We are now developing an in vivo cell delivery system, which is realized by producing aggregates in which bubbles attach onto the surface of cells with acoustic radiation force. We confirmed the controllability of the aggregation of cells with bubbles compared with those without bubbles. However, because of the destruction of bubbles under continuous ultrasound exposure, there is a compatibility problem between controllability and duration of aggregation. Therefore, we introduced the use of a standing wave of an acoustic field to trap the aggregates in the nodes, in which the aggregates are not directly exposed to a high-pressure sound, and manupilation by varying the acoustic field. We prepared the travelling and oscillation motions of the ultrasound standing wave and investigated the mobility of aggregates that were trapped in the nodes of the standing wave to be translated two-dimensionally. Regarding travelling motion, we found that the node velocities of 10 and 20 µm/s showed better performance than that of 40 µm/s. Regarding oscillation motion, we succeeded in the continuous oscillation of aggregates for 36 s. Also, we found the problem of immobile cells, which was considered to be due to the destruction of bubbles.

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Although immunotherapy has been recognized as a new therapeutic method, there is a problem of low cell concentration because of cell dispersion in the bloodstream. We previously developed methods of active induction of cells applicable to future therapeutic methods by using aggregations of bubbles containing Colon-26 cells. However, Colon-26 cells are not appropriate for therapeutic applications. Furthermore, the controllability of aggregation under ultrasound exposure has been confirmed only in still water, not in a flow. Therefore, we utilized T-cell hybridoma (CD8-OVA1.3) and induced the aggregates of bubbles and cells by using acoustic radiation force with the motion of travelling nodes. We improved our observation system including its digital camera for the active induction of aggregates containing therapeutic cells in a flow under ultrasound exposure. We found that the induction performance of bubble-surrounded cells (BSCs) was enhanced with an ultrasound frequency of 5 MHz rather than 3 or 7 MHz. Furthermore, we found that a node velocity of 5 mm/s shows the best performance compared with 1 or 10 mm/s, at which the flow velocity was 10 mm/s. We believe that the parameters realized in this study indicate effective in vivo applications in the future.

We previously reported methods of the active control of the cells by forming bubble-surrounded cells (BSCs), in which bubbles were attached to the surface of the cells, to enhance the controllability of the cells under ultrasound exposure. However, mechanical or biological damage to the cells contained in BSCs according to the conditions of ultrasound exposure has not been clarified. Therefore, we carried out the viability validation of the cells in BSCs versus various conditions of ultrasound exposure. First, we verified the reliability of cell viability by introducing two methods of CCK-8 assay and lactate dehydrogenase assay. Then, we confirmed that the surrounded amount of bubbles decreases the cell viability under continuous wave exposure rather than burst wave. We optimized the parameters to produce BSCs with the concentration of bubbles of 0.3 mg ml −1 and the concentration of the cells of 1.0 × 10 5 ml −1 , to maintain the cell viability under ultrasound exposure. Also, using the maximum sound pressure lower than 300 kPa-pp, and low duty ratio less than 50%, the cell viability would be maintained more than 75% within the emission duration of 60 s.

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