has also been implemented for neuronal gene delivery. BEP uses an
electric field to generate pores in the cell membrane to facilitate the
entry of genetic material [4]. However, BEP typically requires
freshly isolated neurons in suspension, resulting in nonuniform
dosages at the single-cell level, and could result in cytotoxic overstimulation of the cell membrane.
To address these complications, a new nanotechnology platform was developed to efficiently and safely deliver genes to primary
neuronal cultures in situ. Nanochannel-based electroporation
(NEP) uses nanochanneled membranes controllably and benignly
delivers genes to cultured cells in situ [5–12]. These nanochannels
confine the electric field to a small portion of the cell membrane,
preserving the integrity of the rest of the membrane and minimizing cell damage [11]. Here we describe a protocol for the generation and nanoscale electroporation of primary neuronal cultures
from mouse hippocampi.
2 Materials
2.1 Neuronal Culture
Preparation
E18.5 embryos are obtained from Timed-Pregnant Mice (The
Jackson Laboratory). Brains are procured with autoclaved surgical
tools (Fine Science Tools) under a SMZ 745T dissecting microscope (Nikon Inc.) and maintained on ice-cold Hanks’ Balanced
Salt Solution (HBSS) without calcium or magnesium (Thermo
Fisher Scientific) in 150 mm soda lime silica glass petri dish
(Millipore-Sigma). Hippocampal tissue is dissected out in HBSS
without calcium or magnesium (Thermo Fisher Scientific) in sterile
polystyrene P60 petri dish (Fisher Scientific). The tissue is digested
with Trypsin 0.25% (Thermo Fisher Scientific) and DNaseI
(Millipore-Sigma), and dissociated with fire-polished and fetal
bovine serum (FBS)-coated borosilicate glass Pasteur pipettes
(Fisher Scientific) in neuronal culture media composed of Neurobasal™ medium (Thermo Fisher Scientific), 2 mM GlutaMAX™
supplement (Thermo Fisher Scientific), 2% NeuroCult™ SM1
Neuronal Supplement (Stemcell Technologies), and 25 μM L-glutamic acid (Thermo Fisher Scientific). Individual cells are obtained
with a 70 μm cell strainer (Miltenyi Biotec), neurons are isolated
with the Neuron Isolation Kit (Miltenyi Biotec) and seeded directly
on the NEP platform previously coated with poly-D-lysine hydrobromide (Millipore Sigma) prepared in Boric Acid (MilliporeSigma) [2, 13, 14].
2.2 Plasmid
Preparation
The plasmid DNA is expanded using DH5α competent E. coli cells
(Thermo Scientific Inc.) and standard bacterial culture methods.
Plasmid DNA is isolated with ZymoPURE—Express Plasmid Midiprep Kit (Zymo Research) following the procedures described by
the manufacturer. Before use plasmid DNA must be diluted to a
concentration of 0.05 μg/μL in sterile PBS.
146
Diego Alzate-Correa et al.
electric field to generate pores in the cell membrane to facilitate the
entry of genetic material [4]. However, BEP typically requires
freshly isolated neurons in suspension, resulting in nonuniform
dosages at the single-cell level, and could result in cytotoxic overstimulation of the cell membrane.
To address these complications, a new nanotechnology platform was developed to efficiently and safely deliver genes to primary
neuronal cultures in situ. Nanochannel-based electroporation
(NEP) uses nanochanneled membranes controllably and benignly
delivers genes to cultured cells in situ [5–12]. These nanochannels
confine the electric field to a small portion of the cell membrane,
preserving the integrity of the rest of the membrane and minimizing cell damage [11]. Here we describe a protocol for the generation and nanoscale electroporation of primary neuronal cultures
from mouse hippocampi.
2 Materials
2.1 Neuronal Culture
Preparation
E18.5 embryos are obtained from Timed-Pregnant Mice (The
Jackson Laboratory). Brains are procured with autoclaved surgical
tools (Fine Science Tools) under a SMZ 745T dissecting microscope (Nikon Inc.) and maintained on ice-cold Hanks’ Balanced
Salt Solution (HBSS) without calcium or magnesium (Thermo
Fisher Scientific) in 150 mm soda lime silica glass petri dish
(Millipore-Sigma). Hippocampal tissue is dissected out in HBSS
without calcium or magnesium (Thermo Fisher Scientific) in sterile
polystyrene P60 petri dish (Fisher Scientific). The tissue is digested
with Trypsin 0.25% (Thermo Fisher Scientific) and DNaseI
(Millipore-Sigma), and dissociated with fire-polished and fetal
bovine serum (FBS)-coated borosilicate glass Pasteur pipettes
(Fisher Scientific) in neuronal culture media composed of Neurobasal™ medium (Thermo Fisher Scientific), 2 mM GlutaMAX™
supplement (Thermo Fisher Scientific), 2% NeuroCult™ SM1
Neuronal Supplement (Stemcell Technologies), and 25 μM L-glutamic acid (Thermo Fisher Scientific). Individual cells are obtained
with a 70 μm cell strainer (Miltenyi Biotec), neurons are isolated
with the Neuron Isolation Kit (Miltenyi Biotec) and seeded directly
on the NEP platform previously coated with poly-D-lysine hydrobromide (Millipore Sigma) prepared in Boric Acid (MilliporeSigma) [2, 13, 14].
2.2 Plasmid
Preparation
The plasmid DNA is expanded using DH5α competent E. coli cells
(Thermo Scientific Inc.) and standard bacterial culture methods.
Plasmid DNA is isolated with ZymoPURE—Express Plasmid Midiprep Kit (Zymo Research) following the procedures described by
the manufacturer. Before use plasmid DNA must be diluted to a
concentration of 0.05 μg/μL in sterile PBS.
146
Diego Alzate-Correa et al.