Optimization of Matrigel-based culture for expansion of neural stem cells

Neural stem cells (NSCs) are capable of self-renewal and can differentiate into neurons and two types of glial cells. NSCs have great potential for basic studies of neurogenesis and neurodegenerative diseases, and for therapeutic transplantation applications. NSCs can be derived from the fetal or adult brain. <i>In vitro</i> expansion of NSCs may help to elucidate their properties involved in neurogenesis and is crucial to obtain on-demand the large numbers of these cells needed for clinical transplantation trials. Laminin is an extracellular matrix molecule commonly used to culture NSCs as an attached monolayer. However, laminin is costly if NSCs need to be cultured in large quantities. Matrigel is a soluble basement membrane biomaterial, which has been widely used for feeder-free cultures of human embryonic stem cells. In the present study, we evaluated the ability of Matrigel to support the attachment and long-term proliferation of NSCs. We investigated the growth and adherence of NSCs derived from the fetal mouse brain on Matrigel at various concentrations (0.01–1%). We found that 0.02% Matrigel is sufficient for NSCs to be expanded <i>in vitro</i> in long-term culture. The NSCs cultured on Matrigel-coated plates showed typical cellular and molecular characteristics and had multipotency to be differentiated into neurons and glial cells. The Matrigel-based monolayer culture system established in this study provides a cost-effective approach to maintain NSCs <i>in vitro</i> in long-term cultures.

神经干细胞（Neural stem cells, NSCs）具备自我更新能力，可分化为神经元与两类胶质细胞。其在神经发生、神经退行性疾病的基础研究以及治疗性移植应用中具有巨大潜力。神经干细胞可从胎儿或成体大脑中分离获取。体外（*in vitro*）扩增神经干细胞，不仅有助于阐明其参与神经发生的相关特性，更是按需获取临床移植试验所需大量此类细胞的关键环节。层粘连蛋白（Laminin）是一种常用于神经干细胞贴壁单层培养的细胞外基质分子，但大规模培养时其成本相对高昂。基质胶（Matrigel）作为一种可溶性基底膜生物材料，已被广泛应用于人胚胎干细胞的无饲养层培养体系。本研究评估了基质胶支持神经干细胞贴附与长期增殖的能力：我们探究了胎鼠大脑来源的神经干细胞在0.01%~1%不同浓度基质胶上的生长与贴附情况，发现0.02%的基质胶即可满足神经干细胞长期体外扩增培养的需求。在基质胶包被培养板上培养的神经干细胞，呈现出典型的细胞与分子特征，且具备分化为神经元与胶质细胞的多向分化潜能。本研究构建的基于基质胶的贴壁单层培养体系，为长期体外培养维持神经干细胞提供了一种经济高效的方案。