Co-culture of murine small intestinal organoids with type-3 innate lymphoid cells (ILC3)

Type-3 innate lymphoid cells (ILC3) regulate homeostasis and orchestrate immunity in the intestine, mediated in part by their bi-directional communication with the intestinal epithelium. In order to define specific interactions between these two compartments, we used a reductionist co-culture system of murine epithelial small intestinal organoids (SIO) with ILC3. The transcriptomic profile of epithelial cells from these co-cultures was analysed by bulk RNAseq. Murine SIO cultures were established from small intestine of female 6-10 week old CD45.1 mice and maintained in Matrigel. These were then co-cultured with type-3 innate lymphoid cells isolated from the small intestine. After 4 days of co-culture, the SIO were sorted via FACS into into RLT (QIAGEN) lysis buffer for RNA isolation using the RNeasy MicroRNA isolation kit (QIAGEN).