Page 82 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis
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                                Chapter 4
HUVECs were cultured as described before [48], for two days until 70% confluence to resemble neovasculature endothelial cells.
Targeted microbubbles
Biotinylated lipid-coated microbubbles (mean diameter 2.5 μm) consisting of a coating of DSPC (84.8 mol %; P 6517; Sigma-Aldrich, Zwijndrecht, the Netherlands), PEG-40 stearate (8.2 mol %; P 3440; Sigma-Aldrich), DSPE- PEG(2000) (5.9 mol %; 880125 P; Avanti Polar Lipids, Alabaster, AL, USA), and DSPE- PEG(2000)-biotin (1.1 mol %; 880129 C; Avanti Polar Lipids) with a perfluorobutane (C4F10) gas core (F2 Chemicals, Preson, UK) were made by sonication as previously described [49, 50]. Biotinylated anti-human CD31- antibody (BAM3567; R&D Systems, Europe, Abingdon, United Kingdom) was conjugated to the microbubbles via avidin-biotin bridging as previously described [50, 51]. Specificity of binding of these CD31-targted microbubbles was previously reported by us [48].
Cell treatment
The concentration of tMB was evaluated by Coulter Counter (Multisizer 3, Beckman Coulter, Mijdrecht, the Netherlands) measurements (n = 3) using a 20-μm aperture tube allowing quantification of particle diameters between 0.4 and 12 μm using a linear spacing between the 256 channels. Ten million tMB were added to an OptiCellTM chamber with cells plated on the bottom (cell to bubble ratio of 1:3), which was turned upside down to let microbubbles adhere to the cells by flotation. After 5 min incubation at 37°C, the chamber was reverted for the experiment so the bound tMB were on top of the endothelial cells as shown in Fig 1A. SPIO nanoparticles (EndoremTM, Gerber S.A., Paris, France) were added at four time-points: 5 min before, immediately before (0 min), 5 min after, and 15 min after insonification as illustrated in Fig 1B, at a final concentration of 22.4 μg Fe/ml. Each OptiCellTM chamber was divided into six acoustically non-overlapping areas (25 × 30 mm each; see Fig 1C), which covered the beam area (6.5 mm for -6dB beam width) at the focus of the 1.0 MHz transducer (V303; Panametrics-NDTTM, Olympus NDT, Waltham, MA, USA), as verified in advance with a calibrated 0.2 mm PVDF needle hydrophone (Precision Acoustics Ltd, Dorchester, UK). The OptiCell chamber was placed into a 37 °C water bath and connected to a 2D micropositioner (Fig 1 D). The 1
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