Abstract
In vitro studies on liver diseases, such as non-alcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma, are traditionally performed in two-dimensional (2D) cultures of isolated primary cells or immortalized cell lines. However, this approach has limitations, as 2D cultures inadequately replicate the cell–cell and cell–extracellular matrix interactions found in three-dimensional (3D) environments. To overcome this limitation, various 3D models, such as spheroids, have been developed. These spheroids serve as simplified biomimetic in vitro models for studying liver diseases. They can be generated using a variety of cells from healthy and pathological tissues, including liver cancer. Here, we present a comprehensive protocol for performing immunofluorescent staining and confocal imaging on whole human hepatic multicellular spheroids, utilizing primary cells or cell lines. The immunofluorescence technique is a potent tool to understand the spatial distribution of different cell types within the spheroids and define the interactions that occur among these cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
van Grunsven LA (2017) 3D in vitro models of liver fibrosis. Adv Drug Deliv Rev 121:133–146
Van Norman GA (2019) Limitations of animal studies for predicting toxicity in clinical trials: is it time to rethink our current approach? JACC Basic Transl Sci 4(7)
Alzeeb G, Metges JP, Corcos L, Le Jossic-Corcos C (2020) Three-dimensional culture systems in gastric cancer research. Cancers (Basel) 12(10)
Duriez M et al (2020) A 3D human liver model of nonalcoholic Steatohepatitis. J Clin Transl Hepatol 8(4)
Aoudjehane L, Gautheron J, Le Goff W et al (2020) Novel defatting strategies reduce lipid accumulation in primary human culture models of liver steatosis. Dis Model Mech 13(4)
Aoudjehane L, Boelle YP, Bisch G, Delelo R, Paye F, Scatton O, Housset C, Becquart J, Calmus Y, Conti F (2016) Development of an in vitro model to test antifibrotic drugs on primary human liver myofibroblasts. Lab Investig 96:672–679
Aoudjehane L, Bisch G, Scatton O, Granier C, Gaston J, Housset C, Roingeard P, Cosset FL, Perdigao F, Balladur P, Wakita T, Calmus Y, Conti F (2015) Infection of human liver Myofibroblasts by hepatitis C virus: a direct mechanism of liver fibrosis in hepatitis C. PLoS One 10(7)
Pfeiffer E, Kegel V, Zeilinger K, Hengstler JG, Nüssler AK, Seehofer D, Damm G (2015) Featured article: isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells. Exp Biol Med 240(5)
Acknowledgments
The authors gratefully acknowledge Prof. Olivier Scatton and his team for liver samples, and Romain Morichon from the CRSA imagery facility for his technical support.
Financial Support
LA is supported by the Foundation pour la Recherche Médicale (Equipe FRM 2020 n°EQU202003010517).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Couteau, M., Aoudjehane, L. (2024). Immunofluorescent Staining of Human Hepatic Multicellular Spheroids: A Model for Studying Liver Diseases. In: Kroemer, G., Pol, J., Martins, I. (eds) Liver Carcinogenesis. Methods in Molecular Biology, vol 2769. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3694-7_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3694-7_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3693-0
Online ISBN: 978-1-0716-3694-7
eBook Packages: Springer Protocols