Mimicking of Blood Flow Results in a Distinct Functional Phenotype in Human Non-Adherent Classical Monocytes

Abstract

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented.

Negative Magnetic Sorting Preserves the Functionality of Ex Vivo Cultivated Non-Adherent Human Monocytes

“…The activation of monocytes was induced experimentally by the granulocyte-macrophage colony-stimulating factor (GM-CSF), which is often applied to the culture of mature myeloid cells as it acts as a pro-survival and activating factor [ 25 , 26 ]. According to previous results, the activation was confirmed by the measurement of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) [ 22 ]. Moreover, we evaluated the effect of isolation method on LPS responsiveness by measuring the TNF-α secretion.…”

“…Monocytes were isolated from a total of 19 buffy coat (BC) samples, provided by the Department of Transfusion Medicine, Rostock University Medical Center, Rostock, after written consent for scientific use (approval number: A2011-140, Rostock University Ethics committee) and prepared as previously described [ 22 ]. At the time of monocyte isolation, the BCs were no more than 4 h old.…”

“…Monocytes were isolated from human BCs either by positive or negative selection. The purity of CD14 + cells was analyzed by flow cytometry directly after isolation, as previously described [ 22 ]. After isolation, the cells were adjusted to a final concentration of 1 × 10 6 viable cells/ml and seeded in 24-well cell-repellent plates (Greiner Bio-One, Leipzig, Germany).…”

“…These parameters may be associated with the functionality of monocytes intended for application in cell-based therapeutic products. In contrast to other methodological studies [ 18 , 20 ], we prevented the adherence of monocytes by using cell-repellent surfaces as previously described [ 22 ] to avoid adhesion-induced differentiation into macrophages [ 23 ]. In particular, our study aimed to preserve the migratory capacity of the monocytes, which is severely limited with longer culture duration [ 22 ] and under adherent culture conditions [ 24 ].…”

“…In contrast to other methodological studies [ 18 , 20 ], we prevented the adherence of monocytes by using cell-repellent surfaces as previously described [ 22 ] to avoid adhesion-induced differentiation into macrophages [ 23 ]. In particular, our study aimed to preserve the migratory capacity of the monocytes, which is severely limited with longer culture duration [ 22 ] and under adherent culture conditions [ 24 ]. Furthermore, we evaluated the effects of the isolation method on unstimulated (naïve) and activated monocytes.…”