Page 87 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

~10% at 40 kPa to ~16% at 160 kPa). At the same time, cell viability (Fig 3B) decreased with both the increasing acoustical pressure and the insonification time. For example at 80 kPa PNP, the cell viability decreased from ~70% for 10 s of insonification to ~60% for 30 s of insonification. For a treatment time of 30 s, the cell viability dropped by nearly two-fold from 40 kPa to 160 kPa PNP. In general, the cell viability remained high for up to 40 kPa PNP. Specifically, insonification for 30 s demonstrated the best SPIO uptake and was selected for further experiments. We did not investigate a longer insonification time because after 30 s all tMB were destroyed (see Fig 2E).
Figure 3 The influence of ultrasound insonification time on intracellular SPIO uptake efficiency. A SPIO positive cells. B Cell viability. HUVECs were treated with tMB and no ultrasound (- US) or ultrasound at varying PNP (40, 80, or 160 kPa) for 1, 10, or 30 s; SPIO added 5 min before insonification (-5 min); 1 h of incubation after SPIO addition.
SPIO incubation time
The influence of the SPIO incubation time (5 min, 1 h or 3 h) after the treatment with ultrasound and tMB on SPIO uptake and cell viability is illustrated in Fig 4. In general, SPIO uptake increased prominently with the incubation time, for example for 180 kPa PNP SPIO uptake increased from below ~4% to ~22%. The largest ratio between control and treated uptake was at 1 h of incubation for all PNPs. Cell viability (Fig 4B) remained high (>75%) at 40 kPa PNP for all incubation times. It decreased with the pressure (80 – 160 kPa PNP) for both 1 h and 3 h of incubation time. A longer incubation time did not lower cell viability, as cell viability was slightly higher after 3 h of incubation than after 1 h of incubation. Based on the results from this experiment, 1 h of incubation with SPIO was selected for further investigations.
SPIO cell labeling using ultrasound
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