Page 39 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Using these optimal labeling conditions for HUVEC, we also labeled human chondrocytes and murine myoblast cells (C2C12) with SPIO and MPIO particles. In both cell types, labeling efficiency with SPIO particles was similar to that observed in HUVEC. For MPIO, however, labeling efficiency was considerably less in these cell types. For C2C12, labeling of all cells was only achieved at doses of 12.5 mg iron/2 ml/9.5 cm2 and an incubation time of 24 h. Also different was the reaction of these cells to incorporation of MPIO. Following incorporation of MPIO, morphological changes as observed in HUVEC were far less pronounced in C2C12 cells (Fig. 7). Remarkably, labeling of chondrocytes with MPIO was highly inefficient. No incorporation of label occurred using an incubation time of 4 h. Using a dose of 100 mg iron/2 ml/9.5 cm2 MPIO and an incubation time of 24 h maximally 70% of the cells showed incorporation of MPIO particles.
3. Discussion
This study was set up to learn more about the effect labeling conditions have on the incorporation, distribution and retention of iron oxide nanoparticles. In the vast amount of studies dealing with labeling of cells with iron oxide nanoparticles, a large variation of labeling protocols is encountered; labeling doses varying from 1 to 2800 mg/ml and incubation times varying from 1 to 72 h have been described (28–30). Generally, higher doses, longer incubation times, larger particle size and the use of lipofection techniques result in increased labeling efficiency (15,16,18). While in most of these studies labeling in the absence of major adverse effects is reported, it remains unclear what the influence of different labeling protocols is on label incorporation, label distribution and label retention.
As shown in this study, each of these aspects is strongly influenced by the exact labeling protocol used. In terms of intracellular iron load, the optimal labeling protocol for HUVEC using SPIO (Endorem) consisted of a labeling dose of 12.5 mg SPIO/2 ml/surface area of 9.5 cm2 and an incubation time of 24 h. With this protocol an average iron load of 12.0 pg iron/per cell was obtained. This corresponds to an uptake efficiency of 9.6%. If a significantly shorter labeling time is used (4 h) many SPIOs are seen sticking to the outside of the cell instead of being taken up by the cell. As reported by Metz et al.,
Influence of SPIO cell labeling protocol
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