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                                MSC. High intracellular amounts of iron and/or exposure of MSCs to high iron concentrations also inhibited chondrogenic differentiation capacity of MSCs (31,32). Not only the labeling dose but also exposure time were factors in creating this adverse effect.
For MPIO optimal iron incorporation was obtained with a dose of 50 mg iron/2 ml/9.5 cm2. For MPIO the labeling time was of lesser importance since most of the particles were already taken up within 4 h with a 100% labeling efficiency. Under these conditions, the resulting intracellular iron load is 626 pg/cell. This is significantly higher than reported in other studies using different cell types (9,25). In these studies murine hepatocytes were labeled with MPIOs and they generally contained iron levels of 100 pg. On occasion, some cells had levels as high as 400 pg. Macrophages labeled with 1.63 mm MPIOs had an average cellular iron uptake of 39.1 pg/cell, corresponding to approximately 35 particles per cell.
Because of the high uptake efficiency of MPIO, a relatively low dose of 6.25 mg iron results in the labeling of all cells. However, at this dose an incubation time of 24 h is needed. For a dose of 50 mg MPIO, an incubation time of 4 h suffices to label all cells and uptake of most of the iron particles. This shorter incubation time, however, results in a different intracellular distribution of the iron than longer incubation times. After a 4 h incubation period, MPIO particles are homogenously distributed over the cytoplasm. In contrast, after an incubation time of 24 or 48 h, the MPIO particles are found clustered around the cell nucleus. This latter observation is most likely due to intracellular cell trafficking of the endosomes that occurs in time after uptake of the particles. For HUVEC, increasing labeling doses and consequently increasing intracellular iron loads result in more pronounced changes of morphological features. The cells become more spindle-like, larger in size and more granulated. Such effects were not seen in murine monocytes/macrophages in a study by Valable et al. using similar assays (27). As shown in this paper, the labeling efficiency of SPIO is significantly less than for MPIO. Higher doses and longer incubation times are needed to achieve labeling of all the cells. Also, HUVEC displayed a higher tolerance for MPIO than for SPIO. This means that more iron can be brought inside the cell with MPIO in a short period of time. In terms of labeling dose and incubation time the SPIO labeling efficiency was much
Influence of SPIO cell labeling protocol
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