Effect of electroporation medium conductivity on exogenous molecule transfer to cells in vitro.

In this study we evaluated the influence of medium conductivity to propidium iodide (PI) and bleomycin (BLM) electroporation mediated transfer to cells. Inverse dependency between the extracellular conductivity and the efficiency of the transfer had been found. Using 1 high voltage (HV) pulse, the total molecule transfer efficiency decreased 4.67 times when external medium conductivity increased from 0.1 to 0.9 S/m. Similar results had been found using 2 HV and 3 HV pulses. The percentage of cells killed by BLM electroporation mediated transfer had also decreased with the conductivity increase, from 79% killed cells in 0.1 S/m conductivity medium to 28% killed cells in 0.9 S/m conductivity medium. We hypothesize that the effect of external medium conductivity on electroporation mediated transfer is triggered by cell deformation during electric field application. In high conductivity external medium cell assumes oblate shape, which causes a change of voltage distribution on the cell membrane, leading to lower electric field induced transmembrane potential. On the contrary, low conductivity external medium leads to prolate cell shape and increased transmembrane potential at the electrode facing cell poles.

The dependence of efficiency of transmembrane molecular transfer using electroporation on medium viscosity.

BACKGROUND: In the present study, we aimed to evaluate the efficiency of drug and gene electrotransfer into cells in vitro depending on medium viscosity. METHODS: Experiments were performed using Chinese hamster ovary cells. Efficiency of molecular electrotransfer depending of medium viscosity was evaluated using two different electroporation conditions: a high-voltage (HV) pulse and a combination of a high-voltage pulse and a low-voltage pulse (HV + LV). To evaluate the efficiency of molecular electrotransfer, anticancer drug bleomycin and two different plasmids coding for green fluorescent protein and luciferase were used. RESULTS: We found that a slight increase in medium viscosity from 1.3-1.4 mPa·s significantly decreased the transfection efficiency, both in terms of transfected cells and total protein production, which was abolished completely with an increase in medium viscosity to 6.1 mPa·s. Notably, at this medium viscosity, electrotransfer of the small anticancer drug was still efficient. Using HV and HV + LV pulse combinations, we showed that a decrease of DNA electrotransfer, especially at lower medium viscosities, can be compensated for by the LV pulse to some extent. On the other hand, the addition of the LV pulse after the HV pulse did not have any positive effect on the efficiency of bleomycin electrotransfer. CONCLUSIONS: These findings demonstrate that transfection is very susceptible to medium viscosity and highlights the importance of the electrophoretic component in experiments when a considerable transfection level is needed.

Influence of plasmid concentration on DNA electrotransfer in vitro using high-voltage and low-voltage pulses.

DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 microl of CHO cell suspension containing 100, 10 or 1 microg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 microg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 microg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 micros) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 microg/ml and fivefold at 1 microg/ml. At 10 microg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.

Extent of cell electrofusion in vitro and in vivo is cell line dependent.

BACKGROUND: Electric pulses delivered to cells that are in close contact may induce cell fusion, by a process termed electrofusion. Recently it has been shown that electrofusion in tumours in vivo depends on tumour type. The aim of this work was to examine the differences in electrofusion in various cell lines, both in vivo and in vitro. MATERIALS AND METHODS: LPB, B16F10 and DC-3F cells in vitro and LLC tumours in vivo were exposed to various electric pulses. The number of fused cells was then evaluated. RESULTS: Cell electropermeabilization was confirmed to be a necessary but non-exclusive condition to obtain a high level of cell electrofusion. The extent of electrofusion depends both on the degree of permeabilization and cell type. CONCLUSION: It was observed that metastatic tumour cells easily electrofuse, suggesting that cell type-specific membrane properties and/or secretion of proteases determine the extent of electrofusion.