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

Background: Our goal was to produce granulocyte progenitor (CFU-G) and post-progenitor (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. Methods: To study the effect of cell density on total cell expansion, differentiation and lactate production, 50% daily medium exchange were used in cultures of peripheral blood mononuclear cells (PB MNC) maintained at constant densities (ranging from 5 x 10⁴ cells/mL to 2.5 x 10⁶ cells/mL). Results: We observed a significant increase in total cell expansion when the density was increased from 5 x 10⁴ cells/mL to 1 x 10⁶ cells/mL, but a further increase to 2.5 x 10⁶ cells/mL resulted in a decline in cell expansion. Increasing feeding to 90% daily exchange in cultures with 2.5 x 10⁶ cells/mL did not enhance cell expansion; nor did reducing the extent of feeding in cultures with 5 x 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 (q(lac)) in cultures with 2.5 x 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 x 10⁶ cells/mL. Discussion: 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.