Cell density-dependent proliferation in frequently-fed peripheral blood mononuclear cell cultures

Our goal was to produce granulocyte progenitor (CFU-G) and postprogenitor (CD15+CD11b+/−) cells for subsequent transplantation. We hypothesized that increasing the feeding frequency and maintaining constant densities may overcome inhibitory growth conditions (i.e. low pH) in high-density cultures. To study the effect of cell density on total cell expansion, differentiation and lactate production, 50% daily medium exchanges were used in cultures of peripheral blood mononuclear cells (PB MNC) maintained at constant densities (ranging from 5×104 cells/mL to 2.5×106 cells/mL). We observed a significant increase in total cell expansion when the density was increased from 5×104 cells/mL to 1×106 cells/mL, but a further increase to 2.5×106 cells/mL resulted in a decline in cell expansion. Increasing feeding to 90% daily exchange in cultures with 2.5×106 cells/mL did not enhance cell expansion; nor did reducing the extent of feeding in cultures with 5×104 cells/mL to 10% daily exchange. We did not observe a relationship between cell density and the percentage of granulocyte progenitor and post-progenitor (CD15+CD11b−/+) cells. While specific lactate production (qlac) in cultures with 2.5×106 cells/mL was approximately 60% of those observed in lower density cultures by Day 13, this difference was largely eliminated by increasing the extent of feeding in cultures with 2.5×106 cells/mL. Our results suggest that feeding rates must be adjusted according to cell density to maximize culture performance. They also suggest that cellular crowding on the culture surface can limit expansion in suspension (nonadherent) cultures.