Sonoporation of cultured cells in the rotating tube exposure system

Suspensions of Chinese hamster ovary cells were exposed to ultrasound in the presence of fluorescent dextran to determine the conditions needed for sonoporation with uptake of the large molecules. Albunex, a gas-body- based ultrasound contrast agent, was added to enhance cavitation. Ultrasound was continuous wave at frequencies of 1.0, 1.68, 2.25, 3.3, 5.3, and 7.15 MHz. Sterile 4.5-mL exposure chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. After exposure, cells were tested for sonoporation by counting fluorescent cells and for cell lysis by counting cells stained by trypan blue. Sonoporation was a sensitive bioeffects indicator that was detected at pressure amplitudes lower than were needed for transient cavitation or cavitation-induced cell lysis. For 10% Albunex, apparent thresholds for sonoporation, which were comparable to the levels required to perturb the gas bodies, were 0.084 MPa (spatial peak negative pressure amplitude) from 1.0-3.3 MHz and 0.27 MPa at 5.3 and 7.15 MHz. Sonoporation decreased slightly if the tube was not rotated. The effects increased for increasing Albunex concentration (with rotation). The plating efficiency of cells exposed to 0.2 MPa at 2.25 MHz and sorted by a flow cytometer was 19% (3.6% standard deviation [SD]) for fluorescent cells, compared to 67% (1% SD) for nonfluorescent exposed cells and 62% (6% SD) for sham-exposed cells. The reduced viability represents an important consideration for possible applications of sonoporation.