Page 93 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

of 1.5 MHz, 440 kPa PNP, 300 cycles at 1 kHz repetition rate for 30 s versus 1 MHz, ≤160 kPa PNP, 10,000 cycles at 20 Hz for 30 s in our study) and/or the type of cells studied (C6 rat glioma cells versus HUVECs in our study). Recently, the temporal window was reported to be cell type-dependent by the same group [79]. For HUVECs, the temporal was found to be 1 h for the small molecule SYTOX green.
In our study, we defined a cell as SPIO positive when we detected one or more SPIO particles within the cell. It is unlikely that the SPIO particles we detected were extracellular because the cells were washed three times. We observed different labeling patterns, namely differences in the intensity and the intracellular distribution of the SPIO particles, although we did not quantify this degree of uptake. Some cells took up small spots (submicron) of SPIO as dense granules ~2 nm in diameter, while others had large SPIO aggregates in the cytoplasm. This variation in uptake patterns may also suggest uptake by pore formation and endocytosis as SPIO homogeneously distributed in the cytoplasm may suggest pore formation while small aggregates may indicate endocytic uptake. This is supported by different uptake patterns previously reported for dextrans with aggregates verified to be co-localized with endocytic vesicles [46, 47].
As ultrasound contrast agent-mediated SPIO-labeling of endothelial cells is likely faster clinically approved for labeling tumor vasculature, we decided to mimic the tumor vasculature’s compromised endothelial monolayer with poorly connected and sprouting endothelial cells [80] by culturing the HUVECs till 70% confluence. Our findings of effective tMB-mediated SPIO-labeling may therefore not be applicable to a monolayer of HUVECs, i.e. 100% confluence, applicable to vascular grafts. Different ultrasound pressures may be needed for effective tMB-mediated SPIO-labeling HUVECs in a monolayer as cells in a monolayer are more likely in the senescent cell cycle phase (G0) and have a more organized cytoskeleton. With respect to sensitivity to ultrasound treatment, cells in the mitotis(M)-phase [81, 82] and synthesis(S)-phase [82] have been reported most sensitive whereas cells in the senescent cell cycle phase were least sensitive. By contrast, another study showed the opposite: cells in the M and S-phases were more resistant to ultrasound treatment [83]. However, these ultrasound sensitivity studies were performed without the
SPIO cell labeling using ultrasound
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