Interaction of red blood cells and endothelium at single cell level in vitro

Abstract

Microrheology of blood depends on many different factors, i.e., red blood cells (RBCs) aggregation, interaction between blood cells, endothelium function, etc [1]. Endothelial cells are the cells that line the interior surface of blood arteries, veins, and capillaries. Endothelium not only forms an insulating layer between blood and tissues, but also plays an important role in the regulation of blood flow in vessels due to the atrombogenicity of the cell membrane under physiological conditions. Factors leading to the development of inflammatory process and other pathological conditions change the anticoagulant state of endothelium into procoagulant one. As far as endothelium interacts directly with blood cells, this interaction may change RBCs aggregation, for instance. RBCs aggregation is the reversible process of linear or more complex structures formation under low shear stress forces. Varying RBCs aggregation can change dramatically the viscosity of blood. It is well known that RBCs aggregation can occur only in the solution with high molecular weight molecules. In blood plasma the fibrinogen protein molecule is the main inducer of RBCs aggregation. Increased concentrations of fibrinogen in blood can cause thrombosis and vascular damage in case of inflammation and several diseases [1].
The main goal of this study was to investigate the interaction of RBCs of healthy donors with endothelial cell monolayer as well as the interaction between RBCs at different concentrations of fibrinogen at single cell level in vitro using laser tweezers.
Laser tweezers are a scientific tool allowing to trap and manipulate single living cells and measure the interaction forces between the cells. In this work, we used two channeled home-made laser tweezers based on Nd:YAG laser (1064 nm) [2].
Human umbilical vein endothelial cells were grown on cover glasses placed in 24-well plates in a CO2 incubator at 37 °C until a monolayer was formed [3]. Blood for experiment was drawn from the cubital vein of a healthy donor in order to obtain serum and whole blood with EDTA K3 anticoagulant. The sample to be measured comprised serum with added particular fibrinogen concentration and a small amount of blood (1:1000). The following concentrations of fibrinogen in serum were applied: 0, 2, 4, 6, 8 mg/ml. The sample was placed into the cuvette based on glass slide upon which a cover glass with endothelium monolayer was placed. Ordinary cover glass positioned atop, and vacuum gel was used for air isolation of the cuvette. The sample and the cover glass with endothelium monolayer were placed into the cuvette a few minutes before the measurement. Measurement of each one concentration took 30 min and was performed under room temperature.
Our measurements showed that with increasing fibrinogen concentration the interaction force between RBCs and endothelium increases up to fibrinogen concentration of 4 mg/ml, and in the range from 4 to 8 mg/ml the interaction
orce reaches saturation. The expected results were also obtained that the RBCs aggregation force increases monotonically with increasing fibrinogen
concentration. These results are important for better understanding of RBC and endothelium interaction.
This work was supported by the Russian Science Foundation (Grant No. 22-15-00120) and performed according to the Development program of the Interdisciplinary Scientific and Educational School of Lomonosov Moscow State University «Photonic and Quantum Technologies. Digital medicine».
[1] Baskurt O., Neu B., and Meiselman H. “Red Blood Cell Aggregation”, CRC Press, Boca Raton, United States, 2012.
[2] Priezzhev A. V., Lee K., Firsov N. N., and Lademann J. “Optical Study of RBC Aggregation in Whole Blood Samples and on Single-Cell Level,” Chapter 1 in “Handbook on Optical Biomedical Diagnostics”, V. V. Tuchin – editor, 2nd Edition, SPIE Press Bellingham, WA, United States, 2016.
[3] Shcheglovitova O. N., Babayants A.A., Sklyankina N. N., Boldyreva N. V., Belyaev D. L., and Frolova I. S. "Primary culture of human vascular endothelial cells exhibits interferon-producing, antiviral and immunomodulatory activity under the influence of immunomodulators". Immunology, 33 (3), 116-119, 2012 (in Russian).


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Ermolinskiy Petr
aculty of Physics, Lomonosov Moscow State University, Moscow, Russia
Russia

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