Department of Informatics, Bioengineering, Robotics, and Systems Engineering (DIBRIS), University of Genova, Genova, Italy.
Department of Experimental Medicine, University of Genoa, Genoa, Italy.
J Neural Eng. 2021 Dec 10;18(6). doi: 10.1088/1741-2552/ac3e02.
In this work we adapted a protocol for the fast generation of human neurons to build 3D neuronal networks with controlled structure and cell composition suitable for systematic electrophysiological investigations.We used biocompatible chitosan microbeads as scaffold to build 3D networks and to ensure nutrients-medium exchange from the core of the structure to the external environment. We used excitatory neurons derived from human-induced pluripotent stem cells (hiPSCs) co-cultured with astrocytes. By adapting the well-established NgN2 differentiation protocol, we obtained 3D engineered networks with good control over cell density, volume and cell composition. We coupled the 3D neuronal networks to 60-channel micro electrode arrays (MEAs) to monitor and characterize their electrophysiological development. In parallel, we generated two-dimensional neuronal networks cultured on chitosan to compare the results of the two models.We sustained samples until 60 d(DIV) and 3D cultures were healthy and functional. From the structural point of view, the hiPSC derived neurons were able to adhere to chitosan microbeads and to form a stable 3D assembly thanks to the connections among cells. From a functional point of view, neuronal networks showed spontaneous activity after a couple of weeks.We presented a particular method to generate 3D engineered cultures for the first time with human-derived neurons coupled to MEAs, overcoming some of the limitations related to 2D and 3D neuronal networks and thus increasing the therapeutic target potential of these models for biomedical applications.
在这项工作中，我们改编了一种快速生成人类神经元的方案，以构建具有受控结构和细胞组成的 3D 神经元网络，适合系统的电生理研究。我们使用生物相容性壳聚糖微球作为支架来构建 3D 网络，并确保从结构核心到外部环境的营养物质-培养基交换。我们使用源自人诱导多能干细胞（hiPSC）的兴奋性神经元与星形胶质细胞共培养。通过适应成熟的 NgN2 分化方案，我们获得了具有良好细胞密度、体积和细胞组成控制的 3D 工程网络。我们将 3D 神经元网络与 60 通道微电极阵列（MEA）耦合，以监测和表征它们的电生理发育。同时，我们在壳聚糖上培养二维神经元网络，以比较两种模型的结果。我们将样本维持至 60 天（分化日），3D 培养物健康且功能正常。从结构的角度来看，hiPSC 衍生的神经元能够附着在壳聚糖微球上，并通过细胞之间的连接形成稳定的 3D 组装。从功能的角度来看，神经元网络在几周后表现出自发活动。我们首次提出了一种特别的方法来生成与 MEAs 耦合的 3D 工程培养物，使用源自人类的神经元，克服了与 2D 和 3D 神经元网络相关的一些限制，从而增加了这些模型在生物医学应用中的治疗靶标潜力。
