High Efficiency Lentiviral Transduction of Colon Organoids Using Reversible 2D/3D Culture Techniques

Organoids are a promising research tool for studying tissue development and disease in vitro. While organoids are frequently considered a replacement or complementary model for in vivo mouse experiments, exploiting their full potential often requires genetically engineered mice as a source of transgenic stem cells, also because genetic manipulation of organoids is rather inefficient and cumbersome. Here, we describe an alternative and optimized murine colon organoid manipulation protocol that reversibly and temporarily interrupts the 3D organoid structure for short-term 2D monolayer culture. This approach allows highly efficient viral transduction and genetic manipulation of stem cells in a 2D setting, followed by 3D stem cell embedding and restoration of the original organoid architecture. This method greatly improves the efficiency of lentiviral-mediated genetic manipulation of organoids and increases their potential applications in CRISPR/Cas9 and compound screens, immune-competent co-cultures, and disease modeling.