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.
Blood and Cell Therapy Development Department, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD 20855-2743, USA. Celluzzi@usa.redcross.org
Christina M
CM
Celluzzi
Craig
C
Welbon
D017302:Annexins / Q000032:analysis; D017951:Antigen Presentation / Q000502:physiology; D015703:Antigens, CD / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis; D017209:Apoptosis; D001781:Blood Component Removal; D002448:Cell Adhesion; D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002470:Cell Survival; D002498:Centrifugation; D016207:Cytokines / Q000032:analysis; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D005362:Ficoll / Q000737:chemistry* / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000494:pharmacology; D015847:Interleukin-4 / Q000494:pharmacology; D007378:Interleukins / Q000032:analysis; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000502:physiology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000494:pharmacology
D017302:Annexins; D015703:Antigens, CD; D000954:Antigens, Surface; D016207:Cytokines; D007378:Interleukins; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D010835:Phytohemagglutinins; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4; D005362:Ficoll; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor