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Updated: May 10, 2025
Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres
Published on: February 29, 2016
Generation and Pharmacological Manipulation of 3D-Spheroid Cultures Derived From Zebrafish Adult Neural Stem Cells in
Melina Thetiot1, Sébastien Sart2, Delphine Cussigh1,3
1Zebrafish Neurogenetics Unit, Institut Pasteur - Université Paris Cité, UMR3738, CNRS, Paris, France.
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.
