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.

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.

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.

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.

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.

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.

Controlling drug release from mesoporous silica through an amorphous, nanoconfined 1-tetradecanol layer.

Mesoporous silica materials are promising nano-carriers for drug delivery systems. Even though there are many strategies for controlling the drug release kinetics, these must be adapted through trial and error on a case-by-case basis. Here we explore the possibility of tailoring the release kinetics of hydrophilic, water soluble therapeutic agents from mesoporous silica through addition of a hydrophobic excipient, 1-tetradecanol. In vitro drug release experiments performed at 37 °C, in phosphate buffer solution (pH 7.4) show that the addition of tetradecanol yields slower drug release kinetics, which was correlated with the presence of a liquid fatty alcohol interfacial layer. The layer mass is 11-23 wt.% of the metoprolol-loaded silica sample, and it causes up to 1.6 times decrease of initial release rate with respect to materials without the fatty alcohol. This effect does not depend of carrier pore arrangement, being noticed for both hexagonal MCM-41 and cubic KIT-5 mesoporous silica. The toxicity of tetradecanol-containing materials was evaluated by formazan-based viability assay on Opossum kidney epithelial cell line, and no significant toxicity was observed.

Changes in optical properties of electroporated cells as revealed by digital holographic microscopy.

Changes in optical and shape-related characteristics of B16F10 cells after electroporation were investigated using digital holographic microscopy (DHM). Bipolar rectangular pulses specific for electrochemotherapy were used. Electroporation was performed in an "off-axis" DHM set-up without using exogenous markers. Two types of cell parameters were monitored seconds and minutes after pulse train application: parameters addressing a specifically defined area of the cell (refractive index and cell height) and global cell parameters (projected area, optical phase shift profile and dry mass). The biphasic behavior of cellular parameters was explained by water and mannitol dynamics through the electropermeabilized cell membrane.

Evaluation of the metastatic potential of malignant cells by image processing of digital holographic microscopy data.

The cell refractive index has been proposed as a putative cancer biomarker of great potential, being correlated with cell content and morphology, cell division rate and membrane permeability. We used digital holographic microscopy to compare the refractive index and dry mass density of two B16 murine melanoma sublines of different metastatic potential. Using statistical methods, the distribution of phase shifts within the reconstructed quantitative phase images was analyzed by the method of bimodality coefficients. The observed correlation of refractive index, dry mass density and bimodality profile with the metastatic potential of the cells was validated by real time impedance-based assay and clonogenic tests. We suggest that the refractive index and bimodality analysis of quantitative phase image histograms could be developed as optical biomarkers useful in label-free detection and quantitative evaluation of cell metastatic potential.

In vitro increase of the fluid-phase endocytosis induced by pulsed radiofrequency electromagnetic fields: importance of the electric field component.

Nowadays, due to the wide use of mobile phones, the possible biological effects of electromagnetic fields (EMF) become a public health general concern. Despite intensive research, there are no widely accepted theories about the interactions between EMFs and living cells, and the experimental data are often controversial. We examined the effects of mobile phones EMF (envelope frequency of 217 Hz, carrier frequency of 900 MHz and pulse duration of 580 micros) or its pure, low-frequency pulsed electric field component on fluid-phase endocytosis. In both cases, with exposures exceeding 10 min, an increase of the fluid-phase endocytosis rate was observed ( approximately 1.5-fold), on three different cell types. This increase is an all-or-nothing type of response that is occurring for threshold values comprised between 1.3 and 2.6 W/kg for the delivered EMF powers and between 1.1 and 1.5 V/cm for the electric fields intensities depending upon the cell type. The electric component of these EMFs is shown to be responsible for the observed increase. Variations of frequency or pulse duration of the electric pulses are shown to be without effect. Thus, EMF, via their electrical component, can perturb one of the most fundamental physiological functions of the cells-endocytosis.

Stretching of red blood cells using an electro-optics trap.

The stretching stiffness of Red Blood Cells (RBCs) was investigated using a combination of an AC dielectrophoretic apparatus and a single-beam optical tweezer. The experiments were performed at 10 MHz, a frequency high enough to avoid conductivity losses, but below the second turnover point between positive and negative dielectrophoresis. By measuring the geometrical parameters of single healthy human RBCs as a function of the applied voltage, the elastic modulus of RBCs was determined (µ = 1.80 ± 0.5 µN/m) and compared with similar values of the literature got by other techniques. The method is expected to be an easy-to-use, alternative tool to determine the mechano-elastic properties of living cells, and, on this basis, to distinguish healthy and diseased cells.

Hemorrhagic risk due to platelet dysfunction in myelodysplastic patients, correlations with anemia severity and iron overload.

Platelet function is influenced by changes in membrane fluidity that has an important role in the expression of platelet receptors and in modulating the activity of proteins like phospholipase C or proteinkinase C. In freshly prepared platelets, membrane fluidity modifies the aggregation/agglutination function. Reactive oxygen species (ROS) represent another important parameter involved in platelet receptor activation. There is a certain association of high levels of ROS and iron overload. Patients with hemochromatosis have low platelet aggregation induced by thrombin; little is known about the anemia and effects of iron overload on platelet activation in myelodysplastic syndromes (MDS) patients. Study of platelet membrane fluidity and ROS production changes in patients with MDS and possible correlations with altered platelet function as reflected in aggregation curves and platelet receptor expression. To find out possible correlations of fluidity of platelet membrane and ROS level with hematologic parameters and iron levels. The prospective study included 34 patients with myelodysplastic syndromes classified according to French-American-British cooperative group proposals and 29 healthy volunteers. Platelet membrane fluidity was quantified by fluorescence anisotropy measurements using the marker 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate. ROS production was evaluated by fluorescence measurements using 2',7'-dichlorodihydrofluorescein diacetate. Platelet function was analyzed by optical aggregometry using the agonists adenosine diphosphate, collagen, ristocetin and epinephrine. The expression of platelet receptors CD41/CD61, CD42a/CD42b and CD62P/CD63 was evaluated by flow cytometry. Platelet membrane fluidity in patients with MDS was similar to that of healthy volunteers and did not vary according to the risk category. Patients with MDS had increased platelet ROS production compared with the control group without statistical correlation with membrane fluidity. We found a negative correlation of ROS levels with the severity of anemia (R =  -0.587, P = 0.017). Platelet response was reduced in patients with MDS compared with volunteers, for all reagents. The response was different according to the risk category only in case of ristocetin or collagen. Patients with anemia presented a decreased platelet aggregation induced by collagen or ristocetin (collagen: R = 0.395, P = 0.003; ristocetin: R = 0.420, P = 0.002). The membrane fluidity of platelets from MDS patients appeared unmodified, but the ROS production was increased in all risk categories of MDS. The levels of ROS were correlated with the degree of anemia, which, in turn, had a negative impact on the platelet aggregation function induced by collagen or ristocetin.

Correlation of the intracellular reactive oxygen species levels with textural properties of functionalized mesostructured silica.

Mesostructured silica is frequently used in biomedical applications, being considered nontoxic and biocompatible material, suitable for the development of drug delivery systems (DDS). Four functionalized MCM-41 silica materials with hydrophobic (methyl and vinyl) and hydrophilic (3-aminopropyl and 3-mercaptopropyl) groups were obtained by post-synthesis functionalization and characterized by small-angle X-ray diffraction, infrared spectroscopy, thermal analysis, and nitrogen adsorption-desorption isotherms. The main structural and textural parameters of the obtained silica were determined. The effect of the functionalized silica on fibroblast (NIH3T3) and melanocyte cells (B16F10) was studied with respect to the proliferation rate and the levels of reactive oxygen species (ROS). It was found that the textural properties of all samples influenced the levels of intracellular ROS and consequently, the proliferation rate. Both, healthy and malignant cells exhibited linear dependence of ROS levels with the specific surface area values, but with different response. The contribution of the methyl functionalized silica to the ROS level is apart to the general trend.

Microscopic observation of living cells during their exposure to modulated electromagnetic fields.

Studying cell behaviour under irradiation with radiofrequency electromagnetic fields (RF-EMF) is often impeded by the difficulty to monitor cell characteristics during irradiation. Here we report the design and the application of a complete device for continuous microscopic observation of cells exposed to modulated EMF similar to mobile phones signals. The system allows the follow up of cell progression into mitosis under controlled temperature and CO(2) environment. Protocols are proposed in which the same cells are the controls before and after the EMF exposure and we demonstrate the interest of the "before exposure" controls. The exposure system was validated by cell endocytosis measurements. While the endocytosis rate was increased, no alteration of mitosis progression and mitosis duration was observed in cells exposed to 900 MHz modulated EMF for 1 h, at 30 degrees C and at a Specific Absorption Rate of 2.2 W/kg.