Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System.

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study, a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time, in relation to cellular activities during self-renewal or lineage-specific differentiation, in a non-destructive, label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal, or subjected to mesendodermal or ectodermal differentiation, and correlating them to morphological (size, shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall, the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation

Characterization of oxidative stress in Leishmaniasis-infected or LPS-stimulated macrophages using electrochemical impedance spectroscopy

The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedances spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NO-synthase in the case of LPS-stimulatedmacrophages. This production was not observed in the case of Leishmania-infectedmacrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC)2 as NO radical spin trap were carried out

A Novel Cell-Based Hybrid Acoustic Wave Biosensor with Impedimetric Sensing Capabilities

A novel multiparametric biosensor system based on living cells will be presented. The biosensor system includes two biosensing techniques on a single device: resonant frequency measurements and electric cell-substrate impedance sensing (ECIS). The multiparametric sensor system is based on the innovative use of the upper electrode of a quartz crystal microbalance (QCM) resonator as working electrode for the ECIS technique. The QCM acoustic wave sensor consists of a thin AT-cut quartz substrate with two gold electrodes on opposite sides. For integration of the QCM with the ECIS technique a semicircular counter electrode was fabricated near the upper electrode on the same side of the quartz crystal. Bovine aortic endothelial live cells (BAECs) were successfully cultured on this hybrid biosensor. Finite element modeling of the bulk acoustic wave resonator using COMSOL simulations was performed. Simultaneous gravimetric and impedimetric measurements performed over a period of time on the same cell culture were conducted to validate the device\u27s sensitivity. The time necessary for the BAEC cells to attach and form a compact monolayer on the biosensor was 35∼45 minutes for 1.5 × 104 cells/cm2 BAECs; 60 minutes for 2.0 × 104 cells/cm2 BAECs; 70 minutes for 3.0 × 104 cells/cm2 BAECs; and 100 minutes for 5.0 × 104 cells/cm2 BAECs. It was demonstrated that this time is the same for both gravimetric and impedimetric measurements. This hybrid biosensor will be employed in the future for water toxicity detection

Electrochemical Biosensors and Angiogenesis

Electrochemical methods provide attractive sensing techniques for biology. Electrochemical devices can be easily manufactured, miniaturized and are sometimes the only direct sensing method available. However, stability of these sensors is problematic, as foreign-body type reactions may induce distortions of the signal (biofouling). As a consequence, investigating the interactions with the biological matrix is of paramount importance to achieve reliable sensing. Different types of electrodes (boron doped diamond and different preparations of glassy carbon) and various electrode coatings were tested, in the presence of biological molecules. The results showed that boron doped diamond and fibronectin coated sensors offer good stability, even in the presence of high concentrations of proteins. A generally applicable protocol to assess the quality of electrode materials in biofouling conditions is also presented. Fibronectin has also been found to be a highly biocompatible coating, perfectly suited for cell-based measurements. This fibronectin coating was used on an electrode array to study the pathway leading to angiogenic factor induced nitric oxide release. Vascular endothelial growth factor, a well known angiogenic factor, was initially used and allowed me to setup a reliable and robust protocol for the use of electrode arrays in biology. It was then demonstrated that angiogenin, another angiogenic factor, leads to nitric oxide exocytosis through PI-3 kinase transduction

Microfluidic platform for impedance characterization of endothelial cells under fluid shear stress.

Endothelium dysfunction has been associated with many pathophysiological processes leading to cardiovascular diseases. Studying endothelium behavior is vital to understand the onset, prevention, and treatment of such diseases. Electrical impedance spectroscopy has been shown to provide a real-time in vitro evaluation of cell behavior including cell monolayer permeability. However, the majority of published work has been primarily with static cell culture models or macro-scale models that do not properly represent the physiological sizes, structures, and environmental conditions of human blood vessels. Within this dissertation, the design, fabrication, characterization, and application of a microfluidic impedance platform is presented for the in vitro characterization of HUVECs undergoing different hydrodynamic shear stress conditions (static, 2.5, 17.6 and 58.1 dyne/cm2). Electrodes diameters of 50, 100, and 200 µm were incorporated to monitor different subpopulations sizes of HUVECs. Initial characterization experiments with relevant biological solutions indicated that electrodes smaller than 50 µm in diameter suffered from significant interfacial impedance and were unsuitable for the sensing application. Impedance spectra (102-106 Hz) were collected for HUVECs at the different shear conditions for 14 hours. Equivalent circuit fits were implemented to derive the different electrical cell monolayer parameters including the trans-endothelial resistance, cell membrane capacitance, constant phase element, and the resistance of cell culture medium. Results confirmed that while the trans-endothelial resistance and cell membrane capacitance were suitable measurements for cell permeability and confluency respectively, the constant phase element did not identify any discernible cell behavior. Resistance of cell culture medium was strongly influenced by cell attachment and values should be extracted from control cell-free measurements. Initial trans-endothelial resistance measurements showed a shear magnitude dependent increase at the sudden onset of flow. This increase was greatest for the largest shear condition (58.1 dyne/cm2). After 14 hours of shear, trans-endothelial resistance measurements were largest for HUVECs sheared at 58.1 dyne/cm2 and lowest for the 17.6 dyne/cm2 shear condition and the difference showed to be statically significant (p \u3c0.05). Monitored HUVECs were stained for nuclei, F-actin and VE-cadherin. Quantification of immunofluorescence of VE-cadherin showed a similar trend to the extracted trans-endothelial resistance values. Immunofluorescence images of F-actin showed significant cytoskeleton remodeling of sheared HUVECs. While cells sheared at 17.6 dyne/cm2 aligned parallel to the direction of flow, HUVECs sheared at 58.1 dyne/cm2 were angled in the direction of flow and sometimes even perpendicular to flow direction

Voltage Effects on Cells Cultured On Metallic Biomedical Implants

Electrochemical voltage shifts in metallic biomedical implants occur in-vivo due to a number of processes including mechanically assisted corrosion. Surface potential of biomedical implants and excursions from resting open circuit potential (OCP), which is the voltage they attain while in contact with an electrolyte, can significantly change the interfacial properties of the metallic surfaces and alter the behavior of the surrounding cells, compromising the biocompatibility of metallic implants. Voltages can also be controlled to modulate cell function and fate. To date, the details of the physico-chemical phenomena and the role of different biomaterial parameters involved in the interaction between cells and metallic surfaces under cathodic bias have not been fully elucidated. In this work, changes in the interfacial properties of a CoCrMo biomedical alloy (ASTM F-1537) in phosphate-buffered saline (PBS) (pH 7.4) at different voltages was studied. Step polarization impedance spectroscopy technique was used to apply 50 mV voltage steps to samples, and the time-based current transients were recorded. A new equation was derived based on capacitive discharge through a Tafel element and generalized to deal with non-ideal impedance behavior. The new function compared to the KWW-Randles function, better matched the time-transient response. The results also showed a voltage dependent oxide resistance and capacitance behavior. Additionally, the in-vitro effect of static voltages on the behavior of MC3T3-E1 pre-osteoblasts cultured on CoCrMo alloy (ASTM-1537) was studied to determine the range of cell viability and mode of cell death beyond the viable range. Cell viability and morphology, changes in actin cytoskeleton, adhesion complexes and nucleus, and mode of cell death (necrosis, or intrinsic or extrinsic apoptosis) were characterized at different voltages ranging from -1000 to +500 mV (Ag/AgCl). Moreover, electrochemical currents and metal ion concentrations at each voltage were measured and related to the observed responses. Results show that cathodic and anodic voltages outside the voltage viability range (-400 \u3c V \u3c +500) lead to primarily intrinsic apoptotic and necrotic cell death, respectively. Cell death is associated with cathodic current densities of 0.1 uAcm-2 and anodic current densities of 10 uAcm-2. Significant increase in metallic ions (Co, Cr, Ni, Mo) was seen at +500 mV, and -1000 mV (Cr only) compared to open circuit potential. The number and total projected area of adhesion complexes was also lower on the polarized alloy (p \u3c 0.05). These results show that reduction reactions on CoCrMo alloys leads to apoptosis of cells on the surface and may be a relevant mode of cell death for metallic implants in-vivo. On the other hand, we studied how surface oxide thickness of Ti affects its voltage viability range and cellular response and whether anodic oxidation can serve as a means to extend this range. Cellular behavior (cell viability, cytoskeletal organization, and cellular adhesion) on bare and anodized Ti samples, potentiostatically held at voltages at the cathodic edge of the viability range, were assessed. Surfaces were characterized using contact angle (CA) measurement technique and atomic force microscopy (AFM), and the observed cellular response was related to the changes in the electrochemical properties (electrochemical currents, open circuit potential, and impedance spectra) of the samples. Results show that anodization at a low voltage (9 V) in phosphate buffer saline (PBS) generates a compact surface oxide with comparable surface roughness and energy to the starting native oxide on the bare surface. The anodized surface extends the viability range at 24 hours by about a 100 mV in the cathodic region, and preserved the cytoskeletal integrity and cell adhesion. Broadening of the viability range corresponds to an increase in impedance of the anodized surface at -400 mV(Ag/AgCl) and the resulting low average currents (below 0.1 uAcm-2) at the interface, which diminish the harmful cathodic reactions. Finally, cellular dynamics (size, polarity, movement) and temporal changes in the number and total area of focal adhesions in transiently transfected MC3T3-E1 pre-osteoblasts cultured on a CoCrMo alloy polarized at the cathodic and anodic edges of its voltage viability range (-400 and +500 mV(Ag/AgCl) respectively) were studied. Nucleus dynamics (size, circularity, movement) and the release of reactive oxygen species (ROS) was also studied on the polarized metal at -1000, -400, and +500 mV(Ag/AgCl). The results show that at -400 mV(Ag/AgCl) a gradual loss of adhesion occurs over 24 hours while cells shrink in size during this time. At +500 mV, cells become non-viable after 5 hours without showing any significant changes in adhesion behavior right before cell death. Nucleus size of cells at -1000 mV decreased sharply within 15 minutes after electrochemical polarization, which rendered the cells completely non-viable. No significant amount of ROS was released by cells on the polarized CoCrMo at any of these voltages

Three-Dimensional Impedance Tomographic Mapping of Metabolically Active Endolumen

Real-time detection of vulnerable atherosclerotic lesions, characterized by a high content of oxidized low-density lipoprotein (oxLDL)-laden macrophages or foam cells, remains an unmet clinical need. While fractional flow reserve (FFR)-guided revascularization in angiographically intermediate stenoses is utilized to assess hemodynamic significance, in vivo detection of oxLDL-rich plaques may provide a new paradigm for treating metabolically unstable lesions. Herein, we have demonstrated endoluminal mapping of lipid-laden lesions using 3-D electrical impedance spectroscopy-derived impedance tomography (EIT) in a pre-clinical swine model. We performed surgical banding of the right carotid arteries of Yucatan mini-pigs, followed by 16 weeks of high-fat diet, to promote the development of lipid-rich lesions. We implemented an intravascular sensor combining an FFR pressure transducer with a 6-point micro-electrode array for electrical impedance spectroscopy (EIS) measurements. 3-D EIT mapping was achieved using an EIS-based reconstruction algorithm. We demonstrated that EIT mapping corresponds to endoluminal histology for oxLDL-laden lesions. We further used computational models to theoretically predict and validate EIS measurements. Thus, our 3-D EIS-derived EIT provides in vivo detection of metabolically active plaques with the goal of guiding optimal intravascular intervention