Expansion of dendritic cells derived from human CD34+ cells in static and continuous perfusion cultures

We examined the effects of different cytokine combinations and culture conditions on the expansion and modulation of cell surface antigens of CD34⁺ derived dendritic cells (DCs), the most efficient antigen-presenting cells capable of stimulating resting T cells in the primary immune response. Cells with a dendritic morphology and expressing HLA-DR, CD1a, S100 and CD83 were maximally expanded under serum-free conditions with the addition of SCF, GM-CSF, TNF-α, TGF-β and Flt-3 ligand (fold increase of CD1a⁺ cells = 102 ± 32 after 2 weeks of culture). CD34⁺ cells were also grown under continuous flow conditions in an artificial capillary system; after 14 d of culture, the expansion in the total cell number was lower than that of the static cultures (3.3 ± 2 v 18.9 ± 4) but the percentage of CD1a⁺/CD83⁺/CD80⁺ cells was considerably higher, whereas the CD14⁺ cells were significantly reduced (8.9 ± 2 v 26 ± 13). In continuous perfusion cultures, low levels of DC precursors and of LTC-IC were still present up to day 14. The DCs generated under flow conditions stimulated the mixed leucocyte reaction (MLR) more than the cells grown in static cultures. By electron microscopy, cells grown in the continuous flow system showed an increased number of large cells with numerous dendritic processes and abundant multilamellar complexes. The cells expanded under these conditions were sorted on the basis of their light-scatter properties into two fractions: one containing a predominance of CD1a⁺/S100⁺/CD83⁺/CD80⁺/CD14^?‘large cells’ with great internal complexity (mature DCs); the second including ‘small cells’ either CD33⁺/CD14⁺, CD33⁺/CD15⁺ or CD33⁺/CD13^±/CD14^?. The DCs generated and selected with this method are therefore particularly well suited for immunotherapeutic protocols.