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10IMAGING BLOOD AND ENDOTHELIAL CELLS AND MEASURING THEIR INTERACTION FORCES
WITH LASER TWEEZERS
PETR ERMOLINSKIY1, OLGA SCHEGLOVITOVA2, MATVEY MAKSIMOV1, ANDREI LUGOVTSOV,1
and ALEXANDER PRIEZZHEV1
Faculty of Physics, Lomonosov Moscow State University, Russia 2N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Russia
ABSTARCT
Microrheology of blood depends on many different factors, i.e., red blood cells (RBCs) aggregation, interaction between blood cells, between RBCs and vascular endothelium, 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 [2]. 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 stationary state 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 [3]. The schematic layout of the setup is presented in Fig.1. The basic elements are the diode-pumped Nd:YAG laser (A = 1064 nm, MAX output power = 1 W), and the water-immersion objective OLYMPUS with high numerical aperture (NA = 1.00). The wavelength of the laser is in the range of optical transparency of hemoglobin to
minimize heating effect of the laser trapping [4].
CMOS
polarizing cube
polarizing cube
Nd:YAG
^^ _experimental
endothelium layer led) rbcs Figure 1: The schematic layout of laser tweezers.
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 [5]. Blood for experiment was drawn from the cubital vein of two healthy donors to obtain serum and whole blood with EDTA K3 anticoagulant. The sample to be measured comprised serum with added 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 (see the scheme of the cuvette in the Fig.1.). 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.
In the experiments, the interaction forces between RBCs and endothelium as well as the interaction forces between RBCs were measured. The interaction force between RBCs are the forces leading to the aggregation of RBCs (i.e., aggregation and disaggregation forces [6]). Force calibration was performed using Stokes force calibration [6].
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 force reaches saturation (see Fig.2.). It seems that saturation is reached when the concentration of fibrinogen is above the physiological limits.
Figure 2: The dependence of the interaction force between RBC and endothelium on the fibrinogen concentration. P-values is the
calculations using Mann-Whitney U test.
The expected results [7] were also obtained that the RBCs aggregation force as well as the RBCs disaggregation force increases monotonically with increasing fibrinogen concentration (see Fig.3.). Interestingly, the aggregation force between RBCs is correlated by value with the interaction force between RBCs and endothelium. These results are important for better understanding of RBC and endothelium interaction.
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c
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CD CI fij Cn
o
CD
tr
3-
2-
1 -
0
Equation Intercept Slope
Pearson's r Adi R-Square
</ = 3 + b'x
1-85 + 0-14 0.29 ± 003 0.98 0,98
Jt
l
N=2 healthy donors
I 125%-75%
I Mean ± 1 SD — Median Line Mean
12
10-
Equation y = a + b*x
intercept Slope 2.95 ±0.61 1.26 ±0.16
Pearson's r 0.98
Adj R-Square 0.96
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O m CH
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M=2 healthy donors]
I 125%~75% x Mean ± 1 SD — Median Line Mean
02468 0246
Fibrinogen concentration, mg/ml Fibrinogen concentration, mg/ml
(a) (b)
Figure 3: (a) The dependence of RBCs aggregation force on the fibrinogen concentration, (b) The dependence of RBCs
disaggregation force on the fibrinogen concentration. The endothelial part of 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». The measurements in this work were supported by the grant #21-2-10-59-1 from the Foundation for the Development of Theoretical Physics and Mathematics «BASIS».
REFERENCES
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[2] A. Pizov, N. Pizov, O. Skachkova, N. Pizovа, Endothelial function in normal and pathological conditions, Medical Council. 154-159, 2019 (in Russian).
[3] A. V. Priezzhev, K. Lee, N. N. Firsov, J. Lademann, 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 - ed., 2nd ed., SPIE Press Bellingham, WA, United States, 5-37, 2016.
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[5] O. N. Shcheglovitova, A.A. Babayants, N. N. Sklyankina, N. V. Boldyreva, D. L. Belyaev, I. S. Frolova, 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).
[6] P. B. Ermolinskiy, A. E. Lugovtsov, A. I. Maslyanitsina, A. N. Semenov, L. I. Dyachuk, A. V. Priezzhev, Interaction of erythrocytes in the process of pair aggregation in blood samples from patients with arterial hypertension and healthy donors: measurements with laser tweezers, J. of Biomedical Photonics & Eng., 4(3), 030303-1 - 030303-8, 2018.
[7] A. Semenov, A. Lugovtsov, P. Ermolinskiy, K. Lee, A. Priezzhev, Problems of red blood cell aggregation and deformation assessed by laser tweezers, diffuse light scattering and laser diffractometry, Photonics, 9(4), 238, 2022.
