Abstract
Microcontact printing (CP) is widely used to guide neurons to form 2D networks for neuroscience research. However, it is still difficult to establish 3D neuronal cultures on the CP substrate even though 3D neuronal structures are able to recapitulate critical aspects of native tissue. Here, we demonstrate that the reduced cell-substrate adhesion caused by the CP substrate could conveniently facilitate the aggregate formation of large-scale 3D neuron cluster networks. Furthermore, based on the quantitative analysis of the calcium activity of the resulting cluster networks, the effect of cell seeding density and local restriction of the CP substrate on network dynamics was investigated in detail. The results revealed that cell aggregation degree, rather than cell number, could take on the main role of the generation of synchronized network-wide calcium oscillation (network bursts) in the 3D neuron cluster networks. This finding may provide new insights for easy and cell-saving construction of in vitro 3D pathological models of epilepsy, and into deciphering the onset and evolution of network bursts in developmental nerve systems.