Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation.

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low-frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Highly controlled and usable system for Low-Intensity Pulsed Ultrasound Stimulation of Cells.

This work aims to describe the design and development of an in vitro highly controlled ultrasonic stimulation system able to guarantee, at the same time, high usability and full sterility of the tested samples. After creating the first prototype of an ultrasound-transparent three-chambers culture well, sealing tests were conducted to prove its impermeability to external contaminants and in vitro tests were carried out to verify the usability of this system for ultrasonic stimulation of cells in vitro. No statistically significant differences were found between control and tested samples during sealing tests, thus demonstrating optimal sealing ability towards external contaminants. Furthermore, the thin polystyrene membrane used to guarantee US-transparency guaranteed a good adhesion and viability of both human fibroblasts and induced pluripotent stem cells.