A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases. •We optimized conditioned medium preparation from L cells by lentiviral infection•We modified a protocol to differentiate iPSCs into small intestinal organoids•We succeeded in transducing a gene into organoids by 2D culture•We showed that intestinal human organoids self-proliferate in suspension culture Takahashi et al. developed several user-friendly culture methods for human induced pluripotent stem cell (iPSC)-derived intestinal organoids. The methodological improvements include preparation of conditioned medium for organoid culture, efficient differentiation from iPSCs, gene transduction in organoids, and growth in suspension culture. This system will facilitate high-throughput screening of pathogenic factors and treatments for intestinal diseases using physiologically robust intestinal organoids.