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.
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•Electroporation synchronized with fluorescence and temperature measurements.•Lipid bilayer packing (as generalized polarization) changes due to electroporation.•ROS production and ...lipid peroxidation due to electroporation.•Fast lipid unpacking (thermal) after pulses delivery in higher conductivity buffers.•Slow lipid tightening (non-thermal) related to ROS production.
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.
BackgroundAge-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.MethodsBPEI-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.ResultsIncreasing 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.ConclusionsThe 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.
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.
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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.
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.
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
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.
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 fCO) 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. fCO 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' fCO was found in a domain of 2 to 105kHz, while the electroporated cells' fCO was in a domain of 5 to 350kHz, depending on the external media conductivities. At exterior conductivities above ~0.02S/m, fCO of electroporated cells became significantly higher compared to controls. Even though the possible contribution of membrane permittivity to explain the observed fCO 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.
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► Dielectrophoresis was employed to characterize the cell electroporation. ► Six electric and geometrical cell parameters were analyzed using a single shell model. ► Model indicates cytosol conductivity as major contributor for porated cell behavior. ► Cytosol conductivity is assumed linearly depending on external buffer conductivity.
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.
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.
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