Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers

Abstract Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-d...

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Autores principales:	Egi Kardia, Michael Frese, Elena Smertina, Tanja Strive, Xi-Lei Zeng, Mary Estes, Robyn N. Hall
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/43223eede3be437581dc9245543fa637
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spelling	oai:doaj.org-article:43223eede3be437581dc9245543fa6372021-12-02T13:19:23ZCulture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers10.1038/s41598-021-84774-w2045-2322https://doaj.org/article/43223eede3be437581dc9245543fa6372021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84774-whttps://doaj.org/toc/2045-2322Abstract Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-β inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1, an enterotropic lagovirus that—like many other caliciviruses—does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1.Egi KardiaMichael FreseElena SmertinaTanja StriveXi-Lei ZengMary EstesRobyn N. HallNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Egi Kardia Michael Frese Elena Smertina Tanja Strive Xi-Lei Zeng Mary Estes Robyn N. Hall Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers
description	Abstract Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-β inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1, an enterotropic lagovirus that—like many other caliciviruses—does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1.
format	article
author	Egi Kardia Michael Frese Elena Smertina Tanja Strive Xi-Lei Zeng Mary Estes Robyn N. Hall
author_facet	Egi Kardia Michael Frese Elena Smertina Tanja Strive Xi-Lei Zeng Mary Estes Robyn N. Hall
author_sort	Egi Kardia
title	Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers
title_short	Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers
title_full	Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers
title_fullStr	Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers
title_full_unstemmed	Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers
title_sort	culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/43223eede3be437581dc9245543fa637
work_keys_str_mv	AT egikardia cultureanddifferentiationofrabbitintestinalorganoidsandorganoidderivedcellmonolayers AT michaelfrese cultureanddifferentiationofrabbitintestinalorganoidsandorganoidderivedcellmonolayers AT elenasmertina cultureanddifferentiationofrabbitintestinalorganoidsandorganoidderivedcellmonolayers AT tanjastrive cultureanddifferentiationofrabbitintestinalorganoidsandorganoidderivedcellmonolayers AT xileizeng cultureanddifferentiationofrabbitintestinalorganoidsandorganoidderivedcellmonolayers AT maryestes cultureanddifferentiationofrabbitintestinalorganoidsandorganoidderivedcellmonolayers AT robynnhall cultureanddifferentiationofrabbitintestinalorganoidsandorganoidderivedcellmonolayers
_version_	1718393267386580992

Culture and differentiation of rabbit intestinal organoids and organoid-derived cell monolayers

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