Page 32 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis
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                                Chapter 2
green (480/520 nm). These particles are highly effective T2* contrast agents, and can also be imaged by fluorescence microscopy and used in fluorescent activated cell sorting (FACS) studies. MPIO have an average size of 1630 nm and have been shown to be functionally inert (24,25). Both SPIO and MPIO are efficiently endocytosed by many cell types and passed along to daughter cells during mitosis. Cell cycle analysis demonstrates that the labeling process does not have a negative impact on the cell cycle profile in comparison with non- labeled cells (26,27).
2 Results
2.2. Labeling efficiency
2.1.1. Iron uptake
Labeling efficiency of human umbilical vein endothelial cells (HUVECs) is dependent on various labeling parameters: the dose of iron that the cells are exposed to, the incubation time used and the type of iron oxide nanoparticle used (Fig. 1A, B). Using an incubation time of 4 h and doses of 6.25 mg iron/ 2 ml/9.5 cm2, less than 50% of the cells were labeled as assessed by Prussian blue staining. This was also the case for an incubation time of 24 h and a dose of 3.13 iron/2 ml/9.5 cm2. These conditions were therefore not used to measure the average iron content per cell. High concentrations at long incubation times were also not further analyzed since these conditions led to significant cell death (see further below).
For SPIO a maximal iron load of 1.82 mg per 100 000 cells was obtained, representing an average iron load of 18.2 pg per cell. A much higher iron load was achieved with MPIO particles. With MPIO a maximal iron load of 66.1 mg per 100 000 cells was achieved, corresponding to an average iron load of 661 pg per cell. Note that the conditions under which these maximal iron loads were achieved for the two different iron oxide formulations were not the same. In the case of SPIO, maximal incorporation was achieved when cells were exposed to 25 mg iron/2 ml/9.5 cm2 for 24 h. In the case of MPIO, maximal incorporation was achieved within 4 h of incubation using an iron oxide dose of 100 mg iron/2 ml/9.5 cm2. As displayed in Fig. 1(C–E), uptake of MPIO is much more efficient than uptake of SPIO. For MPIO the uptake efficiency
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