Page 48 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 2
The cytospins were also used to study retention of the iron inside the cell over time. The total number of cells was counted and the number of cells stained with Prussian Blue (blue coloration of the iron) was counted in three randomly selected fields of view (objective 40 x ) per time point.
5.6. Cytotoxicity and viability assays
5.6.1. Distribution of iron and cell morphology: Prussian blue staining
To study the effect of SPIO labeling on cell morphology and label distribution using different labeling protocols, labeled cells were washed with PBS and photographed at a light microscope. The cells were then fixed with 4% formalin. After 10 min of fixation, the samples were washed again and stained for 20 min with a freshly prepared solution containing 0.12 M K4Fe(CN)6 and 1 M HCl (Sigma-Aldrich, Zwijndrecht, The Netherlands). The cells were washed again and the cytoplasm was stained with 1% w/v pararosaniline solution. Samples were washed a final time and examined by light microscopy. The appearance of the cells and the place where the iron was taken up in the cells was qualitatively scored.
5.6.2. Cell functionality: Matrigel test
SPIO- and MPIO-labeled HUVEC were seeded on Matrigel to study the tube- forming capacity of the labeled cells (Matrigel Basement Membrane Matrix, Becton Dickinson, Alphen aan den Rijn, The Netherlands). After 4 and 24 h the cells were screened with a light microscope and photographed.
5.6.3. Visualization by MRI
All data acquisition was performed on a GE (HD) 3 T clinical MRI scanner using unmodified gradients and specially designed reception coils, to provide the best signal-to-noise (SNR) performance for the desired field-of-view.
For in vitro imaging of labeled cells, cells were harvested by trypsinization and a cell suspension was prepared in appropriate culture medium at a cell concentration of 50 cells/10 ml. On the bottom of a four-well culture plate (VWR international NUNC176740; Amsterdam, the Netherlands), a grid with rectangles of about 1.5 mm2 was carved (Fig. 4). This grid served as location marker for subsequent MR imaging sessions. A 10 ml aliquot of the cell suspension was seeded into the well, within the surface area of a single
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