Intestinal Organoids Culture in Synthetic Hydrogels

Derived from adult intestinal stem cells, organoids form intricate three-dimensional structures that closely mimic the cellular diversity and spatial organization of the human gut epithelium. Patient-derived organoids (PDOs) can recapitulate patient-specific tumor heterogeneity, making them highly valuable for colorectal cancer research and personalized medicine. However, their long-term in vitro culture still depends mainly on animal-derived materials (e.g., Matrigel) with undefined composition, batch variability, and limited tunability. To overcome these limitations, we introduced the use of synthetic peptide-based hydrogels, particularly peptide amphiphiles alone and in combination with extracellular matrix macromolecules, as a reproducible and tunable alternative. Here, we present a protocol for embedding PDOs in synthetic peptide-based hydrogels, and describe how to extract their RNA for transcriptomic analysis. This workflow enables the culture of intestinal PDOs in synthetic peptide-based hydrogels in a reproducible way, preserving key proliferation and differentiation lineage markers characteristic of PDOs.