ausblenden:
Schlagwörter:
-
Zusammenfassung:
Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology, or in cell-based therapies such as beta cell transplantation in type I diabetes or stem cell therapy. Traditionally, cell labeling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity, and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapor nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in 50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity. Most interestingly, cytosolic delivery by photoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labeling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labeled with fluorescent dextrans could be tracked for up to two months in Swiss nude mice compared to 2 weeks for cells labeled by endocytosis.
