In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye

Recent advances in the field of tissue engineering have culminated in new t issue substitutes that combine a biomaterial and precursor cells. The effec tiveness of these materials is generally assessed in animals, but few studi es explore the fate of the transplanted cells in vivo, despite its paramoun t importance for understanding the function of the engineered tissues. Curr ent methods that use reporter genes or chimeric animals are not always well suited to solving tissue-engineering problems. We therefore developed a ne w method for irreversible labeling of cells to track their fate in oho. We used a fluorescent lipophilic probe, CM-Dil, that avidly binds to the cell membrane. Human bone marrow stromal fibroblasts could be labeled with 20 mu M CM-Dil in 30 min. The CM-Dil was nor cytotoxic and did not affect cell pr oliferation in vitro. Cells could be monitored for up to 30 days when place d in a coral scaffold and implanted intramuscularly or in a bony site. Howe ver, the fluorescence intensity decreased roughly in parallel with the numb er of cell divisions. This fact needs to be taken into account during the d esign and interpretation of experiments. We believe that this technique is also of interest for other cell types. (C) 2001 John Wiley & Sons, inc.