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Electrical pulse stimulation has been used to enhance the differentiation or proliferation of neuronal progenitor cells in tissue engineering and cancer treatment. Therefore, a comprehensive investigation of the effects caused by its parameters is crucial for improvements in those fields. We propose a study of pulse parameters, to allow the control of N2a cell line fate and behavior. We have focused on designing an experimental setup that allows for the knowledge and control over the environment and the stimulation signals applied. To map the effects of the stimulation on N2a cells, their morphology and the cellular and molecular reactions induced by the pulse stimulation have been analyzed. Immunofluorescence, rt-PCR and western blot analysis have been carried out for this purpose, as well as cell counting. Our results show that low-amplitude electrical pulse stimulation promotes proliferation of N2a cells, whilst amplitudes in the range 250 mV/mm-500 mV/mm induce differentiation. Amplitudes higher than 750 mV/mm produce cell damage at low frequencies. For high frequencies, large amplitudes are needed to cause cell death. An inverse relation has been found between cell density and pulse-induced neuronal differentiation. The best condition for neuronal differentiation was found to be 500 mV/mm at 100 Hz. These findings have been confirmed by up-regulation of the Neurod1 gene. Our preliminary study of the molecular effects of electrical pulse stimulation on N2a offers premonitory clues of the PI3K/Akt/GSK-3ß pathway implications on the neuronal differentiation process through ES. In general, we have successfully mapped the sensitivity of N2a cells to electrical pulse stimulation parameters.
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Obtaining cell concentration measurements from a culture assay by using bioimpedance is a very useful method that can be used to translate impedances to cell concentration values. The purpose of this study was to find a method to obtain the cell concentration values of a given cell culture assay in real time by using an oscillator as the measurement circuit. From a basic cell-electrode model, enhanced models of a cell culture immersed in a saline solution (culture medium) were derived. These models were used as part of a fitting routine to estimate the cell concentration in a cell culture in real time by using the oscillation frequency and amplitude delivered by the measurement circuits proposed by previous authors. Using real experimental data (the frequency and amplitude of oscillations) that were obtained by connecting the cell culture to an oscillator as the load, the fitting routine was simulated, and real-time data of the cell concentration were obtained. These results were compared to concentration data that were obtained by using traditional optical methods for counting. In addition, the error that we obtained was divided and analyzed in two parts: the first part of the experiment (when the few cells were adapting to the culture medium) and the second part of the experiment (when the cells exponentially grew until they completely covered the well). Low error values were obtained during the growth phase of the cell culture (the relevant phase); therefore, the results obtained were considered promising and show that the fitting routine is valid and that the cell concentration can be measured in real time by using an oscillator.
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Técnicas de Cultura de Células , Técnicas de Cultura de Células/métodos , Eletrodos , Impedância ElétricaRESUMO
BACKGROUND: Electrical stimulation is a novel tool to promote the differentiation and proliferation of precursor cells. In this work we have studied the effects of direct current (DC) electrical stimulation on neuroblastoma (N2a) and osteoblast (MC3T3) cell lines as a model for nervous and bone tissue regeneration, respectively. We have developed the electronics and encapsulation of a proposed stimulation system and designed a setup and protocol to stimulate cell cultures. METHODS: Cell cultures were subjected to several assays to assess the effects of electrical stimulation on them. N2a cells were analyzed using microscope images and an inmunofluorescence assay, differentiated cells were counted and neurites were measured. MC3T3 cells were subjected to an AlamarBlue assay for viability, ALP activity was measured, and a real time PCR was carried out. RESULTS: Our results show that electrically stimulated cells had more tendency to differentiate in both cell lines when compared to non-stimulated cultures, paired with a promotion of neurite growth and polarization in N2a cells and an increase in proliferation in MC3T3 cell line. CONCLUSIONS: These results prove the effectiveness of electrical stimulation as a tool for tissue engineering and regenerative medicine, both for neural and bone injuries. Bone progenitor cells submitted to electrical stimulation have a higher tendency to differentiate and proliferate, filling the gaps present in injuries. On the other hand, neuronal progenitor cells differentiate, and their neurites can be polarized to follow the electric field applied.
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Coronary artery disease is one of the most common diseases in developed countries and affects a large part of the population of developing countries. Preventing restenosis in patients with implanted stents is an important current medical problem. The purpose of this work is to analyse the viability of bioimpedance sensing to detect the formation of atheromatous plaque in an implantable stent. Simulations in COMSOL Multiphysics were performed to analyse the performance of the proposed bioimpedance sensing system, based on the Sheffield technique. Both non-pathological and pathological models (with atheromatous plaque), including the flow of blood were considered. Simulations with the non-pathological model showed a homogeneous distribution of the measured current intensity in the different electrodes, for every configuration. On the other hand, simulations with the pathological model showed a significant decrease of the measured current intensity in the electrodes close to the simulated atheromatous plaque. The presence of the atheromatous plaque can, therefore, be detected by the system with a simple algorithm, avoiding the full reconstruction of the image and the subsequent computational processing requirements.
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Placa Aterosclerótica , Humanos , StentsRESUMO
In tissue engineering, of utmost importance is the control of tissue formation, in order to form tissue constructs of clinical relevance. In this work, we present the use of an impedance spectroscopy technique for the real-time measurement of the dielectric properties of skeletal myoblast cell cultures. The processes involved in the growth and differentiation of these cell cultures in skeletal muscle are studied. A circuit based on the oscillation-based test technique was used, avoiding the use of high-performance circuitry or external input signals. The effect of electrical pulse stimulation applied to cell cultures was also studied. The technique proved useful for monitoring in real-time the processes of cell growth and estimating the fill factor of muscular stem cells. Impedance spectroscopy was also useful to study the real-time monitoring of cell differentiation, obtaining different oscillation amplitude levels for differentiated and undifferentiated cell cultures. Finally, an electrical model was implemented to better understand the physical properties of the cell culture and control the tissue formation process.
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Técnicas de Cultura de Células , Estimulação Elétrica , Mioblastos Esqueléticos/citologia , Engenharia Tecidual , Diferenciação Celular , HumanosRESUMO
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.
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Técnicas de Cultura de Células/métodos , Impedância Elétrica , Modelos Teóricos , Animais , Contagem de Células , Técnicas de Cultura de Células/instrumentação , Linhagem Celular , Eletrodos , Camundongos , Mioblastos/citologia , Análise de RegressãoRESUMO
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.
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Potenciais de Ação , Desenvolvimento Muscular , Mioblastos Esqueléticos/metabolismo , Animais , Linhagem Celular , Estimulação Elétrica , Mioblastos Esqueléticos/citologia , RatosRESUMO
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.
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Técnicas de Cultura de Células/métodos , Periodicidade , Telemetria/métodos , Crescimento Celular , Proliferação de CélulasRESUMO
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.
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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.
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Técnicas de Cultura de Células/métodos , Proliferação de Células , Custos e Análise de Custo , Espectroscopia Dielétrica/métodos , Tecnologia de Sensoriamento Remoto/métodos , Animais , Células CHO , Técnicas de Cultura de Células/economia , Cricetinae , Cricetulus , Espectroscopia Dielétrica/economia , Internet , Tecnologia de Sensoriamento Remoto/economiaRESUMO
To follow up the restenosis in arteries stented during an angioplasty is an important current clinical problem. A new approach to monitor the growth of neointimal tissue inside the stent is proposed on the basis of electrical impedance spectroscopy (EIS) sensors and the oscillation-based test (OBT) circuit technique. A mathematical model was developed to analytically describe the histological composition of the neointima, employing its conductivity and permittivity data. The bioimpedance model was validated against a finite element analysis (FEA) using COMSOL Multiphysics software. A satisfactory correlation between the analytical model and FEA simulation was achieved in most cases, detecting some deviations introduced by the thin "double layer" that separates the neointima and the blood. It is hereby shown how to apply conformal transformations to obtain bioimpedance electrical models for stack-layered tissues over coplanar electrodes. Particularly, this can be applied to characterize the neointima in real-time. This technique is either suitable as a main mechanism for restenosis follow-up or it can be combined with proposed intelligent stents for blood pressure measurements to auto-calibrate the sensibility loss caused by the adherence of the tissue on the micro-electro-mechanical sensors (MEMSs).
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Stents , Vasos Coronários , Espectroscopia Dielétrica , NeointimaRESUMO
This paper presents a computer tool for automatic analysis of cell culture images. The program allows the extraction of relevant information from biological images for pre- and postsystem analysis. In particular, this tool is being used for electrical characterization of electrode-solution-cell systems in which bioimpedance is the main parameter to be known. The correct modeling of this kind of systems enables both electronic system characterization for circuit design specifications and data decoding from measurements. The developed program allows cell culture image processing for geographic information extraction and generates cell count and equivalent circuit descriptions useful for system simulations.
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Técnicas de Cultura de Células/estatística & dados numéricos , Simulação por Computador , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Contagem de Células/estatística & dados numéricos , Técnicas de Cultura de Células/instrumentação , Biologia Computacional , Impedância Elétrica , Microeletrodos , Modelos Biológicos , SoftwareRESUMO
This paper proposes a cell-microelectrode model for cell biometry applications, based on the area overlap as main parameter. The model can be applied to cell size identification, cell count, and their extension to cell growth and dosimetry protocols. Experiments performed with comercial electrodes are presented, illustrating a procedure to obtain cell number in both growth and dosimetry processes. Results obtained for the AA8 cell line are promising.
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Microeletrodos , Modelos Teóricos , Animais , Células Cultivadas , CricetinaeRESUMO
This paper analyses the use of Electrical Impedance Spectroscopy (EIS) to efficiently monitor cryoprotectant concentrations in cryopreservation protocols. The proposed technique can improve methods such as Liquidus Tracking (LT), allowing vitrification without exposing tissues to damaging concentrations of cryoprotectant at relatively high temperatures, and avoiding rapid temperature changes. This work is focused to continuous monitoring of cryoprotectant concentrations by detecting changes in electrical impedance. These variations, derived from cryoprotectant perfusion inside cells and tissues, can be efficiently measure by using of EIS. Finite element simulation performed with COMSOL Multiphysics software was used to analyse the frequency response of a two-electrode system to several concentrations of Me2SO, perfused into 3T3 fibroblasts and monolayers of Mesenchymal Stem Cells (MSCs), fundamental in tissue-based therapeutics.
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Criopreservação , Impedância Elétrica , Modelos TeóricosRESUMO
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.