Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation.
Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse length 0.1 ms were applied in 1-16 repetitions with a 10-sec pause interval between pulses. Surviving cells were stained by crystal violet and counted using a confocal microscope. Transfected cells were fixed with 10% formaldehyde and counted as green spots in a fluorescence microscope. In the present investigation we used the method of bioimpedance spectrometry to analyze the effect of EP on survival and transfection ratio of cells in suspension. DC and low-frequency AC currents preferably pass through the medium due to the high impedance of the cell membrane. At frequencies above 10 kHz the impedance of the cell membrane starts to decrease and the impedance value of the cell suspension approach a lower limit value Rinfinity at infinite frequency. Recording of electrical impedance spectra of cells in culture was performed over a frequency range of 10 Hz to 125 kHz, allowing separation of the contribution from extracellular space and that of the cell membranes. A parallel resistance capacitance model of the cell suspension was used to evaluate the response of applying EP pulses. The values of the collective membrane resistance RM decay exponentially (r2=0.995) with the number of applied pulses. The ratio of the extrapolated value of the intact membrane resistance before pulsing, RM,0, and the value RM,N after each pulse makes an index of the effect of electroporation on the cells. The ratio RM,N/RM,0 as well as the relative change of the dissipation factor, tandelta, on the "Loss Change Index" (LCI) fits well a dose-response model (r2=0.98) with the number of applied pulses. The changes in the model parameters membrane resistance DeltaRM=[1-RM,N/RM,o] and loss factor [1-tandelta0/tandeltaN] correlate well with the transfection ratio and fraction of dead cells. Those parameters were used for power-assisted electroporation in monitoring, controlling, and optimizing the EP procedure.
Glahder
Jacob
J
The Protein Laboratory, Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Norrild
Bodil
B
Persson
Mikael B
MB
Persson
Bertil R R
BR
D002462:Cell Membrane / Q000502:physiology / Q000528:radiation effects; D002470:Cell Survival / Q000502:physiology; D003198:Computer Simulation; D017097:Electric Impedance; D004574:Electromagnetic Fields; D018274:Electroporation / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D010957:Plasmids / Q000008:administration & dosage* / Q000493:pharmacokinetics*; D011994:Recombinant Proteins / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins