METHODS FOR STUDYING MACROMOLECULAR CROWDING IN THE CYTOPLASM OF RED BLOOD CELLS

Introduction – the relevance of the study is due to the fact that macromolecular crowding reflects the actual conditions of the intravascular blood environment. Using PEG and dextran as model crowders allows for the study of structural changes in red blood cells, including aggregation and membrane integrity. The aim of the study is a comprehensive investigation of the impact of macromolecular crowding on the condition of human red blood cells.

Materials and methods. A suspension of human RBCs was isolated from donor blood via standardized centrifugation and washing. Structural and morphological changes in individual RBCs were quantitatively analyzed using Laser Interference Microscopy (LIM). This label-free, high-resolution technique allows for the precise, real-time measurement of key cellular parameters, including optical path difference (OPDmean, correlating with intracellular dry mass density), cell volume, phase diameter, and phase height. RBCs were incubated in isotonic solutions containing varying concentrations
(0–300 mg/mL) of the crowding agents.

Results. In the course of the study, the polymers were tested for their ability to induce hemolysis and erythrocyte aggregation, and it was found that PEG 8000 and 40 kDa dextran at concentrations of 200 and 300 mg/mL in samples failed this test. When incubating RBCs in dextran (100 mg/mL), a decrease in OPDmean and cell volume by 20.9 % and 7.2 % relative to the control, as well as an increase in phase diameter, was observed. In the presence of PEG (100 mg/mL), an increase in the phase height of RBCs by 6 % was observed with unchanged volume and a decrease in phase diameter.

Discussion and conclusion. This study demonstrates that macromolecular crowding, simulated by inert polymers, exerts a profound and chemically specific influence on RBC morphology beyond simple osmotic effects. The divergent effects of dextran (inducing dehydration and flattening) and PEG (promoting shape change without volume loss) underscore the critical role of the crowding agent's physicochemical properties. These findings have direct translational implications for the rational design of infusion solutions and blood substitutes, the development of accurate in vitro biomimetic models of blood, and the refined interpretation of RBC morphology in clinical pathologies associated with altered plasma composition. The work provides essential quantitative data for specialists in hematology, biochemistry, biophysics, and clinical medicine, establishing a foundation for further research into the role of molecular crowding in RBC pathophysiology and storage biology.