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Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids

There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during a...

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Autores principales: Gopalakrishnan, Shreya, Bakke, Ingunn, Hansen, Marianne Doré, Skovdahl, Helene Kolstad, Granlund, Atle van Beelen, Sandvik, Arne K., Bruland, Torunn
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9995983/
https://www.ncbi.nlm.nih.gov/pubmed/36911731
http://dx.doi.org/10.3389/fimmu.2023.1097383
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author Gopalakrishnan, Shreya
Bakke, Ingunn
Hansen, Marianne Doré
Skovdahl, Helene Kolstad
Granlund, Atle van Beelen
Sandvik, Arne K.
Bruland, Torunn
author_facet Gopalakrishnan, Shreya
Bakke, Ingunn
Hansen, Marianne Doré
Skovdahl, Helene Kolstad
Granlund, Atle van Beelen
Sandvik, Arne K.
Bruland, Torunn
author_sort Gopalakrishnan, Shreya
collection PubMed
description There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during active inflammatory bowel disease (IBD), which is associated with an increased influx of immune cells into the gut mucosa. Therefore, studies on how chemokines are regulated in the intestinal epithelium may identify putative treatment targets in IBD. More recently, patient-derived ex vivo models such as intestinal organoids have facilitated molecular analysis of epithelial alterations in IBD patients own cells. Here, we describe refined experimental protocols and methods for the generation and maintenance of IBD patient-derived colonic organoids (colonoids) culture. We also give detailed description of medium, and supplements needed for colonoid establishment, growth, and differentiation, including production of Wnt-3A and Rspondin1 enriched media. Further, we present protocols for RNA and protein isolation from human colonoids, and subsequent gene expression analysis and Western blotting for e.g., signal transduction studies. We also describe how to process colonoids for chemokine protein expression analysis such as immunostaining, confocal imaging, and detection of secreted chemokines by e.g., enzyme-linked immunosorbent assay (ELISA). As proof of principle, we give examples of how the chemoattractant CCL20 can be regulated and expressed in colonoids derived from IBD-patients and healthy controls upon ligands-driven inflammation.
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spelling pubmed-99959832023-03-10 Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids Gopalakrishnan, Shreya Bakke, Ingunn Hansen, Marianne Doré Skovdahl, Helene Kolstad Granlund, Atle van Beelen Sandvik, Arne K. Bruland, Torunn Front Immunol Immunology There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during active inflammatory bowel disease (IBD), which is associated with an increased influx of immune cells into the gut mucosa. Therefore, studies on how chemokines are regulated in the intestinal epithelium may identify putative treatment targets in IBD. More recently, patient-derived ex vivo models such as intestinal organoids have facilitated molecular analysis of epithelial alterations in IBD patients own cells. Here, we describe refined experimental protocols and methods for the generation and maintenance of IBD patient-derived colonic organoids (colonoids) culture. We also give detailed description of medium, and supplements needed for colonoid establishment, growth, and differentiation, including production of Wnt-3A and Rspondin1 enriched media. Further, we present protocols for RNA and protein isolation from human colonoids, and subsequent gene expression analysis and Western blotting for e.g., signal transduction studies. We also describe how to process colonoids for chemokine protein expression analysis such as immunostaining, confocal imaging, and detection of secreted chemokines by e.g., enzyme-linked immunosorbent assay (ELISA). As proof of principle, we give examples of how the chemoattractant CCL20 can be regulated and expressed in colonoids derived from IBD-patients and healthy controls upon ligands-driven inflammation. Frontiers Media S.A. 2023-02-23 /pmc/articles/PMC9995983/ /pubmed/36911731 http://dx.doi.org/10.3389/fimmu.2023.1097383 Text en Copyright © 2023 Gopalakrishnan, Bakke, Hansen, Skovdahl, Granlund, Sandvik and Bruland https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
Gopalakrishnan, Shreya
Bakke, Ingunn
Hansen, Marianne Doré
Skovdahl, Helene Kolstad
Granlund, Atle van Beelen
Sandvik, Arne K.
Bruland, Torunn
Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids
title Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids
title_full Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids
title_fullStr Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids
title_full_unstemmed Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids
title_short Comprehensive protocols for culturing and molecular biological analysis of IBD patient-derived colon epithelial organoids
title_sort comprehensive protocols for culturing and molecular biological analysis of ibd patient-derived colon epithelial organoids
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9995983/
https://www.ncbi.nlm.nih.gov/pubmed/36911731
http://dx.doi.org/10.3389/fimmu.2023.1097383
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