Avoiding the side effects of electric current pulse application to electroporated cells in disposable small volume cuvettes assures good cell survival

Abstract

BackgroundThe harmful side effects of electroporation to cells due to local changes in pH, the appearance of toxic electrode products, temperature increase, and the heterogeneity of the electric field acting on cells in the cuvettes used for electroporation were observed and discussed in several laboratories. If cells are subjected to weak electric fields for prolonged periods, for example in experiments on cell electrophoresis or galvanotaxis the same effects are seen. In these experiments investigators managed to reduce or eliminate the harmful side effects of electric current application.MethodsFor the experiments, disposable 20 μl cuvettes with two walls made of dialysis membranes were constructed and placed in a locally focused electric field at a considerable distance from the electrodes. Cuvettes were mounted into an apparatus for horizontal electrophoresis and the cells were subjected to direct current electric field (dcEF) pulses from a commercial pulse generator of exponentially declining pulses and from a custom-made generator of double and single rectangular pulses.ResultsMore than 80% of the electroporated cells survived the dcEF pulses in both systems. Side effects related to electrodes were eliminated in both the flow through the dcEF and in the disposable cuvettes placed in the focused dcEFs. With a disposable cuvette system, we also confirmed the sensitization of cells to a dcEF using procaine by observing the loading of AT2 cells with calceine and using a square pulse generator, applying 50 ms single rectangular pulses.ConclusionsWe suggest that the same methods of avoiding the side effects of electric current pulse application as in cell electrophoresis and galvanotaxis should also be used for electroporation. This conclusion was confirmed in our electroporation experiments performed in conditions assuring survival of over 80% of the electroporated cells. If the amplitude, duration, and shape of the dcEF pulse are known, then electroporation does not depend on the type of pulse generator. This knowledge of the characteristics of the pulse assures reproducibility of electroporation experiments using different equipment.

Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts

“…Though the 500 V with 4 mm cuvettes produce same field strength (1.25 kV/cm) as 250 V with 2 mm cuvettes, the efficiency of electroporation was better in 4 mm cuvettes probably owing to the greater distance of cells from electrodes, that prevents contact of extensive number of cells with electrodes. Previous studies proved that cell distance from electrodes is one of the critical parameters that affects electroporation outcomes [41]. Also, it is only at high electric field strengths that cell membrane progressively become less resistive [42].…”

The protective effect of non-invasive low intensity pulsed electric field and fucoidan in preventing oxidative stress-induced motor neuron death via ROCK/Akt pathway

“…In our study, the non-invasive LIPEF instrument delivered stimulations in a non-contact manner, greatly facilitating the development of clinical therapy. Since there was a dielectric air gap separating the electrodes from the cells, it could avoid the harmful effects of direct contact, such as the toxic electrode products and undesirable leakage current in the body, as mentioned previously [ 42 , 44 ]. The method of the study is safer for application to patients with degenerative brain diseases.…”

High-Capacity Redox Polymer Electrodes: Applications in Molecular and Cellular Processing

“…33 These accomplishments will enable near-term replacement of standard metal electrodes within diagnostic and therapeutic manufacturing workflows using methods compatible with production of fully-closed and disposable systems. 1,9,13,17 Beyond this increased flexibility in fabrication, manufacturing, and integration, the novel electrodes were shown to improve the function of several of the electrokinetic components tested, including increased compatibility with DNA amplification in high-voltage applications, 15,19 increased electroosmotic pumping pressure, 30,31,34 and direct integration of battery-operated EOPs for self-contained fluidic valving and control 32,33 (compared with the external tubes and pressure/power sources currently required). Finally, the use of the redox polymer electrodes led to a new discovery, as the EOPs built with the novel electrodes were precharged with an electrochemical potential that was later used to run the pump in the absence of an applied voltage.…”

“…[12][13][14][15][16] However, the metal electrodes commonly used to maintain electrical current within biological fluids and perform these electrokinetic operations have well-known limitations. [17][18][19] Most of these electrodes employ electrolysis for generating current and produce excess H + and OH -, which can negatively affect cells and biomolecules within the fluid. In addition, gas products (O 2 and H 2 ) produced during electrolysis may block electrode access to the biological fluid, creating electrical shorts during operation.…”