Single organoid droplet-based staining method for high-end 3D
imaging of mammary organoids

C 2022

Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids

SUMBAL, Jakub and Zuzana SUMBALOVÁ KOLEDOVÁ

Basic information

Original name

Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids

Authors

SUMBAL, Jakub (203 Czech Republic, belonging to the institution) and Zuzana SUMBALOVÁ KOLEDOVÁ (703 Slovakia, guarantor, belonging to the institution)

Edition

New York, Mammary Stem Cells, p. 259-269, 11 pp. Methods in Molecular Biology, vol 2471, 2022

Publisher

Springer New York - Humana Press

Other information

Language

English

Type of outcome

Kapitola resp. kapitoly v odborné knize

Field of Study

10601 Cell biology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

Publication form

printed version "print"

References:

URL

RIV identification code

RIV/00216224:14110/22:00128719

Organization unit

Faculty of Medicine

ISBN

978-1-0716-2192-9

DOI

http://dx.doi.org/10.1007/978-1-0716-2193-6_14

Keywords in English

3D culture; Clearing; Confocal imaging; Microenvironment; Organoid; Staining.

Abstract

V originále

In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.

Links

LM2018129, research and development project

Name: Národní infrastruktura pro biologické a medicínské zobrazování Czech-BioImaging

Investor: Ministry of Education, Youth and Sports of the CR

MUNI/A/1382/2019, interní kód MU

Name: Zdroje pro tkáňové inženýrství 10 (Acronym: TissueEng 10)

Investor: Masaryk University, Category A

MUNI/G/1446/2018, interní kód MU

Name: Deciphering the mechanisms of mammary epithelial branched pattern formation through iterative biological and mathematical modelling

Investor: Masaryk University, INTERDISCIPLINARY - Interdisciplinary research projects

ROZV/28/LF19/2020, interní kód MU

Name: Regulace morfogeneze epitelu mléčné žlázy pomocí mechanických sil a dynamiky signalizace

Investor: Ministry of Education, Youth and Sports of the CR, Internal development projects

Citovat

SUMBAL, Jakub and Zuzana SUMBALOVÁ KOLEDOVÁ. Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids (Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids. Methods Mol Biol. 2022; 2471: 259-269.). In Vivanco, M.d. (eds) Mammary Stem Cells. Mammary Stem Cells. New York: Springer New York - Humana Press, 2022, p. 259-269. Methods in Molecular Biology, vol 2471. ISBN 978-1-0716-2192-9. Available from: https://dx.doi.org/10.1007/978-1-0716-2193-6_14.

@inbook{2260977,
   author = {Sumbal, Jakub and Sumbalová Koledová, Zuzana},
   address = {New York},
   booktitle = {Mammary Stem Cells},
   doi = {http://dx.doi.org/10.1007/978-1-0716-2193-6_14},
   editor = {Vivanco, M.d. (eds) Mammary Stem Cells},
   keywords = {3D culture; Clearing; Confocal imaging; Microenvironment; Organoid; Staining.},
   howpublished = {tištěná verze "print"},
   language = {eng},
   location = {New York},
   isbn = {978-1-0716-2192-9},
   pages = {259-269},
   publisher = {Springer New York - Humana Press},
   title = {Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids},
   url = {https://link.springer.com/protocol/10.1007/978-1-0716-2193-6_14},
   year = {2022}
}

TY  - CHAP
ID  - 2260977
AU  - Sumbal, Jakub - Sumbalová Koledová, Zuzana
PY  - 2022
TI  - Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids
VL  - Methods in Molecular Biology, vol 2471
PB  - Springer New York - Humana Press
CY  - New York
SN  - 9781071621929
KW  - 3D culture
KW  - Clearing
KW  - Confocal imaging
KW  - Microenvironment
KW  - Organoid
KW  - Staining.
UR  - https://link.springer.com/protocol/10.1007/978-1-0716-2193-6_14
N2  - In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.
ER  -

SUMBAL, Jakub and Zuzana SUMBALOVÁ KOLEDOVÁ. Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids (Single organoid droplet-based staining method for high-end 3D imaging of mammary organoids. Methods Mol Biol. 2022; 2471: 259-269.). In Vivanco, M.d. (eds) Mammary Stem Cells. \textit{Mammary Stem Cells}. New York: Springer New York - Humana Press, 2022, p.~259-269. Methods in Molecular Biology, vol 2471. ISBN~978-1-0716-2192-9. Available from: https://dx.doi.org/10.1007/978-1-0716-2193-6\_{}14.

Detailed Information on Publication Record