Abstract
Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes. In vitro 3D culture systems simulate the in vivo tumor growth and preserve the biological characteristics of tumors more accurately than conventional monolayer cell cultures. We describe a protocol for the development of 3D tumor organoids using conjunctival melanoma (CM) and uveal melanoma (UM) cell lines as well as the use of hand-held customized electrodes, suitable for in vitro ECT in the culture well without destruction of the tumor environment. This protocol analyzes the culture and growth of 3D CM and UM spheroids and their reaction to bleomycin (2.5 µg/mL) alone, electroporation (EP) (750 Volts/cm, 8 pulses, 100 µs, 5 Hz) alone, and ECT as a combination of EP and bleomycin. The drug concentration and the EP settings used in this protocol were established as preferred ECT conditions according to previous experiments. The assay used to determine the spheroid viability was conducted 3-7 days following treatment. The effect on viability and growth of the 3D tumor spheroids was significant only after ECT. The customized electrodes are described in detail in order to facilitate the application of pulses in the culture well. This novel treatment of 3D UM and CM spheroids sets a steppingstone for future clinical application.