RESUMO
The morphological and functional status of organs, tissues, and cells can be assessed by evaluating their electrical impedance. Fluid shear stress regulates the morphology and function of endothelial cells in vitro. In this study, an electrical biosensor was used to investigate the dynamics of flow-induced alterations in endothelial cell morphology in vitro. Quantitative, real-time changes in the electrical impedance of endothelial monolayers were evaluated using a modified electric cell-substrate impedance sensing (ECIS) system. This ECIS/Flow system allows for a continuous evaluation of the cell monolayer impedance upon exposure to physiological fluid shear stress forces. Bovine aortic endothelial cells grown to confluence on thin film gold electrodes were exposed to fluid shear stress of 10 dynes/cm2 for a single uninterrupted 5 h time period or for two consecutive 30 min time periods separated by a 2 h no-flow interval. At the onset of flow, the monolayer electrical resistance sharply increased reaching 1.2 to 1.3 times the baseline in about 15 min followed by a sustained decrease in resistance to 1.1 and 0.85 times the baseline value after 30 min and 5 h of flow, respectively. The capacitance decreased at the onset of flow, started to recover after 15 min and after slightly overshooting the baseline values, decreased again with a prolonged exposure to flow. Measured changes in capacitance were in the order of 5% of the baseline values. The observed changes in endothelial impedance were reversible upon flow removal with a recovery rate that varied with the duration of the preceding flow exposure. These results demonstrate that the impedance of endothelial monolayers changes dynamically with flow indicating morphological and/or functional changes in the cell layer. This in vitro model system (ECIS/Flow) may be a very useful tool in the quantitative evaluation of flow-induced dynamic changes in cultured cells when used in conjunction with biological or biochemical assays able to determine the nature and mechanisms of the observed changes.
Assuntos
Endotélio Vascular/fisiologia , Animais , Engenharia Biomédica/instrumentação , Bovinos , Células Cultivadas , Impedância Elétrica , Endotélio Vascular/citologia , Hemodinâmica , Modelos CardiovascularesRESUMO
It has long been realized that fibroblastic and epithelial cells establish recognizable patterns in tissue culture. This behavior implies that neighboring cells interact with one another to produce organized populations. Interaction between cells that are separated by many intervening cells is also possible and is demonstrated here using a special configuration of a biosensor referred to as electric cell-substrate impedance sensing (ECIS). Normally the electrical impedance of a single electrode covered with a confluent cell layer is measured, and the morphological changes of the cells are reflected in the impedance. In this case the cells are cultured on two closely spaced electrodes whose impedances are measured independently as a function of time, and communication between the cell populations is revealed as a correlation between these two time series. We also report for the first time another striking manifestation of dynamic cell interaction, where confluent layers of Madin-Darby canine kidney cells (MDCK) on a single electrode are observed to oscillate in synchrony with a period of approximately 2.5 h.
Assuntos
Técnicas Biossensoriais/métodos , Comunicação Celular , Movimento Celular , Animais , Técnicas de Cultura de Células , Células Cultivadas , Cães , Impedância ElétricaRESUMO
This article describes the optimization of an experimental technique referred to as electric cell-substrate impedance sensing (ECIS) to monitor attachment and spreading of mammalian cells quantitatively and in real time. The method is based on measuring changes in AC impedance of small gold-film electrodes deposited on a culture dish and used as growth substrate. Based on experimental data and theoretical considerations we demonstrate that high-frequency capacitance measurements (f = 40 kHz) are most suited to follow the increasing surface coverage of the electrode due to cell spreading. The excellent time resolution of the method allowed an in-depth analysis of cell spreading kinetics under various experimental conditions. Using ECIS we studied the attachment and spreading of epithelial MDCK cells (strain II) on different protein coatings, and investigated the influence of divalent cations on spreading kinetics. We quantified the inhibitory effect of soluble peptides that mimic the recognition sequence of fibronectin and other extracellular matrix proteins (RGDS). We also applied the ECIS technique to monitor the detachment of confluent fibroblastic cell layers (WI38/VA-13) by means of these peptides.
Assuntos
Técnicas Biossensoriais , Impedância Elétrica , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Sistemas Computacionais , Condutividade Elétrica , Células Epiteliais/química , Proteínas da Matriz Extracelular/farmacologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Fibronectinas/farmacologia , Integrinas/fisiologia , Rim/citologia , Cinética , Oligopeptídeos/farmacologia , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
Transepithelial resistance (TER) measurement has often been used to study the paracellular transport properties of epithelia grown on permeable filters, especially the barrier function of tight junctions. However, the TER value includes another source, the resistance caused by cell-substrate contact, that may give rise to a high TER value if cell-substrate separation is small. In this study we use electric cell-substrate impedance sensing (ECIS) to measure both paracellular resistance and the average cell-substrate distance of MDCK (II), HEp-2, and WI-38 VA13 cells. Comparing ECIS data with those from TER measurements of cell layers cultured on polycarbonate filters, we can obtain the approximate extra resistance resulting from cell-substrate contact for each cell type. The value of cell-substrate resistance was also estimated by two theoretical calculations that bracket the true values. Our results demonstrate that cell-substrate contact substantially influences the TER data measured using polycarbonate filters and that the extra resistance due to cell-substrate spaces depends on both cell type and filter property.
Assuntos
Adesão Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Animais , Técnicas de Cultura de Células , Linhagem Celular , Cães , Impedância Elétrica , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Junções Intercelulares/fisiologia , Filtros Microporos , Modelos Biológicos , Cimento de PolicarboxilatoRESUMO
Orbital fibroblasts exhibit a phenotype distinct from that of other types of fibroblasts. Addition of prostaglandin E2 (PGE2) to culture medium elicits a dramatic change in orbital fibroblast morphology. That response is mediated through the generation of cAMP. Orbital fibroblasts can generate high levels of PGE2 through induction by proinflammatory cytokines of prostaglandin endoperoxide H synthase-2 (PGHS-2). Here we compare the influence on fibroblast morphology of exogenous PGE2, forskolin, and 8-br-cAMP to that mediated through PGHS-2 induction by a lymphocyte-derived cytokine. Within a few hours, orbital fibroblasts treated with any of these test compounds appear under phase-contrast microscopy to exhibit a stellate morphology. When these changes were assessed quantitatively by electric cell-substrate impedance sensing (ECIS), it became evident that 8-br-cAMP, forskolin, and PGE2 initiated shape changes within 30 min of addition to the culture medium, while effects of the cytokine were first evident after approximately 3.5 h. Dermal fibroblasts failed to respond to any of these compounds with regard to changes in cellular morphology. Analysis of micromotion, manifested as small impedance fluctuations, revealed that orbital fibroblasts treated with 8-br-cAMP exhibit less motion than did untreated cells. These results suggest that orbital fibroblast shape can be altered by several compounds known to alter intracellular cAMP levels. They demonstrate the utility of ECIS in the assessment of very rapid and dynamic cellular events associated with changes in cell morphology.
Assuntos
AMP Cíclico/metabolismo , Fibroblastos/citologia , Doença de Graves/patologia , Órbita/citologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Abdome , Bucladesina/farmacologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , Ciclo-Oxigenase 2 , Derme , Dinoprostona/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Doença de Graves/metabolismo , Humanos , Isoenzimas/fisiologia , Linfocinas/farmacologia , Proteínas de Membrana , Órbita/efeitos dos fármacos , Órbita/metabolismo , Prostaglandina-Endoperóxido Sintases/fisiologia , Fatores de TempoRESUMO
Previously, we demonstrated the role of nitric oxide (NO) in transforming epithelial cells from a stationary to locomoting phenotype [E. Noiri, T. Peresleni, N. Srivastava, P. Weber, W.F. Bahou, N. Peunova, and M. S. Goligorsky. Am. J. Physiol. 270 (Cell Physiol. 39): C794-C802, 1996] and its permissive function in endothelin-1-stimulated endothelial cell migration (E. Noiri, Y. Hu, W. F. Bahou; C. Keese, I. Giaever, and M. S. Goligorsky, J. Biol: Chem. 272: 1747-1753, 1997). In the present study, the role of functional NO synthase in executing the vascular endothelial growth factor (VEGF)-guided program of endothelial cell migration and angiogenesis was studied in two independent experimental settings. First, VEGF, shown to stimulate NO release from simian virus 40-immortalized microvascular endothelial cells, induced endothelial cell transwell migration, whereas NG-nitro-L-arginine methyl ester (L-NAME) or antisense oligonucleotides to endothelial NO synthase suppressed this effect of VEGF. Second, in a series of experiments on endothelial cell wound healing, the rate of VEGF-stimulated cell migration was significantly blunted by the inhibition of NO synthesis. To gain insight into the possible mode of NO action, we next addressed the possibility that NO modulates cell matrix adhesion by performing impedance analysis of endothelial cell monolayers subjected to NO. The data showed the presence of spontaneous fluctuations of the resistance in ostensibly stationary endothelial cells. Spontaneous oscillations were induced by NO, which also inhibited cell matrix adhesion. This process we propose to term "podokinesis" to emphasize a scalar from of micromotion that, in the presence of guidance cues, e.g., VEGF, is transformed to a vectorial movement. In conclusion, execution of the program for directional endothelial cell migration requires two coexisting messages: NO-induced podokinesis (scalar motion) and guidance cues, e.g., VEGF, which imparts a vectorial component to the movement. Such a requirement for the dual signaling may explain a mismatch in the demand and supply with newly formed vessels in different pathological states accompanied by the inhibition of NO synthase.
Assuntos
Movimento Celular/fisiologia , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/fisiologia , Linfocinas/farmacologia , Óxido Nítrico/fisiologia , Cicatrização/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Microcirculação , Modelos Cardiovasculares , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/biossíntese , Oligonucleotídeos Antissenso/farmacologia , Ratos , Artéria Renal , Veias Umbilicais , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Cicatrização/efeitos dos fármacosRESUMO
Since macromolecular permeability between endothelial cells is regulated by tight junctions (zonula occludens), we wished to determine whether they also regulate neutrophil transendothelial migration. HUVEC monolayers, a commonly used model for studying leukocyte transmigration, were characterized using electric cell substrate impedance sensing and transmission electron microscopy. We show that culture medium containing endothelial cell growth supplement (50 microg/ml) was sufficient and necessary for the development of endothelial tight junctions. The frequency with which tight junctions were observed by transmission electron microscopy was further increased (twofold) by culturing HUVEC monolayers in a 1:1 mixture of endothelial medium and astrocyte-conditioned medium. These astrocyte-conditioned HUVEC monolayers showed a >1.5-fold increase in transcellular electrical resistance. The extent of neutrophil migration across IL-1-treated (10 U/ml for 4 h) HUVEC monolayers was the same whether tight junctions were present or absent, and the molecular requirements for neutrophil transmigration (CD18 and intercellular adhesion molecule-1) were unaffected by culturing in astrocyte-conditioned medium. Immunostaining for proteins associated with the intercellular junctional domain (occludin, ZO-1, cadherin, beta-catenin, gamma-catenin, and platelet-endothelial cell adhesion molecule-1) was localized to the endothelial borders, regardless of the culture conditions. Discontinuities were observed in the border staining for occludin, ZO-1, cadherin, and beta-catenin at the tricellular corner where the borders of three endothelial cells intersected. Significantly, 75% of neutrophil migration across IL-1-treated HUVEC monolayers occurred at tricellular corners. It appears that neutrophils preferentially migrate around endothelial tight junctions by crossing at tricellular corners rather than passing through the tight junctions that lie between two endothelial cells.
Assuntos
Movimento Celular/fisiologia , Endotélio Vascular/citologia , Neutrófilos/citologia , Junções Íntimas/fisiologia , Astrócitos/fisiologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Endotélio Vascular/fisiologia , Humanos , Neutrófilos/fisiologiaRESUMO
Endothelin (ET) synthesis is enhanced at sites of ischemia or in injured vessels. The purpose of this study was to explore the possibility of autocrine stimulation of endothelial cell migration by members of the endothelin family. Experiments with microvascular endothelial cell transmigration in a Boyden chemotactic apparatus showed that endothelins 1 and 3, as well as a selective agonist of ETB receptor IRL-1620, equipotently stimulated migration. Endothelial cell migration was unaffected by the blockade of ETA receptor, but it was inhibited by ETB receptor antagonism. Based on our previous demonstration of signaling from the occupied ETB receptor to constitutive nitric oxide (NO) synthase (Tsukahara, H., Ende, H., Magazine, H. I., Bahou, W. F., and Goligorsky, M. S. (1994) J. Biol. Chem. 269, 21778-21785), we next examined the contribution of ET-stimulated NO production to endothelial cell migration. In three independent cellular systems, 1) migration and wound healing by microvascular endothelial cells, 2) wound healing by Chinese hamster ovary cells stably expressing ETB receptor with or without endothelial NO synthase, and 3) application of antisense oligodeoxynucleotides targeting endothelial NO synthase in human umbilical vein endothelial cells, an absolute requirement for the functional NO synthase in cell migration has been demonstrated. These findings establish the permissive role of NO synthesis in endothelin-stimulated migration of endothelial cells.
Assuntos
Endotelinas/fisiologia , Endotélio Vascular/citologia , Óxido Nítrico/fisiologia , Animais , Células CHO , Movimento Celular/fisiologia , Células Cultivadas , Cricetinae , Humanos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/genética , Oligonucleotídeos Antissenso/farmacologia , Receptor de Endotelina B , Receptores de Endotelina/fisiologia , Proteínas Recombinantes/metabolismo , CicatrizaçãoRESUMO
Transepithelial impedance of Madin-Darby canine kidney cell layers is measured by a new instrumental method, referred to as electric cell-substrate impedance sensing. In this method, cells are cultured on small evaporated gold electrodes, and the impedance is measured in the frequency range 20-50,000 Hz by a small probing current. A model for impedance analysis of epithelial cells measured by this method is developed. The model considers three different pathways for the current flowing from the electrode through the cell layer: (1) in through the basal and out through the apical membrane, (2) in through the lateral and out through the apical membrane, and (3) between the cells through the paracellular space. By comparing model calculation with experimental impedance data, several morphological and cellular parameters can be determined: (1) the resistivity of the cell layer, (2) the average distance between the basal cell surface and substratum, and (3) the capacitance of apical, basal, and lateral cell membranes. This model is used to analyze impedance changes on removal of Ca2+ from confluent Mardin-Darby canine kidney cell layers. The method shows that reduction of Ca2+ concentration causes junction resistance between cells to drop and the distance between the basal cell surface and substratum to increase.
Assuntos
Membrana Celular/fisiologia , Eletrofisiologia/métodos , Modelos Biológicos , Animais , Linhagem Celular , Cães , Eletrofisiologia/instrumentação , Epitélio/fisiologia , Ouro , Rim , Matemática , MicroeletrodosRESUMO
Orbital fibroblasts from patients with Graves' ophthalmopathy, when treated with prostaglandin E2 (PGE2), become stellate and develop prominent cellular processes. In this paper, we describe results of studies designed to characterize the action of PGE2 on orbital fibroblast shape changes in vitro. Orbital and dermal fibroblasts were incubated with PGE2, one of several prostanoid analogues, 8-br-cAMP or forskolin and were then visualized by phase-contrast microscopy. Other studies involved seeding cells in special chambers equipped with electrodes for cell sensing using electric cell-substrate impedance sensing (ECIS) to detect changes in shape. PGE2 (10(-7) mol/L) elicited a rapid and dramatic alteration in the shape of orbital fibroblasts but not those derived from the skin. Cells became stellate and developed prominent cytoplasmic processes that extended out from the central area containing the cell nucleus. The effects were stereoselective in that a number of structurally related compounds, including Sulprostone, PGI2, PGF2 alpha, thromboxane A2, thromboxane B2, and 11 deoxy,16,16 dimethyl PGE2 failed to elicit a similar shape change. Butaprost (10(-5) mol/L), a specific EP2 agonist, elicited a similar shape-change as that observed with PGE2. 16,16-dimethyl PGE2, a nonselective agonist, could mimic the action of PGE2. The effect of PGE2 was apparent at 10(-8) mol/L, maximal at a concentration of 10(-7) mol/L and took 4-8 hr to evolve completely. Cycloheximide (10 micrograms/mL) and actinomycin D (1 micrograms/mL) failed to block the shape change. The morphologic change could be reproduced by addition of 8-br-cAMP (3 mmol/L) and by forskolin (5 mumol/L). Moreover, PGE2 and Butaprost treatment elicited in orbital cultures a massive increase in endogenous cAMP production while analogues not affecting cell shape failed to influence cyclic nucleotide generation. Three strains of orbital fibroblasts from patients with Graves' ophthalmopathy and three from normal orbits were tested and all responded to PGE2 (10(-7) mol/L). Four strains of dermal fibroblasts failed to respond to PGE2. The changes in orbital fibroblast morphology were accompanied by a marked decrease in monolayer impedance as assessed by electric cell-substrate impedance sensing. The earliest effects were apparent within 30 min using this sensitive technique. The widely recognized roles of PGE2 and related compounds in the mediation of the inflammatory response make our current findings in orbital fibroblasts of potential importance to the pathogenesis of Graves' ophthalmopathy.
Assuntos
AMP Cíclico/biossíntese , Dinoprostona/farmacologia , Órbita/efeitos dos fármacos , Órbita/metabolismo , Células Cultivadas , Impedância Elétrica , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Doença de Graves/patologia , Humanos , Microscopia de Contraste de Fase , Órbita/patologia , Valores de ReferênciaRESUMO
It is common in animal cell culture to utilize media that are buffered with bicarbonate ions and elevated CO2 concentrations in the incubator gas. Many modern incubators maintain these concentrations by monitoring the level of CO2 and injecting the gas when the value drops below a certain set point. This feedback system results in small oscillations in the incubator CO2 concentration of about 0.5%, resulting in oscillating pH values in the medium bathing the cells. These oscillations are very small and cannot be detected using a standard pH combination electrode, and until now no one has noticed any effect on the cells. In this study, however, we demonstrate that these periodic changes can result in detectable changes in cell morphology and therefore do affect cell behavior. Using electric cell-substrate impedance sensing (ECIS) to detect subtle alterations in cell morphology, we have measured cellular changes that when analyzed with the Fast Fourier Transform have a periodicity that matches those of the CO2 addition. This effect becomes particularly prominent when the culture medium has been acidified by cell metabolism reducing the liquid's buffer capacity. We have further analyzed the cyclical morphological changes employing a model of cell-electrode interactions in the ECIS measurement. We conclude that: (1) the periodic changes in cell morphology are most apparent if the medium is partially spent and if the surface to volume ratio of the culture vessel is high and (2) for confluent WI-38/VA13 fibroblasts and bovine pulmonary endothelial cells (B3B3), the main periodic morphological changes manifest themselves primarily as alterations in paracellular resistance.
Assuntos
Dióxido de Carbono/metabolismo , Células Cultivadas/citologia , Concentração de Íons de Hidrogênio , Animais , Bovinos , Células Cultivadas/metabolismo , Meios de Cultura , Análise de FourierRESUMO
Advantage was taken of DNA transfection techniques to investigate the effect of the pSV2-neo plasmid and its derivatives on recipient NIH 3T3 cell motion. Cell spreading and motion were followed by a newly developed electrical method to monitor cell morphology, referred to as electric cell-substrate impedance sensing. Using this method, we found that the eukaryotic-prokaryotic shuttle vector pSV2-neo had a strong effect on the recipient NIH 3T3 cell spreading and cell motion. However, two new neo plasmids, pSK-neo and pSP-neo, which were constructed by modifying the pSV2-neo plasmid, did not have a significant effect on the recipient cell activities. The results suggest that there may be some sequences in pSV2-neo which affect recipient cell behavior.
Assuntos
Células 3T3/citologia , Movimento Celular , Vetores Genéticos , Células 3T3/fisiologia , Animais , Adesão Celular , Eletrofisiologia , Camundongos , Mapeamento por Restrição , Relação Estrutura-AtividadeRESUMO
Fibroblasts derived from distinct anatomical regions appear to differ in regard to their behavior in culture. These differences may reflect functions of these cells in vivo that are tissue specific. Moreover, intrinsic differences in fibroblasts may underlie the site-specific connective tissue manifestations associated with systemic disease. We have demonstrated previously that orbital fibroblasts exhibit different cytokine response domains and protein synthetic programs when compared to those emanating from the skin. In the present communication, we demonstrate that prostaglandin E2 (PGE2) elicits in cultured human orbital fibroblasts from patients with Graves ophthalmopathy a rapid and dramatic change in cell morphology in vitro as assessed by phase-contrast and scanning electron microscopy. The central areas of the cells become elevated with respect to the plane of the substratum and are stellate, with long processes that touch neighboring cells. These changes occur within 6 hr of prostanoid addition to culture medium at an apparent concentration threshold of approximately 10 nM. Shape changes are accompanied by marked alterations in monolayer impedance as assessed by electric cell-substrate impedance sensing as described previously. Both morphologic and impedance changes elicited by PGE2 revert over 24 hr toward those found in untreated cells despite the continued presence of the prostanoid in the culture medium. In contrast, dermal fibroblasts fail to respond to PGE2. These observations define a previously unrecognized phenotypic attribute of orbital fibroblasts. Intrinsic differences in these cells may account for the anatomic site-selective vulnerability of the orbit in Graves ophthalmopathy. The culture system described here may be useful for studying the morphogenic actions of prostanoids.
Assuntos
Dinoprostona/farmacologia , Fibroblastos/patologia , Doença de Graves/patologia , Células Cultivadas , Humanos , Especificidade de Órgãos , FenótipoRESUMO
An electrical biosensor is described that can continuously track morphological changes of adherent cells providing quantitative data from both sparse and confluent cultures. The method is capable of detecting vertical motion of cells of the order of 1 nm, much below the resolution of an optical microscope.
Assuntos
Técnicas Biossensoriais , Células Cultivadas/citologia , Células Cultivadas/fisiologia , Adenocarcinoma , Animais , Bovinos , Adesão Celular , Movimento Celular , Eletrofisiologia/instrumentação , Eletrofisiologia/métodos , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Macrófagos/citologia , Macrófagos/fisiologia , Masculino , Mamíferos , Camundongos , Neoplasias da Próstata , Ratos , Células Tumorais CultivadasRESUMO
When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Fenômenos Biofísicos , Biofísica , Adesão Celular , Linhagem Celular , Impedância Elétrica , Estimulação Elétrica , Eletrofisiologia , Fibroblastos/fisiologia , Humanos , Potenciais da Membrana , Modelos BiológicosRESUMO
An electrical resistance method was developed to measure volume changes in substratum-attached monolayer cultures. Astrocytes in primary monolayer cultures prepared from neonatal rat cerebral cortex were placed in a confined channel containing a balanced salt solution, and the electrical resistance of the channel was measured using an applied alternating current. If the volume of the cells increases, then the volume of the solution within the channel available for current flow decreases by the same amount, resulting in an increase in the measured resistance through the channel. If the volume of the cells decreases, a decrease in resistance would be recorded. This method allows continuous measurements of volume changes in real time. When primary astrocyte monolayers were exposed to hyposmotic solutions (93-193 mosmol/kgH2O), they showed a rapid initial swelling and, in the continued presence of hyposmotic media, a characteristic regulatory volume decrease (RVD) in which there was a return to normal cell volume within approximately 20 min. Astrocytes exposed to hyperosmotic media (343-493 mosmol/kgH2O) gave a decrease in electrical resistance, indicating shrinkage. Putative endogenous effectors of astrocytic swelling, such as high extracellular K+ and glutamate, resulted in a much slower onset of swelling and no sign of RVD. This system can reliably measure the average change in cell monolayer volume to 1-2% and thus provides a sensitive means of continuous measurements of changes in cell volume in monolayer cultures.
Assuntos
Astrócitos/fisiologia , Eletrofisiologia/métodos , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Células Cultivadas , Condutividade Elétrica , RatosRESUMO
Cell movements in confluent fibroblastic cell layers have been followed with a new instrumental method. Using the electric cell-substrate impedance sensor (ECIS), cell motions on the scale of nanometers are manifested as fluctuations in the impedance of small gold film electrodes that serve as cell substrata. These fluctuations have been recorded and numerically processed for cells at 27 and 37 degrees C, for cells deprived of all external nutrients over extended periods of time, and for cells exposed to different levels of cytochalasin D. Results suggest that the movements in confluent layers detected via ECIS can be correlated with cell metabolic activity.
Assuntos
Movimento Celular , Técnicas de Cultura , Linhagem Celular , Citocalasina D/farmacologia , Fibroblastos , Glucose/farmacologia , Humanos , Métodos , Temperatura , Fatores de TempoRESUMO
We have developed an electrical method to study endothelial cell shape changes in real time in order to examine the mechanisms of alterations in the endothelial barrier function. Endothelial shape changes were quantified by using a monolayer of endothelial cells grown on a small (10(-3) cm2) evaporated gold electrode and measuring the changes in electrical impedance. Bovine pulmonary microvessel endothelial cells and bovine pulmonary artery endothelial cells were used to study the effects of alpha-thrombin on cell-shape dynamics by the impedance measurement. alpha-Thrombin produced a dose-dependent decrease in impedance that occurred within 0.5 min in both cell types, indicative of retraction of endothelial cells and widening of interendothelial junctions because of "rounding up" of the cells. The alpha-thrombin-induced decrease in impedance persisted for approximately 2 hr, after which the value recovered to basal levels. Pretreatment of endothelial cells with the protein kinase C inhibitor, calphostin C, or with 8-bromoadenosine 3',5'-cyclic monophosphate prevented the decreased impedance, suggesting that the endothelial cell change is modulated by activation of second-messenger pathways. The alpha-thrombin-induced decrease in impedance was in agreement with the previously observed increases in transendothelial albumin permeability and evidence of formation of intercellular gaps after alpha-thrombin challenge. The impedance measurement may be a valuable in vitro method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. Since the rapidly occurring changes in endothelial cell shape in response to mediators such as thrombin are mediated activation of second-messenger pathways, the ability to monitor endothelial cell dynamics in real time may provide insights into the signal-transduction events mediating the increased endothelial permeability.
Assuntos
Endotélio Vascular/fisiologia , Animais , Bovinos , Células Cultivadas , Eletrofisiologia , Endotélio Vascular/citologia , Técnicas In Vitro , Potenciais da Membrana , Permeabilidade , Trombina/farmacologiaRESUMO
A new electrical assay to measure the attachment and spreading of cells in tissue culture has been developed and substantiated by comparison with a more conventional assay. Small gold electrodes are vacuum deposited on the bottom of standard polystyrene culture dishes and coated with various proteins. As mammalian fibroblasts attach and spread on these surfaces, the measured impedance of the electrodes changes. These impedance changes reflect the amount of area blocked by the spreading cells. Since the weak electrical signals used have no noticeable effects on the cells, this is a very convenient method that is both quantitative and sensitive for measuring cell attachment and spreading.
Assuntos
Adesão Celular , Movimento Celular , Técnicas Citológicas , Eletrodos , Animais , Divisão Celular , Linhagem Celular Transformada , Ouro , Cinética , Métodos , PoliestirenosRESUMO
Motility is a fundamental property of mammalian cells that normally is observed in tissue culture by time lapse microscopy where resolution is limited by the wavelength of light. This paper examines a powerful electrical technique by which cell motion is quantitatively measured at the nanometer level. In this method, the cells are cultured on small evaporated gold electrodes carrying weak ac currents. A large change in the measured electrical impedance of the electrodes is observed when cells attach and spread on these electrodes. When the impedance is tracked as a function of time, fluctuations are observed that are a direct measure of cell motion. Surprisingly, these fluctuations continue even when the cell layer becomes confluent. By comparing the measured impedance with a theoretical model, it is clear that under these circumstances the average motions of the cell layer of 1 nm can be inferred from the measurements. We refer to this aspect of cell motility as micromotion.