Effects of cryopreservation on the proliferation and anticoagulant activity of human saphenous vein endothelial cells

Abstract

Human saphenous veins were cryopreserved in 4% human albumin and 10% dimethyl sulfoxide. The effect of cryopreservation on endothelial cells was studied in terms of the anticoagulant activity of thrombomodulin and in terms of cell proliferation. After storage for 2 weeks at -150 degrees C, 0.45 +/- 0.07 x 10(5) endothelial cells/cm2 were detected in cryopreserved veins and 1.03 +/- 0.04 x 10(5) endothelial cells/cm2 in fresh veins (p < 0.01). The thrombin-catalyzed activation of protein C decreased after cryopreservation, indicating altered thrombomodulin activity in the endothelial cells. On a cell number basis, the release of soluble thrombomodulin was three times higher from the cryopreserved endothelium than from the fresh endothelium (p< 0.05). The amount of spontaneous release of von Willebrand factor from the endothelial surface was not significantly different between fresh and cryopreserved veins. Endothelial cells were cultured from fresh veins and from their cryopreserved counterparts. On plating of endothelial cells in primary culture, the number of adhered cells was 0.9 +/- 0.09 x 10(3) cells/cm2 from fresh veins and 0.25 +/- 0.03 x 10(3) cells/cm2 from cryopreserved veins (p < 0.01). The positive immunohistochemical stain for von Willebrand factor indicated that the endothelial cell character was maintained after cryopreservation. The endothelial desquamation with loss of anticoagulant function and the slow proliferation of surviving cells in vitro suggest an impaired endothelial healing in vivo. The loss of anticoagulant activity complicates the problems of the exposure of thrombogenic subendothelial matrix to blood in implanted cryopreserved veins.

Considering cellular thrombomodulin distribution and its modulating factors can facilitate the use of plasma thrombomodulin as a reliable endothelial marker?

“…R e g u l a t i o n f a c t o r s R e f e r e n c e T M e x p r e s s i o n C e l l s u s e d i n t h e e x p e r i m e n t s * The essential information for interpretation of pTM levels (Table 3B) deal with the mecha nisms by which endothelial TM is degraded and released into plasma (5,(70)(71)(72)(73). In the recent study by Boehme et al (71) the coordinated action of TNF-a and neutrophils resulted in TM release from EC, thereby resolving the apparent paradoxical increase of pTM in the presen ce of inflammatory cytokines.…”

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

“…Reduced proliferation rates, at least for the first 24 h of re-culture, were described for human umbilical cord cells 32,33 and human saphenous vein endothelial cells. 34 In our study, cell proliferation could be improved by use of solutions 1 or 2 compared to M199 (Figure 5). …”

Effect of cryopreservation on the properties of human endometrial stromal cells used in embryo co-culture systems

“…Disturbances in cellular functions due to freezing have been also detected in human granulocytes and endothelial cells. Cryopreservation has been shown to result in decreased thrombomodulin activity and increased release of soluble thrombomodulin in endothelial cells, indicating impairment of their anticoagulant function [50]. Boonlayangoor et al (1980) have reported a marked decrease in phagocytosis, killing activity, myeloperoxidase activity, nitroblue tetrazolium reduction, and chemiluminescence of cryopreserved granulocytes c.…”

“…In those patients who have one or more cycles passed between biopsy and ART procedure, the coculture is performed using cryopreserved autologous ESCs. Although fresh and frozen cells might exhibit similar morphological patterns and even comparable proliferation rates [49,50], it is quite possible that the cryopreservation process, as previously demonstrated with other cell types such as hepatocytes [51,52], chondrocytes [53], granulocytes [54], and endothelial cells [50], might affect their functional state and, in the case of ESCs, might influence their ability to support and improve embryo development in vitro.…”