In Vitro Wounding Models Using the Electric Cell-Substrate Impedance Sensing (ECIS)-Z? Technology.
ABSTRACT: Electric Cell-Substrate Impedance Sensing (ECIS) can produce reproducible wounding models by mechanically disrupting a cell monolayer. This study compared in vitro wound-healing using human cerebral microvascular endothelial cells (hCMVEC) with both single electrode (8W1E) and multiple electrodes (8W10E+) arrays. Measurements of hCMVEC migration and barrier functions were conducted, revealing variable levels of barrier disruption could be achieved by altering the duration and magnitude of the applied current. In all scenarios, the barrier (Rb) did not recover the strength observed prior to injury. Localization of junctional proteins following wounding were analyzed by immunocytochemistry. Following wounding, cell migration was generally faster on the 8W10E+ than the 8W1E array. Immunohistochemical analysis revealed non-viable cells remained on the 8W1E electrodes but not the 8W10E+ electrodes. However, viable cells partially remained on the 8W10E+ electrodes following wounding. In addition, the 8W10E+ electrodes demonstrated variation in cell loss across electrodes within the same well. This suggests the type of wounding is different on the two array types. However, our data show both arrays can be used to model incomplete barrier recovery and therefore both have potential for testing of drugs to improve endothelial barrier function. This is the first time that the possibility of using the 8W10E+ array as a wounding model is addressed. We highlight the differences in wounding produced between the two arrays, and can be used to study the underlying causes for impaired barrier function following CNS injuries.
Project description:Degeneration of the retinal pigment epithelium (RPE) plays a central role in age-related macular degeneration (AMD). Throughout life, RPE cells are challenged by a variety of cytotoxic stressors, some of which are cumulative with age and may ultimately contribute to drusen and lipofuscin accumulation. Stressors such as these continually damage RPE cells resulting in a state of chronic wounding. Current cell-based platforms that model a state of chronic RPE cell wounding are limited, and the RPE cellular response is not entirely understood. Here, we used the electric cell-substrate impedance sensing (ECIS) system to induce a state of acute or chronic wounding on differentiated human fetal RPE cells to analyze changes in the wound repair response. RPE cells surrounding the lesioned area employ both cell migration and proliferation to repair wounds but fail to reestablish their original cell morphology or density after repetitive wounding. Chronically wounded RPE cells develop phenotypic AMD characteristics such as loss of cuboidal morphology, enlarged size, and multinucleation. Transcriptomic analysis suggests a systemic misregulation of RPE cell functions in bystander cells, which are not directly adjacent to the wound. Genes associated with the major RPE cell functions (LRAT, MITF, RDH11) significantly downregulate after wounding, in addition to differential expression of genes associated with the cell cycle (CDK1, CDC6, CDC20), inflammation (IL-18, CCL2), and apoptosis (FAS). Interestingly, repetitive wounding resulted in prolonged misregulation of genes, including FAS, LRAT, and PEDF. The use of ECIS to induce wounding resulted in an over-representation of AMD-associated genes among those dysregulated genes, particularly genes associated with advanced AMD. This simple system provides a new model for further investigation of RPE cell wound response in AMD pathogenesis.
Project description:Electric cell-substrate impedance sensing (ECIS) is an impedance-based method for monitoring changes in cell behaviour in real-time. In this paper, we highlight the importance of ECIS in measuring the kinetics of human melanoma cell invasion across human brain endothelium. ECIS data can be mathematically modelled to assess which component of the endothelial paracellular and basolateral barriers is being affected and when. Our results reveal that a range of human melanoma cells can mediate disruption of human brain endothelium, primarily involving the paracellular route, as demonstrated by ECIS. The sensitivity of ECIS also reveals that the paracellular barrier weakens within 30-60 min of the melanoma cells being added to the apical face of the endothelial cells. Imaging reveals pronounced localisation of the melanoma cells at the paracellular junctions consistent with paracellular migration. Time-lapse imaging further reveals junctional opening and disruption of the endothelial monolayer by the invasive melanoma cells all within several hours. We suggest that the ability of ECIS to resolve changes to barrier integrity in real time, and to determine the route of migration, provides a powerful tool for future studies investigating the key molecules involved in the invasive process of cancer cells.
Project description:In this paper, we demonstrate the application of electrical cell-substrate impedance sensing (ECIS) technology for measuring differences in the formation of a strong and durable endothelial barrier model. In addition, we highlight the capacity of ECIS technology to model the parameters of the physical barrier associated with (I) the paracellular space (referred to as Rb) and (II) the basal adhesion of the endothelial cells (α, alpha). Physiologically, both parameters are very important for the correct formation of endothelial barriers. ECIS technology is the only commercially available technology that can measure and model these parameters independently of each other, which is important in the context of ascertaining whether a change in overall barrier resistance (R) occurs because of molecular changes in the paracellular junctional molecules or changes in the basal adhesion molecules. Finally, we show that the temporal changes observed in the paracellular Rb can be associated with changes in specific junctional proteins (CD144, ZO-1, and catenins), which have major roles in governing the overall strength of the junctional communication between neighbouring endothelial cells.
Project description:Pericytes and endothelial cells are critical cellular components of the blood-brain barrier (BBB) and play an important role in neuroinflammation. To date, the majority of inflammation-related studies in endothelia and pericytes have been carried out using immortalised cell lines or non-human-derived cells. Whether these are representative of primary human cells is unclear and systematic comparisons of the inflammatory responses of primary human brain-derived pericytes and endothelia has yet to be performed.To study the effects of neuroinflammation at the BBB, primary brain endothelial cells and pericytes were isolated from human biopsy tissue. Culture purity was examined using qPCR and immunocytochemistry. Electrical cell-substrate impedance sensing (ECIS) was used to determine the barrier properties of endothelial and pericyte cultures. Using immunocytochemistry, cytometric bead array, and ECIS, we compared the responses of endothelia and pericytes to a panel of inflammatory stimuli (IL-1?, TNF?, LPS, IFN-?, TGF-?1, IL-6, and IL-4). Secretome analysis was performed to identify unique secretions of endothelia and pericytes in response to IL-1?.Endothelial cells were pure, moderately proliferative, retained the expression of BBB-related junctional proteins and transporters, and generated robust TEER. Both endothelia and pericytes have the same pattern of transcription factor activation in response to inflammatory stimuli but respond differently at the secretion level. Secretome analysis confirmed that endothelia and pericytes have overlapping but distinct secretome profiles in response to IL-1?. We identified several cell-type specific responses, including G-CSF and GM-CSF (endothelial-specific), and IGFBP2 and IGFBP3 (pericyte-specific). Finally, we demonstrated that direct addition of IL-1?, TNF?, LPS, and IL-4 contributed to the loss of endothelial barrier integrity in vitro.Here, we identify important cell-type differences in the inflammatory response of brain pericytes and endothelia and provide, for the first time, a comprehensive profile of the secretions of primary human brain endothelia and pericytes which has implications for understanding how inflammation affects the cerebrovasculature.
Project description:The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer's tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell(®) tracer assays and two commercial impedance devices (xCELLigence(®), ECIS(®)). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments.
Project description:Toll-Like receptors (TLRs) represent an important early warning mechanism for the immune system to detect infection or tissue damage. The focus of this research was to determine the neuroinflammatory responses to commercial TLR ligands and their effects on brain endothelial barrier strength. Using biosensor technology we screened TLR ligands to all human TLRs and found that the brain endothelial hCMVECs cell line only responded to Poly(I:C) (TLR3-ligand), LPS (TLR4-ligand) and Imiquimod (TLR7 ligand). Both Poly(I:C) and LPS induced pronounced pro-inflammatory cytokine secretion as expected, whereas Imiquimod did not induce secretion of any pro-inflammatory cytokines. Using ECIS technology to measure endothelial barrier function, LPS and Poly(I:C) both acutely reduced barrier-strength, whereas Imiquimod caused immediate and sustained strengthening of the barrier. Further cytokine and ECIS studies showed that Imiquimod could abrogate some of the pro-inflammatory responses to Poly(I:C) and LPS. Most surprisingly, PCR revealed that the hCMVECs lacked TLR7 but expressed both TLR3 and TLR4 and did not respond to other structurally different TLR7 ligands. These data demonstrate that brain endothelial cells can be regulated by TLR 3 and TLR4 ligands in a pro-inflammatory manner and have receptors to Imiquimod, distinct to the classical TLR7, that function in an anti-inflammatory manner.
Project description:Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. Humanized disease models are required to develop new therapies for currently incurable forms of AMD. In this work, a tissue-on-a-chip approach was developed through combining human induced pluripotent stem cells, Electric Cell-substrate Impedance Sensing (ECIS) and reproducible electrical wounding assays to model and quantitatively study AMD. Retinal Pigment Epithelium (RPE) cells generated from a patient with an inherited macular degeneration and from an unaffected sibling were used to test the model platform on which a reproducible electrical wounding assay was conducted to model RPE damage. First, a robust and reproducible real-time quantitative monitoring over a 25-day period demonstrated the establishment and maturation of RPE layers on the microelectrode arrays. A spatially controlled RPE layer damage that mimicked cell loss in AMD disease was then initiated. Post recovery, significant differences (P < 0.01) in migration rates were found between case (8.6 ± 0.46 ?m/h) and control cell lines (10.69 ± 0.21 ?m/h). Quantitative data analysis suggested this was achieved due to lower cell-substrate adhesion in the control cell line. The ECIS cell-substrate adhesion parameter (?) was found to be 7.8 ± 0.28 ?(1/2)cm for the case cell line and 6.5 ± 0.15 ?(1/2)cm for the control. These findings were confirmed using cell adhesion biochemical assays. The developed disease model-on-a-chip is a powerful platform for translational studies with considerable potential to investigate novel therapies by enabling real-time, quantitative and reproducible patient-specific RPE cell repair studies.
Project description:Electromechanical function of cardiac muscle depends critically on the crosstalk of myocytes with non-myocytes. Upon cardiac fibrosis, fibroblasts translocate into infarcted necrotic tissue and alter their communication capabilities. In the present in vitro study, we determined a multiple parameter space relevant for fibrotic cardiac tissue development comprising the following essential processes: (i) adhesion to substrates with varying elasticity, (ii) dynamics of contractile function, and (iii) electromechanical connectivity. By combining electric cell-substrate impedance sensing (ECIS) with conventional optical microscopy, we could measure the impact of fibroblast-cardiomyocyte ratio on the aforementioned parameters in a non-invasive fashion. Adhesion to electrodes was quantified via spreading rates derived from impedance changes, period analysis allowed us to measure contraction dynamics and modulations of the barrier resistance served as a measure of connectivity. In summary, we claim that: (i) a preferred window for substrate elasticity around 7 kPa for low fibroblast content exists, which is shifted to stiffer substrates with increasing fibroblast fractions. (ii) Beat frequency decreases nonlinearly with increasing fraction of fibroblasts, while (iii) the intercellular resistance increases with a maximal functional connectivity at 75% fibroblasts. For the first time, cardiac cell-cell junction density-dependent connectivity in co-cultures of cardiomyocytes and fibroblasts was quantified using ECIS.
Project description:Reliable single unit neuron recordings from chronically implanted microelectrode arrays (MEAs) are essential tools in the field of neural engineering. However, following implantation, MEAs undergo a foreign body response that functionally isolates them from the brain and reduces the useful longevity of the array. We tested a novel electrodeposited platinum-iridium coating (EPIC) on penetrating recording MEAs to determine if it improved recording performance. We chronically implanted the arrays in rats and used electrophysiological and histological measurements to compare quantitatively the single unit recording performance of coated vs. uncoated electrodes over a 12-week period. The coated electrodes had substantially lower impedance at 1?kHz and reduced noise, increased signal-to-noise ratio, and increased number of discernible units per electrode as compared to uncoated electrodes. Post-mortem immunohistochemistry showed no significant differences in the immune response between coated and uncoated electrodes. Overall, the EPIC arrays provided superior recording performance than uncoated arrays, likely due to lower electrode impedance and reduced noise.
Project description:BACKGROUND:The growth and development of plants is deleteriously affected by various biotic and abiotic stress factors. Wounding in plants is caused by exposure to environmental stress, mechanical stress, and via herbivory. Typically, oxidative burst in response to wounding is associated with the formation of reactive oxygen species, such as the superoxide anion radical (O2•-), hydrogen peroxide (H2O2) and singlet oxygen; however, few experimental studies have provided direct evidence of their detection in plants. Detection of O2•- formation in plant tissues have been performed using various techniques including electron paramagnetic resonance spin-trap spectroscopy, epinephrine-adrenochrome acceptor methods, staining with dyes such as tetrazolium dye and nitro blue tetrazolium (NBT); however, kinetic measurements have not been performed. In the current study, we provide evidence of O2•- generation and its kinetics in the leaves of spinach (Spinacia oleracea) subjected to wounding. METHODS:Real-time monitoring of O2•- generation was performed using catalytic amperometry. Changes in oxidation current for O2•- was monitored using polymeric iron-porphyrin-based modified carbon electrodes (? = 1 mm) as working electrode with Ag/AgCl as the reference electrode. RESULT:The results obtained show continuous generation of O2•- for minutes after wounding, followed by a decline. The exogenous addition of superoxide dismutase, which is known to dismutate O2•- to H2O2, significantly suppressed the oxidation current. CONCLUSION:Catalytic amperometric measurements were performed using polymeric iron-porphyrin based modified carbon electrode. We claim it to be a useful tool and a direct method for real-time monitoring and precise detection of O2•- in biological samples, with the potential for wide application in plant research for specific and sensitive detection of O2•-.