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.
more »
« less
This content will become publicly available on June 1, 2025
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.
more » « less- Award ID(s):
- 1936065
- PAR ID:
- 10557214
- Publisher / Repository:
- Small
- Date Published:
- Journal Name:
- Small
- Volume:
- 20
- Issue:
- 25
- ISSN:
- 1613-6810
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract Isolation of cells and their transfection in a controlled manner is an integral step in cell biotechnology. Electric field approaches such as dielectrophoresis (DEP) offers a more viable method for targeted immobilization of cells without any labels. For transfection of cells to incorporate exogenous materials, electrical methods such as electroporation, are preferred over chemical and viral delivery methods since they minimally affect cell viability and can target many types. However prior approaches to both methods required multiple excitation sources, an AC source for DEP-based trapping and another DC source for electroporation. In this paper, we present a first of its kind flow through lab-on-chip platform using a single AC excitation source for combined trapping using negative dielectrophoresis (nDEP) and AC electroporation. Use of AC fields for electroporation eliminates the unwanted side effects of electrolysis or joule heating at electrodes compared to DC electroporation. Adjusting the flow rate and the electrical parameters of the incident AC field precisely controls the operation (trap, trap with electroporation and release). The platform has been validated through trapping and simultaneous transfection of HEK-293 embryonic kidney cells with a plasmid vector containing a fluorescent protein tag. Numerical scaling analysis is provided that indicates promise for individual cell trapping and electroporation using low voltage AC fields.
-
more » « less
Abstract Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m2). The energy of the pulse application primarily dictated cell viability, with Mg2+-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg2+-based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg2+also hinders eTE compared to K+-based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.
-
more » « less
Abstract Background Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translation of EVs into viable therapeutic delivery vehicles is challenged by lengthy and inefficient processes for cargo loading and pre- and post-loading purification of EVs, resulting in limited quantity and consistency of engineered EVs.
Results In this work, we develop a fast and streamlined method to load surface protein-specific subpopulations of EVs with miRNA by electroporating EVs, while they are bound to antibody-coated beads. We demonstrate the selection of CD81+EV subpopulation using magnetic microbeads, facilitating rapid EV manipulations, loading, and subsequent purification processes. Our approach shortens the time per post-electroporation EV wash by 20-fold as compared to the gold standard EV washing method, ultracentrifugation, resulting in about 2.5-h less time required to remove unloaded miRNA. In addition, we addressed the challenge of nonspecific binding of cargo molecules due to affinity-based EV selection, lowering the purity of engineered EVs, by implementing innovative strategies, including poly A carrier RNA-mediated blocking and dissociation of residual miRNA and EV-like miRNA aggregates following electroporation.
Conclusions Our streamlined method integrates magnetic bead-based selection with electroporation, enabling rapid and efficient loading of miRNA into CD81+EVs. This approach not only achieves comparable miRNA loading efficiency to conventional bulk electroporation methods but also concentrates CD81+EVs and allows for simple electroporation parameter adjustment, promising advancements in therapeutic RNA delivery systems with enhanced specificity and reduced toxicity.
-
more » « less
Abstract In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow‐through microfluidics, engineered substrates, and automated probe‐based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate‐based electroporation platforms and high throughput, high control methods in general.
