Our Publications Database

Article 59

Dielectrophoretic characterization of peroxidized retinal pigment epithelial cells as a model of age-related macular degeneration

Dharm Singh Yadav; Ioan Tivig; Tudor Savopol; Mihaela G. Moisescu

Journal: BMC OPHTHALMOLOGY

Year:  Aug. 2024

DOI: 1186/s12886-024-03617-0

 

Age-related macular degeneration; Dielectrophoresis; Retinal pigment epithelium; Membrane permittivity; Lipid peroxidation; Cell purification simulations

Background

Age-related macular degeneration (AMD) is a prevalent ocular pathology affecting mostly the elderly population. AMD is characterized by a progressive retinal pigment epithelial (RPE) cell degeneration, mainly caused by an impaired antioxidative defense. One of the AMD therapeutic procedures involves injecting healthy RPE cells into the subretinal space, necessitating pure, healthy RPE cell suspensions. This study aims to electrically characterize RPE cells to demonstrate a possibility using simulations to separate healthy RPE cells from a mixture of healthy/oxidized cells by dielectrophoresis.

Methods

BPEI-1 rat RPE cells were exposed to hydrogen peroxide to create an in-vitro AMD cellular model. Cell viability was evaluated using various methods, including microscopic imaging, impedance-based real-time cell analysis, and the MTS assay. Healthy and oxidized cells were characterized by recording their dielectrophoretic spectra, and electric cell parameters (crossover frequency, membrane conductivity and permittivity, and cytoplasm conductivity) were computed. A COMSOL simulation was performed on a theoretical microfluidic-based dielectrophoretic separation chip using these parameters.

Results

Increasing the hydrogen peroxide concentration shifted the first crossover frequency toward lower values, and the cell membrane permittivity progressively increased. These changes were attributed to progressive membrane peroxidation, as they were diminished when measured on cells treated with the antioxidant N-acetylcysteine. The changes in the crossover frequency were sufficient for the efficient separation of healthy cells, as demonstrated by simulations.

Conclusions

The study demonstrates that dielectrophoresis can be used to separate healthy RPE cells from oxidized ones based on their electrical properties. This method could be a viable approach for obtaining pure, healthy RPE cell suspensions for AMD therapeutic procedures.

Article 52

OpenDEP: An Open-Source Platform for Dielectrophoresis Spectra Acquisition and Analysis

Ioan Tivig, Mihaela Georgeta Moisescu, Tudor Savopol

Journal: ACS Omega

Year: 2023

DOI:  10.1021/acsomega.3c06052

 

dielectrophoresis, dielectric parameters, open-source, OpenDEP

Dielectrophoretic (DEP) cell separation, which utilizes electric fields to selectively manipulate and separate cells based on their electrical properties, has emerged as a cutting-edge label-free technique. DEP separation techniques rely on differences in the electrical and morphological properties of cells, which can be obtained by a thorough analysis of DEP spectra. This article presents a novel platform, named OpenDEP, for acquiring and processing DEP spectra of suspended cells. The platform consists of lab-on-a-chip and open-source software that enables the determination of DEP spectra and electric parameters. The performance of OpenDEP was validated by comparing the results obtained using this platform with the results obtained using a commercially available device, 3DEP from DEPtech. The lab-on-a-chip design features two indium tin oxide-coated slides with a specific geometry, forming a chamber where cells are exposed to an inhomogeneous alternating electric field with different frequencies, and microscopic images of cell distributions are acquired. A custom-built software written in the Python programing language was developed to convert the acquired images into DEP spectra, allowing for the estimation of membrane and cytoplasm conductivities and permittivities. The platform was validated using two cell lines, DC3F and NIH 3T3. The OpenDEP platform offers several advantages, including easy manufacturing, statistically robust computations due to large cell population analysis, and a closed environment for sterile work. Furthermore, continuous observation using any microscope allows for integration with other techniques.

Article 47

Changes in the packing of bilayer lipids triggered by electroporation: real-time measurements on cells in suspension

 

Journal: Bioelectrochemistry

Year: 2021

DOI: 10.1016/j.bioelechem.2020.107689

 Electroporation; Generalized polarization; Laurdan; Membrane lipid peroxidation; ROS production; Real-time measurements.

     Electropermeabilization of the cell membrane is a technique used to facilitate penetration of impermeant molecules into cells. Although there are studies regarding the mechanism of processes occurring after electropermeabilization, the relationship between electropermeabilization and associated phenomena (e.g. generation of reactive oxygen species, endocytosis, lipid peroxidation, etc.) is yet to be elucidated. This work aimed to get information on the changes in the packing of the bilayer lipids and their peroxidation induced by application of electroporation pulses. We used a specially designed system of electrodes which allowed performing electropermeabilization of cells in suspension simultaneously with time-dependent measurements of fluorescence and temperature. The kinetics of membrane packing and production of reactive oxygen species were studied using various conductivity buffers (0.01, 0.04 and 0.14 S/m) and different number of 1 kV/cm bipolar pulses (1-50). Two categories of effects were observed: a thermal effect, consisting in an increased bilayer disorder (a deeper penetration of water into the hydrophobic core), and a nonthermal effect, leading to a higher degree of lipids packing, the latter being attributed to a peroxidation process. An analysis of the permeabilization conditions in which one of these two processes predominates was performed.

Article 46

Influence of surfactant-tailored Mn-doped ZnO nanoparticles on ROS production and DNA damage induced in murine fibroblast cells

Traian Popescu, Christien Oktaviani Matei, Ioana Dorina Vlaicu, Ioan Tivig, Andrei Cristian Kuncser, Mariana Stefan, Daniela Ghica, Luminita Claudia Miclea, Tudor Savopol, Daniela Cristina Culita, Mihaela Georgeta Moisescu

Journal: Nature Scientific Reports

Year: 2020

DOI: 10.1038/s41598-020-74816-0

Cell biology, Chemistry, Materials science

     The present study concerns the in vitro oxidative stress responses of non-malignant murine cells exposed to surfactant-tailored ZnO nanoparticles (NPs) with distinct morphologies and different levels of manganese doping. Two series of Mn-doped ZnO NPs were obtained by coprecipitation synthesis method, in the presence of either polyvinylpyrrolidone (PVP) or sodium hexametaphosphate (SHMTP). The samples were investigated by powder X-ray Diffraction, Transmission Electron Microscopy, Fourier-Transform Infrared and Electron Paramagnetic Resonance spectroscopic methods, and N2 adsorption-desorption analysis. The observed surfactant-dependent effects concerned: i) particle size and morphology; ii) Mn-doping level; iii) specific surface area and porosity. The relationship between the surfactant dependent characteristics of the Mn-doped ZnO NPs and their in vitro toxicity was assessed by studying the cell viability, intracellular reactive oxygen species (ROS) generation, and DNA fragmentation in NIH3T3 fibroblast cells. The results indicated a positive correlation between the specific surface area and the magnitude of the induced toxicological effects and suggested that Mn-doping exerted a protective effect on cells by diminishing the pro-oxidative action associated with the increase in the specific BET area. The obtained results support the possibility to modulate the in vitro toxicity of ZnO nanomaterials by surfactant-controlled Mn-doping.

Article 42

An experimental system for real-time fluorescence recordings of cell membrane changes induced by electroporation

Ioan Tivig, Tudor Savopol, Eugenia Kovacs, Mihaela G Moisescu

Journal: European Biophysical Journal

Year: 2020

DOI: 10.1007/s00249-019-01417-9

3D prototyping; Electric field modeling; Electroporation; Generalized polarization; Real-time fluorescence recording.

     The electroporation of cells is nowadays used for a large variety of purposes, from basic research to cancer therapy and food processing. Understanding molecular mechanisms of the main processes involved in electroporation is thus of significant interest. In the present work, we propose an experimental system to record in real time the evolution of any cell parameter which can be evaluated by fluorescence (before, during and after application of the electroporation pulses to cells in suspension). The system is based on the design of adequate electroporation electrodes, compatible with a standard spectrofluorometer cuvette housing. The electric field intensity generated when pulses are applied was carefully characterized for different geometries of the electrodes, to choose a construction ensuring the greatest homogeneity of the field in combination with the best possible illumination of the sample. As an example of the method’s application, we present here generalized polarization kinetics for a varying number of electroporation pulses applied to a cell suspension; the general polarization parameter is strongly correlated to water presence in the hydrophobic membrane core. The system may be used for many other fluorescence measurements useful for the characterization of the electroporation process.

Article 34

Oxidative stress in androgenetic alopecia

BE Prie, L Iosif, I Tivig, I Stoian, C Giurcaneanu

Journal: Journal of Medicine and Life

Year: 2016

PMCID: PMC5152608

androgenetic alopecia, antioxidants, erythrocytes, oxidative stress, trolox equivalent antioxidant capacity

     Rationale:Androgenetic alopecia is not considered a life threatening disease but can have serious impacts on the patient’s psychosocial life. Genetic, hormonal, and environmental factors are considered responsible for the presence of androgenetic alopecia. Recent literature reports have proved the presence of inflammation and also of oxidative stress at the level of dermal papilla cells of patients with androgenetic alopecia Objective:We have considered of interest to measure the oxidative stress parameters in the blood of patients with androgenetic alopecia Methods and results:27 patients with androgenetic alopecia and 25 age-matched controls were enrolled in the study. Trolox Equivalent Antioxidant Capacity (TEAC), malondialdehyde (MDA) and total thiols levels were measured on plasma samples. Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) activities, and also non protein thiols levels together with TEAC activity were determined on erythrocytes samples No statistically significant changes were observed for TEAC erythrocytes, non-protein thiols, GPx and CAT activities. Significantly decreased (p<0.01) SOD activity was found in patients with androgenetic alopecia. For plasma samples decreased TEAC activity (p<0.001), increased MDA levels (p<0.001) and no change in total thiols concentration were found in patients when compared with the controls. Discussions:Decreased total antioxidant activity and increased MDA levels found in plasma samples of patients with androgenetic alopecia are indicators of oxidative stress presence in these patients. Significantly decreased SOD activity but no change in catalase, glutathione peroxidase, non protein thiols level and total antioxidant activity in erythrocytes are elements which suggest the presence of a compensatory mechanism for SOD dysfunction in red blood cells of patients with androgenetic alopecia.

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