Applications of Bioimpedance Measurement Techniques in Tissue Engineering

Rapid development in the field of tissue engineering necessitates implementation of monitoring methods for evaluation of the viability and characteristics of the cell cultures in a real-time, non-invasive and non-destructive manner. Current monitoring techniques are mainly histological and require labeling and involve destructive tests to characterize cell cultures. Bioimpedance measurement technique which benefits from measurement of electrical properties of the biological tissues, offers a non-invasive, label-free and real-time solution for monitoring tissue engineered constructs. This review outlines the fundamentals of bioimpedance, as well as electrical properties of the biological tissues, different types of cell culture constructs and possible electrode configuration set ups for performing bioimpedance measurements on these cell cultures. In addition, various bioimpedance measurement techniques and their applications in the field of tissue engineering are discussed.

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title: On-line determination of animal cell concentration in two industrial high-density culture processes by dielectric spectroscopy.
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title: Process control in cell culture technology using dielectric spectroscopy.
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title: Detection of the osteogenic differentiation of mesenchymal stem cells in 2D and 3D cultures by electrochemical impedance spectroscopy.
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title: The Theory and Fundamentals of Bioimpedance Analysis in Clinical Status Monitoring and Diagnosis of Diseases
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title: Impedance-Based Monitoring for Tissue Engineering Applications
abstract: Impedance is a promising technique for sensing the overall process of tissue engineering. Different electrode configurations can be used to characterize the scaffold that supports cell organization in terms of hydrogel polymerization and degree of porosity, monitoring cell loading, cell proliferation as well as the spatial distribution of cell aggregates in 3D. We have previously shown that impedance measurements allow accurate determination of conductivity in physiological solutions independent of validation and analysis of a specific equivalent circuit. Similarly to a physiological solution, cell culture medium conductivity, and hence the measured impedance, can respond to proliferating or dying cells populating the scaffold. Impedance may therefore be a key parameter for monitoring the biochemical dynamics that modulate 3D mammalian cell cultures over time. Furthermore, the conductivity of the medium filling the pores of the scaffold can serve as the basis for porosity determination using Archie’s law. Different networks of structured or random channels and degree of porosity can be detected. In addition, by combining a number of two-, three- and four-terminal (2T, 3T, 4T) configurations, it is possible to obtain complementary information on spatial distribution of cells in a 3D scaffold. 2T- and 3T configurations also reflect the impedance at the interface between an electrode and cell-loaded scaffold (polarization impedance, Zp), which may convey a further degree of information about the biochemical phenomena taking place in that sub-volume.

title: A method for bioimpedance measure with four- and two-electrode sensor systems
abstract: This paper presents an alternative method to measure impedances based on constant amplitude voltage excitation. The method makes use of feedback principle to adapt the measure conditions to load under test, being easily applied to bioimpedance measure with electrode sensors. The method has been tested for several frequencies and loads, employing four and two electrode setups. Results from electrical simulations, using CMOS circuits, fulfil the expected performance. This technique can be extended to wide frequency and load ranges, being an excellent option for impedance spectroscopy and EIT applications.

title: Four Versus Two-Electrode Measurement Strategies for Cell Growing and Differentiation Monitoring Using Electrical Impedance Spectroscopy
abstract: The aim of this work is to provide optimization tools for cell and tissue engineering processes through continuous monitoring of cell cultures. Structural cell properties can be obtained from non-destructive electrical measurements by using electrical impedance spectroscopy (EIS). EIS measurements on monolayer animal cell cultures are usually performed using a two-electrode strategy. Because of this, the measurement is very sensitive to the electrode covering ratio and to the degree of adherence of cells. Of course, these parameters give useful information but can mask the behaviour of the cell layer above the electrodes. In a previous work, preliminary measurements with commercial microelectrode structures were performed with simulated grow processes using the settlement of cell suspensions with two and four microelectrode strategies to validate the technique. In this work, real cell growths of Vero cells are described and the resulting EIS biomass density estimators are compared to cell counts. The four-electrode impedance spectra are fitted to the Cole-Cole impedance model and the time course of their parameters are extracted and studied

title: Four Electrode EIS Measurement on Interdigitated Microelectrodes for Adherent Cell Growing and Differentiation Monitoring
abstract: Protocols employed in cell and tissue engineering usually involve destructive tests to characterize the cell cultures, mainly in the case of adherent cells. Impedance spectroscopy obtained with planar electrodes is an alternative to perform non-destructive, on-line measurements. The antecedents of this technique use the two-electrode strategy, then providing high sensitivity but also a high dependence on the electrode coverage. In order to improve the accuracy and to separate cell density from coverage and adhesion we use a four electrode-strategy with interdigitated microelectrodes. The preliminary results have been previously reported. In this communication a set of results obtained with animal cell cultures are presented. The measurement set-up consists of miniature bioreactors (10 ml) where a double set of interdigitated microelectrodes (AbTech) has been inserted. They are connected to an impedance analyser through a custom front-end. The set is placed in an incubator together with parallel cultures in order to provide control cell counting along the 7 days cell growing. The four-electrode follow-up method provides higher sensitivity (63%) than the two-electrodes method, showing this last one a reduced response mainly near the culture end.

title: Impedance-based tissue discrimination for needle guidance.
abstract: Measurement of electrical impedance can discriminate between tissues of different electrical properties. A measurement system with adequate spatial resolution focused on a volume around the tip of a needle or other invasive clinical equipment can be used to determine in which type of tissue the tip is positioned. We have measured the sensitivity zone of a needle electrode with an active electrode area of 0.3 mm(2), and measured impedance spectra in porcine tissue in vivo. Small electrode impedance data will be influenced by electrode polarization impedance (EPI) at low frequencies. To refine existing methods for needle guidance with higher spatial resolution, we have used multivariate analysis and new interpretations of EPI, and tissue data gathered with selected needle electrodes. The focus of this study is on discrimination between muscle and fat/subdermis for drug administration, but our results also indicate that these refinements will facilitate new clinical applications for impedance-based needle guidance in general.

title: Design and fabrication of a microimpedance biosensor for bacterial detection
abstract: A biosensor for bacterial detection was developed based on microelectromechanical systems, heterobifunctional crosslinkers and immobilized antibodies. The sensor detected the change in impedance caused by the presence of bacteria immobilized on interdigitated gold electrodes and was fabricated from (100) silicon with a 2-/spl mu/m layer of thermal oxide as an insulating layer. The sensor active area is 9.6 mm/sup 2/ and consists of two interdigital gold electrode arrays measuring 0.8 /spl times/ 6 mm. Escherichia coli specific antibodies were immobilized to the oxide between the electrodes to create a biological sensing surface. The impedance across the interdigital electrodes was measured after immersing the biosensor in solution. Bacteria cells present in the sample solution attached to the antibodies and became tethered to the electrode array, thereby causing a change in measured impedance. The biosensor was able to discriminate between different cellular concentrations from 10/sup 5/ to 10/sup 7/ CFU/mL in pure culture. The sample testing process, including data acquisition, required 5 min. The design, fabrication, and testing of the biosensor is discussed along with the implications of these findings toward further biosensor development.

title: Resistivity probing of multi-layered tissue phantoms using microelectrodes.
abstract: We present the use of an array of rectangular microelectrodes to discriminate between different resistivities in a thin, layered sample. Each electrode was 8 mm long and 200 nm thick. The electrode widths ranged from 20 to 500 microm. The electrodes were designed such that all pairs of consecutive electrodes had the same relative geometry, and therefore identical cell constants. A hydrogel-based tissue phantom, made by photopolymerization of 2-hydroxyethyl methacrylate (HEMA), was developed. By changing the hydrogel composition and the ionic strength of the storage medium, the resistivity of the hydrogels could be tuned between 100 omegam and 100 komegam. Using bipolar measurements, the tissue phantoms were characterized in the frequency range from 100 Hz to 30 MHz. The relative resistivity distribution of a three-layered structure composed of 120 microm sheets could be calculated and was shown to agree to within 7% of the bulk measurements. Potential clinical applications for this technique include probing of epithelial tissue and skin cancer screening.

title: Simulation of microelectrode impedance changes due to cell growth
abstract: Observation of impedance changes caused by cell growth on microelectrodes provides information about cell coverage and other important quantities, such as the cell-electrode gap. In this paper, we use finite-element simulations to calculate the impedance changes caused by cell growth on electrodes comparable in size to the cell. Parameters describing the impedance of the electrode without cells are derived from measurement. The results show that the impedance magnitude is expected to increase by at least 100% for partial cell coverage. We also report the frequency dependence of the magnitude and phase of the impedance and compare the simulation results with a lumped-element model. The simulation results are important for the design and modeling of arrays of cell-sized electrodes and also for the interpretation of experiments.

title: Impedance Spectroscopic Characterisation of Porosity in 3D Cell Culture Scaffolds with Different Channel Networks
abstract: We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity (networks of structured or random channels) is discussed in relation to Archie’s law. Guidelines for EIS analysis are presented and demonstrated to provide porosity information in physiological buffer that agrees well with a more conventional weight-based approach. We also propose frequency ranges that may serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures.

title: The polarization impedance of common electrode metals operated at low current density
abstract: AbstractThe objective of this study was to characterize the polarization impedance (resistance and capacitance) of several common metal/0.9% saline interfaces operated at low-current density and to thereby provide a useful reference for those wishing to calculate the impedance of such electrodes. The series-equivalent resistance (R) and capacitive reactance (Xc) of stainless steel, platinum, silver, MP35N, palladium, aluminum, rhodium and copper electrodes, all having a surface areas S=0.005 cm2 and all in contact with 0.9% saline, were measured as a function of frequency (100 Hz to 20 kHz) at low-current density (0.025 mA/cm2). For all the metals tested, both R and Xc decreased with increasing frequency and the relationships were linear on a log-log plot. That is, R and Xc exhibited power-law behavior (R=A/fα and Xc=B/fβ). However, it was not generally true that A=B and α=β=0.5 as stated in the Warburg low-current density model. Furthermore, the Fricke constant phase model in which α=β and ϕ=0.5πβ was found not to be applicable in general. In particular, the constraint that α=β was a good approximation for most of the metals tested in this study, but the constraint that ϕ=0.5πβ did not hold in general. Although the Warburg low-current density model provides a useful conceptual tool, it is not the most accurate representation of the electrode-electrolyte interface. The Fricke constant phase model is a better representation of electrode behavior, but it also may not be valid in general. We have found that a better representation is provided by the general power-law model R=A/fα and Xc=B/fβ, where A, B, α, and β depend on the species of both the metal and electrolyte and A and B depend, in addition, on electrode area. Using this model and the data presented in this study, the impedance of an electrode-electrolyte interface operated at low-current density may be calculated as
$$Z = (0.005/S)\sqrt {(Af^{ - \alpha } )^2 + (Bf^{ - \beta } )^2 } ,$$
 where S is the surface area of the electrode in cm2.

title: Criteria for the Selection of Materials for Implanted Electrodes
abstract: AbstractThere are four criteria that must be considered when choosing material for an implanted electrode: (1) tissue response, (2) allergic response, (3) electrode-tissue impedance, and (4) radiographic visibility. This paper discusses these four criteria and identifies the materials that are the best candidates for such electrodes. For electrodes that make ohmic contact with tissues: gold, platinum, platinum–iridium, tungsten, and tantalum are good candidates. The preferred insulating materials are polyimide and glass. The characteristics of stimulator output circuits and the importance of the bidirectional wave- form in relation to electrode decomposition are discussed. The paper concludes with an analysis, the design criteria, and the special properties and materials for capacitive recording and stimulating electrodes.

title: Electrical Impedance Tomography : Methods, History and Applications
abstract: PART 1 ALGORITHMS The Reconstruction Problem PART 2 HARDWARE EIT Instrumentation PART 3 APPLICATIONS Imaging of the Thorax by EIT EIT of Brain Function Breast Cancer Screening with EIT Applications of EIT in the Gastrointestinal Tract (GIT) Other Clinical Applications of EIT PART 4 NEW DIRECTIONS Magnetic Induction Tomography Magnetic Resonance Electrical Impedance Tomography (MREIT) Electrical Tomography for Industrial Applications EIT: The View from Sheffield EIT for Medical Applications at Oxford Brookes 1985-2003 The Rensselaer Experience APPENDIX A BRIEF INTRODUCTION

title: New Method for Separation of Electrode Polarization Impedance from Measured Tissue Impedance
abstract: In this paper we have shown that electrode polarization impedance (EPI) can be separated from measured tissue impedance as long as the characteristic frequencies of EPI and tissue are not too close, so that the EPI is largely displayed as a separate dispersion. In 2-electrode measurements the EPI and sample are physically connected in series, and commonly modelled by equivalent components in series. We have calculated the parallel equivalent elements and converted the series connected EPI and sample to a parallel admittance model. By curve fitting on the converted model we have shown that this provides a new method for estimating the EPI with enhanced accuracy compared to similar techniques used on the impedance model.

title: Hydrogels as extracellular matrix mimics for 3D cell culture.
abstract: Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two-dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three-dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three-dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three-dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic-biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism.

title: Epithelial cancer cells exhibit different electrical properties when cultured in 2D and 3D environments.
abstract: Many drug development and toxicology studies are performed using cells grown in monolayers in well-plates and flasks, despite the fact that these are widely held to be different to cells found in the native environment. 3D, tissue engineered, organotypical tissue culture systems have been developed to be more representative of the native tissue environment than standard monolayer cultures. Whilst the biochemical differences between cells grown in 2D and 3D culture have been explored, the changes on the electrophysiological properties of the cells have not.



We compared the electrophysiological properties of primary normal oral keratinocytes (nOK) and cancerous abnormal oral keratinocytes (aOK), cultured in standard monolayer and reconstituted 3D organotypical tissue cultures. The electrophysiological properties of populations of the cells were analysed using dielectrophoresis. The intracellular conductivity of aOK was significantly increased when grown in organotypical cultures compared to counterpart cells grown in monolayer cultures.



3D cultured aOK showed almost identical intracellular conductivity to nOK also grown in organotypical cultures, but significantly different to aOK grown in monolayers. The effective membrane capacitance of aOK grown in 3D was found to be significantly higher than nOK, but there was no significant difference between the electrophysiological properties of nOK grown in 2D and 3D cultures.



This work suggests that factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology, and highlights the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.

title: Microengineered physiological biomimicry: organs-on-chips.
abstract: Microscale engineering technologies provide unprecedented opportunities to create cell culture microenvironments that go beyond current three-dimensional in vitro models by recapitulating the critical tissue-tissue interfaces, spatiotemporal chemical gradients, and dynamic mechanical microenvironments of living organs. Here we review recent advances in this field made over the past two years that are focused on the development of 'Organs-on-Chips' in which living cells are cultured within microfluidic devices that have been microengineered to reconstitute tissue arrangements observed in living organs in order to study physiology in an organ-specific context and to develop specialized in vitro disease models. We discuss the potential of organs-on-chips as alternatives to conventional cell culture models and animal testing for pharmaceutical and toxicology applications. We also explore challenges that lie ahead if this field is to fulfil its promise to transform the future of drug development and chemical safety testing.

title: Organ-on-a-chip devices advance to market.
abstract: To curb the high cost of drug development, there is an urgent need to develop more predictive tissue models using human cells to determine drug efficacy and safety in advance of clinical testing. Recent insights gained through fundamental biological studies have validated the importance of dynamic cell environments and cellular communication to the expression of high fidelity organ function. Building on this knowledge, emerging organ-on-a-chip technology is poised to fill the gaps in drug screening by offering predictive human tissue models with methods of sophisticated tissue assembly. Organ-on-a-chip start-ups have begun to spawn from academic research to fill this commercial space and are attracting investment to transform the drug discovery industry. This review traces the history, examines the scientific foundation and envisages the prospect of these renowned organ-on-a-chip technologies. It serves as a guide for new members of this dynamic field to navigate the existing scientific and market space.

title: Microsensor systems for cell metabolism - from 2D culture to organ-on-chip.
abstract: Microsensor systems for cell metabolism are essential tools for investigation and standardization in cell culture. Electrochemical and optical read-out schemes dominate, which enable the marker-free, continuous, online recording of transient effects and deliver information beyond microscopy and end-point tests. There has been much progress in microfluidics and microsensors, but the translation of both into standard cell culture procedures is still limited. Within this critical review, we discuss different cell culture formats ranging from standard culture vessels to dedicated microfluidic platforms. Key aspects are the appropriate supply of cells, mass transport of metabolites to the sensors and generation of stimuli. Microfluidics enable the transition from static to dynamic conditions in culture and measurement. We illustrate the parameters oxygen (respiration), pH (acidification), glucose and lactate (energy metabolism) as well as short-lived reactive species (ROS/RNS) from the perspective of microsensor integration in 2D and 3D cell culture. We discuss different sensor principles and types, along with their limitations, microfabrication technologies and materials. The state-of-the-art of microsensor platforms for cell culture is discussed with respect to sensor performance, the number of parameters and timescale of application. That includes the advances from 2D culture to the increasingly important 3D approaches, with specific requirements for organotypic microtissues, spheroids and solid matrix cultures. We conclude on the current progress, potential, benefits and limitations of cell culture monitoring systems from monolayer culture to organ-on-chip systems.

title: Electrical impedance tomography for real-time and label-free cellular viability assays of 3D tumour spheroids.
abstract: There is currently a need to culture cells in 3D to better mimic the behaviour of cells growing in the natural environment. In parallel, this calls for novel technologies to assess cell growth in 3D cell culture. In this study, we demonstrated both in silico and in vitro that cell viability inside large cell spheroids could be monitored in real time and label-free with electrical impedance tomography (EIT). Simulations using a single shell model and the effective media approximation (EMA) method were performed to prove the performance of EIT on spheroid imaging and viability monitoring. Then in vitro experiments were conducted to measure in real time a loss of cell viability in MCF-7 breast cancer spheroids when exposed to Triton X-100 and validate with conventional biochemical assays. It is shown that EIT has a spatial resolution of 1.14% and it could monitor the cell mortality over 20% of a spheroid under laboratory noise level. The reconstructed conductivity images for cell mortality induced by the chemical are clear and match the result in the cellular metabolic viability assay. Furthermore, the image reconstruction speed in the experiment was less than 0.3 seconds. Taken together, the results show the potential of EIT for non-destructive real-time and label-free cellular assays in the miniature sensor, providing physiological information in the applications of 3D drug screening and tissue engineering.

title: Cell culture: Biology's new dimension
abstract: There's a big difference between a flat layer of cells and a complex, three-dimensional tissue. But until recently, many biologists have glossed over this fact.  Abbott discovers what they've been missing.

title: Matrix Elasticity Directs Stem Cell Lineage Specification
abstract: Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.

title: Capturing complex 3D tissue physiology in vitro
abstract: The emergence of tissue engineering raises new possibilities for the study of complex physiological and pathophysiological processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. We discuss here some of the 'design principles' for recreating the interwoven set of biochemical and mechanical cues in the cellular microenvironment, and the methods for implementing them. We emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.

title: A comparison of imaging methodologies for 3D tissue engineering
abstract: Imaging of cells in two dimensions is routinely performed within cell biology and tissue engineering laboratories. When biology moves into three dimensions imaging becomes more challenging, especially when multiple cell types are used. This review compares imaging techniques used regularly in our laboratory in the culture of cells in both two and three dimensions. The techniques reviewed include phase contrast microscopy, fluorescent microscopy, confocal laser scanning microscopy, electron microscopy, and optical coherence tomography. We compare these techniques to the current “gold standard” for imaging three‐dimensional tissue engineered constructs, histology.  2010.

title: Microfabricated mammalian organ systems and their integration into models of whole animals and humans.
abstract: While in vitro cell based systems have been an invaluable tool in biology, they often suffer from a lack of physiological relevance. The discrepancy between the in vitro and in vivo systems has been a bottleneck in drug development process and biological sciences. The recent progress in microtechnology has enabled manipulation of cellular environment at a physiologically relevant length scale, which has led to the development of novel in vitro organ systems, often termed 'organ-on-a-chip' systems. By mimicking the cellular environment of in vivo tissues, various organ-on-a-chip systems have been reported to reproduce target organ functions better than conventional in vitro model systems. Ultimately, these organ-on-a-chip systems will converge into multi-organ 'body-on-a-chip' systems composed of functional tissues that reproduce the dynamics of the whole-body response. Such microscale in vitro systems will open up new possibilities in medical science and in the pharmaceutical industry.

title: A phenotypic in vitro model for the main determinants of human whole heart function.
abstract: This article details the construction and testing of a phenotypic assay system that models in vivo cardiac function in a parallel in vitro environment with human stem cell derived cardiomyocytes. The major determinants of human whole-heart function were experimentally modeled by integrating separate 2D cellular systems with BioMicroelectromechanical Systems (BioMEMS) constructs. The model features a serum-free defined medium to enable both acute and chronic evaluation of drugs and toxins. The integration of data from both systems produced biologically relevant predictions of cardiac function in response to varying concentrations of selected drugs. Sotalol, norepinephrine and verapamil were shown to affect the measured parameters according to their specific mechanism of action, in agreement with clinical data. This system is applicable for cardiac side effect assessment, general toxicology, efficacy studies, and evaluation of in vitro cellular disease models in body-on-a-chip systems.

title: Integration of a pump and an electrical sensor into a membrane-based PDMS microbioreactor for cell culture and drug testing
abstract: To the extent possible, artificial organs should have characteristics that match those of the in vivo system. To this end, microfabrication techniques allow us to create microenvironments that can help maintain cell organization and functionality in in vitro cultures. We present three new microbioreactors, each of which allows cells to be cultured in a perfused microenvironment similar to that found in vivo. Our microbioreactors use new technology that permits integration onto the chip (35 mm × 20 mm) of an electrical sensor, in addition to one or more pumping systems and associated perfusion circuitry. The monitoring of Caco-2 cell cultures using electrical impedance spectroscopy (EIS) has allowed us to measure the effects of cell growth, cellular barrier formation and the presence of chemical compounds and/or toxins. Specifically, we have investigated the ability of the electrical sensor to maintain appropriate sensitivity and precision. Our results show that the sensor was very sensitive not only to the presence or the absence of the cells, but also to changes in cell state. Our perfused microbioreactors are highly efficient miniaturized tools that are easy to operate. We anticipate that they will offer promising new opportunities in many types of cell culture research, including drug screening and tissue engineering.

title: Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments.
abstract: Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory.

title: Microfluidic 3D cell culture: potential application for tissue-based bioassays.
abstract: Current fundamental investigations of human biology and the development of therapeutic drugs commonly rely on 2D monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function or physiology of living tissues, nor the highly complex and dynamic 3D environments in vivo. Microfluidic technology can provide microscale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in the microfluidic technology for 3D cell culture and their biological applications.

title: Surface electrochemical impedance in situ monitoring of cell-cultured titanium with a nano-network surface layer
abstract: A TiO2 layer with a nano-network size under 100 nm was developed on titanium (Ti) substrate using galvanodynamic anodization treatment. The network-structured TiO2 layer on Ti substrate showed a higher (1.8–3.8 times) electrochemical impedance (2–4.7 × 106 Ω cm2) and better cell spreading, namely better biocompatibility, than the untreated Ti surface.

title: Monitoring living cell assays with bio-impedance sensors
abstract: This work proposes a cell–microelectrode model to be used on cell culture assays as an alternative to end-point protocols employed in cell growth and cell biometry applications. The microelectrode model proposed is based on the area overlap between the microelectrode sensor and the living cells as main parameter. This model can be applied to cell size identification, cell count, and their extension to cell growth, motility and dosimetry protocols. A procedure to fit the proposed model to microelectrode electrical performance is presented, enabling the decoding of empirical measurements and its interpretation in terms of number of cells. This fitting procedure depends on three parameters: microelectrode geometry, gap resistance between substrate attached cells and microelectrode and, mainly, on microelectrode area covered by cells. The model has been implemented employing Analog Hardware Descriptions Language (AHDL) to be incorporated also to mixed-mode simulation processes during circuit design flow. Experiments performed with commercial electrodes are described, illustrating a procedure to obtain cell number in real time in both, growth and dosimetry assays, employing an established cell line (AA8). The results are displayed in the form of growth curves (cells were growing during a week), as well as dosimetric response after treatment with MG132, a proteasome inhibitor. The results agree with the expected performances, with errors around 10–20% in the number of cells measured, therefore we think that these results are promising.

title: Practical Characterization of Cell-Electrode Electrical Models in Bio-Impedance Assays
abstract: This paper presents the fitting process followed to adjust the parameters of the electrical model associated to a cell-electrode system in Electrical Cell-substrate Impedance Spectroscopy (ECIS) technique, to the experimental results from cell-culture assays. A new parameter matching procedure is proposed, under the basis of both, mismatching between electrodes and time-evolution observed in the system response, as consequence of electrode fabrication processes and electrochemical performance of electrode-solution interface, respectively. The obtained results agree with experimental performance, and enable the evaluation of the cell number in a culture, by using the electrical measurements observed at the oscillation parameters in the test circuits employed.

title: Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue.
abstract: We describe a method for monitoring cell proliferation in a small-scale hollow-fiber bioreactor (culture volume: 1 ml) by use of the Alamar Blue dye. Alamar Blue is a non-fluorescent compound, which yields a fluorescent product after reduction, e.g. by living cells. In contrast to the MTT-assay, the Alamar Blue assay does not lead to cell death. However, when not removed from the cells, the Alamar Blue dye shows a reversible, time- and concentration-dependent growth inhibition as observed for the leukemic cell lines CCRF-CEM, HL-60 and REH. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows discriminating between cell densities of 10(5), 10(6) and 10(7) of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 10(6) cells/ml, in particular. We conclude that the Alamar Blue-assay combined with a hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system.

title: Impedance Spectroscopy: A Powerful Tool for Rapid Biomolecular Screening and Cell Culture Monitoring
abstract: Dielectric spectroscopy or Electrochemical impedance spectroscopy (EIS) is traditionally used in corrosion monitoring, coatings evaluation, batteries, and electrodeposition and semiconductor characterization. However, in recent years, it is gaining widespread application in biotechnology, tissue engineering, and characterization of biological cells, disease diagnosis and cell culture monitoring. This article discusses the principles and implementation of dielectric spectroscopy in these bioanalytical applications. It provides examples of EIS as label-free, mediator-free strategies for rapid screening of biocompatible surfaces, monitoring pathogenic bacteria, as well as the analysis of heterogeneous systems, especially biological cells and tissues. Descriptions are given of the application of nanoparticles to improve the analytical sensitivities in EIS. Specific examples are given of the detection of base pair mismatches in the DNA sequence of Hepatitis B disease, TaySach's disease and Microcystis spp. Others include the EIS detection of viable pathogenic bacteria and the influence of nanomaterials in enhancing biosensor performance. Expanding applications in tissue engineering such as adsorption of proteins onto thiolated hexa(ethylene glycol)-terminated (EG6) self-assembled monolayer (SAM) are discussed.

title: Automated, Multiplexed Electrical Impedance Spectroscopy Platform for Continuous Monitoring of Microtissue Spheroids.
abstract: Microtissue spheroids in microfluidic devices are increasingly used to establish novel in vitro organ models of the human body. As the spheroids are comparably sizable, it is difficult to monitor larger numbers of them by optical means. Therefore, electrical impedance spectroscopy (EIS) emerges as a viable alternative to probing spheroid properties. Current spheroid EIS systems are, however, not suitable for investigating multiple spheroids in parallel over extended time in an automated fashion. Here we address this issue by presenting an automated, multiplexed EIS (AMEIS) platform for impedance analysis in a microfluidic setting. The system was used to continuously monitor the effect of the anticancer drug fluorouracil (5-FU) on HCT116 cancer spheroids. Simultaneous EIS monitoring of up to 15 spheroids was performed in parallel over 4 days at a temporal resolution of 2 min without any need for pumps. The measurements were continuous in nature, and the setup was kept in a standard incubator under controlled conditions during the measurements. A baseline normalization method to improve robustness and to reduce the influence of slow changes in the medium conductivity on the spheroid EIS readings has been developed and validated by experiments and means of a finite-element model. The same method and platform was then used for online monitoring of cardiac spheroids. The beating frequency of each cardiac spheroid could be read out in a completely automated fashion. The developed system constitutes a promising method for simultaneously evaluating drug impact and/or toxic effects on multiple microtissue spheroids.

title: Electrical Impedance Spectroscopy for Microtissue Spheroid Analysis in Hanging-Drop Networks.
abstract: Electrical impedance spectroscopy (EIS) as a label free and noninvasive analysis method receives growing attention for monitoring three-dimensional tissue constructs. In this Article, we present the integration of an EIS readout function into the hanging-drop network platform, which has been designed for culturing microtissue spheroids in perfused multitissue configurations. Two pairs of microelectrodes have been implemented directly in the support of the hanging drops by using a small glass inlay inserted in the microfluidic structure. The pair of bigger electrodes is sensitive to the drop size and allows for drop size control over time. The pair of smaller electrodes is capable of monitoring, on the one hand, the size of microtissue spheroids to follow, for example, the growth of cancer microtissues, and, on the other hand, the beating of cardiac microtissues in situ. The presented results demonstrate the feasibility of an EIS readout within the framework of multifunctional hanging-drop networks.

title: Monitoring of yeast cell concentration using a micromachined impedance sensor
abstract: The paper describes the design, modelling and experimental characterization of a micromachined impedance sensor for on-line monitoring of the viable yeast cell concentration (biomass) in a miniaturized cell assay. Measurements in a Saccharomyces cerevisiae cell culture show that the permittivity of the cell suspension depends linearly on the biomass concentration within the range of 0 to 9 g/l. In order to compensate the measurements for changes in the dielectric properties of the background electrolyte, the use of a three-electrode configuration in combination with a semi-permeable pHEMA membrane was explored. Measurements showed that the impedance of the hydrated pHEMA varies with only the background electrolyte conductivity, and not with the concentration of cells, indicating that pHEMA is suitable for this purpose. The optimal pHEMA membrane thickness was determined using finite element modelling.

title: On-chip microelectrode impedance analysis of mammalian cell viability during biomanufacturing.
abstract: The characterization of cell viability is a challenging task in applied biotechnology, as no clear definition of cell death exists. Cell death is accompanied with a change in the electrical properties of the membrane as well as the cell interior. Therefore, changes in the physiology of cells can be characterized by monitoring of their dielectric properties. We correlated the dielectric properties of industrially used mammalian cells, sedimented over interdigitated microelectrodes, to the AC signal response across the chip. The voltage waveforms across the electrodes were processed to obtain the circuit impedance, which was used to quantify the changes in cell viability. We observed an initial decrease in impedance, after which it remained nearly constant. The results were compared with data from the dye exclusion viability test, the cell specific oxygen uptake rate, and the online viable cell density data from capacitance probes. The microelectrode technique was found to be sensitive to physiological changes taking place inside the cells before their membrane integrity is compromised. Such accurate determination of the metabolic status during this initial period, which turned out to be less well captured in the dye exclusion tests, may be essential for several biotechnology operations.

title: Impedance analysis of adherent cells after in situ electroporation: non-invasive monitoring during intracellular manipulations.
abstract: In this study adherent animal cells were grown to confluence on circular gold-film electrodes of 250 μm diameter that had been deposited on the surface of a regular culture dish. The impedance of the cell-covered electrode was measured at designated frequencies to monitor the behavior of the cells with time. This approach is referred to as electric cell-substrate impedance sensing or short ECIS in the literature. The gold-film electrodes were also used to deliver well-defined AC voltage pulses of several volts amplitude and several hundred milliseconds duration to the adherent cells in order to achieve reversible membrane electroporation (in situ electroporation=ISE). Electroporation-assisted introduction of membrane impermeable molecules into the cytoplasm was studied by using FITC-labeled dextran molecules of different molecular weights. Probes as big as 2MDa were successfully loaded into the cells residing on the electrode surface. Time-resolved impedance measurements before and immediately after the electroporation pulse revealed the kinetics of membrane resealing as well as subsequent changes in cell morphology. Cells recovered from the electroporation pulse within less than 90 min. When membrane-impermeable, bioactive compounds like N(3)(-) or bleomycin were introduced into the cells by in situ electroporation, concomitant ECIS readings sensitively reported on the associated response of the cells to these toxins as a function of time (ISE-ECIS).

title: Integrated 3-D Silicon Electrodes for Electrochemical Sensing in Microfluidic Environments: Application to Single-Cell Characterization
abstract: A microtechnology allowing the integration of thin metal electrodes and three dimensional highly doped bulk silicon electrodes on a hybrid PDMS/glass fluidic microchip has been developed. The fabrication involved anodic bonding of a silicon wafer onto glass substrate, deep reactive ion etching of 3-D bulk silicon electrodes, and plasma bonding of a PDMS microfluidic structure on a silicon/gold/glass substrate. The devices realized using this technology have been used for electrical impedance characterization of chemical and biological material. Microdevices with typical dimensions of hundreds of micrometers have been fabricated and tested in the determination of the conductivity of NaCl solutions. Smaller sensors, with critical dimensions under 10 m, have been achieved for single-cell characterization. Human hepatocellular liver carcinoma cells have been introduced in the microimpedance sensors. Measurements show the interfacial relaxation of the cellular membrane in the range. It is expected that other electrochemical sensors and electrokinetic actuators can benefit from this technology.

title: Dielectric spectroscopy in a micromachined flow cytometer: theoretical and practical considerations.
abstract: We propose a model to determine the influence of different cell properties, such as size, membrane capacitance and cytoplasm conductivity, on the impedance spectrum as measured in a microfabricated cytometer. A dielectric sphere of equivalent complex permittivity is used as a simplified model to describe a biological cell. The measurement takes place between a pair of facing microelectrodes in a microchannel filled with a saline solution. The model incorporates various cell parameters, such as dielectric properties, size and position in the channel. A 3D finite element model is used to evaluate the magnitude of the electric field in the channel and the resultant changes in charge densities at the measurement electrode boundaries as a cell flows past. The charge density is integrated on the electrode surface to determine the displacement current and the channel impedance for the computed frequency range. The complete impedance model combines the finite element model, the electrode-electrolyte interface impedance and stray impedance, which are measured from a real device. The modeled dielectric complex spectra for various cell parameters are discussed and a measurement strategy for cell discrimination with such a system is proposed. We finally discuss the amount of noise and measurement fluctuations of the sensor.

title: Microfluidic device for cell capture and impedance measurement
abstract: This work presents a microfluidic device to capture physically single cells within microstructures inside a channel and to measure the impedance of a single HeLa cell (human cervical epithelioid carcinoma) using impedance spectroscopy. The device includes a glass substrate with electrodes and a PDMS channel with micro pillars. The commercial software CFD–ACE+ is used to study the flow of the microstructures in the channel. According to simulation results, the probability of cell capture by three micro pillars is about 10%. An equivalent circuit model of the device is established and fits closely to the experimental results. The circuit can be modeled electrically as cell impedance in parallel with dielectric capacitance and in series with a pair of electrode resistors. The system is operated at low frequency between 1 and 100 kHz. In this study, experiments show that the HeLa cell is successfully captured by the micro pillars and its impedance is measured by impedance spectroscopy. The magnitude of the HeLa cell impedance declines at all operation voltages with frequency because the HeLa cell is capacitive. Additionally, increasing the operation voltage reduces the magnitude of the HeLa cell because a strong electric field may promote the exchange of ions between the cytoplasm and the isotonic solution. Below an operating voltage of 0.9 V, the system impedance response is characteristic of a parallel circuit at under 30 kHz and of a series circuit at between 30 and 100 kHz. The phase of the HeLa cell impedance is characteristic of a series circuit when the operation voltage exceeds 0.8 V because the cell impedance becomes significant.

title: On-chip electrical impedance tomography for imaging biological cells.
abstract: Electrical impedance tomography is an imaging technology that spatially characterizes the electrical properties of an object. We present a miniaturized electrical impedance tomography system that can image the electrical conductivity distribution within a two-dimensional cell culture. A chip containing a circular 16-electrode array was fabricated using printed circuit board developing technology and used to inject current and to measure spatial voltage across the object. The signal stimulation and voltage data acquisition were performed using an impedance analyzer, operating in four-electrode mode. An open source software, EIDORS was used for image reconstruction. Finite element modelling was used to simulate the image reconstruction process by imaging two ellipsoidal phantoms in the circular 16-electrode array. The effect of the regularization parameter in the reconstruction algorithm and the influence from noise on the fidelity of the images has been numerically analyzed. Experimentally, we show reconstructed images of a multi-nuclear single cellular organism, Physarum Polycephalum, demonstrating the first step towards impedance imaging of single cells in culture. Our system provides a non-invasive lab-on-a-chip technology for spatially mapping the electrical properties of single cells, which would be significant and useful for diagnostic and clinical applications.

title: Continuous differential impedance spectroscopy of single cells
abstract: A device for continuous differential impedance analysis of single cells held by a hydrodynamic cell trapping is presented. Measurements are accomplished by recording the current from two closely-situated electrode pairs, one empty (reference) and one containing a cell. We demonstrate time-dependent measurement of single cell impedance produced in response to dynamic chemical perturbations. First, the system is used to assay the response of HeLa cells to the effects of the surfactant Tween, which reduces the impedance of the trapped cells in a concentration dependent way and is interpreted as gradual lysis of the cell membrane. Second, the effects of the bacterial pore-forming toxin, Streptolysin-O are measured: a transient exponential decay in the impedance is recorded as the cell membrane becomes increasingly permeable. The decay time constant is inversely proportional to toxin concentration (482, 150, and 30 s for 0.1, 1, and 10 kU/ml, respectively).

title: Leukocyte analysis and differentiation using high speed microfluidic single cell impedance cytometry.
abstract: Miniature high speed label-free cell analysis systems have yet to be developed, but have the potential to deliver fast, inexpensive and simple full blood cell analysis systems that could be used routinely in clinical practice. We demonstrate a microfluidic single cell impedance cytometer that performs a white blood cell differential count. The device consists of a microfluidic chip with micro-electrodes that measure the impedance of single cells at two frequencies. Human blood, treated with saponin/formic acid to lyse erythrocytes, flows through the device and a complete blood count is performed in a few minutes. Verification of cell dielectric parameters was performed by simultaneously measuring fluorescence from CD antibody-conjugated cells. This enabled direct correlation of impedance signals from individual cells with phenotype. Tests with patient samples showed 95% correlation against commercial (optical/Coulter) blood analysis equipment, demonstrating the potential clinical utility of the impedance microcytometer for a point-of-care blood analysis system.

title: Cell-based biosensors : principles and applications
abstract: Cell Culture on Chips Mechanisms and Models of Cell-Based Biosensors Microelectrodes Arrays (MEA) as Cell-Based Biosensors Field Effect Transistor (FET) as Cell-Based Biosensors Light Addressable Potential Sensor (LAPS) as Cell-Based Biosensors Electric Cell-Substrate Impedance Sensor (ECIS) as Cell-Based Biosensors Patch Clamp Chip as Cell-Based Biosensors Other Cell-Based Biosensors Developments of Cell-Based Biosensors.

title: Electrode models in electrical impedance tomography
abstract: This paper presents different views on electrode modelling, which include electrode electrochemistry models for modelling the effects of electrode-electrolyte interface, electric field electrode models for modelling electrode geometry, and electrode models for modelling the effects of electrode common mode voltage and double layer capacitance. Taking the full electrode models into consideration in electrical impedance tomography (EIT) will greatly help the optimised approach to a good solution and further understanding of the measurement principle.

title: Label-Free Impedance Biosensors: Opportunities and Challenges.
abstract: Impedance biosensors are a class of electrical biosensors that show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in surface impedance when a target molecule binds to an immobilized probe. The affinity capture step leads to challenges shared by all label-free affinity biosensors; these challenges are discussed along with others unique to impedance readout. Various possible mechanisms for impedance change upon target binding are discussed. We critically summarize accomplishments of past label-free impedance biosensors and identify areas for future research.

title: Bioelectrical Impedance Methods for Noninvasive Health Monitoring: A Review
abstract: Under the alternating electrical excitation, biological tissues produce a complex electrical impedance which depends on tissue composition, structures, health status, and applied signal frequency, and hence the bioelectrical impedance methods can be utilized for noninvasive tissue characterization. As the impedance responses of these tissue parameters vary with frequencies of the applied signal, the impedance analysis conducted over a wide frequency band provides more information about the tissue interiors which help us to better understand the biological tissues anatomy, physiology, and pathology. Over past few decades, a number of impedance based noninvasive tissue characterization techniques such as bioelectrical impedance analysis (BIA), electrical impedance spectroscopy (EIS), electrical impedance plethysmography (IPG), impedance cardiography (ICG), and electrical impedance tomography (EIT) have been proposed and a lot of research works have been conducted on these methods for noninvasive tissue characterization and disease diagnosis. In this paper BIA, EIS, IPG, ICG, and EIT techniques and their applications in different fields have been reviewed and technical perspective of these impedance methods has been presented. The working principles, applications, merits, and demerits of these methods has been discussed in detail along with their other technical issues followed by present status and future trends.

title: High accurate and wideband current excitation for bioimpedance health monitoring systems
abstract: Current sources are critical for the performance of bioimpedance health monitoring device. The challenging features for a high accuracy and wide bandwidth voltage controlled current source are stable and safe current magnitudes. Corresponding to the typical measurement requirements, it should allow typical load impedances in the range from 100 Ω to 10 kΩ and have high output impedance of approximately 10 MΩ at 5 kHz and 1 MΩ at high frequencies up to 1 MHz without harmonics and influence of stray capacitances. Generalized impedance converters and negative impedance converters help to reduce the influence of stray capacitances. In this study, we compare between two types of voltage controlled current sources for grounded loads: Howland and Tietze circuits. Based on this study, we suggest an improved Howland circuit in inverting dual configuration with a negative feedback using the high bandwidth amplifier AD8021. With this configuration, a better accuracy is reached at higher frequencies up to 1 MHz. With the addition of compensation capacitor to the operational amplifier, this circuit configuration is capable to maintain approximately constant current amplitude within an accuracy of less than 0.022% at both low and high frequencies up to 1 MHz.

title: Electrical impedance spectroscopy of the breast: clinical imaging results in 26 subjects
abstract: Electrical impedance spectroscopy (EIS) is a potential, noninvasive technique to image women for breast cancer. Studies have shown characteristic frequency dispersions in the electrical conductivity and permittivity of malignant versus normal tissue. Using a multifrequency EIS system, we imaged the breasts of 26 women. All patients had mammograms ranked using the American College of Radiology (ACR) BIRADS system. Of the 51 individual breasts imaged, 38 were ACR 1 negative, six had ACR 4-5 suspicious lesions, and seven had ACR 2 benign findings such as fibroadenomas or calcifications. A radially translatable circular array of 16 Ag/AgCl electrodes was placed around the breast while the patient lay prone. We applied trigonometric voltage patterns at ten frequencies between 10 and 950 kHz. Anatomically coronal images were reconstructed from this data using nonlinear partial differential equation methods. Typically, ACR 1-rated breasts were interrogated in a single central plane whereas ACR 2-5-rated breasts were imaged in multiple planes covering the region of suspicion. In general, a characteristic homogeneous image emerged for mammographically normal cases while focal inhomogeneities were observed in images from women with malignancies. Using a specific visual criterion, EIS images identified 83% of the ACR 4-5 lesions while 67% were detected using a numerical criterion. Overall, multifrequency electrical impedance imaging appears promising for detecting breast malignancies, but improvements must be made before the method reaches its full potential.

title: Skin cancer identification using multifrequency electrical impedance-a potential screening tool
abstract: Electrical bio-impedance can be used to assess skin cancers and other cutaneous lesions. The aim of this study was to distinguish skin cancer from benign nevi using multifrequency impedance spectra. Electrical impedance spectra of about 100 skin cancers and 511 benign nevi were measured. Impedance of reference skin was measured ipsi-laterally to the lesions. The impedance relation between lesion and reference skin was used to distinguish the cancers from the nevi. It was found that it is possible to separate malignant melanoma from benign nevi with 75% specificity at 100% sensitivity, and to distinguish nonmelanoma skin cancer from benign nevi with 87% specificity at 100% sensitivity. The power of skin cancer detection using electrical impedance is as good as, or better than, conventional visual screening made by general practitioners.

title: Non-invasive assessment of radiation injury with electrical impedance spectroscopy.
abstract: A detailed understanding of non-targeted normal tissue response is necessary for the optimization of radiation treatment plans in cancer therapy. In this study, we evaluate the ability of electrical impedance spectroscopy (EIS) to non-invasively determine and quantify the injury response in soft tissue after high dose rate (HDR) irradiation, which is characterized by large localized dose distributions possessing steep spatial gradients. The HDR after-loading technique was employed to irradiate small volumes of muscle tissue with single doses (26-52 Gy targeted 5 mm away from the source). Impedance measurements were performed on 29 rats at 1, 2 and 3 month post-irradiation, employing 31 frequencies in the 1 kHz to 1 MHz range. Over the first 3 months, conductivity increased by 48% and 26% following target doses of 52 Gy and 26 Gy 5 mm from the HDR source, respectively. Injury, assessed independently through a grid-based scoring method showed a quadratic dependence on distance from source. A significant injury (50% of cells atrophied, necrotic or degenerating) in 1.2% of the volume, accompanied by more diffuse injury (25% of cells atrophied, necrotic or degenerating) in 9% of the tissue produced a conductivity increase of 0.02 S m(-1) (8% over a baseline of 0.24 S m(-1)). This was not statistically significant at p = 0.01. Among treatment groups, injury differences in 22% of the volume led to statistically significant differences in conductivity of 0.07 S m(-1) (23% difference in conductivity). Despite limitations, the success of EIS in detecting responses in a fraction of the tissue probed, during these early post-irradiation time-points, is encouraging. Electrical impedance spectroscopy may provide a useful metric of atrophy and the development of fibrosis secondary to radiation that could be further developed into a low-cost imaging method for radiotherapy monitoring and assessment.

title: Detection of cervical intraepithelial neoplasia using impedance spectroscopy: a prospective study
abstract: To compare cervical impedance spectrometry in the cervical epithelium of women with cervical intraepithelial neoplasia (CIN) and normal epithelium.

title: Intravascular electric impedance spectroscopy of atherosclerotic lesions using a new impedance catheter system
abstract: Newer techniques are required to identify atherosclerotic lesions that are prone to rupture. Electric impedance spectroscopy (EIS) can characterize biological tissues by measuring the electrical impedance over a frequency range. We tested a newly designed intravascular impedance catheter (IC) by measuring the impedance of different stages of atherosclerosis induced in an animal rabbit model. Six female New Zealand White rabbits were fed for 17 weeks with a 5% cholesterol–enriched diet to induce early forms of atherosclerotic plaques. All aortas were prepared from the aortic arch to the renal arteries and segments of 5–10 mm were marked by ink spots. A balloon catheter system with an integrated polyimide–based microelectrode structure was introduced into the aorta and the impedance was measured at each spot by using an impedance analyzer. The impedance was measured at frequencies of 1 kHz and 10 kHz and compared with the corresponding histomorphometric data of each aortic segment.Forty–four aortic segments without plaques and 48 segments with evolving atherosclerotic lesions could be exactly matched by the histomorphometric analysis. In normal aortic segments (P0) the change of the magnitude of impedance at 1 kHz and at 10 kHz (|Z|1 kHz – |Z|10 kHz, = ICF) was 208.5 ± 357.6 Ω. In the area of aortic segments with a plaque smaller than that of the aortic wall diameter (PI), the ICF was 137.7 ± 192.8 Ω. (P 0 vs. P I; p = 0.52), whereas in aortic segments with plaque formations larger than the aortic wall (PII) the ICF was significantly lower –22.2 ± 259.9 Ω. (P0 vs. PII; p = 0.002). Intravascular EIS could be successfully performed by using a newly designed microelectrode integrated onto a conventional coronary balloon catheter. In this experimental animal model atherosclerotic aortic lesions showed significantly higher ICF in comparison to the normal aortic tissue.

title: On-line monitoring of yeast cell growth by impedance spectroscopy.
abstract: The application of impedance spectroscopy to estimate on-line cell concentration was studied. The estimation was based on the relative variation between electrical impedance measured at low (10 kHz) and high frequencies (10 MHz). Studies were carried out to characterise the influence of changes in physical and chemical parameters on the impedance measurement. Two different possibilities to perform on-line measurements were tested: a simple set-up, based on an in situ probe, gave good results but was not suitable for high agitation and aeration rates. An ex situ flow-through on-line measuring cell was used to overcome these problems, showing a better performance. The use of this set-up for the growth monitorisation of a Saccharomyces cerevisiae culture showed an efficient performance, having the correlation between estimated and measured S. cerevisiae a Pearson coefficient of 0.999.

title: Small intestinal ischemia and reperfusion-bioimpedance measurements.
abstract: Trans-intestinal bioimpedance measurements have previously been used to investigate changes in electrical parameters during 6 h of ischemia in the small intestine. Knowledge is lacking regarding the time course of trans-intestinal bioimpedance parameters during reperfusion. As reperfusion is an important part in the clinical treatment of intestinal ischemia, we need to know how it affects the bioimpedance measurements.



We performed bioimpedance measurements, using a two-electrode setup on selected segments of the jejunum in 15 pigs. A controlled voltage signal was applied while measuring the resulting current. In each pig, five or six 30 cm segments of the jejunum were made ischemic by clamping the mesenteric arteries and veins creating segments with ischemia from 1-16 h duration. Reperfusion was initiated at selected time intervals of ischemia, and measured for 5-15 h afterwards.



The tan δ parameter (loss tangent) was different (p  <  0.016) comparing ischemic and control tissue for the duration of the experiment (16 h). Comparing the control tissue 30 cm from the ischemic area with the control tissue 60 cm from the ischemic tissue, we found that the mean tan δ amplitude in the frequency range (3900-6300 Hz) was significantly higher (p  <  0.036) in the proximal control after 10 h of experiment duration. After reperfusion, the time development of tanδm (loss tangent maximum over a frequency range) amplitude and frequency overlapped and periodically increased above the tanδm in the ischemic intestine. Dependent on the ischemic duration pre-reperfusion, the initial increase in tan δ stabilizes or increases drastically over time, compared to the tan δ amplitude of the ischemic tissue.



As during ischemia, the electrical parameters during reperfusion also follow a characteristic time-course, depending on the ischemic exposure before pre-reperfusion. The temporal changes in electrical parameters during small intestinal ischemia followed by reperfusion provides important information for assessment of tissue injury.

title: Non-invasive Characterization of the Osteogenic Differentiation of hMSCs in 3D by Impedance Spectroscopy
abstract: Considering the fact that human mesenchymal stem cells (hMSCs) replicate and differentiate multipotently, the provide a cell source for tissue engineering and cell-based test systems.

title: Monitoring Muscle Stem Cell Cultures with Impedance Spectroscopy
abstract: The aim of this work is to present a new circuit for the real-time monitoring the processes of cellular growth and differentiation of skeletal myoblast cell cultures. An impedance spectroscopy Oscillation-Based technique is proposed for the test circuit, converting the biological system into a voltage oscillator, and avoiding the use of very high performance circuitry or equipment. This technique proved to be successful in the monitoring of cell cultures growth levels and could be useful for determining the degree of differentiation achieved, of practical implications in tissue engineering.

title: Electrical Impedance of Cultured Endothelium Under Fluid Flow
abstract: AbstractThe 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. 
PAC01: 8719Nn, 8719Uv, 8717-d

title: Impedance spectroscopy-based cell/particle position detection in microfluidic systems.
abstract: An impedance spectroscopy-based cell/particle position detection method in microfluidic systems is presented. A single pair of non-parallel surface microelectrodes was utilized to detect the transverse positions of particles/cells flowing in a microchannel without the need for a multi-electrode multi-channel impedance detection. This method can be a simple solution for high-throughput and low-cost position detection in microfluidic sorting and separation applications.

title: Electrical impedance spectroscopy - a potential method for the study and monitoring of a bone critical-size defect healing process treated with bone tissue engineering and regenerative medicine approaches.
abstract: The development of strategies of bone tissue engineering and regenerative medicine has been drawing considerable attention to treat bone critical-size defects (CSDs). Notably, new strategies and/or treatment approaches always require appropriate tools to track the healing process so as to evaluate their success. In this paper, we present the development of a novel approach for the non-invasive, yet real-time, monitoring and assessment of bone CSDs treated with biomaterials and biomedical approaches. For this, we employed the technique of electrical impedance spectroscopy (EIS) to quantitatively monitor and assess the changes in electrical impedance, and thus the regeneration process. In our in vitro tests, we examined the biochemical changes of the fracture area and investigated the influence of collagen and hydroxyapatite on the changes in electrical impedance by EIS, thus inferring the changes in bone regeneration and structure. Based on this success, we further demonstrated, in real time, the process of regeneration of the traumatic area in an in vivo rabbit model. Our electrical-impedance data of the experiment groups, i.e., the ones treated with natural coral and bone morphogenetic protein-2 (BMP-2), revealed that each group has its unique impedance graph characteristics, which are directly associated with the degree of regeneration. For comparison, we also employed radiography, gross anatomy, and histological analyses in examination. Our results illustrate that EIS holds considerable potential as a non-invasive tool for monitoring, in real time, the healing of bone CSDs by allowing for quantitatively characterizing the changes of both hydroxyapatite and collagen.

title: On-line biomass measurements in bioreactor cultivations: comparison study of two on-line probes
abstract: Two on-line probes for biomass measurement in bioreactor cultivations were evaluated. One probe is based on near infrared (NIR) light absorption and the other on dielectric spectroscopy. The probes were used to monitor biomass production in cultivations of several different microorganisms. Differences in NIR probe response compared to off-line measurement methods revealed that the most significant factor affecting the response was cell shape. The NIR light absorption method is more developed and reliable for on-line in situ biomass estimation than dielectric spectroscopy. The NIR light absorption method is, however, of no significant use, when the cultivation medium is not clear, and especially in processes using adsorbents or solid matrix for the microorganism to grow on. The possibilities offered by dielectric spectroscopy are impressive, but the on-line probe technology needs to be improved.

title: Online impedance monitoring of yeast cell culture behaviors
abstract: Online impedance monitoring of cell suspensions can be performed with integrated microelectrode array in microfluidic devices. However, its accuracy is affected by the formation of cell aggregates on the surface of the electrode array. To make this technical more reliable, we deposited a thin film of agarose which is highly permeable to soluble ions on the electrodes. The growth behavior of yeast cells in YPD medium was studied for more than 24h and the recorded impedance spectra were used to determinate the cell metabolism induced changes of the medium resistance and the double layer capacitance as a function of time. Our results showed a much more important variation of the double layer capacitance, indicating the importance of online monitoring at low frequencies.

title: Development of a conductometric biocompatible sensor for detecting ischemia
abstract: Insufficient blood supply to the organs is the major cause of morbidity and mortality worldwide. This condition must be detected early to avoid irreversible loss of function and tissue death as occurs with for instance myocardial infarction, stroke and many other diseases. It has been shown that CO2 tension in tissue is a valid variable for detecting insufficient blood supply in any organ, yet there exists no commercially available sensor capable of performing this measurement continuously, selectively and in real-time. In this paper we present a sensor prototype for measuring tissue tension of CO2 (PCO2) based on a conductometric principle. It has a diameter of 1mm, and is made purely of biocompatible materials. The sensor has been produced by injection moulding, laser patterning, electroplating and manual assembly. Its challenges of drift and long-time survivability are discussed. We will also discuss optimisation of electrode polarisation and stray capacitance of miniaturised electrodes, challenges that are relevant also for a larger span of miniaturised devices. We describe different in vivo experiments where the sensor has shown a lack of blood supply in several types of tissues with high sensitivity and specificity. It is expected that this approach will help to save the lives of patients and in the future become part of the standard of organ monitoring.

title: Micromotion of mammalian cells measured electrically.
abstract: 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.

title: A study of electrochemical biosensor for analysis of three-dimensional (3D) cell culture.
abstract: Cell culture has a fundamental role not only in regenerative medicine but also in biotechnology, pharmacology, impacting both drug discovery and manufacturing. Although cell culture has been generally developed for only two-dimensional (2D) culture systems, three-dimensional (3D) culture is being spotlighted as the means to mimic in vivo cellular conditions. In this study, a method for cytotoxicity assay using an electrochemical biosensor applying 3D cell culture is presented. In order to strengthen the advantage of a 3D cell culture, the experimental condition of gelation between several types of sol-gels (alginate, collagen, matrigel) and cancer cells can be optimized to make a 3D cell structure on the electrode, which will show the reproducibility of electrical measurement for long-term monitoring. Moreover, cytotoxicity test results applying this method showed IC(50) value of A549 lung cancer cells to erlotinib. Thus, this study evaluates the feasibility of application of the electrochemical biosensor for 3D cell culture to cytotoxicity assay for investigation of 3D cell response to drug compounds.

title: Impedance analysis of epithelial and endothelial cell monolayers cultured on gold surfaces.
abstract: The present study describes a new method to determine transepithelial and transendothelial electrical resistances (TER) of cultured cell monolayers which is based on impedance analysis. To obtain impedance data of the epithelia or endothelia under investigation, we developed special measuring chambers that allow to culture the cells on gold surfaces that are used as measuring electrodes. Impedance analysis is carried out in the frequency range from 1 to 10(5) s-1 under normal culture conditions using a self-developed continuous wave impedance spectrometer. Evaluation of impedance data is achieved by fitting (NLSQ) the parameters of appropriate equivalent circuits to the experimental data. We investigated cell monolayers of primary cultured endothelial cells isolated from porcine brain microvessels, epithelial cells from porcine choroid plexus as well as those of the epithelial cell line MDCK. Transepithelial resistances were found to be in good agreement with published data. The main advantages of the new technique are the ability (i) to use multi-electrode arrays that allow to determine TERs at different locations of a given cell monolayer; (ii) to carry out impedance analysis under normal culture conditions; and (iii) to obtain TER values of cell monolayers grown on impermeable supports, which means that conditions cells are normally exposed to in ordinary culture dishes are maintained.

title: Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility
abstract: Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

title: Monitoring motion of confluent cells in tissue culture.
abstract: 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.

title: Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing.
abstract: 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.

title: A real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells.
abstract: Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host(1-3). Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors(4,5). The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process(6). Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells. Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes(7,8). The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance(9-12). The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density. The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer . The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

title: Electrical Cell‐Substrate Impedance Spectroscopy Can Monitor Age‐Grouped Human Adipose Stem Cell Variability During Osteogenic Differentiation
abstract: Human adipose stem cells (hASCs) are an attractive cell source for bone tissue engineering applications. However, a critical issue to be addressed before widespread hASC clinical translation is the dramatic variability in proliferative capacity and osteogenic potential among hASCs isolated from different donors. The goal of this study was to test our hypothesis that electrical cell‐substrate impedance spectroscopy (ECIS) could track complex bioimpedance patterns of hASCs throughout proliferation and osteogenic differentiation to better understand and predict variability among hASC populations. Superlots composed of hASCs from young (aged 24–36 years), middle‐aged (aged 48–55 years), and elderly (aged 60–81 years) donors were seeded on gold electrode arrays. Complex impedance measurements were taken throughout proliferation and osteogenic differentiation. During osteogenic differentiation, four impedance phases were identified: increase, primary stabilization, drop phase, and secondary stabilization. Matrix deposition was first observed 48–96 hours after the impedance maximum, indicating, for the first time, that ECIS can identify morphological changes that correspond to late‐stage osteogenic differentiation. The impedance maximum was observed at day 10.0 in young, day 6.1 in middle‐aged, and day 1.3 in elderly hASCs, suggesting that hASCs from younger donors require a longer time to differentiate than do hASCs from older donors, but young hASCs proliferated more and accreted more calcium long‐term. This is the first study to use ECIS to predict osteogenic potential of multiple hASC populations and to show that donor age may temporally control onset of osteogenesis. These findings could be critical for development of patient‐specific bone tissue engineering and regenerative medicine therapies. Stem Cells Translational Medicine 2017;6:502–511

title: Impedance spectroscopy and biosensing.
abstract: This chapter introduces the basic terms of impedance and the technique of impedance measurements. Furthermore, an overview of the application of this transduction method for analytical purposes will be given. Examples for combination with enzymes, antibodies, DNA but also for the analysis of living cells will be described. Special attention is devoted to the different electrode design and amplification schemes developed for sensitivity enhancement. Finally, the last two sections will show examples from the label-free determination of DNA and the sensorial detection of autoantibodies involved in celiac disease.

title: Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces.
abstract: 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.

title: Electrode-skin contact impedance: In vivo measurements on an ovine model
abstract: The problem of electrical impedance between the skin and the electrode is an on-going challenge in bio-electronics. This is particularly true in the case of Electrical Impedance Tomography (EIT), which uses a large number of skin-contact electrodes and is very sensitive to noise. In the present article, contact impedance is measured and compared for a range of electrodes placed on the thorax of an ovine model. The study has been approved by the Westmead Hospital Animal Ethics Committee. The electrode models that were employed in the research are Ag/AgCl electrodes (E1), commonly used for ECG and EIT measurements in both humans and animal models, stainless steel crocodile clips (E2), typically used on animal models, and novel multi-point dry electrodes in two modifications: bronze plated (E3) and nickel plated (E4). Further, since the contact impedance is mostly attributed to the acellular outer layer of the skin, in our experiment, we attempted to study the effect of this layer by comparing the results when the skin is intact and when electrodes are introduced underneath the skin through small cuts. This boundary effect was assessed by comparison of measurements obtained during E2 skin surface contact, and sub-cutaneous contact (E5). Twelve gauge intradermal needles were also tested as an electrode (E6). The full impedance spectrum, from 500 Hz to 300 kHz, was recorded, analysed and compared. As expected, the contact impedance in the more invasive cases, i.e the electrodes under the skin, is significantly lower than in the non-invasive cases. At the frequency of 50 kHz which is commonly used in lung EIT acquisition, electrodes E3, E4 and E6 demonstrated contact impedance of less than 200 Ω, compared to more than 400 Ω measured for electrodes E1, E2 and E5. In conclusion, the novel multipoint electrodes proved to be best suited for EIT purposes, because they are non-invasive and have lower contact impedance than Ag/AgCl and crocodile clips, in both invasive and non-invasive cases. This further prompted us to design a flexible electrode belt using the novel multi-point electrodes for lung EIT on animal models.

title: TEER Measurement Techniques for In Vitro Barrier Model Systems
abstract: Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. TEER values are strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. TEER measurements can be performed in real time without cell damage and generally are based on measuring ohmic resistance or measuring impedance across a wide spectrum of frequencies. The measurements for various cell types have been reported with commercially available measurement systems and also with custom-built microfluidic implementations. Some of the barrier models that have been widely characterized using TEER include the blood–brain barrier (BBB), gastrointestinal (GI) tract, and pulmonary models. Variations in these values can arise due to factors such as temperature, medium formulation, and passage number of cells. The aim of this article is to review the different TEER measurement techniques and analyze their strengths and weaknesses, determine the significance of TEER in drug toxicity studies, examine the various in vitro models and microfluidic organs-on-chips implementations using TEER measurements in some widely studied barrier models (BBB, GI tract, and pulmonary), and discuss the various factors that can affect TEER measurements.

title: Transepithelial/endothelial Electrical Resistance (TEER) theory and applications for microfluidic body-on-a-chip devices
abstract: Transepithelial/endothelial electrical resistance (TEER) is a valuable method for assaying in vitro barrier tissue integrity, and is becoming an important measurement for body-on-a-chip barrier tissue devices due to its usefulness and non-invasive nature. The measurement concept is relatively straightforward, with TEER measurements performed by applying an AC electrical signal across electrodes placed on both sides of a cellular monolayer and measuring voltage and current to calculate the electrical resistance of the barrier. However, details of the setup, measurement circuit, and applied electrical signal must be properly designed for accurate measurements. Several main factors contribute to errors and variability in the measurement of TEER values, and while many of these factors can be reasonably controlled with little effort in Transwell®-type culture conditions, these factors can become major issues in body-on-a-chip devices without proper design. This minireview outlines several important aspects of TEER measurements, including the basic theory, commercial systems used to perform measurements, major factors that contribute to measurement errors, and the application of TEER measurements to current body-on-a-chip barrier tissue devices, with the aim of providing guidance for the design of novel body-on-a-chip systems.

title: Impedance-based cell monitoring: barrier properties and beyond
abstract: In multicellular organisms epithelial and endothelial cells form selective permeable interfaces between tissue compartments of different chemical compositions. Tight junctions which connect adjacent cells, control the passage of molecules across the barrier and, in addition, facilitate active transport processes. The cellular barriers are not static but can be deliberately modulated by exposure to specific external stimuli. In vitro models representing the essential absorption barriers of the body are nowadays available, thus allowing investigation of the parameters that control permeability as well as transport processes across those barriers. Independent of the origin of the barrier forming cells, techniques are needed to quantify their barrier integrity. One simple assay is to measure the permeability for given hydrophilic substrates possessing different molecular weights like sucrose or dextrans. However, this technique is time-consuming and labor-intensive. Moreover, radioactive or fluorescently-labeled substrates are needed to allow easy analytical detection. Finally, if transport processes are investigated, the standard permeant may interfere with the transport process under investigation or might even alter the barrier integrity by itself. Thus, independent, non-invasive techniques are needed to quantify the barrier integrity continuously during the experiment. Such techniques are available and are mainly based on the measurement of the transendothelial or transepithelial electrical resistance (TEER) of barrier forming cells grown on porous membranes. Simple devices using two sets of electrodes (so-called Voltohmeters) are widely used. In addition, an easy-to-use physical technique called impedance spectroscopy allows the continuous analysis of both the TEER and the electrical capacitance giving additional information about the barrier properties of cells grown on permeable membranes. This technique is useful as a quality control for barrier forming cells. Another impedance-based approach requires cells to be grown directly on solid, micro-structured electrodes. Here, we will discuss the physical background of the different techniques; advantages, disadvantages, and applications will be scrutinized. The aim is to give the reader a comprehensive understanding concerning the range and limits of the application, mainly focusing on endothelial cells.

title: An Inter-laboratory Study to Evaluate the Effects of Medium Composition on the Differentiation and Barrier Function of Caco-2 Cell Lines
abstract: Differentiated human intestinal Caco-2 cells are frequently used in toxicology and pharmacology as in vitro models for studies on intestinal barrier functions. Since several discrepancies exist among the different lines and clones of Caco-2 cells, comparison of the results obtained and optimisation of models for use for regulatory purposes are particularly difficult, especially with respect to culture conditions and morphological and biochemical parameters. An inter-laboratory study has been performed on the parental cell line and on three clonal Caco-2 cell lines, with the aim of standardising the culture conditions and identifying the best cell line with respect to parameters relevant to barrier integrity, namely, transepithelial electrical resistance (TEER) and mannitol passage, and of epithelial differentiation (alkaline phosphatase activity). Comparison of the cell lines maintained in traditional serum-supplemented culture medium or in defined medium, containing insulin, transferrin, selenium and lipids, showed that parameter performance was better and more reproducible with the traditional medium. The maintenance of the cell lines for 15 days in culture was found to be sufficient for the development of barrier properties, but not for full epithelial differentiation. Caco-2/TC7 cells performed better than the other three cell lines, both in terms of reproducibility and performance, exhibiting low TEER and mannitol passage, and high alkaline phosphatase activity.

title: Bioimpedance imaging: an overview of potential clinical applications.
abstract: Electrical Impedance Tomography (EIT) is an imaging technique based on multiple bio impedance measurements to produce a map (image) of impedance or changes in impedance across a region. Its origins lay in geophysics where it is still used to today. This review highlights potential clinical applications of EIT. Beginning with a brief overview of the underlying principles behind the modality, it describes the background research leading towards the development of the application of EIT for monitoring pulmonary function, detecting and localising tumours and monitoring brain function.

title: Generalized optimal current patterns and electrical safety in EIT.
abstract: There are a number of constraints which limit the current and voltages which can be applied on a multiple drive electrical imaging system. One obvious constraint is to limit the maximum ohmic power dissipated in the body. Current patterns optimizing distinguishability with respect to this constraint are singular functions of the difference of transconductance matrices with respect to the power norm (the optimal currents of Isaacson). If one constrains the total current (L1 norm) the optimal patterns are pair drives. On the other hand if one constrains the maximum current on each drive electrode (an L(infinity) norm), the optimal patterns have each drive channel set to the maximum source or sink current value. In this paper we consider appropriate safety constraints and discuss how to find the optimal current patterns with those constraints.

title: Analysis of the Peak Resistance Frequency Method
abstract: This study analyzes the peak resistance frequency (PRF) method described by Mercanzini et al., a method that can easily extract the tissue resistance from impedance spectroscopy for many neural engineering applications but has no analytical description thus far.  Mathematical analyses and computer simulations were used to explore underlying principles, accuracy, and limitations of the PRF method.  The mathematical analyses demonstrated that the PRF method has an inherent but correctable deviation dependent on the idealness of the electrode-tissue interface, which is validated by simulations. Further simulations show that both frequency sampling and noise affect the accuracy of the PRF method, and in general, it performs less accurately than least squares methods. However, the PRF method achieves simplicity and reduced measurement and computation time at the expense of accuracy.  From the qualitative results, the PRF method can work with reasonable precision and simplicity, although its limitation and the idealness of the electrode-tissue interface involved should be taken into consideration.  This paper provides a mathematical foundation for the PRF method and its practical implementation.

title: Cell Culture Imaging Using Microimpedance Tomography
abstract: We present a novel, inexpensive, and fast microimpedance tomography system for two-dimensional imaging of cell and tissue cultures. The system is based on four-electrode measurements using 16 planar microelectrodes (5 mum x 4 mm) integrated into a culture chamber. An Agilent 4294A impedance analyzer combined with a front-end amplifier is used for the impedance measurements. Two-dimensional images are obtained using a reconstruction algorithm. This system is capable of accurately resolving the shape and position of a human hair, yielding vertical cross sections of the object. Human epithelial stem cells (YF 29) are also grown directly on the device surface. Tissue growth can be followed over several days. A rapid resistivity decrease caused by permeabilized cell membranes is also monitored, suggesting that this technique can be used in electroporation studies.

title: On-chip electrical impedence tomography for monitoring the kinetics in the cell culture
abstract: In this paper, electrical impedance tomography (EIT) is proposed to monitor the kinetics in substrate-based cell culture due to the spatial conductivity distribution within the culturing environment. A circular 16-electrode array was designed for imaging the culturing of Physarum. Polycephalum. The theoretical modelling and preliminarily reconstructed image of Physarum cell are presented.

title: Biodegradable magnesium alloys for orthopaedic applications: A review on corrosion, biocompatibility and surface modifications.
abstract: Magnesium (Mg) and its alloys have been extensively explored as potential biodegradable implant materials for orthopaedic applications (e.g. Fracture fixation). However, the rapid corrosion of Mg based alloys in physiological conditions has delayed their introduction for therapeutic applications to date. The present review focuses on corrosion, biocompatibility and surface modifications of biodegradable Mg alloys for orthopaedic applications. Initially, the corrosion behaviour of Mg alloys and the effect of alloying elements on corrosion and biocompatibility is discussed. Furthermore, the influence of polymeric deposit coatings, namely sol-gel, synthetic aliphatic polyesters and natural polymers on corrosion and biological performance of Mg and its alloy for orthopaedic applications are presented. It was found that inclusion of alloying elements such as Al, Mn, Ca, Zn and rare earth elements provides improved corrosion resistance to Mg alloys. It has been also observed that sol-gel and synthetic aliphatic polyesters based coatings exhibit improved corrosion resistance as compared to natural polymers, which has higher biocompatibility due to their biomimetic nature. It is concluded that, surface modification is a promising approach to improve the performance of Mg-based biomaterials for orthopaedic applications.

title: Design of a microscopic electrical impedance tomography system for 3D continuous non-destructive monitoring of tissue culture
abstract: BackgroundNon-destructive continuous monitoring of regenerative tissue is required throughout the entire period of in vitro tissue culture. Microscopic electrical impedance tomography (micro-EIT) has the potential to monitor the physiological state of tissues by forming three-dimensional images of impedance changes in a non-destructive and label-free manner. We developed a new micro-EIT system and report on simulation and experimental results of its macroscopic model.MethodsWe propose a new micro-EIT system design using a cuboid sample container with separate current-driving and voltage sensing electrodes. The top is open for sample manipulations. We used nine gold-coated solid electrodes on each of two opposing sides of the container to produce multiple linearly independent internal current density distributions. The 360 voltage sensing electrodes were placed on the other sides and base to measure induced voltages. Instead of using an inverse solver with the least squares method, we used a projected image reconstruction algorithm based on a logarithm formulation to produce projected images. We intended to improve the quality and spatial resolution of the images by increasing the number of voltage measurements subject to a few injected current patterns. We evaluated the performance of the micro-EIT system with a macroscopic physical phantom.ResultsThe signal-to-noise ratio of the developed micro-EIT system was 66 dB. Crosstalk was in the range of -110.8 to -90.04 dB. Three-dimensional images with consistent quality were reconstructed from physical phantom data over the entire domain. From numerical and experimental results, we estimate that at least 20 × 40 electrodes with 120 μm spacing are required to monitor the complex shape of ingrowth neotissue inside a scaffold with 300 μm pore.ConclusionThe experimental results showed that the new micro-EIT system with a reduced set of injection current patterns and a large number of voltage sensing electrodes can be potentially used for tissue culture monitoring. Numerical simulations demonstrated that the spatial resolution could be improved to the scale required for tissue culture monitoring. Future challenges include manufacturing a bioreactor-compatible container with a dense array of electrodes and a larger number of measurement channels that are sensitive to the reduced voltage gradients expected at a smaller scale.

title: Two-dimensional impedance imaging of cell migration and epithelial stratification.
abstract: We present a miniaturized impedance imaging system, developed for 2D imaging of cell and tissue culture. The system is based on 16 microelectrodes (5 microm x 4 mm). An equivalent circuit for four-point (tetrapolar) impedance spectra was developed and validated. The system uses an Agilent 4294A impedance analyser combined with a front-end amplifier for the impedance measurements. Human epithelial stem cells (YF 29) were grown on the device surface. Cell migration speeds of 300 nm min(-1) following a "scratch" wound closure assay could be established. Using a commercial software developed for geophysical prospecting, we could generate impedance tomography images at 10 kHz revealing cell migration, increase of epithelial thickness and changes in tissue resistivity over a time course of several days.

title: A Miniature Electrical Impedance Tomography Sensor and 3-D Image Reconstruction for Cell Imaging
abstract: Real-time quantitative imaging is becoming highly desirable to study nondestructively the biological behavior of 3-D cell culture systems. In this paper, we investigate the feasibility of quantitative imaging/monitoring of 3-D cell culture processes via electrical impedance tomography (EIT), which is capable of generating conductivity images in a non-destructive manner with high temporal resolution. To this end, a planar miniature EIT sensor amenable to standard cell culture format is designed, and a 3-D forward model for the sensor is developed for 3-D imaging. Furthermore, a novel 3-D-Laplacian and sparsity joint regularization algorithm is proposed for enhanced 3-D image reconstruction. Simulation phantoms with spheres at various vertical and horizontal positions were imaged for 3-D performance evaluation. In addition, experiments on human breast cancer cell spheroid and a triangular breast cancer cell pellet were carried out for experimental verification. The results have shown that the stable measurement on high conductive cell culture medium and the significant improvement of image quality based on the proposed regularization method are achieved. It demonstrates the feasibility of using the miniature EIT sensor and 3-D image reconstruction algorithm to visualize 3-D cell cultures, such as spheroids or artificial tissues and organs. The established work would expedite real-time quantitative imaging of 3-D cell culture for assessment of cellular dynamics.

title: Bipolar resistivity profiling of 3D tissue culture.
abstract: We describe a new low-cost technique for continuous monitoring of the thickness of biofilms and tissue cultures, and we demonstrate the advantage of using electrodes of different dimensions to probe different depths of a sample. We have used electric impedance spectroscopy to monitor keratinocyte stem cells (YF29) growing on an array of Ti/Pt coplanar microelectrodes. The thickness of the sample was reconstructed by fitting the measurements to theoretical curves. We have developed an algorithm for the rapid calculation of the resistance through a multilayered sample. This algorithm is based on conformal mapping and the serial partial capacitance technique. The validity of the technique was tested by measuring the sedimentation rate of an alumina powder. Sample thicknesses between 10 and 80 microm could be measured with a resolution of a few microns using the device.

title: Design and calibration of a compact multi-frequency EIT system for acute stroke imaging.
abstract: A new, compact UCLH Mk 2.5 EIT system has been developed and calibrated for EIT imaging of the head. Improvements include increased input and output impedances, increased bandwidth and improved CMRR (80 dB) and linearity over frequencies and load (0.2% on a single channel, +/-0.7% on a saline tank over 20 Hz-256 kHz and 10-65 Omega). The accuracy of the system is sufficient to image severe acute stroke according to the specification from recent detailed anatomical modelling (Horesh et al 2005 3rd European Medical and Biological Engineering Conference EMBEC'05). A preliminary human study has validated the main specifications of the modelling, the range of trans-impedance from the head (8-70 Omega) using a 32 electrode, 258 combination protocol and contact impedances of 300 Omega to 2.7 kOmega over 20 Hz to 256 kHz.

title: Effects of Electromagnetic Field Exposure on Electromagnetic Properties of Biological Tissues*: Effects of Electromagnetic Field Exposure on Electromagnetic Properties of Biological Tissues*
abstract: Electromagnetic radiations affect human health seriously. Bio-effects of electromagnetic fields mechanisms closely depend on the properties of themselves. Moreover, electromagnetic properties of signaling molecules, radicals and magnetic particles within body will be changed in electromagnetic fields, especially the conductivity, dielectric and magnetic properties of biological tissues will be significant difference when biological tissues exposure to various frequency electromagnetic fields. Identifying the evolution of electromagnetic properties of biological tissues is a key issue to explore underlying mechanisms and prevent electromagnetic bio-effects. Effects and applications of biological tissues in electromagnetic fields are summarized, and several future research directions are put forward.

title: Low frequency dielectric characteristics of human blood: A non-equilibrium thermodynamic approach
abstract: In this paper we apply some recent results of non-equilibrium thermodynamic to human blood. In particular we focus our attention on dielectric α-dispersion region which presents some not clear aspects. In the contest of Kluitenberg's non-equilibrium thermodynamic theory we determine the phenomenological and state coefficients that appear in the aforementioned theory in the case of a harmonic perturbation. This is possible by invoking a classical linear response theory and correlating dielectric storage and loss moduli to the aforementioned coefficients. These coefficients allow us to determine the elastic and inelastic parts of the polarization P so as to study some dielectric effects of blood in the α dispersion region. By taking into account these results we ascribe some relevant polarization effects, as the dispersion aforementioned, to white blood cells in agreement to some authors as Abdalla. Moreover we determine the trend of entropy production observing more and more irreversibility as increasing of the frequency of perturbation.

title: Microfluidic biosensors for microwave dielectric spectroscopy
abstract: This paper deals with the development and characterization of label-free microfluidic biosensors ded-icated to the microwave dielectric spectroscopy of small volumes and flowing micro-scale particles.Especially, microwave dielectric spectroscopy offers unique capabilities to investigate intracellular prop-erties of biological cells, which are informative on many biological processes. This paper presents twoprototypes of high frequency biosensors able to operate in the microwave frequency range. Both arebased on capacitive detection either in a broadband or resonant mode. Thick gold metal electrodes havebeen investigated to perform high sensitive analysis, while a microfluidic system was implemented overthe RF circuit so that flowing liquid minimally impacts global losses of the system and has hence a limitedeffect on sensor sensitivity. Particles to be tested are manipulated to be accurately located in the detectionarea using dielectrophoresis techniques. To demonstrate the proposed approach and assess the detec-tion capabilities of such sensors, HF measurements (up to 8 GHz) have been performed on calibratedsize polystyrene beads (12 m diameter) and compared with full-wave electromagnetic simulations.Measured capacitance contrasts are typically around 1 fF at 5 GHz. Preliminary experimentations werethen achieved on cancerous cells. The presented results demonstrate the high potential of the proposedapproach to develop new label-free analysis tools with a large application range, either in the diagnosisor treatment fields.

title: A novel technique for complex permittivity measurement based on a planar four port device
abstract: A novel four port device for complex permittivity measurement is presented consisting of the cascade of two 3 dB branch line directional couplers connected through two U-shaped transmission line lengths. The material under test (MUT) is brought into contact with one U-shaped section. The complex dielectric permittivity of the MUT is evaluated by simply measuring the amplitudes of the two outputs, thus obtaining a simple and cheap measurement procedure. A prototype has been fabricated and experimentally tested exhibiting very good accuracy.

title: Dielectric properties of a protein-water system in selected animal tissues.
abstract: Dielectric spectroscopy has been applied to study aspects of the organization of water in selected animal tissues (tendon, bone and horn). The measurements of the relative permittivity epsilon' and the dielectric loss epsilon'' were carried over the frequency range of 10-100 kHz and at temperatures from 22 to 240 degrees C. The water content was 10% for bone and horn, and 22% for tendon by mass at room temperature at a relative humidity of 70%. The temperature dependencies of epsilon' and epsilon'' reveal distinctively the temperature ranges corresponding to the release of water in temperatures up to about 200 degrees C for all tissues and the melting of the crystalline structure only for tendon and horn, above this temperature. The frequency dependencies of epsilon' and epsilon'' show a remarkable dispersion in the low-frequency at selected temperatures up to 200 degrees C for all tissues due to the release of the loosely and strongly bound water. The results were discussed in terms of the interfacial (Maxwell-Wagner) polarization and polarization mechanism involving hopping charge carriers interacting with the bound water molecules. The information on the effect of temperature, water content and frequency of the electromagnetic field on the dielectric behaviour of the tissues studied is of importance in the design and construction of medical diagnostic or therapeutic instruments based on the use of electric signals.

title: In Vivo Blood Characterization From Bioimpedance Spectroscopy of Blood Pooling
abstract: Characterization of blood impedance properties is important to estimate clinical diagnostic indexes such as hematocrit, glucose level, and hydration. Current in vivo bioimpedance spectroscopy methods are performed on a body appendage and thus represent a combined measurement of all tissues in the measurement field, rather than the blood individually. This paper describes a novel in vivo measurement technique to calculate bioelectrical properties of blood while excluding the disturbances from surrounding tissues, based on analysis of the impedance changes caused by blood accumulation. The forearm was modeled as a cylinder containing anatomical structures such as skin-fat layer, muscles, and bones. Blood volume was modeled as the inner cylinder. A tetrapolar electrode system was applied to a human forearm, and the impedance curves measured with and without blood pooling were processed to calculate the impedance parameters of arterial blood. The bioelectrical parameters of blood were estimated by fitting the blood curve to a Cole-Cole model using the Levenberg-Marquardt (LM) nonlinear curve-fitting method. The proposed approach was verified using an experimental phantom, an equivalent circuit model, and a preliminary human experiment. Results show that electrical properties of blood and surrounding tissues can successfully be separated. Of Cole-Cole parameters, the characteristic frequency fc is the most reliable parameter to characterize blood bioelectrical properties. This method may allow a simplified measurement of blood characteristic parameters for many biomedical and clinical monitoring applications.

title: Towards on‐line monitoring of cell growth in microporous scaffolds: Utilization and interpretation of complex permittivity measurements
abstract: Here we demonstrate the ability to characterize microporous scaffolds and evaluate cell concentration variation via the utilization and interpretation of complex permittivity measurements (CP), a direct and nondestructive method. Polymer‐based microporous scaffolds are of importance to tissue engineering, particularly in the promotion of cell adhesion, proliferation, and differentiation in predefined shapes. Chitosan gel scaffolds were seeded with increasing concentrations of macrophages to simulate cell growth. Complex permittivity measurements were performed using a dielectric probe and a vector network analyzer over a frequency ranging from 200 MHz to 2 GHz. An effective medium theory was applied to interpret the data obtained; respectively, Looyenga and Maxwell‐Wagner‐Hanai functions were used to retrieve the porosity and the variation of the cell concentration from the CP measurements. Calculated porosities were in agreement with experimental evaluation—porosity ranged from 81–96%. Changes in cell concentration inside the scaffolds upon injection of differing cell concentrations into the scaffold were detected distinguishably. Variations resulting from the cumulative injection of 400–1800 μL of 106 cells/mL solution into the scaffold were monitored. Results suggest that CP measurements in combination with an appropriate effective medium approximation can enable on‐line monitoring of cell growth within scaffolds.

title: Complex permittivity measurement as a new noninvasive tool for monitoring in vitro tissue engineering and cell signature through the detection of cell proliferation, differentiation, and pretissue formation
abstract: In in vitro tissue engineering, microporous scaffolds are commonly used to promote cell proliferation and differentiation in three-dimensional structures. Classic measurement methods are particularly time consuming, difficult to handle, and destructive. In this study, a new nondestructive method based on complex permittivity measurement (CPM) is proposed to monitor and track the osteoblast and macrophage differentiation through their morphological variation upon cell attachment and proliferation inside the microporous scaffolds. CPM is performed using a vector network analyzer and a dielectric probe under sterile conditions in a laminar-flow hood. A suitable effective medium approximation (EMA) is applied to fit the data in order to extract the parameters of the different constituents. Our data show that the EMA depolarization factor can be monitored to assess the variation of cell morphology characterizing cell attachment. Discrimination between two batches of scaffolds seeded, respectively, with 2 million and 1 million osteoblast cells is possible; the ratio of their CPM-derived cell volume fractions is in agreement with the ratio of their cell seeding numbers. In addition, cell proliferation inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium and inside scaffolds seeded with osteoblasts cultured in alpha minimum essential medium supplemented to induce the formation of extracellular matrix is monitored via CPM over several days. CPM-determined cell volume fraction is compared to DNA assay cell counts. Extracellular matrix formation and cell presence was confirmed by scanning electron microscopy. A set of three signature parameters () characteristic of cell line is extracted from CPM. Distinct signatures are recorded for osteoblasts and macrophages, thus confirming the ability of CPM to discriminate between different cell types. This study demonstrates the potential of CPM as a diagnostic tool to monitor quickly and noninvasively cell growth and differentiation inside microporous scaffolds. Our findings suggest that the use of CPM could be extended to many biomedical applications, such as drug detection and automation of tissue and bacterial cultures in bioreactors.

title: Non-invasive Imaging of Tissue-Engineered Vascular Endothelium with Iron Oxide Nanoparticles
abstract: J. Frese, L. Hrdlicka, M. E. Mertens, L. Rongen, S. Koch, P. Schuster, V.N. Gesché, T. Lammers, P. Mela, F. Kiessling, S. Jockenhoevel Department of Tissue Engineering & Textile Implants, Institute of Applied Medical Engineering, RWTH Aachen University, Aachen, Germany, frese@hia.rwth-aachen.de Department of Experimental Molecular Imaging, RWTH-Aachen University, Aachen, Germany, Department of Tissue Engineering & Textile Implants, Institut für Textiltechnik, RWTH Aachen University, Aachen Germany

title: Bioimpedance Measurement Setup for the Assessment of Viability and Number of Human Adipose Stem Cells Cultured as Monolayers
abstract: There is a clear need for non-invasive methods for monitoring tissue-engineered constructs prior to clinical use since presently used analysis techniques usually require destruction of the developed construct. We have developed a novel, non-invasive electric impedance measurement system to assess the properties of the tissue-engineered constructs. In this paper we introduce the first part of the study where we monitored the adipose stem cells cultured as monolayers. By measuring the impedance value of the cell cultures, it would be possible to evaluate cell viability and cell number. In addition, cell cultures were modeled with finite element model (FEM) system.