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

Pavel Měřička,Radka Lainková,Myroslav Salmay,Miroslav Špaček,Ján Sabó,Miroslava Jandová,Libor Janoušek,Petra Prokšová,Rudolf Špunda,Jaroslav Lindner,Ondřej Pecha,Lubomír Štěrba,Jiří Gregor,Aleš Benda,Markéta Dalecká

doi:10.3390/ijms221910653

Abstract

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.

Highlights

Introduction conditions of the Creative CommonsThe authors present their contribution to the improvement of methods suitable for detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures
Slightly lower viability was found in longitudinal cuts (LC) (Table 1, Figure 1), but the difference was still not significant (p = 0.0996)
The post-thaw cell viability was relatively stable during the 48 h culture of tissue slices, the interval in which the delayed onset of cell death regularly occurs [7]

Summary

Introduction

Introduction conditions of the Creative CommonsThe authors present their contribution to the improvement of methods suitable for detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. One approach is based on the isolation of cells by tissue digestion, followed by the application of the earlier mentioned cell viability and phenotyping assays Another approach is based on in situ vital staining of cells [5]. Innovative approaches to cell viability assessment consider the results of tests performed immediately after thawing, and the changes in the post-thaw cell viability through time [6,7,8] This approach enables the detection of cryopreservation and storage induced apoptosis [9,10,11] and/or the delayed onset of cell death, leading to cell necrosis, apoptosis, or a combination of both [2,3,11]. In the case of solid tissue transplantation, the graft immunogenicity and/or the intensity of the graft rejection can be modified [2,15]

Objectives

Methods

Results

Discussion

Conclusion