Page 81 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

1.2 MI, i.e. 3.5 MPa acoustic pressure), which induced considerable arterial wall damage. To the best of our knowledge, no in-depth studies have been performed to characterize the parameters (e.g., the acoustic settings, the SPIO addition time and incubation time) that strongly influence the efficacy and safety of SPIO-labeling of endothelial cells using tMB at low MI (<0.2).
The aim of our in vitro study was to find optimal parameters for non-invasive, tMB-mediated, SPIO-labeling of endothelial cells for the future application of MRI tracking of tumor vasculature and tissue engineered vasculature structures. We used lipid-coated tMB targeted against CD31 (i.e. platelet/endothelial cell adhesion molecule-1 (PECAM1)), a biomarker constitutively expressed on endothelial cell membranes [44], as proof of concept. Iron specific Prussian Blue staining in combination with calcein-AM based cell viability assays were applied to define the most efficient and safe conditions for SPIO-labeling of endothelial cells in vitro. We investigated a fixed ultrasound driving frequency of 1 MHz and a series of low diagnostic acoustical pressures (<200 kPa; MI<0.2) and treatment duration times (0 – 30 s). In our study we used 1 MHz as the ultrasound frequency because it is commonly used for microbubble-mediated drug delivery studies and is close to the resonance frequency of microbubbles [26]. Although the exact link between the type of microbubble behavior and drug uptake is not yet known [26], it was reported that endocytosis was stimulated at longer (2,000 – 10,000 cycles) acoustic cycles [45-47]. SPIO are typically 80 – 150 nm [17] nanoparticles which may require uptake by endocytosis, as this has been shown to be the main uptake mechanism for therapeutics larger than ~17 nm in radius [46]. This is the reason why we chose to study 10,000 acoustic cycles.
Materials and Methods
Endothelial cells
Human umbilical vein endothelial cells (HUVECs) (Lonza, Verviers, Belgium) were cultured in EGM-2 (Lonza) medium in T75 flasks (BD, Breda, the Netherlands) in a humidified incubator at 37°C with 5% CO2. Cells were detached with 0.25% Trypsin in EDTA (Lonza) and replated on one side of the acoustically transparent OptiCellTM (NUNC, Wiesbaden, Germany) chambers.
SPIO cell labeling using ultrasound
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