porous-substrate-based-electroporation-with-transepithelial

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

University of Nebraska-Lincoln

In vitro intracellular delivery is a fundamental challenge with no widely adopted methods capable of both delivering to millions of cells and controlling that delivery to a high degree of accuracy. One promising method is porous substrate electroporation (PSEP), where cells are cultured on porous substrates and electric fields are used to permeabilize discrete portions of the cell membrane for delivery. A major obstacle to the widespread use of PSEP is a poor understanding of the various impedances that constitute the system, including the impedances of the porous substrate and the cell monolayer, and how these impedances are influenced by experimental parameters. In response, we used impedance measurements to develop an equivalent circuit model that closely mimics the behavior of each of the main components of the PSEP system. This circuit model reveals for the first time the distribution of voltage across the electrode-electrolyte interface impedances, the channels of the porous substrate, the cell monolayer, and the transmembrane potential during PSEP. We applied sample waveforms through our model to understand how waveforms can be improved for future studies. Our model was validated from intracellular delivery of protein using PSEP.

An equivalent circuit model for localized electroporation on porous substrates.

Complex patterns of hemodynamic wall shear stress occur in regions of arterial branching and curvature. Areas within these regions can be highly susceptible to atherosclerosis. Although many studies have characterized the response of vascular endothelial cells to shear stress in a categorical manner, our study herein addresses the need of characterizing endothelial behaviors over a continuous range of shear stress conditions that reflect the extensive variations seen in the vasculature. We evaluated the response of human umbilical vein endothelial cell monolayers to orbital flow at 120, 250, and 350 revolutions per minute (RPM) for 24 and 72 h. The orbital shaker model uniquely provides a continuous range of shear stress conditions from low and multidirectional at the center of each well of a culture plate to high and unidirectional at the periphery. We found distinct patterns of endothelial nuclear area, nuclear major and minor diameters, nuclear aspect ratio, and expression of endothelial nitric oxide synthase over this range of shear conditions and relationships were fit with linear and, where appropriate, power functions. Nuclear area was particularly sensitive with increases in the low and multidirectional WSS region that incrementally decreased as WSS became higher in magnitude and more unidirectional over the radius of the cell layers. The patterns of all endothelial behaviors exhibited high correlations (positive and negative) with metrics of shear stress magnitude and directionality that have been shown to strongly associate with atherosclerosis. Our findings demonstrate the exquisite sensitivity of these endothelial behaviors to incremental changes in shear stress magnitude and directionality, and provide critical quantitation of these relationships for predicting the susceptibility of an arterial segment to diseases such as atherosclerosis, particularly within complex flow environments in the vasculature such as around bifurcations.

Characterizing nuclear morphology and expression of eNOS in vascular endothelial cells subjected to a continuous range of wall shear stress magnitudes and directionality.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of the PSEP delivery process are not well understood, partly because most PSEP studies rely solely on imaging for evaluating delivery. Although effective, imaging alone limits understanding of intermediate processes leading to delivery. PSEP is an electrical process, so electrical impedance measurements naturally complement imaging for PSEP characterization. In this study, we developed a device capable of measuring impedance and performing PSEP and we monitored changes in transepithelial electrical impedance (TEEI). Our measurements show TEEI increases following PSEP, unlike other electroporation methods. We then demonstrated how cell culture conditions and electrical waveforms influence this response. More importantly, we correlated TEEI response features with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which we expect will aid PSEP optimization for new cell types and cargos.

Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery.

Porous substrate electroporation (PSEP) is a promising new method for intracellular delivery, yet fundamentals of PSEP are not well understood, especially the intermediate processes leading to delivery. PSEP is an electrical method, yet the relationship between PSEP and electrical impedance remains underexplored. In this study, a device capable of measuring impedance and performing PSEP is developed and the changes in transepithelial electrical impedance (TEEI) are monitored. These measurements show TEEI increases following PSEP, unlike other electroporation methods. The authors then demonstrate how cell culture conditions and electrical waveforms influence this response. More importantly, TEEI response features are correlated with viability and delivery efficiency, allowing prediction of outcomes without fluorescent cargo, imaging, or image processing. This label-free delivery also allows improved temporal resolution of transient processes following PSEP, which the authors expect will aid PSEP optimization for new cell types and cargos.

Article	Year
An equivalent circuit model for localized electroporation on porous substrates. Biosensors & bioelectronics\| PubMed ID: 34923307	2022
Characterizing nuclear morphology and expression of eNOS in vascular endothelial cells subjected to a continuous range of wall shear stress magnitudes and directionality. Journal of the mechanical behavior of biomedical materials\| PubMed ID: 36368188	2023
Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery. bioRxiv : the preprint server for biology\| PubMed ID: 37905105	2023
Transepithelial Electrical Impedance Increase Following Porous Substrate Electroporation Enables Label-Free Delivery. Small (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 38396158	2024

Justin R. Brooks

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