The methodological approach for the generation of human dendritic cells from monocytes affects the maturation state of the resultant dendritic cells.
Dendritic cells (DCs) are effective as antigen-presenting cells in the immune system and are present at two functional stages depending on their maturation state. For experimental investigation of this concept, CD14(+) monocytes from blood are isolated and cultured to generate in vitro the DCs needed for functional analysis. For positive selection of CD14(+) monocytes we compared two immunomagnetic bead technologies: MACS Separation, created by Miltenyi Biotec, and EasySep Selection, created by StemCell Technologies. The monocytes provided dendritic cells for their functional analysis. Lipopolysaccharide was added to cultured DCs to induce maturation. Although both systems generated DCs from the positively selected CD14(+) cells, there were certain differences between them. Morphological, phenotypic, and functional analysis showed that MACS-selection provided DCs that have typical features corresponding to day 6 or 7 of maturation. EasySep-DCs exist in a partially-mature state from day 6 onward, even without the addition of a maturation stimulus. The reason behind this partial maturation is possibly based on the dextran-coated beads that are associated with the EasySep product. Both methods provide pure and viable DCs, but we would recommend using the MACS system for obtaining DCs suitable for functional studies.
Mucci
Ilaria
I
Department of Pediatrics and Reproductive Medicine, University of Pisa, via Roma, 67, 56100 Pisa, Italy. ilaria.mucci@med.unipi.it
Legitimo
Annalisa
A
Compagnino
Marta
M
Consolini
Rita
R
Migliaccio
Pasquale
P
Metelli
Maria Rita
MR
Scatena
Fabrizio
F
D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D016207:Cytokines / Q000032:analysis / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D016130:Immunophenotyping; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
D017077:Culture Media, Conditioned; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides