2.4 DC-Derived
Exosome Isolation
Exosomes are isolated from the culture medium of NEP-transfected
DCs using an ExoQuick kit (System Bioscience, USA) [14]. To
measure exosome loading efficiency, RNA isolation is performed
using mirVana kit (ThermoFisher, USA) following the procedure
described by the manufacturer. Prepare cDNA using SuperScript III
(ThermoFisher, USA) following the methodology described by the
manufacturer. qRT-PCR is performed to characterize exosome
loading using TaqMan fast advance master Mix (ThermoFisher,
USA) with specific primers/oligos. Analyze exosome sizes and concentration via Nanosight (Malvern Panalytical, USA).
3 Methodology
3.1 DC Isolation
1. Sacrifice mice by CO 2 inhalation and disinfect them with 75%
ethanol.
2. With dissection scissors, remove the tibias and femurs under
sterile conditions, clean off as much muscle as possible. Transfer clean femurs into a plastic dish filled with RPMI medium
supplemented with 1% FBS.
3. From here, all the following steps should be carried out in a
sterile cell culture hood. Media, instruments and tissue culture
material should be sterile.
4. In the hood, transfer bones from the RPMI media to a small
culture dish filled with 70% ethanol on ice. Allow bones to soak
in ethanol for 5 min.
5. Remove the ethanol with phosphate buffer saline (PBS) 1Â and
cut both ends of the bone off with scissors and insert a 1-mL
syringe (26–28-gauge needle) into the bone cavity to rinse the
bone marrow (BM). Draw about 100–200 mL of sterile RPMI
without FBS into a sterile culture dish with complete DC
media.
6. Repeat this step as many times as needed to completely wash
the marrow out of the bone until the bone looks white and
translucent.
7. Transfer the cell suspension to a sterile falcon tube. Very gently
pipette media up and down in the tube to break up the marrow
into a single cell suspension. This may take several minutes and
should be done slowly to avoid killing cells.
8. Centrifuge the cell suspension at 220 Â g for 5 min at room
temperature and discard the supernatant.
9. Remove the tube from the centrifuge after the spin is done and
remove the supernatant.
10. Resuspend the cell pellet with 3 mL red blood cell (RBC) lysis
solution (Miltenyi Biotec, Germany) to lyse the RBCs. Centrifuge at 220 Â g for 5 min at room temperature.
Nanoelectroporation and Collection of Genetically Modified Exosomes. . .
81
Exosome Isolation
Exosomes are isolated from the culture medium of NEP-transfected
DCs using an ExoQuick kit (System Bioscience, USA) [14]. To
measure exosome loading efficiency, RNA isolation is performed
using mirVana kit (ThermoFisher, USA) following the procedure
described by the manufacturer. Prepare cDNA using SuperScript III
(ThermoFisher, USA) following the methodology described by the
manufacturer. qRT-PCR is performed to characterize exosome
loading using TaqMan fast advance master Mix (ThermoFisher,
USA) with specific primers/oligos. Analyze exosome sizes and concentration via Nanosight (Malvern Panalytical, USA).
3 Methodology
3.1 DC Isolation
1. Sacrifice mice by CO 2 inhalation and disinfect them with 75%
ethanol.
2. With dissection scissors, remove the tibias and femurs under
sterile conditions, clean off as much muscle as possible. Transfer clean femurs into a plastic dish filled with RPMI medium
supplemented with 1% FBS.
3. From here, all the following steps should be carried out in a
sterile cell culture hood. Media, instruments and tissue culture
material should be sterile.
4. In the hood, transfer bones from the RPMI media to a small
culture dish filled with 70% ethanol on ice. Allow bones to soak
in ethanol for 5 min.
5. Remove the ethanol with phosphate buffer saline (PBS) 1Â and
cut both ends of the bone off with scissors and insert a 1-mL
syringe (26–28-gauge needle) into the bone cavity to rinse the
bone marrow (BM). Draw about 100–200 mL of sterile RPMI
without FBS into a sterile culture dish with complete DC
media.
6. Repeat this step as many times as needed to completely wash
the marrow out of the bone until the bone looks white and
translucent.
7. Transfer the cell suspension to a sterile falcon tube. Very gently
pipette media up and down in the tube to break up the marrow
into a single cell suspension. This may take several minutes and
should be done slowly to avoid killing cells.
8. Centrifuge the cell suspension at 220 Â g for 5 min at room
temperature and discard the supernatant.
9. Remove the tube from the centrifuge after the spin is done and
remove the supernatant.
10. Resuspend the cell pellet with 3 mL red blood cell (RBC) lysis
solution (Miltenyi Biotec, Germany) to lyse the RBCs. Centrifuge at 220 Â g for 5 min at room temperature.
Nanoelectroporation and Collection of Genetically Modified Exosomes. . .
81