Ultrasound-mediated devices are in rapid development to facilitate cellular trapping, manipulation and uptake of drugs in a non-invasive manner. However, the possible destructive effects that ultrasound and second-order factors i.e., cavitation, acoustic streaming, temperature and radiation forces have on the subjected (viable) cells are not fully known. A 1D ultrasonic device was designed and manufactured to explore particle and cell trapping using ultrasonic standing waves (USW). The operation and characterization of the ultrasonic device was carried out in order to obtain in a reliable cellular and microparticle trapping. Also, systematic optimisation studies were performed to evaluate cell viability and metabolic activity in a cancer cell line (HeLa) and in a healthy cell line Human Dermal Fibroblasts (HDF) as a function of time and applied voltage with a temperature regulator. The results demonstrate that high cell viability for both cell lines is achieved when the ultrasonic device is operated at the minimum trapping voltage while increasing in small steps 0.01 MHz the frequency to enhance the acoustic standing wave field at the trapping area. In addition, the optimised conditions to keep cell viability and metabolic activity close to control levels for both cell lines are 34°C with a driving voltage of 8 Vpp and acoustically exposed for 15 min. Furthermore, the mechanisms of particle uptake through ultrasound mediation are also not fully known, however, several studies suggest the involvement of a rearrangement of the cytoskeletal network as well as the particle internalization via endocytosis and transitional or lethal pore formation in the cell membrane. In this thesis, the dependence of cellular uptake depending on microparticle surface charge, employing the 1D custom-built ultrasonic device was investigated. Fluorescent carboxyl-polystyrene microspheres (~1μm) were coated with different formulations to obtain cationic, anionic and sugar-coated microspheres. The uptake of the conjugated microspheres by HeLa cells was evaluated using flow cytometry and, confocal fluorescence microscopy. Furthermore, transmission electron microscopy (TEM) was employed to visualize the cell’s plasma membrane integrity after particle internalization. It was found that the cellular uptake by ultrasound exposure occurs through the transient formation of plasma membrane pores which reseal after treatment. Microparticle uptake of cells takes place via macropinocytosis and all treatments maintained their viability with no presence of toxicity from the conjugated microparticles. In general, this thesis highlights the importance of characterizing ultrasonic devices and the development of systematic and reliable acoustic protocols. In addition, it provided evidence of the mechanisms that support ultrasound-mediated delivery of microparticles.
Ultrasonic standing wave device for cellular and drug delivery applications and multipolar mitosis present in hPSCs
Levario Diaz, V. (Author). 24 Jun 2021
Student thesis: Doctoral Thesis › Doctor of Philosophy (PhD)