Dendritic cell culture: A simple closed culture system using ficoll, monocytes, and a table-top centrifuge

Dendritic cells (DCs) are potent antigen-presenting cells involved in the induction of T cell-mediated immune responses and as such have emerged as important candidates for cellular-based therapies. Critical to safe clinical use is the easy manipulation of DCs and their precursors in a closed system. We have developed a serum-free, closed culture system applying a simple wash-Ficoll centrifugation method to reduce platelet and red blood cell (RBC) contamination. This procedure optimized adherence of monocytes (44 +/- 10.9% recovery, >85% expressed CD14(+)/CD163(+)) for the generation of DCs from mononuclear cell (MNC) apheresis units. Most RBCs and up to 98% of platelets were removed. Following density sedimentation, cell viability remained high (98 +/- 2%) with only minimal loss of monocytes (3 +/- 3%). Importantly, Ficoll-treated monocytes retained their ability to differentiate to mature DCs demonstrated by morphology, phenotype (MHC class II+, CD1a(+), CD80(+), CD86(+), and CD83(+)), ability to stimulate mixed lymphocyte responses ( MLR), present antigen, and produce interleukin-12 (IL-12). Nonadherent CD3(+) (80 +/- 4%) were also isolated for functional assays. Ficoll can be easily incorporated into a simple adherence-based closed system for collection of lymphocytes and adherent monocytes for DC culture. The procedure is relatively fast ( effective working time 5 - 6 h), does not impair monocyte function or induce substantial cell activation, and can be performed economically using equipment found in a typical blood banking environment.