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1. A Low-Cost Electroporator for Genetically Modifying Social Amoeba Dictyostelium Discoideum

   https://doi.org/10.5334/joh.52  

   Cauchy, Michael C.; Khan, Ali A.; Artemenko, Yulia; Dunn, David A. (January 2023, Journal of Open Hardware)

   The social amoeba Dictyostelium discoideum is a commonly used eukaryotic model organism for the study of cell division, chemotaxis, differentiation, phagocytosis, and other cellular processes. Electroporation is an effective and efficient method for delivering plasmid DNA into D. discoideum, an invaluable tool for studying intracellular processes. The technology is readily available but often prohibitively expensive. Although several custom-built electroporation devices have been developed, none deliver the specific 8.5kV/cm exponentially decaying waveform required for D. discoideum transformation. The present study examined whether a simple, inexpensive device can be built to produce this waveform through a simple resistor-capacitor (RC) circuit. A pulse generator RC circuit was built incorporating inexpensive electronic components and a 3D printed cuvette chamber. All four possible combinations of custom-built and commercial pulse generators and custom-built and commercial cuvette chambers were used to transform D. discoideum cells with a plasmid encoding green fluorescent protein (GFP). There were no significant differences in the number of surviving cells immediately following or 24 hours post-transformation between the systems. All combinations of custom-built and commercial systems achieved comparably high transformation efficiency shown by percent of cells expressing GFP six days after the transformation. Since the waveform-specific electroporation system we present here can be built by non-experts with easily obtainable materials and 3D printing, we envision this device to benefit investigators in areas with low research budgets and educators in multiple STEM fields. 

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2. 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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3. 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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4. 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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5. CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

   https://doi.org/10.1093/nar/gkac049  

   Lin-Shiao, Enrique; Pfeifer, Wolfgang G.; Shy, Brian R.; Saffari Doost, Mohammad; Chen, Evelyn; Vykunta, Vivasvan S.; Hamilton, Jennifer R.; Stahl, Elizabeth C.; Lopez, Diana M.; Sandoval Espinoza, Cindy R.; et al (February 2022, Nucleic Acids Research)

   Abstract DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei. 

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