Development of a new protocol for 2-day generation of mature dendritic cells from human monocytes

Abstract

We developed a new 2-day protocol for the generation of dendritic cells (DCs) from human monocytes in vitro. First, we demonstrated that 24 hours of culture with GM-CSF and IL-4 are sufficient to generate immature DCs capable of antigen uptake. We then compared two different strategies for DC maturation: proinflammatory mediators were either added together with GM-CSF and IL-4 from the beginning of cell culture or added after 24 hours of differentiation with GM-CSF and IL-4. After 48 hours of total culture period, expression of activation markers was more pronounced in cells generated by the 2-step differentiation and activation method. Our new protocol for 2-day DC differentiation reduces labor, cost and time and also reliably renders high numbers of mature and viable DCs.

Strategic use of an adenoviral vector for rapid and efficient ex vivo‐generation of cytomegalovirus pp65‐reactive cytolytic and helper T cells

“…Therefore, it normally takes 7-9 d to produce 'conventional' DCs for T-cell stimulation studies. Recent studies have implied the effectiveness of 'fast' DCs which are generated in only 48-72 h Obermaier et al, 2003;Xu et al, 2003;Ho et al, 2006).…”

Generation of functional monocyte-derived fast dendritic cells suitable for clinical application in the absence of interleukin-6

“…Also, it is up-regulated during bacterial and viral infection as well as trauma (Heinrich et al 2003). TNF-a, IL-1b, IL-6 and PGE 2 were supposed to be essential cytokines for in vitro maturation of DCs and their production of interferon (IFN)-c and IL-12 (Blanco et al 2008;Bohnenkamp and Noll 2003;Feuerstein et al 2000;Obermaier et al 2003). In addition, PGE 2 was able to induce the final maturation of IL-12-deficient CD1a…”

“…Recently, a new strategy was described for differentiation and maturation of human Mo-derived fastDCs with full T cell stimulatory capacity within only 48-72 h of in vitro culture Obermaier et al 2003;Ramadan et al 2004). In this strategy adherent blood Mo were cultured for 1 day with GM-CSF and IL-4 to generate CD14 -CD1a…”

“…Then, they were activated and converted to functional APCs (2nd step of fast-DCs generation: maturation) by culturing with the following proinflammatory mediators for either 1 or 2 days: tumour necrosis factor (TNF)-a, IL-1b, IL-6 and prostaglandin (PG)-E 2 Obermaier et al 2003;Ramadan et al 2004). Mature DCs exhibit increased expression of the major histocompatibility complex (MHC) and costimulatory molecules (such as CD86) as well as secrete large amounts to immunostimulatory cytokines such as IL-12 thus rendering them highly effective inducers of antigenspecific T cell responses (Cella et al 1997;Schnurr et al 2000).…”

Adoptively Transferred Dendritic Cells Restore Primary Cell-Mediated Inflammatory Competence to Acutely Malnourished Weanling Mice

“…Forward angle light scatter was examined as an index proportional to cellular surface area and volume, 19 both of which increase as dendritic cells mature. 20 This index increased in JAWS II cells only in response to the combined influences of granulocytemacrophage colony-stimulating factor and endotoxin (Table 2). During 5 days of culture, unstimulated JAWS II cells were unable to generate concentrations of either IL-10 or IL-12p70 that exceeded the detection limits of the respective assays (Figure 3).…”