3 Electroporation
3.1 NIH-3T3 Cells
Culture
1. The microfluidic platform (Fig. 4) is sterilized by 60 s of plasma
oxygen (100% O
2 , 100 W) treatment followed by 20 min of
UV exposure in laminar flow hood.
2. The NIH-3T3 cells are seeded on the microfluidic platform at a
concentration of 1.5 Â 10
4 cells/cm
2
.
3. The cell seeded platform is incubated at fixed temperature and
CO 2 concentration (37
C, 5%) for 36 h.
4. A daily change of culture medium is carried out until 100%
confluence is reached.
3.2 Culture of
Cardiac Muscle Cells
HL-1
1. Similar as step 1 in Subheading 3.1.
2. The microfluidic platform is treated with 0.01% poly-L-lysine
solution (Sigma-Aldrich) for 5 min to increase the cell adhesion.
3. The cardiac muscle cells HL-1 with a concentration of
8.5 Â 10
4 cells/cm
2 are seeded and grown with Claycomb
culture medium (Sigma-Aldrich) supplemented with 10% fetal
bovine serum (FBS), 100 μm norepinephrine, 300 μM ascorbic
acid, 2 mM L-glutamine, and 100 μg/ml penicillin/streptomycin (Sigma-Aldrich).
4. Similar as step 3 in Subheading 3.1.
5. Similar as step 4 in Subheading 3.1.
3.3 Electroporation
1. All the electroporation experiments are carried out in DMEM
with 1% of Pen/Strep antibiotic and 10% of FBS (Sigma-Aldrich).
2. A pulse train of 2 V, 100 μs, 20 Hz (standard softelectroporation protocol) for 10 s between the 3D hollow
nanoelectrodes and a platinum electrode immersed in the electrolyte (PBS, Sigma-Aldrich) is applied (see Note 7).
3. The cells are stained both with Calcein AM and propidium
iodide (PI) to perform a vitality test.
3.4 Observation
1. The cells are fixed with glutaraldehyde 2.5% in 0.1 M of sodium
cacodylate (Sigma-Aldrich) for 2 h on ice.
2. The cells are stained with 1% osmium tetroxide by EMS (2 h)
and 1% of uranyl acetate (4 h).
3. The cells are dehydrated in ethanol (30%, 50%, 70%, 90%, 96%,
and 100%) (Sigma-Aldrich) and dried by the critical point dryer
(CPD) technique.
4. The samples are coated with 5 nm of gold by sputter coating
technique.
5. The SEM images are performed with a FEI Helios Nanolab
650 dual beam.
Soft Electroporation Through 3D Hollow Nanoelectrodes
17
3.1 NIH-3T3 Cells
Culture
1. The microfluidic platform (Fig. 4) is sterilized by 60 s of plasma
oxygen (100% O
2 , 100 W) treatment followed by 20 min of
UV exposure in laminar flow hood.
2. The NIH-3T3 cells are seeded on the microfluidic platform at a
concentration of 1.5 Â 10
4 cells/cm
2
.
3. The cell seeded platform is incubated at fixed temperature and
CO 2 concentration (37
C, 5%) for 36 h.
4. A daily change of culture medium is carried out until 100%
confluence is reached.
3.2 Culture of
Cardiac Muscle Cells
HL-1
1. Similar as step 1 in Subheading 3.1.
2. The microfluidic platform is treated with 0.01% poly-L-lysine
solution (Sigma-Aldrich) for 5 min to increase the cell adhesion.
3. The cardiac muscle cells HL-1 with a concentration of
8.5 Â 10
4 cells/cm
2 are seeded and grown with Claycomb
culture medium (Sigma-Aldrich) supplemented with 10% fetal
bovine serum (FBS), 100 μm norepinephrine, 300 μM ascorbic
acid, 2 mM L-glutamine, and 100 μg/ml penicillin/streptomycin (Sigma-Aldrich).
4. Similar as step 3 in Subheading 3.1.
5. Similar as step 4 in Subheading 3.1.
3.3 Electroporation
1. All the electroporation experiments are carried out in DMEM
with 1% of Pen/Strep antibiotic and 10% of FBS (Sigma-Aldrich).
2. A pulse train of 2 V, 100 μs, 20 Hz (standard softelectroporation protocol) for 10 s between the 3D hollow
nanoelectrodes and a platinum electrode immersed in the electrolyte (PBS, Sigma-Aldrich) is applied (see Note 7).
3. The cells are stained both with Calcein AM and propidium
iodide (PI) to perform a vitality test.
3.4 Observation
1. The cells are fixed with glutaraldehyde 2.5% in 0.1 M of sodium
cacodylate (Sigma-Aldrich) for 2 h on ice.
2. The cells are stained with 1% osmium tetroxide by EMS (2 h)
and 1% of uranyl acetate (4 h).
3. The cells are dehydrated in ethanol (30%, 50%, 70%, 90%, 96%,
and 100%) (Sigma-Aldrich) and dried by the critical point dryer
(CPD) technique.
4. The samples are coated with 5 nm of gold by sputter coating
technique.
5. The SEM images are performed with a FEI Helios Nanolab
650 dual beam.
Soft Electroporation Through 3D Hollow Nanoelectrodes
17