KR20240057238A - Kit for clearing biological tissue comprising solution for accelerating biological tissue clearing and mounting solution not producing crystal, and method for clearing biological tissue - Google Patents

Abstract

The present invention relates to a kit for clearing biological tissue, which includes an accelerating solution for clearing biological tissue and a mounting solution that does not generate crystals, and a method for transparentizing biological tissue. The tissue clearing solution according to the present invention can be used to remove biological tissue by electrophoresis. It can make it transparent and accelerate the speed of transparency. In addition, the mounting solution according to the present invention solves the problem of crystallization of the mounting solution used for existing biological tissue transparency and does not crystallize even at low temperatures, so that high tissue transparency is observed when biological tissue is transparent, and the amount of fluorescence is reduced during 3D imaging. It has the effect of increasing and maintaining fluorescence for a long time. Accordingly, the tissue clearing solution and mounting solution according to the present invention are compositions of a biological tissue transparency kit used for three-dimensional fluorescence imaging of biological tissues, and are expected to be useful in making various biological tissues transparent.

Description

Kit for clearing biological tissue comprising solution for accelerating biological tissue clearing and mounting solution not producing crystals, and method for clearing biological tissue for clearing biological tissue}

The present invention relates to a kit for biological tissue transparency, including a biological tissue transparency acceleration solution and a mounting solution that does not generate crystals, and a method for biological tissue transparency.

Tissue transparency is a method that can identify the causes of various diseases by analyzing structural images created by antibody staining in tissues, and is a research topic that has recently become an issue in the field of biology.

This method prevents tissue damage that occurs during the process of cutting the tissue thinly for microscopic observation by making the entire tissue transparent without damaging the protein, and allows the observation of protein changes in living tissue, such as brain neural networks, through three-dimensional images.

Existing tissue clearing technologies include the Spatleholz, BABB, Scale S, and iDISCO methods, which are tissue clearing methods using organic solvents, and the ACT (active CLARITY technology) method, which is a polymer injection method, and the antigen preservation of treated tissues has been reported. Other methods except ACT have the problem of reduced fluorescence and antigen preservation. ACT has an antigen preservation of over 90%, which shows higher preservation compared to methods such as CLARITY that require binding to a hydrogel polymer in addition to the immobilized protein. However, the strong tissue fixation process causes loss of antigenicity, and problems such as a decrease in usable antibodies must be considered, so various improvements in technology are necessary.

Additionally, the CLARITY method uses a method of inserting hydrogel into the tissue to create a type of mesh support that holds substances important for diagnosis, such as DNA or proteins, and then selectively removing only the lipids. Tissue transparency technologies, such as CLARITY and Perfusion Assisted Reagent Release (PARS), which can produce optically transparent, polymer-transparent images, have provided major advances in greatly improved organ system imaging.

However, the Clarity method has the disadvantage that it takes a long time to become transparent because as the hydrogel concentration increases, the degree of binding to proteins increases, the tissue becomes harder, and lipid removal becomes difficult. This causes deposition of air and black particles on the tissue surface. In addition, the existing clarity method is a complicated process and requires a lot of additional equipment. In addition, only one tissue can be made transparent at a time, causing economic and time losses, and staining using antibodies was unstable.

Meanwhile, after the biological tissue is made transparent, it is observed using a confocal microscope and a lightsheet microscope. In particular, for microscopic observation, the tissue must be immersed in a mounting solution to adjust the refractive index while being processed and observed. However, in the space where the confocal microscope is set up, the temperature is low even in winter because the heater is not turned on, and in this case, crystals form momentarily. If the temperature is low or the cover is open, crystals form in the mounting solution and cause serious damage to the tissue. There was a problem in that the tissue could not be observed because it caused a condition in which imaging could not be performed.

Accordingly, the present inventors have developed an accelerated solution for biological tissue transparency that can make biological tissues transparent in a short period of time using electrophoresis, and a solution that solves the problem of crystallization of the mounting solution used for existing transparency and provides microscopic observation for three-dimensional imaging of biological tissues. We have invented a kit for biological tissue transparency that includes a non-crystallization mounting solution that increases the transparency of the tissue, increases the amount of fluorescence, and maintains the fluorescence for a long time.

Korean Patent Publication No. 10-1563826

The present inventors have developed an accelerated solution for biological tissue transparency that can make biological tissue transparent in a short period of time using an electrophoresis method, and have solved the problem of crystallization of the mounting solution used for existing biological tissue transparency, thereby solving the problem of crystallization. We have developed a mounting solution that does not crystallize at any temperature, so that high tissue transparency can be observed when making biological tissue transparent, increases the amount of fluorescence during 3D imaging, and has the effect of maintaining fluorescence for a long time. Based on this, the present invention was completed.

Accordingly, the object of the present invention is to provide a kit for making biological tissue transparent,

The kit contains N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, CHAPS), and at least one selected from the group consisting of urea, and

Selected from the group consisting of sorbitol, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, and Triton A tissue clearing solution containing one or more active ingredients; and

N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, Providing a kit, characterized in that it includes a mounting solution containing at least one selected from the group consisting of CHAPS), urea, and sodium chloride (NaCl), and sorbitol as an active ingredient. will be.

Another object of the present invention is to provide a transparency method for three-dimensional imaging of biological tissue using the kit for biological tissue transparency, comprising the following steps:

(a) fixing a biological tissue sample separated from a subject with a fixation solution;

(b) a clearing step of making the fixed biological tissue sample transparent with a tissue clearing solution;

(c) a washing step of washing away organic matter attached to the transparent biological tissue sample with a washing solution; and

(d) fixing the washed sample with a mounting solution containing sorbitol.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art to which the present invention belongs from the description below. There will be.

In order to achieve the above object, the present invention provides a kit for biological tissue transparency,

The kit contains N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, CHAPS), and at least one selected from the group consisting of urea, and

Selected from the group consisting of sorbitol, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, and Triton A tissue clearing solution containing one or more active ingredients; and

N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, A kit is provided, characterized in that it includes a mounting solution containing at least one selected from the group consisting of CHAPS), urea, and sodium chloride (NaCl), and sorbitol as an active ingredient. .

In one embodiment of the present invention, in the tissue clearing solution, N-lauroylsarcosine has a concentration of 0.01% (w/v) to 5% (w/v), and CHAPS has a concentration of 10% (w/v) to 10% (w/v). 40% (w/v), and the urea concentration may be from 10% (w/v) to 40% (w/v), but is not limited thereto.

In another embodiment of the present invention, in the tissue clearing solution, sorbitol has a concentration of 1% (w/v) to 10% (w/v), and 3-(N,N-dimethylmyristylammonio)propanesulfonate has a concentration of 1% (w/v) to 10% (w/v). The concentration may be 0.5% (w/v) to 5% (w/v), and Triton X-100 may have a concentration of 0.1% (w/v) to 3% (w/v), but is not limited thereto.

As another embodiment of the present invention, in the mounting solution, N-lauroylsarcosine has a concentration of 0.01% (w/v) to 5% (w/v), and CHAPS has a concentration of 20% (w/v) to 60% (w/v), urea may have a concentration of 20% (w/v) to 60% (w/v), and sodium chloride may have a concentration of 0.01% (w/v) to 2% (w/v). However, it is not limited to this.

As another embodiment of the present invention, the concentration of sorbitol in the mounting solution may be from 1% (w/v) to 19% (w/v), but is not limited thereto.

As another embodiment of the present invention, the tissue clearing solution may accelerate biological tissue transparency, but is not limited thereto.

As another embodiment of the present invention, the mounting solution may not crystallize even at a refrigerated temperature of 4°C to 10°C, but is not limited thereto.

As another embodiment of the present invention, the mounting solution can increase the amount of fluorescence and fluorescence retention time during 3D imaging of biological tissue, but is not limited thereto.

As another embodiment of the present invention, the biological tissue may be brain, liver, lung, spleen, kidney, intestine, heart, pancreas, muscle, blood vessel, or whole tissue, but is not limited thereto.

As another embodiment of the present invention, the kit includes a fixation solution containing sucrose as an active ingredient; and

It may further include, but is not limited to, a washing solution containing as an active ingredient one or more selected from the group consisting of phosphate buffer saline (PBS) and sodium azide.

As another embodiment of the present invention, the concentration of sucrose may be 20% (w/v) to 100% (w/v), but is not limited thereto.

As another embodiment of the present invention, the concentration of sodium azide may be 0.01% (w/v) to 1% (w/v), but is not limited thereto.

In addition, the present invention provides a transparency method for three-dimensional imaging of biological tissue using the kit for biological tissue transparency, comprising the following steps:

(a) fixing a biological tissue sample separated from a subject with a fixation solution;

(b) a clearing step of making the fixed biological tissue sample transparent with a tissue clearing solution;

(c) a washing step of washing away organic substances attached to the transparent biological tissue sample with a washing solution; and

(d) fixing the washed sample with a mounting solution containing sorbitol.

In one embodiment of the present invention, step (b) may be a step of making the biological tissue sample transparent by electrophoresis, but is not limited thereto.

As another embodiment of the present invention, the biological tissue that has undergone step (d) may have increased fluorescence amount and fluorescence retention time during 3D imaging, but is not limited thereto.

The tissue clearing solution according to the present invention can make biological tissue transparent through electrophoresis and accelerate the speed of transparency. In addition, the mounting solution according to the present invention solves the problem of crystallization of the mounting solution used for existing biological tissue transparency and does not crystallize even at low temperatures, so that high tissue transparency is observed when biological tissue is transparent, and the amount of fluorescence is reduced during 3D imaging. It has the effect of increasing and maintaining fluorescence for a long time. Accordingly, the tissue clearing solution and mounting solution according to the present invention are compositions of a biological tissue transparency kit used for three-dimensional fluorescence imaging of biological tissues, and are expected to be useful in making various biological tissues transparent.

Figure 1 is a diagram showing the results of clearing mouse brain tissue by electrophoresis using a tissue clearing solution that accelerates biological tissue clearing according to an embodiment of the present invention.
Figure 2 is a diagram comparing crystallization of a mounting solution containing sorbitol according to an embodiment of the present invention and a conventional mounting solution not containing sorbitol.
Figure 3 is a diagram comparing the transmittance upon transparency of various biological tissues using a mounting solution containing sorbitol according to an embodiment of the present invention and a conventional mounting solution not containing sorbitol.
Figure 4 is a diagram comparing the amount of fluorescence observed when biological tissue is made transparent using a mounting solution containing sorbitol according to an embodiment of the present invention and a conventional mounting solution not containing sorbitol.
Figure 5 is a diagram comparing the fluorescence retention effect when biological tissue was made transparent and imaged in three dimensions using a mounting solution containing sorbitol according to an embodiment of the present invention and a conventional mounting solution not containing sorbitol.

In one embodiment of the present invention, for transparency and three-dimensional imaging of biological tissue, the ingredients of Table 1 include a fixing solution, a tissue clearing solution, a washing solution, and a mounting solution ( A kit for biological tissue transparency was prepared, including a tissue clearing solution that accelerates biological tissue transparency and a mounting solution that does not generate crystals (see Example 1).

In another embodiment of the present invention, the biological tissue was made transparent by electrophoresis using a tissue clearing solution, which is an accelerating solution for biological tissue transparency. As a result, the transparency step took 7 days due to the ingredients of the tissue clearing solution of the present invention. It was confirmed that can be shortened to about 6 hours (see Example 2).

In another embodiment of the present invention, crystallization of the mounting solution was confirmed in a mounting solution containing sorbitol and a mounting solution not containing sorbitol. As a result, when brain tissue was made transparent using a mounting solution not containing sorbitol, the mounting solution While crystallization occurred and low transparency was observed when observing the brain tissue, it was confirmed that when a mounting solution containing sorbitol was used, crystallization of the mounting solution did not occur and the brain tissue was observed transparently (see Example 3).

In another embodiment of the present invention, various biological tissues of a mouse were each made transparent using a biological tissue transparency method, and the crystallization of the mounting solution according to the presence or absence of sorbitol was confirmed by observing this. As a result, when the biological tissue was made transparent, mounting solution not containing sorbitol was confirmed. It was confirmed that when a mounting solution containing sorbitol was used compared to when a solution was used, crystallization of the mounting solution was suppressed and tissue permeability was generally observed to be higher (see Example 4).

In another embodiment of the present invention, mouse brain tissue was each made transparent using a biological tissue transparency method and three-dimensional fluorescence imaging was performed. As a result, compared to the case of using a mounting solution that does not contain sorbitol when making brain tissue transparent, sorbitol was included. It was confirmed that when using the mounting solution, the amount of fluorescence increased and the fluorescence was maintained for a long time (see Example 5).

Accordingly, the present invention relates to a kit for making biological tissue transparent,

The kit contains N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, CHAPS), and at least one selected from the group consisting of urea, and

Selected from the group consisting of sorbitol, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, and Triton A tissue clearing solution containing one or more active ingredients; and

N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, A kit is provided, characterized in that it includes a mounting solution containing at least one selected from the group consisting of CHAPS), urea, and sodium chloride (NaCl), and sorbitol as an active ingredient. .

In the present invention, the concentration of N-lauroylsarcosine in the tissue clearing solution is 0.01% (w/v) to 5% (w/v), 0.01% (w/v) to 4% (w/v). , 0.01 % (w/v) to 3 % (w/v), 0.01 % (w/v) to 2 % (w/v), 0.01 % (w/v) to 1 % (w/v), 0.01 % (w/v) to 0.5 % (w/v), 0.05 % (w/v) to 5 % (w/v), 0.05 % (w/v) to 4 % (w/v), 0.05 % ( w/v) to 3%(w/v), 0.05%(w/v) to 2%(w/v), 0.05%(w/v) to 1%(w/v), 0.05%(w/v) v) to 0.5 % (w/v), 0.1 % (w/v) to 5 % (w/v), 0.1 % (w/v) to 4 % (w/v), 0.1 % (w/v) to 3 % (w/v), 0.1 % (w/v) to 2 % (w/v), 0.1 % (w/v) to 1 % (w/v), 0.1 % (w/v) to 0.5 %(w/v), or 0.1 %(w/v), but is not limited thereto.

In the present invention, the concentration of CHAPS in the tissue clearing solution is 10% (w/v) to 40% (w/v), 10% (w/v) to 35% (w/v), and 10% (w). /v) to 30% (w/v), 10% (w/v) to 25% (w/v), 15% (w/v) to 40% (w/v), 15% (w/v) ) to 35% (w/v), 15% (w/v) to 30% (w/v), 15% (w/v) to 25% (w/v), 20% (w/v) to 40%(w/v), 20%(w/v) to 35%(w/v), 20%(w/v) to 30%(w/v), 20%(w/v) to 25% (w/v), or 25% (w/v), but is not limited thereto.

In the present invention, the concentration of urea in the tissue clearing solution is 10% (w/v) to 40% (w/v), 10% (w/v) to 35% (w/v), and 10% (w). /v) to 30% (w/v), 10% (w/v) to 25% (w/v), 15% (w/v) to 40% (w/v), 15% (w/v) ) to 35% (w/v), 15% (w/v) to 30% (w/v), 15% (w/v) to 25% (w/v), 20% (w/v) to 40%(w/v), 20%(w/v) to 35%(w/v), 20%(w/v) to 30%(w/v), 20%(w/v) to 25% (w/v), or 25% (w/v), but is not limited thereto.

In the present invention, the concentration of sorbitol in the tissue clearing solution is 1% (w/v) to 10% (w/v), 1% (w/v) to 9% (w/v), and 1% (w). /v) to 8 % (w/v), 1 % (w/v) to 7 % (w/v), 1 % (w/v) to 6 % (w/v), 1 % (w/v) ) to 5% (w/v), 3% (w/v) to 10% (w/v), 3% (w/v) to 9% (w/v), 3% (w/v) to 8%(w/v), 3%(w/v) to 7%(w/v), 3%(w/v) to 6%(w/v), 3%(w/v) to 5% (w/v), 4%(w/v) to 10%(w/v), 4%(w/v) to 9%(w/v), 4%(w/v) to 8%(w) /v), 4 % (w/v) to 7 % (w/v), 4 % (w/v) to 6 % (w/v), 4 % (w/v) to 5 % (w/v) ), or 5% (w/v), but is not limited thereto.

In the present invention, the concentration of 3-(N,N-dimethylmyristylammonio)propanesulfonate in the tissue clearing solution is 0.5% (w/v) to 5% (w/v), 0.5% (w/ v) to 4 % (w/v), 0.5 % (w/v) to 3 % (w/v), 0.5 % (w/v) to 2 % (w/v), 1 % (w/v) to 5 % (w/v), 1 % (w/v) to 4 % (w/v), 1 % (w/v) to 3 % (w/v), 1 % (w/v) to 2 %(w/v), or 2 %(w/v), but is not limited thereto.

In the present invention, the concentration of Triton % (w/v) to 2 % (w/v), 0.1 % (w/v) to 1.5 % (w/v), 0.1 % (w/v) to 1 % (w/v), 0.5 % ( w/v) to 3 % (w/v), 0.5 % (w/v) to 2.5 % (w/v), 0.5 % (w/v) to 2 % (w/v), 0.5 % (w/ v) to 1.5 % (w/v), 0.5 % (w/v) to 1 % (w/v), or 1 % (w/v), but is not limited thereto.

In the present invention, the concentration of N-lauroylsarcosine in the mounting solution is 0.01% (w/v) to 5% (w/v), 0.01% (w/v) to 4% (w/v), 0.01 % (w/v) to 3 % (w/v), 0.01 % (w/v) to 2 % (w/v), 0.01 % (w/v) to 1 % (w/v), 0.01 % (w/v) to 0.5%(w/v), 0.05%(w/v) to 5%(w/v), 0.05%(w/v) to 4%(w/v), 0.05%(w) /v) to 3 % (w/v), 0.05 % (w/v) to 2 % (w/v), 0.05 % (w/v) to 1 % (w/v), 0.05 % (w/v) ) to 0.5 % (w/v), 0.1 % (w/v) to 5 % (w/v), 0.1 % (w/v) to 4 % (w/v), 0.1 % (w/v) to 3 % (w/v), 0.1 % (w/v) to 2 % (w/v), 0.1 % (w/v) to 1 % (w/v), 0.1 % (w/v) to 0.5 % (w/v), or 0.1 % (w/v), but is not limited thereto.

In the present invention, the concentration of CHAPS in the mounting solution is 20% (w/v) to 60% (w/v), 20% (w/v) to 55% (w/v), 20% (w/ v) to 50% (w/v), 20% (w/v) to 45% (w/v), 20% (w/v) to 40% (w/v), 25% (w/v) to 60% (w/v), 25% (w/v) to 55% (w/v), 25% (w/v) to 50% (w/v), 25% (w/v) to 45% %(w/v), 25%(w/v) to 40%(w/v), 30%(w/v) to 60%(w/v), 30%(w/v) to 55%( w/v), 30 % (w/v) to 50 % (w/v), 30 % (w/v) to 45 % (w/v), 30 % (w/v) to 40 % (w/ v), 35 % (w/v) to 60 % (w/v), 35 % (w/v) to 55 % (w/v), 35 % (w/v) to 50 % (w/v) , 35 % (w/v) to 45 % (w/v), 35 % (w/v) to 40 % (w/v), 40 % (w/v) to 60 % (w/v), 40 %(w/v) to 55%(w/v), 40%(w/v) to 50%(w/v), 40%(w/v) to 45%(w/v), or 40% It may be (w/v), but is not limited thereto.

In the present invention, the concentration of urea in the mounting solution is 20% (w/v) to 60% (w/v), 20% (w/v) to 55% (w/v), and 20% (w/v). v) to 50% (w/v), 20% (w/v) to 45% (w/v), 20% (w/v) to 40% (w/v), 25% (w/v) to 60% (w/v), 25% (w/v) to 55% (w/v), 25% (w/v) to 50% (w/v), 25% (w/v) to 45% %(w/v), 25%(w/v) to 40%(w/v), 30%(w/v) to 60%(w/v), 30%(w/v) to 55%( w/v), 30 % (w/v) to 50 % (w/v), 30 % (w/v) to 45 % (w/v), 30 % (w/v) to 40 % (w/ v), 35 % (w/v) to 60 % (w/v), 35 % (w/v) to 55 % (w/v), 35 % (w/v) to 50 % (w/v) , 35 % (w/v) to 45 % (w/v), 35 % (w/v) to 40 % (w/v), 40 % (w/v) to 60 % (w/v), 40 %(w/v) to 55%(w/v), 40%(w/v) to 50%(w/v), 40%(w/v) to 45%(w/v), or 40% It may be (w/v), but is not limited thereto.

In the present invention, the concentration of sodium chloride in the mounting solution is 0.01 % (w/v) to 2 % (w/v), 0.01 % (w/v) to 1.5 % (w/v), 0.01 % (w/ v) to 1 % (w/v), 0.01 % (w/v) to 0.5 % (w/v), 0.05 % (w/v) to 2 % (w/v), 0.05 % (w/v) to 1.5 % (w/v), 0.05 % (w/v) to 1 % (w/v), 0.05 % (w/v) to 0.5 % (w/v), 0.1 % (w/v) to 2 % (w/v), 0.1 % (w/v) to 1.5 % (w/v), 0.1 % (w/v) to 1 % (w/v), 0.1 % (w/v) to 0.5 % ( w/v), 0.1 % (w/v), or 1 % (w/v), but is not limited thereto.

In the present invention, the concentration of sorbitol in the mounting solution is 1% (w/v) to 19% (w/v), 1% (w/v) to 17% (w/v), and 1% (w/v). v) to 15 % (w/v), 1 % (w/v) to 13 % (w/v), 1 % (w/v) to 11 % (w/v), 3 % (w/v) to 19% (w/v), 3% (w/v) to 17% (w/v), 3% (w/v) to 15% (w/v), 3% (w/v) to 13 % (w/v), 3 % (w/v) to 11 % (w/v), 5 % (w/v) to 19 % (w/v), 5 % (w/v) to 17 % ( w/v), 5%(w/v) to 15%(w/v), 5%(w/v) to 13%(w/v), 5%(w/v) to 11%(w/v) v), 7 % (w/v) to 19 % (w/v), 7 % (w/v) to 17 % (w/v), 7 % (w/v) to 15 % (w/v) , 7 % (w/v) to 13 % (w/v), 7 % (w/v) to 11 % (w/v), 9 % (w/v) to 19 % (w/v), 9 % (w/v) to 17 % (w/v), 9 % (w/v) to 15 % (w/v), 9 % (w/v) to 13 % (w/v), 9 % ( w/v) to 11 % (w/v), or 10 % (w/v), but is not limited thereto.

In the present invention, the tissue clearing solution may be a biological tissue clearing acceleration solution that accelerates biological tissue clearing, but is not limited thereto.

In the present invention, the mounting solution may not crystallize even at low temperatures, and may not crystallize within a temperature range of at least 4°C, for example, 4°C to 10°C, 4°C to 9°C, 4°C to 8°C. It may not crystallize at refrigerated temperatures of, but is not limited to, 4°C to 7°C, 4°C to 6°C, 4°C to 5°C, or 4°C.

In the present invention, the mounting solution can increase the amount of fluorescence and fluorescence retention time during the three-dimensional imaging process of biological tissue, and according to one embodiment of the present invention, fluorescence can be maintained even after 12 days, but is not limited thereto. No.

In the present invention, “biological tissue” refers to tissue existing in the living body of an animal and tissue formed by blood vessels and other cells, and the biological tissue includes the brain, liver, lung, spleen, kidney, intestine, heart, and pancreas. , may be, but are not limited to, muscles, blood vessels, or whole tissues.

In the present invention, “mounting” means fixing a biological tissue sample during microscope observation, minimizing photobleaching, and homogenizing the refractive index.

In the present invention, the composition for the biological tissue mounting is 1.42 to 1.48, 1.42 to 1.47, 1.42 to 1.46, 1.43 to 1.48, 1.43 to 1.47, 1.43 to 1.46, 1.44 to 1.48, 1.44 to 1.47, 1.44 It can be adjusted to 1.46, 1.45 to 1.48, 1.45 to 1.47, 1.45 to 1.46, or 1.46, but is not limited thereto.

In the present invention, the kit includes a fixation solution containing sucrose as an active ingredient; and

It may further include, but is not limited to, a washing solution containing as an active ingredient one or more selected from the group consisting of phosphate buffer saline (PBS) and sodium azide.

In the present invention, the sucrose contained in the fixing solution has a concentration of 20% (w/v) to 100% (w/v), 20% (w/v) to 90% (w/v), 20% ( w/v) to 80%(w/v), 20%(w/v) to 70%(w/v), 20%(w/v) to 60%(w/v), 20%(w/v) v) to 50% (w/v), 20% (w/v) to 40% (w/v), 20% (w/v) to 30% (w/v), 30% (w/v) to 100% (w/v), 30% (w/v) to 90% (w/v), 30% (w/v) to 80% (w/v), 30% (w/v) to 70 %(w/v), 30%(w/v) to 60%(w/v), 30%(w/v) to 50%(w/v), 30%(w/v) to 40%( w/v), 25 % (w/v) to 35 % (w/v), 25 % (w/v) to 30 % (w/v), or 30 % (w/v), but Not limited.

The fixation solution according to the present invention contains sucrose at a concentration of 20% (w/v) or more, and according to one embodiment of the present invention, 30% (w/v), thereby dehydrating the tissue sample and removing organic matter with PFA (paraformaldehyde). Covalently bonded samples can be fixed more strongly.

In the present invention, the sodium azide contained in the washing solution has a concentration of 0.01% (w/v) to 1% (w/v), 0.01% (w/v) to 0.8% (w/v), 0.01 % (w/v) to 0.6 % (w/v), 0.01 % (w/v) to 0.4 % (w/v), 0.01 % (w/v) to 0.2 % (w/v), 0.01 % (w/v) to 0.1%(w/v), 0.05%(w/v) to 1%(w/v), 0.05%(w/v) to 0.8%(w/v), 0.05%(w) /v) to 0.6 % (w/v), 0.05 % (w/v) to 0.4 % (w/v), 0.05 % (w/v) to 0.2 % (w/v), 0.05 % (w/v) ) to 0.1 % (w/v), 0.1 % (w/v) to 1 % (w/v), 0.1 % (w/v) to 0.8 % (w/v), 0.1 % (w/v) to It can be 0.6 % (w/v), 0.1 % (w/v) to 0.4 % (w/v), 0.1 % (w/v) to 0.2 % (w/v), or 0.1 % (w/v). However, it is not limited to this.

The washing solution according to the present invention contains sodium azide, so it increases the moisture content of the biological tissue sample dehydrated after transparency by up to 30% and then dehydrates 15% to remove organic substances that interfere with imaging attached to the tissue. can be washed.

In addition, the present invention relates to N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (3-[(3-cholamidopropyl)dimethylammonio] -1-propanesulfonate, CHAPS), urea, sorbitol, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, and Triton Provided is a tissue clearing composition that accelerates biological tissue transparency and includes -100 (Triton X-100) as an active ingredient.

In addition, the present invention relates to N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (3-[(3-cholamidopropyl)dimethylammonio] Provided is a mounting composition that does not form crystals and includes -1-propanesulfonate (CHAPS), urea, sodium chloride (NaCl), and sorbitol as active ingredients.

In addition, the present invention provides a fixative solution containing sucrose as an active ingredient;

N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS), urea, sorbitol, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, and Triton A tissue clearing solution containing -100) as an active ingredient;

A washing solution containing phosphate buffer saline (PBS) and sodium azide; and

N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, A kit for biological tissue transparency is provided, including a mounting solution containing CHAPS), urea, sodium chloride (NaCl), and sorbitol.

In the present invention, “transparency or clearing” refers to a technique of making an organ or tissue transparent enough to optically observe the inside of the tissue while minimizing incision or structural damage to the organ or tissue to be observed. it means.

In the present invention, “kit” refers to a tool that includes the fixing solution, tissue clearing solution, cleaning solution, and mounting solution, thereby making biological tissue transparent and enabling three-dimensional imaging. In addition to the above solution, the kit of the present invention may include other components, solutions, compositions, devices, etc. commonly required for biological tissue transparency and three-dimensional imaging. At this time, the solutions can be applied one or more times without limitation, there is no limit to the order in which each solution is applied, and the application of each solution may be carried out simultaneously or with a time difference.

In the present invention, the kit includes a container; directions; and the fixing solution, tissue clearing solution, washing solution, and mounting solution. The container may serve to package the preparation, and may also serve to store and secure the preparation. The material of the container may take the form of, for example, a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., which may be partially or entirely made of plastic, glass, paper, or foil. , wax, etc. The container may be equipped with a completely or partially removable closure that may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also provide access to the contents by needle. A stopper can be installed. The kit may include an external package, and the external package may include instructions for use of the components.

In addition, the present invention provides a transparency method for three-dimensional imaging of biological tissue using the kit for biological tissue transparency, comprising the following steps:

(a) fixing a biological tissue sample separated from a subject with a fixation solution;

(b) a clearing step of making the fixed biological tissue sample transparent with a tissue clearing solution;

(c) a washing step of washing away organic matter attached to the transparent biological tissue sample with a washing solution; and

(d) fixing the washed sample with a mounting solution containing sorbitol.

In the present invention, the (b) transparency step may be a step of making the biological tissue sample transparent by electrophoresis, for example, at a voltage of 50 to 300 v, 50 to 250 v, 50 to 200 v, 100 to 300 v, 100 to 250 v, or 100 to 200 v for 4 to 30 hours, 4 to 27 hours, 4 to 24 hours, 4 to 20 hours, 4 to 16 hours, 4 to 12 hours, 4 to 8 hours, 8 to 30 hours, This may be a step of applying electricity for 8 to 27 hours, 8 to 24 hours, 8 to 20 hours, 8 to 16 hours, 8 to 12 hours, 4 to 6 hours, 4 hours, 5 hours, or 6 hours to make it transparent. It is not limited to this.

In the present invention, the method of making biological tissue transparent can be used for observation through three-dimensional fluorescence imaging of biological tissue, and can be used by administering a fluorescent substance to a subject for three-dimensional fluorescence imaging, or before or during transparency of biological tissue, Alternatively, the step of treating the biological tissue with an antibody labeled with a fluorescent substance after transparency may be further included, but is not limited thereto. At this time, the fluorescent material known in the art can be used, and there is no limitation on its type. In the present invention, the biological tissue that has gone through step (d) may have increased fluorescence and fluorescence retention time during 3D imaging, but is not limited thereto.

In the present invention, the fixation in step (a) or (d) is 12 to 48 hours, 12 to 40 hours, 12 to 32 hours, 12 to 24 hours, 20 to 48 hours, 20 to 40 hours, 20 to 40 hours. It may be carried out for 32 hours, 20 to 24 hours, 24 to 48 hours, 24 to 40 hours, 24 to 32 hours, or 24 hours, but is not limited thereto.

In the present invention, the subject may be one or more selected from the group consisting of humans, non-human primates, fish, mice, dogs, cats, rabbits, horses, and cattle, but is not limited thereto.

When the term “comprising” is used in the present invention, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary. The term “step of” or “step of” used throughout the specification of the present invention does not mean “step for.”

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Preparation of a kit for making biological tissue transparent including an accelerating solution that can quickly make biological tissue transparent by electrophoresis and a non-crystallizing mounting solution

For transparency and three-dimensional imaging of biological tissues, a fixing solution, a tissue clearing solution that can quickly make biological tissues transparent through electrophoresis, a washing solution, and a non-crystallized solution are used. A kit for biological tissue transparency containing a mounting solution was prepared, and the components of each solution are shown in Table 1 below.

Specifically, in order to dehydrate biological tissue samples and more strongly fix samples covalently bonded between organic substances with PFA (paraformaldehyde), a fixation solution containing sucrose at a concentration of 30% (w/v) as a component is used. (fixing solution) was prepared.

In addition, in order to minimize denaturation of fluorescent substances in biological tissue samples when administering fluorescent substances for 3D fluorescence imaging by stabilizing tissue deformation and ionic strength due to osmotic pressure, 0.1% (w/v) N -lauroylsarcosine, 25% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 25% (w/v) urea By adding 5% (w/v) Sorbitol, 2% (w/v) 3-N,N-Dimethylmyristyammonio) propanesulfonate, and 1% (w/v) Triton A tissue clearing solution, an accelerated solution that can make the tissue transparent over time, was prepared.

Next, the moisture content of the dehydrated biological tissue sample after transparency was increased to a maximum of 30%, and then 15% was dehydrated, and organic substances adhering to the biological tissue sample that interfered with imaging were added to phosphate buffer saline (PBS). A washing solution was prepared by adding 0.1% (w/v) of sodium azide.

In addition, 0.1% (w/v) N-lauroylsarcosine, 40% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfo A mounting solution was prepared by adding 10% sorbitol to nate), 40% (w/v) urea, and 0.1 to 1% (w/v) sodium chloride (NaCl), and the refractive index was set to 1.46. In subsequent experiments, a NaCl concentration of 1% (w/v) was used.

composition composition One Fixing solution Sucrose (30%(w/v)) 2 Tissue clearing solution N-lauroylsarcosine (0.1% (w/v)), CHAPS (25% (w/v)), Urea (25% (w/v)), Sorbitol (5% (w/v)), 3-(N ,N-Dimethylmyristylammonio) propanesulfonate (2% (w/v)), Triton X-100 (1% (w/v)) 3 Washing solution PBS, sodium azide (0.1%(w/v)) 4 Mounting solution N-lauroylsarcosine (0.1% (w/v)), CHAPS (40% (w/v)), Urea (40% (w/v)), NaCl (0.1-1% (w/v)), Sorbitol ( 10%(w/v))

Example 2. Mouse brain tissue transparency using biological tissue transparency acceleration solution

Mouse brain tissue was made transparent using the biological tissue transparency kit prepared in Example 1.

Specifically, the brain tissue of a C57BL/6 5-week-old mouse was placed in a fixation solution containing sucrose, and the tissue was fixed by shaking incubation at 4°C/50 rpm for 24 hours until it settled.

Thereafter, the tissue was placed in a chamber dedicated to transparency and electrophoresed for 4-6 hours at 100 v and 10 mA using the tissue clearing solution shown in Table 1 above. Next, 50 ml of washing solution was added and cultured with shaking at 4°C/50 rpm for 1 hour, and this was repeated once more to wash the tissue.

The tissue samples that were cleared and washed were placed in the mounting solution and cultured with shaking at 35°C/50 rpm for 24 hours.

As a result, as shown in Figure 1, while it takes approximately 7 days to make transparent using the existing passive method, when making transparent using the tissue clearing solution according to the present invention, the brain is removed in about 6 hours at the clearing stage. It was confirmed that the organization can be made transparent.

Example 3. Confirmation of crystallization of mounting solution with or without sorbitol

The kit for biological tissue transparency prepared in Example 1 contains N-lauroylsarcosine, CHAPS, urea, NaCl, and sorbitol as components of the mounting solution, and compared to the mounting solution that does not contain sorbitol, the mounting solution Crystallization was confirmed.

For this purpose, a kit for clearing biological tissue prepared without sorbitol as a mounting solution and all remaining components were the same as those in Table 1, and a kit for clearing biological tissue containing sorbitol prepared in Example 1 were used, respectively. Thus, biological tissue transparency was performed.

Specifically, the brain tissue of a C57BL/6 5-week-old mouse was placed in a fixation solution and incubated with shaking at 4°C/50 rpm for 24 hours until it settled, thereby fixing the tissue.

Afterwards, the settled tissue sample was placed in a tissue clearing solution, applied with electricity at a voltage of 100-200 v for 4-24 hours, and made transparent by electrophoresis. Next, 50 ml of washing solution was added and cultured with shaking at 4°C/50 rpm for 1 hour, and this was repeated once more to wash the tissue.

The tissue samples that were cleared and washed were placed in the mounting solution and cultured with shaking at 35°C/50 rpm for 24 hours.

As a result of observing the brain tissue after making it transparent with each biological tissue transparency kit through the above method, as shown in FIG. 2, when a mounting solution that does not contain sorbitol was used, the mounting solution crystallized at a refrigeration temperature of 4 to 10 ° C. occurred, and low transparency was observed when observing brain tissue. However, when a mounting solution containing sorbitol was used, it was confirmed that crystallization of the mounting solution did not occur and the brain tissue was observed transparently.

From this, the biological tissue transparency kit prepared in Example 1 prevents crystallization from occurring in the mounting solution by including sorbitol as a component of the mounting solution, and thereby obtains highly transparent biological tissue observation results when transparent the biological tissue. I found out that it was possible.

Example 4. Confirmation of crystallization of mounting solution with or without sorbitol in various biological tissues

Using the biological tissue transparency method of Example 2, various biological tissues of a mouse were each made transparent and observed to confirm crystallization of the mounting solution with or without sorbitol.

The kidneys, liver, spleen, and heart of a C57BL/6 5-week-old mouse were removed and each was made transparent by the method of Example 2. As shown in Figure 3, when each biological tissue was made transparent, a mounting solution containing no sorbitol was used. It was confirmed that when a mounting solution containing sorbitol was used compared to the case where sorbitol was used, crystallization of the mounting solution was suppressed and tissue permeability was generally observed to be higher.

Example 5. Confirmation of fluorescence amount and fluorescence retention effect depending on the presence or absence of sorbitol during 3D imaging of biological tissue

The purchased Thy-1 YFP transgenic mouse brain tissues were each transparent using the biological tissue transparency method of Example 2, and the cleared brain tissues were imaged using a confocal microscope to measure the amount of fluorescence.

3D images were imaged using arivis vison 4D software (arivis-AG, Rostock, Germany) and Imaris software (Bitplane, USA).

The brain tissue was made transparent using a mounting solution containing and not containing sorbitol, and the transparency was compared by measuring the tissue permeability. As shown in Figure 4, when the brain tissue was made transparent, the mounting solution not containing sorbitol was used. Compared to using a solution (Control), when a mounting solution containing sorbitol was used (Sorbitol), the transparency of the tissue was higher, and the fluorescence intensity was higher at 78% compared to Control, which was 63.1% based on 100%. confirmed.

In addition, as shown in Figure 5, as a result of checking the three-dimensional image after brain tissue transparency, when using a mounting solution that does not contain sorbitol, almost no fluorescence appears after 12 days, whereas when using a mounting solution containing sorbitol, 12 It was confirmed that fluorescence was maintained for a long time even after one day.

From the above, it can be seen that the mounting solution according to the present invention prevents damage to biological tissues due to crystallization and at the same time increases the transparency of biological tissues to help obtain clearer fluorescence images.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims (15)

In the kit for biological tissue transparency,
The kit contains N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, CHAPS), and at least one selected from the group consisting of urea, and
Selected from the group consisting of sorbitol, 3-(N,N-Dimethylmyristylammonio)propanesulfonate, and Triton A tissue clearing solution containing one or more active ingredients; and
N-lauroylsarcosine, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, A kit comprising a mounting solution containing at least one selected from the group consisting of CHAPS), urea, and sodium chloride (NaCl), and sorbitol as an active ingredient.
According to paragraph 1,
In the tissue clearing solution, N-lauroylsarcosine has a concentration of 0.01% (w/v) to 5% (w/v), and CHAPS has a concentration of 10% (w/v) to 40% (w/v). A kit, characterized in that urea has a concentration of 10% (w/v) to 40% (w/v).
According to paragraph 1,
In the tissue clearing solution, sorbitol has a concentration of 1% (w/v) to 10% (w/v), and 3-(N,N-dimethylmyristylammonio)propanesulfonate has a concentration of 0.5% (w/v). ) to 5% (w/v), and Triton X-100 has a concentration of 0.1% (w/v) to 3% (w/v).
According to paragraph 1,
In the mounting solution, N-lauroylsarcosine has a concentration of 0.01% (w/v) to 5% (w/v), CHAPS has a concentration of 20% (w/v) to 60% (w/v), and urea. The kit is characterized in that the concentration is 20% (w/v) to 60% (w/v), and the sodium chloride concentration is 0.01% (w/v) to 2% (w/v).
According to paragraph 1,
The kit, characterized in that the concentration of sorbitol in the mounting solution is 1% (w/v) to 19% (w/v).
According to paragraph 1,
The tissue clearing solution is a kit characterized in that it accelerates biological tissue transparency.
According to paragraph 1,
The kit is characterized in that the mounting solution does not crystallize even at a refrigerated temperature of 4 ℃ to 10 ℃.
According to paragraph 1,
The kit is characterized in that the mounting solution increases the amount of fluorescence and fluorescence retention time during the three-dimensional imaging process of biological tissue.
According to paragraph 1,
The kit, wherein the biological tissue is brain, liver, lung, kidney, intestine, spleen, heart, pancreas, muscle, blood vessel, or whole tissue.
According to paragraph 1,
The kit includes a fixation solution containing sucrose as an active ingredient; and
A kit, characterized in that it further comprises a washing solution containing at least one selected from the group consisting of phosphate buffer saline (PBS) and sodium azide as an active ingredient.
According to clause 10,
The kit, characterized in that the sucrose has a concentration of 20% (w/v) to 100% (w/v).
According to clause 10,
The kit, wherein the sodium azide has a concentration of 0.01% (w/v) to 1% (w/v).
A clearing method for three-dimensional imaging of biological tissue using the biological tissue clearing kit of any one of claims 1 to 12, comprising the following steps:
(a) fixing a biological tissue sample separated from a subject with a fixation solution;
(b) a clearing step of making the fixed biological tissue sample transparent with a tissue clearing solution;
(c) a washing step of washing away organic matter attached to the transparent biological tissue sample with a washing solution; and
(d) fixing the washed sample with a mounting solution containing sorbitol.
According to clause 13,
The method (b) is characterized in that the biological tissue sample is made transparent by electrophoresis.
According to clause 13,
A method, wherein the amount of fluorescence and fluorescence retention time of the biological tissue that has passed through step (d) are increased during three-dimensional imaging.