Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations

In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).     

Design and performance of an electrical stimulator for long-term contraction of cultured muscle cells

Excitability in muscle cells manifests itself as contractility and may be evoked by electrical stimulation. Here we describe an electrical stimulator device applicable to cells seeded on standard multiwell plates and demonstrate how it effectively stimulates synchronous contraction of skeletal muscle C2C12 cells without damaging them. The electrical stimulator of cultured cells (ESCC) consists of two connection cards and a network of platinum electrodes positioned in such way that each well in a row is uniformly stimulated. The ESCC may produce a range of outputs based on the stimulation parameters it receives from a commercial pulse generator and can be placed in a standard cell incubator, allowing for long-term stimulation as required for biochemical and molecular biological assays. We show that a 90-min stimulation of C2C12 myotubes at 50 V, 30 ms of pulse duration, and 3 Hz of frequency enhances glucose metabolism and glycogen mobilization while oppositely modulating the activity ratio of glycogen metabolizing enzymes. Thus, we demonstrate that long-term electrical stimulation of C2C12 myotubes with the ESCC results in contractility and metabolic changes, as seen in exercising muscle.     

Recovery of adherent cells after in situ electroporation monitored electrically

Here we describe various experiments that address the efficiency of loading extracellular probes into the cytoplasm of adherent mammalian cells (normal rat kidney, Madin-Darby canine kidney, and African green monkey) by means of in situ electroporation. Subsequent cell recovery from the electroporation pulse was monitored electrically in real time for each condition. In this study, small, gold-film electrodes (5 x 10(-4) cm2) are used as culture substrates and at the same time as an electrode for both the application of the electroporating voltage pulse and the noninvasive electrical monitoring of cell recovery, using a technique referred to as ECIS. Electroporation has been performed by using ac sinusoidal voltage pulses of varying frequency, amplitude, and duration. Permeabilization and re-closure of the plasma membrane were evaluated by the uptake of the fluorescence probe, Lucifer Yellow, from the extracellularfluid. With the experimental setup described here, efficient electroporation was achieved with voltages less than 5 V. Using ECIS, we followed the morphological response of the cells to the electricfield-induced membrane permeabilization. For optimized electroporation conditions, cell recovery was completed in less than 1 h. The introduction of membrane-impermeable substances by electroporation and in situ monitoring of the cellular response mayfind many applications in cell biology.     

A novel electroporation method using a capillary and wire-type electrode

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.     

In situ bipolar electroporation for localized cell loading with reporter dyes and investigating gap junctional coupling

Electroporation is generally used to transfect cells in suspension, but the technique can also be applied to load a defined zone of adherent cells with substances that normally do not permeate the plasma membrane. In this case a pulsed high-frequency oscillating electric field is applied over a small two-wire electrode positioned close to the cells. We compared unipolar with bipolar electroporation pulse protocols and found that the latter were ideally suited to efficiently load a narrow longitudinal strip of cells in monolayer cultures. We further explored this property to determine whether electroporation loading was useful to investigate the extent of dye spread between cells coupled by gap junctions, using wild-type and stably transfected C6 glioma cells expressing connexin 32 or 43. Our investigations show that the spatial spread of electroporation-loaded 6-carboxyfluorescein, as quantified by the standard deviation of Gaussian dye spread or the spatial constant of exponential dye spread, was a reliable approach to investigate the degree of cell-cell coupling. The spread of reporter dye between coupled cells was significantly larger with electroporation loading than with scrape loading, a widely used method for dye-coupling studies. We conclude that electroporation loading and dye transfer is a robust technique to investigate gap-junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-cell communication.     

Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation

The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Electrotransfection of anchorage-dependent mammalian cells

Reversible electropermeabilization (or electroporation) of cell membranes is a very efficient method for intracellular delivery of xenomolecules, particularly of DNA. In the case of anchorage-dependent cells, however, enzymatic or mechanical detachment from the substratum is required prior to electropulsing. This can damage the plasma membrane and lead to low transfection yields. Here we present an efficient method for in situ electroporation of mammalian cells while they are attached to a solid substratum. For this purpose an electroporation chamber was constructed that housed a cell culture insert with a cell monolayer grown on a porous filter. By real-time monitoring the transmonolayer resistance, the field pulse parameters resulting in transient and reversible permeabilization of cell membranes were determined for two adherent cell lines, which were found to differ markedly in their sensitivity to electropulsing. Based on the transmonolayer resistance data, the pulsing conditions for optimum electrotransfection of two murine cell lines with plasmid DNA could be established in a very short time. The transfection yield and gene expression were significantly higher in cell monolayers facing the cathode compared to those exposed to field pulses of the reverse direction. This might be due to contribution of the electrophoresis to the translocation of the polyanionic plasmid DNA across the electropermeabilized cell membrane. The experimental setup presented here appears to be a promising tool not only for rapid optimization of in situ electrotransfection of anchorage-dependent cells but also for studying the molecular/biophysical mechanisms of the membrane breakdown and resealing.     

Suspended-drop electroporation for high-throughput delivery of biomolecules into cells

We present a high-throughput method that enables efficient delivery of biomolecules into cells. The device consists of an array of 96 suspended electrode pairs, where small sample volumes are top-loaded, electroporated and bottom-ejected into 96-well plates. We demonstrate the use of this suspended-drop electroporation (SDE) device to effectively introduce fluorescent dextran, small interfering RNA (siRNA) or cDNA into primary neurons, differentiated neutrophils and other cell types with conventionally low transfection rates.     

High efficiency,Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes^1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies^9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies¹³. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells^14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies^{19, 24-26}. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection²⁷. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures^17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.     

In situ electroporation of radioactive compounds into adherent cells

We previously developed a technique, termed in situ electroporation, where nonpermeant molecules are introduced through an electrical pulse into adherent cells, while they grow on electrically conductive, optically transparent, indium-tin oxide (ITO). Careful control of the electric field intensity results in essentially 100% of the cells taking up the introduced material, without any detectable effect upon the physiology of the cell, presumably because the pores reseal rapidly so that the cellular interior is restored to its original state. Electroporation of radioactive material is faced with two important considerations: (1) potential for exposure of personnel to irradiation, and (2) the requirement for electroporation of a large number of cells. In this report, we describe a modification in the geometry of the slides and electrodes which permits the use of inexpensive ITO-coated glass of lower conductivity that can be discarded after use, to electroporate large numbers of cells using a minimum volume of radioactive nucleotide solution. The results demonstrate that, using this assembly, the determination of the Ras-bound GTP/GTP+GDP ratios through electroporation of [alpha32P]GTP can be conducted using approximately five times lower amounts of isotope than in previous designs. Moreover, this assembly permits efficient upscaling, which makes the determination of Ras-GTP binding in cells which are deficient in Ras activity possible. In addition, we demonstrate the labeling of two viral phosphoproteins--the Simian Virus 40 Large Tumor antigen, and Adenovirus E1A--through [gamma32P]ATP electroporation using this setup. In both cases, electroporation of the nucleotide can achieve a great increase in the efficiency and specificity of labeling compared to the addition of [32P]-orthophosphate to the culture medium, presumably because the immediate phosphate donor nucleotide itself is introduced, which can directly bind to the target proteins.     

High-throughput RNAi screening in vitro: from cell lines to primary cells

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.     

Low-temperature pausing of cultivated mammalian cells

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a variety of cultivation vessels. High cell viability and exponential growth were observed after pausing a recombinant Chinese hamster ovary cell line (CHO-Clone 161) in a temperature range of 6-24 degrees C in microcentrifuge tubes for up to 3 weeks. After pausing in T-flasks at 4 degrees C for 9 days, adherent cultures of CHO-DG44 and human embryonic kidney (HEK293 EBNA) cells resumed exponential growth when incubated at 37 degrees C. Adherent cultures of CHO-DG44 cells paused for 2 days at 4 degrees C in T-flasks and suspension cultures of HEK293 EBNA cells paused for 3 days at either 4 degrees C or 24 degrees C in spinner flasks were efficiently transfected by the calcium phosphate-DNA coprecipitation method, yielding reporter protein levels comparable to those from nonpaused cultures. Finally, cultures of a recombinant CHO cell line (CHO-YIgG3) paused for 3 days at 4 degrees C, 12 degrees C, or 24 degrees C in bioreactors achieved the same cell mass and recombinant protein productivity levels as nonpaused cultures. The success of this approach to cell storage with rodent and human cell lines points to a general biological phenomenon which may have a wide range of applications for cultivated mammalian cells.     

A system to impose prescribed homogenous strains on cultured cells.

There is presently significant interest in cellular responses to physical forces, and numerous devices have been developed to apply stretch to cultured cells. Many of the early devices were limited by the heterogeneity of deformation of cells in different locations and by the high degree of anisotropy at a particular location. We have therefore developed a system to impose cyclic, large-strain, homogeneous stretch on a multiwell surface-treated silicone elastomer substrate plated with pulmonary epithelial cells. The pneumatically driven mechanism consists of four plates each with a clamp to fix one edge of the cruciform elastomer substrate. Four linear bearings set at predetermined angles between the plates ensure a constant ratio of principal strains throughout the stretch cycle. We present the design of the device and membrane shape, the surface modifications of the membrane to promote cell adhesion, predicted and experimental measurements of the strain field, and new data using cultured airway epithelial cells. We present for the first time the relationship between the magnitude of cyclic mechanical strain and the extent of wound closure and cell spreading.     

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo . The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo . The device can be sterilized for cell culture using common methods without loss of function. The device''s applicability to studying the in vitro wound healing response will be demonstrated.Open in a separate windowClick here to view.^{(69M, flv)}     

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.     

GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode.

This study correlates the fluorescent signal from stable recombinant CHO cell lines expressing the green fluorescent protein (GFP) at high levels with biomass or cell number, extending the use of fluorescent proteins to applications and assays where cell growth rates are important. Using a standard fluorometer, growth of these cells can be quantified noninvasively in multiwell plates, and because signals are obtained without preparation, the same culture samples can be measured repeatedly. Even with a small relative change in biomass, the specific growth rate can be determined in a few hours. The dynamics of cell populations can now be studied with high sensitivity, low error rate, and minimum sample preparation.     

A high-throughput respirometric assay for mitochondrial biogenesis and toxicity

Mitochondria are a common target of toxicity for drugs and other chemicals and result in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality, and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTCs) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Bioscience analyzer to measure mitochondrial function in real time in multiwell plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl₂, and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone)-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTCs. The merger of the RPTC model and multiwell respirometry results in a single high-throughput assay to measure mitochondrial biogenesis and toxicity and nephrotoxic potential.