Alan B. Moy

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces.

cAMP protects endothelial barrier function independent of inhibiting MLC20-dependent tension development

Exposure of cultured human umbilical vein endothelial cells to the cAMP agonists theophylline and forskolin decreased constitutive isometric tension of a confluent monolayer inoculated on a collagen membrane, but it did not prevent increased tension in cells exposed to thrombin. The inability of cAMP agonists to prevent tension development correlated with an inability of cAMP stimulation to prevent increased 20-kDa myosin light chain (MLC20) phosphorylation in response to thrombin. Although cAMP did not prevent tension development or increased MLC20phosphorylation, cAMP attenuated the effect of thrombin on transendothelial electrical resistance across a confluent monolayer inoculated on a gold microelectrode. Activation of cAMP-dependent signal transduction did not prevent a decline in resistance in thrombin-treated cells, but it more promptly restored transendothelial resistance to initial basal levels (10 min) compared with thrombin only (60 min). ML-7, an MLC kinase antagonist, at doses that attenuate increased MLC20 phosphorylation and tension development, did not prevent a decline in resistance in thrombin-treated cells. Yet, ML-7 also restored transendothelial resistance more rapidly than thrombin alone (20 min) but at a slower rate than cAMP. These data demonstrate that activation of cAMP-dependent signal transduction protects barrier function independent of inhibition of MLC20-dependent tension development.

Endothelial Cell Electrical Impedance Parameter Artifacts Produced by a Gold Electrode and Phase Sensitive Detection

Frequency dependent cellular micro-impedance estimates obtained from a gold two-electrode configuration using phase sensitive detection have become increasingly used to evaluate cellular barrier model parameters. The results of this study show that cellular barrier function parameter estimates optimized using measurements obtained from this biosensor are highly susceptible to both time dependent and systematic instrumental artifacts. Based on a power spectral analysis of experimentally measured microelectrode voltages, synchronous, 60 Hz, and white Gaussian noise were identified as the most significant time dependent instrumental artifacts. The reduction of these artifacts using digital filtering produced a corresponding reduction in the optimized model parameter fluctuations. Using a series of instrumental circuit models, this study also shows that electrode impedance voltage divider effects and circuit capacitances can produce systematic deviations in cellular barrier function parameter estimates. Although the implementation of an active current source reduced the voltage divider effects, artifacts produced by coaxial cable and other circuit capacitive elements at frequencies exceeding 1 kHz still remained. Reducing time dependent instrumental fluctuations and systematic errors produced a significant reduction in cellular model barrier parameter errors and improved the model fit to experimental data

Modelling and parameter estimation of a cell system

A non-linear identification approach for modelling endothelial cells is mathematically derived and experimentally tested under the realistic alternating current voltage and a square-like shape assumption. Results demonstrate the applicability and effectiveness of the approach. The derived model is an important step in understanding a cell system and in developing controlling and treating certain disorders by new drugs.

Instrumental noise estimates stabilize and quantify endothelial cell micro-impedance barrier function parameter estimates

Abstract A novel transcellular micro-impedance biosensor, referred to as the electric cell-substrate impedance sensor or ECIS, has become increasingly applied to the study and quantification of endothelial cell physiology. In principle, frequency dependent impedance measurements obtained from this sensor can be used to estimate the cell–cell and cell–matrix impedance components of endothelial cell barrier function based on simple geometric models. Few studies, however, have examined the numerical optimization of these barrier function parameters and established their error bounds. This study, therefore, illustrates the implementation of a multi-response Levenberg–Marquardt algorithm that includes instrumental noise estimates and applies it to frequency dependent porcine pulmonary artery endothelial cell impedance measurements. The stability of cell–cell, cell–matrix and membrane impedance parameter estimates based on this approach is carefully examined, and several forms of parameter instability and refinement illustrated. Including frequency dependent noise variance estimates in the numerical optimization reduced the parameter value dependence on the frequency range of measured impedances. The increased stability provided by a multi-response non-linear fit over one-dimensional algorithms indicated that both real and imaginary data should be used in the parameter optimization. Error estimates based on single fits and Monte Carlo simulations showed that the model barrier parameters were often highly correlated with each other. Independently resolving the different parameters can, therefore, present a challenge to the experimentalist and demand the use of non-linear multivariate statistical methods when comparing different sets of parameters.

Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells

Aim: Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28. We describe a robust Myc and Lin28-free IPSC reprogramming approach using reprogramming molecules. Methods: IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. Results: While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28-free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28. Conclusion: This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.

A multiplexed optical micro-impedance biosensor array for quantitaitive cancer drug assays

Optically based assays have traditionally been used to quantify cancer cell proliferation and cytotoxicity in response to chemotherapeutic agents. Cellular micro-impedance measurements provide complementary information that is a function of cellular adhesion and membrane properties. The ability to simultaneously acquire both optical and micro-impedance data has significant synergistic diagnostic potential. This study therefore proposes a multiplexed optical micro-impedance assay system compatible with routine pharmaceutical testing. Optically thin micro-impedance electrodes were designed for twenty-four and ninety-six well tissue culture plate readers. Electrode arrays were designed using micro-fabrication methods. The electrode arrays were then assembled into modified twenty-four and ninety-six well cell culture plates designed using computer aided design. Pharmaceutical research and personalized medicine applications are briefly discussed.

Cellular electrical micro-impedance parameter artifacts produced by passive and active current regulation.

This study analyzes the cellular microelectrode voltage measurement errors produced by active and passive current regulation, and the propagation of these errors into cellular barrier function parameter estimates. The propagation of random and systematic errors into these parameters is accounted for within a Riemannian manifold framework consistent with information geometry. As a result, the full non-linearity of the model parameter state dependence, the instrumental noise distribution, and the systematic errors associated with the voltage to impedance conversion, are accounted for. Specifically, cellular model parameters are treated as the coordinates of a model space manifold that inherits a Riemannian metric from the data space. The model space metric is defined in terms of the pull back of an instrumental noise-dependent Fisher information metric. Additional noise sources produced by the evaluation of the cell-covered electrode model that is a function of a naked electrode random variable are also included in the analysis. Based on a circular cellular micro-impedance model in widespread use, this study shows that cellular barrier function parameter estimates are highly model state dependent. Systematic errors produced by coaxial lead capacitances and circuit loading can also lead to significant and model state-dependent parameter errors and should, therefore, be either reduced or corrected for analytically.

Real time monitoring of endothelial cell actin filament disruption by cytochalasin D using a cellular impedance biosensor

Using a novel cellular impedance biosensor and confocal microscopy, this study has examined the dynamic and steady state cellular impedance response of porcine pulmonary endothelial cells to varying doses of cytochalasin D. Endothelial cell monolayer impedances were obtained using an array of gold microelectrodes coated with fibronectin to facilitate endothelial cell adhesion. Impedance measurements were acquired at 5.6 kHz by phase sensitive detection from a lock-in amplifier. The electrically measured cytochalasin D dose dependant actin disruption was successfully correlated with actin stained confocal microscopy images quantified using image-processing techniques. Based on this study, the cellular kinetic response to cytochalasin D increased systematically with the dose and saturated at a critical concentration. The real time quantification of pharmacological agents that target specific elements of the cytoskeleton using electrical impedance measurements therefore has a number of important applications in understanding the dynamic and steady state response of endothelial cells to toxins and drug induced cellular cytoskeletal micromechanics

A microfluidic biosensor array for endothelial and cancer cell co-culture interaction studies

Understanding the interaction between cancer and endothelial cells is a key element to more effective chemotherapeutics. A microfluidic cellular co-culture biosensor capable of simultaneous optical and electrical impedance measurements is therefore proposed. Using micro-fabrication methods, an array of optically thin electrically conductive biosensors is designed capable of monitoring cancer and endothelial cell interactions. Computer aided design methods were used to layout out a microfluidic channel compatible with the working bio-electrode array. The micro-fluidic channel allowed cancer cell spheroids and pharmaceutical agents to be injected into an inlet port. Both the optical and electrical properties of the micro-fluidic device have been examined. The proposed system has a number of applications where cancer and endothelial cell interactions need to be studied. The results of this study demonstrate the feasibility of the multiplexed biosensor for studying cancer and endothelial cell interactions under static and dynamic shear flow conditions.