Dendritic Cell Culture: A Simple Closed Culture System Using Ficoll,Monocytes,and a Table-Top Centrifuge

Abstract

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.