Functional Comparison of monocytes isolated from culture flask by lidocaine/EDTA, trypsin and cold-PBS/EDTA

Easy access, quick recovery and high therapeutic potential of monocyte cells, led to special attention to this cell in cell therapy. Monocytes are flask-adherent cells. Therefore, it is important to find a suitable separation methods with minimal damage to the cells and their functions. This study was done to investigate the comparison between monocytes functions after isolation with three different protocols including: lidocaine / EDTA, trypsin and cold PBS / EDTA.
In this experimental study, isolated mononuclear cells (1×107 cell/ml) from the peripheral blood of BALB/c mice were incubated for 4 hours in T25 culture flasks in RPMI culture mediua. After the incubation time, non-adherent cells (mainly lymphocytes) were separated by two washes. Then, tree different method including trypsin, lidocaine and cold PBS, were used to isolate adherent monocytes. The functional activity of monocytes was evaluated after isolating cells by each method. The data were analyzed using one-way ANOVA. p The results showed that the isolation rate, viability, metabolic activity, phagocytosis percent respiratory burst, yeast cidal and the levels of nitric oxide in cells that were isolated using lidocaine were significantly higher than other groups. However, trypsin led to isolate more cells than the cooled PBS but these cells had more physiologic impairment compared to lidocaine / EDTA or cold PBS / EDTA. Neutral red uptake by trypsin isolated monocyte was lower the cells isolated by other procedures. The results indicate there is no significant difference for neutral red intake between cold PBS and lidocaine.
lidocaine / EDTA is a valuable procedure to achieve higher calls with appropriated efficacy compared to trypsin or cold PBS / EDTA procedure.