Thetiot Melina, Sart Sébastien, Cussigh Delphine, Coolen Marion, Baroud Charles N, Bally-Cuif Laure
Zebrafish Neurogenetics Unit, Institut Pasteur - Université Paris Cité, UMR3738, CNRS, Paris, France.
Physical Microfluidics and Bioengineering Unit, Institut Pasteur - Université Paris Cité, Paris, France.
Biol Cell. 2025 Apr;117(4):e70007. doi: 10.1111/boc.70007.
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.
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.
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.
神经干细胞（NSCs）在成年脊椎动物大脑中生成神经元和神经胶质细胞，这对组织维持和可塑性至关重要。它们通过在静止、激活和谱系进展之间的转换来平衡神经发生与自我更新。这些过程背后的分子和细胞机制仍未完全了解。
在此，我们描述了一种从成年斑马鱼大脑皮层（一个主要的神经干细胞生态位）中分离和扩增神经干细胞的方案。我们介绍了增殖神经干细胞原代培养物的步骤，随后是三维球体的生成及其在液滴微流控平台中的调控。然后，我们详细说明了在药物处理后分析三维球体中成年神经干细胞命运的程序。
我们表明，7微升的液滴足以形成尺寸可控的三维球体，其中神经干细胞维持自我更新，并能够平衡静止和激活状态。我们概述了潜在的应用，包括对参与成年神经干细胞激活的因素的研究以及对其可溶性环境的监测，对于这些应用而言，密闭培养系统具有优势。
