The neuron patterning method has been applied to cultured neuronal networks to achieve better reproducibility and various experimental conditions in brain research. However, as models of dynamic environments, conventional patterned neural networks having an unalterable structure formed by a pre- programmed substrate have limitations, such as replicating the changes in the physical structure of networks in vivo. This study presents a novel culture technique that can control the location of the neuronal cell bodies constituting the network and the connection of the isolated neuronal networks at a desired time point. Alginate hydrogel, which has a cell-repellent property and is dissolvable by weak chemical treatment, is used as a background masking material for patterning, Axon growth from neural clusters to the background area formed by a microwell-patterned alginate layer is possible due to removal of the alginate layer, triggering synchronized spontaneous firing of the neuronal clusters. This technique was combined with a microelectrode array system to observe dynamic changes in neuronal activity according to the transformed morphology of the network. Finally, the clustered network structure formed by this technique showed greatly improved spontaneous firing rates and spike amplitudes compared to the network model prepared using an existing uniform structure.