Optimal incubating time of in vitro bromodeoxyuridine labeling of human umbilical cord blood- mononuclear cells and their functional assessment in ICH rats

Bromodeoxyuridine (BrdU) is a thymidine analog that is ready to be incorporated into DNA and can be used as a marker for in vitro pre-labeling of isolated stem cells. The objective of this study was to explore the optimal incubating time of in vitro BrdU labeling for human umbilical cord blood - derived mononuclear cells (HUCB-MCs). The mononuclear cells were isolated by the standard density gradient technique and were labeled with 10 µM/L BrdU. Then, the cells were incubated at 37°C for 1, 6, 12, 24, 48 and 72 h, respectively. Immunocytochemistery was performed respectively to calculate the labeling index and the cells' activity for different time. Efficacy of BrdU positive HUCB-MCs was evaluated by in vivo study in intra-cerebral hemorrhage (ICH) rat model. The current findings indicated that labeling ratio significantly increased after 24 incubating hours. Cell viability remained high (75%) after 24 h. There was a significant recovery in behavioral performance 2 weeks after HUCB-MCs infusion to ICH rats and BrdU+ cells were localized by immunohistochemistry in the injured area of brain. This study shows that the most appropriate time for BrdU labeling HUCB-MCs is 24 h and these BrdU+ cells have high efficacy and stability in cell therapy. Key words: