Electric Cell-substrate Impedance Sensing Research Articles

Modulation of the Blood–Brain Barrier by Sigma-1R Activation

Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood–brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood–brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.

Development of a label‐free, impedance‐based biosensor to identify harmful effects of pesticides on insect cells

AbstractInsects are a major part of the planet´s ecosystem and their vital role as pollinators for agriculture is undisputed. Alongside factors as climate change or loss of habitats, rising use of pesticides emerges as a key threat to insect populations. For fighting this man‐made problem, development of an easy, fast, sensitive and non‐invasive biosensor for determining pesticide toxicity may help to ban harmful substances and formulations. Here, a biosensor based on Sf21 (Spodoptera frugiperda) insect cells as sensors and electric cell‐substrate impedance sensing (ECIS) as physical transducer is described. Sf21 cell suspensions and well‐defined pesticide solutions were mixed immediately before seeding on planar gold‐film electrodes. The capacitance at 20 kHz was recorded as a function of time as a measurand for cell adhesion providing dose‐response profiles of pesticide impact. For future in‐field applications, decoupling of the cell culture routines from the actual cytotoxicity assay is mandatory. Thus, suspensions of Sf21 cells were cryopreserved at –80°C in the wells of multi‐electrode arrays and thawed anytime for conducting the assays. Five pesticides were tested for their concentration dependent cytotoxicity expressed as EC50 values by ECIS and validated using the well‐established WST‐1 cell viability assay. Results were found to be in good agreement. Our studies revealed cytotoxic effects of some pesticides sold for home usage far below the recommended concentration and were found to be more toxic than formulations sold for agricultural industry only.This article is protected by copyright. All rights reserved.

Metal-organic frameworks as an active substrate for cell-interaction studies and cell-on-a-chip platforms

Metal-organic frameworks (MOFs) are an emerging class of materials with immense biomedical potential for their unique interactions with biological and organic materials. In this work, we select two candidate two-dimensional (2D) MOF systems based on Fe3+ and Ni2+ metal centers and 2-aminoterephthalate acid ligand (Fe-MOF and Ni-MOF) and evaluate their performance as an active interface for study of cell-interactions. 2D Fe-MOF and Ni-MOF were synthesized onto hydroxyl-modified gold and glass substrates using a layer-by-layer liquid-phase-epitaxy (LbL-LPE) growth at room temperature and used as active substrates (Fe-MOF/glass, Fe-MOF/Au, Ni-MOF/glass and Ni-MOF/Au, respectively) for MTT cell-proliferation and reactive oxygen species tests using the PC-12 cell-line in order to investigate the biocompatibility. Immunostaining and morphological analyses of PC-12 cells on MOF interfaces suggested a stronger cell-substrate interaction in comparison to glass and were further characterized using the Electrical Cell-substrate Impedance Sensing (ECIS) technique, here for the first time, employed to study cell attachment, spreading and proliferation on 2D Fe-MOF. The 2D Fe-MOF showed superior long-term stability in cell culture medium by recording impedance over 24 hours, crucial to monitor cell-dynamics at a solid-liquid interface. A significant increase of interfacial impedance was observed in ECIS, due to PC-12 cells adhering onto 2D Fe-MOF, which was also confirmed by the focused ion beam etching followed by scanning electron microscopy. Our novel findings, therefore, suggest 2D MOFs as highly suitable platform for the study of cell-related interactions using electrical techniques and potentially pave the way for future use of MOFs for bioelectronics and biosensor applications.

Analysis of Impedance Based Sensor to Discover the Invasive Nature of A549 Lung Cancer Cell

Numerous studies have been conducted to investigate the effectiveness of impedance-based sensors in detecting the invasive behavior of cancer cells, specifically through the use of Electric Cell-substrate Impedance Sensing (ECIS) methodology. However, the current equivalent circuit models used to represent the invasive nature of cancer cells have limitations and inaccuracies, and there has been a lack of utilization of mathematical and data analysis software to better understand the growth and invasive behavior of these cells. To address these gaps, this research aims to measure the impedance of A549 lung cancer cells, develop a simplified equivalent circuit model, and analyze the results using mathematical and data analysis software. The invasive behavior of A549 cells will first be studied through impedance measurements, and then a circuit model will be designed and simulated using software tools to reveal the true invasive nature of these cells. Finally, rigorous mathematical analysis and the use of suitable data analysis software (such as Matlab Powergui and Simulink, EIS Spectrum Analyzer, and Plotly) will be applied to gain a comprehensive understanding of the morphological behavior of the cells. The experimental results of this research are expected to align with previous findings, and a quadratic equation will be derived to predict the body's resistance of the A549 lung cancer cell using mathematical and data analysis approaches.

Modulation of the expression of connexins 37, 40, and 43 in endothelial cells in a culture.

Connexin (Cx) 37, 40, and 43 are implicated in vascular function, specifically in the electrical coupling of endothelial cells and vascular smooth-muscle cells. In the present study, we investigated whether factors implicated in vascular dysfunction can modulate the gene expression of Cx37, Cx40, and Cx43 and whether this is associated with changes in endothelial layer barrier function in human microvascular endothelial cells (HMEC-1). First, HMEC-1 were subjected to stimuli for 4 and 8h. We tested their responses to DETA-NONOate, H2O2, high glucose, and angiotensin II, none of which relevantly affected the transcription of the connexin genes. Next, we tested inflammatory factors IL-6, interferon gamma (IFNγ), and TNFα. IFNγ (10ng/mL) consistently induced Cx40 expression at 4 and 8 h to 10-20-fold when corrected for the control. TNFα and IL-6 resulted in small but significant depressions of Cx37 expression at 4h. Two JAK inhibitors, epigallocatechin-3-gallate (EGCG) (100-250μM) and AG490 (100-250μM), dose-dependently inhibited the induction of Cx40 expression by IFNγ. Subsequently, HMEC-1 were subjected to 10ng/mL IFNγ for 60h, and intercellular and transcellular impedance was monitored by electric cell-substrate impedance sensing (ECIS). In response to IFNγ, junctional-barrier impedance increased more than cellular-barrier impedance; this was prevented by AG490 (5μM). In conclusion, IFNγ can strongly induce Cx40 expression and modify the barrier properties of the endothelial cell membrane through the JAK/STAT pathway. Moreover, the Cx37, Cx40, and Cx43 expression in endothelial cells is stable and, apart from IFNγ, not affected by a number of factors implicated in endothelial dysfunction and vascular diseases.

Porous Membrane Electrical Cell-Substrate Impedance Spectroscopy for Versatile Assessment of Biological Barriers In Vitro.

Cell culture models of endothelial and epithelial barriers typically use porous membrane inserts (e.g., Transwell inserts) as a permeable substrate on which barrier cells are grown, often in coculture with other cell types on the opposite side of the membrane. Current methods to characterize barrier function in porous membrane inserts can disrupt the barrier or provide bulk measurements that cannot isolate barrier cell resistance alone. Electrical cell-substrate impedance sensing (ECIS) addresses these limitations, but its implementation on porous membrane inserts has been limited by costly manufacturing, low sensitivity, and lack of validation for barrier assessment. Here, we present porous membrane ECIS (PM-ECIS), a cost-effective method to adapt ECIS technology to porous substrate-based in vitro models. We demonstrate high fidelity patterning of electrodes on porous membranes that can be incorporated into well plates of a variety of sizes with excellent cell biocompatibility with mono- and coculture set ups. PM-ECIS provided sensitive, real-time measurement of isolated changes in endothelial cell barrier impedance with cell growth and barrier disruption. Barrier function characterized by PM-ECIS resistance correlated well with permeability coefficients obtained from simultaneous molecular tracer permeability assays performed on the same cultures, validating the device. Integration of ECIS into conventional porous cell culture inserts provides a versatile, sensitive, and automated alternative to current methods to measure barrier function in vitro, including molecular tracer assays and transepithelial/endothelial electrical resistance.

Transmembrane Protein CMTM6 Alleviates Ocular Inflammatory Response and Improves Corneal Epithelial Barrier Function in Experimental Dry Eye.

To investigate the impact of transmembrane protein CMTM6 on the pathogenesis of dry eye disease (DED) and elucidate its potential mechanisms. CMTM6 expression was confirmed by database analysis, real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Tear secretion was measured using the phenol red thread test. Immune cell infiltration was assessed through flow cytometry. Barrier function was evaluated by fluorescein sodium staining, immunofluorescence staining of zonula occludens 1 (ZO-1), and electric cell-substrate impedance sensing (ECIS) assessment. For silencing CMTM6 expression, siRNA and shRNA were employed, along with lentiviral vector-mediated overexpression of CMTM6. Proinflammatory cytokine levels were analyzed by RT-PCR and cytometric bead array (CBA) analysis. CMTM6 showed high expression in healthy human and mouse corneal and conjunctival epithelium but was notably reduced in DED. Notably, this downregulation was correlated with disease severity. Cmtm6-/- dry eye (DE) mice displayed reduced tear secretion, severe corneal epithelial defects, decreased conjunctival goblet cell density, and upregulated inflammatory response. Additionally, Cmtm6-/- DE mice and CMTM6 knockdown human corneal epithelial cell-transformed (HCE-T) cells showed more severe barrier disruption and reduced expression of ZO-1. Knockdown of CMTM6 in HCE-T cells increased inflammatory responses induced by hyperosmotic stress, which was significantly mitigated by CMTM6 overexpression. Moreover, the level of phospho-p65 in hyperosmolarity-stimulated HCE-T cells increased after silencing CMTM6. Nuclear factor kappa B (NF-κB) p65 inhibition (JSH-23) reversed the excessive inflammatory responses caused by hyperosmolarity in CMTM6 knockdown HCE-T cells. The reduction in CMTM6 expression on the ocular surface contributes to the pathogenesis of DED. The CMTM6-NF-κB p65 signaling pathway may serve as a promising therapeutic target for DED.

Monitoring Intestinal Organoid-Derived Monolayer Barrier Functions with Electric Cell-Substrate Impedance Sensing (ECIS).

The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro. So far, the ECIS technology has exclusively been performed on cell lines. Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids. Key features • We present a protocol that allows the assessment of various cell functions, such as proliferation and barrier formation, with ECIS on organoid-derived monolayers. • The protocol facilitates intestinal barrier research on patient tissue-derived organoids, providing a valuable tool for disease modeling.

Lidocaine inhibits migrationof tenocytesby downregulating focal adhesion kinaseand paxillin phosphorylation.

Lidocaine is the most frequently applied local infiltration anesthetic agent for treating tendinopathies. However, studies have discovered lidocaine to negatively affect tendon healing. In the current study, the molecular mechanisms and effects of lidocaine on tenocyte migration were evaluated. We treated tenocytes intrinsic to the Achilles tendons of Sprague-Dawley rats with lidocaine. The migration ability of cells was analyzed using electric cell-substrate impedance sensing (ECIS) and scratch wound assay. We then used a microscope to evaluate the cell spread. We assessed filamentous actin (F-actin) cytoskeleton formation through immunofluorescence staining. In addition, we used Western blot analysis to analyze the expression of phospho-focal adhesion kinase (FAK), FAK, phospho-paxillin, paxillin, and F-actin. We discovered that lidocaine had an inhibitory effect on the migration of tenocytes in the scratch wound assay and on the ECIS chip. Lidocaine treatment suppressed cell spreading and changed the cell morphology and F-actin distribution. Lidocaine reduced F-actin formation in the tenocyte during cell spreading; furthermore, it inhibited phospho-FAK, F-actin, and phospho-paxillin expression in the tenocytes. Our study revealed that lidocaine inhibits the spread and migration of tenocytes. The molecular mechanism potentially underlying this effect is downregulation of F-actin, phospho-FAK, and phospho-paxillin expression when cells are treated with lidocaine.

Upper glycolytic components contribute differently in controlling retinal vascular endothelial cellular behavior: Implications for endothelial-related retinal diseases.

Retinal degenerative diseases such as diabetic retinopathy and diabetic macular edema are characterized by impaired retinal endothelial cells (RECs) functionality. While the role of glycolysis in glucose homeostasis is well-established, its contributions to REC barrier assembly and cell spreading remain poorly understood. This study aimed to investigate the importance of upper glycolytic components in regulating the behavior of human RECs (HRECs). Electric cell-substrate impedance sensing (ECIS) technology was employed to analyze the real-time impact of various upper glycolytic components on maintaining barrier functionality and cell spreading of HRECs by measuring cell resistance and capacitance, respectively. Specific inhibitors were used: WZB117 to inhibit Glut1/3, lonidamine to inhibit hexokinases, PFK158 to inhibit the PFKFB3-PFK axis, and TDZD-8 to inhibit aldolases. Additionally, the viability of HRECs was evaluated using the lactate dehydrogenase (LDH) cytotoxicity assay. The most significant reduction in electrical resistance and increase in capacitance of HRECs resulted from the dose-dependent inhibition of PFKFB3/PFK using PFK158, followed by aldolase inhibition using TDZD-8. LDH level analysis at 24- and 48-hours post-treatment with PFK158 (1 μM) or TDZD-8 (1 and 10 μM) showed no significant difference compared to the control, indicating that the disruption of HRECs functionality was not attributed to cell death. Conversely, inhibiting Glut1/3 with WZB117 had minimal impact on HREC behavior, except at higher concentrations (10 μM) and prolonged exposure. Lastly, inhibiting hexokinase with lonidamine did not noticeably alter HREC cell behavior. This study illustrates the unique impacts of components within upper glycolysis on HREC functionality, emphasizing the crucial role of the PFKFB3/PFK axis in regulating HREC behavior. Understanding the specific contributions of each glycolytic component in preserving normal REC functionality will facilitate the development of targeted interventions for treating endothelial cell dysfunction in retinal disorders while minimizing effects on healthy cells.

Abstract 13070: Interleukin-19 Regulates Lymphatic Permeability and Induces Prox1-Dependent Lymphangiogenesis

Introduction: Increased lymphangiogenesis is associated with atherosclerosis, but whether it is disease-causative or a compensatory attempt to attenuate disease is currently unresolved. Recent studies suggest lymphatic permeability plays a role in reverse cholesterol transport (RCT) as a possible mechanism for attenuating atherosclerosis. We previously reported that that Interleukin-19 (IL-19), an immunomodulatory cytokine, is unique by inducing angiogenesis while reducing atherosclerosis. The purpose of this study was to test the hypothesis that IL-19 could be atheroprotective by inducing functional lymphangiogenesis and promoting lymphatic flux. Methods & Results: RNA sequencing of IL-19-treated human Lymphatic Endothelial Cells (hLEC) identified induction of chemokines, pro-lymphangiogenic, and permeability-associated genes. Prox1, the master regulator of lymphatic formation, was increased by six-fold, validated by qPCR and western blot. Cellular assays further demonstrated IL-19-associated lymphangiogenesis through hLEC proliferation, migration, and tube formation, each mitigated by Prox1 siRNA knockdown. Angpt2, a regulator of permeability through VE-cadherin, is also upregulated by IL-19 and mitigated by Prox1 siRNA knockdown. Electric Cell-substrate Impedance Sensing (ECIS) showed that IL-19 mitigates oxLDL-associated decreases in permeability, suggestive of preventing lymphatic barrier malfunction during atherosclerosis. Preliminary in vivo lymphatic permeability assays suggest injection of rmIL-19 improves lymphatic function in conditional lymphatic-specific Prox1 heterozygous mice. Conversely, similar experiments show that Il19 / LDLR double knockout mice, which are known to have exacerbated atherosclerosis, also have significantly decreased lymphatic permeability compared to LDLR -/- mice after 16-weeks of high fat diet. Conclusion: These data suggest that IL-19 is a previously unrecognized regulator of lymphatic function as it increases lymphangiogenesis and permeability, with the potential to improve RCT. Together, these effects may regulate atherosclerosis, and influence our understanding of the association between the lymphatic system and atherosclerosis.

A Novel Combination Therapy Tβ4/VIP Protects against Hyperglycemia-Induced Changes in Human Corneal Epithelial Cells.

Despite the prevalence of diabetic retinopathy, the majority of adult diabetic patients develop visually debilitating corneal complications, including impaired wound healing. Unfortunately, there is limited treatment for diabetes-induced corneal damage. The current project investigates a novel, peptide-based combination therapy, thymosin beta-4 and vasoactive intestinal peptide (Tβ4/VIP), against high-glucose-induced damage to the corneal epithelium. Electric cell-substrate impedance sensing (ECIS) was used for real-time monitoring of barrier function and wound healing of human corneal epithelial cells maintained in either normal glucose (5 mM) or high glucose (25 mM) ± Tβ4 (0.1%) and VIP (5 nM). Barrier integrity was assessed by resistance, impedance, and capacitance measurements. For the wound healing assay, cell migration was also monitored. Corneal epithelial tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were assessed to confirm our findings. Barrier integrity and wound healing were significantly impaired under high-glucose conditions. However, barrier function and cell migration significantly improved with Tβ4/VIP treatment. These findings were supported by high-glucose-induced downregulation of tight junction proteins that were effectively maintained similar to normal levels when treated with Tβ4/VIP. These results strongly support the premise that Tβ4 and VIP work synergistically to protect corneal epithelial cells against hyperglycemia-induced damage. In addition, this work highlights the potential for significant translational impact regarding the treatment of diabetic patients and associated complications of the cornea.

Determination of Endothelial Barrier Resistance by Electric Cell-Substrate Impedance Sensing (ECIS) System.

Endothelial cells lining the inner surface of blood vessels and lymphatic vessels play an indispensable role in vascular homeostasis. Apart from regulating vessel tone and forming an anti-thrombotic and anti-atherosclerotic surface, the dynamic endothelial barrier controls transport of solutes and fluid in and out of tissues at the capillary bed. Transit of circulating leukocytes into and out of circulation during inflammation and tissue repair is also regulated by the endothelium. Dysregulation of this barrier function of endothelial cells is a hallmark feature of multiple diseases and conditions such as sepsis, cancer metastasis, and edema. In this chapter we describe a detailed methodology to perform an in vitro experiment to monitor changes in barrier properties of human umbilical vein endothelial cells (HUVECs) in real time, in response to thrombin with electrical cell-substrate impedance sensing (ECIS) biosensor system.

Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines.

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising pericytes, astrocytic foot processes, and tightly adhered endothelial cells. Here, the brain endothelial cells form the first line of barrier against blood-borne pathogens. In conditions like cancer and neuroinflammation, circulating factors in the blood can disrupt this barrier. Disease progression significantly worsens post barrier disruption, which permits access to or impairment of regions of the brain. This significantly worsens the prognoses, particularly due to limited treatment options available at the level of the brain. Hence, emerging studies aim to investigate potential therapeutics that can prevent these detrimental factors in the blood from interacting with the brain endothelial cells. The commercially available Electric Cell-Substrate Impedance Sensing (ECIS) and cellZscope instruments measure the impedance across cellular monolayers, such as the BBB endothelium, to determine their barrier strength. Here we detail the use of both biosensors in assessing brain endothelial barrier integrity upon the addition of various stimuli. Crucially, we highlight the importance of their high-throughput capability for concurrent investigation of multiple variables and biological treatments.

Mitigation of trauma-induced endotheliopathy by activated protein C: A potential therapeutic for postinjury thromboinflammation.

Activated Protein C (aPC) plays dual roles after injury, driving both trauma-induced coagulopathy (TIC) by cleaving, and thus inactivating, factors Va and VIIIa and depressing fibrinolysis while also mediating an inflammomodulatory milieu via protease activated receptor-1 (PAR-1) cytoprotective signaling. Because of this dual role, it represents and ideal target for study and therapeutics after trauma. A known aPC variant, 3K3A-aPC, has been engineered to preserve cytoprotective activity while retaining minimal anticoagulant activity rendering it potentially ideal as a cytoprotective therapeutic after trauma. We hypothesized that 3K3A-aPC would mitigate the endotheliopathy of trauma by protecting against endothelial permeability. We used electric cell-substrate impedance sensing to measure permeability changes in real time in primary endothelial cells. These were cultured, grown to confluence, and treated with a 2 μg/mL solution of 3K3A-aPC at 180 minutes, 120 minutes, 60 minutes, 30 minutes prior to stimulation with ex vivo plasma taken from severely injured trauma patients (Injury Severity Score > 15 and BD < -6) (trauma plasma [TP]). Cells treated with thrombin and untreated cells were included in this study as control groups. Permeability changes were recorded in real time via electric cell-substrate impedance sensing for 30 minutes after treatment with TP. We quantified permeability changes in the control and treatment groups as area under the curve (AUC). Rac1/RhoA activity was also compared between these groups. Statistical significance was determined by one-way ANOVA followed by a post hoc analysis using Tukey's multiple comparison's test. Treatment with aPC mitigated endothelial permeability induced by ex vivo trauma plasma at all pre-treatment time points. The AUC of the 30-minute 3K3A-aPC pretreatment group was higher than TP alone (mean diff. 22.12 95% CI [13.75, 30.49], p < 0.0001) (Figure). Moreover, the AUC of the 60-minute, 120-minute, and 180-minute pretreatment groups was also higher than TP alone (mean diff., 16.30; 95% confidence interval [CI], 7.93-24.67; 19.43; 95% CI, 11.06-27.80, and 18.65; 95% CI, 10.28-27.02;, all p < 0.0001, respectively). Rac1/RhoA activity was higher in the aPC pretreatment group when compared with all other groups ( p < 0.01). Pretreatment with 3K3A-aPC, which retains its cytoprotective function but has only ~5% of its anticoagulant function, abrogates the effects of trauma-induced endotheliopathy. This represents a potential therapeutic treatment for dysregulated thromboinflammation for injured patients by minimizing aPC's role in trauma-induced coagulopathy while concurrently amplifying its essential cytoprotective function. Prognostic and Epidemiological; Level III.

The contribution of reticular basement membrane proteins to basal airway epithelial attachment, spreading and barrier formation: implications for airway remodeling in asthma.

In the healthy lung, the pseudostratified conducting airway epithelium is anchored to the reticular basement membrane (RBM) via hemidesmosome junction complexes formed between basal cells and the extracellular matrix (ECM). The RBM within the healthy lung is composed of the ECM proteins laminin and collagen-IV. In patients with asthma, the RBM is remodeled with collagen-I, -III and fibronectin deposition. The goal of this study was to assess the effect of RBM ECM proteins on basal airway epithelial cell attachment, spreading and barrier formation using real-time electrical cell-substrate impedance sensing (ECIS). ECIS 8-well arrays were coated with 50 μg/mL of fibronectin, collagen-I, collagen-III, collagen-IV, or laminin and compared to bovine serum albumin (BSA) or uncoated controls. The airway epithelial cell line (1HAEo-) was seeded 40, 50, 60, and 70 k cells/well and continuously monitored over 70 h to assess cell attachment, spreading and barrier formation using high (64 k Hz) and low (500 Hz) frequency resistance and capacitance. Data were analyzed using a one-phase decay model from which half-life (time cells cover half of the electrode area) and rate-constant (cell-spreading rate/h) were determined and a generalized additive mixed effect model (GAMM) was used to assess ECM proteins over the entire experiment. High-frequency (64 kHz) capacitance measures demonstrated the half-life for 1HAEo-cells to attach was fastest when grown on fibronectin (6.5 h), followed by collagen-I (7.2 h) and collagen-III (8.1 h), compared to collagen-IV (11.3 h), then laminin (13.2 h) compared to BSA (12.4 h) and uncoated (13.9 h) controls. High-frequency (64 kHz) resistance measures demonstrated that the rate of 1HAEo- cell spreading was significantly faster on fibronectin and collagen-I compared to collagen-III, collagen-IV, laminin, BSA and the uncoated control. Low-frequency (500 Hz) resistance measures demonstrated that 1HAEo-cells formed a functional barrier fastest when grown on fibronectin and collagen-I, compared to the other ECM conditions. Lastly, the distance of 1HAEo-cells from the ECM substrates was the smallest when grown on fibronectin reflecting high cell-matrix adhesion. Airway epithelial cells attach, spread and form a barrier fastest on fibronectin, and collagen-I and these reticular basement membrane ECM proteins may play a protective role in preserving the epithelial barrier during airway remodeling in asthma.

Classifying contaminated cell cultures using time series features

We examine the use of time series data, derived from Electric Cell-substrate Impedance Sensing (ECIS), to differentiate between standard mammalian cell cultures and those infected with a mycoplasma organism. With the goal of easy visualization and interpretation, we perform low-dimensional feature-based classification, extracting application-relevant features from the ECIS time courses. We can achieve very high classification accuracy using only two features, which depend on the cell line under examination. Initial results also show the existence of experimental variation between plates and suggest types of features that may prove more robust to such variation. Our paper is the first to perform a broad examination of ECIS time course features in the context of detecting contamination; to combine different types of features to achieve classification accuracy while preserving interpretability; and to describe and suggest possibilities for ameliorating plate-to-plate variation.

BSBM-13 MELANOMA CELLS PRODUCE A SUSPENSION SUB-POPULATION THAT DRASTICALLY DISRUPTS THE BLOOD-BRAIN BARRIER ENDOTHELIUM

Abstract Melanoma metastasizes to the brain with high propensity. Brain metastasis requires cancer cells to breach the blood-brain barrier (BBB) endothelium, which forms the first line of defence against blood-borne pathogens. An increasing number of molecules are discussed in BBB disruption, and their importance varies across melanoma and endothelial cell lines. This diversity in literature proposes that cancer cells of the same cancer type employ different modes of metastasis. This study shows an example of the difference in migration capabilities of adherent melanoma cells and a suspension sub-population. We used Electric Cell-substrate Impedance Sensing (ECIS) to assess changes in human brain endothelial barrier function upon adding human melanoma lines. Several melanoma lines induced brain endothelial barrier disruption within the first few hours of addition. Intriguingly, the suspension population of melanoma cells disrupted the endothelial barrier faster and more drastically than the adherent counterpart. Time-lapse imaging revealed that the suspension population used a transcellular route of invasion compared to the paracellular route used by the adherent cells. Confocal microscopy supported this, showing clear differences in the physical interaction of the suspension subtype with the brain endothelium, compared to the adherent cells. Flow cytometry and NanoString technology allowed characterization of the melanoma lines, showing that the suspension subtype protein composition differs from their adherent counterpart. We hypothesize that melanoma cells may produce specific subtypes that are more invasive, which can accelerate migration through the brain endothelium. We stress the importance of investigating the composition of melanoma cells and their bi-products when researching cancer metastasis. Once successfully within the brain parenchyma, these cancer cells are effectively separated from the peripheral adaptive immune system, designed to detect and eliminate them. Therefore, preventing cancer cell migration into the brain is an attractive consideration, as they are considerably more targetable whilst still in the blood.

#5930 ENDOTHELIAL CELL PROTEOME AND SECRETOME ALTERATION UNDER CHRONIC UREMIC EXPOSURE

Abstract Background and Aims Dialysis therapies including peritoneal dialysis (PD) are described to lead to premature vascular aging, atherosclerosis and associated cardiomyopathies, which are associated with high cardiovascular morbidity and mortality. Chronic inflammation, incomplete clearance of uremic toxins, and dyslipidaemia, are factors affecting the vasculature system. However, how these factors affect the endothelium, the fist cell layer in contact with these molecules, and the molecular mechanisms triggered by uremic toxins remain poorly understood. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PD fluids reduce peritoneal damage in mesothelial and endothelial (EC) cells. Their potential systemic effects were not studied so far. Here, we demonstrate, in a newly developed model system, the changes induced by chronic exposure to serum of PD patients in the proteome and secretome of endothelial cells, and the effects of cytoprotective additives. Method For modelling systemic conditions of PD patients, primary EC were cultured for 5 passages in medium containing 10% serum collected from PD patients (n = 26) during regular PET tests, or from healthy donors (n = 12). Cytoprotective additives were added in parallel experiments. Cells were stably labelled (SILAC) to differentiate cell and donor serum proteins and the cellular proteome and secretome profiles were analysed by quantitative mass spectrometry. Prior to analysis of the secretome, equalizer beads, to enrich low abundant and deplete high abundant proteins, were used. ECIS (Electric Cell-substrate Impedance Sensing), was used to measure barrier function, growth rate, and permeability. Results Proteome analysis revealed perturbation of major cellular processes by serum of PD patients including inflammatory related processes such TLR regulation and complement activation, as well membrane related processes such as extracellular matrix interactions and junctions, and plasma lipoprotein remodelling. Addition of LiCl counteracted cell-adhesion related proteins. ECIS analysis showed that in uremic conditions the EC monolayer has a decreased barrier function compared to healthy conditions, and LiCl partially restores the tightness of the membrane. Secretome analysis showed differentially regulated proteins related to oxidative stress, senescence-associated secretory phenotype (SASP), and apoptosis. Interestingly, in the secretome, LiCl addition counter regulated INHBA and tissue factor pathway inhibitor 2, both proteins related to vascular calcification. Conclusion Surprisingly few studies have analysed uremic effects on EC using proteomics approaches, and no reports of chronic settings, modelling the patient situation, are available. Our data demonstrates that EC react to serum factors of PD patients with increased inflammation, permeability and a secretory profile. Interestingly, our model reflects many of the known effects on the vasculature, but unravels the molecular mechanism that may induce those processes. We have also identified potential mechanisms by which the addition of cytoprotective additives may counteract some of the uremic effects systemically. Secretomics data identified several proteins secreted by EC that are regulated in uremia with potential for cellular crosstalk with other cells of the vasculature, showing the potential to identify therapeutic targets to reduce the cardiovascular risk of PD patients and current limitations of the therapy.

A simplified workflow with end-point validation of real-time electrical cell-substrate impedance sensing of retinoic acid stimulated neurogenesis in human SH-SY5Y cells in vitro

ObjectiveRetinoic acid (RA) is known to transition proliferating SH-SY5Y neuroblastoma cells towards functional neurons. However, the activity of RA is restricted due to its photolability where any findings from prolonged time course observations using microscopy may alter outcomes. The aim of the study was to establish a real-time, long-term (9-day) protocol for the screening of differentiation events using Electrical cell-substrate impedance sensing (ECIS).Results and discussionA differentiation baseline for SH-SY5Y cells was established. Cells were seeded and exposed to repeated spikes of RA using the xCELLigence real-time cell analyser single plate (RTCA-SP) for real-time monitoring and identification of differentiation activity over a 9 day period in order to be more representative of differentiation over a prolonged timeline. Specific features associated with differentiation (growth inhibition, neurite outgrowths) were confirmed by end-point analysis.RA-induced growth inhibition and assumed phenotypic changes (i.e. neurite outgrowth) were identified by the xCELLigence analysis and further confirmed by end-point metabolic and phenotypic assays. Change in cellular morphology and neurite outgrowth length was identified by end-point fluorescence detection followed by computational analysis. Based on this it was possible to identify SH-SY5Y phenotypic differentiation with distinct phases observed over 9 days using Electric cell-substrate impedance sensing (ECIS) cell index traces providing a path to application in larger scale neurotrophic factor screening using this scalable technology.