Intestinal Organoid-Derived Monolayer System

Introduction

The loss of epithelial barrier function is a hallmark of IBD. TNFα is considered a key driver of IBD, directly causing barrier disfunction. Although TNFα-neutralizing therapy has been used for decades to treat IBD, a subset of patients stop responding to the therapy. This highlights the importance of developing a better understanding of the role of this cytokine in IBD progression.

Current in vitro models of barrier disfunction are based on the use of cancer cell lines. These lack the cell differentiation complexity of the human intestine and harbor mutations that might affect the response. To overcome this issue, the authors developed a novel monolayer system using human gut organoids.

Main Points

• The authors generated gut organoids derived from the ileum of adult donors.
• Organoids were dissociated into single cells and plated in 96-well transwells to develop monolayer cultures.
• The monolayers were differentiated by withdrawing Wnt pathway agonists from culture medium.
• The differentiation status was determined by gene expression analysis and confirmed by immunofluorescence: differentiated culture showed lower expression of the stem cell marker LGR5 and increased expression of secretory and absorptive cell markers (secretory: MUC2, CHGA, MMP7; absorptive: CYP3A4, SLC15A1).
• Diffrerentiated monolayers were treated with TNFα and barrier function was quantified by measuring TEER.
• A dose-dependent reduction of TEER was observed upon treatment with TNFα, together an increase in p65 phosphorylation and activation of NFKB pathway (measured by qPCR)
• Addition of an anti-TNF-α antibody to 96-well monolayer cultures treated with TNF-α efficiently rescued barrier damage in a dose-dependent manner and inhibited p65 phosphorylation.
• Cytokine secretion was analysed in both apical and basolateral compartments by Luminex, and results were in agreement with the gene expression results, indicating activation of the NFKB pathway.
• IL-8 expression was upregulated by TNFα treatment in a dose-dependent manner, indicating that IL-8 secretion analysis could be used as a method to quantitatively assess the effect of TNF-α pathway modulators.
• To note, the stimulation of the monolayer with TNFα induced a a strong damage-response only when administered in the basolateral compartment.

Concluding Remarks

In conclusion the 96-well human gut organoid derived monolayer system is a reliable model that can facilitate multiple readouts to quantify the effects of drugs on barrier integrity, recapitulating key aspects of the human intestine physiology.

Epistem Services

Epistem offers organoid-derived monolayer assays, an in vitro solution to study drug-induced effects on the small intestinal barrier. The model provides direct access to both sides of the epithelium, maintaining differentiated cell types. Key measurements include trans-epithelial electrical resistance (TEER), permeability, cell death, and tight junction integrity assessed through immunofluorescence.

Epistem provides 3D organoid models that mimic the intestine, offering valuable insights into biology, disease, and compound effects. These structures allow for multiple assessments, including viability, branching, and gene expression.

Both models provide a comprehensive array of readouts, encompassing assessments of cell viability, detailed immunofluorescence analyses, in-depth examination of gene expression patterns, and a variety of other informative data points.