REFRACTIVE INDEX MATCHING COMPOSITION FOR BIOLOGICAL TISSUE

Abstract

The present invention is about biological tissue refractive index (RI) matching composition and the method for clearing biological tissue. Specifically, the RI matching composition of the present invention shows remarkable RI matching effects when clearing biological tissue, and has excellent fluorescent signal preservation, and can be used for long-term storage of biological tissue since the biological tissue remains clear during in low temperature storage.

Claims

1. A refractive index matching composition for biological tissue, which comprises iopromide or its active derivative as an active ingredient.

2. The refractive index matching composition of claim 1, wherein the iopromide has the following chemical structure of Formula 1: ##STR00003##

3. The refractive index matching composition of claim 1, wherein the composition comprises 0.1 g to 5 g of iopromide in 1 ml of a solvent.

4. A method for preparing a refractive index matching composition for biological tissue, which comprises dissolving and stirring iopromide in a solvent.

5. The method for preparing a refractive index matching composition for biological tissue of claim 4, wherein the stirring is conducted at between 20° C. and 40° C.

6. A composition for mounting biological tissue, which comprises the refractive index matching composition of claim 1.

7. The composition for mounting biological tissue of claim 6, which further comprises antibodies, fluorescent probes, or nucleic acids.

8. A tissue clearing method which comprises, i) embedding hydrogel into in vitro separated biological tissue or biological tissue of an animal except human beings; ii) removing lipids from the tissue embedded with the hydrogel; and iii) submerging the delipidated tissue into the composition of claim 1.

9. The tissue clearing method of claim 8, wherein lipids are removed by electrophoresis.

10. The tissue clearing method of claim 9, wherein the electrophoresis is carried out for hours to days.

11. The tissue clearing method of claim 8, wherein the lipids are removed by incubating the tissue in a detergent.

12. The tissue clearing method of claim 11, wherein the detergent is selected from the group consisting of triton-X, saponin, NP-40 (nonyl phenoxypolyethoxylethanol), tween20 and SDS (sodium dodecyl sulfate).

13. The tissue clearing method of claim 8, which further comprises slicing the tissue into sections, after extracting the biological tissue.

14. The tissue clearing method of claim 8, which further comprises immunolabeling the biological tissue, before submerging the tissue in the composition of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 shows the result of RI matching of biological tissue by using the present RI homogenization composition, after imbedding acrylamide into the lung tissues of a mouse and then removing lipids therefrom using electrophoresis.

[0023] FIG. 2 shows the refractive indexes according to the concentration of iopromide in the RI homogenization composition of the present invention.

[0024] FIG. 3 is a photo representing the recovery times of the tissues swollen by biological tissue clearing.

[0025] FIG. 4 shows the relative size and light intensity of the swollen tissues according to times.

[0026] A: the comparison of tissue sizes before/after the tissue clearing; and

[0027] B: the comparison of light intensities before/after the tissue clearing

[0028] FIG. 5 shows the preservation, 2 weeks after the biological tissue clearing with the present RI homogenization composition.

[0029] FIG. 6 shows the transparency preservation during the storage in low temperature, after the biological tissue clearing by using the present composition.

DETAILED DESCRIPTION

[0030] Hereinafter, the present invention is explained in more detail with the examples. However, the present invention is not subjected to any limitations in the following description.

[0031] In one aspect, the present invention is about a biological tissue RI homogenization composition comprising iopromide as an active ingredient.

[0032] The present biological tissue RI homogenization composition is used for a method for clearing tissue or its fragments (slices or sections). That is, the present invention relates to a RI matching composition which is used for RI homogenization of the components of biological tissue or its fragments.

[0033] The words, “refractive index homogenization”, “refractive index matching” and “RI matching” mean clearing tissue by homogenizing refractive indexes of the tissue components.

[0034] The “active derivative” of the present invention comprises, without limitation, chemical compounds which have refractive index homogenization effects comparable to those of iopromide.

[0035] The “biological tissue clearing” means to make a tissue transparent to three dimensionally observe it or its fragments with an optical microscope. The biological tissue clearing technology comprises, for example, CLARITY and PARS technologies. According to the CLARITY, the hydrogel (for example, acrylamide) is embedded into the tissue, followed by delipidation using electrophoresis, and then the refractive index is homogenized to make the tissue transparent. Further, according to the PARS, the hydrogel (for example, acrylamide) is embedded into the tissue and then SDS buffer solution is circulated thorough the tissue for a long time to have the tissue transparent.

[0036] The tissue specimen made transparent by the refractive index matching composition of the present invention can be detected with an optical microscope, a fluorescence microscope, a confocal microscope, a single-photon microscope or two-photon microscope, or a light-sheet microscope.

[0037] In one embodiment, iopromide is the following chemical compound of Formula 1 or its active derivatives:

##STR00002##

[0038] 1-N,3-N-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-(2-methoxyacetamido)-1-N-methylbenzene-1,3-dicarboxamide

[0039] In one embodiment, the refractive index homogenization composition of the present invention comprises 0.15 g to 3 g of iopromide in 1 ml of a solvent which is DDW (double distilled water), PBS (phosphate buffered saline), saline solution (Normal saline) or ethanol, but not limited thereto.

[0040] In one aspect, the present invention is about a method for preparing a refractive index homogenization composition.

[0041] In one embodiment, the refractive homogenization composition for biological tissue can be prepared by dissolving iopromide in a solvent and then stirring the mixture at between 20° C. and 40° C.

[0042] In one aspect, the present invention relates to a tissue mounting composition which comprises the refractive index homogenization composition for biological tissue.

[0043] The words, “composition for mounting a tissue” mean a mounting solution to fill in biological tissue for imaging thereof, since the tissue or its fragments left as they are loose transparency due to dehydrogenation.

[0044] The composition for mounting a tissue can be prepared by additionally adding glycerin, gelatin, rubber syrup, balsam, or biolite to the composition. In addition, antibodies, fluorescent probes or nucleic acids can be further included for detecting specific materials.

[0045] In one aspect, the present invention relates to a method for clearing biological tissue, which comprises, i) embedding hydrogel into a tissue in vitro separated from a living organism or a tissue of an animal except human beings; ii) removing lipids from the tissue embedded with the hydrogel; iii) adding a biological tissue refractive index homogenization composition to the delipidated tissue.

[0046] In one embodiment, the removal of lipids can be performed by electrophoresis, and the electrophoresis can be carried out for hours or for days, if necessary, by the use of commercially available devices or self-manufactured devices.

[0047] In one embodiment, the delipidation can be performed by incubating the tissue in a detergent but not electrophoresis, and the detergent can be selected from the group consisting of triton-X, saponin, NP-40, tween20 and SDS (Sodium dodecyl sulfate). For example, 1 to 8% SDS buffer can be employed.

[0048] In another embodiment, the method for clearing biological tissue comprises, i) perfusing a tissue in vitro separated from a living organism or a tissue of an animal except human being by using hydrogel; ii) extracting the tissue perfused with the hydrogel; and iii) removing lipids from the extracted tissue, iv) submersing the tissue above iii) in the refractive index homogenization composition for biological tissue of the present invention.

[0049] In yet another embodiment, the extracted biological tissue is further processed to fragments.

[0050] In yet another embodiment, immunolabeling process is further performed before submerging tissue samples in the biological tissue refractive index matching composition of the present invention. Antibodies, fluorescent probes, nucleic acids or proteins can be used for the immunolabeling.

[0051] The present invention will be further explained based on the examples hereunder. The examples hereunder, however, are only for the purpose of illustrating the present invention, and the scope of the present invention is not limited to the examples hereunder.

Example

Preparation 1: Preparation of a Biological Tissue Refractive Index Matching Composition Comprising Iopromide

[0052] 35 g of iopromide (Ultravist, Bayer Healthcare) was dissolved and mixed in 30 ml of DDW (double distilled water)

Example 1: Biological Tissue Clearing by Using the Biological Tissue RI Matching Composition of the Preparation 1 Above

[0053] The lung of a mouse (ICR, female, 12 week old, Daehanbiolink Co., Ltd.) was extracted, and then the extracted lung was imbedded with hydrogel solution comprising arylamide for 24 hrs at 4° C., and thereafter the polymerization of hydrogel solution was allowed for 3 hrs at 37° C. Next, electrophoresis was carried out for the lung having the polymerized acrylamide by using the X-CLATRITY (Trademark, Logosbiosystems Inc., Korea). The specific operating conditions for the electrophoresis was as follows: 15 A, 60V, 4% SDS buffer solution, 30 rpm, for 12 hrs. After completing the electrophoresis, the lung tissues were treated with 5 ml of the RI matching solution prepared above, for 300 hrs, at room temperature to clear the tissues. Subsequently, the lung tissues of the mouse were detected with a stereoscopic microscope (Nikon, SMZ 745T, photos were taken by EP1 camera, Olympus). The results show that the composition of the present invention has excellent refractive index matching effects (see FIG. 1).

Example 2: Assessment of Refractive Index According to the Concentration of Iopromide in the RI Matching Composition

[0054] As shown in Table 1 below, 0 (control), 5 g, 10 g, 15 g, 20 g, 25 g, 30 g, 35 g, 37 g, 38 g, 39 g, 40 g, 42 g, 45 g or 48 g of iopromide was dissolved and mixed well in 30 ml of DDW at 25° C. or 37° C., to prepare refractive index matching compositions having different iopromide concentrations. The lung tissues were cleared with the RI matching compositions having different iopromide concentrations as described in the Example 1, and then the refractive indexes were assessed. The RIs were detected with a refractive index detector (ATAGO, RX-9000a). As a result, the RI increased in proportion to the concentration of iopromide in the RI matching composition (see Table 1 and FIG. 2).

TABLE-US-00001 TABLE 1 iopromide (g)/DDW 30 ml Refractive Index (R.I) dissolving condition (° C.) 0 1.333 25° C. 5 1.357 25° C. 10 1.379 25° C. 15 1.398 25° C. 20 1.414 25° C. 25 1.428 25° C. 30 1.444 25° C. 35 1.455 25~37° C.    37 1.460 37° C. 38 1.461 37° C. 39 1.463 37° C. 40 1.466 37° C. 42 1.469 37° C. 45 1.474 37° C. 48 1.480 37° C. *DDW: double distilled water

Example 3: Comparison of the Recovery Times of the Tissues Swollen after Tissue Clearing Treatments

[0055] As disclosed in the Example 1, the lung tissues of mouse was perfused with acrylamide, and then lipids were removed by electrophoresis. Next, the tissue clearing was carried out by submerging the tissues in the RI matching composition of the present invention, or the conventional RI matching composition of RIMS (Yang et al., 2014) or FocusClear (Chiang et al., 2002, Cell Explorer, Taiwan). Then, tissue recovery was assessed by determining the size of the swollen tissue at each time point of 0 hr (control), 0.5 hr, 1 hr, 2 hrs, and 3 hrs after the completion of the tissue clearing, and detecting the light transmission intensities.

[0056] As a result, regarding the recovery effects of returning the size of the biological tissues swollen by the tissue clearing to the original size, the RI matching composition comprising iopromide of the present invention was remarkable in comparison to the composition of RIMS or FocusClear (FIG. 3 and FIG. 4A). Further, the tissues, which were cleared with the RI matching composition comprising iopromide of the present invention, showed the most excellent light transmission property (FIG. 4B).

[0057] Therefore, it was confirmed that the composition comprising iopromide of the present invention has significantly outstanding RI matching effects, in comparison to the conventional RI matching composition (FocusClear or RIMS).

Example 4: Confirmation of Fluorescent Signal Preservation

[0058] Lipids were removed from the mouse brain tissue slices (1 mm thick) by using 4% SDS solution and electrophoresis device as disclosed in the above Example 1, and then the primary antibody (anti-collagen IV, Bio Rad Company) staining was performed at 37° C. for 24 hrs. Next, the primary antibodies were washed off with PBS, and the antibodies (Cy-3, Bio Rad Company) labeled with the second fluorescent materials were stained under the same conditions as for the primary antibodies. The washed sample was made to be transparent by matching the RI with the RI composition of the present invention, RIMS or FocusClear, and thereafter the tissue staining was assessed and imaged using a confocal microscope (ZEISS, LSM 710). Subsequently, after 2 weeks, the preservation of the fluorescent staining was confirmed under the same experimental conditions as above.

[0059] As a result, while the fluorescent signal of the tissue treated with the FocusClear for the tissue clearing was rapidly decreased, the tissues treated with the composition comprising iopromide minimized the fluorescence bleaching, thereby presenting excellent fluorescence preservation capacity (FIG. 5).

Example 5: Assessment of the Maintenance of Transparency During in Low Temperature Storage

[0060] As disclosed in Example 1, the RI matching composition of the present invention, RIMS or FocusClear was used to make the lung tissues of a mouse optically transparent. After the tissue clearing treatment, the tissues were immersed in 5 ml of each RI matching composition and stored for 24 hrs, at 25° C. and 18° C., and then the tissue clearing was quantified with a stereoscopic microscope (Nikon, SMZ 745T, equipped with Olympus, EP1 camera). As a result, crystalline materials were developed in the biological tissue stored in the RIMS, which resulted in rapid decrease of the RI of the tissue. To the contrary, no crystalline material was detected in the biological tissue stored in the RI composition of the present invention (see FIG. 6).