Film-based Cell Culture Device and In Vitro Setup for Ultrasound Modulation.

Conventional ultrasound in vitro systems, such as petri dishes and well plates often introduce acoustic reflections and pressure accumulation, compromising the reliability and reproducibility of experimental results. Custom sonication vessels and setups, while addressing some of these issues, often involve complicated assembly processes and can be compatible only with specific experimental setups. To address these limitations, we developed an easy-to-use 3D-printed device that utilizes parafilm on the top and bottom, enabling the device chamber to be optimized for quick assembly, contamination prevention and ultrasound wave propagation. Pressure field mapping with a needle-hydrophone confirmed a predictable ultrasound pressure distribution within the device. In addition to parafilm, Mylar and polystyrene films were tested showing minimal interference when measuring the pressure field. Rat-derived primary astrocytes and microglial cells, as well as immortalized human embryonic kidney-derived HEK293t cells, were cultured directly onto pre-coated Mylar films, which exhibit superior optical and acoustic transparency. These cell types were selected due to their wide range of potential applications, especially in the emerging field of ultrasound modulation and sonogenetics. Cell viability was assessed using trypan blue exclusion and the results demonstrate the feasibility of seeding cells onto Mylar film. The device maintained sterility with no leakage, confirming its efficacy and reliability for cell culture experiments. This novel 3D-printed device provides more control over the ultrasound parameters delivered to cells. Its adaptable design supports flexible modifications, allowing researchers to tailor it to specific experimental needs, thereby improving the accuracy and reproducibility of in vitro ultrasound modulation studies.