Page 37 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

2.2. Toxicity
2.2.1. Cell survival
Toxicity of the labeling procedure was dependent on both the labeling dose and the incubation times used. At incubation times of 24 and 48 h significant cell death of more than 50% of the cells was seen at doses of 50 and 25 mg, respectively, for SPIO and at doses of 100 and 50 mg, respectively, for MPIO. These conditions were therefore not used in other studies. For the other doses and incubation times tested, cell viability was between 80 and 100% (Fig. 5C). For both MPIO and SPIO higher doses were tolerated at shorter incubation times. At equal incubation times, MPIO was better tolerated than SPIO. For example, at incubation times of 24 h, a dose of SPIO 50 mg resulted in a loss of cell viability of 50% while for MPIO cell viability was 80% at this dose.
2.2.2. Cell morphology
At the highest doses tested for each of the incubation times that did not affect cell survival too severely (cell viability 80%), a change in morphology of the cells was observed. This change involved a more spindle-like appearance of the cells in culture. In FACS studies, clear changes in forward scatter and side scatter plots, corresponding to changes in cell size and cell granularity, respectively, were observed following labeling
(Fig. 5A and B). Both effects were more pronounced after labeling with MPIO than with SPIO. At the highest doses of MPIO tested cell sizes increased 4–7 times in cell volume compared with unlabeled control cells. Cell volumes were calculated from the average measured length and the width of labeled and unlabeled cells and the assumed height of 0.5x the cell width using the formula to calculate the volume of an ellipsoid.
2.2.3. Cell function
Unlabeled (control) HUVECs show tube formation when seeded on matrigel (Fig. 6). This phenomenon is already apparent after 4 h. After 24 h the tubular network is much finer of structure. The effects of SPIO and MPIO labeling on tube forming capacity of HUVECS were tested at all doses that did not significantly affect cell survival. For all these conditions tested, HUVECs still displayed tube forming capacity (Fig. 6).
Influence of SPIO cell labeling protocol
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