DCs exhibit the highest potency for antigen uptake and presentation among the immune cells [11, 12]. They show many immune regulation features that balance the complex system of inflammatory and inhibitory immune reactions in the tumor tissue microenvironment . DCs are involved in innate and adaptive immunity. They can modulate immune function, reverse immunosuppression, and decrease cancer immunotolerance .
Human monocytes isolated from PBMCs differentiate to produce iDCs so that DCs are usually generated from blood monocytes in vitro. iDC maturation to mature DCs occurs through an antigen-loading step .
Human DCs are loaded with recombinant proteins, lysates of tumor cells, or peptides, the latter representing the most common process .
DC vaccines aim to stimulate cancer-specific effector T cells to eradicate tumor cells and to stimulate immunological memory to control cancer recurrence . Peptide-loaded DCs can present Ags to naïve T lymphocytes . The proliferative and cytolytic function of tumor-specific cytotoxic T-lymphocytes requires Ag identification by the T cell receptor (TCR). These antigens are linked to MHC class I molecules on APCs .
This study evaluated the DC generation from monocytes isolated from whole peripheral blood for immunotherapy in an in vitro model. These DCs were isolated using Ficoll-Hypaque gradient centrifugation.
There are multiple methods for collecting monocytes from whole peripheral blood, including flask adherence , density gradient centrifugation , and separations using a specific marker such as magnetic-activated cell sorting  or monocytes separation by bipolar tetrameric antibody . In our study, we used the flask adhesion method for monocyte separation. Delirezh and Shojaeefar  demonstrated that magnetic-activated cell sorted monocytes’ viability was slightly higher than Flask-DCs. They express higher levels of CD14− compared with Flask-DCs but a higher IFN-γ to IL-4 ratio, IL-12, and an IL-12 to IL-10 ratio in the flask group. Also, Flask-DCs polarized the immune response toward a Th1 cytokine profile and cell-mediated immunity, a desired feature in cancer immunotherapy.
For MoDC generation through human monocytes isolation, Posch et al.  used the anti-human CD14 magnetic nanoparticle for the positive selection of CD14 leukocytes. The advantages of this technique compared with other protocols are the high purity and speed. MoDCs are the most popular model used for DC generation because the direct isolation of DCs from biopsies or cord blood CD34+ stem cells is a more complex technique. Also, it leads to inefficient cell numbers.
Elkord et al.  showed that the positive selection of monocytes by anti-CD14-coated microbeads inhibits lipopolysaccharide (LPS)-induced production of IL-12, IL-10, and TNF-α from DCs. However, for Flask-DCs, LPS induced much higher levels of IL-12, IL-10, and TNF-α cytokines and CTLs.
DCs were phenotypically and genetically confirmed. HLA-DR, CD 83, and dextran uptake were detected by flow cytometry. Also, the expression of CD80 and CD86 genes was measured by quantitative real-time PCR.
Our results demonstrated DC-positive expression of CD83 and HLA-DR by flow cytometry, DCs showed high positive uptake of dextran and significant upregulation of CD80 and CD86 genes expression in DCs compared to monocytes.
Pan et al.  and Elkord et al.  reported similar findings.
Pan et al.  observed positive CD83 and HLA-DR and positive expression of CD80 and CD86. In their model, PBMCs were isolated from healthy donors' peripheral blood using Ficoll-Hypaque gradient centrifugation, and the monocytes were separated by the flask adherence method. Non-adherent cells (T cells) were removed. Monocytes were harvested with media enriched with IL-4 (400 U/ml) and GM-CSF (1000 U/ml) to produce DCs under conditions identical to our study.
Elkord et al.  demonstrated that monocytes exhibit lower levels of CD14 expression and higher levels of HLA-DR and CD86 expression. iDCs express CD1a and low levels of CD80 on their cell surface but do not express CD83.
Our study used IL-4 and GM-CSF at concentrations of 10 ng/mL and 50 ng/mL, respectively, and the cells were cultivated for 6 days, consistent with El-Sahrigy et al. . They demonstrated that iDCs were generated by culturing monocytes selected by the flask adhesion method and examining their viability. The gated cells (iDCs) showed positive CD1a, HLA-DR, CD11c, and CD83 expression. The monocyte culture media was enriched with 50 ng/ml GM-CSF and 20 ng/ml IL-4.
However, Colić et al.  provided evidence that GM-CSF (100 ng/ml) with IL-4 (5 ng/ml) was effective in iDC generation from monocytes at the same concentration of GM-CSF and ten times higher concentration of IL-4 (50 ng/ml). iDCs were characterized by HLA-DR, CD80, CD86, and CD1a positive expression and downregulation of CD14 and an absence of CD83. At lower concentrations of IL-4, a high number of cells were adherent, and DC generated at low concentrations of IL-4 (5 ng/ml) showed more robust anti-tumor capacity against the Jurkat cell line than DC generated at higher IL-4 concentrations. Adherent cells cultured with only GM-CSF were primarily macrophages, as confirmed by CD14-positive expression.
El Ashmawy et al.  reported that cultured cells were supplemented with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL). Cells were characterized by morphological change and appeared to be semi-adherent with branched projections, and they showed positive expression of CD83 and CD86 on the cell surface.
Other researchers used variable growth factor concentrations, and their trials were successful regarding DC generation. The concentrations were as follows: GM-CSF 50 ng/ml and IL-4 50 ng/ml , GM-CSF 100 ng/ml and IL-4 20 ng/ml , GM-CSF 100 ng/ml and IL-4 20 ng/ml , and GM-CSF 10 ng/ml and IL-4 20 ng/ml .
In our study, we cultured the cells in a 6-well culture plate. Each well contained 4 ml of culture media (RPMI-1640 containing 10% FCS, 2% penicillin-streptomycin with GM-CSF (50 ng/mL) and IL-4 (10 ng/mL)). Tkachenko et al.  used different media for the generation of iDCs. They used RPMI 1640 with 2% human serum albumin, RPMI 1640 with 2% TCH serum replacement, Panserin 501, and X-VIVO 15. Flow cytometry showed that in all previous media, the iDCs were CD45+ CD83+ and lost CD14.
Our results were also in line with Osugi et al. , who reported that MoDCs derived in in vitro culture did not express the CD1a surface antigen but expressed high levels of the HLA-DR CD86, CD83, and CD40 surface antigens. Kolli et al.  also characterized the phenotype of DCs by flow cytometry for other surface markers, including MHC class II, CD11b, CD11c, CD86, and CD80.
DCs have mannose receptors on their cell membrane that facilitates dextran uptake and phagocytosis . In a functional study of DCs using FITC-dextran, we demonstrated that DCs showed a high capacity for FITC-dextran uptake, which is in line with Encabo et al. , who confirmed that fresh monocytes and immature MODCs showed an increased ability for accumulating FITC-dextran. After exposure to TNF-α for 2 days, mature Mo-derived DCs reduced their ability to internalize FITC-dextran.