Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O-2 concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O-2 solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O-2 concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34(+) cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O-2 for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34(+) cells at 5% O-2, using 21%-O-2 cultures as a control. In hypoxia we found that CD34(+) cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen-and c-kit-dependent pathways at the molecular level. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1154-1162, 2011