Objective. The construction of in vitro three-dimensional (3D) neural tissue has to overcome two main types of challenges: (1) How to obtain enough number of functional neurons from stem cells in 3D culture; (2) How to wire those lately developed neurons into functional neural networks. Here, we describe the potential of using direct current (DC) electric field (EF) together with basic fibroblast growth factor (bFGF) synergistically in promoting neural stem cell (NSC) neuronal differentiation following by directing neurite outgrowth in the 3D neural tissue construction. Approach. By adjusting the electrical stimulation setup in this study, long-term electrical stimulation could be present in vitro. At an EF strength of 150 mV mm⁻¹, cell responses, including cell viability, neuronal differentiation, cell morphology, the length of neuronal processes, synaptic structure and neural network formation, were quantified and analyzed. Main results. Analysis revealed that NSCs showed no significant cell death after certain EF treatments. EF-stimulated NSCs in 3D Matrigel mainly differentiated into neurons, but unlike NSCs in two-dimensional conditions, their processes were flat and stunted. When combined with bFGF, EF stimulation provided appropriate bioactive cues to establish engineered neural tissue with a proper neuronal cell number, highly branched neurites, and a well-developed neuronal network. Significance. It is for the first time the synergistic effects of EF and bFGF stimulation have been evaluated in inducing the differentiation of NSCs into neurons and the acquisition of long neurites in a culture environment of in vitro 3D model. These optimized conditions may allow a well-developed neuronal network to be established within hydrogel droplets.

客观。体外三维（3D）神经组织的构建必须克服两大类挑战：（1）如何从3D培养的干细胞中获得足够数量的功能神经元；（2）如何将那些新开发的神经元连接成功能神经网络。在这里，我们描述了使用直流 (DC) 电场 (EF) 与碱性成纤维细胞生长因子 (bFGF) 协同促进神经干细胞 (NSC) 神经元分化的潜力，方法是在 3D 神经组织构建中引导神经突生长。方法。通过调整本研究中的电刺激设置，可以在体外进行长期电刺激。在 150 mV mm ^{-1的 EF 强度下}, 细胞反应, 包括细胞活力、神经元分化、细胞形态、神经元过程的长度、突触结构和神经网络形成, 被量化和分析。主要结果。分析表明，在某些 EF 治疗后，NSCs 没有显示出明显的细胞死亡。3D Matrigel 中 EF 刺激的 NSC 主要分化为神经元，但与二维条件下的 NSC 不同，它们的过程平坦且发育不良。当与 bFGF 结合时，EF 刺激提供了适当的生物活性线索，以建立具有适当神经元细胞数量、高度分支的神经突和发达的神经元网络的工程神经组织。意义. 这是首次在体外3D 模型的培养环境中评估了 EF 和 bFGF 刺激在诱导 NSC 分化为神经元和获得长神经突方面的协同作用。这些优化的条件可以允许在水凝胶液滴内建立发达的神经元网络。