Kinetic study of the 5-bromodeoxiuridine interaction with monononucleated cells: CD45+ and CD34- for cell therapy an experimental murine model

Abstract

The 5-bromodeoyuridine is an analogous marker for the timidine, allowing to perform techniques such as immunocitochemstry, and widely used also to identify cells that proliferate in phase S of the cellular cycle. Purpose: The aim is to set the protocol to set the time of incubation to cell marker using the 5-bromodeoxyuridine of mononuclear bone marrow derived from stem cells through a kinetic study focusing the use in tissue bioengineering. Methods: 16 male Wistar rats with weight of 300g were used. The bone marrow stem cells gathering was carried through the iliac crest punction-aspiration. The isolation was through the density gradient (Ficoll-Hypaque). Bone marrow stem cell (CD45+ and CD34-) suspensions were incubated with 5-bromodeoxyuridine in the final concentration of 10mM to 37º C in the settled periods of 1, 5, 16, and 24h for each sample, and followed by direct immunofluorescence. Results: All incubation periods of such cells had a positive marking for the 5-bromodeoxyuridine, meaning evidences in the proliferation phase of these cell suspensions, but from 16h on, we have demonstrated a better marker (55%±4%, 16h and 53±6%, 24h versus 12%±3, 1h and 23±4, 5h). Conclusions: The best incubation time for mononucleated cells was that of 16hs seeking to use such marker for those cells, and this protocol could be adopted in the routine analysis of the cell sampling for the cell therapy, being able to perform a prognostic for the cell integration potential predictor in the host tissue as well substituting the radioactive thimidine.