Influence of deep freezing on cellular structure of the human red blood cells

Abstract
Atomic force microscopy (AFM) technique was used for assessment of structural changes in red blood cells (RBCs) subjected to cryopreservation with 40% glycerol. This freezing regimen has been shown to exert some controversial effects upon RBCs structure in final cell concentrates. According to AFM scanning of RBCs suspensions, two subpopulations of the cells could be revealed. One of them represents glycerol-absorbing cells, whereas second subpopulation seems to exhibit glycerol-free cell surfaces. The glycerol-absorbing RBCs are subject to picnotic transformation, being compressed, mainly, at their short axis and disk height. By contrary, RBCs with relatively clean surface have an increased volume, with their linear dimensions exceeding control values. Glycerol is adsorbed at the cell surface as discrete particles of up to 1,5 microns in size. The particles are assembled into typical  branched structures which are detectable both at RBCs surfaces, as well as  at substrate layer. Some RBCs strongly adsorb and entrap glycerol at the surface of their cytoplasmatic membranes. Following a routine technological washing, the RBCs, while being devoid of glycerol, seem to open their membranes pores, thus accelerating transmembrane exchange and promoting their functional recovery (endocellular hypostasis). Hence, application of high glycerol concentrations for cryopreservation requires an optimised washing technology to obtain glycerol-free suspensions of red blood cells.