Gemcitabine

For mEHT treatment, Lab-EHY 200 device (Oncotherm Kft, Budaors, Hungary) was used with all accessories customized for cell suspension treatment. The treatment bag containing 1.5 ml of 10⁶ cells/ml suspension was submerged into the treatment cuvette filled with distilled water. One temperature sensor was inserted directly inside the treatment bag and another one into the surrounding water. The treatments lasted for 65 min including 5–10 min preheating period at 10 W average input power, and 55–60 min maintaining period using 2.4 W average input power. The amplitude modulated electric field generated 42 ± 0.3°C inside the treatment bag.
For gemcitabine (Fresenius Kabi Oncology Plc., Hampshire, United States) treatment, a 1,000 µM stock solution was made in 0.1 M neutral phosphate-buffered saline (PBS) buffer. This stock solution was diluted in cell culture media to achieve the preferred concentration and added to the cells, or added right after the 60 min mEHT treatment when combined. Samples were collected and tested at 0, 24, 48 or 72 h from the start of any treatment. In the 60 min mEHT monotherapy group tumor cells were grown in normal culture media, until sampling. In the GEM treatment groups cells were grown in GEM containing media either from the beginning, or after 60 min mEHT, when the two treatments were combined (mEHT + GEM), for the rest of time.