Page 44 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 2
optimized protocols for HUVECS to different cell lines shows that no standard labeling protocol is useful for all the cell types growing in vitro. Consequently, an optimal labeling protocol has to be determined for each cell type in combination with the specific particle separately.
5. Experimental procedures
5.1. Cell culture
For extensive assessment of the effects of labeling dose and duration primary culture HUVECs were used. HUVECs were grown in endothelial growth medium (EGM-2 bullet kit CC3156 with CC4176; Cambrex, Verviers, Belgium) in a six- well plate (Corning Incorporated, New York, USA) with a surface growth area of 9.5 cm2. For assessment of the general applicability of our findings, optimal labeling protocols as found for HUVEC were used for labeling of a murine myoblast cell line, C2C12 and human chondrocytes. C2C12 cells were cultured in Dulbecco’s modified Eagle medium + 4500 mg/l D-glucose (Invitrogen, Breda, The Netherlands) + 1% (v/v) penicilline/streptomycine [10.000 U penicilline/ml + 10.000 mg streptomycine/ml (Cambrex, Verviers, Belgium)] + 10% (v/v) fetal bovine serum (Cambrex, Verviers, Belgium). Chondrocytes were cultured in chondrocyte expansion medium as described previously (38).
5.2. SPIO labeling
Labeling of cells with SPIO was performed at 90% confluence using Endorem (Guerbet S.A., Paris, France) and lipofectamine 2000 (Invitrogen, Breda, The Netherlands) (21). A labeling stock solution of 215 mg Fe/ml was prepared as follows: 10 ml Endorem was added to 250 ml Opti-MEM (Invitrogen, Breda, The Netherlands) and 10 ml of lipofectamine was added to 250 ml Opti-MEM. After 5 min the two solutions were mixed together and the resulting suspension was incubated at room temperature for 20 min. After washing the cells with phosphate-buffered saline (PBS) (Invitrogen, Breda, The Netherlands), the culture medium was replaced by 2 ml Opti-MEM and the SPIO–lipofectamine suspension (0, 3.13, 6.25, 12.5, 25, 50 mg) was added drop-wise to the medium such that an equal distribution of SPIO–lipofectamine complexes was obtained in the well. The cells were then incubated for 4, 24 or 48 h at 37°C/ 5% CO2. Before further use of labeled cells, the monolayer cultures were rinsed three times with
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