RESUMO
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).
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Bleomicina/administração & dosagem , Eletroporação/instrumentação , Corantes Fluorescentes/administração & dosagem , Animais , Adesão Celular , Linhagem Celular , Impedância Elétrica , Desenho de Equipamento , Humanos , CinéticaRESUMO
Using an electrical measurement known as electric cell-substrate impedance sensing (ECIS), we have recorded the dynamics of viral infections in cell culture. With this technique, cells are cultured on small gold electrodes where the measured impedance mirrors changes in attachment and morphology of cultured cells. As the cells attach and spread on the electrode, the measured impedance increases until the electrode is completely covered. Viral infection inducing cytopathic effect results in dramatic impedance changes, which are mainly due to cell death. In the current study, two different fish cell lines have been used: chinook salmonid embryonic (CHSE-214) cells infected with infectious pancreatic necrosis virus (IPNV) and epithelioma papulosum cyprini (EPC) carp cells infected with infectious hematopoeitic necrosis virus (IHNV). The impedance changes caused by cell response to virus are easily measured and converted to resistance and capacitance. An approximate linear correlation between log of viral titer and time of cell death was determined.
Assuntos
Técnicas Biossensoriais/métodos , Células-Tronco Embrionárias/citologia , Vírus da Necrose Hematopoética Infecciosa/crescimento & desenvolvimento , Vírus da Necrose Pancreática Infecciosa/crescimento & desenvolvimento , Animais , Técnicas Biossensoriais/instrumentação , Carpas , Morte Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Impedância Elétrica , Eletrodos , Células-Tronco Embrionárias/virologia , Salmonidae , Fatores de TempoRESUMO
A new method combining optical and electrical impedance measurements is described that enables submicroscopic cell movements to be monitored. The cells are grown on small gold electrodes that are transparent to light. This modified electrical cell-substrate impedance sensor (ECIS) allows simultaneous microscopic recording of both growth and motility, thus enabling cell confluence on the electrodes to be systematically correlated to the impedance in regular time intervals of seconds and for extended periods of time. Furthermore, the technique provides an independent measure of monolayer cell densities that we compare to calculated values from a theoretical model. We have followed the attachment and spreading behavior of epithelial Madin-Darby canine kidney strain I (MDCK-I) cell cultures on microelectrodes for up to 40 h. The studies reveal a high degree of correlation between the measured resistance at 4 kHz and the corresponding cell confluence in 4- to 6-h intervals with typical linear cross-correlation factors of r equaling approximately 0.9. In summary, the impedance measured with the ECIS technique provides a good quantitative measure of cell confluence.
Assuntos
Impedância Elétrica , Óptica e Fotônica , Animais , Células Cultivadas , Cães , EletrodosRESUMO
Confluent cell monolayers in tissue culture are fragile and can easily be mechanically disrupted, often leaving an area devoid of cells. This opening in the cell sheet is then repopulated, because the cells on the fringe of the damage, which are no longer contact-inhibited, move into the available space. This mechanical disruption is often done deliberately in a "wound-healing" assay as a means to assess the migration of the cells. In such assays, a scrape is made in the cell layer followed by microscopy to monitor the advance of the cells into the wound. We have found that these types of assays can also be accomplished electrically. In this approach, cells growing on small electrodes and monitored by using electric cell-substrate impedance sensing are subjected to currents, resulting in severe electroporation and subsequent cell death. After this invasive treatment, the electrode's impedance is again monitored to chart the migration and ultimate healing of the wound. We report here that this procedure to study cell behavior is both highly reproducible, quantitative, and provides data similar to that acquired with traditional measurements.
Assuntos
Cicatrização , Animais , Linhagem Celular , Chlorocebus aethiops , Cães , Eletroporação , Técnicas In Vitro , RatosRESUMO
Here we present research detecting the invasive activities of metastatic cells in vitro using electric cell-substrate impedance sensing (ECIS). The assay is based on previous microscopic observations, where metastatic cells added over established endothelial cell layers were observed to attach to and invade the cell layer. Human umbilical vein endothelial cells (HUVECs) werefirst grown to confluence on small gold electrodes. The impedance of these electrodes was followed after the addition of suspensions of different sublines of the Dunning murine prostatic adenocarcinoma series (G, AT1, AT2, AT3, ML, and MLL). For highly metastatic sublines, within an hour after being challenged, the impedance of the confluent HUVEC layer was substantially reduced. The effect of the weakly metastatic sublines was less pronounced, and the extent and the rate of this drop in impedance could be correlated with the metastatic potential of each of six sublines tested. The real-time assay is effective in both normal and low (1%) serum concentrations, and the detected activity requires the presence of viable transformed cells. In addition to the murine cell lines, similar behavior was observed using four established human prostatic cancer lines (DU145, PC3, TSU, and PPC1). These results suggest that this ECIS-based assay might be used with primary human cultures to establish the metastatic abilities of cells isolated from biopsies.
Assuntos
Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/fisiopatologia , Células Cultivadas , Impedância Elétrica , Endotélio/citologia , Humanos , Masculino , Neoplasias da Próstata , Células Tumorais Cultivadas , Veias Umbilicais/citologiaRESUMO
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.