CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistoc...

Full description

Saved in:
Bibliographic Details
Main Authors: T. A. Shnaider, I. E. Pristyazhnyuk
Format: Article
Language:English
Published: Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders 2022-01-01
Series:Вавиловский журнал генетики и селекции
Subjects:
Online Access:https://vavilov.elpub.ru/jour/article/view/3208
Tags: Add Tag
No Tags, Be the first to tag this record!
_version_ 1832575097498501120
author T. A. Shnaider
I. E. Pristyazhnyuk
author_facet T. A. Shnaider
I. E. Pristyazhnyuk
author_sort T. A. Shnaider
collection DOAJ
description Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.
format Article
id doaj-art-837ea6320de94e06aeb98a499be3a7c9
institution Kabale University
issn 2500-3259
language English
publishDate 2022-01-01
publisher Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders
record_format Article
series Вавиловский журнал генетики и селекции
spelling doaj-art-837ea6320de94e06aeb98a499be3a7c92025-02-01T09:58:10ZengSiberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and BreedersВавиловский журнал генетики и селекции2500-32592022-01-0125888989510.18699/VJ21.1031228CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoidsT. A. Shnaider0I. E. Pristyazhnyuk1Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesInstitute of Cytology and Genetics of the Siberian Branch of the Russian Academy of SciencesCerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.https://vavilov.elpub.ru/jour/article/view/3208cerebral organoidsclaritylight-sheet microscopyimmunohistochemistrytissue clearingtissue imaging
spellingShingle T. A. Shnaider
I. E. Pristyazhnyuk
CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids
Вавиловский журнал генетики и селекции
cerebral organoids
clarity
light-sheet microscopy
immunohistochemistry
tissue clearing
tissue imaging
title CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids
title_full CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids
title_fullStr CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids
title_full_unstemmed CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids
title_short CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids
title_sort clarity and light sheet microscopy sample preparation in application to human cerebral organoids
topic cerebral organoids
clarity
light-sheet microscopy
immunohistochemistry
tissue clearing
tissue imaging
url https://vavilov.elpub.ru/jour/article/view/3208
work_keys_str_mv AT tashnaider clarityandlightsheetmicroscopysamplepreparationinapplicationtohumancerebralorganoids
AT iepristyazhnyuk clarityandlightsheetmicroscopysamplepreparationinapplicationtohumancerebralorganoids