An improved organ explant culture method reveals stem cell lineage dynamics in the adult Drosophila intestine

Abstract: In recent years, live-imaging techniques have been developed for the adult midgut of Drosophila melanogaster that allow temporal characterization of key processes involved in stem cell and tissue homeostasis. However, current organ culture techniques are limited to imaging sessions of <16 hours, an interval too short to track dynamic processes such as damage responses and regeneration, which can unfold over several days. therefore, we developed a new organ explant culture protocol capable of sustaining midguts ex vivo for up to 3 days. This was made possible by the formulation of a culture medium specifically designed for adult Drosophila tissues with an increased Na⁺/K⁺ ratio and trehalose concentration, and by placing midguts at an air-liquid interface for enhanced oxygenation. We show that midgut progenitor cells can respond to gut epithelium damage ex vivo, proliferating and differentiating to replace lost cells, but are quiescent in healthy intestines. Using ex vivo gene induction to promote stem cell proliferation, we demonstrate that intestinal stem lineages can be traced through multiple cell divisions using live imaging. Both asymmetric and symmetric divisions can be identified in the reconstructed lineages. We find that daughter cells of asymmetric divisions remain in close proximity of each other, while the progeny of symmetric divisions actively move apart, with implications for cell differentiation and tissue organization. We show that the same culture set-up is useful for imaging adult renal tubules and ovaries for up to 72 hours. By enabling both long-term imaging and real-time ex vivo gene manipulation, our simple culture protocol provides a powerful tool for studies of epithelial biology and cell lineage behavior.