Optically transparent polymer devices for in situ assessment of cell electroporation

Abstract

In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 μl was quantified in situ as a function of applied voltage from 10 to 90 V in a series of ~2-ms pulses across 0.5-mm electrode spacing. The electric field at the interface and device current was calculated using a model that takes into account bulk screening of the transient pulse. The voltage dependence of the number of electroporated cells could be explained using a stochastic model for the electroporation kinetics, and the free energy for pore formation was found to be 45.6 ± 0.5 kT at room temperature. With this device, the optimum electroporation conditions can be quickly determined by monitoring the uptake of PI marker dye in situ under the application of millisecond voltage pulses. The electroporation efficiency was also quantified using an ex situ fluorescence-assisted cell sorter, and the morphology of cultured cells was evaluated after the pulsing experiment. Importantly, the efficacy of the developed device was tested independently using two cell lines (C2C12 mouse myoblast cells and yeast cells) as well as in three different electroporation buffers (phosphate buffer saline, electroporation buffer and 10% glycerol).

Estimation of activation energy for electroporation and pore growth rate in liquid crystalline and gel phases of lipid bilayers using molecular dynamics simulations

“…Electroporation is a stochastic process that can be reversible or irreversible depending on the field strength and duration of the applied field. 5,[70][71][72] As reported by other groups, 19 our all atomistic molecular dynamics simulations suggest that interfacial water plays a key role in electroporation. 19,73 In our study, the water defect formed at the water/lipid interface and entered the bilayer from opposing leaflets to form the pore inside the bilayer.…”

“…During electroporation, the membrane permeability increases as a function of time. [1][2][3][4][5][6] Although electroporation is extensively used in biology, biotechnology and medicine, [7][8][9] the cascade of events that occur during this phenomenon remain elusive. There exist several molecular dynamics (MD) simulation studies [10][11][12][13][14] which reveal microscopic level insights into the mechanism of pore formation in model lipid bilayers.…”