Showing papers on "Electroporation published in 2013"

Transient Mammalian Cell Transfection with Polyethylenimine (PEI)

Abstract: Standard protein expression systems, such as E. coli, often fail to produce folded, monodisperse, or functional eukaryotic proteins (see Small-scale Expression of Proteins in E. coli). The expression of these proteins is greatly benefited by using a eukaryotic system, such as mammalian cells, that contains the appropriate folding and posttranslational machinery. Here, we describe methods for both small- and large-scale transient expression in mammalian cells using polyethylenimine (PEI). We find this procedure to be more cost-effective and quicker than the more traditional route of generating stable cell lines. First, optimal transfection conditions are determined on a small-scale, using adherent cells. These conditions are then translated for use in large-scale suspension cultures. For further details on generating stable cell lines please (see Rapid creation of stable mammalian cell lines for regulated expression of proteins using the Gateway® Recombination Cloning Technology and Flp-In T-REx® lines or Generating mammalian stable cell lines by electroporation).

Nanostraw–Electroporation System for Highly Efficient Intracellular Delivery and Transfection

Abstract: Nondestructive introduction of genes, proteins, and small molecules into mammalian cells with high efficiency is a challenging, yet critical, process. Here we demonstrate a simple nanoelectroporation platform to achieve highly efficient molecular delivery and high transfection yields with excellent uniformity and cell viability. The system is built on alumina nanostraws extending from a track-etched membrane, forming an array of hollow nanowires connected to an underlying microfluidic channel. Cellular engulfment of the nanostraws provides an intimate contact, significantly reducing the necessary electroporation voltage and increasing homogeneity over a large area. Biomolecule delivery is achieved by diffusion through the nanostraws and enhanced by electrophoresis during pulsing. The system was demonstrated to offer excellent spatial, temporal, and dose control for delivery, as well as providing high-yield cotransfection and sequential transfection.

Microfluidic electroporation for cellular analysis and delivery

Abstract: Electroporation is a simple yet powerful technique for breaching the cell membrane barrier. The applications of electroporation can be generally divided into two categories: the release of intracellular proteins, nucleic acids and other metabolites for analysis and the delivery of exogenous reagents such as genes, drugs and nanoparticles with therapeutic purposes or for cellular manipulation. In this review, we go over the basic physics associated with cell electroporation and highlight recent technological advances on microfluidic platforms for conducting electroporation. Within the context of its working mechanism, we summarize the accumulated knowledge on how the parameters of electroporation affect its performance for various tasks. We discuss various strategies and designs for conducting electroporation at the microscale and then focus on analysis of intracellular contents and delivery of exogenous agents as two major applications of the technique. Finally, an outlook for future applications of microfluidic electroporation in increasingly diverse utilities is presented.

Modeling of electric field distribution in tissues during electroporation

Abstract: Electroporation based therapies and treatments (e.g. electrochemotherapy, gene electrotransfer for gene therapy and DNA vaccination, tissue ablation with irreversible electroporation and transdermal drug delivery) require a precise prediction of the therapy or treatment outcome by a personalized treatment planning procedure. Numerical modeling of local electric field distribution within electroporated tissues has become an important tool in treatment planning procedure in both clinical and experimental settings. Recent studies have reported that the uncertainties in electrical properties (i.e. electric conductivity of the treated tissues and the rate of increase in electric conductivity due to electroporation) predefined in numerical models have large effect on electroporation based therapy and treatment effectiveness. The aim of our study was to investigate whether the increase in electric conductivity of tissues needs to be taken into account when modeling tissue response to the electroporation pulses and how it affects the local electric distribution within electroporated tissues. We built 3D numerical models for single tissue (one type of tissue, e.g. liver) and composite tissue (several types of tissues, e.g. subcutaneous tumor). Our computer simulations were performed by using three different modeling approaches that are based on finite element method: inverse analysis, nonlinear parametric and sequential analysis. We compared linear (i.e. tissue conductivity is constant) model and non-linear (i.e. tissue conductivity is electric field dependent) model. By calculating goodness of fit measure we compared the results of our numerical simulations to the results of in vivo measurements. The results of our study show that the nonlinear models (i.e. tissue conductivity is electric field dependent: σ(E)) fit experimental data better than linear models (i.e. tissue conductivity is constant). This was found for both single tissue and composite tissue. Our results of electric field distribution modeling in linear model of composite tissue (i.e. in the subcutaneous tumor model that do not take into account the relationship σ(E)) showed that a very high electric field (above irreversible threshold value) was concentrated only in the stratum corneum while the target tumor tissue was not successfully treated. Furthermore, the calculated volume of the target tumor tissue exposed to the electric field above reversible threshold in the subcutaneous model was zero assuming constant conductivities of each tissue. Our results also show that the inverse analysis allows for identification of both baseline tissue conductivity (i.e. conductivity of non-electroporated tissue) and tissue conductivity vs. electric field (σ(E)) of electroporated tissue. Our results of modeling of electric field distribution in tissues during electroporation show that the changes in electrical conductivity due to electroporation need to be taken into account when an electroporation based treatment is planned or investigated. We concluded that the model of electric field distribution that takes into account the increase in electric conductivity due to electroporation yields more precise prediction of successfully electroporated target tissue volume. The findings of our study can significantly contribute to the current development of individualized patient-specific electroporation based treatment planning.

Safety and Comparative Immunogenicity of an HIV-1 DNA Vaccine in Combination with Plasmid Interleukin 12 and Impact of Intramuscular Electroporation for Delivery

Abstract: DNA-based immunization offers several advantages [1]. DNA vaccines contain nonliving, nonreplicating, and nontransmissible material, providing an improved safety profile over live attenuated viral vectors. They do not elicit antivector immunity, retaining potency through multiple boost cycles. In theory, DNA vaccines are simple and relatively inexpensive to construct, readily produced in large quantities, easy to characterize, and stable and can be combined into complex formulations. Despite the early enthusiasm from results of studies in small animals, DNA vaccines have not generated robust immune responses in humans [2–6]. The amount of antigen produced by each transfected cell is low because of the low transcription rate of antigen sequences being driven off the cytomegalovirus promoter [7–9]. One approach to augment the immunogenicity of DNA is to combine the DNA vaccine with a plasmid cytokine adjuvant [10–13]. Interleukin 12 (IL-12) is a key cytokine for the induction of cellular immune responses [14, 15]. Interleukin 15 (IL-15) is a member of the common cytokine receptor γ-chain family [16–18] that fosters development of long-lived memory T-cell responses [19–21]. A newer strategy for increasing immune potency has been to deliver the plasmids with in vivo electroporation. Electroporation enhances uptake of DNA into cells by temporarily generating an electrical field that increases the permeability of cell membranes and moves the macromolecules through the briefly open membrane pores. Clinical applications of electroporation have been tested, especially in cancer treatment and gene therapy [22–24]. Electroporation has elicited HIV-specific cellular immune responses in mice [25] and simian immunodeficiency virus–specific immune responses in macaques [26]. In macaque studies, genetic optimization, electroporation, and IL-12 plasmid adjuvant have improved the immunogenicity of DNA vaccines in vivo [26]. More recently, Vasan et al reported on a trial that showed the potential to increase vaccine-induced cellular responses to a DNA vaccine relative to intramuscular injection alone [27]. However, electroporation remains investigational [28] and has not been licensed by the Food and Drug Administration for clinical use. This is the first report on the combination of these approaches in humans. Here, we summarize the results of 2 trials of an HIV plasmid DNA vaccine, PENNVAX®-B (PV), one investigating HIV consensus clade B Gag, Pol, and Env with IL-12 or IL-15 plasmid cytokine adjuvants delivered by intramuscular injection without electroporation (HIV Vaccine Trials Network [HVTN] study 070) and the other investigating the same vaccine with plasmid IL-12 delivered intramuscularly with electroporation (HVTN study 080). The results illustrate the power of these combined DNA approaches to generate impressive immune responses in humans.

Physical non-viral gene delivery methods for tissue engineering.

Abstract: The integration of gene therapy into tissue engineering to control differentiation and direct tissue formation is not a new concept; however, successful delivery of nucleic acids into primary cells, progenitor cells, and stem cells has proven exceptionally challenging. Viral vectors are generally highly effective at delivering nucleic acids to a variety of cell populations, both dividing and non-dividing, yet these viral vectors are marred by significant safety concerns. Non-viral vectors are preferred for gene therapy, despite lower transfection efficiencies, and possess many customizable attributes that are desirable for tissue engineering applications. However, there is no single non-viral gene delivery strategy that “fits-all” cell types and tissues. Thus, there is a compelling opportunity to examine different non-viral vectors, especially physical vectors, and compare their relative degrees of success. This review examines the advantages and disadvantages of physical non-viral methods (i.e., microinjection, ballistic gene delivery, electroporation, sonoporation, laser irradiation, magnetofection, and electric field-induced molecular vibration), with particular attention given to electroporation because of its versatility, with further special emphasis on Nucleofection™. In addition, attributes of cellular character that can be used to improve differentiation strategies are examined for tissue engineering applications. Ultimately, electroporation exhibits a high transfection efficiency in many cell types, which is highly desirable for tissue engineering applications, but electroporation and other physical non-viral gene delivery methods are still limited by poor cell viability. Overcoming the challenge of poor cell viability in highly efficient physical non-viral techniques is the key to using gene delivery to enhance tissue engineering applications.

Synthetic DNA Vaccines: Improved Vaccine Potency by Electroporation and Co-Delivered Genetic Adjuvants

Abstract: In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.

Cell membrane electroporation-Part 2: the applications

Abstract: Electroporation can be used as a tool for extracting or introducing molecules from or into a cell. The most important and promising applications of electroporation in medicine and biotechnology are described.

Improving dendritic cell vaccine immunogenicity by silencing PD-1 ligands using siRNA-lipid nanoparticles combined with antigen mRNA electroporation

Abstract: Dendritic cell (DC)-based vaccination boosting antigen-specific immunity is being explored for the treatment of cancer and chronic viral infections. Although DC-based immunotherapy can induce immunological responses, its clinical benefit has been limited, indicating that further improvement of DC vaccine potency is essential. In this study, we explored the generation of a clinical-grade applicable DC vaccine with improved immunogenic potential by combining PD-1 ligand siRNA and target antigen mRNA delivery. We demonstrated that PD-L1 and PD-L2 siRNA delivery using DLin-KC2-DMA-containing lipid nanoparticles (LNP) mediated efficient and specific knockdown of PD-L expression on human monocyte-derived DC. The established siRNA-LNP transfection method did not affect DC phenotype or migratory capacity and resulted in acceptable DC viability. Furthermore, we showed that siRNA-LNP transfection can be successfully combined with both target antigen peptide loading and mRNA electroporation. Finally, we demonstrated that these PD-L-silenced DC loaded with antigen mRNA superiorly boost ex vivo antigen-specific CD8(+) T cell responses from transplanted cancer patients. Together, these findings indicate that our PD-L siRNA-LNP-modified DC are attractive cells for clinical-grade production and in vivo application to induce and boost immune responses not only in transplanted cancer patients, but likely also in other settings.

Long-term channelrhodopsin-2 (ChR2) expression can induce abnormal axonal morphology and targeting in cerebral cortex

Abstract: Long-term expression of optogenetic proteins including channelrhodopsin-2 (ChR2) is widely used to study neural circuit function, but whether ChR2 expression itself perturbs circuits is not known. We expressed a common construct, CAG::ChR2 (H134R)-EYFP-WPRE, in L2/3 pyramidal cells in rat somatosensory cortex via in utero DNA electroporation (IUE). L2/3 pyramidal cells expressed ChR2-EYFP, but histology revealed abnormal morphology and targeting of ChR2-EYFP expressing axons, beginning at postnatal day (P) 33 and increasing with age. Axonal abnormalities included cylinders that enveloped pyramidal cell proximal apical dendrites, and spherical, calyx-like structures that surrounded neuronal cell bodies, including in L4. These are abnormal subcellular and laminar targets for L2/3 pyramidal cell synapses. Abnormalities did not occur in cells expressing GFP instead of ChR2, or in intermixed ChR2-negative axons. Long-term viral-mediated expression (80 d) did not cause axonal abnormalities when the CAG promoter was used, but produced some abnormalities with the stronger αCaMKII promoter (albeit much less than with in utero electroporation). Thus, under some circumstances high-level, long-term expression of ChR2-EYFP can perturb the structural organization of cortical circuits.

Nanofountain Probe Electroporation (NFP-E) of Single Cells

Abstract: The ability to precisely deliver molecules into single cells is of great interest to biotechnology researchers for advancing applications in therapeutics, diagnostics, and drug delivery toward the promise of personalized medicine. The use of bulk electroporation techniques for cell transfection has increased significantly in the past decade, but the technique is nonspecific and requires high voltage, resulting in variable efficiency and low cell viability. We have developed a new tool for electroporation using nanofountain probe (NFP) technology, which can deliver molecules into cells in a manner that is highly efficient and gentler to cells than bulk electroporation or microinjection. Here we demonstrate NFP electroporation (NFP-E) of single HeLa cells within a population by transfecting them with fluorescently labeled dextran and imaging the cells to evaluate the transfection efficiency and cell viability. Our theoretical analysis of the mechanism of NFP-E reveals that application of the voltage creates a localized electric field between the NFP cantilever tip and the region of the cell membrane in contact with the tip. Therefore, NFP-E can deliver molecules to a target cell with minimal effect of the electric potential on the cell. Our experiments on HeLa cells confirm that NFP-E offers single cell selectivity, high transfection efficiency (>95%), qualitative dosage control, and very high viability (92%) of transfected cells.

Irreversible Electroporation: Treatment Effect Is Susceptible to Local Environment and Tissue Properties

Abstract: The outcome of irreversible electroporation ablation is substantially affected by tissue properties and structure, the local environment, and the orientation of the electrodes.

Snap-, CLIP- and Halo-Tag Labelling of Budding Yeast Cells

Abstract: Fluorescence microscopy of the localization and the spatial and temporal dynamics of specifically labelled proteins is an indispensable tool in cell biology. Besides fluorescent proteins as tags, tag-mediated labelling utilizing self-labelling proteins as the SNAP-, CLIP-, or the Halo-tag are widely used, flexible labelling systems relying on exogenously supplied fluorophores. Unfortunately, labelling of live budding yeast cells proved to be challenging with these approaches because of the limited accessibility of the cell interior to the dyes. In this study we developed a fast and reliable electroporation-based labelling protocol for living budding yeast cells expressing SNAP-, CLIP-, or Halo-tagged fusion proteins. For the Halo-tag, we demonstrate that it is crucial to use the 6′-carboxy isomers and not the 5′-carboxy isomers of important dyes to ensure cell viability. We report on a simple rule for the analysis of 1H NMR spectra to discriminate between 6′- and 5′-carboxy isomers of fluorescein and rhodamine derivatives. We demonstrate the usability of the labelling protocol by imaging yeast cells with STED super-resolution microscopy and dual colour live cell microscopy. The large number of available fluorophores for these self-labelling proteins and the simplicity of the protocol described here expands the available toolbox for the model organism Saccharomyces cerevisiae.

Micro-/nanofluidics based cell electroporation

Abstract: Non-viral gene delivery has been extensively explored as the replacement for viral systems. Among various non-viral approaches, electroporation has gained increasing attention because of its easy operation and no restrictions on probe or cell type. Several effective systems are now available on the market with reasonably good gene delivery performance. To facilitate broader biological and medical applications, micro-/nanofluidics based technologies were introduced in cell electroporation during the past two decades and their advances are summarized in this perspective. Compared to the commercially available bulk electroporation systems, they offer several advantages, namely, (1) sufficiently high pulse strength generated by a very low potential difference, (2) conveniently concentrating, trapping, and regulating the position and concentration of cells and probes, (3) real-time monitoring the intracellular trafficking at single cell level, and (4) flexibility on cells to be transfected (from single cell to large scale cell population). Some of the micro-devices focus on cell lysis or fusion as well as the analysis of cellular properties or intracellular contents, while others are designed for gene transfection. The uptake of small molecules (e.g., dyes), DNA plasmids, interfering RNAs, and nanoparticles has been broadly examined on different types of mammalian cells, yeast, and bacteria. A great deal of progress has been made with a variety of new micro-/nanofluidic designs to address challenges such as electrochemical reactions including water electrolysis, gas bubble formation, waste of expensive reagents, poor cell viability, low transfection efficacy, higher throughput, and control of transfection dosage and uniformity. Future research needs required to advance micro-/nanofluidics based cell electroporation for broad life science and medical applications are discussed.

Flow-through comb electroporation device for delivery of macromolecules.

Abstract: We present a microfluidic electroporation device with a comb electrode layout fabricated in polydimethylsiloxane (PMDS) and glass. Characterization experiments with HeLa cells and fluorescent dextran show efficient delivery (∼95%) with low toxicity (cell viability ∼85%) as well as rapid pore closure after electroporation. The activity of delivered molecules is also verified by silencing RNA (siRNA) studies that demonstrate gene knockdown in GFP expressing cells. This simple, scalable approach to microfluidic, flow-through electroporation could facilitate the integration of electroporation modules within cell analysis devices that perform multiple operations.

Performance of High Quality Minicircle DNA for In Vitro and In Vivo Gene Transfer

Abstract: Plasmid DNA is frequently used particularly for nonviral gene therapy. Conventional plasmid DNA contains bacterial backbone and resistance gene sequences, as well as immunogenic CpG motifs. These components are not required for transgene expression. They represent a potential risk for safe clinical application and reduce gene transfer rates as well as transgene expression. To overcome these drawbacks, the minicircle technology is removing such sequences, to improve performance and also to reduce DNA size. Here, we show the effective production of luciferase, GFP, or lacZ-carrying minicircle DNA with high yield and reproducible high quality. They are used for lipofection or electroporation gene transfer into human melanoma and colon carcinoma cell lines. Comparison of respective parental plasmid and minicircle-mediated luciferase gene transfer shows improved luciferase expression by minicircle in all cell lines. This is not associated with increase in intracellular minicircle copy numbers after lipofection or electroporation. The minicircles rather mediate enhanced transgene mRNA transcription compared to their parental plasmids. In addition, FACS analysis revealed increase in counts of GFP positive cells after minicircle gene transfer, indicating higher gene transfer rates. Furthermore, minicircle showed also improved performance in vivo after jet-injection gene transfer. Therefore, availability of minicircles with reproducible high quality and sufficient amount makes them an applicable and effective alternative to conventional plasmid gene vectors.

Ultra-localized single cell electroporation using silicon nanowires

Abstract: Analysis of cell-to-cell variation can further the understanding of intracellular processes and the role of individual cell function within a larger cell population. The ability to precisely lyse single cells can be used to release cellular components to resolve cellular heterogeneity that might be obscured when whole populations are examined. We report a method to position and lyse individual cells on silicon nanowire and nanoribbon biological field effect transistors. In this study, HT-29 cancer cells were positioned on top of transistors by manipulating magnetic beads using external magnetic fields. Ultra-rapid cell lysis was subsequently performed by applying 600–900 mVpp at 10 MHz for as little as 2 ms across the transistor channel and the bulk substrate. We show that the fringing electric field at the device surface disrupts the cell membrane, leading to lysis from irreversible electroporation. This methodology allows rapid and simple single cell lysis and analysis with potential applications in medical diagnostics, proteome analysis and developmental biology studies.

Electroporation of Intracellular Liposomes Using Nanosecond Electric Pulses—A Theoretical Study

Abstract: Nanosecond (ns) electric pulses of sufficient amplitude can provoke electroporation of intracellular organelles. This paper investigates whether such pulses could provide a method for controlled intracellular release of a content of small internalized artificial lipid vesicles (liposomes). To estimate the pulse parameters needed to selectively electroporate liposomes while keeping the plasma and nuclear membranes intact, we constructed a numerical model of a biological cell containing a nucleus and liposomes of different sizes (with radii from 50 to 500 nm), which were placed in various sites in the cytoplasm. Our results show that under physiological conditions selective electroporation is only possible for the largest liposomes and when using very short pulses (few ns). By increasing the liposome interior conductivity and/or decreasing the cytoplasmic conductivity, selective electroporation of even smaller liposomes could be achieved. The location of the liposomes inside the cell does not play a significant role, meaning that liposomes of similar size could all be electroporated simultaneously. Our results indicate the possibility of using ns pulse treatment for liposomal drug release.

Recent Trends on Micro/Nanofluidic Single Cell Electroporation

Abstract: The behaviors of cell to cell or cell to environment with their organelles and their intracellular physical or biochemical effects are still not fully understood. Analyzing millions of cells together cannot provide detailed information, such as cell proliferation, differentiation or different responses to external stimuli and intracellular reaction. Thus, single cell level research is becoming a pioneering research area that unveils the interaction details in high temporal and spatial resolution among cells. To analyze the cellular function, single cell electroporation can be conducted by employing a miniaturized device, whose dimension should be similar to that of a single cell. Micro/nanofluidic devices can fulfill this requirement for single cell electroporation. This device is not only useful for cell lysis, cell to cell fusion or separation, insertion of drug, DNA and antibodies inside single cell, but also it can control biochemical, electrical and mechanical parameters using electroporation technique. This device provides better performance such as high transfection efficiency, high cell viability, lower Joule heating effect, less sample contamination, lower toxicity during electroporation experiment when compared to bulk electroporation process. In addition, single organelles within a cell can be analyzed selectively by reducing the electrode size and gap at nanoscale level. This advanced technique can deliver (in/out) biomolecules precisely through a small membrane area (micro to nanoscale area) of the single cell, known as localized single cell membrane electroporation (LSCMEP). These articles emphasize the recent progress in micro/nanofluidic single cell electroporation, which is potentially beneficial for high-efficient therapeutic and delivery applications or understanding cell to cell interaction.

In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation.

Abstract: Brain structures such as the outer subventricular zone (OSVZ) and the inner fiber layer (IFL) in the developing cerebral cortex are especially prominent in higher mammals. However, the molecular mechanisms underlying the formation of the OSVZ are still largely unknown, mainly because genetic manipulations that can be applied to the OSVZ in higher mammals had been poorly available. Here we developed and validated a rapid and efficient genetic manipulation technique for germinal zones including the OSVZ using in utero electroporation in developing gyrencephalic carnivore ferrets. We also determined the optimal conditions for using in utero electroporation to express transgenes in germinal zones. Using our electroporation procedure, the morphology of GFP-positive cells in the OSVZ was clearly visible even without immunostaining, and multiple genes were efficiently co-expressed in the same cells. Furthermore, we uncovered that fibers, which seemed to correspond to those in the IFL of monkeys, also existed in ferrets, and were derived from newly generated cortical neurons. Our technique promises to be a powerful tool for investigating the fundamental mechanisms underlying the formation and abnormalities of the cerebral cortex in higher mammals.

Electroporation: past, present and future.

Abstract: Gene transfer by electroporation has become an indispensable method for the study of developmental biology. The technique is applied not only in chick embryos but also in mice and other organisms. Here, a short history and perspectives of electroporation for gene transfer in vertebrates are described.

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

Abstract: Background Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. Methods A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. Results 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. Conclusion This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Abstract: Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

High-density distributed electrode network, a multi-functional electroporation method for delivery of molecules of different sizes

Abstract: We present a multi-functional electroporation method for delivery of biomolecule utilizing a high-density distributed electrode network (HDEN) under tri-phase electric stimulation. The HDEN device, with which drastic pH change during the electroporation was avoided,was demonstrated to be highly effective for transfection of not only DNA plasmids and small interfering RNAs (siRNA), but also a small molecular anti-cancer drug, into cells in adjustable volumes of cell suspension. The method constitutes a very flexible electroporation approach in a wide range of in vitro or ex vivo scenarios in various tubes, standard multi-well plates as well as flow chambers.

Targeting endogenous nuclear antigens by electrotransfer of monoclonal antibodies in living cells.

Abstract: Antibodies are valuable tools for functional studies in vitro, but their use in living cells remains challenging because they do not naturally cross the cell membrane. Here, we present a simple and highly efficient method for the intracytoplasmic delivery of any antibody into cultured cells. By following the fate of monoclonal antibodies that bind to nuclear antigens, it was possible to image endogenous targets and to show that inhibitory antibodies are able to induce cell growth suppression or cell death. Our electrotransfer system allowed the cancer cells we studied to be transduced without loss of viability and may have applications for a variety of intracellular immuno-interventions.

Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level

Abstract: Background Gene electrotransfer is a nonviral method used for DNA delivery into cells. Several steps are involved. One of them is the interaction of DNA with the cell membrane, which is crucial before DNA can enter the cell. We analysed the level of DNA–membrane interaction in relation to electrotransfer efficiency and the importance of the electrophoretic accumulation of DNA at the cell membrane. Systematic comparison of long-duration, short-duration and combinations of electropermeabilizing short (high-voltage; HV) and electrophoretic long (low-voltage; LV) pulses were performed. The effect of Mg2+ ion concentrations on electrotransfer and their effect on DNase activity were explored. Methods To visualize the DNA–membrane interaction, TOTO-1 labeled DNA was used. Transfection efficiency was assessed with plasmid DNA coding for green fluorescent protein. Results Higher relative electrotransfer efficiency was obtained by using longer pulses, whereas shorter pulses preserved cell viability. Short-duration pulses enabled higher (24%) overall transfection yield compared to long-duration pulses (12%), although a higher DNA–membrane interaction was observed. No significant difference in transfection was obtained between different HV-LV pulsing protocols, although the highest DNA–membrane interaction was observed with HV + LV pulses. The formation of the DNA–membrane complex depended on the Mg2+ concentration, whereas DNase inhibitor did not affect gene expression. Conclusions Gene electrotransfer is a complex phenomenon, where many factors mutually affect the process and the DNA–membrane interaction only comprises the first step. We showed that longer electric pulses are optimal for higher transfection efficiency but reduce viability, whereas shorter pulses enable moderate transfection efficiency and preserve viability. Thus, each application needs a careful choice of pulsing protocol. Copyright © 2013 John Wiley & Sons, Ltd.