Cell counting tool parameters optimization approach for electroporation efficiency determination of attached cells in phase contrast images.

In this paper a novel parameter optimization approach for cell detection tool and counting cells procedure in phase contrast images are presented. Manual counting of the attached cells in phase contrast images is time-consuming and subjective. For evaluation of electroporation efficiency of attached cells, we often perform manual counting of the cells which is needed to determine the percentage of electroporated cells under different experimental conditions. Here we present an automated cell counting procedure based on novel artificial neural network optimization of Image-based Tool for Counting Nuclei algorithm parameters to fit the training image set based on counts from an expert. Comparing the results of automated cell counting to user manual counting a 90,31% average agreement was achieved which is reasonably good especially taking into account inter-person error which can be up to 10%. Even more, our procedure can also be used for fluorescent cell images with similar counting accuracy (>90%) enabling us to determine electroporation efficiency. In our experiments, the electroporation efficiency determined by manual cell counting was virtually the same as the one obtained by the automated procedure.

The role of electrophoresis in gene electrotransfer.

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LV + HV and HV + LV pulses. We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced for HV + LV, while for the reversed sequence, LV + HV, there was only a minor effect of the LV pulse. For the highest plasmid concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect.

The temperature effect during pulse application on cell membrane fluidity and permeabilization.

Cell membrane permeabilization is caused by the application of high intensity electric pulses of short duration. The extent of cell membrane permeabilization depends on electric pulse parameters, characteristics of the electropermeabilization media and properties of cells exposed to electric pulses. In the present study, the temperature effect during pulse application on cell membrane fluidity and permeabilization was determined in two different cell lines: V-79 and B16F-1. While cell membrane fluidity was determined by electron paramagnetic resonance (EPR) method, the cell membrane electropermeabilization was determined by uptake of bleomycin and clonogenic assay. A train of eight rectangular pulses with the amplitude of 500 V/cm, 700 V/cm and 900 V/cm in the duration of 100 micros and with repetition frequency 1 Hz was applied. Immediately after the pulse application, 50 microl droplet of cell suspension was maintained at room temperature in order to allow cell membrane resealing. The cells were then plated for clonogenic assay. The main finding of this study is that the chilling of cell suspension from physiological temperature (of 37 degrees C) to 4 degrees C has significant effect on cell membrane electropermeabilization, leading to lower percent of cell membrane permeabilization. The differences are most pronounced when cells are exposed to electric pulse amplitude of 900 V/cm. At the same time with the decreasing of temperature, the cell membranes become less fluid, with higher order parameters in all three types of domains and higher proportion of domain with highest order parameter. Our results indicate that cell membrane fluidity and domain structure influence the electropermeabilization of cells, however it seems that some other factors may have contributing role.

Shape transformation of giant phospholipid vesicles at high concentrations of C12E8.

Giant unilamellar phospholipid vesicles were prepared by the method of electroformation from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). We studied the influence of different concentrations of the surfactant octaethyleneglycol dodecylether (C(12)E(8)) on the spontaneous shape transformations of POPC vesicles at room temperature. In accordance with previous results, we observed that low concentration of C(12)E(8) increased the speed of the characteristic vesicle shape transformation, starting from the initial shape with thin tubular protrusion, through beaded protrusion where the number of beads gradually decreased, to final spherical shapes with invagination, whereby the average mean curvature of the vesicle membrane monotonously decreased. In contrast, higher concentration of C(12)E(8) initially induced the shape transformation in the "opposite direction": in the protrusion, the number of beads gradually increased and eventually a tube was formed whereby the average mean curvature of the vesicle membrane gradually increased. However, at a certain point, an abrupt shape change took place to yield the vesicle with invagination. In this transition, the average mean curvature of the vesicle membrane discontinuously decreased. After this transition, the vesicle began to shrink and finally disappeared. We discuss possible mechanisms involved in the observed transformations.

The influence of medium conductivity on electropermeabilization and survival of cells in vitro.

Electropermeabilization and cell death caused by the exposure to high voltage electric pulses depends on the parameters of pulses, as well as the composition of the extracellular medium. We studied the influence of extracellular conductivity on electropermeabilization and survival of cells in vitro. For this purpose, we used a physiological medium with a conductivity of 1.6 S/m and three artificial media with conductivities of 0.14, 0.005, and 0.001 S/m. Measurements of pH, osmolarity, and cell diameter were made to estimate possible side effects of the media on the cells. Our study shows that the percentage of surviving cells increases with the decreasing medium conductivity, while the percentage of electropermeabilized cells remains unaffected. Our results show that cell survival in experiments involving electropermeabilization can be improved by decreasing the medium conductivity. To provide an interpretation of experimental results, we have theoretically estimated the resting transmembrane voltage, the induced transmembrane voltage, the time constant of the voltage inducement, and heating of the cell suspension for each of the media used. These calculations imply that for accurate interpretation of experimental results, both the induced and the resting transmembrane voltage must be considered, taking into account the conductivity and the ionic composition of the extracellular medium.

Model-based automated detection of mammalian cell colonies.

Manually counting cell colonies, especially those that originate from fibroblast cell lines, is a time-consuming, eye-straining and tedious task in which consistency of counting is difficult to maintain. In this paper we present a novel model-based image segmentation method, which employs prior knowledge about the shape of a colony with the aim to automatically detect isolated, touching and overlapping cell colonies of various sizes and intensities. First, a set of hypothetical model instances is generated by using a robust statistical approach to estimate the model parameters and a novel confidence measure to quantify the difference between a model instance and the underlying image. Second, the model instances matching the individual colonies in the image are selected from the set by a minimum description length principle. The procedure was applied to images of Chinese hamster lung fibroblast cell line DC3F, which forms poorly defined or 'fuzzy' colonies. The correlation with manual counting was determined and the cell survival curves obtained by automated and manual counting were compared. The results obtained show that the proposed automatic procedure was capable to correctly identify 91% of cell colonies typical of mammalian cell lines.