Gd loading by hypotonic swelling: an efficient and safe route for cellular labeling

Cells incubated in hypo-osmotic media swell and their membranes become leaky. The flow of water that enters the cells results in the net transport of molecules present in the incubation medium directly into the cell cytoplasm. This phenomenon has been exploited to label cells with MRI Gd-containing contrast agents. It has been found that, in the presence of 100 mM Gd-HPDO3A in an incubation medium characterized by an overall osmolarity of 160 mOsm l⁻¹, each cell is loaded with amounts of paramagnetic complex ranging from 2 × 10⁹ to 2 × 10¹⁰ depending on the cell type. To obtain more insight into the determinants of cellular labeling by the 'hypo-osmotic shock' methodology, a study on cell viability, proliferation rate and cell morphology was carried out on J774A.1 and K562 cells as representative of cells grown in adhesion and suspended ones, respectively. Moreover a comparison of the efficiency of the proposed method with established cell labeling procedures such as pinocytosis and electroporation was carried out. Finally, the effects of the residual electric charge, the size and some structural features of the metal complex were investigated. In summary, the 'hypotonic shock' methodology appears to be an efficient and promising tool to pursue cellular labeling with paramagnetic complexes. Its implementation is straightforward and one may foresee that it will be largely applied in in vitro cellular labeling of many cell types.