Quantification of Electroporative Uptake Kinetics and Electric Field Heterogeneity Effects in Cells

Abstract

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 mus pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.

Electric Field Orientation for Gene Delivery Using High-Voltage and Low-Voltage Pulses

“…Permeabilization was quantified by monitoring propidium iodide uptake in CHO cells (Kennedy et al 2008). We observed a higher rate of permeabilized cells with HVLV pulses when compared to classical pulses (EGT) (Fig.…”

“…Cells were resuspended in an electropulsation buffer (10 mM phosphate, 1 mM MgCl 2 , 250 mM sucrose, pH 7.4) complemented with propidium iodide (100 lM) (Sigma-Aldrich, St. Louis, MO) at 5 9 10 6 cells/ml. Penetration of the propidium iodide was used to monitor cell permeability (Kennedy et al 2008). This suspension (100 ll) was poured between two plated parallel stainless-steel electrodes (distance between electrodes 4 mm).…”

Blumlein Configuration for High-Repetition-Rate Pulse Generation of Variable Duration and Polarity Using Synchronized Switch Control

“…Nevertheless, thick electrodes with sharp edges are needed to achieve a well-defined and homogeneous electric field in upper layers. Developers of such electrodes have made 20-µm-thick electrodes from nickel [29] and silver [30]. We have made similar electrodes but from more biocompatible material-gold.…”

Peningkatan Ketebalan Miokardium Mencit (Mus musculus L.) Akibat Paparan Medan Listrik Tegangan Tinggi

“…Menurut laporan Kennedy dkk., 12 mekanisme medan listrik dapat merusak sel melalui proses elektroporasi. Proses energi listrik merusak otot jantung melalui proses elektroporasi, panas (joule heating), denaturasi protein, dan hiperkontraksi otot rangka.…”

“…Perubahan yang terjadi akan menyebabkan cedera dan juga mengganggu fungsi membran sel. 12 Membran sel yang mengalami cedera menyebabkan membran sel tidak mampu memompa ion natrium yang cukup, dengan demikian kenaikan konsentrasi natrium dalam sel menarik air masuk ke dalam sel. Mekanisme ini yang diduga dapat merusak otot jantung dan dapat bermanifestasi pada perubahan ukuran ketebalan sel miokardium.…”