Isolation and Nanoscale Electroporation of Primary Neuronal Cultures In Situ

Developing effective gene therapies for disorders of the central nervous system (CNS) is extremely challenging due to the lack of safe and efficient gene delivery methods to neurons and glial cells, hampering the study of CNS physiology and the identification of novel therapeutic targets. Current gene transfer methodologies for neuronal cultures rely on synthetic nanoparticles or viral transduction. These approaches present low gene transfer efficiency, are highly toxic, and may induce adverse immune responses. Electroporation has been implemented as an alternative approach; however, this method is restricted for the most part to cells in suspension, and electrical overstimulation of the neuronal membrane may have detrimental consequences. To overcome these barriers, here we describe the implementation of nanochannel-based electroporation for gene delivery into primary neural cultures safely and efficiently. We outline the preparation of viable primary neuronal cultures from the hippocampus of E18.5 mouse embryos and describe the optimal parameter for transfection using a nanochannel-based electroporation platform.