transduction, for example, could increase immunogenicity, reduce
therapeutic effectiveness [5, 6], and pose safety risks for clinical
implementation [7, 8]. Standard nonviral transfection avoids these
issues, but it is limited by low efficiencies and severely hampered cell
viability (~20%) [9, 10]. These drawbacks have thus limited widespread implementation of DC-based therapeutics.
Nanotechnology-driven transfection has emerged as promising
alternative to status quo strategies. Nanotechnology is increasingly
being used to develop a number of improved drug and gene
delivery approaches for different conditions [11, 12]. Nanochannel-based electroporation (NEP) is a deterministic transfection
technology that yields high efficiencies and cell viabilities [1–7],
and as such could be used to promote effective and benign gene
delivery to DCs for fundamental or therapeutic applications. Here
we describe a protocol for NEP-based gene delivery to DCs, and
subsequent collection of genetically engineered exosomes for
downstream analyses or use.
2 Materials
2.1 DC Isolation
and Differentiation
Complete media for isolation and differentiation of mouse bone
marrow derived-DCs is RPMI-1640 medium supplemented with
10% fetal bovine serum (FBS), 1% antibiotics, 2-mercaptoethanol
50 μM (Gibco, USA), granulocyte-macrophage colony stimulating
factor (GM-CSF) 20–30 ng/mL (PeproTech, USA), and
interleukin-4 (IL-4) 10–40 ng/mL (PeproTech, USA).
Bone marrow-derived DCs can be isolated from adult mice of
different strains. Characterization of DCs can be conducted using
flow cytometry Becton Dickson FACSAria (BD Bioscience, San
Jose, CA). The antibody panel for flow cytometry analysis includes
fluorescently labeled rat anti-mouse monoclonal CD11c (APC),
CD80 (FITC), CD86 (PercPCy5), CD40 (Pacific blue), MHC-II
(PE), and the IgG isotypes (eBioscience, USA).
2.2 Plasmid Isolation
For plasmid preparation, use ZymoPure II kit (Zymo research,
USA) following the procedure described by the manufacturer.
Quantify DNA plasmid concentration using a two-wavelength
spectrophotometric method on a NanoDrop spectrophotometer
(Thermo Fisher Scientific, Waltham, MA). DNA plasmid can be
stored at À80
C until use. Plasmid DNA is used at concentration
of 0.05 μg/μL.
2.3 NEP of DCs
NEP devices are fabricated as described before [13–17]. DCs are
seeded in the apical portion of the device, while the basal portion is
filled with the plasmid solution.
80
Silvia Duarte-Sanmiguel et al.
therapeutic effectiveness [5, 6], and pose safety risks for clinical
implementation [7, 8]. Standard nonviral transfection avoids these
issues, but it is limited by low efficiencies and severely hampered cell
viability (~20%) [9, 10]. These drawbacks have thus limited widespread implementation of DC-based therapeutics.
Nanotechnology-driven transfection has emerged as promising
alternative to status quo strategies. Nanotechnology is increasingly
being used to develop a number of improved drug and gene
delivery approaches for different conditions [11, 12]. Nanochannel-based electroporation (NEP) is a deterministic transfection
technology that yields high efficiencies and cell viabilities [1–7],
and as such could be used to promote effective and benign gene
delivery to DCs for fundamental or therapeutic applications. Here
we describe a protocol for NEP-based gene delivery to DCs, and
subsequent collection of genetically engineered exosomes for
downstream analyses or use.
2 Materials
2.1 DC Isolation
and Differentiation
Complete media for isolation and differentiation of mouse bone
marrow derived-DCs is RPMI-1640 medium supplemented with
10% fetal bovine serum (FBS), 1% antibiotics, 2-mercaptoethanol
50 μM (Gibco, USA), granulocyte-macrophage colony stimulating
factor (GM-CSF) 20–30 ng/mL (PeproTech, USA), and
interleukin-4 (IL-4) 10–40 ng/mL (PeproTech, USA).
Bone marrow-derived DCs can be isolated from adult mice of
different strains. Characterization of DCs can be conducted using
flow cytometry Becton Dickson FACSAria (BD Bioscience, San
Jose, CA). The antibody panel for flow cytometry analysis includes
fluorescently labeled rat anti-mouse monoclonal CD11c (APC),
CD80 (FITC), CD86 (PercPCy5), CD40 (Pacific blue), MHC-II
(PE), and the IgG isotypes (eBioscience, USA).
2.2 Plasmid Isolation
For plasmid preparation, use ZymoPure II kit (Zymo research,
USA) following the procedure described by the manufacturer.
Quantify DNA plasmid concentration using a two-wavelength
spectrophotometric method on a NanoDrop spectrophotometer
(Thermo Fisher Scientific, Waltham, MA). DNA plasmid can be
stored at À80
C until use. Plasmid DNA is used at concentration
of 0.05 μg/μL.
2.3 NEP of DCs
NEP devices are fabricated as described before [13–17]. DCs are
seeded in the apical portion of the device, while the basal portion is
filled with the plasmid solution.
80
Silvia Duarte-Sanmiguel et al.