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

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.

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

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.

Changes of cell electrical parameters induced by electroporation. A dielectrophoresis study.

Dielectrophoresis was employed to distinguish the electroporated from non-electroporated cells. It was found that the electric field frequency at which cells change the direction of their movement (the crossover frequency f(CO)) is higher when cells are electroporated. The contribution to the cell dielectrophoretic behavior of four electric and geometrical cell parameters was analyzed using a single shell model. f(CO) measurements were performed in media with conductivities of 0.001-0.09S/m, on B16F10 cells which were electroporated in a Mannitol solution (0.001S/m), using rectangular or exponential pulses. The control cells' f(CO) was found in a domain of 2 to 105 kHz, while the electroporated cells' f(CO) was in a domain of 5 to 350 kHz, depending on the external media conductivities. At exterior conductivities above ~0.02S/m, f(CO) of electroporated cells became significantly higher compared to controls. Even though the possible contribution of membrane permittivity to explain the observed f(CO) shift toward higher values cannot be excluded, the computations highlight the fact that the variation of cytosol conductivity might be the major contributor to the dielectrophoretic behavior change. Our experimental observations can be described by considering a linear dependence of electroporated cells' cytosol conductivity against external conductivity.

Cost-effective optimized method to process 3D tumoral spheroids in microwell arrays for immunohistochemistry analysis.

This study presents an improved method for obtaining spheroids microwell arrays for histological processing and analysis, focusing on glioblastoma (U87 MG) and breast adenocarcinoma (MCF-7) tumor models. By transitioning from traditional 2D cell cultures to 3D systems, this approach overcomes the limitations of 2D cultures by more accurately replicating the tumor microenvironment. The method consists of producing homotypic and heterotypic spheroids using low-adherence agarose-coated wells, embedding these spheroids in agarose microwell arrays, and conducting immunohistochemistry (IHC) to analyze cellular and molecular profiles. Morphological analyses were performed using OrganoSeg software, and IHC staining confirmed marker expressions consistent with respective tumor types. The study details the workflow from 2D cell culture to IHC analysis, including agarose well coating, spheroid embedding, and IHC staining for markers such as EMA, p53, Ki-67, ER, PR, and HER2. Results demonstrated compact, round U87 MG spheroids and fibroblast-stabilized MCF-7 spheroids, with both types exhibiting specific marker expressions. This innovative approach significantly enhances the efficiency of producing and analyzing large volumes of spheroids, making it both quick and cost-effective. It offers a robust drug screening and cancer research platform, maintaining spheroid traceability even in bulk workflow conditions. Furthermore, this methodology supports advances in personalized medicine by providing a more physiologically relevant model than 2D cultures, which is crucial for investigating tumor behavior and therapeutic responses through IHC.

Early differentiation of mesenchymal stem cells is reflected in their dielectrophoretic behavior.

The therapeutic use of mesenchymal stem cells (MSCs) becomes more and more important due to their potential for cell replacement procedures as well as due to their immunomodulatory properties. However, protocols for MSCs differentiation can be lengthy and may result in incomplete or asynchronous differentiation. To ensure homogeneous populations for therapeutic purposes, it is crucial to develop protocols for separation of the different cell types after differentiation. In this article we show that, when MSCs start to differentiate towards adipogenic or osteogenic progenies, their dielectrophoretic behavior changes. The values of cell electric parameters which can be obtained by dielectrophoretic measurements (membrane permittivity, conductivity, and cytoplasm conductivity) change before the morphological features of differentiation become microscopically visible. We further demonstrate, by simulation, that these electric modifications make possible to separate cells in their early stages of differentiation by using the dielectrophoretic separation technique. A label free method which allows obtaining cultures of homogenously differentiated cells is thus offered.

Grading of glioma tumors using digital holographic microscopy.

Gliomas are the most common type of cerebral tumors; they occur with increasing incidence in the last decade and have a high rate of mortality. For efficient treatment, fast accurate diagnostic and grading of tumors are imperative. Presently, the grading of tumors is established by histopathological evaluation, which is a time-consuming procedure and relies on the pathologists' experience. Here we propose a supervised machine learning procedure for tumor grading which uses quantitative phase images of unstained tissue samples acquired by digital holographic microscopy. The algorithm is using an extensive set of statistical and texture parameters computed from these images. The procedure has been able to classify six classes of images (normal tissue and five glioma subtypes) and to distinguish between gliomas types from grades II to IV (with the highest sensitivity and specificity for grade II astrocytoma and grade III oligodendroglioma and very good scores in recognizing grade III anaplastic astrocytoma and grade IV glioblastoma). The procedure bolsters clinical diagnostic accuracy, offering a swift and reliable means of tumor characterization and grading, ultimately the enhancing treatment decision-making process.

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

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.

Mechanosensitive Ion Channels and Their Role in Cancer Cells.

Mechanical forces are an inherent element in the world around us. The effects of their action can be observed both on the macro and molecular levels. They can also play a prominent role in the tissues and cells of animals due to the presence of mechanosensitive ion channels (MIChs) such as the Piezo and TRP families. They are essential in many physiological processes in the human body. However, their role in pathology has also been observed. Recent discoveries have highlighted the relationship between these channels and the development of malignant tumors. Multiple studies have shown that MIChs mediate the proliferation, migration, and invasion of various cancer cells via various mechanisms. This could show MIChs as new potential biomarkers in cancer detection and prognosis and interesting therapeutic targets in modern oncology. Our paper is a review of the latest literature on the role of the Piezo1 and TRP families in the molecular mechanisms of carcinogenesis in different types of cancer.

Method for nanoparticles uptake evaluation based on double labeled fluorescent cells scanned in enhanced darkfield microscopy.

We present a method that integrates the standard imaging tools for locating and detecting unlabeled nanoparticles (NPs) with computational tools for partitioning cell volumes and NPs counting within specified regions to evaluate their internal traffic. The method uses enhanced dark field CytoViva optical system and combines 3D reconstructions of double fluorescently labeled cells with hyperspectral images. The method allows the partitioning of each cell image into four regions: nucleus, cytoplasm, and two neighboring shells, as well as investigations across thin layers adjacent to the plasma membrane. MATLAB scripts were developed to process the images and to localize NPs in each region. Specific parameters were computed to assess the uptake efficiency: regional densities of NPs, flow densities, relative accumulation indices, and uptake ratios. The results of the method are in line with biochemical analyses. It was shown that a sort of saturation limit for intracellular NPs density is reached at high extracellular NPs concentrations. Higher NPs densities were found in the proximity of the plasma membranes. A decrease of the cell viability with increasing extracellular NPs concentration was observed and explained the negative correlation of the cell eccentricity with NPs number.

Evaluation of intracellular distribution of folate functionalized silica nanoparticles using fluorescence and hyperspectral enhanced dark field microscopy.

Using nanoparticles as carriers for drug delivery systems has become a widely applied strategy in therapeutics and diagnostics. However, the pattern of their intracellular distribution is yet to be clarified. Here we present an in vitro study on the incorporation of mesoporous silica nanoparticles conjugated with folate and loaded with a cytotoxic drug, Irinotecan. The nanoparticles count and distribution within the cell frame were evaluated by means of enhanced dark field microscopy combined with hyperspectral imagery and 3D reconstructions from double-labeled fluorescent samples. An original post-processing procedure was developed to emphasize the nanoparticles' localization in 3D reconstruction of cellular compartments. By these means, it has been shown that the conjugation of mesoporous silica nanoparticles with folate increases the efficiency of nanoparticles entering the cell and their preferential localization in the close vicinity of the nucleus. As revealed by metabolic viability assays, the nanoparticles functionalized with folate enhance the cytotoxic efficiency of Irinotecan.

Facile synthesis of low toxicity iron oxide/TiO2 nanocomposites with hyperthermic and photo-oxidation properties.

The present study aimed to assess the feasibility of developing low-cost multipurpose iron oxide/TiO2 nanocomposites (NCs) for use in combined antitumor therapies and water treatment applications. Larger size (≈ 100 nm) iron oxide nanoparticles (IONPs) formed magnetic core-TiO2 shell structures at high Fe/Ti ratios and solid dispersions of IONPs embedded in TiO2 matrices when the Fe/Ti ratio was low. When the size of the iron phase was comparable to the size of the crystallized TiO2 nanoparticles (≈ 10 nm), the obtained nanocomposites consisted of randomly mixed aggregates of TiO2 and IONPs. The best inductive heating and ROS photogeneration properties were shown by the NCs synthesized at 400 °C which contained the minimum amount of α-Fe2O3 and sufficiently crystallized anatase TiO2. Their cytocompatibility was assessed on cultured human and murine fibroblast cells and analyzed in relation to the adsorption of bovine serum albumin from the culture medium onto their surface. The tested nanocomposites showed excellent cytocompatibility to human fibroblast cells. The results also indicated that the environment (i.e. phosphate buffer or culture medium) used to disperse the nanomaterials prior to performing the viability tests can have a significant impact on their cytotoxicity.

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

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.

Digital holographic microscopy evaluation of dynamic cell response to electroporation.

Phase-derived parameters and time autocorrelation functions were used to analyze the behavior of murine B16 cells exposed to different amplitudes of electroporation pulses. Cells were observed using an off-axis digital holographic microscope equipped with a fast camera. Series of quantitative phase images of cells were reconstructed and further processed using MATLAB codes. Projected area, dry mass density, and entropy proved to be predictors for permeabilized cells that swell or collapse. Autocorrelation functions of phase fluctuations in different regions of the cell showed a good correlation with the local effectiveness of permeabilization.

Lutein Treatment Effects on the Redox Status and Metalloproteinase-9 (MMP-9) in Oral Cancer Squamous Cells-Are There Therapeutical Hopes?

Carotenoids loaded in nanoparticles should be regarded as a promising way to increase the availability in healthy cells and to induce apoptosis in cancer. Lutein is a carotenoid that, in contrast to beta-carotene, has no known toxicities. Oral cancer represents one of the most frequent types of cancer world-wide with an incidence rate of about 9% of all types of cancer. Almost 95% of all oral cancers are represented by squamous cell carcinomas (OSCC). The aim of this study was to review and analyse the effects of lutein and Poly(d,l-lactide-co-glycolide) (PLGA) Nps containing lutein (Lut Nps) on oxidative stress biomarkers (OXSR-1, FOXO-3, TAC) and collagen degradation biomarker-MMP-9, in human cells BICR10 of buccal mucosa squamous carcinoma. Lut Nps were prepared by the emulsion-solvent evaporation method. MMP, OXSR-1, TAC, FOXO-3 and MMP-9 were measured in tumour cell lysates by the ELISA technique. Our results have shown that in Lut 100 cells and Lut Nps the OXSR1 (p < 0.001, p < 0.001) and TAC (p < 0.001, p < 0.001) values were significantly higher than in control cells. The Lut 100 and Lut Nps FOXO-3 levels revealed no significant differences versus the control. MMP-9 levels were significantly reduced (p < 0.001) in the Lut Nps cells versus control cells. In our study conditions, lutein and lutein Nps did not trigger an oxidative stress by ROS induction. However, lutein Nps treatment seemed to have a positive effect, by downregulating the MMP-9 levels. Loaded in Nps, lutein could be regarded as a protective factor against local invasiveness, in whose molecular landscape MMPs, and especially MMP-9 are the main actors.

Effects of Ibrutinib on biophysical parameters of platelet in patients with chronic lymphocytic leukaemia.

Patients with chronic lymphocytic leukemia (CLL) treated with Ibrutinib often present hemorrhagic complications. Platelets dysfunction is well documented by aggregometry and flow cytometry, but the mechanisms by which Ibrutinib treatment influences the platelets status is yet to be evaluated. The aim of this study is to identify platelet membrane parameters in chronic lymphocytic leukemia (CLL) that could be altered by Ibrutinib administration. In this paper we propose a set of fluorescence measurements of the following parameters: membrane fluidity, resting membrane potential, and reactive oxygen species production of platelets suspensions obtained from CLL patients treated or not with Ibrutinib as markers for platelets status in this pathological situation. Platelets from CLL patients treated with Ibrutinib have higher membrane fluidity, lower resting membrane potential and higher level of reactive oxygen species production compared to the untreated CLL patients. These patients are also presenting higher membrane fluidity and lower resting membrane potential compared to healthy volunteers.

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

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.

Effect of Nanoconfinement of Polyphenolic Extract from Grape Pomace into Functionalized Mesoporous Silica on Its Biocompatibility and Radical Scavenging Activity.

The aim of this paper is to assess the properties of Mamaia (MM) grape pomace polyphenolic extract loaded onto pristine and functionalized MCM-41 mesoporous silica as potential ingredients for nutraceuticals or cosmetics. The chemical profile of hydroalcoholic polyphenolic extracts, prepared either by conventional extraction or microwave-assisted method, was analyzed by reverse-phase high-performance liquid chromatography with photodiode array detector (HPLC-PDA) analysis, while their radical scavenger activity (RSA) was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl radical) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assays. The extract-loaded materials were characterized by Fourier transform infrared (FTIR) spectroscopy, N2 adsorption-desorption isotherms, thermogravimetric analysis, as well as RSA (DPPH and ABTS assays). The polyphenols release profiles from pristine and functionalized (with mercaptopropyl, propyl sulfonic acid, cyanoethyl and propionic acid moieties) MCM-41-type supports were determined in phosphate buffer solution (PBS) pH 5.7. For selected materials containing embedded phytochemicals, cellular viability, and oxidative stress level on immortalized mouse embryonic fibroblast cell line (NIH3T3) were evaluated. A more acidic functional groups linked on silica pore walls determined a higher amount of phytochemicals released in PBS. The extract-loaded materials showed a good cytocompatibility on tested concentrations. The embedded extract preserved better the RSA over time than the free extract. The polyphenols-loaded MCM-41-type silica materials, especially MM@MCM-COOH material, demonstrated a good in vitro antioxidant effect on NIH3T3 cells, being potential candidates for nutraceutical or cosmetic formulations.

900 MHz modulated electromagnetic fields accelerate the clathrin-mediated endocytosis pathway.

We report new data regarding the molecular mechanisms of GSM-induced increase of cell endocytosis rate. Even though endocytosis represents an important physical and biological event for cell physiology, studies on modulated electromagnetic fields (EMF) effects on this process are scarce. In a previous article, we showed that fluid phase endocytosis rate increases when cultured cells are exposed to 900 MHz EMF similar to mobile phones' modulated GSM signals (217 Hz repetition frequency, 576 micros pulse width) and to electric pulses similar to the GSM electrical component. Trying to distinguish the mechanisms sustaining this endocytosis stimulation, we exposed murine melanoma cells to Lucifer Yellow (LY) or to GSM-EMF/electric pulses in the presence of drugs inhibiting the clathrin- or the caveolin-dependent endocytosis. Experiments were performed at a specific absorption rate (SAR) of 3.2 W/kg in a wire patch cell under homogeneously distributed EMF field and controlled temperature (in the range of 28.5-29.5 degrees C). Thus, the observed increase in LY uptake was not a thermal effect. Chlorpromazine and ethanol, but not Filipin, inhibited this increase. Therefore, the clathrin-dependent endocytosis is stimulated by the GSM-EMF, suggesting that the cellular mechanism affected by the modulated EMF involves vesicles that detach from the cell membrane, mainly clathrin-coated vesicles.

Combined microwave and electron beam exposure facilities for medical studies and applications.

The paper presents two radiation exposure facilities (REFs) which permit separate and simultaneous irradiation with microwaves (MW) of 2.45 GHz and electron beams (EB) of 6.23 MeV for malignant melanoma (MM) cell investigations, in vitro (MW+EB-REF-vitro) and in vivo (MW+EB-REF-vivo). The REFs are specifically designed for the following medical studies: 1) The effects of separate and combined (successive and simultaneous) MW and EB irradiation on the B16F10 mouse--MM cell cultures without/with drugs incubation, 2) The effects of separate and combined MW and EB irradiation on human blood components irradiated in samples of integral blood from healthy donors and from donors with MM; 3) The effects of separate and combined MW and EB whole body irradiation on the C57 BL/6 mice bearing MM without/with drugs administration. Several representative results obtained by experiments with REFs in vitro and in vivo are discussed. The most important conclusion of the experimental results is that low dose-total body MW+EB irradiation combined with drugs administration could present a valuable potential for an advanced study in malignant melanoma therapy.

Cell investigations simultaneously with exposure to 2.45 GHz microwaves.

The paper presents two microwave (MW) exposure systems (MWESs) that permit observations and measurements on cell cultures during their exposure to MW of 2.45 GHz: MWES-1 and MWES-2. MWES-1 is designed for the measurement of the cell membrane fluorescence anisotropies (MFA) simultaneously with MW exposure. MWES-2 is designed for the cells culture exploration under an inverted microscope before, during and after MW exposure. MWES-1 consists mainly of a 2.45 GHz microwave generator (MWG-2.45 GHz-SAIREM) of 0-25 W, equipped with forward power and reflected power displaying, and an adjustable coaxial antenna immersed directly into the cuvette with the cells-suspension of a Spex type spectrofluorometer. The MW effect on membrane fluidity of B16F10 malignant melanoma (B16F10-MM) cells in suspension were investigated with MWES-1, by MFA measurements. We observed a MW induced transition temperature (ITT) rising strongly during the MW exposure as compared with ITT obtained by classical heating (CH). The MWES-2 consists of the MWG-2.45 GHz-SAIREM generator and a rectangular waveguide applicator with traveling wave placed between the condenser and the objective of a Zeiss Axiovert 200 microscope, equipped with a fluorescence device and image acquisition. The MW effects on shape and apoptosis of the B16F10-MM cells were investigate with MWES-2. The B16F10-MM cells exhibited visible shape changes during MW exposure up to 37 degrees C. The MW exposure induced cells apoptosis/necrosis in several seconds after that MW are applied, beginning with SAR = 1.5 W/sample, compared to CH controls exposed at the same temperature dynamics.