COMPOSITION FOR ADJUSTING BIOLOGICAL TISSUE SIZE, AND METHOD FOR ADJUSTING SIZE OF BIOLOGICAL TISSUE USING SAID COMPOSITION

Abstract

The present invention provides a composition for adjusting biological tissue size and a method for adjusting the size of biological tissue using the said composition. The composition for adjusting the size of biological tissue according to the present invention can adjust the size of biological tissue according to the specifications of a microscope and the needs of a researcher, and can be used as a mounting solution to easily acquire an image of the biological tissue. Therefore, the composition can be usefully used to reveal the causes of and find treatment methods for various disorders.

Claims

1. A composition for adjusting biological tissue size comprising a compound represented by formula 1 below, an optical isomer thereof, a hydrate thereof, or a salt thereof and an alkali metal halide: ##STR00004## (In formula 1 above, R.sup.1 and R.sup.2 are independently C.sub.1-10 straight or branched alkyl; and p, q and r are independently an integer of 010).

2. The composition for adjusting biological tissue size according to claim 1, wherein R.sup.1 and R.sup.2 are independently C.sub.1-5 straight or branched alkyl; and p, q and r are independently an integer of 05.

3. The composition for adjusting biological tissue size according to claim 1, wherein R.sup.1 and R.sup.2 are independently methyl; and p, q and r are independently an integer of 1.

4. The composition for adjusting biological tissue size according to claim 1, wherein the compound represented by formula 1 is a compound represented by the following formula 2 or a hydrate thereof: ##STR00005##

5. The composition for adjusting biological tissue size according to claim 1, wherein the composition additionally includes one or more compounds selected from the group consisting of urea, CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), sucrose, fructose, glycerol, diatrizoic acid, Triton X-100, Tween-20, 2,2-thiodiethanol, iohexol and chloral hydrate.

6. The composition for adjusting biological tissue size according to claim 1, wherein the composition for adjusting biological tissue size additionally includes a simulated body fluid in which the concentration of the compound represented by formula 1, the optical isomer thereof, the hydrate thereof or the salt thereof is 3060 w/v % and the concentration of the alkali metal halide is 15 w/v %.

7. The composition for adjusting biological tissue size according to claim 1, wherein the biological tissue is brain, blood vessel, liver, lung, kidney, pancreas or intestine.

8. A method for adjusting the size of biological tissue which includes a step of adjusting the size of the tissue by contacting the biological tissue with the composition of claim 1.

9. The method for adjusting the size of biological tissue according to claim 8, wherein the method is performed at the temperature range of 10 C. to 50 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1 to 5 are photographs illustrating the biological tissues obtained from each step performed to prepare a vitrified biological tissue according to an example of the present invention.

[0017] FIG. 6(a) and FIG. 6(b) are photographs illustrating the biological tissues vitrified according to an example of the present invention whose sizes had been reduced by using a mounting solution. FIG. 6(c) is an image of the size-reduced biological tissue obtained through a microscope.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention provides a composition for adjusting biological tissue size comprising a compound represented by formula 1 below, an optical isomer thereof, a hydrate thereof, or a salt thereof and an alkali metal halide.

##STR00002##

[0019] In formula 1 above,

[0020] R.sup.1 and R.sup.2 are independently C.sub.1-10 straight or branched alkyl; and

[0021] p, q and r are independently an integer of 010.

[0022] Preferably,

[0023] R.sup.1 and R.sup.2 are independently C.sub.1-5 straight or branched alkyl; and

[0024] p, q and r are independently an integer of 05.

[0025] More preferably,

[0026] R.sup.1 and R.sup.2 are methyl; and

[0027] p, q and r are an integer of 1.

[0028] Most preferably, the compound represented by formula 1 is a compound represented by the following formula 2 or a hydrate thereof.

##STR00003##

[0029] Hereinafter, the composition for adjusting biological tissue size according to the present invention is described in detail.

[0030] The composition for adjusting biological tissue size according to the present invention can adjust the size of biological tissue by controlling the content and the concentration of each constituent.

[0031] In the present invention, the composition for adjusting biological tissue size is prepared for easy observation of a biological tissue under a microscope, in which the biological tissue can be a vitrified biological tissue.

[0032] The composition for clearing biological tissue used for obtaining a vitrified biological tissue can be a composition that is commonly used, and preferably the composition for clearing biological tissue can include the compound represented by formula 1. At this time, the concentration of the compound is 255 w/v % (weight/volume %) and preferably 2050 w/v %. The solution for the concentration can be a simulated body fluid which has been generally used in this field. More specifically, distilled water, PBS (phosphate buffer saline or TBS (tris buffer solution) can be used, but not always limited thereto.

[0033] If the concentration of the compound represented by formula 1 above is less than 2 w/v %, the rate of clearing the biological tissue would be slowed. On the other hand, if the concentration of the compound is more than 55 w/v %, the CHAPS represented by formula 1 above would not be dissolved completely.

[0034] Further, the composition for clearing biological tissue can additionally include a substance that can accelerate the progress of biological tissue clearing by controlling osmotic pressure. At this time, the substance that can accelerate the progress of biological tissue clearing is exemplified by urea, CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), sucrose, fructose, glycerol, diatrizoic acid, Triton X-100, Tween-20, 2,2-thiodiethanol, iohexol, chloral hydrate, or a combination thereof, but not always limited thereto.

[0035] At this time, the substance accelerating biological tissue clearing can be included at the concentration of 580 w/v %, preferably 575 w/v %, more preferably 1070 w/v %, 550 w/v %, and most preferably 3560 w/v. At this time, if the concentration of the substance is less than 5 w/v %, the rate of tissue clearing would be slowed. On the other hand, if the concentration of the substance is more than 80 w/v %, crystals would be formed or not dissolved in the solution. As an example, in the case of using urea as the substance accelerating biological tissue clearing, the concentration of urea can be 1070 w/v % and more preferably 2060 w/v %. In addition, the concentration of the substance accelerating biological tissue clearing can be appropriately adjusted to the preferable concentration range of the compound represented by formula 1 above.

[0036] Once the biological tissue is vitrified by the composition for clearing biological tissue, the size of the biological tissue can be adjusted by using the composition for adjusting biological tissue size of the present invention.

[0037] The composition for adjusting biological tissue size according to the present invention can include a compound represented by formula 1, an optical isomer thereof, a hydrate thereof, or a salt thereof and an alkali metal halide.

[0038] At this time, the said composition can additionally include urea, CHAPSO (3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), sucrose, fructose, glycerol, diatrizoic acid, Triton X-100, Tween-20, 2,2-thiodiethanol, iohexol, chloral hydrate, or a combination thereof, and more preferably include urea.

[0039] The composition for adjusting biological tissue size can additionally include a simulated body fluid. In the mixture of the composition for adjusting biological tissue size and the simulated body fluid, the concentration of the compound represented by formula 1 above, the optical isomer thereof, the hydrate thereof or the salt thereof can be 3060 w/v % and the concentration of the alkali metal halide can be 15 w/v %.

[0040] The alkali metal halide can comprise any form of alkali metal and halogen element combined, which is preferably sodium chloride.

[0041] As described hereinbefore, the composition for adjusting biological tissue size according to the present invention can adjust the size of the vitrified biological tissue without damaging the tissue; can be applied to various biological tissues such as brain, liver, lung, kidney, intestine, heart, muscle, and blood vessel without damaging them; can prevent tissues from swelling, bubble formation, discoloration and black deposit; and is not expensive, so that it can be effectively used as a composition for adjusting biological tissue size.

[0042] The present invention also provides a method for adjusting the size of biological tissue which includes a step of adjusting the size of the tissue by contacting the biological tissue with the said composition for adjusting biological tissue size.

[0043] Hereinafter, the method for adjusting biological tissue size according to the present invention is described in more detail.

[0044] The biological tissue according to the present invention can be a vitrified tissue, and the method for adjusting the size of the vitrified tissue includes a step of adjusting the size of the tissue by contacting the vitrified tissue with the composition above.

[0045] Particularly, the method for adjusting the size of the vitrified tissue according to the present invention is accomplished by contacting the vitrified tissue with the composition comprising the compound represented by formula 1 to adjust the size by inducing changes in physiochemical characteristics of the biological tissue, by which images thereof can be easily obtained through a microscope.

[0046] In this invention, fixation of the biological tissue is necessary in order to prepare the vitrified biological tissue. The method for fixing biological tissue can be used without any particular limitation.

[0047] More particularly, the method for fixing biological tissue can be performed by the conventional method using paraformaldehyde, ethylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, glycerol polyglycidyl ether, glutaraldehyde, polyacrylamide or a combination thereof, but not always limited thereto.

[0048] In a preferred embodiment of the present invention, the tissue size can be adjusted by treating the tissue with the composition for adjusting biological tissue size which is the mixture of CHAPS, urea and an alkali metal halide.

[0049] A solution for regulating the concentration above can be a simulated body fluid generally used in this field, or can be distilled water, PBS (phosphate buffer saline) or TBS (tris buffer solution), but not always limited thereto. Soaking can be performed at the temperature range of 10 C. to 50 C., preferably at the temperature range of 12 C. to 48 C., at the temperature range of 14 C. to 46 C., at the temperature range of 16 C. to 44 C., at the temperature range of 18 C. to 42 C., at the temperature range of 20 C. to 40 C., at the temperature range of 24 C. to 39 C. , at the temperature range of 28 C. to 38 C. , at the temperature range of 30 C. to 37 C., and at the temperature range of 33 C. to 34 C.

[0050] In the case that the composition for adjusting biological tissue size includes a simulated body fluid additionally, the concentration of CHAPS in the mixed solution comprising the composition for adjusting biological tissue size and the simulated body fluid can be 30 w/v %60 w/v % and the concentration of the alkali metal halide can be 1 w/v %5 w/v %.

[0051] At this time, the alkali metal halide can comprise any form of alkali metal and halogen element combined, which is preferably sodium chloride.

[0052] The method for adjusting biological tissue size according to the present invention can be applied to various vertebrate tissues, particularly to brain, blood vessel, liver, lung, kidney, pancreas, intestine, heart, etc, and the composition can adjust the size of the entire tissue at once.

[0053] The method of the present invention facilitates the regulation of the size of the vitrified tissue without damaging the tissue and thereby imaging the three dimensional distribution of cells and molecules for better observation. Therefore, the method of the present invention is advantageous for the observation and study of various biological tissues having a complicated structure as a whole structure in the size of hundreds of micrometers and also for obtaining useful information from the tissue to be useful for finding causes of various diseases including brain disease.

[0054] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

[0055] However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

EXPERIMENTAL EXAMPLE 1: CHANGES OF VITRIFIED BIOLOGICAL TISSUE SIZE

[0056] The following experiment was performed to investigate whether the composition according to the present invention was able to change the size of the vitrified tissue. All the animal test processes in this specification were performed in accordance with the guideline (Approval No. RS17003) of the Institutional Animal Care and Use Committee, Korea Institute of Toxicology.

1. Preparation of Vitrified Biological Tissue (Step 1)

[0057] First, adult mice (at 8 weeks) were anesthetized with isoflurane (1 cc/min), an inhalation anesthetic, followed by perfusion with 50 mL of cold 1 PBS and cold 4% PFA (paraformaldehyde) stepwise. Then, organs were extracted and immersed in 4% PFA solution, followed by incubation at 4 C. for 24 hours. At this time, the temperature above was not limited to a specific range but preferably 010 C.

[0058] Next, the samples were incubated in the presence of 30% sucrose at 010 C. for 24 hours. The overall brain size was 1 cm1.3 cm (widthlength) except the part of olfactory bulb. The results are shown in FIGS. 1(a) and 1(b).

[0059] To clarify the tissue sample, shaking incubation was performed with a mixed solution comprising 25 w/v % of CHAPS, 50 w/v % of urea and 50 mM sodium azide at 35 C. at 150 rpm for 24 hours. The incubation time can be extended or shortened if necessary. The overall brain size was 1.2 cm1.3 cm (widthlength) except the part of olfactory bulb. The results are shown in FIGS. 2(a) and 2(b).

[0060] Then, incubation was performed three times with 50 mL of sterile distilled water containing PBS (phosphate buffer saline, 0.01%) at 010 C. at 150 rpm for 24 hours. The overall brain size was 1.3 cm2 cm (widthlength) except the part of olfactory bulb. The results are shown in FIGS. 3(a) and 3(b).

[0061] Incubation was performed again with a mixed solution comprising 20 w/v % of CHAPS, 50 w/v % of urea and 50 mM sodium azide at 35 C. at 150 rpm for 24 hours. The incubation time can be extended or shortened if necessary. The overall brain size was 1.2 cm1.3 cm (widthlength) except the part of olfactory bulb. The results are shown in FIGS. 4(a) and 4(b).

[0062] Then, incubation was also performed with a mixed solution comprising 40 w/v % of CHAPS and 40 w/v % of urea at 37 C. at 220 rpm for 24 hours. The incubation time can be extended or shortened if necessary. The overall brain size was 1.2 cm1.3 cm (widthlength) except the part of olfactory bulb. The results are shown in FIGS. 5(a) and 5(b).

[0063] The sample was vitrified through the above procedure with raising the concentration of CHAPS slowly.

2. Reduction of the Size of the Sample Prepared in Step 1 (step 2)

[0064] The vitrified sample prepared in step 1 was incubated in a mixed solution comprising 40 w/v % of CHAPS, 40 w/v % of urea and 50 mM sodium chloride at 35 C. at 220 rpm for 24 hours. The overall brain size was 0.8 cm1 cm (widthlength) except the part of olfactory bulb. The results are shown in FIGS. 6(a) and 6(b).

[0065] As a result, it was confirmed that the size of the vitrified tissue was reduced by the treatment of the composition of the present invention.

EXPERIMENTAL EXAMPLE 2: OBTAINING MICROSCOPE IMAGES OF THE VITRIFIED TISSUE REDUCED IN SIZE

[0066] The size of the vitrified tissue was reduced by using the composition of the present invention, so that the sample was changed in size suitable for the observation under microscope. The vitrified brain tissue reduced in size was observed under microscope using lx objective lens, from which green fluorescent protein (GFP) of the mouse brain was confirmed (FIG. 6(c)).

[0067] As a result, it was confirmed that the desired image can be easily obtained by reducing the size of the vitrified tissue to fit the microscope size.

INDUSTRIAL APPLICABILITY

[0068] The composition for adjusting biological tissue size according to the present invention can be used as a mounting solution so that it can be effectively used to obtain biological tissue images, to reveal the causes of and find treatment methods for various disorders.