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Comparison Between Direct and Reverse Electroporation of Cells in Situ: a Simulation Study Leila Towhidi Imperial College London

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Comparison Between Direct and Reverse Electroporation of Cells in Situ: a Simulation Study Leila Towhidi Imperial College London View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UNL | Libraries University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Mechanical & Materials Engineering Faculty Mechanical & Materials Engineering, Department Publications of 2016 Comparison between direct and reverse electroporation of cells in situ: a simulation study Leila Towhidi Imperial College London Delaram Khodadadi Imperial College London Nataly Maimari Imperial College London Ryan M. Pedrigi University of Nebraska-Lincoln, [email protected] Henry Ip Imperial College London See next page for additional authors Follow this and additional works at: http://digitalcommons.unl.edu/mechengfacpub Part of the Mechanics of Materials Commons, Nanoscience and Nanotechnology Commons, Other Engineering Science and Materials Commons, and the Other Mechanical Engineering Commons Towhidi, Leila; Khodadadi, Delaram; Maimari, Nataly; Pedrigi, Ryan M.; Ip, Henry; Kis, Zoltan; Kwak, Brenda R.; Petrova, Tatiana W.; Delorenzi, Mauro; and Krams, Rob, "Comparison between direct and reverse electroporation of cells in situ: a simulation study" (2016). Mechanical & Materials Engineering Faculty Publications. 296. http://digitalcommons.unl.edu/mechengfacpub/296 This Article is brought to you for free and open access by the Mechanical & Materials Engineering, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Mechanical & Materials Engineering Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Leila Towhidi, Delaram Khodadadi, Nataly Maimari, Ryan M. Pedrigi, Henry Ip, Zoltan Kis, Brenda R. Kwak, Tatiana W. Petrova, Mauro Delorenzi, and Rob Krams This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/mechengfacpub/296 Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Comparison between direct and reverse electroporation of cells in situ: a simulation study Leila Towhidi1, Delaram Khodadadi1, Nataly Maimari1, Ryan M. Pedrigi1, Henry Ip1, Zoltan Kis1, Brenda R. Kwak2,3, Tatiana W. Petrova4, Mauro Delorenzi4 & Rob Krams1 1 Department of Bioengineering, Imperial College London, London, United Kingdom 2 Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland 3 Department of Medical Specializations – Cardiology, University of Geneva, Geneva, Switzerland 4 Department of Oncology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland Keywords Abstract Electroporation, high-throughput techniques, transfection efficiency. The discovery of the human genome has unveiled new fields of genomics, transcriptomics, and proteomics, which has produced paradigm shifts on how Correspondence to study disease mechanisms, wherein a current central focus is the under- Rob Krams, Chair in Molecular standing of how gene signatures and gene networks interact within cells. These Bioengineering, Department of gene function studies require manipulating genes either through activation or Bioengineering, Imperial College London, inhibition, which can be achieved by temporarily permeabilizing the cell Room 3.15, Royal School of Mines, Exhibition membrane through transfection to deliver cDNA or RNAi. An efficient trans- Road SW7 2AZ, London. Tel: +442075941473 fection technique is electroporation, which applies an optimized electric pulse Fax: +442075949817 to permeabilize the cells of interest. When the molecules are applied on top of E-mail: [email protected] seeded cells, it is called “direct” transfection and when the nucleic acids are printed on the substrate and the cells are seeded on top of them, it is termed Funding Information “reverse” transfection. Direct transfection has been successfully applied in pre- We acknowledge support from the British vious studies, whereas reverse transfection has recently gained more attention Heart Foundation (RG/11/13/29055). This in the context of high-throughput experiments. Despite the emerging impor- work was supported by Swiss National Science Foundation (CRSII3-141811). tance, studies comparing the efficiency of the two methods are lacking. In this study, a model for electroporation of cells in situ is developed to address this Received: 14 September 2015; Revised: 19 deficiency. The results indicate that reverse transfection is less efficient than November 2015; Accepted: 10 December direct transfection. However, the model also predicts that by increasing the 2015 concentration of deliverable molecules by a factor of 2 or increasing the applied voltage by 20%, reverse transfection can be approximately as efficient doi: 10.14814/phy2.12673 as direct transfection. Physiol Rep, 4 (6), 2016, e12673, doi: 10.14814/phy2.12673 Introduction Wes et al. 2016). These gene networks often consist of more than 1000 genes with a huge amount of interactions The discovery of the human genome has unveiled new between them. As an approach to understand the topol- fields of genomics, transcriptomics, and proteomics ogy and complex interactions of these gene networks, one (Duscher et al. 2016; Stegle et al. 2015; Taher et al. 2015; needs to be able to manipulate genes either through acti- Wes et al. 2016). This has produced a paradigm shift on vation or inhibition. However, the cell membrane is fairly understanding the pathogenesis of disease, which is now impermeable to external molecules, including DNA and increasingly defined by gene signatures and gene networks RNA (Xiang and Chen 2000; Wu et al. 2002; Dorsett and (Duscher et al. 2015; Stegle et al. 2015; Taher et al. 2015; Tuschi 2004). To overcome this permeability barrier, ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2016 | Vol. 4 | Iss. 6 | e12673 the American Physiological Society and The Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Comparison Between Direct and Reverse Electroporation of Cells L. Towhidi et al. transfection methods such as lipofection, viral transduc- for electroporation, optical transparency provides the tion, and electroporation have been developed (Rama- capability to observe and examine the cells by microscope moorth and Narvekar 2015; Silva et al. 2015). Lipofection (Raptis and Firth 2008; Raptis et al. 2008; Santra et al. is a lipid-mediated transfection method that delivers 2013). molecules to the cell by means of a liposome that easily There are two types of in situ electroporation tech- merges with the cell membrane (Ramamoorth and Narve- niques: direct and reverse. In the case of direct transfec- kar 2015; Silva et al. 2015). Despite numerous efforts, the tion, the nucleic acids are in the medium above the efficiency of this technique to transfer molecules is low in cells, and a pulse with appropriate parameters is applied primary mammalian cells (Ramamoorth and Narvekar to the adherent cells for transfection. For reverse trans- 2015; Silva et al. 2015). Viral transduction involves the fection, the nucleic acids are first added to the substrate transfer of molecules encapsulated in viral vectors such as and then the cells are seeded on top of them. A pulse lentivirus or adenovirus. The limitations of viral transduc- with optimized parameters is applied at the desired time tion include labor insensitivity causing low throughput, to the cells seeded on the substrate covered by nucleic need for safety measures, insertional mutagenesis, DNA acids. Direct electroporation has been successfully package size limit, and immunogenicity (Ramamoorth applied in previous studies (Li and Ma 2001; Li 2004); and Narvekar 2015; Silva et al. 2015). For electroporation, on the other hand, reverse transfection has gained more an electric pulse is applied to the cell to permeabilize the attention recently. Reverse transfection requires fewer cell membrane and allow uptake of external molecules. nucleic acids for experiments, so it is more cost-effec- Under optimized pulse parameters, the membrane returns tive, and it has the potential advantage of allowing high- to its intact state (Neumann et al. 1982; Felgner et al. throughput experiments (Li and Ma 2001; Li 2004). The 1987; Plank et al. 2003; Bonetta 2005). Recent studies approach is to seed cells onto a surface that is typically have shown that electroporation or nucleofection are coated with either siRNA or cDNA/CRISPr, which needs indeed the most efficient transfection methods for diffi- to be transfected into the cells by then applying an elec- cult-to-transfect primary cell types (Gilbert et al. 2008; Lu tric pulse (Li and Ma 2001; Li 2004). For a high- et al. 2008). throughput experimental design, different nucleic acids In conventional electroporation, cells are first detached need to be dispensed at different spots over a suitable from the substrate and then exposed to electric pulses in substrate to allow the cells attached to these spots to be the presence of the desired extracellular molecules which efficiently reverse transfected without cross contamina- are dissolved in the cell suspension. The problem with tion. The conditions leading to optimal transfection effi- this approach is that electroporation of cells in suspension ciency are currently unknown and need to be further can adversely affect cell viability and normal cellular func- studied. tion (Gowrishankar
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