Abstract
BackgroundDendritic cells (DCs), which can be used as anti-cancer vaccines, are generally obtained in vitro from isolated CD14+ monocytes (MoDCs). This generates high cell numbers and allows instructing DCs to guarantee effective antitumor responses. However, the impact of the monocyte isolation step in the antitumor effectiveness of the generated MoDCs is still unknown. Here, we compared the most used immunomagnetic technologies for monocyte isolation: magnetic activated cell sorting (MACS) from Miltenyi Biotec and EasySep from STEM CELL.ResultsMACS technology allowed a higher monocyte yield and purity and, by flow cytometry, monocytes displayed higher size and lower granularity. In the resting state, EasySep_MoDCs showed a higher basal expression of HLA-DR, and no significant response to stimulation by LPS and TNF-α. When stimulated with whole tumor cells lysates, both MoDCs expressed similar levels of maturation and co-stimulatory markers. However, when cultured with autologous T cells, MACS_MoDCs induced significantly higher IFN-γ secretion than EasySep_MoDCs, indicating a stronger induction of Th1 cell response profile. Concordantly, T cells induced by MACS_MoDCs also showed a higher release of cytotoxic granules when in contact with tumor cells.ConclusionsOverall, both the MACS and the EasySep isolation immunomagnetic technologies provide monocytes that differentiate into viable and functional MoDCs. In our experimental settings, resting EasySep_MoDCs showed a higher basal level of maturation but show less responsivity to stimuli. On the other hand, MACS_MoDCs, when stimulated with tumor antigens, showed better ability to stimulate Th1 responses and to induce T cell cytotoxicity against tumor cells. Thus, monocyte isolation techniques crucially affect MoDCs’ function and, therefore, should be carefully selected to obtain the desired functionality.
Highlights
Dendritic cells (DCs), which can be used as anti-cancer vaccines, are generally obtained in vitro from isolated CD14+ monocytes (MoDCs)
To determine the isolation yield, Peripheral blood mononuclear cells (PBMCs) and CD14+ cells were counted in the optic microscope, after each isolation step, and the number of CD14+ cells was divided by the initial number of PBMCs
EasySep monocytes showed an side scattered (SSC) of 6.58 ± 0.22 × 106, which was significantly higher than the SSC of 4.04 ± 0.15 × 106 observed in magnetic activated cell sorting (MACS)
Summary
Dendritic cells (DCs), which can be used as anti-cancer vaccines, are generally obtained in vitro from isolated CD14+ monocytes (MoDCs). We compared the most used immunomagnetic technologies for monocyte isolation: magnetic activated cell sorting (MACS) from Miltenyi Biotec and EasySep from STEM CELL. To obtain high cell purity, the immunomagnetic separation is preferred and two of the widely used immunomagnetic separation technologies are magnetic activated cell sorting (MACS) technology (Miltenyi Biotec) and EasySep technology (StemCell Technologies). Both frequently use positive selection where leukocytes are incubated with magnetic beads linked to an antibody that recognizes the CD14 antigen. EasySep technology is column-free and uses tetrameric antibody complexes recognizing CD14 and dextran-coated magnetic particles
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