Kit for clearing biological tissue comprising biological tissue clearing acceleration solution, electrophoresis device for clearing biological tissue, and method for clearing biological tissue using same

Abstract

The present invention relates to a kit for clearing a biological tissue including a biological tissue clearing acceleration solution; an electrophoresis device for clearing a biological tissue; and a method for clearing a biological tissue using same. The kit for clearing a biological tissue according to the present invention comprises a tissue clearing acceleration solution and a mounting solution, and if a biological tissue is cleared by an electrophoresis device using the kit for clearing a biological tissue, more rapid clearing is possible due to the tissue clearing acceleration solution, and fluorescence preservation is improved by the mounting solution having a high refractive index, thereby obtaining a high-quality three-dimensional image result. Therefore, the kit for clearing biological tissue and the electrophoresis device according to the present invention are expected to be usefully employed for efficiently clearing various biological tissues for three-dimensional fluorescence imaging of biological tissues.

Description

A kit for making biotissue transparent, comprising a solution for accelerating making biotissue transparent, an electrophoresis device for making biotissue transparent, and a method for making biotissue transparent using the same.

[0001] The present invention relates to a kit for making biological tissue transparent, including a solution for accelerating making biological tissue transparent, an electrophoresis device for making biological tissue transparent, and a method for making biological tissue transparent using the same.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0170004, filed November 29, 2023, the entire disclosure of which is incorporated herein by reference.

[0003]

[0004] Tissue transparency is a research method that has recently become an issue in the field of biology, as it can identify the causes of various diseases by analyzing structural images created by antibody staining within tissues.

[0005] This method prevents tissue damage that occurs during the process of thinly slicing tissue for microscopic observation by making the entire tissue transparent without damaging proteins, and allows for the observation of protein changes in biological tissues, such as brain neural networks, as three-dimensional images.

[0006] Existing tissue clearing technologies have reported antigen preservation in tissues treated with organic solvent-based methods such as Spatleholz, BABB, Scale S, and iDISCO, as well as polymer-injected ACT (active CLARITY technology). Other methods, excluding ACT, suffer from reduced fluorescence and antigen preservation. ACT boasts over 90% antigen preservation, which is higher than methods such as CLARITY, which require additional binding to hydrogel polymers on immobilized proteins. However, the strong tissue fixation process can cause a loss of antigenicity, which reduces the amount of usable antibodies, necessitating further improvements in various technologies.

[0007] Furthermore, the CLARITY method utilizes a method that selectively removes lipids by injecting hydrogels into tissues to create a mesh-like scaffold that captures diagnostically important substances such as DNA and proteins. Tissue transparency technologies, such as CLARITY and perfusion-assisted reagent release (PARS), which produce optically transparent and polymer-permeable images, have provided significant advances in highly advanced organ imaging.

[0008] However, the clarity method has the disadvantage of taking a long time to clear because as the hydrogel concentration increases, the degree of protein binding increases, making the tissue harder and more difficult to remove lipids. This causes the deposition of air and black particles on the tissue surface. Furthermore, the existing clarity method is complex and requires a lot of additional equipment. Furthermore, only one tissue can be cleared at a time, resulting in economic and time losses, and the problem of unstable antibody staining.

[0009] 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 processed and immersed in a mounting solution for refractive index control during observation.

[0010] Accordingly, the inventors of the present invention invented a kit for making biological tissue transparent, which includes a solution for accelerating transparency that can make biological tissue transparent in a short period of time and a mounting solution that has a high refractive index to improve the transparency of the tissue and enable clearer three-dimensional imaging, and developed a method for making biological tissue transparent more quickly and with higher transparency using the kit for making biological tissue transparent and an electrophoresis device.

[0011]

[0012] The present inventors have developed a kit for making biological tissue transparent, including a solution for accelerating making biological tissue transparent, which can make biological tissue transparent in a short period of time by an electrophoresis method, and a mounting solution that increases the refractive index to observe high transparency of the tissue when making biological tissue transparent, increases the amount of fluorescence of a target protein during three-dimensional imaging, and exhibits the effect of maintaining fluorescence for a long time, and have developed an electrophoresis system for making biological tissue transparent quickly using the kit for making biological tissue transparent, and have completed the present invention based on this.

[0013] Accordingly, the purpose of the present invention is to provide a kit for making biological tissue transparent.

[0014] Another object of the present invention is to provide an electrophoresis device for making biological tissue transparent and an electrophoresis system for making biological tissue transparent.

[0015] Another object of the present invention is to provide a method for making biological tissue transparent using a kit for making biological tissue transparent.

[0016]

[0017] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0018]

[0019] In order to achieve the above purpose, the present invention provides a kit for making biological tissue transparent,

[0020] The above kit comprises a rapid tissue clearing solution comprising at least one selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and

[0021] A kit is provided, characterized in that it includes a mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as an active ingredient.

[0022] As one embodiment of the present invention, in the tissue transparency acceleration solution, CHAPS may have a concentration of 10% (w / v) to 40% (w / v), urea may have a concentration of 10% (w / v) to 40% (w / v), and THEED may have a concentration of 0.1% (w / v) to 3% (w / v), but is not limited thereto.

[0023] As another embodiment of the present invention, in the mounting solution, TDE may have a concentration of 10% (w / v) to 18% (w / v), DMSO may have a concentration of 10% (w / v) to 18% (w / v), iohexol may have a concentration of 45% (w / v) to 55% (w / v), and sodium chloride may have a concentration of 0.1% (w / v) to 1% (w / v), but is not limited thereto.

[0024] As another embodiment of the present invention, the biological tissue may be, but is not limited to, brain, liver, lung, kidney, intestine, spleen, heart, pancreas, muscle, blood vessel, or whole tissue.

[0025] As another embodiment of the present invention, the kit comprises a fixing solution comprising sucrose as an active ingredient; and

[0026] The washing solution may further include, but is not limited to, one or more active ingredients selected from the group consisting of phosphate buffer saline (PBS) and sodium azide.

[0027] As another embodiment of the present invention, the concentration of the sucrose may be, but is not limited to, 20% (w / v) to 100% (w / v).

[0028] As another embodiment of the present invention, the concentration of the sodium azide may be, but is not limited to, 0.01% (w / v) to 1% (w / v).

[0029] As another embodiment of the present invention, the mounting solution may have a refractive index of 1.47 to 1.55, but is not limited thereto.

[0030] In addition, the present invention relates to an electrophoresis device for making biological tissue transparent,

[0031] An electrophoresis chamber capable of containing a sample;

[0032] A main body equipped with the above electrophoresis chamber;

[0033] A cover that is mounted on the upper part of the main body on which the electrophoresis chamber is mounted and can be opened and closed;

[0034] Dial for time adjustment;

[0035] A display showing time or temperature; and

[0036] An electrophoresis device for biotissue transparency is provided, characterized in that it includes a switch for changing power or mode.

[0037] As one embodiment of the present invention, the electrophoresis chamber may include a sample injection unit capable of introducing a sample, but is not limited thereto.

[0038] In addition, the present invention provides an electrophoresis device for making the biological tissue transparent; and

[0039] An electrophoresis system for biotissue transparency is provided, comprising a cassette mounted in an electrophoresis chamber.

[0040] In addition, the present invention provides a method for making a living tissue transparent using the kit for making a living tissue transparent, comprising the following steps:

[0041] (a) a step of fixing a biological tissue sample separated from a subject with a fixing solution;

[0042] (b) a clearing step of clearing the fixed biological tissue sample with a tissue clearing acceleration solution;

[0043] (c) a washing step of washing away organic substances attached to the above-mentioned transparent biological tissue sample with a washing solution; and

[0044] (d) A step of fixing the washed sample with a mounting solution.

[0045] As one embodiment of the present invention, the step (b) may be a step of making a biological tissue sample transparent using the electrophoresis device, but is not limited thereto.

[0046] As another embodiment of the present invention, step (b) may be performed for 4 to 8 hours, but is not limited thereto.

[0047] In addition, the present invention provides a rapid tissue clearing solution comprising at least one selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and

[0048] Provided is a kit for use in clearing biological tissue, comprising a mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as active ingredients.

[0049] In addition, the present invention provides a rapid tissue clearing solution comprising at least one selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and

[0050] Provided is a use for the preparation of a mounting solution for making a living tissue transparent, the mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as active ingredients.

[0051]

[0052] The kit for making biological tissue transparent according to the present invention includes a tissue clearing acceleration solution and a mounting solution. When making biological tissue transparent using an electrophoresis device using the kit for making biological tissue transparent, the tissue clearing acceleration solution enables faster clearing, and the mounting solution with a high refractive index improves fluorescence retention, thereby enabling the acquisition of high-quality three-dimensional image results. Therefore, the kit for making biological tissue transparent and the electrophoresis device according to the present invention are expected to be useful for efficiently making various biological tissues transparent for three-dimensional fluorescence imaging of biological tissues. In addition, while the existing CLARITY method makes tissue transparent by electrophoresis after hydrogel embedding, the method for making biological tissue transparent of the present invention allows electrophoresis immediately after tissue fixation without embedding, thereby having the effect of making biological tissue transparent very simply and minimizing tissue damage.

[0053]

[0054] Figure 1 is a drawing showing the results before (left) and after (right) clearing of mouse brain tissue using electrophoresis for 6 hours using a solution accelerating clearing of biological tissue according to one embodiment of the present invention.

[0055] FIG. 2 is a drawing showing the results of electrophoresis for 6 hours to make mouse spleen, heart, lung, and kidney tissues transparent using a solution for accelerating the transparency of biological tissue according to one embodiment of the present invention.

[0056] FIG. 3 is a drawing comparing the results of transparentizing mouse brain tissue using a mounting solution having a refractive index of 1.52 according to one embodiment of the present invention (right) with the results of transparentizing using a mounting solution having a refractive index of 1.46 (left).

[0057] FIG. 4 is a perspective view of an electrophoresis device for making biological tissue transparent according to one embodiment of the present invention.

[0058] FIG. 5 is a plan view (left), a perspective view (middle), and a front view (right) of an electrophoresis chamber for making biological tissue transparent according to one embodiment of the present invention.

[0059] FIG. 6 is a drawing showing a bottom view (left), a back view (middle), and a perspective view (right) of a cassette for making biological tissue transparent according to one embodiment of the present invention.

[0060]

[0061] In one embodiment of the present invention, a fixing solution, a rapid tissue clearing solution, a washing solution, and a mounting solution were prepared using the components of Table 1, thereby preparing a kit for clearing biological tissues including a tissue clearing acceleration solution that accelerates clearing biological tissues and a mounting solution having a high refractive index (see Example 1).

[0062] In another embodiment of the present invention, mouse brain tissue and various organ tissues were made transparent using the kit for making biotissue transparent and the electrophoresis system manufactured in Example 1, and as a result, it was confirmed that brain, spleen, heart, lung, and kidney tissues could be made transparent in about 6 hours in the making transparent step using the tissue making accelerated solution according to the present invention through the electrophoresis device (see Example 2).

[0063] In another embodiment of the present invention, as a result of confirming tissue transparency according to the difference in refractive index of the mounting solution, it was confirmed that when a mounting solution having a refractive index of 1.52 was used, transparency and fluorescence preservation were improved compared to when a mounting solution having a refractive index of 1.46 was used, and thus high-quality 3D image results could be obtained (see Example 3).

[0064]

[0065] Hereinafter, the present invention will be described in detail.

[0066]

[0067] The present invention relates to a kit for making biological tissue transparent,

[0068] The above kit comprises a rapid tissue clearing solution comprising at least one selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and

[0069] A kit is provided, characterized in that it includes a mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as an active ingredient.

[0070] In the present invention, in the tissue transparency acceleration solution, the concentration of CHAPS is 10% (w / v) to 40% (w / v), 10% (w / v) to 35% (w / v), 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.

[0071] In the present invention, the concentration of urea in the tissue transparency acceleration solution is 10% (w / v) to 40% (w / v), 10% (w / v) to 35% (w / v), 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.

[0072] In the present invention, THEED in the tissue transparency acceleration solution has a concentration of 0.1% (w / v) to 3% (w / v), 0.1% (w / v) to 2.7% (w / v), 0.1% (w / v) to 2.5% (w / v), 0.1% (w / v) to 2.3% (w / v), 0.1% (w / v) to 2% (w / v), 0.2% (w / v) to 3% (w / v), 0.2% (w / v) to 2.7% (w / v), 0.2% (w / v) to 2.5% (w / v), 0.2% (w / v) to 2.3% (w / v), 0.2% (w / v) to 2.3% (w / v), 0.2% (w / v) to 2% (w / v), 0.3% (w / v) to 3% (w / v), 0.3 %(w / v) to 2.7 %(w / v), 0.3 %(w / v) to 2.5 %(w / v), 0.3 %(w / v) to 2.3 %(w / v), or 0.3 %(w / v) to 2 %(w / v), but is not limited thereto.

[0073] In the present invention, the TDE in the mounting solution has a concentration of 10% (w / v) to 18% (w / v), 10% (w / v) to 16% (w / v), 10% (w / v) to 15% (w / v), 10% (w / v) to 14.56% (w / v), 11% (w / v) to 18% (w / v), 11% (w / v) to 16% (w / v), 11% (w / v) to 15% (w / v), 11% (w / v) to 14.56% (w / v), 12% (w / v) to 18% (w / v), 12% (w / v) to 16% (w / v), 12% (w / v) to 15% (w / v), 12% (w / v) to 14.56 %(w / v), 13 %(w / v) to 18 %(w / v), 13 %(w / v) to 16 %(w / v), 13 %(w / v) to 15 %(w / v), 13 %(w / v) to 14.56 %(w / v), 14 %(w / v) to 18 %(w / v), 14 %(w / v) to 16 %(w / v), 14 %(w / v) to 15 %(w / v), 14 %(w / v) to 14.56 %(w / v), or 14.56 %(w / v), but is not limited thereto.

[0074] In the present invention, the concentration of DMSO in the mounting solution is 10% (w / v) to 18% (w / v), 10% (w / v) to 16% (w / v), 10% (w / v) to 15% (w / v), 10% (w / v) to 14.56% (w / v), 11% (w / v) to 18% (w / v), 11% (w / v) to 16% (w / v), 11% (w / v) to 15% (w / v), 11% (w / v) to 14.56% (w / v), 12% (w / v) to 18% (w / v), 12% (w / v) to 16% (w / v), 12% (w / v) to 15% (w / v), 12% (w / v) to 14.56 %(w / v), 13 %(w / v) to 18 %(w / v), 13 %(w / v) to 16 %(w / v), 13 %(w / v) to 15 %(w / v), 13 %(w / v) to 14.56 %(w / v), 14 %(w / v) to 18 %(w / v), 14 %(w / v) to 16 %(w / v), 14 %(w / v) to 15 %(w / v), 14 %(w / v) to 14.56 %(w / v), or 14.56 %(w / v), but is not limited thereto.

[0075] In the present invention, the concentration of iohexol in the mounting solution is 45% (w / v) to 55% (w / v), 45% (w / v) to 54% (w / v), 45% (w / v) to 53% (w / v), 45% (w / v) to 52% (w / v), 45% (w / v) to 51.46% (w / v), 47% (w / v) to 55% (w / v), 47% (w / v) to 54% (w / v), 47% (w / v) to 53% (w / v), 47% (w / v) to 52% (w / v), 47% (w / v) to 51.46% (w / v), 49% (w / v) to 55% (w / v), 49% (w / v) to 54 %(w / v), 49 %(w / v) to 53 %(w / v), 49 %(w / v) to 52 %(w / v), 49 %(w / v) to 51.46 %(w / v), 51 %(w / v) to 55 %(w / v), 51 %(w / v) to 54 %(w / v), 51 %(w / v) to 53 %(w / v), 51 %(w / v) to 52 %(w / v), 51 %(w / v) to 51.46 %(w / v), or 51.46 %(w / v), but is not limited thereto.

[0076] In the present invention, the concentration of sodium chloride in the mounting solution is 0.1% (w / v) to 1% (w / v), 0.1% (w / v) to 0.8% (w / v), 0.1% (w / v) to 0.6% (w / v), 0.1% (w / v) to 0.4% (w / v), 0.1% (w / v) to 0.3% (w / v), 0.2% (w / v) to 1% (w / v), 0.2% (w / v) to 0.8% (w / v), 0.2% (w / v) to 0.6% (w / v), 0.2% (w / v) to 0.4% (w / v), 0.2% (w / v) to 0.3% (w / v), 0.3% (w / v) to 1% (w / v), It may be, but is not limited to, 0.3 %(w / v) to 0.8 %(w / v), 0.3 %(w / v) to 0.6 %(w / v), 0.3 %(w / v) to 0.4 %(w / v), or 0.3 %(w / v).

[0077] In the present invention, the tissue transparency acceleration solution may be one that makes biological tissue transparent and accelerates transparency, but is not limited thereto.

[0078] In the present invention, the mounting solution has a refractive index of 1.47 to 1.55, 1.47 to 1.54, 1.47 to 1.53, 1.47 to 1.52, 1.48 to 1.55, 1.48 to 1.54, 1.48 to 1.53, 1.48 to 1.52, 1.49 to 1.55, 1.49 to 1.54, 1.49 to 1.53, 1.49 to 1.52, 1.50 to 1.55, 1.50 to 1.54, 1.50 to 1.53, 1.50 to 1.52, 1.51 to 1.55, 1.51 to 1.54, 1.51 to 1.53, It may be, but is not limited to, 1.51 to 1.52, 1.52 to 1.55, 1.52 to 1.54, 1.52 to 1.53, or 1.52.

[0079] In the present invention, the mounting solution has a refractive index in the above range, so that when making biological tissue transparent and performing three-dimensional imaging, transparency and fluorescence preservation are improved, and high-quality three-dimensional image results can be obtained, but are not limited thereto.

[0080] In the present invention, “living tissue” refers to a tissue existing in the body of an animal, and refers to a tissue formed by blood vessels and other cells, etc., and the living tissue may be, but is not limited to, the brain, liver, lungs, spleen, kidney, intestine, heart, pancreas, muscle, blood vessels, or entire tissue.

[0081] In the present invention, “mounting” means fixing a biological tissue sample, minimizing photobleaching, and homogenizing the refractive index during microscopic observation.

[0082] In the present invention, the kit comprises a fixing solution containing sucrose as an active ingredient; and

[0083] The washing solution may further include, but is not limited to, one or more active ingredients selected from the group consisting of phosphate buffer saline (PBS) and sodium azide.

[0084] 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) 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 is not limited thereto.

[0085] In the present invention, the fixing solution 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 more strongly fixing the sample covalently bonded between organic substances with PFA (paraformaldehyde).

[0086] In the present invention, the concentration of sodium azide included in the washing solution is 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 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), but is not limited thereto.

[0087] The washing solution according to the present invention contains sodium azide, so that the moisture content of a dehydrated biological tissue sample after transparency can be increased by up to 30%, and then 15% can be dehydrated to wash away organic substances that interfere with imaging attached to the tissue.

[0088] In addition, the present invention provides a rapid tissue clearing solution that accelerates the clearing of living tissue, comprising 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), urea, and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients.

[0089] In addition, the present invention provides a mounting solution comprising 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), sodium chloride (NaCl), and iohexol as active ingredients.

[0090] In addition, the present invention provides a fixed solution comprising sucrose as an active ingredient;

[0091] A tissue clearing accelerating solution containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), urea, and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients;

[0092] A washing solution containing phosphate buffer saline (PBS) and sodium azide; and

[0093] A kit for clearing biological tissue is provided, comprising a mounting solution containing 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), sodium chloride (NaCl), and iohexol as active ingredients.

[0094] In the present invention, “transparenting or clearing” means a technique for making an organ or tissue transparent to a degree that allows optical observation of the inside of the tissue while minimizing incision or structural damage to the organ or tissue to be observed.

[0095] In the present invention, the “kit” refers to a tool that enables transparentization of biological tissue and three-dimensional imaging by including the fixation solution, tissue clearing acceleration solution, washing solution, and mounting solution. In addition to the above solutions, the kit of the present invention may include other components, solutions, compositions, devices, etc. that are typically required for transparentization of biological tissue and three-dimensional imaging. In this case, the solutions may be applied one or more times without limitation, and there is no limitation on the order in which each solution is applied, and the application of each solution may be performed simultaneously or with a time difference.

[0096] In the present invention, the kit may include a container; instructions; and the fixative solution, tissue clearing acceleration solution, washing solution, and mounting solution. The container may serve to package the formulation, and may also serve to store and fix it. 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., and these may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may be initially equipped with a completely or partially detachable stopper, which may be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper for allowing access to the contents by means of a syringe needle. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0097]

[0098] In addition, the present invention relates to an electrophoresis device (100) for making biological tissue transparent,

[0099] An electrophoresis chamber (110) capable of containing a sample;

[0100] A main body (130) equipped with the above electrophoresis chamber;

[0101] A cover (120) that is mounted on the upper part of the main body on which the electrophoresis chamber is mounted and can be opened and closed;

[0102] Dial for time adjustment (140);

[0103] A display (150) showing time or temperature; and

[0104] An electrophoresis device for biotissue transparency is provided, characterized in that it includes a switch (160) for changing power or mode.

[0105] In the present invention, the electrophoresis chamber (110) may include a sample injection portion (111) into which a sample can be inserted, and may include an electrode portion (112) made of a banana cord and a platinum wire to conduct electricity, but is not limited thereto. The capacity of the electrophoresis chamber may be a maximum of 30 ml for mice, and a maximum of 50 ml for rats, but is not limited thereto. For example, for mice, electrophoresis can be performed by adding a solution of 10 ml to 30 ml, 10 ml to 25 ml, 10 ml to 20 ml, 10 ml to 15 ml, 15 ml to 30 ml, 15 ml to 25 ml, 15 ml to 20 ml, 20 ml to 30 ml, 20 ml to 25 ml, 10 ml, 15 ml, 20 ml, 25 ml, or 30 ml, and for rats, 10 ml to 50 ml, 10 ml to 45 ml, 10 ml to 40 ml, 10 ml to 35 ml, 10 ml to 30 ml, 10 ml to 25 ml, 10 ml to 20 ml, 10 ml to 15 ml, 20 ml to 50 ml, 20 ml to 45 ml, 20 ml to Electrophoresis can be performed by adding a solution of 40 ml, 20 ml to 35 ml, 20 ml to 30 ml, 20 ml to 25 ml, 30 ml to 50 ml, 30 ml to 45 ml, 30 ml to 40 ml, 30 ml to 35 ml, 40 ml to 50 ml, 40 ml to 45 ml, 10 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 40 ml, 45 ml, or 50 ml, but is not limited thereto.

[0106] In the present invention, the electrophoresis chamber (110) may be made of acrylic, but is not limited thereto.

[0107] In the present invention, the cover (120) may be formed in a form in which one side is fixed to the body so as to cover the upper part of the body in which the electrophoresis chamber is mounted and can be opened and closed, and the entire surface may be made of a transparent material so that the inside can be observed from the outside. The material of the cover may be, for example, plastic or glass, but is not limited thereto.

[0108] In the present invention, the dial (140) can be turned left and right to adjust the time, and the time can be adjusted from 1 minute to 99 hours and 59 minutes, but is not limited thereto.

[0109] In the present invention, the display (150) can display the electrophoresis time or the temperature inside the electrophoresis chamber, and may be formed of an LCD with a back light function, but is not limited thereto.

[0110] In the present invention, the switch (160) is for changing power or mode, and may be in the form of a touch button, but is not limited thereto.

[0111] In the present invention, the electrophoresis device can minimize tissue damage while making the tissue transparent by setting the current value not to exceed a specific value when making the tissue transparent. In this case, the specific current value may be, for example, 100 mA, but is not limited thereto.

[0112] In addition, the present invention comprises an electrophoresis device (100) for making the biological tissue transparent; and

[0113] An electrophoresis system for making biological tissue transparent is provided, including a cassette (200) mounted in an electrophoresis chamber.

[0114] In the present invention, the cassette (200) can be mounted in an electrophoresis chamber for transparency after placing a fixed biological tissue therein, and may include 1 to 50, 1 to 45, 10 to 50, 10 to 45, 20 to 50, 20 to 45, 30 to 50, 30 to 45, or 36 holes of a certain size on each of the upper and lower surfaces so that the tissue transparency acceleration solution of the present invention can penetrate well into the interior of the cassette, but is not limited thereto. At this time, the inner diameter of the hole may be, but is not limited to, 3 mm to 15 mm, 3 mm to 10 mm, 3 mm to 5 mm, 5 mm to 15 mm, 5 mm to 10 mm, 10 mm to 15 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm.

[0115] The size of the above cassette (200) can be appropriately manufactured according to the size of the biological tissue, and as an embodiment of the present invention, it can be manufactured to the length described in the drawing of Fig. 6, but is not limited thereto. In this case, the unit of the length described in the drawing of Fig. 6 may be mm.

[0116] In addition, the present invention provides a method for making a living tissue transparent using the kit for making a living tissue transparent, comprising the following steps:

[0117] (a) a step of fixing a biological tissue sample separated from a subject with a fixing solution;

[0118] (b) a clearing step of clearing the fixed biological tissue sample with a tissue clearing acceleration solution;

[0119] (c) a washing step of washing away organic substances attached to the above-mentioned transparent biological tissue sample with a washing solution; and

[0120] (d) A step of fixing the washed sample with a mounting solution.

[0121] In addition, the present invention provides a method for making a living tissue transparent using the kit for making a living tissue transparent, comprising the following steps:

[0122] (a) a step of fixing a biological tissue sample separated from a subject with a fixing solution;

[0123] (b) a clearing step of clearing the fixed biological tissue sample with a tissue clearing acceleration solution; and

[0124] (c) A step of fixing the washed sample with a mounting solution.

[0125] In the present invention, the step (b) may be a step of making a biological tissue sample transparent using the electrophoresis device, for example, a voltage of 100 to 800 V, 100 to 700 V, 100 to 600 V, 100 to 500 V, 100 to 400 V, 100 to 300 V, 100 to 200 V, 200 to 800 V, 200 to 600 V, 200 to 400 V, 200 to 300 V, 300 to 800 V, 300 to 600 V, 300 to 400 V, 400 to 800 V, 400 to 600 V, 400 to 500 V, 500 to 800 V, 500 to 600 V, 600 to Electrophoresis can be performed at 800 V, or 700 to 800 V, but is not limited thereto.

[0126] In the present invention, the step (b) may be performed for 4 to 8 hours, 4 to 7 hours, 4 to 6 hours, 5 to 8 hours, 5 to 7 hours, 5 to 6 hours, 6 to 8 hours, 6 to 7 hours, 4 hours, 5 hours, or 6 hours, but is not limited thereto.

[0127] In the present invention, the biological tissue that has gone through the step (b) can be made transparent in just 4 to 8 hours.

[0128] In the present invention, the method for making biological tissue transparent can be used for observation of biological tissue through three-dimensional fluorescence imaging, and may further include a step of administering a fluorescent substance to a subject for three-dimensional fluorescence imaging or treating the biological tissue with an antibody labeled with a fluorescent substance before, during, or after making the biological tissue transparent, but is not limited thereto. At this time, the fluorescent substance may be a fluorescent substance known in the art, and there is no limitation on its type. In the present invention, the biological tissue that has gone through the step (d) may have increased fluorescence retention during three-dimensional imaging, but is not limited thereto.

[0129] In the present invention, in step (a) or step (d), fixation may be performed for 12 to 60 hours, 12 to 50 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 18 to 60 hours, 18 to 50 hours, 18 to 48 hours, 18 to 36 hours, 18 to 24 hours, 24 to 60 hours, 24 to 50 hours, 24 to 48 hours, 24 to 36 hours, 24 hours, or 48 hours, but is not limited thereto.

[0130] 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 cows, but is not limited thereto.

[0131] In addition, the present invention provides a rapid tissue clearing solution comprising at least one selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and

[0132] Provided is a kit for use in clearing biological tissue, comprising a mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as active ingredients.

[0133] In addition, the present invention provides a rapid tissue clearing solution comprising at least one selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and

[0134] Provided is a use for the preparation of a mounting solution for making a living tissue transparent, the mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as active ingredients.

[0135] When the term "comprising" is used in the present invention, it means that other components may be included rather than excluding other components unless specifically stated otherwise. As used throughout the specification of the present invention, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0136]

[0137] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0138]

[0139] [Example]

[0140] Example 1. Preparation of a kit for making biological tissue transparent, including an accelerating solution capable of rapidly making biological tissue transparent by electrophoresis and a mounting solution with a high refractive index.

[0141] A kit for clearing biological tissues was prepared, including a fixing solution for clearing biological tissues and for three-dimensional imaging, a rapid tissue clearing solution for rapidly clearing biological tissues by electrophoresis, a washing solution, and a mounting solution with a high refractive index. The components of each solution are shown in Table 1.

[0142] Specifically, a fixing solution containing 30% (w / v) sucrose was prepared to dehydrate a biological tissue sample and more strongly fix the covalently bonded organic material sample with PFA (paraformaldehyde).

[0143] In addition, for the clarity of biological samples, a rapid tissue clearing solution, which is an accelerated solution that can quickly clarify biological tissues, was prepared by adding 0.3 to 2% (w / v) N,N,N',N'-Tetrakis(2-hydroxyethyl)ethylenediamine(2,2′,2″,2″′-(Ethylenedinitrilo)tetraethanol, THEED) to 25% (w / v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 25% (w / v) urea. In the subsequent experiments, 0.3% (w / v) THEED was used for mouse brain, and 2% (w / v) THEED was used for other organ tissues.

[0144] Next, after clearing, the moisture content of the dehydrated tissue sample was increased to a maximum of 30%, and then 15% was dehydrated. To wash away organic substances that interfere with imaging attached to the tissue sample, a washing solution was prepared by adding 0.1% (w / v) sodium azide to phosphate buffer saline (PBS).

[0145] Additionally, a mounting solution was prepared by adding 0.1 to 1% (w / v) sodium chloride (NaCl) to 14.56% (w / v) TDE (2,2'-Thiodiethanol), 14.56% (w / v) demethylsolfoxide (DMSO), and 51.46% (w / v) Iohexol, and the refractive index was adjusted to 1.52. In subsequent experiments, the NaCl concentration was used as 0.3% (w / v).

[0146] Components1Fixing solutionSucrose (30 %(w / v))2Rapid tissue clearing solutionCHAPS(25%(w / v)), Urea (25%(w / v)), THEED (0.3-2%(w / v))3Washing solutionPBS, sodium azide(0.1%(w / v))4Mounting solutionTDE (14.56%(w / v)), DMSO (14.56%(w / v)), Iohexol (51.46%(w / v)), NaCl (0.1-1%(wv))

[0147]

[0148] Example 2. Biotissue transparency using a biotissue transparency accelerator solution and an electrophoresis system.

[0149] Mouse brain tissue and various organ tissues were made transparent using the kit for making biotissue transparent and the electrophoresis system manufactured in Example 1 above.

[0150] Specifically, various organ tissues, including the brain, spleen, heart, lung, and kidney of 8-week-old C57BL / 6 mice, were placed in a fixative solution containing sucrose and reacted in a refrigerator at 4°C for 1-2 days. Then, the fixed samples were transferred to cassettes appropriate for the size of the tissue and mounted in an electrophoresis chamber. After adding a tissue clearing acceleration solution, the tissues were cleared using an electrophoresis apparatus at 100-200 V for 6 hours.

[0151] The tissue samples that had been cleared were placed in a mounting solution and cultured with shaking at 35°C / 50 rpm for 24 hours.

[0152] As a result, in the case of the conventional passive method of transparency without using the tissue transparency acceleration solution according to the present invention, the brain tissue hardly becomes transparent after 6 hours and takes approximately 7 days to become transparent, whereas, as shown in Fig. 1, when the tissue transparency acceleration solution according to the present invention is used for transparency through an electrophoresis device, it was confirmed that the brain tissue becomes transparent in about 6 hours in the transparency step compared to before transparency (see the left drawing of Fig. 1) (see the right drawing of Fig. 1).

[0153] In addition, as a result of confirming the transparency of the spleen, heart, lung, and kidney tissues of the mouse, as shown in Fig. 2, it was confirmed that the various biological tissues could be made transparent in about 6 hours in the transparency step when made transparent through an electrophoresis device using the tissue transparency acceleration solution according to the present invention.

[0154]

[0155] Example 3. Confirmation of tissue transparency according to the difference in refractive index of the mounting solution.

[0156] The kit for making biological tissue transparent manufactured in Example 1 above was manufactured with Iohexol as the main component of the mounting solution and was manufactured to have a refractive index of 1.52. In order to confirm tissue transparency according to the difference in the refractive index of the mounting solution, the components of the mounting solution were changed in the kit for making biological tissue transparent including the components of Table 1 above, and a mounting solution having a refractive index of 1.46 was manufactured, and then compared with the case of using the mounting solution of the present invention having a refractive index of 1.52.

[0157] The components of the mounting solution with a refractive index of 1.46 are as follows: N-lauroylsarcosine (0.1% (w / v)), CHAPS (40% (w / v)), Urea (40% (w / v)), NaCl (0.1-1% (w / v)), and Sorbitol (10% (w / v).

[0158]

[0159] Specifically, the brain tissue of the purchased Thy-1 YFP transgenic mouse was placed in a fixative solution and reacted in a refrigerator at 4°C until it settled, and then transparentized using an electrophoresis device in the same manner as in Example 2.

[0160] Next, 50 ml of washing solution was added and the tissue was washed by shaking and incubating at 4°C / 50 rpm for 1 hour and repeating this once more.

[0161] After clearing and washing, the tissue samples were placed in a mounting solution and cultured with shaking at 35°C / 50 rpm for 24 hours.

[0162] The brain tissues made transparent by the above method were imaged using a confocal microscope and the fluorescence amount was measured. Three-dimensional images were imaged using arivis vison 4D software (arivis-AG, Rostock, Germany) and Imaris software (Bitplane, USA).

[0163] As a result of comparing the cases of using a mounting solution with a refractive index of 1.46 and a mounting solution with a refractive index of 1.52 using the above method to confirm the transparency and fluorescence of the tissue, as shown in Fig. 3, it was found that when a mounting solution with a refractive index of 1.52 was used, the transparency and fluorescence preservation ability were improved, and a high-quality 3D image result could be obtained.

[0164]

[0165] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0166]

[0167] [Explanation of symbols]

[0168] 100: Electrophoresis device for making biological tissue transparent

[0169] 110: Electrophoresis chamber

[0170] 111: Sample injection unit

[0171] 112: Electrode section

[0172] 120: Cover

[0173] 130: Body

[0174] 140: Dial

[0175] 150: Display

[0176] 160: Switch

[0177] 200: Cassette

[0178]

[0179] The kit and electrophoresis device for making biological tissue transparent according to the present invention are expected to be useful for efficiently making various biological tissues transparent for three-dimensional fluorescent imaging of biological tissues, and thus have industrial applicability.

Claims

1. In a kit for making biological tissue transparent, The above kit comprises a rapid tissue clearing solution comprising at least one selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and A kit comprising a mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as an active ingredient.

2. In paragraph 1, A kit, characterized in that in the above tissue transparency acceleration solution, CHAPS has a concentration of 10% (w / v) to 40% (w / v), urea has a concentration of 10% (w / v) to 40% (w / v), and THEED has a concentration of 0.1% (w / v) to 3% (w / v).

3. In paragraph 1, A kit, characterized in that in the mounting solution, TDE has a concentration of 10% (w / v) to 18% (w / v), DMSO has a concentration of 10% (w / v) to 18% (w / v), iohexol has a concentration of 45% (w / v) to 55% (w / v), and sodium chloride has a concentration of 0.1% (w / v) to 1% (w / v).

4. In paragraph 1, A kit, characterized in that the biological tissue is brain, liver, lung, kidney, intestine, spleen, heart, pancreas, muscle, blood vessel, or whole tissue.

5. In paragraph 1, The above kit comprises a fixing solution containing sucrose as an active ingredient; and A kit characterized by further comprising a washing solution comprising at least one active ingredient selected from the group consisting of phosphate buffer saline (PBS) and sodium azide.

6. In paragraph 5, A kit, characterized in that the sucrose has a concentration of 20% (w / v) to 100% (w / v).

7. In paragraph 5, A kit, characterized in that the sodium azide has a concentration of 0.01% (w / v) to 1% (w / v).

8. In paragraph 1, A kit, wherein the mounting solution has a refractive index of 1.47 to 1.

55.

9. In an electrophoresis device for making biological tissue transparent, An electrophoresis chamber capable of containing a sample; A main body equipped with the above electrophoresis chamber; A cover that is mounted on the upper part of the main body in which the electrophoresis chamber is mounted and can be opened and closed; Dial for time control; A display showing time or temperature; and An electrophoresis device for making biological tissue transparent, characterized by including a switch for changing power or mode.

10. In paragraph 9, An electrophoresis device for making biological tissue transparent, characterized in that the electrophoresis chamber includes a sample injection portion capable of inserting a sample.

11. Electrophoresis device for making biological tissue transparent according to clause 9 or 10; and An electrophoresis system for making biological tissue transparent, comprising a cassette mounted in an electrophoresis chamber.

12. A method for making a living tissue transparent using a kit for making a living tissue transparent according to any one of claims 1 to 8, comprising the following steps: (a) a step of fixing a biological tissue sample separated from a subject with a fixing solution; (b) a clearing step of clearing the fixed biological tissue sample with a tissue clearing acceleration solution; (c) a washing step for washing away organic substances attached to the above-mentioned transparent biological tissue sample with a washing solution; and (d) A step of fixing the washed sample with a mounting solution.

13. In paragraph 12, A method for making a living tissue transparent, characterized in that the step (b) above is a step of making a living tissue sample transparent using the electrophoresis device of claim 9.

14. In paragraph 12, A method for making a living tissue transparent, characterized in that the step (b) is performed for 4 to 8 hours.

15. In paragraph 12, A method for making biological tissue transparent, wherein the mounting solution has a refractive index of 1.47 to 1.

55. A rapid tissue clearing solution comprising at least one selected from the group consisting of 16.3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and A kit for use in clearing biological tissue, comprising a mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as active ingredients. A rapid tissue clearing solution comprising at least one selected from the group consisting of 17.3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and urea and N,N,N',N'-tetrakis(2-hydroxyethyl)ethylenediamine (THEED) as active ingredients; and Use for the manufacture of a preparation for transparentizing living tissue, comprising a mounting solution comprising at least one selected from the group consisting of 2,2'-thiodiethanol (TDE), dimethyl sulfoxide (DMSO), and sodium chloride (NaCl) and iohexol as active ingredients.

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