A protocol for rapid monocyte isolation and generation of singular human monocyte-derived dendritic cells.

Abstract

The monocyte-derived dendritic cells (moDCs) are a subset of dendritic cells widely used in immunological studies as a convenient and easy approach after isolation of mononuclear cells directly from peripheral blood mononuclear cells (PBMC). Both the purification and cell culture of monocytes impact on the differentiation of monocytes into moDCs. The methodology to isolate and differentiate monocytes into moDCs is still controversial. We aimed to compare three different protocols for monocyte isolation from PBMC: 1) Cold-aggregation; 2) Percoll gradient; and 3) Magnetic beads cell-enrichment. Additionally we also compared four different monocyte differentiation and culture techniques: 1) Cell culture media; 2) Serum sources; 3) required GM-CSF and IL-4 concentrations; 4) Cell culture systems. We used flow cytometry analysis of light scattering and/or expression of pan surface markers, such as CD3, CD14 and CD209 to determine isolation/differentiation degree. Purified PBMC followed by two steps of cold aggregation, yielded cell viability around 95% with poor monocyte enrichment (monocytes increase vs. lymphocytes reduction was not statistically significant, p>0.05). Conversely, monocyte isolation from PBMC with discontinuous Percoll gradient generated around 50% cell viability. Albeit, we observed a significant reduction (p≤0.05) of lymphocytes contaminants. The magnetic beads cell-enrichment yield cell viability higher than 95%, as high as a significant lymphocyte depletion (p≤0.005) when compared to all other techniques employed. The moDCs showed better differentiation based on increased CD209 expression, but lower CD14 levels, when cells were cultured in RPMI medium plus 500IU/mL of both GM-CSF and IL-4 in a semi-adherent fashion. Serum sources showed no influence on the culture performance. In conclusion, the magnetic beads cell-enrichment showed superior cell viability, indicating that this approach is a better choice to isolate monocytes, and moDCs cultured afterwards in appropriate medium, serum, cytokines and culture system might influence the monocytes differentiation into moDC.