Intracellular delivery of trehalose renders mesenchymal stromal cells viable and immunomodulatory competent after cryopreservation

Cytotechnology - Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible...     

Effect of Blood Vessel Segmentation on the Outcome of Electroporation-Based Treatments of Liver Tumors

Electroporation-based treatments rely on increasing the permeability of the cell membrane by high voltage electric pulses applied to tissue via electrodes. To ensure that the whole tumor is covered with sufficiently high electric field, accurate numerical models are built based on individual patient anatomy. Extraction of patient''s anatomy through segmentation of medical images inevitably produces some errors. In order to ensure the robustness of treatment planning, it is necessary to evaluate the potential effect of such errors on the electric field distribution. In this work we focus on determining the effect of errors in automatic segmentation of hepatic vessels on the electric field distribution in electroporation-based treatments in the liver. First, a numerical analysis was performed on a simple ''sphere and cylinder'' model for tumors and vessels of different sizes and relative positions. Second, an analysis of two models extracted from medical images of real patients in which we introduced variations of an error of the automatic vessel segmentation method was performed. The results obtained from a simple model indicate that ignoring the vessels when calculating the electric field distribution can cause insufficient coverage of the tumor with electric fields. Results of this study indicate that this effect happens for small (10 mm) and medium-sized (30 mm) tumors, especially in the absence of a central electrode inserted in the tumor. The results obtained from the real-case models also show higher negative impact of automatic vessel segmentation errors on the electric field distribution when the central electrode is absent. However, the average error of the automatic vessel segmentation did not have an impact on the electric field distribution if the central electrode was present. This suggests the algorithm is robust enough to be used in creating a model for treatment parameter optimization, but with a central electrode.     

Dual-porosity model of solute diffusion in biological tissue modified by electroporation

In many electroporation applications mass transport in biological tissue is of primary concern. This paper presents a theoretical advancement in the field and gives some examples of model use in electroporation applications. The study focuses on post-treatment solute diffusion.     

Electroporator with automatic change of electric field direction improves gene electrotransfer <Emphasis Type="Italic">in-vitro</Emphasis>

Background  

Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses.     

Analysis and Comparison of Electrical Pulse Parameters for Gene Electrotransfer of Two Different Cell Lines

Knowledge of the parameters which influence the efficiency of gene electrotransfer has importance for practical implementation of electrotransfection for gene therapy as well as for better understanding of the underlying mechanism. The focus of this study was to analyze the differences in gene electrotransfer and membrane electropermeabilization between plated cells and cells in a suspension in two different cell lines (CHO and B16F1). Furthermore, we determined the viability and critical induced transmembrane voltage (ITV_c) for both cell lines. In plated cells we obtained relatively little difference in electropermeabilization and gene electrotransfection between CHO and B16F1 cells. However, significant differences between the two cell lines were observed in a suspension. CHO cells exhibited a much higher gene electrotransfection rate compared to B16F1 cells, whereas B16F1 cells reached maximum electropermeabilization at lower electric fields than CHO cells. Both in a suspension and on plated cells, CHO cells had a slightly better survival rate at higher electric fields than B16F1 cells. Calculation of ITV_c in a suspension showed that, for both electropermeabilization and gene electrotransfection, CHO cells have lower ITV_c than B16F1 cells. In all cases, ITV_c for electropermeabilization was lower than ITV_c for gene electrotransfer, which is in agreement with other studies. Our results show that there is a marked difference in the efficiency of gene electrotransfer between suspended and plated cells.     

Cell membrane fluidity related to electroporation and resealing

In this paper, we report the results of a systematic attempt to relate the intrinsic plasma membrane fluidity of three different cell lines to their electroporation behaviour, which consists of reversible and irreversible electroporation. Apart from electroporation behaviour of given cell lines the time course required for membrane resealing was determined in order to distinguish the effect of resealing time from the cell’s ability to survive given electric pulse parameters. Reversible, irreversible electroporation and membrane resealing were then related to cell membrane fluidity as determined by electron paramagnetic resonance spectroscopy and computer characterization of membrane domains. We found that cell membrane fluidity does not have significant effect on reversible electroporation although there is a tendency for the voltage required for reversible electroporation to increase with increased membrane fluidity. Cell membrane fluidity, however, may affect irreversible electroporation. Nevertheless, this effect, if present, is masked with different time courses of membrane resealing found for the different cell lines studied. The time course of cell membrane resealing itself could be related to the cell’s ability to survive.     

Assessing the radiological load on the environment in the middle Danube river basin on the basis of a study of the Kopački Rit Nature Park,Croatia

A study of the environmental radioactivity in the Kopa?ki Rit Nature Park, Croatia, is presented. This wildlife reserve is part of the Middle Danube River Basin, and it is exposed to various pollutants due to a number of human activities in the surroundings, where there is a nuclear power plant and also urban centres and areas of intense agricultural production. Results for the activity concentrations of soil and surface water samples do not indicate any elevated radioactivity level, which is confirmed by on-site measurements of ambient dose rate equivalent. An assessment of the radiological load on the local biota, carried out using the ERICA tool, implies an overall low radiological risk even if conservative values of the risk quotient are used. Therefore, human activities do not have a significant effect on the radiological load on the Kopa?ki Rit area. A similar conclusion might be made with regard to numerous similar environments in the Middle Danube River Basin.     

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by ΔΦ, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of ΔΦ on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of ΔΦ. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of ΔΦ.