The effects of electroporation buffer composition on cell viability and electro-transfection efficiency

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/m 2). The energy of the pulse application primarily dictated cell viability, with Mg 2+-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg 2+-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, Mg 2+ 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.

Optimized guide RNA selection recommendations for usingspCas9 gene editing in human hematopoietic stem and progenitor cells

“…The copyright holder for this this version posted December 19, 2020. ; https://doi.org/10.1101/2020.12.18.423329 doi: bioRxiv preprint ideal in the 1M buffers that are used for CD34+ cells, and can possibly be improved by lowering the Mg2+ concentration and increase the K+ concentration, as Mg2+ and K+ have been linked to survival and transfer efficacy respectively [49]. Previously, ATP leaks are observed during electroporation as well as other cellular substances that are depleted from the cytosol [50,51].…”

Transfection types, methods and strategies: a technical review

“…For instance, the use of fetal bovine serum was shown to yield better transduction efficiency than bovine calf serum during transduction (Denning et al, 2013). (Haas et al, 2000;Ikeda et al, 2002;Denning et al, 2013) Potter & Heller, 2003;Molnar et al, 2004;Yao et al, 2009;Hornstein et al, 2016;Chopra et al, 2020;Sherba et al, 2020) (Stevenson et al, 2006;Terakawa et al, 2006;Tsen et al, 2009) (Wells et al, 1998;Dahlhoff et al, 2012;Mellott, Forrest & Detamore, 2013;Chow et al, 2016) (Ogawa, Tachibana & Kondo, 2006;Kinoshita & Hynynen, 2007;Zhou et al, 2012) (Mykhaylyk et al, 2007;Fouriki et al, 2010;Kardos & Rabussay, 2012;Wang et al, 2014) Likewise, polycations such as DEAE-dextran were shown to minimize repulsion forces between negatively-charged cells and facilitated viral transduction (Denning et al, 2013).…”

The Ins and Outs of Messenger RNA Electroporation for Physical Gene Delivery in Immune Cell-Based Therapy

“…The conductivity of the electroporation buffer, marked by its salt content, and the cell concentration are two of the main parameters that affect the resistance of the sample during electroporation [ 14 ]. Moreover, the composition of the buffer, especially the content in salts and sugars, may have a negative effect on cell viability and transfection efficiency [ 15 , 16 , 17 ]. Related to this, the presence of remaining salts in the cell and nucleic acid suspension may increase the final concentration of salts in the electroporation mixture leading to arcing [ 18 ].…”