Chapter 15
Isolation and Nanoscale Electroporation of Primary
Neuronal Cultures In Situ
Diego Alzate-Correa, William Lawrence, Natalia Higuita-Castro,
and Daniel Gallego-Perez
Abstract
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.
Key words Neuronal primary cultures, Gene delivery, Nanochannel-based electroporation
1 Introduction
Gene therapies could potentially provide the foundation for the
treatment of numerous neurological disorders [1]. Given the high
complexity of the CNS, the establishment of primary neuronal
cultures from defined regions of the embryonic brain has allowed
for the manipulation and analysis of neurons [2], increasing our
understanding of key biochemical and pathophysiological processes
underlying many CNS disorders. However, genetic manipulation
of neurons with status quo approaches such as lipofection or calcium
phosphate precipitation has resulted in low efficiencies and high
toxicity. While efficiencies have been improved with the use of viral
vectors, this approach can lead to insertional mutagenesis and is
severely limited by the capsid size [3]. Bulk electroporation (BEP)
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_15,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
145
Isolation and Nanoscale Electroporation of Primary
Neuronal Cultures In Situ
Diego Alzate-Correa, William Lawrence, Natalia Higuita-Castro,
and Daniel Gallego-Perez
Abstract
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.
Key words Neuronal primary cultures, Gene delivery, Nanochannel-based electroporation
1 Introduction
Gene therapies could potentially provide the foundation for the
treatment of numerous neurological disorders [1]. Given the high
complexity of the CNS, the establishment of primary neuronal
cultures from defined regions of the embryonic brain has allowed
for the manipulation and analysis of neurons [2], increasing our
understanding of key biochemical and pathophysiological processes
underlying many CNS disorders. However, genetic manipulation
of neurons with status quo approaches such as lipofection or calcium
phosphate precipitation has resulted in low efficiencies and high
toxicity. While efficiencies have been improved with the use of viral
vectors, this approach can lead to insertional mutagenesis and is
severely limited by the capsid size [3]. Bulk electroporation (BEP)
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_15,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
145