Tomás García-Sánchez

Interpulse multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes

In this study, electrical impedance spectroscopy measurements are performed during electroporation of monolayers of differentiated myotubes. The time resolution of the system (1 spectrum/ms) enable 860 full spectra (21 frequencies from 5 kHz to 1.3 MHz) to be acquired during the time gap between consecutive pulses (interpulse) of a classical electroporation treatment (8 pulses, 100 μs, 1 Hz). Additionally, the characteristics of the custom microelectrode assembly used allow the experiments to be performed directly in situ in standard 24 multi-well plates. The impedance response dynamics are studied for three different electric field intensities (400, 800 and 1200 V/cm). The multifrequency information, analysed with the Cole model, reveals a short-term impedance recovery after each pulse in accordance with the fast resealing of the cell membrane, and a long-term impedance decay over the complete treatment in accordance with an accumulated effect pulse after pulse. The analysis shows differences between the lowest electric field condition and the other two, suggesting that different mechanisms that may be related with the reversibility of the process are activated. As a result of the multifrequency information, the system is able to measure simultaneously the conductivity variations due to ion diffusion during electroporation. Finally, in order to reinforce the physical interpretation of the results, a complementary electrical equivalent model is used.

Design and Implementation of a Microelectrode Assembly for Use on Noncontact In Situ Electroporation of Adherent Cells

In situ electroporation of adherent cells provides significant advantages with respect to electroporation systems for suspension cells, such as causing minimal stress to cultured cells and simplifying and saving several steps within the process. In this study, a new electrode assembly design is shown and applied to in situ electroporate adherent cell lines growing in standard multiwell plates. We designed an interdigitated array of electrodes patterned on copper with printed circuit board technology and covered with nickel/gold. Small interelectrode distances were used to achieve effective electroporation with low voltages. Epoxy-based microseparators were constructed to avoid direct contact with the cells and to create more uniform electric fields. The device was successful in the electropermeabilization of two different adherent cell lines, C2C12 and HEK 293, as assessed by the intracellular delivery of the fluorescent dextran FD20S. Additionally, as a collateral effect, we observed cell electrofusion in HEK 293 cells, thus making this device also useful for performing cell fusion. In summary, we show the effectiveness of this minimally invasive device for electroporation of adherent cells cultured in standard multiwell plates. The cheap technologies used in the fabrication process of the electrode assembly indicate potential use as a low-cost, disposable device.

Recognition of Fibrotic Infarct Density by the Pattern of Local Systolic-Diastolic Myocardial Electrical Impedance

Myocardial electrical impedance is a biophysical property of the heart that is influenced by the intrinsic structural characteristics of the tissue. Therefore, the structural derangements elicited in a chronic myocardial infarction should cause specific changes in the local systolic-diastolic myocardial impedance, but this is not known. This study aimed to characterize the local changes of systolic-diastolic myocardial impedance in a healed myocardial infarction model. Six pigs were successfully submitted to 150 min of left anterior descending (LAD) coronary artery occlusion followed by reperfusion. 4 weeks later, myocardial impedance spectroscopy (1–1000 kHz) was measured at different infarction sites. The electrocardiogram, left ventricular (LV) pressure, LV dP/dt, and aortic blood flow (ABF) were also recorded. A total of 59 LV tissue samples were obtained and histopathological studies were performed to quantify the percentage of fibrosis. Samples were categorized as normal myocardium (<10% fibrosis), heterogeneous scar (10–50%) and dense scar (>50%). Resistivity of normal myocardium depicted phasic changes during the cardiac cycle and its amplitude markedly decreased in dense scar (18 ± 2 Ω·cm vs. 10 ± 1 Ω·cm, at 41 kHz; P < 0.001, respectively). The mean phasic resistivity decreased progressively from normal to heterogeneous and dense scar regions (285 ± 10 Ω·cm, 225 ± 25 Ω·cm, and 162 ± 6 Ω·cm, at 41 kHz; P < 0.001 respectively). Moreover, myocardial resistivity and phase angle correlated significantly with the degree of local fibrosis (resistivity: r = 0.86 at 1 kHz, P < 0.001; phase angle: r = 0.84 at 41 kHz, P < 0.001). Myocardial infarcted regions with greater fibrotic content show lower mean impedance values and more depressed systolic-diastolic dynamic impedance changes. In conclusion, this study reveals that differences in the degree of myocardial fibrosis can be detected in vivo by local measurement of phasic systolic-diastolic bioimpedance spectrum. Once this new bioimpedance method could be used via a catheter-based device, it would be of potential clinical applicability for the recognition of fibrotic tissue to guide the ablation of atrial or ventricular arrhythmias.

Early detection of acute transmural myocardial ischemia by the phasic systolic-diastolic changes of local tissue electrical impedance

Myocardial electrical impedance is influenced by the mechanical activity of the heart. Therefore, the ischemia-induced mechanical dysfunction may cause specific changes in the systolic-diastolic pattern of myocardial impedance, but this is not known. This study aimed to analyze the phasic changes of myocardial resistivity in normal and ischemic conditions. Myocardial resistivity was measured continuously during the cardiac cycle using 26 different simultaneous excitation frequencies (1 kHz-1 MHz) in 7 anesthetized open-chest pigs. Animals were submitted to 30 min regional ischemia by acute left anterior descending coronary artery occlusion. The electrocardiogram, left ventricular (LV) pressure, LV dP/dt, and aortic blood flow were recorded simultaneously. Baseline myocardial resistivity depicted a phasic pattern during the cardiac cycle with higher values at the preejection period (4.19 ± 1.09% increase above the mean, P < 0.001) and lower values during relaxation phase (5.01 ± 0.85% below the mean, P < 0.001). Acute coronary occlusion induced two effects on the phasic resistivity curve: 1) a prompt (5 min ischemia) holosystolic resistivity rise leading to a bell-shaped waveform and to a reduction of the area under the LV pressure-impedance curve (1,427 ± 335 vs. 757 ± 266 Ω·cm·mmHg, P < 0.01, 41 kHz) and 2) a subsequent (5-10 min ischemia) progressive mean resistivity rise (325 ± 23 vs. 438 ± 37 Ω·cm at 30 min, P < 0.01, 1 kHz). The structural and mechanical myocardial dysfunction induced by acute coronary occlusion can be recognized by specific changes in the systolic-diastolic myocardial resistivity curve. Therefore these changes may become a new indicator (surrogate) of evolving acute myocardial ischemia.

A new spiral microelectrode assembly for electroporation and impedance measurements of adherent cell monolayers

In this study, a new microelectrode assembly based on spiral geometry applicable to in situ electroporation of adherent cell monolayers on standard multiwell plates is presented. Furthermore, the structure is specially conceived to perform electrical impedance spectroscopy (EIS) measurements during electroporation. Its performance for cell membrane permeabilization is tested with a fluorescent probe. Gene electrotransfer is also assayed using a plasmid DNA encoding GFP in four different cell lines (CHO, HEK293, 3T3-L1 and FTO2B). Additionally, siRNA α-GFP electrotransfection is tested in GFP gene-expressing CHO cells. Our data show considerable differences between permeabilization and gene transfer results and cell line dependence on gene expression rates. Successful siRNA electro-mediated delivery is also achieved. We demonstrate the applicability of our device for electroporation-mediated gene transfer of adherent cells in standard laboratory conditions. Finally, electrical impedance measurements during electroporation of CHO and 3T3-L1 cells are also given.

Electrical impedance measurements on electropermeabilized cells attached to microelectrodes

The aim of this study is to use fast electrical impedance spectroscopy to measure the process of electroporation applied on cell monolayers growing attached to a specifically designed set of microelectrodes. The frequency response of the impedance can provide useful information about the extent of permeabilization in the cell membranes exposed to high electric fields and also the time dynamics of creation and resealing of the ”‘pores’” created. Cell line CHO-K1 was cultured as a monolayer on a microelectrode assembly fabricated on Indium Tin Oxide substrates. Additionally, propidium iodide fluorescent dye was used to asses the success of permeabilization of cells. Results show two different resealing dynamics corresponding with the presence of two different type of pores (short-lived VS long-lived pores) and how information at different frequencies is valuable to separate different effects namely pore dynamics and release of intracellular contents.

Automatic system for electroporation of adherent cells growing in standard multi-well plates

In this study an automatic system is presented to perform electroporation, also known as electropermeabilization, on adherent cells. It is an intention of this system to apply electric field pulses directly to cells growing in standard multi-well plates as a step forward to include this technique in standard laboratory protocols. An interdigitated microelectrode assembly constructed with Printed Circuit Board (PCB) is placed closely above the cell monolayer, and in order to avoid direct contact with cells, small micro-separators were included in the structure. Additionally, distribution of current density was modified by filling the gap between adjacent electrodes with a non conductive material as predicted by electric field simulations. This modification helps to concentrate the electric field intensity in the region where cells are present. The device was tested using C2C12 cell line growing adhered in 24 multi-well plates and fluorescent labeled dextran FD20S as the molecule to be delivered. Successful transfection was observed with minimal invasiveness of the operation reducing the stress caused to cells.

Myocardial contractility assessed by dynamic electrical impedance measurements during dobutamine stress

In this study, the electrical impedance of myocardial tissue is measured dynamically during the cardial cycle. The multisine-based approach used to perform electrical impedance spectroscopy (EIS) measurements allows acquiring complete spectral impedance information of the tissue dynamics during contraction. Measurements are performed in situ in the left ventricule of swines during contractility stress tests induced by dobutamine infusion. Additionally, the ECG and the left ventricular (LV) pressure are also acquired synchronously to the impedance signals. The calculated impedance magnitude exhibits a periodic behavior during tissue contraction. The amplitude (peak-to-peak) of this signal is quantified and the compared to the maximum first derivative of the LV pressure (dP/dtmax) that is used as an indicator of contractility variations. The results show a linear correlation between impedance amplitude and dP/dtmax during dobutamine-increased contractility. The present work demonstrates how fast EIS measurements during heart contraction can represent a feasible method to assess changes in myocardial contractility.

Modeling Dynamic Electrical Impedance Spectroscopy Measurements on Electroporated Cells

In the present work, electrical impedance spectroscopy (EIS) measurements were performed during electroporation of C2C12 cells monolayers. The measuring strategy, based on multisine excitations, allows acquiring full impedance spectra at a rate of 1 spectrum/ms during the interval between pulses of a traditional electroporation treatment. The multifrequency information provided by the measuring system was then studied using models. In this study, the Cole model and an electrical equivalent circuit were used. The results show the ability of the system for monitoring the fast impedance dynamics of cells during electroporation. The differences in the information shown at different frequencies suggest the ability of the system to measure different phenomena at the same time, namely: membrane changes and medium conductivity variations. The use of the Cole model reveals the need of more complex models to correctly separate and interpret the results. The use of the proposed circuital model enables studying the dynamics of pore formation independently of the conductivity variations. This work confirms the advantages of performing EIS measurements in order to have a more complete snapshot of the system behavior.

Interpulsed multifrequency electrical impedance measurements during electroporation of adherent differentiated myotubes

Plasma membrane of living cells constitutes the barrier between the intracellular and extracellular media and regulates the transport of chemical species from or into the cell cytoplasm. Electroporation, also called electropermeabilization, is a phenomenon occurring when cell membranes are exposed to high electric field pulses. When the parameters of such electric field are appropriate, a transient state of permeability to molecular species is generated. The technique is currently used as a tool to deliver membrane impermeable molecules into the cells both in vitro and in vivo with its main interest focused in electrochemotherapy of tumors and nucleic acids electrotransfer for gene therapy or DNA vaccination.