Cells can become cancerous because of multiple gene mutations that regulate cellular proliferation and cellular death. Their excessive growth demands higher energy uptake when compared to healthy cells, this means that their metabolic rate is higher. The required energy comes from glycolysis, the metabolic pathway that converts glucose to pyruvate, in the form of ATP. Cancerous cells, that are in the vicinity of blood vessels, have access to molecular oxygen, which means that they can use the process of aerobic glycolysis. As cancerous tissue grows some of the cells lose the access to molecular oxygen, since the distance for oxygen diffusion becomes too big. That leads to anaerobic glycolysis, the process responsible for production of lactic acid, which in turn lowers the pH of the tissue.
In practice some cancers can be treated with electrochemotherapy, that allows local delivery of drugs into the cancerous tissue with electroporation. In this thesis we tested the effect of cell medium, with lowered pH, on the electroporation of cancer cells B16F1 in vitro. We changed the mediums pH by adding acetic acid and tested cell viability in mediums with pH 8,1 (normal medium without acetic acid), 7,45, 6,87, 6,47, 5,95, 5,33. Cells were exposed to these mediums for 24 hours. As we expected the cell viability was lower in mediums with lower pH. In the following experiments we tested the effect of an acidic medium (pH 6,72) on cell electroporation and cell recovery. We used the following parameters for electroporation: 8 voltage pulses (180 V (120 V/mm), 270 V (180 V/mm), 360 V (240 V/mm) and 450 V (300 V/mm)), with length of 100 µs and frequency of repetition 1 Hz. We carried out 4 combinations of experiments, where we electroporated cells in normal or acidic medium and let them recover for 10 minutes in normal or acidic medium. 24 hours after this process we tested the cell viability. The results showed that electroporation and recovery in acidic medium increases the cell viability compared to electroporation and recovery in normal medium. We have also done tests with transporting the propidium iodine into cells with electroporation, where electroporation and recovery period were done in the same combinations of mediums as in the previous experiments. We measured the presence of propidium iodine with flow cytometry. As was expected, the results showed that we need higher voltages for electroporation while in acidic medium, to get the same propidium iodine uptake as in normal medium.
|
