Estudo sobre a eletroporação de células biológicas com pulsos de curta duração e alta intensidade

Abstract

The aim of this work was to build an electroporator of biological cells, capable of generating pulses from 2$\mu$s to 100$\mu$s in duration and amplitudes ranging from 100V to 900V, to perform cell viability experiments and evaluate the effect of these pulses in the entry of fluorescent molecules in LLC-MK2 cells. The electroporator consists of three modules: a high voltage d.c. generator, a control module and a high voltage switching module. The high voltage generator was built with capacitor voltage doublers, the control module uses a PIC18F4550 and drivers to control power semiconductor devices and the high voltage switching module was built with capacitors and a power IGBT. The electroporator circuit was used to generate 1kV/cm to 9kV/cm pulses with 100$\mu$s duration and 2$\mu$s to 100$\mu$s pulses with 9kV/cm amplitude in viability and electroporation tests. Electroporation cuvettes with 1mm distance between the electrodes were used to submit a suspension of LLC-MK2 cells to the pulses. Viability tests were made using the MTT colorimetric technique. In the electroporation tests, flow cytometry was used to evaluate the ammount of propidium iodide and FITC-Dextran into the cells. Cells were submited to the pulses in culture medium with the fluorescent molecules previously added. There was also a test in which cells were submited to the pulses in medium without the fluorescent molecules and propidium iodide was added 30 minutes after the pulses. The electroporator was capable of generating pulses that significantly changed the fractions of permeable or dead cells with satisfatory performance. In the viability tests, it was observed that 100$\mu$s pulses with amplitudes of 5kV/cm or 9kV/cm and 9kV/cm pulses with duration of 50$\mu$s decreased the cell viability up to 20$\%$, revealing that these pulses are aggressive to the type of cell used. The proportion of labeled cells with propidium iodide previously added to the medium reached 80$\%$ for 100$\mu$s and 9kV/cm pulses. The proportion was smaller for FITC-Dextran, reaching 20$\%$ to 30$\%$ for the same kind of pulse, with no statistical difference between sizes of 20kDa and 2MDa. After an interval of 30 minutes, cells kept permeable to propidium iodide with a drecrease in labeled cell proportion of 25$\%$ to 30$\%$ compared to the cells submited to the pulses with propidium iodide prevously added to the cell suspension. The lack of statistical significance of this 30 minutes interval effect for 100$\mu$s and 9kV/cm pulses indicates that these have a longer effect. The increase of amplitude for 100$\mu$s pulses decreased cell viability and increased the proportion of cells labeled with fluorescent molecules. The increase in pulse duration for 9kV/cm pulses decreased cell viability, inctreased the proportion of cells labeled with propidium iodide but had no effect over the proportion of cells labeled with FITC-Dextran. The proportion of labeled cells were statistically similar for the two sizes of FITC-Dextran, and different of the proportion of cells lebeled with propidium iodide. It was observed that the pulses used in this work greatly decrease the cell viability or are inefficient to allow the entrance of molecules into the cytoplasm. A low proportion of cells labeled with FITC-Dextran was obtained. Therefore, the pulses used in this work would be indicated for applications in which a high cell death associated with the insertion of small molecules in the cytoplasm is desired.