Transparentizing pretreatment method of biological sample having size of at most 1 mm, and transparentizing method of biological sample including same

Patent metadata

Field	Value
Publication number	US-12399090-B2
Application number	US-202118010906-A
Country	US
Kind code	B2
Filing date	May 28, 2021
Priority date	Jun 16, 2020
Publication date	Aug 26, 2025
Grant date	Aug 26, 2025

Abstract

A clearing pretreatment method of a biological sample having a size of at most 1 mm, that is, a spheroid or organoid, according to the present invention uses a phosphate-buffered saline (PBS) rather than a conventional sucrose solution as a pretreatment solution, thus solving the problem in which the spheroid or organoid floats above the surface of water due to the density difference such that the structure of the sample is damaged. Therefore, there is an effect in that the spheroid or organoid can be transparentized while maintaining the original shape thereof, thus making it possible to image deep parts.

First claim

The invention claimed is:

1. A clearing method of a biological sample having a size of 1 mm or less, the method comprising:
fixing a biological sample having a size of 1 mm or less;
pretreating the fixed biological sample by contacting the fixed biological sample with a pretreatment solution consisting of phosphate-buffered saline (PBS) and incubating at 0° C. to 10° C.; and
clearing the resulting pretreated biological sample by bringing the pretreated biological sample into contact with a clearing agent comprising a 3-[(3-cholamidopropyl)-dimethylammonio]-propane-sulfonate (CHAPS) compound of Formula 2 or a hydrate thereof in an amount of 2 to 55 w/v %, and urea in an amount of 10 to 70 w/v %,

2. The method of claim 1 , wherein the biological sample is a spheroid or an organoid.

3. The method of claim 2 , wherein the spheroid or organoid is formed from a cell derived from brain, blood vessels, liver, lungs, kidneys, pancreas, stomach, or intestines.

4. The method of claim 1 , wherein the biological sample has an average diameter in a range of 0.05 mm to 1 mm.

5. The method of claim 1 , wherein the biological sample is fixed by contacting the biological sample with at least one selected from the group consisting of paraformaldehyde, ethylene glycol diglycidyl ether, dipropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, glycerol polyglycidyl ether, glutaraldehyde, and polyacrylamide.

6. The method of claim 1 , further comprising contacting the pretreated biological sample, which has been removed or transferred from the PBS pretreatment solution, with agarose after the pretreating of the biological sample with PBS and before the clearing of the biological sample.

Assignees

• Korea Res Inst Chemical Tech
• Seoul Nat Univ R&Db Foundation
• Seoul National Univ R&Dbfoundation

Inventors

• Park Sun-Hyun
• Kim Ki-Suk
• Lee Jae-Meun
• Lee Jae-Hyeok
• Park Seong-Hoon
• Lee Ji Yeoun
• Park Jinju

Classifications

• G01N2001/305

  Fixative compositions · CPC title

• G01N1/36

  Embedding or analogous mounting of samples · CPC title

• G01N1/30 Primary

  Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} · CPC title