Confocal Microscopy Improves 3D Microdosimetry Applied to Nanoporation Experiments Targeting Endoplasmic Reticulum

Frontiers in Bioengineering and Biotechnology

Micaela Liberti¹  Francesca Apollonio¹  Annalisa De Angelis¹  Agnese Denzi²  Caterina Merla³  Lluis M. Mir⁴  Frank M. Andre⁴ 

[1] Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy;Inter University Center for the Study of Electromagnetic Fields and Biological Systems (ICEmB) at Department of Electronic Engineering and Telecommunications (DIET), University of Rome “La Sapienza”, Rome, Italy;National Italian Agency for Energy, New Technologies and Sustainable Economic Development – Department of Sustainability (ENEA, SSPT) – Division of Health Protection Technologies, Rome, Italy;Université Paris-Saclay, Institut Gustave Roussy, CNRS, Metabolic and Systemic Aspects of Oncogenesis, Villejuif, France;

关键词: confocal fluorescence microscopy;    microdosimetry;    electroporation;    electropermeabilization;    nanosecond pulses;    nanoporation experiments;   

DOI  :  10.3389/fbioe.2020.552261

来源: DOAJ

【 摘 要 】

In the last years, microdosimetric numerical models of cells including intracellular compartments have been proposed, aiming to investigate the poration induced by the application of nanosecond pulsed electric fields (nsPEFs). A limitation of such models was the extremely approximate cell and organelle shapes, leading to an incorrect estimation of the electric field or transmembrane potential distribution in the studied domain. In order to obtain a reliable model of in vitro experiments and a one-to-one comparison between experimental and simulated results, here, a realistic model of 12 human mesenchymal stem cells was built starting from their optical microscopy images where different cell compartments were highlighted. The microdosimetric analysis of the cells group was quantified in terms of electric field and transmembrane potentials (TMPs) induced by an externally applied 10-ns trapezoidal pulse with rise and fall times of 2 ns, with amplitudes ranging from 2 to 30 MV/m. The obtained results showed that the plasma and endoplasmic reticulum (ER) membrane of each cell respond in a different way to the same electric field amplitude, depending on differences in shape, size, and position of the single cell with respect to the applied electric field direction. Therefore, also the threshold for an efficient electroporation is highly different from cell to cell. This difference was quantitatively estimated through the cumulative distribution function of the pore density for the plasma and ER membrane of each cell, representing the probability that a certain percentage of membrane has reached a specific value of pore density. By comparing the dose-response curves resulted from the simulations and those from the experimental study of De Menorval et al. (2016), we found a very good matching of results for plasma and ER membrane when 2% of the porated area is considered sufficient for permeabilizing the membrane. This result is worth of noting as it highlights the possibility to effectively predict the behavior of a cell (or of a population of cells) exposed to nsPEFs. Therefore, the microdosimetric realistic model described here could represent a valid tool in setting up more efficient and controlled electroporation protocols.

【 授权许可】

Unknown