Page 34 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 2
2.1.2. Label distribution
Distribution of label within the cell population and within the cell is also dependent on the labeling protocol used. For MPIO, cell labeling efficiency in terms of the percentage of cells labeled as assessed by Prussian blue staining soon reached a plateau. As shown in Fig. 2(A), >85% of the cells were labeled at doses of 6.25 mg iron/2 ml/9.5 cm . For SPIO a minimum amount of 12.5 mg iron/2 ml/9.5 cm2 was needed to ensure that all cells were labeled. Differences in labeling efficiency between SPIO and MPIO were also found in terms of distribution of iron oxide complexes per cell. Following an incubation time of 4 h, iron oxide complexes were mostly attached to the outside of the cell, in case of SPIO, while for MPIO, most of the label was found within the cell (Fig. 2B). Differences seen in the location of MPIO particles depending on the incubation time used were also seen. Following 4 h of incubation, MPIO particles were spread throughout the cell as opposed to a more perinuclear clustering of the particles following incubation for 24 or 48 h (Fig. 2C). This latter observation probably reflects intracellular trafficking of the endosomes occurring after internalization of the cells, and most likely also occurs at later time points following a short incubation time.
2.1.3. Label retention
As shown above, the amount of label incorporated by HUVECs is strongly dependent on both the labeling dose and the incubation time used. This effect was most pronounced for SPIO. For instance at a dose of 12.5 mg iron/2 ml/9.5 cm2 the average iron load increased from 7.5 to 12.0 mg iron/cell when incubation times were increased from 4 to 24 or 48 h. For MPIO, longer incubation times generally did not result in significantly higher intracellular iron loads. For instance, at a dose of 25 mg MPIO the average intracellular iron load was similar for incubation times of 4, 24 and 48 h. As can be appreciated from in Fig. 1, for SPIO comparable iron loads were obtained when cells were labeled with 50 mg for 4 h, or with 25 mg for 24 h, or with 12.5 mg for 48 h. To assess whether the potentially different kinetics of iron uptake under these conditions may result in differences in label retention we counted the percentage iron-positive cells over time, for cells labeled according to these three protocols. These experiments revealed that, 1 week post-labeling, more cells contained detectable amounts of iron oxide when a short incubation time
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