Electrical stimulation of cardiac adipose tissue-derived progenitor cells modulates cell phenotype and genetic machinery.

A major challenge of cardiac tissue engineering is directing cells to establish the physiological structure and function of the myocardium being replaced. Our aim was to examine the effect of electrical stimulation on the cardiodifferentiation potential of cardiac adipose tissue-derived progenitor cells (cardiac ATDPCs). Three different electrical stimulation protocols were tested; the selected protocol consisted of 2 ms monophasic square-wave pulses of 50 mV/cm at 1 Hz over 14 days. Cardiac and subcutaneous ATDPCs were grown on biocompatible patterned surfaces. Cardiomyogenic differentiation was examined by real-time PCR and immunocytofluorescence. In cardiac ATDPCs, MEF2A and GATA-4 were significantly upregulated at day 14 after stimulation, while subcutaneous ATDPCs only exhibited increased Cx43 expression. In response to electrical stimulation, cardiac ATDPCs elongated, and both cardiac and subcutaneous ATDPCs became aligned following the linear surface pattern of the construct. Cardiac ATDPC length increased by 11.3%, while subcutaneous ATDPC length diminished by 11.2% (p = 0.013 and p = 0.030 vs unstimulated controls, respectively). Compared to controls, electrostimulated cells became aligned better to the patterned surfaces when the pattern was perpendicular to the electric field (89.71 ± 28.47º for cardiac ATDPCs and 92.15 ± 15.21º for subcutaneous ATDPCs). Electrical stimulation of cardiac ATDPCs caused changes in cell phenotype and genetic machinery, making them more suitable for cardiac regeneration approaches. Thus, it seems advisable to use electrical cell training before delivery as a cell suspension or within engineered tissue.

Differentiation of the intracellular structure of slow- versus fast-twitch muscle fibers through evaluation of the dielectric properties of tissue.

Slow-twitch (type 1) skeletal muscle fibers have markedly greater mitochondrial content than fast-twitch (type 2) fibers. Accordingly, we sought to determine whether the dielectric properties of these two fiber types differed, consistent with their distinct intracellular morphologies. The longitudinal and transverse dielectric spectrum of the ex vivo rat soleus (a predominantly type 1 muscle) and the superficial layers of rat gastrocnemius (predominantly type 2) (n = 15) were measured in the 1 kHz-10 MHz frequency range and modeled to a resistivity Cole-Cole function. Major differences were especially apparent in the dielectric spectrum in the 1 to 10 MHz range. Specifically, the gastrocnemius demonstrated a well-defined, higher center frequency than the soleus muscle, whereas the soleus muscle showed a greater difference in the modeled zero and infinite resistivities than the gastrocnemius. These findings are consistent with the fact that soleus tissue has larger and more numerous mitochondria than gastrocnemius. Evaluation of tissue at high frequency could provide a novel approach for assessing intracellular structure in health and disease.

Harmonic impedance spectra identification from time-varying bioimpedance: theory and validation.

The harmonic impedance spectra (HIS) of a time-varying bioimpedance Z(ω, t) is a new tool to better understand and describe complex time-varying biological systems with a distinctive periodic character as, for example, cardiovascular and respiratory systems. In this paper, the relationship between the experimental setup and the identification framework for estimating Z(ω, t) is set up. The theory developed applies to frequency response based impedance measurements from noisy current-voltage observations. We prove theoretically and experimentally that a voltage source (VS) and a current source (CS) analogue front end-based measurement lead, respectively, to a closed-loop and an open-loop HIS identification problem. Next, we delve into the estimation of the HIS by treating Z(ω, t), on the one hand, as a linear time-invariant (LTI) system within a short time window; and, on the other hand, as a linear periodically time-varying (PTV) system within the entire measurement interval. The LTI approach is based on the short-time Fourier transform (STFT), while the PTV approach relies on the information that is present in the skirts of the voltage and/or current spectra. In addition, direct and indirect methods are developed for estimating the HIS by using simple as well as more sophisticated techniques. Ultimately, the HIS and their uncertainty bounds are estimated from real measurements conducted on a periodically varying dummy impedance.

A new measuring and identification approach for time-varying bioimpedance using multisine electrical impedance spectroscopy.

The bioimpedance measurement/identification of time-varying biological systems Z(ω, t) by means of electrical impedance spectroscopy (EIS) is still a challenge today. This paper presents a novel measurement and identification approach, the so-called parametric-in-time approach, valid for time-varying (bio-)impedance systems with a (quasi) periodic character. The technique is based on multisine EIS. Contrary to the widely used nonparametric-in-time strategy, the (bio-)impedance Z(ω, t) is assumed to be time-variant during the measurement interval. Therefore, time-varying spectral analysis tools are required. This new parametric-in-time measuring/identification technique has experimentally been validated through three independent sets of in situ measurements of in vivo myocardial impedance. We show that the time-varying myocardial impedance Z(ω, t) is dominantly periodically time varying (PTV), denoted as ZPTV(ω, t). From the temporal analysis of ZPTV(ω, t), we demonstrate that it is possible to decompose ZPTV(ω, t) into a(n) (in)finite sum of fundamental (bio-)impedance spectra, the so-called harmonic impedance spectra (HIS) Zk(ω)s with [Formula: see text]. This is similar to the well-known Fourier series of a periodic signal, but now understood at the level of a periodic system's frequency response. The HIS Zk(ω)s for [Formula: see text] actually summarize in the bi-frequency (ω, k) domain all the temporal in-cycle information about the periodic changes of Z(ω, t). For the particular case k = 0 (i.e. on the ω-axis), Z0(ω) reflects the mean in-cycle behavior of the time-varying bioimpedance. Finally, the HIS Zk(ω)s are directly identified from noisy current and voltage myocardium measurements at the multisine measurement frequencies (i.e. nonparametric-in-frequency).

Minimally invasive in vivo human lung tissue bioimpedance measurements during the bronchoscopy procedure.

Respiratory diseases, which include diseases of the lung, pleura, bronchial tree, trachea, upper respiratory tract and of the respiratory muscles and nerves, are a common and important cause of illness and death among the population. Experimental evidences have shown that tissue lesions have different electrical properties compared with normal tissue. Therefore, lung tissues lesions may be differentiated from lung normal tissue by comparing the tissue passive electrical properties. The manuscript reports a feasibility study for minimally invasive in vivo human lung tissue tetrapolar bioimpedance measurements using a catheter during the bronchoscopy procedure based on multisine Electrical Impedance Spectroscopy (EIS) at 10 kHz - 1 MHz.

Towards on line monitoring the evolution of the myocardium infarction scar with an implantable electrical impedance spectrum monitoring system.

The human heart tissue has a limited capacity for regeneration. Tissue and cellular therapies based on the use of stem cells may be useful alternatives to limit the size of myocardial infarction. In this paper, the preliminary results from an experimental campaign for on-line monitoring of myocardium scar infarction are presented. This study has been carried out under a research project that has as main objective the development and application of a bioactive patch implant for regeneration of myocardial infarction. Electrical Impedance Spectroscopy (EIS) has been chosen as a tissue state monitoring technique. What is presented in this communication is the first results of an implantable EIS measurement system which has been implanted in a subset of the animals corresponding to the control group, along one month. In all the animals, the myocardial infarction was induced by the ligation of the first circumflex marginal artery. In the animal group presented, the bioactive patch scaffold and the electrodes were implanted without the stem cells load. The scaffold is a piece of decellularized human pericardium, lyophilized and rehydrated with hydrogel RAD16-I. Nanogold particles were also placed near the electrodes to improve the electrode area conductivity. The results presented correspond to the subset of animals (n = 5), which had implanted the bioimpedance system monitoring the electrical impedance spectrum in vivo during 1 month. Two electrodes were connected to the bioactive patch implant. A total of 14 logarithmically spaced frequencies were measured every 5 minutes, from 100 Hz to 200 kHz. Results show a convergence of low-frequency and high frequency impedance magnitudes along the measurement period, which is coherent with the scar formation.

Influence of electrical stimulation on 3D-cultures of adipose tissue derived progenitor cells (ATDPCs) behavior.

Tissue engineering has a fundamental role in regenerative medicine. Still today, the major motivation for cardiac regeneration is to design a platform that enables the complete tissue structure and physiological function regeneration of injured myocardium areas. Although tissue engineering approaches have been generally developed for two-dimensional (2D) culture systems, three-dimensional (3D) systems are being spotlighted as the means to mimic better in vivo cellular conditions. This manuscript examines the influence of electrical stimulation on 3D cultures of adipose tissue-derived progenitor cells (ATDPCs). ATDPCs cells were encapsulated into a self-assembling peptide nanoscaffold (RAD16-I) and continuously electro stimulated during 14-20 days with 2-ms pulses of 50mV/cm at a frequency of 1 Hz. Good cellular network formation and construct diameter reduction was observed in electro stimulated samples. Importantly, the process of electro stimulation does not disrupt cell viability or connectivity. As a future outlook, differentiation studies to cardiomyocytes-like cells will be performed analyzing gene profile and protein expression.

Electrical impedance spectroscopy measurements using a four-electrode configuration improve on-line monitoring of cell concentration in adherent animal cell cultures.

This paper describes the improvement in the use of electrical impedance spectroscopy (EIS) for animal cell concentration monitoring of adherent cultures by using a four-electrode configuration instead of the commonly used two-electrode configuration. This four-electrode configuration prevents cell concentration measurements from external masking effects such as the electrode covering ratio, the degree of cellular adherence to the electrodes and the impedance of the measuring electrodes. Cell concentration was monitored using both four-electrode and two-electrode configurations in vero cell and human mesenchymal stem cell cultures in order to analyze the attained improvement in two cell lines with opposite growth characteristics. The experiments performed with vero cell cultures evidenced that the four-electrode configuration enables cell concentration measurements along all culture phases, even once the culture reached cell confluence (over 2×10(5) cells/cm(2)), confirming that this configuration is less effected by all the external influences. The experiments performed with human mesenchymal stem cells demonstrated good sensitivity of the measurement at very low cell concentrations, as well as a very good robustness all over the 12-days experiment. Finally, off-line cell measurements during cell cultures proved good accuracy of impedance measurements carried out with a four-electrode configuration along all cell growth phases, enabling determination of relevant cell growth parameters.

Implantable bioimpedance monitor using ZigBee.

In this paper, a novel implantable bioimpedance monitor using a free ZigBee protocol for the transmission of the measured data is described. The application field is the tissue and organ monitoring through electrical impedance spectroscopy in the 100 Hz - 200 kHz range. The specific application is the study of the viability and evolution of engineered tissue in cardiac regeneration. Additionally to the telemetric feature, the measured data are stored in a memory for backup purposes and can be downloaded at any time after an RF link break. In the debugging prototype, the system autonomy exceeds 1 month when a 14 frequencies impedance spectrum is acquired every 5 minutes. In the current implementation, the effective range of the RF link is reduced and needs for a range extender placed near the animal. Current work deals with improving this range.

Measurement errors in multifrequency bioelectrical impedance analyzers with and without impedance electrode mismatch.

The purpose of this study is to compare measurement errors in two commercially available multi-frequency bioimpedance analyzers, a Xitron 4000B and an ImpediMed SFB7, including electrode impedance mismatch. The comparison was made using resistive electrical models and in ten human volunteers. We used three different electrical models simulating three different body segments: the right-side, leg and thorax. In the electrical models, we tested the effect of the capacitive coupling of the patient to ground and the skin-electrode impedance mismatch. Results showed that both sets of equipment are optimized for right-side measurements and for moderate skin-electrode impedance mismatch. In right-side measurements with mismatch electrode, 4000B is more accurate than SFB7. When an electrode impedance mismatch was simulated, errors increased in both bioimpedance analyzers and the effect of the mismatch in the voltage detection leads was greater than that in current injection leads. For segments with lower impedance as the leg and thorax, SFB7 is more accurate than 4000B and also shows less dependence on electrode mismatch. In both devices, impedance measurements were not significantly affected (p > 0.05) by the capacitive coupling to ground.

Four versus two-electrode measurement strategies for cell growing and differentiation monitoring using electrical impedance spectroscopy.

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.

Multiple automated minibioreactor system for multifunctional screening in biotechnology.

The current techniques applied in biotechnology allow to obtain many types of molecules that must be tested on cell cultures (high throughput screening HTS). Although such tests are usually carried out automatically on mini or microwell plates, the procedures in the preindustrial stage are performed almost manually on higher volume recipients known as bioreactors. The growth conditions in both stages are completely different. The screening system presented in this work is based on the multiwell test plates philosophy, a disposable multiple minibioreactor that allows reproduction of industrial bioreactor culture conditions: aeration, stirring, temperature, O2, pH and visible range optical absorbance measurements. It is possible to reproduce the growth conditions for both suspended and adherent animal cell types using 1 to 10 ml vol. bioreactors. In the case of bacteria or yeast, it is not possible to achieve a high biomass concentration, due to the reduced head volume air supply.

On-line monitoring of yeast cell growth by impedance spectroscopy.

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.

Percutaneous electrocatheter technique for on-line detection of healed transmural myocardial infarction.

Healed myocardial infarction has been recognized by its particular tissue electrical impedance spectrum measured with intramural needle electrodes in animal models. The aim of this study was to develop a percutaneous approach for in vivo recognition of areas of healed myocardial infarction by measuring myocardial electrical impedance with an intracavitary contact electrocatheter. Electrical impedance (resistance and phase angle) of normal myocardium and of a 2-month-old anterior transmural infarction were measured in nine chloralose anesthetized pigs by applying alternating currents from 1 kHz to 1 MHZ between a bipolar intracavitary catheter and a reference electrode placed on the epicardium (group I, n = 4) or on the precordium (group II, n = 5). Resistance of the infarcted myocardium was lower than that of healthy tissue at all current frequencies (ANOVA, P < 0.001) (i.e., at 1 kHz: 15 +/- 4 omega vs 50 +/- 19 omega in group I, and 64 +/- 13 omega vs 76 +/- 13 omega in group II). Phase angle at 316 kHz best differentiated transmural infarction from normal tissue (group I: -2.5 +/- 1.9 degrees vs -14.8 +/- 4.6 degrees, P < 0.001; group II: +0.7 +/- 1.0 degrees vs -2.7 +/- 1.4 degrees, P < 0.001). This study shows that analysis of myocardial impedance spectrum using a percutaneous intracavitary contact catheter approach permits on-line recognition of areas of healed transmural myocardial infarction. This technique may be useful to optimize clinical application of energy sources (i.e., radiofrequency ablation, laser myocardial revascularization).

Algorithms for parametric images in MEIT systems.

In this work we show the algorithms developed to extract the Cole parameters from multi-frequency EIT. With these parameters it is possible to obtain information about various different tissues and their pathologies. The algorithms developed obtain the Cole-model parameters from the real and imaginary parts of impedance, or using only the real part, without problems of convergence. A study of the influence of noise is performed with simulations. We find a correct solution in all cases with signal to noise ratio in the data higher than 40 dB. Finally, we show parametric images of the human abdomen obtained with these algorithms.

In vivo and in situ ischemic tissue characterization using electrical impedance spectroscopy.

The investigation of processes of ischemia in different organ tissues is very important for the development of methods of protection and preservation during surgical procedures. Electrical impedance spectroscopy was used to distinguish between different tissues and their degree of ischemia. We describe mathematical methods used to adjust experimental data to Cole-Cole models for one-circle and two-circle impedance loci and a study of the main parameters for representing the behavior of ischemia in time. In vivo and in situ postmortem measurements of different tissues from pigs are shown in the 100 Hz to 1 MHz range. The Cole parameters that best characterize the ischemia are R0 and fc.

Biomass monitoring using impedance spectroscopy.

The biomass density in biotechnological processes is often determined by indirect and manual methods. Electrical impedance spectroscopy can provide online viable biomass density estimators. In this work, we present two linear estimators obtained with this technique. Four different microorganisms were measured. The detection threshold was approximately 1 g/L (dry weight) for bacteria and 0.5 g/L for yeast. Liposome suspensions were also used to validate the methods. The monitoring of the continuous growth of a yeast culture is also presented.

Passive transmission of ischemic ST segment changes in low electrical resistance myocardial infarct scar in the pig.

OBJECTIVES: To analyze the passive electrical properties of a healed infarction and assess their role on transmission of contiguous ischemic ST segment potential changes. METHODS: We measured tissue resistivity (omega cm) at 1 kHz and the epicardial ST segment during 1 h of proximal reocclusion of the left anterior descending (LAD) coronary artery in 12 anesthetized pigs with one-month-old transmural infarction elicited by LAD ligature below the first branch. The impedance spectrum (1 to 1000 kHz) of normal and infarcted myocardium was measured in seven other pigs with similar infarctions. Electrical transmission of current pulses (30 microA) in infarcted tissue and in test solutions was also investigated. RESULTS: The infarct scar has a lower than normal resistivity (110 +/- 30 omega cm vs. 235 +/- 60 omega cm, p < 0.0001) and, unlike the normal myocardium, resistivity and phase angle of the scar did not change at increasing current frequencies, reflecting no capacitative response. LAD reocclusion induced a resistivity rise (510 +/- 135 omega cm, p < 0.01) and a ST segment elevation (0.6 +/- 0.7 to 9.5 +/- 5.1 mV, p = 0.002) in the ischemic peri-infarction zone, whereas the infarcted area showed ST segment elevation (0.5 +/- 0.5 to 3.8 +/- 2.6 mV, p = 0.03) with no resistivity changes. Potential decay of both ST segment and current pulses in the scar and in 0.9% NaCl solution was less than 1 mV/mm. Transmural deposition of connective tissue was seen in the center of the infarction. CONCLUSIONS: A one-month-old transmural infarction is a low resistance, noncapacitative medium that allows a good transmission of current pulses and of ST segment potential changes generated by contiguous peri-infarction ischemia.

A parallel broadband real-time system for electrical impedance tomography.

This paper deals with the design, implementation and performance of TIE-4sys, an electrical impedance tomograph. This instrument is a parallel broad-band real-time system. It measures impedance using an array of 16 electrodes and reconstructs the images using a weighted back-projection technique. The objective of this development is to enable multifrequency EIT clinical studies to be undertaken. The system is capable of acquiring 25 frames/s and makes multifrequency cardiac-gated images. The frequency range is from 10 kHz to 250 kHz and the signal to noise ratio for the real component is better than 60 dB.

Broadband quasi-differential multifrequency electrical impedance imaging system.

Dynamic and multifrequency imaging methods have been demonstrated both theoretically and experimentally. Multifrequency methods are able to produce images of static structures inside the measured object. Data collection systems, however, are affected by errors due to their non-ideal frequency behaviour. If the frequencies used in the measurement were close enough, the system would behave in almost the same way. In this case, however, the impedance change displayed by biological tissues is small, so the situation is similar to dynamic imaging. We call this method the quasi-differential imaging method. We have designed and built an instrument able to apply signals from 1 kHz to 1 MHz, with frequency increments of 10 Hz. Patient interface circuits and demodulators were designed to display a flat response in the full frequency range of operation. Signals are digitized with 16 bit resolution and sent to the host computer using a high-speed serial interface. This allows a maximum measurement speed of about 8 images/s. All the system parts were full characterized out of the system and the results of these measurements are given as an indication of the limits of its use as a quasi-static imaging or quasi-differential imaging data collection system.