Optimized culture conditions for the generation of dendritic cells from peripheral blood monocytes

Dendritic cells (DCs) are promising adjuvants for clinical immunotherapy, but they are scantily distributed. Therefore, numerous in vitro methods have been developed to expand these cells while maintaining their normal functions. Current culture systems generally require the use of fetal bovine serum (FBS)-supplemented media in order to attain DCs with high immunostimulatory activity. However, the presence of exogenous animal proteins sets limits for their use in clinical trials. The purpose of this study was to establish a simple, efficient and FBS-free method for the generation of human DCs for clinical application.

We compared monocyte-derived DCs generated in a standard FBS-supplemented medium vs. DCs generated in an autologous plasma (AutoPl)-supplemented medium, with regard to their yield, function and longevity. Peripheral blood monocytes were isolated from buffy coats by two consecutive 2-h adherence steps in tissue culture flasks. The adherent cells were differentiated into DCs within 2 weeks by adding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), c-kit ligand and tumour necrosis factor-alpha (TNF-alpha). Every 2-3 days, the cells in suspension were analysed for their immunophenotype and apoptosis rate by flow cytometry. Their function was demonstrated by their allostimulatory and migratory capacity, as well as by their proteolytic activity.

We show that more than 30 x 10(6) DCs can be achieved per unit of buffy coat using either AutoPl- or FBS-supplemented media. The purity of the DCs was 53.4% and 65% (P > 0.05) in AutoPl- and FBS-based medium, respectively. DCs grown in AutoPl media showed a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1aneg phenotype, while FBS-generated DCs exhibited a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1ahigh phenotype. The apoptosis rate in both culture conditions increased from 10% to 25% over 1 week. AutoPl-generated DCs were shown to be equally strong stimulators for proliferation of allogeneic T lymphocytes as FBS-generated DCs. In addition, the capacity to migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha) and stromal-cell-derived factor 1alpha (SDF-1alpha) was similar in both groups, whereas the response to MIP-3beta was reduced in AutoPl-derived cells. Zymography analysis of supernatants from 5-day-old cultures demonstrated that AutoPl-generated DCs produced higher amounts of matrix metalloproteinases, suggesting that they have an enhanced capability to traffic through peripheral tissues.

Our findings indicate that plastic-adherent peripheral blood cells, when cultured with GM-CSF, IL-4, c-kit-ligand and TNF-alpha in autologous human plasma-supplemented media, are a potent source of functional DCS that may be of value for human therapy.