KR102673166B1 - 3D imaging method in tissue of cell therapy products using tissue clearing - Google Patents

Abstract

The present invention relates to a three-dimensional imaging method within a tissue of a cell therapy product using tissue transparency. More specifically, the present invention relates to a three-dimensional imaging method of the distribution of a cell therapy agent, such as stem cells, within a tissue using a tissue transparency composition. It relates to a method of monitoring movement and distribution to target tissue. The tissue-clearing composition according to the present invention can greatly improve tissue transparency, and performs pharmacokinetic analysis in target tissue after administration of a cell therapy agent. It can be easily used.

Description

{3D imaging method in tissue of cell therapy products using tissue clearing}

The present invention relates to a three-dimensional imaging method within a tissue of a cell therapy product using tissue transparency. More specifically, the present invention relates to a three-dimensional imaging method of the distribution of a cell therapy agent, such as stem cells, within a tissue using a tissue transparency composition. It concerns a method of monitoring movement and distribution to target tissue.

The in vivo movement, distribution, and residual properties after administration of stem cells provide important information in ensuring the effectiveness and safety of therapeutic agents, but it is difficult to predict and measure pharmacokinetic aspects such as absorption, distribution, metabolism, and excretion in the body. Although complex imaging techniques are used using markers using fluorescence, it is very difficult to obtain high-magnification, three-dimensional images.

Recently, tissue transparency technology has been developed as a method for observing gene expression in the entire biological tissue, and technologies that can make tissue transparent in a variety of ways have been developed. 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.

The recently developed CLARITY method is a method of inserting hydrogel into the tissue to create a network support that holds important diagnostic materials such as DNA or proteins, and then performing electrophoresis to selectively remove lipids. Use it. 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 of taking a long time to make it transparent because as the hydrogel concentration increases, the degree of binding to the protein increases, making the tissue harder and removing lipids more 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, which causes economic and time losses, and staining using antibodies is unstable.

Accordingly, the present inventors have developed a tissue transparency composition and a tissue transparency method that can make the entire tissue transparent using a transparent solution that can increase the transparency of biological tissue without damaging various tissues without requiring an expensive electrophoresis device. wanted to invent it.

The present inventors have made extensive research efforts to develop a tissue clearing solution and a tissue clearing method using the same that can increase the transparency of biological tissues without requiring an expensive electrophoresis device and without damaging various tissues. As a result, N-laurylsarcosine, CHAPS [3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate], urea, Tris base; 2-Amino-2- The present invention was completed after confirming that the transparency of tissue can be significantly improved by using a composition containing (hydroxymethyl)-1,3-propanediol) and NaCl.

Accordingly, the purpose of the present invention is to provide a tissue transparency composition.

Another object of the present invention is to provide a kit for tissue transparency.

Another object of the present invention is to provide a method for producing a transparent tissue.

Another object of the present invention relates to the use of a tissue clearing composition.

The present invention relates to a three-dimensional imaging method within the tissue of a cell therapy product using tissue transparency. The tissue transparency composition according to the present invention can significantly improve tissue transparency compared to existing compositions.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is N-laurylsarcosine, CHAPS [3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate], urea, Tris base (Tris base; 2-Amino It is a tissue clearing composition containing -2-(hydroxymethyl)-1,3-propanediol) and NaCl.

The term “transparency” in this specification refers to a technology that makes 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.

The term "tissue" used herein may be, but is not limited to, the brain, spinal cord, heart, muscle, liver, lung, kidney, large intestine, or small intestine, and the tissue clearing method according to the present invention can be used for various tissues. It can be applied.

In one embodiment of the invention, the composition has 0.01-10, 0.1-9, 0.1-8, 0.1-7, 0.1-6, 0.1-5, 0.1-4, 0.1-3, 0.1-2, 0.1-1, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-10, 2-9, 2-8, 2- 7, 2-6, 2-5, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 4-10, 4-9, 4-8, 4-7, It may contain 4-6, 4-5, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v) of N-laurylsarcosine.

In one embodiment of the invention, the composition has 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 16-24, 16-23, 16-22, 16-21, 17-24, 17-23, 17-22, 17-21, 18-24, 18-23, 18-22, 18- 21, 19-20, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25% (w/v) of CHAPS.

In one embodiment of the invention, the composition has 30-70, 30-65, 30-60, 30-55, 35-70, 35-65, 35-60, 35-55, 40-70, 40-65, 40-60, 40-55, 45-70, 45-65, 45-60, 45-55, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55% (w/v ) may contain urea.

In one embodiment of the invention, the composition comprises 0.01-10% (w/v) N-laurylsarcosine, 10-40% (w/v) CHAPS, and 30-70% (w/v) It may contain urea.

In one embodiment of the invention, the composition has 0.001-1, 0.01-1, 0.1-1, 0.001-0.9, 0.001-0.8, 0.001-0.7, 0.001-0.6, 0.001-0.5, 0.001-0.4, 0.001-0.3, 0.001-0.2, 0.001-0.1, 0.01-0.9, 0.01-0.8, 0.01-0.7, 0.01-0.6, 0.01-0.5, 0.01-0.4, 0.01-0.3, 0.01-0.2, 0.01-0.1, 0.01, , 0.03, It may contain 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 (w/v) sodium chloride (NaCl).

In the present invention, NaCl (sodium chloride) can minimize denaturation of fluorescent substances in the sample by stabilizing tissue deformation and ionic strength due to osmotic pressure.

In one embodiment of the invention, the composition has 0.1-10, 0.1-9, 0.1-8, 0.1-7, 0.1-6, 0.2-9, 0.2-8, 0.2-7, 0.2-6, 0.3-9, 0.3-8, 0.3-7, 0.3-6, 0.4-9, 0.4-8, 0.4-7, 0.4-6, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, It may contain 3, 4, 5, 6, 7, 8, 9 or 10% (w/v) of Tris base.

according to the invention As the tissue transparency composition contains Tris base, pH can be adjusted and the efficiency and speed of tissue transparency can be improved. In addition, the present inventors confirmed that by adding Tris base to the tissue transparency composition, tissue transparency can be significantly improved compared to existing tissue transparency compositions (see Figures 1 and 2).

In one embodiment of the present invention, the pH of the Tris base may be 10 to 11.

Another aspect of the present invention is a kit for tissue clearing comprising:

fixative solution;

N-lauroylsarcosine, CHAPS [3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate], urea, Tris base; 2-Amino-2-(hydroxymethyl) A first tissue clearing solution containing -1,3-propanediol) and NaCl;

a secondary tissue clearing solution comprising N-laurylsarcosine, CHAPS, urea, and NaCl;

cleaning solution; and

Mounting solution.

In one embodiment of the invention, the fixation solution may include sucrose. Sucrose 20-100 %(w/v), 20-90 %(w/v), 20-80 %(w/v), 20-70 %(w/v), 20-60 %(w/v) ), 20-50 %(w/v), 20-40 %(w/v), 20-30 %(w/v), 30-100 %(w/v), 30-90 %(w/v) ), 30-80 %(w/v), 30-70 %(w/v), 30-60 %(w/v), 30-50 %(w/v), 30-40 %(w/v) ), 40-100 %(w/v), 40-90 %(w/v), 40-80 %(w/v), 40-70 %(w/v), 40-60 %(w/v) ), or may be included in the fixation solution at a concentration of 40-50% (w/v), but is not limited thereto.

As the fixation solution according to the present invention contains sucrose at a concentration of 20% (w/v) or more, it can dehydrate tissue samples and more strongly fix samples covalently bonded between organic substances with PFA (paraformaldehyde).

In one embodiment of the present invention, the cleaning solution may contain 0.001-0.5% (w/v) of sodium azide.

Sodium azide is added to the cleaning solution at concentrations of 0.01-0.4 % (w/v), 0.01-0.3 % (w/v), 0.01-0.2 % (w/v), 0.01-0.1 % (w/v). It may be included, or may be included at a concentration of 0.01-0.5% (w/v), 0.1-0.5% (w/v), but is not limited thereto.

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 one embodiment of the present invention, the washing solution may include phosphate buffered saline.

In one embodiment of the invention, the mounting solution may include N-laurylsarcosine, CHAPS, urea, and sodium chloride (NaCl).

In one embodiment of the invention, the mounting solution is 0.001-1, 0.01-1, 0.1-1, 0.001-0.9, 0.001-0.8, 0.001-0.7, 0.001-0.6, 0.001-0.5, 0.001-0.4, 0.001-0.3. , 0.001-0.2, 0.001-0.1, 0.01-0.9, 0.01-0.8, 0.01-0.7, 0.01-0.6, 0.01-0.5, 0.01-0.4, 0.01-0.3, 0.01-0.2, 0.01-0.1, 0.01, 0. 02, 0.03 , may include N-laurylsarcosine at a concentration of 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 (w/v).

In one embodiment of the invention, the mounting solution is 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 25-60, 25-55, 25-50, 25-45. , 25-40, 36-44, 36-43, 36-42, 36-41, 37-44, 37-43, 37-42, 37-41, 38-44, 38-43, 38-42, 38 It may include -41, 39-40, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45% (w/v) of CHAPS, but is not limited thereto.

In one embodiment of the invention, the mounting solution is 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 25-60, 25-55, 25-50, 25-45. , 25-40, 36-44, 36-43, 36-42, 36-41, 37-34, 37-43, 37-42, 37-41, 38-44, 38-43, 38-42, 38 -41, 39-40, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45% (w/v) of urea.

In one embodiment of the invention, the mounting solution is 0.001-1, 0.01-1, 0.1-1, 0.001-0.9, 0.001-0.8, 0.001-0.7, 0.001-0.6, 0.001-0.5, 0.001-0.4, 0.001-0.3. , 0.001-0.2, 0.001-0.1, 0.01-0.9, 0.01-0.8, 0.01-0.7, 0.01-0.6, 0.01-0.5, 0.01-0.4, 0.01-0.3, 0.01-0.2, 0.01-0.1, 0.01, 0. 02, 0.03 , may include sodium chloride (NaCl) at a concentration of 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 (w/v).

The mounting solution according to the present invention may contain 40% (w/v) and 40% (w/v) of CHAPS and urea, respectively, to adjust the refractive index of the solution to 1.45, and the concentration of sodium chloride (NaCl) may be 0.1. It may contain from 1% (w/v), but is not limited thereto.

Another aspect of the present invention is a method for producing a cleared tissue comprising the following steps:

A fixation step of fixing the collected tissue by immersing it in a fixation solution;

The fixed tissue was treated with N-lauroylsarcosine, CHAPS [3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate], urea, Tris base; 2-Amino-2 A first clearing step of immersing in a first tissue clearing solution containing -(hydroxymethyl)-1,3-propanediol) and NaCl;
A washing step of washing organic matter attached to the tissue with a washing solution;

A second clearing step of immersing the fixed tissue in a second tissue clearing solution containing N-laurylsarcosine, CHAPS, urea, and NaCl;

A washing step of washing organic matter attached to the tissue with a washing solution; and

Mounting step where the tissue is fixed with a mounting solution.

In one embodiment of the invention, the fixation phase is 20-100 % (w/v), 20-90 % (w/v), 20-80 % (w/v), 20-70 % (w/v) , 20-60 %(w/v), 20-50 %(w/v), 20-40 %(w/v), 20-30 %(w/v), 30-100 %(w/v) , 30-90 %(w/v), 30-80 %(w/v), 30-70 %(w/v), 30-60 %(w/v), 30-50 %(w/v) , 30-40 %(w/v), 40-100 %(w/v), 40-90 %(w/v), 40-80 %(w/v), 40-70 %(w/v) , may include fixing the tissue by immersing it in a solution containing sucrose at a concentration of 40-60% (w/v), or 40-50% (w/v).

In one embodiment of the invention, the first clearing step is to coat the fixed tissue with 0.01-10% (w/v) N-laurylsarcosine, 10-40% (w/v) CHAPS, 30-70% It may be immersed in a first tissue clearing solution containing (w/v) urea, 0.1-10% (w/v) Tris base, and 0.001-1% (w/v) NaCl.

delete

In one embodiment of the invention, the second clearing step is performed by adding 0.01-10% (w/v) of N-laurylsarcosine, 10-40% (w/v) of CHAPS and 30-70% (w/v) of ) of urea and 0.001-1% (w/v) of NaCl.

In one embodiment of the present invention, the washing step removes organic matter attached to the tissue with a washing solution containing 0.001-0.5% (w/v) sodium azide and phosphate buffered saline. It could be washing.

In one embodiment of the present invention, the mounting step may be fixing the tissue with a mounting solution containing N-laurylsarcosine, CHAPS, urea, and sodium chloride (NaCl).

In one embodiment of the invention, the mounting step includes 0.01-10% (w/v) of N-laurylsarcosine, 20-60% (w/v) of CHAPS, 10-60% (w/v) of The tissue may be fixed with a mounting solution containing urea.

In one embodiment of the invention, the mounting step includes 0.01-10% (w/v) of N-laurylsarcosine, 20-60% (w/v) of CHAPS, 10-60% (w/v) of The tissue may be fixed with a mounting solution containing urea and 0.001-3% (w/v) NaCl.

In one embodiment of the present invention, after the first clearing step and the washing step are performed, the second clearing step and the washing step may be sequentially repeated two or more times. Accordingly, after the fixing step is performed, the first clearing step and the washing step are performed, and the second clearing step and the washing step are repeated twice and then the mounting step may be performed.

In one embodiment of the invention, the fixation phase is 12 to 48 hours, 12 to 44 hours, 12 to 40 hours, 12 to 36 hours, 12 to 32 hours, 12 to 28 hours, 16 to 48 hours, 16 to 44 hours. , may be carried out for 16 to 40 hours, 16 to 36 hours, 16 to 32 hours, 16 to 28 hours or 24 to 48 hours.

In one embodiment of the invention, the first clearing step is performed for 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 36 to 96 hours, 36 to 84 hours, 36 to 84 hours. It may be carried out for 72 hours, 36 to 60 hours, 36 to 48 hours or 48 hours.

In one embodiment of the present invention, the second clearing step is performed for 24 to 96 hours, 24 to 84 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, 36 to 96 hours, 36 to 84 hours, 36 to 84 hours. It may be carried out for 72 hours, 36 to 60 hours, 36 to 48 hours or 48 hours.

In one embodiment of the invention, the mounting step lasts from 12 to 48 hours, from 12 to 44 hours, from 12 to 40 hours, from 12 to 36 hours, from 12 to 32 hours, from 12 to 28 hours, from 16 to 48 hours, from 16 to 44 hours. , may be carried out for 16 to 40 hours, 16 to 36 hours, 16 to 32 hours, 16 to 28 hours or 24 to 48 hours.

Another aspect of the present invention is a method for imaging the distribution of a cell therapy agent in a tissue after administration comprising the following steps:

The tissue of the subject administered the cell therapy agent was treated with a clearing solution containing N-laurylsarcosine, CHAPS, urea, Tris base (2-Amino-2-(hydroxymethyl)-1,3-propanediol), and NaCl. A transparency step of making transparent using;

Analysis step to analyze pharmacokinetic information of cell therapy products in cleared tissue.

In the present invention, the subject may be a human, non-human primate, mouse, dog, cat, rabbit, horse, or cow, but is not limited thereto.

In one embodiment of the present invention, the clearing step includes a fixing step of fixing the collected tissue by immersing it in a fixing solution; The fixed tissue was immersed in a second tissue clearing solution containing N-laurylsarcosine, CHAPS [3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate], urea, and NaCl. Additional transparency steps required; A washing step of washing organic matter attached to the tissue with a washing solution; And it may further include a mounting step of fixing the tissue with a mounting solution.

In the present invention, the cell therapy agent may include stem cells.

The term “stem cell” as used herein refers to an undifferentiated cell that has the ability to self-replicate and differentiate into two or more different types of cells.

In one embodiment of the invention, the stem cells may be autologous or allogeneic, may be derived from any type of animal, including humans and non-human mammals, may be stem cells derived from adults, or may be derived from embryos. It could be a stem cell. For example, stem cells may be embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPSC), and human umbilical cord derived mesenchymal stem cells (human Wharton's jelly derived MSC), but are not limited thereto. no.

In one embodiment of the present invention, the stem cells may be labeled, for example, may be fluorescently labeled. The labeling agent may be used without limitation as long as it is a fluorescent labeling agent known in the art, but may be, for example, DID [1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine].

In one embodiment of the present invention, the analysis step may further include an imaging step of constructing an image using a microscope.

In the present invention, the imaging step may use the ImarisTM program, but is not limited to this, and any program known in the art that can three-dimensionally detect and analyze fluorescent signals can be used without limitation. there is.

In one embodiment of the present invention, human umbilical cord-derived mesenchymal stem cells fluorescently labeled with DID were administered to a mouse model, a clearing solution was prepared to make the tissue of the mouse transparent, and the clearing solution was used to administer the stem cells to the mouse. Three-dimensional immunostaining images of the model were acquired (see Example 3).

The present invention relates to a three-dimensional imaging method within a tissue of a cell therapy product using tissue transparency. More specifically, the present invention relates to a three-dimensional imaging method of the distribution of a cell therapy agent, such as stem cells, within a tissue using a tissue transparency composition. It relates to a method of monitoring movement and distribution to target tissue. The tissue-clearing composition according to the present invention can greatly improve tissue transparency, and performs pharmacokinetic analysis in target tissue after administration of a cell therapy agent. It can be easily used.

Figure 1 is a photograph showing the results of tissue clearing of mouse kidney tissue using a conventional tissue clearing solution.
Figure 2 is a photograph showing the results of tissue clearing of mouse kidney tissue using a tissue clearing solution prepared according to an embodiment of the present invention.
Figure 3 shows human umbilical cord-derived mesenchymal stem cells administered to mice within the lung tissue of transgenic mice and wild type (WT) mice using a tissue clearing solution prepared according to an embodiment of the present invention. This photo shows the results of imaging the distribution of (human Wharton's jelly derived MSC).
Figure 4 shows human umbilical cord-derived mesenchymal stem cells administered to mice within the liver tissues of transgenic mice and wild type (WT) mice using a tissue clearing solution prepared according to an embodiment of the present invention. This photo shows the results of imaging the distribution of (human Wharton's jelly derived MSC).
Figure 5 shows human umbilical cord-derived mesenchymal stem cells administered to mice within the muscle tissues of transgenic mice and wild type (WT) mice using a tissue clearing solution prepared according to an embodiment of the present invention. This photo shows the results of imaging the distribution of (human Wharton's jelly derived MSC).
Figure 6 shows a human umbilical cord administered to a mouse in a 1 mm-thick lung slice of a transgenic mouse and a wild type (WT) mouse using a tissue clearing solution prepared according to an embodiment of the present invention. This is a photograph showing the results of imaging the distribution of derived mesenchymal stem cells (human Wharton's jelly derived MSC) (red) and the distribution of blood vessels (green).
Figure 7 shows human umbilical cord-derived mesenchymal stems administered to mice in the whole lung lobe of transgenic mice and wild type (WT) mice using a tissue clearing solution prepared according to an embodiment of the present invention. This is a photograph showing the results of imaging the distribution of cells (human Wharton's jelly derived MSC) (red) and the distribution of blood vessels (green).

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Comparative Example 1: Preparation of existing tissue clearing composition

A clearing composition was prepared by preparing a fixing solution, a tissue clearing solution, a washing solution, and a mounting solution necessary for tissue clearing, and the components of the composition are shown in Table 1 below.

serial number composition composition One Fixing solution Sucrose (30%(w/v)) 2 Tissue clearing solution N-lauroylsarcosine (0.1%(w/v)), CHAPS (20%(w/v)), Urea (50%(w/v)), NaCl (0.1%(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%(w/v))

Specifically, in order to dehydrate biological tissue samples and more strongly fix samples covalently bonded between organic substances with PFA (paraformaldehyde), a fixing solution containing sucrose at a concentration of 30% (w/v) was used. solution) was prepared. In addition, in order to minimize denaturation of fluorescent substances in biological tissue samples by stabilizing tissue deformation and ionic strength due to osmotic pressure, 20% (w/v) CHAPS (3-[ (3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 0.1 to 0.5% (w/v) sodium chloride (NaCl) added to 50% (w/v) urea. , a tissue clearing solution was prepared.

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

Additionally, the mounting solution ( mounting solution) was prepared and the refractive index was set to 1.45.

Example 1: Preparation of tissue clearing composition

A clearing composition was prepared by preparing a fixing solution, a first tissue clearing solution, a second tissue clearing solution, a washing solution, and a mounting solution necessary for tissue clearing. The components of the composition are shown in Table 2 below.

serial number composition composition One Fixing solution Sucrose (30%(w/v)) 2 Tissue clearing solution 1 N-lauroylsarcosine (0.1%(w/v)), CHAPS (20%(w/v)), Urea (50%(w/v)), NaCl (0.1%(w/v)), pH 10 Tris- base (5% (w/v)) 3 Tissue clearing solution 2 N-lauroylsarcosine (0.1%(w/v)), CHAPS (20%(w/v)), Urea (50%(w/v)), NaCl (0.1%(w/v)) 4 Washing solution PBS, sodium azide (0.1 %(w/v)) 5 Mounting solution N-lauroylsarcosine (0.1%(w/v))CHAPS(40%(w/v)), Urea(40%(w/v)), NaCl(0.1%(w/v))

Specifically, the fixation solution, second tissue clearing solution, washing solution, and mounting solution were prepared in the same manner as in Comparative Example 1.

And, to maximize the tissue transparency effect, 20% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 50% (w/v) urea ( urea) to a separate solution by adding Tris-base (2-Amino-2-(hydroxymethyl)-1,3-propanediol) at pH 10 to sodium chloride (NaCl) at a concentration of 0.1 to 0.5% (w/v). A first tissue clearing solution was prepared.

Example 2: Comparison of tissue transparency with existing clarifying compositions

2-1. Tissue transparency through existing transparent compositions

Mouse tissue transparency was performed using the clarifying composition prepared in Comparative Example 1 above. Kidney tissue from 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.

Afterwards, the sank tissue sample was placed in a tissue clearing solution and cultured with shaking at 35°C/50 rpm for 48 hours to clear the tissue. 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. Then, the tissue clearing and washing process was repeated three times.

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.

2-2. Tissue transparency through a transparent composition

Mouse tissue transparency was performed using the clarifying composition prepared in Example 1. Kidney tissue from 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.

Afterwards, the settled tissue sample was placed in the first tissue clearing solution and cultured with shaking at 35°C/50 rpm for 48 hours to clear the tissue. Then, 50 ml of washing solution was added and incubated at 4°C/50 rpm for 1 hour. The culture was shaken, and this was repeated once more to wash the tissue.

Then, as before, tissue transparency was performed by placing it in the second tissue clearing solution and culturing it with shaking at 35°C/50 rpm for 48 hours. 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. Then, the tissue clearing and washing process was repeated twice.

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.

2-3. Comparison of organizational transparency

The degree of transparency of the kidney tissue of the mouse whose tissue was made transparent in Example 2-1 was compared with the kidney tissue of the mouse whose tissue was made transparent in Example 2-2.

As a result of the experiment, as can be seen in Figures 1 and 2, when a separate clearing process was performed using the first tissue clearing solution to which Tris base was added, the tissue was transparent compared to the method of clearing the tissue using the existing method without the first tissue clearing solution. It was confirmed that transparency can be significantly improved.

Example 3: Acquisition of 3D images using tissue transparency after stem cell administration

Transgenic mice that do not express Dystrophin, C57BL/10ScSn- Dmdmdx/J (jackson laboratory stock #: 001801), 25-week-old male triplets and Wild Type (WT) mouse (C57BL/10J), 25-week-old male ( male) was used in the following experiment. To obtain brain tissue samples from mice, isoflurane was used as an anesthetic gas and mixed with 100% oxygen to inhale anesthesia.

Human umbilical cord-derived mesenchymal stem cells (human Wharton's jelly derived MSC) were fluorescently labeled using DiD lyphophilic reagent (ThermoFisher, V22887) and injected into the mouse tail vein (IV injection) at a concentration of 1Х10 ⁶ cells/200 ul per head. Brain tissue of mice was sampled 1 hour and 1 week after injection.

Specifically, in the negative control, stem cells were not administered to one transgenic mouse, and in the positive control, one transgenic mouse was sacrificed immediately after stem cell administration, and one week after transfection. In the group (Tg 1 week), one transgenic mouse was sacrificed 1 hour or 1 week after administration of stem cells, and in the wild type 1 week group (WT 1 week), one wild type mouse was sacrificed 1 week after administration of stem cells.

Before sacrifice, each mouse was perfused with PBS after staining blood vessels with lectin, and then perfused with 4% paraformaldehyde using a peristaltic pump. The tissue was fixed. Then, the liver, lung, and calf muscle (Gastronecmius) were sampled. Afterwards, the sampled tissue was made transparent as in Example 2-2.

After transparency, the results were observed at a wavelength of 505-545 nm for green (blood vessels) and 660 nm for red (stem cells) using a lightsheet microscope, using arivis vison 4D software (arivis-AG, Rostock, Germany) and Imaris. A 3D image was constructed by imaging with software (Bitplane, USA).

As a result of the experiment, as can be seen in FIGS. 3 to 7, when the clearing composition according to the present invention was used, the distribution of the injected stem cells in the cleared brain tissue and the resulting changes in the surrounding environment could be accurately observed.

Claims (14)

delete delete delete delete delete Method for preparing a transparent tissue comprising the following steps:
A fixation step of fixing the collected tissue by immersing it in a fixation solution;
The fixed tissue was treated with N-laurylsarcosine, CHAPS [3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate], urea, Tris base; 2-Amino-2 A first clearing step of immersing in a first tissue clearing solution containing -(hydroxymethyl)-1,3-propanediol) and NaCl;
A washing step of washing organic matter attached to the tissue with a washing solution;
A second clearing step of immersing the fixed tissue in a second tissue clearing solution containing N-laurylsarcosine, CHAPS, urea, and NaCl;
A washing step of washing organic matter attached to the tissue with a washing solution; and
A mounting step of fixing the tissue with a mounting solution. The method of claim 6, wherein the first tissue clearing solution contains 0.01-10% (w/v) of N-laurylsarcosine, 10-40% (w/v) of CHAPS, and 30-70% (w/v) of CHAPS. A method comprising the urea of v). The method of claim 6, wherein the first tissue clearing solution comprises 0.1-10% (w/v) of Tris base. 7. The method of claim 6, wherein the fixative solution comprises 20-100% (w/v) sucrose. The method of claim 6, wherein the washing solution contains 0.001-0.5% (w/v) sodium azide. The method of claim 6, wherein the washing solution contains phosphate buffered saline. The method of claim 6, wherein the mounting solution includes N-laurylsarcosine, CHAPS, urea, and sodium chloride (NaCl). 7. The method of claim 6, wherein the mounting solution comprises 0.01-10% (w/v) of N-laurylsarcosine, 20-60% (w/v) of CHAPS, 10-60% (w/v) of urea. A method comprising (urea). The method of claim 6, wherein the mounting solution contains 0.001-3% (w/v) NaCl.