TY - JOUR
T1 - Intestinal organoid cocultures with microbes
AU - Puschhof, Jens
AU - Pleguezuelos-Manzano, Cayetano
AU - Martinez-Silgado, Adriana
AU - Akkerman, Ninouk
AU - Saftien, Aurelia
AU - Boot, Charelle
AU - de Waal, Amy
AU - Beumer, Joep
AU - Dutta, Devanjali
AU - Heo, Inha
AU - Clevers, Hans
N1 - Funding Information:
We thank E. Allen-Vercoe and A. Robinson for provision of bacterial strains and discussions on bacterial culturing conditions. The development of the methods was supported by CRUK grant OPTIMISTICC (C10674/A27140) (J.P., C.P.-M. and H.C.), the Gravitation projects CancerGenomiCs.nl, and the Netherlands Organ-on-Chip Initiative (024.003.001) from the Netherlands Organisation for Scientific Research (NWO) funded by the Ministry of Education, Culture and Science of the government of the Netherlands (J.P., C.P.-M. and H.C.), the Oncode Institute (partly financed by the Dutch Cancer Society), the European Research Council under ERC Advanced Grant Agreement no. 67013 (J.P., D.D., I.H. and H.C.) and NETRF/Petersen Accelerator (J.B.).
Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature Limited.
PY - 2021/10
Y1 - 2021/10
N2 - Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host–microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.
AB - Adult-stem-cell-derived organoids model human epithelial tissues ex vivo, which enables the study of host–microbe interactions with great experimental control. This protocol comprises methods to coculture organoids with microbes, particularly focusing on human small intestinal and colon organoids exposed to individual bacterial species. Microinjection into the lumen and periphery of 3D organoids is discussed, as well as exposure of organoids to microbes in a 2D layer. We provide detailed protocols for characterizing the coculture with regard to bacterial and organoid cell viability and growth kinetics. Spatial relationships can be studied by fluorescence live microscopy, as well as scanning electron microscopy. Finally, we discuss considerations for assessing the impact of bacteria on gene expression and mutations through RNA and DNA sequencing. This protocol requires equipment for standard mammalian tissue culture, or bacterial or viral culture, as well as a microinjection device.
UR - https://www.scopus.com/pages/publications/85112249078
U2 - 10.1038/s41596-021-00589-z
DO - 10.1038/s41596-021-00589-z
M3 - Review article
C2 - 34381208
AN - SCOPUS:85112249078
SN - 1754-2189
VL - 16
SP - 4633
EP - 4649
JO - Nature protocols
JF - Nature protocols
IS - 10
ER -