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Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier.

ABSTRACT: Here, we describe the use of polarized patient enteroid-derived monolayers (EDMs) to assess the impact of e-cigarettes on the human gut barrier. These EDMs can be adapted to culture in a 96-well plate for high-throughput screening. We model the effect of e-cigarettes by combining pathogens, enteroids, and e-cigarette vapor-infused media and assess gut barrier integrity, bacterial internalization, and inflammatory response of the gut epithelium. This protocol can be used to assess the effects of e-cigarette components on gut functions. For complete details on the use and execution of this protocol, please refer to Sharma et al. (2021).

SUBMITTER: Sayed IM

PROVIDER: S-EPMC8313751 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier.

Sayed Ibrahim M IM Tindle Courtney C Fonseca Ayden G AG Ghosh Pradipta P Das Soumita S

STAR protocols 20210717 3

Here, we describe the use of polarized patient enteroid-derived monolayers (EDMs) to assess the impact of e-cigarettes on the human gut barrier. These EDMs can be adapted to culture in a 96-well plate for high-throughput screening. We model the effect of e-cigarettes by combining pathogens, enteroids, and e-cigarette vapor-infused media and assess gut barrier integrity, bacterial internalization, and inflammatory response of the gut epithelium. This protocol can be used to assess the effects of ...[more]

PMID: 34337445

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Project description:BackgroundPatient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier.MethodsPatient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib.ResultsIn response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib.ConclusionsWe describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.

Dataset Information

Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier.

Publications

Functional assays with human patient-derived enteroid monolayers to assess the human gut barrier.

Similar Datasets

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