[Experimental model of in vitro generation of human dendritic cells]

Dendritic cells (DCs) are considered the most powerful antigen-presenting cells (APC) playing a strategic role in triggering the immune response. Monocytes are the physiological precursors of myeloid DCs.

This paper aims to develop an in vitro experimental model of human dendritic cells generation and phenotypic and functional analysis starting from CD14+ monocyte precursors isolated from peripheral blood.

CD14+ monocytes were isolated from venous peripheral blood which was harvested from consenting healthy donors. By the means of GM-CSF and IL-4 immature DCs were generated from monocytes. In the sixth day we added TNF-alpha and PGE-2 maturation factors and, after 48 hours we harvested mature DCs. Their morphology was monitored by optical microscopy, while the phenotypes were determined by flow cytometry.

After three days of culture, the monocytes developed a specific morphology, gathering in clusters; cells presenting specific features start to detach in the sixth day. Treatment with TNF-alpha and PGE-2 induces the maturation of DCs and up-regulation of CD80, CD83 and CD86 expressions. Phenotypic analysis of mature DCs was HLA-DR+++/CD86- CD80+++/CD14(-)/CD11a+/CD83+++. DCs obtained are able to stimulate in vivo allolymphocyte capacity. Functional analysis test showed 87% CD4+ CFSE+(DCs+PBMC) cells vs CD4+ CFSE+(PBMC) 15% cells.

This experiment developed an effective and viable way to generate DCs from PBMCs exposed to GM-CSF and IL-4. The cells obtained presented morphological, phenotypic and functional features as DCs described in the literature.