Protocol for generating and analyzing organ-on-chip using human and mouse intestinal organoids

Area of Science:

• Biomedical Engineering
• Cell Biology
• Gastroenterology

Background:

• Organ-on-chip technology offers advanced in vitro modeling capabilities.
• Intestinal organoids preserve cellular diversity and function but face lumen inaccessibility.
• Combining organ-on-chip technology with organoids addresses these limitations.

Purpose of the Study:

• To present a comprehensive protocol for generating and analyzing human and mouse intestinal organoids on-chip.
• To enable independent stimulation and sampling of apical and basal sides of the intestinal epithelium.
• To provide a robust platform for studying intestinal physiology and disease in vitro.

Main Methods:

• Establishment of intestinal organoid lines.
• Single-cell dissociation and preparation of organoids for chip seeding.
• Chip preparation, including surface treatment and assembly.
• Seeding of organoids into microfluidic chips.
• Conducting permeability assays.
• RNA isolation, staining, and imaging for analysis.
• Independent stimulation and sampling from apical and basal compartments.

Main Results:

• Successful generation and integration of intestinal organoids within microfluidic chips.
• Demonstration of preserved cellular diversity and epithelial function in the organ-on-chip system.
• Validation of permeability assays to assess barrier integrity.
• Successful independent stimulation and sampling from both sides of the epithelium.
• High-quality imaging and RNA analysis for detailed characterization.

Conclusions:

• The developed protocol enables the creation of functional intestinal organ-on-chips using human and mouse organoids.
• This model system overcomes lumen inaccessibility, preserving key features of the intestinal epithelium.
• The platform facilitates advanced in vitro studies of intestinal physiology, drug permeability, and disease modeling.