Spanish National Research Council · University of Seville
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Author: Pérez García, Pablo
Year: Since 2002
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Article Data-Analytics Modeling of Electrical Impedance Measurements for Cell Culture Monitoring
E. García, P. Pérez, A. Olmo, R. Díaz, G. Huertas and A. Yúfera
Journal Paper - Sensors, vol. 19, no. 21, art. 4639, 2019
MDPI    DOI: 10.3390/s19214639    ISSN: 1424-8220    » doi
[abstract]
High-throughput data analysis challenges in laboratory automation and lab-on-a-chip devices´ applications are continuously increasing. In cell culture monitoring, specifically, the electrical cell-substrate impedance sensing technique (ECIS), has been extensively used for a wide variety of applications. One of the main drawbacks of ECIS is the need for implementing complex electrical models to decode the electrical performance of the full system composed by the electrodes, medium, and cells. In this work we present a new approach for the analysis of data and the prediction of a specific biological parameter, the fill-factor of a cell culture, based on a polynomial regression, data-analytic model. The method was successfully applied to a specific ECIS circuit and two different cell cultures, N2A (a mouse neuroblastoma cell line) and myoblasts. The data-analytic modeling approach can be used in the decoding of electrical impedance measurements of different cell lines, provided a representative volume of data from the cell culture growth is available, sorting out the difficulties traditionally found in the implementation of electrical models. This can be of particular importance for the design of control algorithms for cell cultures in tissue engineering protocols, and labs-on-a-chip and wearable devices applications.

A 32 Input Multiplexed Channel Analog Front-End with Spatial Delta Encoding Technique and Differential Artifacts Compression
N. Pérez-Prieto, R. Fiorelli, J.L. Valtierra, P. Pérez-García, M. Delgado-Restituto and A. Rodríguez-Vázquez
Conference - IEEE Biomedical Circuits and Systems Conference BioCAS 2019
[abstract]
This paper describes a low-noise, low-power and high dynamic range analog front-end intended for sensing neural signals. In order to reduce interface area, a 32-channel multiplexer is implemented on circuit input. Furthermore, a spatial delta encoding is proposed to compress the signal range. A differential artifact compression algorithm is implemented to avoid saturation in the signal path, thus enabling reconstruct or suppressing artifacts in digital domain. The proposed design has been implemented using 0.18 μm TSMC technology. Experimental results shows a power consumption per channel of 1.0 μW, an input referred noise of 1.1 μVrms regarding the bandwidth of interest and a dynamic range of 91 dB.

Characterization of Implanted Stents through Neointimal Tissue Bioimpedance Simulations
J.M. Portillo-Anaya, P. Pérez, G. Huertas, A. Olmo, J.A. Serrano, A. Maldonado-Jacobi and A. Yúfera
Conference - International Conference of the IEEE Engineering in Medicine and Biology Society EMBC2019
[abstract]
This work describes how is possible the definition of the light hole or lumen in implanted stents affected by restenosis processes using the BioImpedance (BI) as biomarker. The main approach is based on the fact that neointimal tissues implied in restenosis can be detected and measured thanks to their respective conductivity and dielectric properties. For this goal, it is proposed a four-electrode setup for bioimpedance measurement. The influence of the several involved tissues in restenosis: fat, muscle, fiber, endothelium and blood, have been studied at several frequencies, validating the setup and illustrating the sensitivity of each one. Finally, a real example using a standard stent, has been analyzed for stable and vulnerable plaques in restenosis test cases, demonstrating that the proposed method is useful for the stent obstruction test. Bioimpedance simulation test has been performed using the electric physics module in COMSOL Multiphysics®.

Electrical pulse stimulation of skeletal myoblasts cell cultures with simulated action potentials
P. Villanueva, S. Pereira, A. Olmo, P. Pérez, Y. Yuste, A. Yúfera and F. de la Portilla
Journal Paper - Journal of Tissue Engineering and Regenerative Medicine, vol. 13, no. 7, pp 1265-1269, 2019
JOHN WILEY & SONS    DOI: 10.1002/term.2869    ISSN: 1932-6254    » doi
[abstract]
Electrical pulse stimulation has an important effect on skeletal muscle development and maturation. However, the methodology for controlling these stimulation parameters to develop in vitro functional skeletal muscle tissues remains to be established. In this work, we have studied the effect of simulated action potentials on the growth and differentiation of skeletal myoblast cell cultures. A circuit simulating action potentials of 0.15 and 0.3 V/mm, at a frequency of 1 Hz and with a 4-ms pulse width, is proposed. Results show an important improvement of the growth rate and differentiation of myoblasts at a voltage of 0.15 V/mm. Parameters such as electrodes geometry or type of signals must be considered in the development of in vitro skeletal muscle.

Remote Cell Growth Sensing using Self-Sustained Bio-Oscillations
P. Pérez, G. Huertas, A. Olmo, A. Maldonado-Jacobi, J. Serrano, M. Martín, P. Daza and A. Yúfera
Journal Paper - Sensors, vol. 18, no. 8, art. 2550, 2018
MDPI    DOI: 10.3390/s18082550    ISSN: 1424-8220    » doi
[abstract]
A smart sensor system for cell culture real-time supervision is proposed, allowing for a significant reduction in human effort applied to this type of assay. The approach converts the cell culture under test into a suitable "biological" oscillator. The system enables the remote acquisition and management of the "biological" oscillation signals through a secure web interface. The indirectly observed biological properties are cell growth and cell number, which are straightforwardly related to the measured bio-oscillation signal parameters, i.e., frequency and amplitude. The sensor extracts the information without complex circuitry for acquisition and measurement, taking advantage of the microcontroller features. A discrete prototype for sensing and remote monitoring is presented along with the experimental results obtained from the performed measurements, achieving the expected performance and outcomes.

An empirical-mathematical approach for calibration and fitting cell-electrode electrical models in bioimpedance tests
J.A. Serrano, G. Huertas, A. Maldonado-Jacobi, A. Olmo, P. Pérez, M.E. Martín, P. Daza and A. Yúfera
Journal Paper - Sensors, vol. 18, no. 7, article 2354, 2018
MDPI AG    DOI: 10.3390/s18072354    ISSN: 1424-8220    » doi
[abstract]
This paper proposes a new yet efficient method allowing a significant improvement in the on-line analysis of biological cell growing and evolution. The procedure is based on an empirical-mathematical approach for calibration and fitting of any cell-electrode electrical model. It is valid and can be extrapolated for any type of cellular line used in electrical cell-substrate impedance spectroscopy (ECIS) tests. Parameters of the bioimpedance model, acquired from ECIS experiments, vary for each cell line, which makes obtaining results difficult and -to some extent-renders them inaccurate. We propose a fitting method based on the cell line initial characterization, and carry out subsequent experiments with the same line to approach the percentage of well filling and the cell density (or cell number in the well). To perform our calibration technique, the so-called oscillation-based test (OBT) approach is employed for each cell density. Calibration results are validated by performing other experiments with different concentrations on the same cell line with the same measurement technique. Accordingly, a bioimpedance electrical model of each cell line is determined, which is valid for any further experiment and leading to a more precise electrical model of the electrode-cell system. Furthermore, the model parameters calculated can be also used by any other measurement techniques. Promising experimental outcomes for three different cell-lines have been achieved, supporting the usefulness of this technique.

Sensing Cell-Culture Assays with Low-Cost Circuitry
P. Pérez, G. Huertas, A. Maldonado-Jacobi, M. Martín, J.A. Serrano, A. Olmo, P. Daza and A. Yúfera
Journal Paper - Scientific Reports, vol. 8, no. 1, article 8841, 2018
NATURE PUBLISHING GROUP    DOI: 10.1038/s41598-018-27295-3    ISSN: 2045-2322    » doi
[abstract]
An alternative approach for cell-culture end-point protocols is proposed herein. This new technique is suitable for real-time remote sensing. It is based on Electrical Cell-substrate Impedance Spectroscopy (ECIS) and employs the Oscillation-Based Test (OBT) method. Simple and straightforward circuit blocks form the basis of the proposed measurement system. Oscillation parameters - frequency and amplitude - constitute the outcome, directly correlated with the culture status. A user can remotely track the evolution of cell cultures in real time over the complete experiment through a web tool continuously displaying the acquired data. Experiments carried out with commercial electrodes and a well-established cell line (AA8) are described, obtaining the cell number in real time from growth assays. The electrodes have been electrically characterized along the design flow in order to predict the system performance and the sensitivity curves. Curves for 1-week cell growth are reported. The obtained experimental results validate the proposed OBT for cell-culture characterization. Furthermore, the proposed electrode model provides a good approximation for the cell number and the time evolution of the studied cultures.

Practical Characterization of Cell-Electrode Electrical Models in Bio-Impedance Assays
J.A. Serrano, P. Pérez, A. Maldonado, M. Martín, A. Olmo, P. Daza, G. Huertas and A. Yúfera
Conference - International Conference on Biomedical Electronics and Devices BIODEVICES 2018
[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.

Monitoring Muscle Stem Cell Cultures with Impedance Spectroscopy
Y. Yuste, J.A. Serrano, A. Olmo, A. Maldonado-Jacobi, P. Pérez, G. Huertas, S. Pereira, F. de la Portilla and A. Yúfera
Conference - International Conference on Biomedical Electronics and Devices BIODEVICES 2018
[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.

Remote Sensing of Cell-Culture Assays
P. Pérez, A. Maldonado-Jacobi, A.J. López, C. Martínez, A. Olmo, G. Huertas and A. Yufera
Book Chapter - New Insights into Cell Culture Technology, pp 135-155, 2017
INTECH    DOI: 10.5772/67496    ISBN: 978-953-51-3133-5    » doi
[abstract]
This chapter describes a full system developed to perform the remote sensing of cell-culture experiments from any access point with internet connection. The proposed system allows the real-time monitoring of cell assays thanks to bioimpedance measurement circuits developed to count the number of cell present in a culture. Cell-culture characterization is performed through the measurement of the increasing bioimpedance parameter over time. The circuit implementation is based on the oscillation-based test (OBT) methodology. Bioimpedance of cell cultures is measured in terms of the oscillation parameters (frequency, amplitude, phase, etc.) and used as empirical markers to carry out an appropriate interpretation in terms of cell size identification, cell counting, cell growth, growth rhythm, etc. The device is capable of managing the whole sensing task and performs wireless communication through a Bluetooth module. Data are interpreted and displayed on a computer or a mobile phone through a web application. The system has its practical application in drug development processes, offering a label-free, high-throughput, and high-content screening method for cellular research, avoiding the classical end-point techniques and a significant workload and cost material reduction.

A Tracking Algorithm For Cell Motility Assays in CMOS Systems
C. Martínez-Gómez, A. Olmo, G. Huertas, P. Pérez, A. Maldonado-Jacobi and A. Yúfera
Conference - International Conference of the IEEE Engineering in Medicine and Biology Society EMBC 2017
[abstract]
This work proposes a method for the study and real-time monitorization of a single cell on a 2D electrode matrix, of great interest in cell motility assays and in the characterization of cancer cell metastasis. A CMOS system proposal for cell location based on occupation maps data generated from Electrical Cell-substrate Impedance Spectroscopy (ECIS) has been developed. From experimental assays data, an algorithm based on analysis of the eight nearest neighbours has been implemented to find the cell center of mass. The path followed by a cell, proposing a Brownian route, has been simulated with the proposed algorithm. The presented results give an accuracy over 95% in the determination of the coordinates (x, y) from the expected cell center of mass.

A CMOS Tracking System Approach for Cell Motility Assays
C. Martínez-Gómez, A. Olmo, G. Huertas, P. Pérez, A. Maldonado-Jacobi and A. Yufera
Conference - International Conference on Biomedical Electronics and Devices BIODEVICES 2017
[abstract]
This work proposes a method for studying and monitoring in real-time a single cell on a 2D electrode matrix, of great interest in cell motility assays and in the characterization of cancer cell metastasis. A CMOS system proposal for cell location based on occupation maps data generated from Electrical Cell-substrate Impedance Spectroscopy (ECIS) has been developed. From this cell model, obtained from experimental assays data, an algorithm based on analysis of the 8 nearest neighbors has been implemented, allowing the evaluation of the cell center of mass. The path followed by a cell, proposing a Brownian route, has been simulated with the proposed algorithm. The presented results show the success of the approach, with accuracy over 95% in the determination of any coordinate (x, y) from the expected center of mass.

Microcontroller-Based Sinusoidal Voltage Generation for Electrical Bio-Impedance Spectroscopy Applications
J.A. Castro, A. Olmo, P. Pérez and A. Yúfera
Journal Paper - Journal of Computer and Communications, vol. 4, no. 17, pp 51-58, 2016
SCIENTIFIC RESEARCH PUBLISHING    DOI: 10.4236/jcc.2016.417003    ISSN: 2327-5219    » doi
[abstract]
A sinusoidal voltage wave generator is proposed based on the use of micro-processor digital signals with programmable duty-cycles, with application to real-time Electrical Cell-substrate Impedance Spectroscopy (ECIS) assays in cell cultures. The working principle relies on the time convolution of the programmed microcontroller (μC) digital signals. The expected frequency is easily tuned on the bio-impedance spectroscopy range [100 Hz, 1 MHz] thanks to the μC clock frequency selection. This system has been simulated and tested on the 8 bits μC ArduinoTM Uno with ATmega328 version. Results obtained prove that only three digital signals are required to fit the general specification in ECIS experiments, below 1% THD accuracy, and show the appropriateness of the system for the real-time monitoring of this type of biological experiments.

Monitoring Tissue Evolution on Electrodes with Bio-Impedance Test
A. Maldonado, P. Pérez, G. Huertas, A. Yúfera, A. Rueda, and J.L. Huertas
Conference - Conference on Design of Circuits and Integrated Systems DCIS 2016
[abstract]
A technique for real-time monitoring of bio-impedances using a Voltage Oscillation (VO) methodology is proposed. The main idea relies on connecting the bio-system in such a way that a suitable electrical oscillator, which only uses a DC power source, is built. Thanks to the employed electrical models, the oscillation parameters can be directly related to the biological test. System simulations show that the impedance values of a tissue, called herein Zx, can be determined by measuring the actual frequency and amplitude of the proposed VO system, being possible to select the frequency range to optimize the system sensitivity.

Cell-Culture Measurements using Voltage Oscillations
A. Maldonado, P. Pérez, G. Huertas, A. Yúfera, A. Rueda and J.L. Huertas
Conference - IEEE Latin American Symposium on Circuits and Systems LASCAS 2016
[abstract]
A comprehensive system for real-time monitoring of a set of cell-cultures using a Voltage Oscillation (VO) methodology is proposed. The main idea is to connect the bioelectrical elements (electrodes&cell-culture) in such a way that sequentially a suitable electrical oscillator, which only uses a DC power source, is built. Using the employed electrical models given in [1, 2], the attained oscillation parameters (frequency and amplitude) can be directly related to the biological test. A digital circuitry is developed to pick-up the experimental measurements, which are gathered and shown in real-time in a web application.

From voltage oscillations to tissue-impedance measurements
A. Maldonado, P. Perez, G. Huertas, A. Yufera, A. Rueda and J.L. Huertas
Conference - IEEE Biomedical Circuits and Systems Conference BioCAS 2015
[abstract]
A technique for real-time monitoring of bio-impedances using a Voltage Oscillation (VO) methodology is proposed. The main idea relies on connecting the bio-system in such a way that a suitable electrical oscillator, which only uses a DC power source, is built. Thanks to the employed electrical models, the oscillation parameters can be directly related to the biological test. System simulations show that the impedance values of a tissue, called herein Zx, can be determined by measuring the actual frequency and amplitude of the proposed VO system, being possible to select the frequency range to optimize the system sensitivity.

Towards Bio-Impedance Based Labs: A Review
P. Pérez, A. Maldonado, A. Yúfera, G. Huertas, A. Rueda and J.L. Huertas
Conference - Conference on Design of Circuits and Integrated Systems DCIS 2015
[abstract]
Conference Paper

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