An Integrated Workflow for Culturing and AI-Based Profiling of Mouse Intestinal Epithelium

Two-dimensional (2D) culture systems are powerful in vitro tools that have revolutionized the study of intestinal stem cell biology. These culture systems derived from mouse intestinal and colonic epithelia enable the modeling of tissue architecture, barrier function, and disease mechanisms under physiologically relevant conditions. This protocol details methods for isolating and maintaining mouse intestinal crypts as 2D monolayers. Monolayers are established via enzymatic dissociation and seeding onto ECM-functionalized hydrogels. This protocol also describes a deep learning pipeline that performs virtual fluorescent staining of label-free phase-contrast images of live organoids. This protocol enables the reproducible generation and computational analysis of murine intestinal epithelial cultures for studies in epithelial biology, drug testing, and disease modeling. This integrated system is designed for facilitating high-throughput imaging, immunolabeling, and functional analysis, expanding its use in regenerative medicine, tissue engineering, and gastrointestinal research.