (en)
Japanese (ja)
Inventor
哲朗 成相
秀一 中本
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Sumitomo Chemical Co Ltd
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Sumitomo Chemical Co Ltd
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Priority to JP2022561943A priority Critical patent/JPWO2022102623A1/ja
Publication of WO2022102623A1 publication Critical patent/WO2022102623A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Definitions

  • One aspect of the present invention relates to a method and a kit for making a biological material transparent.
  • a technique for improving the light transmission of a biological material and making the biological material transparent is known. This technique has been applied to advanced observations such as gene expression and substance localization in biomaterials. To date, various reagents, devices, treatment methods, etc. for making biological materials transparent have been proposed.
  • Patent Document 1 is a first solution containing (i) an amino alcohol, (ii) at least one compound selected from the group consisting of urea and a urea derivative, and (iii) a nonionic surfactant.
  • a method for preparing a biological material having excellent light transmission which comprises a first infiltration step of infiltrating the biological material.
  • One aspect of the present invention includes the following configurations. ⁇ 1> It ’s a way to make biomaterials transparent. The step of immersing the above biological material in the following reagent (2) and A step of immersing the biological material in the reagent (1) after immersing in the reagent (2), and a step of immersing the biological material in the reagent (1).
  • ⁇ 4> The method according to any one of ⁇ 1> to ⁇ 3>, wherein the biological material is a cancer tissue.
  • ⁇ 5> Selected from the group consisting of (i) the following reagents (1) and (2), (ii) the following reagents (3) and (4), (iii) the following reagents (5), and (iv) the following reagents (6).
  • the present invention also includes the following configurations.
  • ⁇ 6> It is a method of manufacturing transparent biomaterials.
  • ⁇ 7> A method to assist in the inspection of biomaterials
  • the step of making the biological material transparent by the method according to any one of ⁇ 1> to ⁇ 4>, and The process of applying visualization treatment to the target contained in the above biomaterial Including A method in which the biomaterial is a biomaterial collected from a living body or derived from a non-human organism.
  • ⁇ 8> It ’s a method of inspecting biomaterials.
  • the process of applying visualization treatment to the target contained in the above biomaterial A process of performing a pathological examination using the above-mentioned biological material that has been subjected to visualization treatment with an arbitrary configuration, and Including, how.
  • a method for making a biological material transparent which has an improved degree of transparency as compared with the prior art.
  • a method for making a biological material transparent which is improved in speed of transparency as compared with the prior art.
  • FIG. 3 is a conceptual diagram showing a sphere (1) and a sphere (2) (or a sphere (3) and a sphere (4)) in HSP space. It is a figure which shows the time-dependent change of the human cancer tissue which became transparent by the method which concerns on one Embodiment of this invention. It is a figure which shows the time-dependent change of the human cancer tissue which was made transparent by the prior art. It is a figure which shows the appearance of the nuclear staining of the transparent human cancer tissue.
  • the present inventors have compared and examined various reagents as to what kind of reagent has the effect of making the biological material transparent. As a result, it was found that there is a certain tendency in the Hansen solubility parameter of the reagent and the level of the clearing action of the reagent. Specifically, it has been found that a group of reagents located close to each other in the HSP space may exhibit a high transparency effect (not an accurate figure, but see FIG. 1). From this, the present inventors conceived that the reagent located inside or on the surface of a specific Hansen sphere may exhibit a high clearing effect (see FIG. 2 although it is not an accurate figure).
  • the present inventors have repeated studies to further improve the transparent action of biomaterials. As a result, it was found that a particularly high clearing effect can be obtained by sequentially immersing the biological material in the reagents located inside or on the surface of a plurality of Hansen spheres. In addition to this, the present inventors can obtain a particularly high transparency effect by immersing the biological material in a reagent located inside or on the surface of a specific Hansen sphere without changing the reagent to be immersed in the middle. I found.
  • One aspect of the present invention has been completed based on these findings.
  • a method for making a biomaterial transparent comprises immersing the biomaterial in a reagent whose Hansen solubility parameter is within a specific region.
  • the Hansen solubility parameter has been specified for a large number of substances. Assuming that the Hansen solubility parameters of two different substances are ⁇ X and ⁇ Y , substances with a short distance between ⁇ X and ⁇ Y in the HSP space tend to have high compatibility, and substances with a long distance tend to have low compatibility. It is in.
  • the distance R between the point ⁇ X ( ⁇ D X , ⁇ P X , ⁇ H X ) and the point ⁇ Y ( ⁇ DY , ⁇ P Y , ⁇ HY ) in the HSP space is calculated by the following equation.
  • reagent (1) examples include 2-phenoxyethanol, 1-phenyl-2-propanol, 2-o-tolylethanol, phenylethyl alcohol, trichloroethanol, benzyl alcohol, cyclohexanemethanol, cyclopentylacetic acid, and 2-phenyl-1.
  • Preferred reagents (1) include 2-phenoxyethanol, 1-phenyl-2-propanol, 2-o-tolylethanol, phenylethyl alcohol, trichloroethanol, benzyl alcohol, cyclohexanemethanol, cyclopentylacetic acid, 2-phenyl-1-propanol. , 4-Phenyl-2-butanol, 3-phenyl-1-propanol, pentaethylenehexamine, 3-chloro-1-propanol, benzylamine, 2- (m-tolyl) ethanol, 3-methylbenzyl alcohol, 4-phenyl -1-Butanol, and mixtures thereof.
  • More preferable reagents (1) include 2-phenoxyethanol, 1-phenyl-2-propanol, 2-o-tolylethanol, phenylethyl alcohol, benzyl alcohol, 2-phenyl-1-propanol and 3-phenyl-1-propanol. , And mixtures thereof.
  • reagent (1) is 2-phenoxyethanol, 2-o-tolylethanol, benzylamine, or a mixture thereof.
  • reagent (2) examples include 1-methyl-2-pyrrolidone, cinnamaldehyde, 2-methylthiazolin, chlorodimethylacetamide, N-methylformanilide, 2-nitroanisole, ⁇ -caprolactone, and tris phosphate. Included are (2-chloroethyl), tetramethylene sulfoxide, cyclohexanone, acrylonitrile, and mixtures thereof.
  • Preferred reagents (2) include N-methylformanilide.
  • reagent (2) is 2-methylthiazolin, chlorodimethylacetamide, tetramethylenesulfoxide, or a mixture thereof.
  • the reagent (3) is one or more reagents located inside the sphere (3).
  • the reagent (3) include 2-phenoxyethanol, 1-phenyl-2-propanol, 2-otrilethanol, phenylethyl alcohol, trichloroethanol, benzyl alcohol, cyclopentylacetic acid, 2-phenyl-1-propanol, 4 -Phenyl-2-butanol, 3-phenyl-1-propanol, pentaethylenehexamine, 3-chloro-1-propanol, benzylamine, 1- (p-tolyl) ethanol, 2- (m-tolyl) ethanol, 3- Examples include methylbenzyl alcohol, 1-phenyl-1-propanol, 4-phenyl-1-butanol, and mixtures thereof.
  • Preferred reagents (3) include 2-phenoxyethanol, 1-phenyl-2-propanol, 2-o-tolylethanol, trichloroethanol, benzyl alcohol, 2-phenyl-1-propanol, pentaethylenehexamine and 3-chloro-1. Includes propanol, benzylamine, 1- (p-tolyl) ethanol, 2- (m-tolyl) ethanol, 3-methylbenzyl alcohol, and mixtures thereof. More preferable reagents (3) include 2-phenoxyethanol, 2-o-tolylethanol, trichloroethanol, 3-chloro-1-propanol, benzylamine, 1- (p-tolyl) ethanol, and 2- (m-tolyl). Examples include ethanol and mixtures thereof.
  • the reagent (3) is 2-o-tolylethanol. In one embodiment, the reagent (3) is benzyl alcohol. In one embodiment, reagent (3) is 2-phenoxyethanol, 2-o-tolylethanol, benzylamine, or a mixture thereof.
  • the reagent (4) is one or more reagents located inside the sphere (4).
  • reagent (4) examples include dimethyl sulfoxide, ⁇ -butyrolactone, 1-methyl-2-pyrrolidone, chlorodimethylacetamide, ⁇ -caprolactone, tris phosphate (2-chloroethyl), tetramethylene sulfoxide, and the like. Mixtures of. Preferred reagents (4) include dimethyl sulfoxide, 1-methyl-2-pyrrolidone, chlorodimethylacetamide, tetramethylene sulfoxide, and mixtures thereof. More preferred reagents (4) include chlorodimethylacetamide, tetramethylene sulfoxide, and mixtures thereof.
  • the reagent (4) is chlorodimethylacetamide. In one embodiment, the reagent (4) is 1-methyl-2-pyrrolidone. In one embodiment, reagent (4) is 2-methylthiazolin, chlorodimethylacetamide, tetramethylenesulfoxide, or a mixture thereof.
  • the reagent (5) is one or more reagents located inside the sphere (5).
  • Specific examples of the reagent (5) include 2-o-tolylethanol, benzylamine, 1- (p-tolyl) ethanol, 2- (m-tolyl) ethanol, and mixtures thereof.
  • the reagent (5) is 2-o-tolylethanol.
  • the reagent (5) is benzylamine.
  • reagent (5) is 1- (p-tolyl) ethanol. In one embodiment, reagent (5) is 2- (m-tolyl) ethanol. In one embodiment, reagent (5) is 2-o-tolylethanol, benzylamine, or a mixture thereof.
  • One or more reagents located In one embodiment, the reagent (6) is one or more reagents located inside the sphere (6).
  • the reagent (6) is chlorodimethylacetamide.
  • the reagent (6) is tetramethylene sulfoxide.
  • reagent (6) is chlorodimethylacetamide, tetramethylenesulfoxide, or a mixture thereof.
  • the reagent for immersing the biological material is exchanged on the way.
  • These reagents are located inside or on the surface of different spheres in the HSP space, as described above. There are no overlapping regions for the spheres (1) and (2), and there are no overlapping regions for the spheres (3) and (4).
  • the sphere (1) and the sphere (3) are spread over substantially the same region, and the sphere (2) and the sphere (4) are spread over substantially the same region. Therefore, the solubility of the substance is different between the reagent (1) and the reagent (2), but the solubility of the substance is similar between the reagent (1) and the reagent (3).
  • the solubility of the substance is different between the reagent (3) and the reagent (4), but the solubility of the substance is similar between the reagent (2) and the reagent (4).
  • the class of lipids removed from the biological material by the reagents (1) and (3) is different from the class of lipids removed from the biological material by the reagents (2) and (4).
  • the method according to one embodiment of the present invention can remove a wider variety of lipids from biological materials by exchanging the reagents to be immersed. In the method of exchanging the reagent for immersing the biological material in the middle, it is considered that the lipid can be removed more efficiently, and therefore high transparency can be realized.
  • the residual liquid after immersing the biological material in the reagent (1) or the reagent (3) tends to contain a large amount of lipids such as triacylglycerol (TG). Further, the residual liquid after immersing the biological material in the reagent (2) or the reagent (4) tends to contain a large amount of lipids such as free fatty acid (FFA) and lysophosphatidylethanolamine (LPE).
  • lipids such as triacylglycerol (TG).
  • TG triacylglycerol
  • the residual liquid after immersing the biological material in the reagent (2) or the reagent (4) tends to contain a large amount of lipids such as free fatty acid (FFA) and lysophosphatidylethanolamine (LPE).
  • FFA free fatty acid
  • LPE lysophosphatidylethanolamine
  • the lipid dissolution properties of reagent (5) are similar to reagents (1) and reagent (3).
  • the lipid dissolution properties of reagent (6) are similar to reagents (2) and reagent (4). Since the reagent (5) and the reagent (6) have high solubility of lipids, it is considered that the reagent (5) has an action of making the biological material transparent without exchanging the reagent in the middle.
  • the method of not exchanging the reagent for immersing the biological material in the middle has an advantage of simplifying the work.
  • the method for clearing a biological material is to immerse it in a reagent having a specific Hansen solubility parameter.
  • the reagent in which the biological material is immersed is replaced in the middle. That is, the reagent (2) is exchanged with the reagent (1), or the reagent (4) is exchanged with the reagent (3).
  • the reagents that immerse the biological material are not replaced prematurely. That is, it is immersed in the reagent (5) or the reagent (6), and the reagent is not replaced in the middle.
  • One aspect of the present invention is a method for producing a transparent biomaterial, which comprises a step of making the biomaterial transparent by the method described in Section [2].
  • This manufacturing method may include a known method related to the present technical field (a step of collecting a biological material from a living body, a step of fixing the biological material, a step of slicing the biological material, etc.).
  • biomaterial generally refers to a material of biological origin.
  • biological materials include individuals (excluding humans), organs, tissues, cell masses, and body fluids. Examples of individuals include adults, juveniles, fetuses and embryos of animals and plants.
  • organs and tissues include the brain, heart, lungs, liver and kidneys.
  • the organ or tissue to be made transparent may be the whole organ or tissue, or may be a part of the organ or tissue.
  • cell masses include cell masses collected from an organism and cultured cell masses.
  • body fluids include blood, saliva, serum, plasma, urine, synovial fluid, and cerebrospinal fluid.
  • the biomaterial is a biomaterial taken from a living body and is separated from a living organism.
  • the biomaterial is a biomaterial derived from an organism other than human.
  • the biomaterial may be derived from any organism.
  • the biological material is of plant or animal origin.
  • animals include fish, amphibians, reptiles, birds and mammals. Of these, mammals are preferred.
  • mammals include laboratory animals (mice, rats, rabbits, guinea pigs, non-human primates, etc.), pet animals (dogs, cats, etc.), domestic animals (cattle, horses, etc.), wildlife, and humans.
  • the biomaterial is cancer tissue.
  • the method according to one embodiment of the present invention can (i) have a high degree of transparency of cancer tissue and / or (ii) can clear cancer tissue more quickly.
  • the cancer tissue may be derived from any cancer (solid cancer, blood cancer, etc.).
  • the cancer tissue is a solid cancer tissue.
  • the cancer tissue is a carcinoma tissue.
  • the cancer tissue is sarcoma tissue. Examples of cancers include kidney cancer, prostate cancer, breast cancer, lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, liver cancer, colon cancer, esophageal cancer, Examples include small bowel cancer, bladder cancer, brain tumor, cervical cancer, thyroid cancer, laryngeal cancer, and skin cancer.
  • the reagent for immersing the biological material is exchanged on the way.
  • the biological material is immersed in the reagent (2), the biological material is removed from the reagent (2), and the biological material is immersed in the reagent (1).
  • the biological material is immersed in the reagent (4), the biological material is removed from the reagent (4), and the biological material is immersed in the reagent (3).
  • the method according to the embodiment of the present invention does not replace the reagent for immersing the biological material in the middle.
  • the biological material is immersed in reagent (5) or (6).
  • the liquid in which the biological material is immersed does not substantially contain the reagent (2).
  • the liquid in which the biological material is immersed does not substantially contain the reagent (1).
  • the liquid in which the biological material is immersed does not substantially contain the reagent (4).
  • the liquid in which the biological material is immersed does not substantially contain the reagent (3).
  • the liquid in which the biological material is immersed does not substantially contain the reagent (6).
  • the liquid in which the biological material is immersed does not substantially contain the reagent (5).
  • the liquid in which the biomaterial is immersed does not substantially contain components other than the reagent (1). In one embodiment, in the step of immersing the biomaterial in the reagent (2), the liquid in which the biomaterial is immersed does not substantially contain components other than the reagent (2). In one embodiment, in the step of immersing the biomaterial in the reagent (3), the liquid in which the biomaterial is immersed does not substantially contain components other than the reagent (3). In one embodiment, in the step of immersing the biomaterial in the reagent (4), the liquid in which the biomaterial is immersed does not substantially contain components other than the reagent (4).
  • the liquid in which the biomaterial is immersed does not substantially contain components other than the reagent (5). In one embodiment, in the step of immersing the biomaterial in the reagent (6), the liquid in which the biomaterial is immersed does not substantially contain components other than the reagent (6).
  • substantially free of X means that the abundance of X is 10% by weight or less, 5% by weight or less, 3% by weight or less, and 1% by weight or less of the liquid in which the biological material is immersed. Or it means that it is 0% by weight.
  • the biological material may be immersed in a reagent other than the reagents (1) to (6).
  • the biological material may be immersed in a clearing reagent other than the reagents (1) to (6).
  • the biological material is not immersed in any clearing reagents other than the reagents (1)-(6).
  • the biological material may be immersed in a reagent other than the clearing reagent.
  • the biological material may be immersed in a fixing solution.
  • the time for immersing the biological material in any one of the reagents (1) to (6) is preferably 1 hour or longer, more preferably 4 hours or longer.
  • the total time for immersing the biological material in the reagents (1) and (2) or the reagents (3) and (4) is preferably 2 hours or more, more preferably 8 hours or more. If the immersion time is within the above range, the biological material can be sufficiently made transparent.
  • the time for immersing the biological material in any one of the reagents (1) to (6) is preferably 8 hours or less, and more preferably 4 hours or less.
  • the total time for immersing the biological material in the reagents (1) and (2) or the reagents (3) and (4) is preferably 24 hours or less, more preferably 8 hours or less. If the immersion time is within the above range, it can be said that the biological material can be made transparent more quickly than the conventional technique (such as the technique disclosed in Patent Document 1).
  • the degree of transparency of the biomaterial is defined by a comparison of the biomaterial that has undergone the transparency treatment with a negative control.
  • Negative control is a biological material that has been treated in the same manner as a normal sample, except that it has been treated with a reagent that does not have a clearing effect (PBS, Tris, ethanol, saline, etc.) in the clearing treatment step. ..
  • the degree of transparency of the biological material is defined by comparison before and after the transparency treatment.
  • the optical concentration of the sample can be set to the degree of transparency (transparency score) under the test conditions where the optical concentration of the negative control is constant. In this case, it can be said that the lower the transparency score, the higher the degree of transparency. See Example 1 of the present application for an example of a specific calculation method of the transparency score.
  • the degree of transparency is "relative intensity of light transmitted through the sample after the transparency treatment-relative intensity of light transmitted through the sample before the transparency treatment". Can be. In this case, it can be said that the higher the transparency score, the higher the degree of transparency. See Example 2 of the present application for an example of a specific calculation method of the transparency score.
  • the transparency score (wavelength: optical density (OD) of light rays at 600 nm) is preferably 1.4 or less, more preferably 1.1 or less, still more preferably 0.3 or less. ..
  • the lower limit of the transparency score in this case can be, for example, 0 or more.
  • the transparency score (see Example 2 of the present application for the calculation method) is preferably 0.1 or more, more preferably 15 or more, and even more preferably 24 or more.
  • the upper limit of the transparency score in this case can be, for example, 80 or less.
  • the clarified biomaterial can be used, for example, for microscopic observation.
  • the biological material Prior to microscopic observation, the biological material may be visualized (staining, marking, etc.).
  • the visualization treatment may be performed before the transparency treatment, during the transparency treatment, or after the transparency treatment. The visualization process facilitates the analysis of biomaterials by microscopic observation.
  • the fluorescent protein gene may be introduced into a living biological material to express the fluorescent protein prior to the clearing treatment.
  • a chemical substance fluorescent chemical substance, etc.
  • the chemical substance may be injected into the biomaterial after the clearing treatment.
  • an optical microscope is usually used.
  • the type of optical microscope is not particularly limited.
  • three-dimensional super-decomposition microscopy techniques STD, 3D PALM, FPALM, 3D STORM, SIM, etc.
  • STD three-dimensional super-decomposition microscopy techniques
  • microscopic observation may be performed using a multi-photon excitation type (generally two-photon excitation type) optical microscopy technique.
  • One aspect of the present invention includes a step of making a biological material collected from a living body transparent by the method described in Section [2], and a step of subjecting a target contained in the biological material to a visualization treatment. It is a method of assisting the inspection of (or a method of inspecting a biological material). Examples of visualized targets include cancer cells, immune cells, and nerve cells. Alternatively, markers indicating these cells (proteins, lipids, sugar chains, nucleic acids, etc.) may be targeted. The method for visualizing the target is as described above.
  • the visualization treatment may be performed before the transparency treatment, during the transparency treatment, or after the transparency treatment.
  • the method of assisting the inspection of the biological material may include a step of performing a pathological examination using the biological material subjected to the visualization treatment. Since the specific method for performing a pathological examination is well known in the present art, the description thereof will be omitted.
  • One aspect of the present invention comprises a step of making a biological material collected from a living body transparent by the method described in Section [2] and a step of subjecting a target contained in the biological material to a visualization treatment. It is a diagnostic method.
  • This diagnostic method may include a step of performing a pathological examination using a biomaterial that has been visualized. Examples of diseases diagnosed by the diagnostic method include cancer, inflammatory diseases, neurodegenerative diseases and the like.
  • kits for making biological materials transparent consisting of (i) reagents (1) and (2), (ii) reagents (3) and (4), (iii) the following reagents (5), and (iv) the following reagents (6). It has one or more selected.
  • the kit is of (i) Reagents (1) and (2), (ii) Reagents (3) and (4), (iii) Reagents (5), or (iv) Reagents (6). It has one of them.
  • Specific examples of the reagents (1) to (6) are as described in the items of [Reagent (1)] to [Reagent (6)] in Section [1], and therefore the description is omitted in this item.
  • the "kit” refers to a package including a container (bottle, plate, tube, dish, etc.) containing a specific element (reagents (1) to (6), etc.).
  • “having" an element of the kit means that the element is stored in one or more of the containers constituting the kit.
  • the kit may be a package in which a plurality of different compositions are packaged together.
  • the composition may be stored in a plurality of different containers.
  • the kit includes a plurality of components, the plurality of components may be mixed and stored in the same container, or may be stored separately in different containers.
  • the kit is equipped with instructions.
  • the instructions describe how to make the biomaterial transparent using each element included in the kit.
  • the instruction sheet may be written on a medium such as paper, or may be stored on an electronic medium (magnetic tape, computer-readable disk, CD-ROM, etc.).
  • the kit may include a container containing a diluent, solvent, cleaning solution or other reagent.
  • the kit may be equipped with the necessary equipment to make the biological material transparent.
  • the second aspect of the present invention also includes the following aspects.
  • One or more reagents to do. [2] It ’s a way to make biomaterials transparent. The step of immersing the above biological material in the following reagent (4a) and A step of immersing the biological material in the reagent (3a) after immersing in the reagent (4a), and a step of immersing the biological material in the reagent (3a).
  • a method for making a biological material transparent which comprises a step of immersing the biological material in one kind of reagent selected from the following reagents (5a) or (6a):
  • [4] The method according to any one of [1] to [3], wherein the biological material is a cancer tissue.
  • [5] (i) Selected from the group consisting of the following reagents (1a) and (2a), (ii) the following reagents (3a) and (4a), (iii) the following reagents (5a), and (iv) the following reagents (6a).
  • Example 1 Using a suspension of cancer cells, the clearing action of various reagents was investigated. The specific procedure is as follows. 1. 1. 1 g of human cancer cells (human colon cancer cell line HCT116 transplanted into mice) were placed in a microtube, and 4 mL of ethanol was further injected. Next, a cell suspension was prepared with a homogenizer. 2. 2. 100 ⁇ L of cell suspension and 550 ⁇ L of various reagents were stirred and mixed. As a negative control, ethanol was used instead of the reagent. 3. 3.
  • the results are shown in Table 1.
  • the item "Score” represents the transparency score. The larger the number of +, the greater the clearing effect.
  • the item "sphere” indicates whether or not each reagent is located inside or on the surface of the sphere (1) or sphere (2) in the HSP space. The reagent whose item is 1 is located inside or on the surface of the sphere (1), and the reagent whose item is 2 is located inside or on the surface of the sphere (2).
  • spheres (1) and spheres (2) could be prepared.
  • the centers and radii of the spheres (1) and (2) are set such that the reagents located inside or on the surface of the spheres exert a clearing effect on the cancer cell suspension.
  • Example 2 Using sliced specimens of cancer tissue, the clearing action of various reagents was investigated. The specific procedure is as follows. 1. 1. A human cancer tissue (human colon cancer cell line HCT116 transplanted into a mouse) was cut into an appropriate size to prepare a slice specimen having a thickness of 2 mm. 2. 2. The obtained sliced specimens were immersed in 3 mL of various reagents and incubated. Incubation conditions were set at 37 ° C. for 24-48 hours. 3. 3. A sliced specimen was placed on a colored film, and an image was taken in a state of irradiating light from the lower side of the film. 4. The obtained image was processed by image processing software (Image J) to obtain a monochrome image.
  • Image J image processing software
  • the average intensity of transmitted light in a certain area was determined. 5.
  • the value of "(average intensity of transmitted light in slice sample / average intensity of transmitted light in background) x 100" was defined as the degree of transparency. Further, for each reagent, the value represented by "transparency before transparency treatment-transparency after transparency treatment” was used as a transparency score. The larger the clearing score, the greater the clearing effect of each reagent.
  • the results are shown in Table 2.
  • the item "Score” represents the transparency score. The larger the number of +, the greater the clearing effect.
  • the item "sphere” indicates whether or not each reagent is contained in a sphere (3) or a sphere (4) in the HSP space. Reagents with this item 3 are located inside or on the surface of the sphere (3), and reagents 4 are located inside or on the surface of the sphere (4).
  • spheres (3) and spheres (4) could be prepared.
  • the centers and radii of the spheres (3) and (4) are set such that reagents located inside or on the surface of the spheres exert a clearing effect on sliced specimens of cancerous tissue.
  • Example 3 The clearing effect of the biomaterial was investigated by the method of exchanging the reagent for immersing the biomaterial on the way.
  • Example 3a The biological material was made transparent using 2-o-tolylethanol as the reagent (3) and chlorodimethylacetamide as the reagent (4).
  • the specific procedure is as follows. 1. 1. A human cancer tissue (human colon cancer cell line HCT116 transplanted into a mouse) was cut into an appropriate size to prepare a slice specimen having a thickness of 2 mm. 2. 2. The obtained sliced specimen was immersed in 3 mL of reagent (4) and incubated. The incubation conditions were 37 ° C. for 4 hours. 3. 3. Sliced specimens were removed from reagent (4), immersed in 3 mL of reagent (3) and incubated. The incubation conditions were 37 ° C. for 4 hours.
  • CUBIC-L is an amino alcohol-based reagent utilizing the technique disclosed in Patent Document 1.
  • FIG. 4 shows the state of the cancer tissue before the treatment, 4 hours after the start of the treatment, 8 hours after the start of the treatment, and 2 weeks after the start of the treatment.
  • the method according to the embodiment of the present invention has a higher degree of transparency than the prior art.
  • the method according to the embodiment of the present invention has become more transparent than the prior art.
  • Example 3b Nuclear staining (FIG. 5) or cytokeratin staining (FIG. 6) was applied to the sliced specimen of the cancer tissue that had been clarified by the same procedure as in Example 3a. As shown in these figures, good stained images were obtained in each case.
  • Example 3c Various cancerous tissues and non-cancerous tissues were made transparent by the same procedure as in Example 3a.
  • FIG. 7 is a human lung cancer tissue
  • FIG. 8 is a human breast cancer tissue
  • FIG. 9 is a mouse brain tissue
  • FIG. 10 is a result of clearing mouse lung tissue (top) and liver tissue (bottom).
  • the method according to one embodiment of the present invention it is possible to make the tissue highly transparent regardless of whether it is a cancerous tissue or a non-cancerous tissue. This suggests that the method according to one embodiment of the present invention can be applied to a wide range of biological species and materials.
  • Example 3d The transparency scores of the method according to the embodiment of the present invention and the method using the prior art were quantitatively compared. Specifically, the cancer tissue was made transparent by the following three kinds of transparency methods.
  • Method A A method using 2-o-tolylethanol as the reagent (3) and chlorodimethylacetamide as the reagent (4).
  • Method B A method using benzyl alcohol as the reagent (3) and 1-methyl-2-pyrrolidone as the reagent (4).
  • Method C A method using a commercially available animal clearing reagent "CUBIC-L" (manufactured by Tokyo Chemical Industry Co., Ltd.).
  • the results are shown in FIG.
  • the right side of A, the right side of B and C in the bar graph correspond to methods A, B and C.
  • a photograph of the cancer tissue after the clearing treatment is shown at the top of the bar graph (control is the cancer tissue treated with PBS).
  • the methods A and B according to the embodiment of the present invention had a higher transparency score than the method C which is the prior art.
  • the transparency scores when the cancer tissue is first immersed in the reagent (3) and then immersed in the reagent (4) are shown on the left side of A and the left side of B in the bar graph. This suggests that the order of immersion in the reagents is important in the method according to the embodiment of the present invention. That is, it is suggested that the order in which the biological material is first immersed in the reagent (4) and then immersed in the reagent (3) is important.
  • Example 4 The clearing action of the biomaterial was investigated by a method in which the reagent for immersing the biomaterial was not replaced in the middle.
  • reagent (5) 2-o-tolylethanol or benzylamine was used.
  • reagent (6) chlorodimethylacetamide or tetramethylenesulfoxide was used.
  • the specific procedure is as follows. 1. 1. A human cancer tissue (human colon cancer cell line HCT116 transplanted into a mouse) was cut into an appropriate size to prepare a slice specimen having a thickness of 2 mm. 2. 2. The obtained sliced specimen was immersed in 3 mL of reagent (5) or (6) and incubated. The incubation conditions were 37 ° C. for 24 hours. As a conventional technique for comparison, a commercially available animal clearing reagent "CUBIC-L" (manufactured by Tokyo Chemical Industry Co., Ltd.) was used to clear the cancer tissue for 24 hours.
  • CBIC-L animal clearing reagent
  • FIGS. 12-18 show the results of treatment with 2-o-tolylethanol.
  • FIG. 13 shows the result of treatment with benzylamine.
  • FIG. 14 shows the result of treatment with 1- (p-tolyl) ethanol.
  • FIG. 15 shows the result of treatment with 2- (m-tolyl) ethanol.
  • FIG. 16 shows the result of treatment with chlorodimethylacetamide.
  • FIG. 17 shows the result of treatment with tetramethylene sulfoxide.
  • FIG. 18 shows the result of processing using CUBIC-L.
  • the method according to the embodiment of the present invention has a higher degree of transparency 24 hours after the start of the treatment than the prior art. From this, it can be said that the method according to the embodiment of the present invention can make the biological material transparent more quickly than the prior art.
  • FIG. 19 A comparison of the transparency scores of each of the reagents (5) and (6) used in Example 4 and CUBIC-L is shown in FIG. 19 (comparison 24 hours after the start of treatment).
  • the figure quantitatively shows that the method according to one embodiment of the present invention rapidly makes a biological material transparent.
  • One aspect of the present invention can be used for observing biological materials and the like. To give a more specific example, one aspect of the present invention can be used for pathological diagnosis of cancer and the like.

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Abstract

L'invention concerne un procédé pour rendre transparent un matériau biologique améliorant la vitesse et/ou l'étendue de la transparence par rapport à l'état de la technique. Un aspect de la présente invention concerne un procédé pour rendre transparent un matériau biologique comprenant les étapes suivantes : (i) immersion d'un matériau biologique dans un ou plusieurs réactifs (2) situés à l'intérieur ou à la surface d'une sphère spécifique (2) dans un espace HSP ; et (ii) immersion du matériau biologique, après immersion dans le ou les réactifs (2), dans un ou plusieurs réactifs (1) situés à l'intérieur ou à la surface d'une sphère spécifique (1) dans un espace HSP.
PCT/JP2021/041219 2020-11-10 2021-11-09 Procédé et kit pour rendre transparent un matériau biologique Ceased WO2022102623A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003066035A (ja) * 2001-08-06 2003-03-05 Anse Ko 水溶性組織清澄溶液
JP2015049101A (ja) * 2013-08-30 2015-03-16 オリンパス株式会社 生体透明化剤
CN106556582A (zh) * 2016-10-31 2017-04-05 华中科技大学 一种光透明化生物组织的方法
WO2017188264A1 (fr) * 2016-04-28 2017-11-02 国立研究開発法人理化学研究所 Composition pour préparer un matériel biologique présentant une excellente transmissivité de la lumière et utilisation de la composition
JP2019536005A (ja) * 2016-09-27 2019-12-12 ラトガース，ザ ステート ユニバーシティ オブ ニュー ジャージー 顕微鏡検査用透明化剤および封入媒体

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003066035A (ja) * 2001-08-06 2003-03-05 Anse Ko 水溶性組織清澄溶液
JP2015049101A (ja) * 2013-08-30 2015-03-16 オリンパス株式会社 生体透明化剤
WO2017188264A1 (fr) * 2016-04-28 2017-11-02 国立研究開発法人理化学研究所 Composition pour préparer un matériel biologique présentant une excellente transmissivité de la lumière et utilisation de la composition
JP2019536005A (ja) * 2016-09-27 2019-12-12 ラトガース，ザ ステート ユニバーシティ オブ ニュー ジャージー 顕微鏡検査用透明化剤および封入媒体
CN106556582A (zh) * 2016-10-31 2017-04-05 华中科技大学 一种光透明化生物组织的方法

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