Comprehensive Isolation Strategy for Intestinal Epithelial and Fibroblast Cells from Healthy and Tumor Human Tissues

Organoids and fibroblasts derived from patient tissue serve as physiologically meaningful in vitro models to investigate tissue biology, diseases, and treatment responses. In this study, we present a robust protocol for the simultaneous isolation and long-term culture of patient-derived organoids (PDOs) and fibroblasts from both healthy and colorectal cancer samples. The workflow uses enzymatic dissociation followed by efficient separation into epithelial and stromal cell fractions. Organoids are embedded in Matrigel or Matrigel-Collagen I mixtures to support three-dimensional growth, whereas fibroblasts are maintained on conventional two-dimensional culture dishes. This dual-culture approach facilitates a broad range of downstream applications.