RESUMO
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.
Assuntos
Impedância Elétrica , Eletroporação , Eletroporação/métodos , Porosidade , Animais , Humanos , Sobrevivência CelularRESUMO
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.
RESUMO
Non-colloidal suspensions undergoing dipolar interactions in an electric field have been extensively studied and are also known as smart materials as they share similarities with electrorheological (ER) fluids. Although the macroscopic responses are well-documented, the multiscale nature of such suspensions is still lacking. In this study, a large-scale Stokesian dynamics simulation is used to investigate the structural formation of such suspensions in an electric field up to highly concentrated regimes across different length scales: from particle-level (microscale) to particle cluster-level (mesoscale) and stress response-level (macroscale). It is observed that at a volume fraction of Ï ≈ 30%, the steady-state structures are the most isotropic at the microscale, but at the macroscale, their normal stress fields are the most anisotropic. Interestingly, these structures are also the most heterogeneous at both the microscale and mesoscale. Furthermore, the effects of confinement on the multiscale responses are explored, revealing that there could be a strong link between the mesoscale and macroscale. This multiscale nature can offer the potential for precisely controlling or designing ER fluids in practical applications.
RESUMO
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.
