TY - JOUR
T1 - Human colonoid monolayers to study interactions between pathogens, commensals, and host intestinal epithelium
AU - In, Julie
AU - Foulke-Abel, Jennifer
AU - Clarke, Elizabeth
AU - Kovbasnjuk, Olga N
N1 - Funding Information:
This work was supported by NIH grants P01 AI125181, K01 DK106323 (JGI), and K01 DK113043 (JFA). We thank James Kaper (University of Maryland, Baltimore, MD, USA) for providing E. coli strain HS and EHEC. We also acknowledge the Integrated Physiology and Imaging Cores of the Hopkins Conte Digestive Disease Basic and Translational Research Core Center (P30 DK089502) and the Johns Hopkins Mass Spectrometry and Proteomics Core.
Publisher Copyright:
© 2019 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.
PY - 2019/4
Y1 - 2019/4
N2 - Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.
AB - Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.
KW - Apical and basolateral secretion
KW - Apical infection
KW - Colonic mucus
KW - Host-pathogen interaction
KW - Human enteroid or colonoid monolayers
KW - Immunology and Infection
KW - Intestinal organoid monolayer
KW - Issue 146
UR - http://www.scopus.com/inward/record.url?scp=85065413374&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85065413374&partnerID=8YFLogxK
U2 - 10.3791/59357
DO - 10.3791/59357
M3 - Article
C2 - 31033964
AN - SCOPUS:85065413374
SN - 1940-087X
VL - 2019
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 146
M1 - e59357
ER -