Page 90 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 4
Ultrasound and tMB treated cells demonstrated much higher SPIO uptake as shown in Figs 6C–G. Iron particles were detected as aggregates of different sizes in the cytoplasm (Figs 6C, 6D). Other typical individual examples of intracellular SPIO distribution patterns after ultrasound and tMB treatment are shown in Figs 6E-F. These staining patterns included distribution of aggregates varying in size and blue intensity throughout the cytoplasm (Fig 6E) and one large aggregate mainly located near the nucleus (Fig 6F) having a higher blue intensity in comparison to the aggregates in Fig 6E. The intensity differences of the blue stain suggest different concentrations of SPIO particles.
Discussion
Tracking of endothelial cells is important for cancer and cardiovascular disease. There are several ways of SPIO cell labeling in vitro [22, 54]. Most of these techniques require different transfection agents, which cannot be used in vivo due to the associated high toxicity and systemic effects. We therefore studied a technique, based on ultrasound-activated ultrasound contrast agents that will be compatible for in vivo use. The SPIO uptake was dependent on multiple factors, including the ultrasound settings, the time of SPIO addition, and the incubation time of SPIO with cells after the ultrasound treatment. Optimal labeling at 1 MHz ultrasound frequency was observed when the ultrasound parameters were 40 kPa peak negative pressure (MI 0.04), 10,000 cycles and repetition rate of 20 Hz, applied for 30 s when SPIO were added at 0 min. Compared to the control, this resulted in an approximate 12 times increase of SPIO uptake with 85% cell viability.
Microbubble dynamics
We found an increasing trend of both SPIO positive cells and cell death with the acoustic PNP increase. Note that we studied acoustic pressures up to 160 kPa PNP, a regime in which the amplitude of radial lipid-coated microbubble oscillations increases with pressure [55, 56]. A previous study by Vos et al. [57] has reported that highly non-spherical microbubble vibrations can be induced at pressures as low as 140 kPa PNP for lipid-coated microbubbles at resonance. In this regime, the acoustic streaming generated by oscillating microbubbles and the produced shear stresses [58-61] can be one of the mechanisms for
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