Browsing by Author "Caldeira, Viviana Modesto"
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- Effect of pulsed electric fields on cell viability and metabolismPublication . Caldeira, Viviana Modesto; Redondo, Luís; Calado, Cecília Ribeiro da CruzABSTRACT - Pulsed Power (PP) is used in industry and biotechnology research due to its high voltage singularities. In biological systems, electroporation based on Pulsed Electric Fields (PEF) is an effective way of energy delivery, allowing controlled pulses with specific electrical parameters. Electroporation is the name given to the phenomenon where occurs permeabilization of the plasma membrane and this process could be reversible or irreversible. PEF on mammalian cells promotes diverse biological processes based on non-ligand agonists, that bypass receptors at the plasma membrane while inducting diverse intracellular signalling pathways. The final biological effect will depend on the quantity and type of the targeted cells and PEF conditions and can be as diverse as the enhancement of cells proliferation capacity or in opposition, cell death. The main goal of this experimental work, based on in vitro techniques using a fibroblast L929 cell line, was to evaluate three different conditions under PEF applications that may influence fibroblast metabolism. Firstly, the impact of electric fields magnitude (between 10 and 15kV/cm) with different pulses, secondly the effect of different fibroblasts concentration on the assay (between 50,000 to 1500 x103 cells per 800μL of Dulbecco’s Phosphate-Buffered Saline - DPBS) and finally to evaluate the effect of repeated PEF applications (between 1 to 3 repetitions) on cells viability and proliferative capacity. To achieve the latter, three experiments were designed. The first experiment evaluated the impact of PEF on cell viability immediately, at 48 hours, and at 72 hours after the assay. The second experiment studied the influence of cell density and its interference on the electric field applied, cell lysis, and on cell viability immediately and 72 hours after the assay. The third experiment entails three assays that assessed the weight of applying consecutively PEF with 48 hours intervals on different cell density samples and the maintenance of cell viability upon each application – immediately, at 48 hours, and 96 hours after the assay. The specific energy (Ws) and temperature increase (ΔT) of each biological sample were calculated after each experiment to guarantee that was no cell overheating. The DPBS used as a conductive solution, the cell pellet and the culture medium of all samples after PEF application were submitted to Fourier Transformed Infrared (FTIR) spectroscopy, the data obtained was then processed by Unscrumbler X software and submitted to Principal Component Analysis (PCA). Regarding cell count and cell viability common statistical analysis (average and standard deviation) and Analysis of Variance – ANOVA – single factor evaluation was performed. The data obtained demonstrates that a high electric field (15kV/cm with 1 pulse) promoted almost whole cell lysis, representing non-thermal irreversible electroporation (NTIRE), whereas the lowest electric field (10kV/cm with 2 pulses or 10 pulses) did not affect with statical significance cell viability. In addition, the lowest field with 5 pulses (10kV/cm with 5 pulses) was the most promising since the cells showed acceptable FTIR spectroscopy signals without major effects on initial cell viability. Moreover, it was observed that larger cell densities during PEF application guarantee higher cell viability, being the ideal number between 1,500x103 and 500x103 cells per 800μL of the assay. Furthermore, the repeated application of PEF in fibroblasts affected cell viability, being acceptable for one or two repetitions with intervals not inferior to 48h between applications. According to statistical data the assays are verifiable and reproducible, but in the future, more control samples are needed. This work confirms the potential of PEF in mammalian cell research, since it avoids overheating in living matter, showing that further studies are needed, in this area.