Comparative characteristics of monocytes isolation conditions by adhesion method for in vitro experiments

Introduction. Monocytes and macrophages are cells that play an important role in the function of the innate immune system and may be involved in the pathological process in various diseases. For this reason, in vitro studies of the functional activity of these cells are of great scientific interest. The literature indicates a large number of methods used to isolate primary monocytes but the approaches are often controversial.
Aim. To perform a comparative analysis of the influence of various conditions on monocytes adhesion efficiency to determine the most optimal combinations of factors providing the highest yield and purity of the obtained cells.
Materials and methods. Peripheral venous blood samples were obtained from 6 healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by standard centrifugation on Ficoll density gradient (1.077 g/ml). After the centrifugation, PBMC were plated in wells of a 24-well plate, coated with collagen or uncoated, and incubated for 2 or 24 hours in RPMI-1640 medium containing 10% fetal calf (FCS) or autologous human serum (AHS). The number of attached monocytes was assessed by epifluorescence microscopy after staining with Hoechst 33342 with automatic counting of nuclei in ten fields of view.
Results. It has been established that incubation of cells for 2 hours in wells without coating in the presence of 10% FCS allows obtaining the largest number of monocytes with median purity of 80.9 (66.5-89.1)%. The number of monocytes obtained by adhesion in collagen-coated wells tended to be lower, regardless of the type of serum used. In our experiment, the adhesion capacity of monocytes was reduced when cells were plated in uncoated wells with 10% AHS. Extended to 24 hours incubation time was accompanied by a significant decrease in the number of attached monocytes in most cases.
Conclusion. Incubation of PBMC for 2 hours on uncoated tissue culture plastic in the presence of 10% FCS provides the highest yield and purity of monocytes in comparison with other tested conditions.

monocytes, macrophages, adhesion, inflammation

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