Poster U14 - A Novel Acoustic Levitation Device for YAP Mechanotransduction

Introduction: With space travel and exploration becoming increasingly feasible for humans, there is a growing need to investigate the effects of low gravity environments on living organisms and humans. Current microgravity research methods for mechanobiology are minimal, preventing scientists from investigating the effects of microgravity on individual cells. Previously, low-cost technology enabling acoustic levitation of liquid droplets (TinyLev1) has been developed. Fluid currents present in levitated liquid droplets generate near microgravity environments2 and create opportunities to investigate the effects of microgravity on cells. Before utilizing the acoustic levitator TinyLev to investigate microgravity effects on morphology and F-actin structures in mouse mesenchymal stem cells, protective cases as well as a controlled method for loading liquid droplets into TinyLev were developed.

Materials and

Methods: Cell media droplets containing C3H10T1/2 murine MSCs (ATCC, CCL-226) were levitated inside TinyLev. Droplet levitation times were controlled by the researcher and varied between 0 and 30 minutes. Post levitation, cells were seeded on the Poly-L-Lysine (PLL) coated coverslips and incubated for 18 hours. Anti-YAP and Alexa Fluor 488-conjugated secondary antibodies were added to enable YAP viewing. F-actin was stained with rhodamine phalloidin and nuclei were stained with DAPI. Processed cells were imaged with a Zeiss LSM800 confocal microscope. Nuclear morphology and yes-associated-protein concentrations were analyzed using FIJI (ImageJ). An ANOVA coupled with a Tukey test was used to statistically analyze the measurements.

Results, Conclusions, and Discussions: It was found that levitation significantly affected nucleus size and YAP nuclear translocation. Qualitative changes in actin structures were observed. While the actin structures displayed in the confocal microscope images have yet to be quantitatively analyzed, they do suggest that levitation in TinyLev may result in cytoskeletal structural changes in the cell. YAP nuclear translocation and F-actin cytoskeletal changes have been shown to be connected to mesenchymal stem cell osteogenesis3. While in space, humans can experience microgravity environments that alter osteogenic homeostasis, leading to decreased bone density and strength. The connection between microgravity and decreased bone density can be further studied to better inform future space travel decisions. These findings also demonstrate the potential of current acoustic levitation technology for use in mechanobiology research and beyond. Future work will be done to lengthen the time that cells can continuously spend in TinyLev as well as investigate additional research areas where TinyLev could be utilized.

Acknowledgements (Optional): We thank the UCARE program at UNL as well as NE DHHS Stem Cell Research Project (2023-04).