Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

The Effect of Different Thawing Rates on Cryopreserved Human Iliac Arteries Allograft's Structural Damage and Mechanical Properties.

INTRODUCTION: The rate of thawing of cryopreserved human iliac arteries allografts (CHIAA) directly affects the severeness of structural changes that occur during this process. METHOD: The experiment was performed on ten CHIAA. The 10% dimethylsulphoxide in 6% hydroxyethyl starch solution was used as the cryoprotectant; all CHIAA were cooled at a controlled rate and stored in the vapor phase of liquid nitrogen (-194°C). Two thawing protocols were tested: (1) placing the CHIAA in a water bath at 37°C, and (2) the CHIAA were thawed in a controlled environment at 5°C. All samples underwent analysis under a scanning electron microscope. Testing of the mechanical properties of the CHIAA was evaluated on a custom-built single axis strain testing machine. Longitudinal and circumferential samples were prepared from each tested CHIAA. RESULTS: Ultrastructural analysis revealed that all five CHIAA thawed during the thawing protocol 1 which showed significantly more damage to the subendothelial structures when compared to the samples thawed in protocol 2. Mechanical properties: Thawing protocol 1-longitudinal UTS 2, 53 ± 0, 47 MPa at relative strain 1, 27 ± 0, 12 and circumferential UTS 1, 94 ± 0, 27 MPa at relative strain 1, 33 ± 0, 09. Thawing protocol 2-longitudinal ultimate tensile strain (UTS) 2, 42 ± 0, 34 MPa at relative strain 1, 32 ± 0, 09 and circumferential UTS 1, 98 ± 0, 26 MPa at relative strain 1, 29 ± 0, 07. Comparing UTS showed no statistical difference between thawing methods. CONCLUSION: Despite the significant differences in structural changes of presented thawing protocols, the ultimate tensile strain showed no statistical difference between thawing methods.