WO2020110404A1

WO2020110404A1 - Microtube for preparing paraffin block

Info

Publication number: WO2020110404A1
Application number: PCT/JP2019/034333
Authority: WO
Inventors: 武寿磯田; 円央原田
Original assignee: コニカミノルタ株式会社
Priority date: 2018-11-28
Filing date: 2019-09-02
Publication date: 2020-06-04
Also published as: JPWO2020110404A1

Abstract

Provided is a microtube for preparing a paraffin block in which masses of cultured cells rarely lose the shape and are uniformly dispersed.　The present invention involves a microtube for preparing a paraffin block, said microtube being filled with paraffin at the bottom.

Description

Microtube for paraffin block production

The present invention relates to a microtube for producing a paraffin block.

In morphological observation, paraffin blocks are prepared by embedding tissues and cells with paraffin when preparing specimen slides while retaining the structures of tissues and cells such as microorganisms, plants or animals to be observed. The method of doing is widely used.

For example, Patent Document 1 discloses a paraffin block having a columnar body having an identifiable cross-sectional shape. When the paraffin block is sliced, the color of the columnar body and the identification shape of the fragment shape are formed in the thin piece, and as a result, it is an effect of the invention to eliminate the trouble of confusing the sample.

Further, Patent Document 2 discloses a paraffin block in which paraffin embedding a specimen adheres to the bottom surface of a cassette body for microscope specimen preparation. Since the paraffin has no seam, it is an effect of the invention to solve the problem of cracking or peeling when slicing.

On the other hand, a paraffin section was cut out from a paraffin block using a microtome, the paraffin section was attached to a slide glass, and then deparaffinization treatment, antigen activation treatment, blocking treatment, staining treatment, encapsulation treatment, etc. were performed to prepare a specimen slide. The method of making specimens is widely used, and various methods have been devised for making specimen slides.

For example, Patent Document 3 discloses a method for producing a biological sample specimen using a paraffin block. In the preparation method, one or more through-holes are opened in a paraffin block, the through-holes are filled with a tissue and embedded, and a thin section is cut out from the embedded solid sample and attached to a slide glass, Paraffin is removed to make thin sections for tissue observation.

JP, 2011-136128, A JP, 2005-129739, A Japanese Patent Laid-Open No. 2018-013050

When producing paraffin blocks of cultured cells using paraffin, it is necessary to disperse the cultured cells uniformly in paraffin in order to obtain high reproducibility. However, when the cell mass of the cultured cells was immersed in paraffin by a known method, the shape of the cell mass collapsed and the cultured cells could not be dispersed uniformly, so that highly reproducible results could not be obtained. Therefore, at the time of producing the paraffin block, it has been required to produce the paraffin block in which the cultured cells are uniformly dispersed.

The present invention has been completed in view of the above problems, and includes, for example, [1] to [6] below.
[1] A paraffin block-producing microtube having a bottom filled with paraffin.
[2] The paraffin block-producing microtube according to [1], wherein the thickness of the paraffin is 1 to 10 mm from the bottom end of the bottom.
[3] The microtube for paraffin block production according to [1] or [2], wherein the bottom has an opening surface and the paraffin is exposed from the opening surface.
[4] A method for producing a paraffin block, which uses the microtube for producing a paraffin block according to any one of [1] to [3],
A cell filling step of filling a cell mass to be observed in the paraffin block making microtube,
A cell immobilization step of fixing the cell mass filled in the cell filling step with a fixative,
A coating step of preparing a coated cell by coating the fixed cell mass obtained in the cell fixing step with a thickening polysaccharide,
A method for producing a paraffin block, comprising a film forming step of reacting the coated cells obtained in the coating step with a gelling agent to form a gel film around the coated cells.
[5] The method for producing a paraffin block according to [4], wherein the thickening polysaccharide is at least one selected from the group consisting of sodium alginate, pectin, and carrageenan.
[6] The paraffin block production method according to [4] or [5], wherein the gelling agent is at least one selected from the group consisting of calcium chloride, calcium phosphate, and calcium carbonate.

According to the paraffin block-producing microtube of the present invention, the shape collapse of the cell mass of the cultured cells does not easily occur during the production of the paraffin block of the cultured cells, and the cultured cells are embedded in the paraffin block while maintaining uniform dispersibility. can do.

FIG. 1 is a sectional view schematically showing an embodiment of a microtube for producing a paraffin block of the present invention. FIG. 2 is a cross-sectional view schematically showing another embodiment of the paraffin block-producing microtube of the present invention. FIG. 3 is a photomicrograph of a hematoxylin-stained specimen slide of a thin section obtained from a paraffin block of cultured cells SKBR3 (HER2 expressing cells), which was prepared by the method for preparing a paraffin block of the present invention. FIG. 4 is a photomicrograph of a comparative slide obtained by hematoxylin-staining a thin section obtained from a paraffin block of cultured cells SKBR3 (HER2 expressing cells), which was prepared by a conventional microtube not filled with paraffin as a comparative example. FIG. 5 is a fluorescence image of a specimen slide prepared by the method for producing paraffin blocks of the present invention, in which thin sections of paraffin blocks of cultured cells SKBR3 (HER2 expressing cells) were immunostained with anti-HER2 antibody-bound fluorescent particles. It is a bar graph showing the number of fluorescent particles per cell obtained from the analysis. FIG. 6 is a comparative slide in which a thin section of a paraffin block of cultured cells SKBR3 (HER2-expressing cells) prepared using a conventional microtube not filled with paraffin was immunostained with anti-HER2 antibody-bound fluorescent particles. 2 is a bar graph showing the number of fluorescent particles per cell obtained from the fluorescence image analysis.

The present invention will be described in detail below. The paraffin block manufacturing microtube of the present invention is characterized in that the bottom of the microtube is filled with paraffin. The paraffin block-producing microtube of the present invention may be filled with paraffin at the bottom. As the members other than the paraffin forming the paraffin block-producing microtube of the present invention, members similar to ordinary microtubes can be used, and there is no particular limitation, and for example, known microtubes themselves can be used. .. Hereinafter, members other than paraffin that compose the microtube for producing a paraffin block will be simply referred to as microtube members.

<Micro tube member>
The microtube member used in the microtube for producing a paraffin block of the present invention is not particularly limited, and a known microtube or the like can be used. For example, the internal shape of the microtube member is a cylindrical shape from the opening to the upper end of the bottom, and the upper end of the bottom to the lower end of the bottom are substantially conical (see FIG. 1), or a cylindrical shape from the opening to the upper end of the bottom. To a bottom end having a substantially hemispherical shape, and a columnar shape from the opening to the bottom. In addition, since the paraffin block-producing microtube of the present invention is filled with paraffin at the bottom, the microtube member itself may have an opening surface at the bottom.

The internal volume of the microtube member is not particularly limited, but for example, 0.1 mL to 5 mL can be used. From the viewpoint of operability, 0.2 mL to 2 mL is preferable, and 0.5 mL to 1.5 mL is more preferable.

The material of the microtube member is not particularly limited, and the material used for known microtubes can be used. Examples of the material include polypropylene, fluororesin such as PFA (perfluoroalkoxyalkane), and glass.

The presence or absence of silicon coating on the microtube member, applicable temperature, presence or absence of autoclave, presence or absence of light-shielding treatment, presence or absence of lid, and the shape of the lid are free-standing or non-freestanding, the paraffin block manufacturing method of the present invention described later. It is not particularly limited as long as it can be used.

<paraffin>
The paraffin used in the microtube for producing a paraffin block of the present invention is not particularly limited as long as it is a paraffin wax that is melted by heating in a solid state at room temperature, and known ones can be used. For example, purified paraffin, paraffin to which a polymer is added for the purpose of increasing tissue permeability, semitransparent or colored paraffin can be used. The melting point of paraffin is not particularly limited, but from the viewpoint of operability, the melting point is preferably 40°C to 70°C, more preferably 60°C to 65°C.

<Microtube for producing paraffin block and method for producing the same>
The paraffin block-producing microtube of the present invention is one in which the paraffin is filled in the microtube member, and preferably 1 mm to 10 mm in the vertical direction from the lower end of the bottom of the microtube member to the opening. The thickness is more preferably 4 mm to 6 mm.

The above-mentioned microtube for paraffin block production is such that solid paraffin is put into the microtube member, heated above the melting point of the paraffin to melt the paraffin, and the paraffin is collected at the bottom of the microtube member. It can be prepared by placing it and solidifying the paraffin.

In the paraffin block-producing microtube of the present invention, the bottom of the microtube member may have an opening surface, and the paraffin block may be exposed from the opening surface (see FIG. 2 ). The opening surface is preferably circular or elliptical, and more preferably circular. The diameter (minor diameter) of the opening surface is not particularly limited, but is preferably 2 mm to 10 mm, more preferably 3 mm to 9 mm, and further preferably 4 mm to 8 mm from the viewpoint of operability. The paraffin is preferably exposed in contact with the opening surface.

In this way, because the paraffin is exposed from the bottom in advance, it becomes easier to take out the cells from the microtube when making the paraffin block described later.

The microtube for making a paraffin block having the above-mentioned opening surface can be made as follows. The bottom of the microtube for paraffin block production prepared by filling with paraffin as described above is cut with a knife such as scissors or a cutter knife to produce a microtube for paraffin block production having an opening surface. To do. In addition, a microtube member having an opening on the bottom is made in advance, and the opening is covered with a peelable film or metal plate, and then paraffin is added to dissolve and solidify, and finally the lid is closed. By removing the, it is possible to manufacture a paraffin block-producing microtube having an opening surface.

Place the cell mass of the cultured cells to be observed on the upper surface of the paraffin of the microtube for paraffin block production, coat the paraffin and the cell mass with the thickening polysaccharide described below, and further form a film with a gelling agent. By pushing out from the bottom toward the opening of the microtube, paraffin, cells and the like can be taken out in an integrated state.

<How to make paraffin block>
A paraffin block can be produced as follows using the microtube for producing a paraffin block of the present invention.

(1) Cell Filling Step The paraffin block making microtube is filled with a paraffin block making microtube, usually paraffin in the microtube, by a cell mass to be observed using a pipette or the like. Then, after washing with an arbitrary buffer, centrifugation may be performed to remove the supernatant.

(2) Cell Immobilization Step After the cell packing step, the packed cell mass is fixed with a fixative. The fixing solution is not particularly limited, and examples thereof include formalin, zinc formalin, ethanol, a mixed solution of ethanol and acetone, and bouin. Fixation can be performed according to a conventional method. For example, when 10% neutral buffered formalin or 4% paraformaldehyde is used, the fixation is performed for 8 hours or longer, and when zinc formalin is used, the fixation is performed for 4 to 48 hours.

(3) Coating Step After the cell immobilization step, the fixed cell mass is coated with a thickening polysaccharide to prepare coated cells. That is, after removing the fixative solution used in the cell immobilization step, the fixed cell mass and paraffin are covered with a thickening polysaccharide. The thickening polysaccharide is not particularly limited as long as it reacts with a gelling agent described below to form a film. Examples of the thickening polysaccharide include sodium alginate, pectin, and carrageenan, and sodium alginate is preferable.

Prior to coating, if there is no open surface at the bottom of the paraffin block making microtube, cut the bottom with a knife such as scissors or a cutter knife as described above to expose the paraffin to the open surface. Is preferred. From the opening of the paraffin block-producing microtube, the thickened polysaccharide solution diluted to an arbitrary concentration is poured into the fixed cell mass on the upper surface of paraffin. Thereafter, a rod-shaped member smaller than the size of the opening surface, such as a cotton swab, gently moves up and down the paraffin exposed on the opening surface to spread the thickening polysaccharide solution evenly around the cells and paraffin, It is preferred to coat the paraffin with a thickening polysaccharide.

(4) Membrane forming step After the coating step, the coated cells are reacted with a gelling agent to form a gel film around the coated cells. That is, a gelling agent is added to a microtube for paraffin block production and reacted with the coated thickening polysaccharide to form a gel film around the cell mass and paraffin. The gelling agent is not particularly limited as long as it reacts with the thickening polysaccharide to form a gel. Examples of gelling agents include calcium chloride, calcium phosphate, and calcium carbonate. It is preferable that the coating step and the film forming step are alternately performed to form a superposed gel film around cells and paraffin. This makes it possible to take out the paraffin block in which the cell aggregate is not collapsed and is integrated with the paraffin from the paraffin block-producing microtube.

(5) Optional step Paraffin is removed from the paraffin block using a displacing solvent such as xylene or limonene, and only the cell mass fixed by the gel membrane is once removed, and then the cell mass is embedded again with paraffin. It is preferably a paraffin block.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to these examples.
[Example 1]
(1) Preparation of microtube for paraffin block preparation A 0.5 ml microtube (product number: TBI-0501, manufactured by Bio Rad) was filled with 0.07 g of paraffin pellets (Tissue Tech Paraffin Wax; manufactured by Sakura Fine Tech Japan). did. Hot air was blown to the microtubes using a dryer to dissolve the paraffin pellets in the microtubes and collect them at the bottom. Then, the microtube for paraffin block production was produced by standing at room temperature for 1 hour or more while standing on the microtube stand. Paraffin had a thickness of 5 mm from the bottom edge.

(2) Filling and fixing of cells From the opening of the microtube prepared above, the SKBR3 cultured cell suspension was added to the upper part of paraffin so that the suspension amount was 1.0×10 ⁷ to 3.0×10 ⁷ cells/mL. Filled. Fetal bovine serum FBS (product number: SH30910.03, manufactured by Hyclone) (300 μL) was added thereto, and after gently pipetting, the mixture was allowed to stand at room temperature for 10 minutes. The supernatant was removed by centrifugation at 1000 xg and 4°C for 5 minutes. Here, 10% neutral buffered formalin solution (product number: 20211
(For tissue process; manufactured by Muto Kagaku Co., Ltd.) 400 μL was added, and the mixture was left to stand overnight in a refrigerator to fix the cultured cells existing on the upper part of paraffin.

(3) Coating with sodium alginate The immobilized microtube was centrifuged at 1000 xg and 4°C for 5 minutes to remove the supernatant. A part of the bottom of the tube was cut with scissors to expose the cut surface of paraffin. After 200 μL of 1% sodium alginate solution was gently added through the opening of the 200 μL tube, the cut surface of the paraffin exposed by the above cutting was pushed up with a cotton swab to spread the sodium alginate solution to the bottom of the microtube, and paraffin and its upper part. The cultured cells (hereinafter, referred to as "paraffin cell mass") present in 1. were coated with sodium alginate.

(4) Film formation by calcium chloride solution After the above coating treatment, 200 μL of 1M calcium chloride solution was added from the opening of the microtube, and the cut surface of paraffin exposed by the above cutting was pushed up with a cotton swab to add the calcium chloride solution to the microtube. The pellet was spread to the bottom and reacted with sodium alginate coating the paraffin cell mass to prepare a pellet in which a film of calcium alginate was formed around the paraffin cell mass (hereinafter referred to as “cell pellet”). The cell pellet was transferred from a microtube to a container containing a 1 M calcium chloride solution so that the paraffin side of the cell pellet was on the top and the cell side was on the bottom, and the cell pellet was allowed to stand for 10 minutes. The cell pellet is once taken out of the container, dipped in a 1% sodium alginate solution, and again transferred to a container containing a 1M calcium chloride solution so that the paraffin side of the cell pellet is the upper side and the cell side is the lower side, and left still for 10 minutes. Then, a film of calcium alginate was again formed on the cell pellet.

(5) Paraffin Blocking After washing the cell pellet obtained above with Milli-Q water, place the paraffin side of the cell pellet on top in the embedding cassette (Asia Kikai System Cassette II-M (6 fractions)). The cell pellet was covered with a sponge moistened with MilliQ water, covered with a SUS frame for an embedded cassette, and allowed to stand for 5 minutes.

Next, the embedding cassette was set in the dedicated basket of the processor (product number: TISSUE-TEK VIP5 Junior, made by Sakura Seiki Co., Ltd.) and processed under the conditions shown in Table 1 below. The paraffin process was performed at 65°C.

After the above treatment, the embedding cassette is transferred to the paraffin tank for standby of the embedding device (product number: TISSUE-TEK VIP5 Junior, manufactured by Sakura Seiki Co., Ltd.) with the paraffin side of the cell pellet facing up and the cell side facing down. A paraffin block was prepared according to the operating method of the embedding device. The produced paraffin block was left on the cooling plate for 1 hour or more to solidify the paraffin.

[Comparative example]
A paraffin block of a comparative example was produced in the same manner as in Example 1 except that a microtube not filled with paraffin was used instead of the microtube filled with paraffin of the present invention. At that time, the cell mass at the bottom of the microtube and the portion of the gel film covering the periphery of the microtube were cut with scissors, so that the shapes of the cell mass and gel collapsed.

[Production example]
Specimen slides stained with fluorescent particles were prepared from the paraffin blocks prepared in the above Examples and Comparative Examples as follows.
(1) Preparation of Fluorescent Particles 14.4 mg of Sulfo Rhodamine 101 (manufactured by Sigma-Aldrich) as a fluorescent dye was dissolved in 22 mL of water. To this, 2 mL of a 5% aqueous solution of Emulgen (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation), which is an emulsifier for emulsion polymerization, was added. This was heated to 70° C. with stirring, and then 0.65 g of melamine resin raw material Nikalac MX-035 (manufactured by Nippon Carbide Industry Co., Ltd.) was added.

1000 μL of a 10% aqueous solution of dodecylbenzenesulfonic acid (Kanto Chemical Co., Inc.) was added to this solution, and the mixture was stirred at 70° C. for 50 minutes. Then, the temperature was raised to 90° C. and the mixture was stirred for 20 minutes to obtain a dispersion liquid of fluorescent particles.

(2) Introduction of antibody to fluorescent particles An anti-HER2 antibody was bound to the fluorescent particles by the following method.
First, 1 mg of fluorescent particles recovered from the above dispersion liquid by centrifugation was dispersed in 5 mL of pure water, and 100 μL of an aqueous dispersion of aminopropyltriethoxysilane (product number: LS-3150, manufactured by Shin-Etsu Chemical Co., Ltd.) Was added and stirred at room temperature for 12 hours.

After centrifugation at 10,000 g for 60 minutes to remove the supernatant, ethanol was added to disperse the precipitate, and centrifugation was performed again. The same procedure using ethanol and pure water was repeated once. FT-IR measurement of the obtained particles confirmed that amino groups were introduced into the fluorescent particles.

Fluorescent particles having an amino group introduced therein were adjusted to 3 nM with PBS containing 2 mM EDTA (ethylenediaminetetraacetic acid). SM (PEG) 12 (manufactured by Thermo Fisher Scientific) was added thereto so that the final concentration was 10 mM, and the mixture was stirred to react the fluorescent particles with the SM (PEG) 12 to introduce a maleimide group into the fluorescent particles. ..

The above reaction solution was centrifuged at 10,000×g for 60 minutes to remove the supernatant. PBS containing 2 mM EDTA (ethylenediaminetetraacetic acid) was added to disperse the precipitate, followed by centrifugation again to remove the supernatant. The same procedure was repeated 3 times. Then, the precipitate was redispersed using 500 μL of PBS to obtain a dispersion liquid of the maleimide group-introduced fluorescent particles.

100 μg of anti-HER2 antibody was dissolved in 100 μL of PBS. 1 M dithiothreitol (DTT) was added to the solution and reacted for 30 minutes to obtain a reduced anti-HER2 antibody solution.

The above-mentioned maleimide group-introduced fluorescent particle dispersion and a reduced anti-HER2 antibody solution were mixed in PBS and reacted for 1 hour. The reaction was stopped by adding 4 μL of 10 mM mercaptoethanol. The reaction solution was centrifuged at 10,000×g for 60 minutes to remove the supernatant. PBS containing 2 mM EDTA was added to redisperse the precipitate, and centrifugation was performed again. After performing this operation 3 times, 500 μL of PBS was added to obtain a dispersion liquid of the anti-HER2 antibody-bound fluorescent particles.

(3) Immunostaining Using Anti-HER2 Antibody-Binding Fluorescent Particles From the paraffin blocks prepared in Examples and Comparative Examples, 4 μM thin sections were cut using a microtome and attached to slides, and specimens derived from the paraffin blocks of Examples were prepared. A slide and a comparative slide derived from the paraffin block of the comparative example were prepared. By the following method, both slides were immunostained with the fluorescent particles to which the anti-HER2 antibody prepared above was bound.

The slide was placed in a container containing xylene, and while changing the xylene three times on the way, the slide was immersed in xylene for a total of 30 minutes to remove the paraffin on the slide. Next, the slide was placed in a container containing ethanol, and the slide was immersed in ethanol for a total of 30 minutes while changing ethanol three times during the process, and xylene was replaced with ethanol. Then, the slide was placed in a container containing pure water, and the slide was immersed in pure water for a total of 30 minutes while changing the pure water three times during the process, and ethanol was replaced with water.

Subsequently, the slide was immersed in a 10 mM citrate buffer solution (pH 6.0) for 30 minutes and then heat-treated at 121° C. for 10 minutes to activate the antigen. After that, the slide was immersed in a container containing PBS for 30 minutes, then PBS containing 1% BSA was placed on the thin section on the slide and left for 1 hour for blocking treatment.

Next, a staining solution prepared by adjusting anti-HER2 antibody-bound fluorescent particles to 0.02 nM in PBS containing 1% BSA was placed on a thin section on a slide and reacted for 3 hours. After the reaction, the slide was placed in a container containing PBS and immersed for 30 minutes. The slide was fixed with a 4% paraformaldehyde solution for 10 minutes and then stained with hematoxylin. A mounting agent Aquatex (Merck Chemicals) was dropped and a cover glass was placed to mount a thin section, and a specimen slide after immunostaining and a comparative slide were prepared. The specimen slide was prepared by dividing the immunostaining day into 6 times and 3 times each time. Similarly, the above-mentioned comparative slides were prepared by dividing the immunostaining day into 6 times, 4 to 5 times each time.

[Evaluation]
The bright field image and the fluorescence image of the sample slide and the comparative slide prepared in the above preparation example were acquired using Axio Imager M2 (manufactured by Carl Zeiss). Representative brightfield images of sections on specimen slides and representative slides on comparative slides are shown in FIGS. 3 and 4, respectively.

Fluorescence images were acquired by irradiating excitation light with a wavelength of 580 nm and imaging the fluorescence with a wavelength of 610 nm emitted from the section. From the fluorescence image, the number of fluorescent particles was measured using the bright spot measurement software “G-count” manufactured by Di-Angstrom. Then, the number of bright spots in the cell region was calculated by superimposing it on the bright field image, and the number of fluorescent particles per cell and the CV value (coefficient of variation) of each section of the sample slide and the comparative slide were calculated. The results are shown in FIGS. 5 and 6, respectively. From this, it was shown that the thin section obtained by the method for producing a paraffin block of the present invention had a lower CV value and improved dispersibility as compared with the comparative example.

Claims

Microtube for paraffin block production with paraffin filled in the bottom.
The paraffin block manufacturing microtube according to claim 1, wherein the thickness of the paraffin is 1 to 10 mm from the bottom end of the bottom.
The microtube for paraffin block production according to claim 1 or 2, wherein the bottom portion has an opening surface, and the paraffin is exposed from the opening surface.
A paraffin block production method using the paraffin block production microtube according to any one of claims 1 to 3.
A cell packing step of packing cells to be observed in the paraffin block making microtube,
A cell immobilization step of fixing the cells filled in the cell filling step with a fixative,
A coating step of preparing a coated cell by coating the fixed cells obtained in the cell fixing step with a thickening polysaccharide,
A method for producing a paraffin block, comprising a film forming step of reacting a gelling agent with the coated cells obtained in the coating step to form a gel film around the coated cells.
The paraffin block manufacturing method according to claim 4, wherein the thickening polysaccharide is at least one selected from the group consisting of sodium alginate, pectin, and carrageenan.
The paraffin block manufacturing method according to claim 4 or 5, wherein the gelling agent is at least one selected from the group consisting of calcium chloride, calcium phosphate, and calcium carbonate.