TY - JOUR
KW - Coculture Techniques
KW - Intestines
KW - organoids
AU - Jens Puschhof
AU - Cayetano Pleguezuelos-Manzano
AU - Adriana Martinez-Silgado
AU - Ninouk Akkerman
AU - Aurelia Saftien
AU - Charelle Boot
AU - Amy de Waal
AU - Joep Beumer
AU - Devanjali Dutta
AU - Inha Heo
AU - Hans Clevers
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.
BT - Nature Protocols
DA - 2021-10
DO - 10.1038/s41596-021-00589-z
IS - 10
LA - eng
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.
PY - 2021
SP - 4633
EP - 4649
T2 - Nature Protocols
TI - Intestinal organoid cocultures with microbes
VL - 16
SN - 1750-2799
ER -