High-frequency ultrasound (HFUS) signals backscattered from RBL-2H3 cell pellets prepared under different centrifugal forces were analyzed to investigate the packing effect of cell aggregates. The measurements were performed in a pulse-echo setup with a 40-MHz transducer. The changes of ultrasound signals from cell pellet in backscattered power, statistical parameter, and pellet thickness were monitored after centrifugation at between 100g and 1600g. Experimental results showed that the HFUS backscattered power from cell pellets was inversely proportional to centrifugal force and increased to a plateau within 1-2h after centrifugation. The initial thickness of cell pellets decreased with higher centrifugal force, but the changes in thickness and time that took to reach a plateau increased at higher centrifugal force. The envelope statistics of backscattered signals with Nakagami distribution indicates that the centrifugal force and elapsed time after centrifugation affected the backscattering characteristics. The present study suggests that centrifugal force and data acquisition time after cell pellet formation should be considered in in vitro cell packing method with centrifugation to emulate the tissue in vivo.

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