Dendritic cells (DCs) have received much attention as a therapeutic tool for infections and immunotherapy of advanced malignancies, due to their unique ability of antigen-presentation and initiation of the T-cell-dependent immune response. However, the broader application of vaccine- or antitumor specific T cell–based immunotherapy is limited by DC’s scarcity in the peripheral blood and the complex and costly methodology required for their production. To date, peripheral blood monocytes are the most common source for DCs generation and recently hematopoietic stem cell (CD34+)-derived DCs have also been produced. However, the accessibility to stem cell sources is limited and the number of generated DCs are barely enough for wide clinical use.


The non-transplantable umbilical cord blood (UCB) units could be a readily available and promising source for large scale DC production, by offering adequate number of CD34+ cells. In the present study, we developed a new culture method for the generation of high numbers of myeloid DCs from non-transplantable UCB units by using new generation bioreactors (G-rex) and estimated the minimum required volume of a UCB unit to produce adequate number of DCs for clinical application.


CD34+cells from non-transplantable UCBs were purified by immunomagnetic separation, cultured in the presence of a cytokine cocktail [stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin-4 (IL-4)) for 10 days in tissue culture plates and subsequently expanded in G-rex bioreactors for 25 additional days. Control cultures were completed in conventional plates. DCs were matured either with a combination of Toll-like receptor ligands [(TLR-Ls), Poly I:C and R848] or with commonly used vaccines containing TLR-Ls [Act-HIV (bacterial form of influenza), Influvac (viral form of influenza), Typhim Vi, HBV and BCG ]. DCs phenotype was analyzed by flow cytometry (CD40, HLA-DR, CD83 and CD86) and the cytokine secretion values were measured by ELISA. The endophagocytic activity of DCs was assessed by a dead yeast engulfment assay.


DCs derived from UCB CD34+ cells with more than 85% purity, were cultured in bioreactors or in conventional plates. DCs cultured in Grexes reached a median fold expansion 20.875 and a median absolute number of 2 billions, in sharp contrast to a median 100-fold increase of conventionally cultured DCs. The expanded DCs had the typical morphology and surface markers of myeloid DCs (CD33+/CD11+: 70.47%).To address whether those highly expanded DCs are functional and have the ability, upon maturation, to induce Th1 response, the cells were matured with either a cocktail of TLR-Ls or commonly used vaccines, and tested for the expression of molecules needed for antigen presentation, endophagocytic activity and cytokine production. Similar to conventional DCs, the highly-expanded and matured with vaccines or TLR-Ls DCs, showed dendrites and endocytotic capacity. The expanded and matured with either way DCs, expressed also HLA-DR and co-stimulatory molecules (CD40, CD86 and CD83) whereas they produced increased levels of IL-12p70, TNF-α and IL-6 but undetectable IL-10, thus suggesting a strong potential for Th1 response. We tested different volumes and UCB units for CD34+cell-derived DCs and showed that the minimum volume of a UCB unit that could be used for large scale DCs generation with our optimized culture system, is 9ml, corresponding to approximately 10% of the volume of a transplantable UCB unit.


We established an optimized and simple culture system to expand at clinically relevant numbers, the production of human, myeloid DCs from UCB-derived CD34+ cells. These DCs have the functional properties that are necessary to obtain therapeutic gains against malignancies and viral infections. Billions of DCs were derived from a minimum of 10% of the volume of an average transplantable UCB unit. This new method could be one step closer to a broader clinical application of DC- and T-cell based immunotherapy for various malignancies.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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