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1. Transepithelial electrical impedance increase following porous substrate electroporation enables label-free delivery

   Brooks, Justin R; Heiman, Tyler C; Lorenzen, Sawyer R; Mungloo, Ikhlaas; Mirfendereski, Siamak; Park, Jae Sung; Yang, Ruiguo (February 2024, Small)

   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. 

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2. High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery

   https://doi.org/10.1002/smll.202004917  

   Brooks, Justin; Minnick, Grayson; Mukherjee, Prithvijit; Jaberi, Arian; Chang, Lingqian; Espinosa, Horacio D.; Yang, Ruiguo (November 2020, Small)

   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. 

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3. Robust Three-dimensional Nanotube-in-Micropillar Array Electrodes to Facilitate Size Independent Electroporation in Blood Cell Therapy

   https://doi.org/10.1039/D1LC00690H  

   Liu, Xuan; Chang, An-Yi; Ma, Yifan; Hua, Liping; Yang, Zhaogang; Wang, Shengnian (September 2021, Lab on a Chip)

   null (Ed.)

   Blood is an attractive carrier for plasmid and RNA based medicine in cell therapy. Electroporation serves as its favorable delivery tool for simple operation, quick internalization, minimum cell culture involvement, and low contamination risk. However, the delivery outcomes of electroporation heavily depend on the treated cells such as their type, size, and orientation to the electric field, not ideal for highly heterogeneous blood samples. Herein a new electroporation system was developed towards effective transfection to cells in blood regardless their large diversity. By coupling replica molding and infiltration coating processes, we successfully configured a three-dimensional electrode comprised of a polymer micropillar array on which carbon nanotubes (CNTs) are partially embedded. During electroporation, cells sag between micropillars and deform to form conformal contact with their top and side surface. The implanted CNTs not only provide a robust conductive coating for the polymer micropattern, but also have their protruded ends face the cell membrane vertically everywhere with maximum transmembrane potential. Regardless their largely varied sizes and random dispersion, both individual blood cell type and whole blood samples were effectively transfected with plasmid DNA (85% after 24 hrs and 95% after 72 hrs, or 2.5-3.0 folds enhancement). High-dose RNA probes were also introduced which regulate better the expression levels of exogenous and endogenous genes in blood cells. Besides its promising performance on non-viral delivery route to cell-related studies and therapy, the invovled new fabrication method also provides a convenient and effective way to construct flexible electronics with stable micro/nanofeatures on the surface. 

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4. A portable impedance microflow cytometer for measuring cellular response to hypoxia

   https://doi.org/10.1002/bit.27879  

   Dieujuste, Darryl; Qiang, Yuhao; Du, E. (July 2021, Biotechnology and Bioengineering)

   Abstract This article presents the development and testing of a low‐cost (<$60), portable, electrical impedance‐based microflow cytometer for single‐cell analysis under a controlled oxygen microenvironment. The system is based on an AD5933 impedance analyzer chip, a microfluidic chip, and an Arduino microcontroller operated by a custom Android application. A representative case study on human red blood cells (RBCs) affected by sickle cell disease is conducted to demonstrate the capability of the cytometry system. Impedance values of sickle blood samples exhibit remarkable deviations from the common reference line obtained from two normal blood samples. Such deviation is quantified by a conformity score, which allows for the measurement of intrapatient and interpatient variations of sickle cell disease. A low conformity score under oxygenated conditions or drastically different conformity scores between oxygenated and deoxygenated conditions can be used to differentiate a sickle blood sample from normal. Furthermore, an equivalent circuit model of a suspended biological cell is used to interpret the electrical impedance of single flowing RBCs. In response to hypoxia treatment, all samples, regardless of disease state, exhibit significant changes in at least one single‐cell electrical property, that is, cytoplasmic resistance and membrane capacitance. The overall response to hypoxia is less in normal cells than those affected by sickle cell disease, where the change in membrane capacitance varies from −23% to seven times as compared with −17% in normal cells. The results reported in this article suggest that the developed method of testing demonstrates the potential application for a low‐cost screening technique for sickle cell disease and other diseases in the field and low‐resource settings. The developed system and methodology can be extended to analyze cellular response to hypoxia in other cell types. 

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5. Microfluidic Electroporation Coupling Pulses of Nanoseconds and Milliseconds to Facilitate Rapid Uptake and Enhanced Expression of DNA in Cell Therapy

   https://doi.org/10.1038/s41598-020-63172-8  

   Chang, An-Yi; Liu, Xuan; Tian, Hong; Hua, Liping; Yang, Zhaogang; Wang, Shengnian (April 2020, Scientific Reports)

   Abstract Standard electroporation with pulses in milliseconds has been used as an effective tool to deliver drugs or genetic probes into cells, while irreversible electroporation with nanosecond pulses is explored to alter intracellular activities for pulse-induced apoptosis. A combination treatment, long nanosecond pulses followed by standard millisecond pulses, is adopted in this work to help facilitate DNA plasmids to cross both cell plasma membrane and nuclear membrane quickly to promote the transgene expression level and kinetics in both adherent and suspension cells. Nanosecond pulses with 400–800 ns duration are found effective on disrupting nuclear membrane to advance nuclear delivery of plasmid DNA. The additional microfluidic operation further helps suppress the negative impacts such as Joule heating and gas bubble evolution from common nanosecond pulse treatment that lead to high toxicity and/or ineffective transfection. Having appropriate order and little delay between the two types of treatment with different pulse duration is critical to guarantee the effectiveness: 2 folds or higher transfection efficiency enhancement and rapid transgene expression kinetics of GFP plasmids at no compromise of cell viability. The implementation of this new electroporation approach may benefit many biology studies and clinical practice that needs efficient delivery of exogenous probes. 

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