Generation and Pharmacological Manipulation of 3D-Spheroid Cultures Derived From Zebrafish Adult Neural Stem Cells in a Droplet-Based Microfluidic Platform

AIMS: Neural stem cells (NSCs) generate neurons and glia in the adult vertebrate brain, crucial for tissue maintenance and plasticity. They balance neurogenesis with self-renewal, regulated through transitions between quiescence, activation, and lineage progression. The molecular and cellular mechanisms behind these processes remain incompletely understood. METHODS: Here, we describe a protocol to isolate and expand NSCs from the adult zebrafish pallium, a major NSC niche. We present the procedures to propagate primary cultures of NSCs, followed by the generation of 3D spheres and their regulation in a droplet microfluidic platform. We then detail the procedure to analyze adult NSC fate within the 3D spheroids following drug treatment. RESULTS: We show that 7 µL droplets are sufficient to allow the formation of size-controlled 3D spheroids, in which NSCs sustain self-renewal and are able to balance quiescence and activation. We outline potential applications, including investigation of factors involved in adult NSC activation and monitoring of their soluble environment, for which a confined culture system is advantageous.