Avoiding the side effects of electric current pulse application to electroporated cells in disposable small volume cuvettes assures good cell survival

Abstract Background The harmful side effects of electroporation to cells due to local changes in pH, the appearance of toxic electrode products, temperature increase, and the heterogeneity of the electric field acting on cells in the cuvettes used for electroporation were observed and discussed in several laboratories. If cells are subjected to weak electric fields for prolonged periods, for example in experiments on cell electrophoresis or galvanotaxis the same effects are seen. In these experiments investigators managed to reduce or eliminate the harmful side effects of electric current application. Methods For the experiments, disposable 20\ua0\u3bcl cuvettes with two walls made of dialysis membranes were constructed and placed in a locally focused electric field at a considerable distance from the electrodes. Cuvettes were mounted into an apparatus for horizontal electrophoresis and the cells were subjected to direct current electric field (dcEF) pulses from a commercial pulse generator of exponentially declining pulses and from a custom-made generator of double and single rectangular pulses. Results More than 80% of the electroporated cells survived the dcEF pulses in both systems. Side effects related to electrodes were eliminated in both the flow through the dcEF and in the disposable cuvettes placed in the focused dcEFs. With a disposable cuvette system, we also confirmed the sensitization of cells to a dcEF using procaine by observing the loading of AT2 cells with calceine and using a square pulse generator, applying 50\ua0ms single rectangular pulses. Conclusions We suggest that the same methods of avoiding the side effects of electric current pulse application as in cell electrophoresis and galvanotaxis should also be used for electroporation. This conclusion was confirmed in our electroporation experiments performed in conditions assuring survival of over 80% of the electroporated cells. If the amplitude, duration, and shape of the dcEF pulse are known, then electroporation does not depend on the type of pulse generator. This knowledge of the characteristics of the pulse assures reproducibility of electroporation experiments using different equipment

High aspect ratio electrodes for high yield electroporation of cells

Electroporation is a widely used process in cell biology studies. It uses an electric field to create pores on the cell membrane in order to either insert exogenous molecules inside the cells or disrupt the cell membrane to kill the cells. Current micro-fluidic electroporation devices use the planar electrodes situated at the bottom of a microchannel. These planar electrodes i) require a high voltage and ii) generate a nonuniform electric field which result in low yield of the electroporation. The standard silicon microfabrication technologies are not suitable to fabricate non-planar electrodes required to increase the yield of electroporation. In this research, an electroporation device is fabricated with an array of five pairs of three dimensional (3D) electrodes situated along the sidewalls of a microchannel. These 3D electrodes are fabricated by filling the molten indium inside the chosen microchannels. The indium filling method allows the fabrication of microstructures with planar dimensions larger than ~30 µm regardless of their height, integrated into the PDMS device. The selective electroporation of fibroblast cells is successfully demonstrated using a fabricated device by applying a low voltage (1.67 V). The uniform electric field generated in cross sections of microchannel by 3D electrodes will avoid the limitations of planar electrodes by i) preventing cell death due to an excessive electric field and ii) preventing lack of electroporation due to a low electric field. As a result, these 3D electrodes should be capable of increasing the yield of electroporation

High Throughput and Highly Controllable Methods for in Vitro Intracellular Delivery

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

Sistemas microfuidicos de gotas para incorporaçao de acidos nucleicos em lipossomas cationicos e para transfecçao in vitro de células de mamiferos

Orientadores: Lucimara Gaziola de la Torre, Charles BaroudTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química, École Polytechnique - FrançaResumo: Este trabalho visou o uso de um sistema microfluídico de gotas para incorporar ácidos nucleicos em lipossomas catiônicos e outro para estudar o processo de transfecção em células de mamíferos. A primeira etapa do projeto utilizou um microdispositivo para incorporar pDNA em lipossomas catiônicos de modo a obter lipoplexos reprodutíveis e adequados para transfectar células dendríticas (DCs). Com esta finalidade, alguns parâmetros experimentais foram investigados, tais como vazões de entrada, manutenção das propriedades dos lipossomas após processamento no microdispositivo, características dos lipoplexos (tamanho, polidispersidade e carga) em função da razão molar de carga (R+/-) e do desenho do microdispositivo. Lipoplexos produzidos em microdispositivo com canal de serpentina largo e região de divisão de gotas que diminuem a polidispersidade dos lipoplexos, operando à razão de vazão água/óleo 0,25 e R+/- 1,5; 3; 5; 7 e 10 foram utilizados para transfectar DCs in vitro. Todos os lipoplexos foram capazes de transfectar as DCs e ao mesmo tempo proporciaonar a ativação das células. A segunda etapa do trabalho utilizou uma plataforma microfluídica de célula única para investigar e controlar as condições de transfecção, tendo em vista a otimização dos rendimentos de produção de proteínas recombinantes. Neste contexto, as células de ovário de hamster Chinês (CHO-S) foram transfectadas no microdispositivo com diferentes tipos de lipoplexos (R+/- 1,5; 3; 5) e monitoradas em relação à produção de proteína verde fluorescente (GFP) e viabilidade celular. A plataforma de célula única permite avaliar a heterogeneidade celular, revelando a presença de uma subpopulação que produz níveis elevados de GFP. Essas células com alta produção de GFP (HP) mostraram um aumento do tamanho celular em comparação à média da população. Além disso, a carga dos lipoplexes apresenta um importante papel na transfecção das células CHO-S, visto que os únicos lipoplexos com carga positiva R+/- 5 produziram mais HPs. A quantidade de pDNA entregue às células afeta a produção de proteína, já que os lipoplexos com mais pDNA R+/- 1,5 aumentaram a produtividade específica de GFP das HPs. Esta tese foi desenvolvida no âmbito de um programa de co-tutela entre a Universidade Estadual de Campinas, Brasil e a École Polytechnique, França. Em geral, este trabalho apresenta contribuições originais para as áreas da microfluídica e da entrega de genesAbstract: This work aimed at using one droplet-based microfluidic systems to incorporate nucleic acids into cationic liposomes and another one to study the mammalian cell transfection process. In the first part of this study we used a droplet-based microfluidic system to complex cationic liposomes with pDNA in order to obtain reproducible and suitable lipoplexes to dendritic cells (DCs) transfection. For this purpose, some experimental parameters were investigated, such as inlet flow rates, the maintenance of liposomes¿ properties after microfluidic processing, lipoplex characteristics (size, polydispersity and zeta potential) as function of molar charge ratio (R+/-) and microchip design. Lipoplexes produced in a microchip with large serpentine channel and split region, which decreases lipoplex polydispersity, operating at ratio aqueous/oil flow rate 0.25 and R+/- 1.5, 3, 5, 7 and 10 were used to transfect DCs in vitro. All lipoplexes transfected DCs and resulted in cell activation. In the second part of this study we used a single-cell microfluidic platform to investigate and control transfection conditions, in view of optimizing the recombinant protein production by transfected cells. Chinese hamster ovary cells (CHO-S) were transfected in microchip with different types of lipoplexes (R+/- 1.5, 3, 5) and monitored by green fluorescent protein (GFP) production and cell viability. The single-cell platform enables to assess the heterogeneities of CHO-S population, revealing the presence of a subpopulation producing significantly high levels of GFP. These high producers (HP) showed increased cell size in comparison to the average population. Moreover, the charge of lipoplexes shows an important role to transfect CHO-S, since the unique positive charged lipoplex R+/- 5 produced more HPs. Additionally, the amount of pDNA delivered affects protein production, since R+/- 1.5 with more pDNA increased GFP specific productivity of HPs. This thesis was developed under the joint graduate program of the University of Campinas, Brazil and École Polytechnique, France. In general, this work presents original contributions in the areas of microfluidics and gene deliveryDoutoradoDesenvolvimento de Processos BiotecnologicosDoutora em Engenharia Quimica2012/24797-2, 2014/10557-5FAPES

Electromanipulation of Ellipsoidal Cells in Fluidic Micro-Electrode Systems

Recently, electromanipulation technologies for handling and characterizing individual cells or particles have been applied to lab-on-chip devices. These devices play a role in pharmacological and clinical applications as well as environmental and nanotechnologies. Electromanipulation of ellipsoidal cells in fluidic micro-electrode systems has been studied by numerical simulations, theoretical analysis and experiment. The field distributions in electrorotation chip chambers were analyzed using numerical field simulations in combination with analytical post-processing. The optimal design for two-dimensional electrorotation chips features electrodes with pyramidal rounded tips. Moreover, the three-dimensional electric field distributions in the electroporation and electrorotation chambers were analyzed. The advantage of electroporation chip chambers is to avoid strongly increasing temperatures after pulse application. New chips may be developed for nanoscale applications in the future. New simplified analytical equations have been developed for the transmembrane potential (delta_phi) induced in cells resembling ellipsoids of rotation, i.e. spheroids, by homogeneous DC or AC fields. The new equations avoid the complicated description by the depolarizing factors. Also the dielectrophoretic force expression for spheroidal objects has been simplified. Furthermore, the effects of cell orientation and electric field frequency on the delta_phi induced in ellipsoidal cells were studied. Simplified equations were derived. They show that the membrane surface points for the maximum of delta_phi depend on cell shape, cell orientation, electric cell parameters and field frequency. The theoretical results were compared to electropermeabilization experiments with chicken red blood cells. Experiments confirmed that equations for the transmembrane potential were advantageous for describing the transmembrane potential induced in arbitrarily oriented ellipsoidal cells.In letzter Zeit sind Elektromanipulations-Technologien für die Manipulation und die Charakterisierung von einzelnen Zellen oder Partikeln in Lab-on-Chip Systeme integriert worden. Die neuen Systeme spielen eine Rolle in pharmakologischen und klinischen Anwendungen sowie in Umwelt- und Nanotechnologien. Die Elektromanipulation von ellipsoiden Zellen in fluidischen Mikro-Elektrodensystemen wurde mit Hilfe numerischer Simulation, theoretischer Analyse sowie Experimenten beschrieben. Die Feldverteilung in Elektrorotationskammern wurde mit numerischen Simulationen analysiert und optimert. Als geeignetes Elektrodendesign in zweidimensionalen Elektrorotationskammern erwiesen sich pyramidale, abgerundete Elektrodenspitzen. Zusätzlich wurden die drei-dimensionalen Feldverteilungen in den Elektroporations- und Elektrorotationskammern analysiert, um starke Temperaturerhöhungen durch den elektrischen Puls zu vermeiden. Mit diesen Ergebnissen könnten neue Chips für Anwendungen im Nanometerbereich entwickelt werden. Neue und vereinfachte analytische Gleichungen für das Transmembranpotential (delta_phi), welches in einem homogenen Gleich- oder Wechselfeld in Zellen ähnlich Rotationsellipsoiden, d.h. Spheroide, induziert wird, wurden unter Vermeidung der Depolarisierungsfaktoren hergeleitet. Ebenso wurde die Gleichung für die dielektrophoretische Kraft auf spheroide Objekte vereinfacht, sowie die Effekte von Zellorientierung und Frequenz des Wechselfeldes auf das delta_phi von ellipsoiden Zellen untersucht und vereinfachte Gleichungen abgeleitet. Sie zeigen, dass die Membranpunkte mit maximalem delta_phi abhängig sind von der Zellform, der Zellorientierung, den elektrischen Eigenschaften der Zelle und der Frequenz des Wechselfeldes. Die theoretischen Ergebnisse wurden mit Experimenten zur Elektropermeabilität von Hühnererythrozyten verglichen, die bestätigten, dass die vereinfachten Gleichungen das in beliebig orientierten elliptischen Zellen induzierte Transmembranpotential richtig beschreiben

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research

SINGLE-CELL ELECTROPORATION USING ELECTROLYTE-FILLED CAPILLARIES -EXPERIMENTAL AND MODELING INVESTIGATIONS

Electrolyte-filled capillaries (EFCs) with fine tips provide a highly concentrated electric field for local single-cell electroporation (SCEP) with high spatial resolution. A complete circuit for SCEP experiments was built that consisted of a test circuit and an electroporation circuit, with the ability to monitor electrically the electroporation pulses. SCEP itself was monitored in real time by observing the loss of a fluorescent adduct of glutathione (Thioglo-1-GSH) from the intracellular space. SCEP can be applied for transfection of individual adherent cells. We hypothesize that transfection of single cells can be accomplished with the plasmid contained in a single capillary. During SCEP, electroosmotic flow can pump electrolyte out of the capillary enhancing plasmid transfer into cells. This was confirmed from both simulation and transfection experiments. Cells were successfully transfected with EGFP-C2 plasmid when the loss of Thioglo-1-GSH upon SCEP (ΔF) is larger than 10% and its mass transfer rate (M) through the membrane exceeds 0.03 s-1. A series of SCEP experiments has been carried out on PC-3 cells (with 2-µm tip opening) and A549 cells (with 4~5-µm tip opening) to investigate how the parameters such as cell-to-tip distance (dc), cell size (dm) and shape, temperature, current, and the cell cycle affect SCEP outcomes (M and resealing rate α) via statistical analysis. A good linear regression is achieved only at a low temperature of 15℃. The main factors affecting small molecule transport across cell membrane are dc, dm and electric current. A range of M (0.03 s-1 ~ 0.4 s-1 for PC-3 cells, or 0.03 s-1 ~ 0.5 s-1 for A549 cells) gives the best linear regressions. M is also affected by the cell cycle of A549 cells, and correlated with cell roundness only for PC-3 cells. Cells reseal faster at higher temperature; while lower temperature provides better survivability with identical ΔF. Lastly, numerical models were elaborated as a platform for better understanding of the SCEP process and prediction of the trends of SCEP under various experimental conditions. A mass transport model involving potential distribution, diffusion, convection and electrokinetic flow was extended to study mass transport at a buffer-filled pipette tip/porous medium interface

Electrokinetic Transport Process in Nanopores Generated on Cell Membrane during Electroporation

In this thesis, underlying concepts of transport phenomena through generated nanopores on a cell membrane during electroporation were studied. A comprehensive literature review was performed to find the pros and cons of the previous works and consequently extensive studies were accomplished to explain shortcomings of the former studies on this topic. The membrane permeabilization of the single cell located in the microchannel was studied, and the effects of microchannel’s wall and electrode size were investigated on cell electroporation. It was studied how the electrical (e.g., strength of the electric pulse) and geometrical parameters (e.g., microchannel height and electrode size) affect size, location, and number of created hydrophilic pores on the cell membrane. Because of a transmembrane potential, the electrokinetic effects have decisive influence on the transport process through the created nanopores. A comprehensive study was performed to explain the electrokinetic transport through the nanochannels. Effects of surface electric charge and radius of the nanochannel on the electric potential, liquid flow, and ionic transport were investigated. Unlike microchannels, the electric potential field, ionic concentration field, and velocity field are strongly size-dependent in the nanochannels. They are also affected by the surface electric charge of the nanochannel. More counter ions than co-ions are transported through the nanochannel. The ionic concentration enrichment at the entrance and the exit of the nanochannel is completely evident from the simulation results. The study also shows that the fluid velocity in the nanochannel is higher when the surface electric charge is stronger, or the radius of the nanochannel is larger. The obtained model of the electrokinetic effects in the nanochannels was utilized to examine the ionic mass transfer and the fluid flow through the generated hydrophilic nanopores of the cell membrane during electroporation. The results showed how the electric potential, velocity field, and ionic concentration vary with the size and angular position of the generated nanopores of the cell membrane. It was also shown that, in the presence of the electric pulse, the electrokinetic effects (the electroosmosis and the electrophoresis) had significant influences on the ionic mass transfer through the nanopores, while the effect of diffusion on the ionic mass flux was negligible in comparison with the electrokinetics. Increasing the radius of the nanopores intensified the effect of convection (electroosmosis) in comparison with the electrophoresis on the ionic flux. Furthermore, the electrokinetic motion of the nanoparticle through the nanochannel was investigated to mimic inserting the nanoscale biological samples, such as QDots and DNAs, through the created nanopores on the cell membrane. It was proved that, because of the large applied electric field over the nanochannel, the impact of the Brownian force was negligible in comparison with the electrophoretic and the hydrodynamic forces. It was demonstrated that increasing the bulk ionic concentration or the surface charge of the nanochannel will increase the electroosmotic flow, and hence affect the particle’s motion. It was also shown that, unlike the microchannels with thin EDL, the change in the nanochannel size will change the EDL field and the ionic concentration field in the nanochannel, affecting the particle’s motion. If the nanochannel size is fixed, a larger particle will move faster than a smaller particle under the same conditions. Finally, it was examined how the nanoscale biological samples (nanoparticles) reach openings of the generated nanopores on the cell membrane during electroporation. It was examined what forces (electrophoresis, diffusion, and convection) brings the nanoparticles into the nanopores and how the size and the surface electric charge of the nanoparticle affect its transport to the opening of the nanopores.1 yea