Cell origins and fates following transplantation of cryopreserved allografts

Efforts in preservation of allograft heart valves aim at remaining cellular viability. Viability in this respect always means fibroblast viability as endothelial cells are almost completely lost in the sequence of dissection, sterilization, cryopreservation, thawing and implantation.1,2 Cryopreservation is now considered to be the method of choice of allograft heart valve preservation and banking. Cellular viability after cryopreservation has been well documented. 3'5 The length of donor ischemia time is one of the factors that influence the quantity and quality of remaining viable donor cells.6,7 Cytotoxicity of antibiotics and DMSO, and to a lesser degree cryopreservation itself also are of negative influence on cellular viability.8'10 The fate of the donor cells after allograft implantation has been object of discussion. It has been stated that the presence of viable donor cells after implantation is essential for graft function and longevity.Viable donor cells were believed to play a key role in maintaining the integrity of matrix and fiber structures in the valve leaflets.3,11The number of publications contradicting this theory is increasing. Explant studies commonly show little or no cellularity. 2,12,13 Furthermore, the presumed presence of viable donor cells increases antigenicity, which is thought to be detrimental to allograft function and longevity.13'15 Cellular elements presenting in explanting allografts may be of donor or recipient origin. Before questions concerning the role of surviving donor cells can be answered we must be able to distinguish between donor and recipients cells. The origin of fibroblasts and other cells in allograft explants can be determined if a sex difference between donor and recipient is present. Many techniques are dependent on tissue culture. Demonstration of Barr bodies in cultured fibroblasts is another sex-related technique that has been used to demonstrate the presence of remaining donor fibroblasts.11 DNA fingerprinting is a third method to distinguish between donor and recipient cells. DNA fingerprinting can distinguish between cells without the need of a sex difference.2 These three techniques (Barr body demonstration, chromosome banding and DNA fingerprinting) share one major disadvantage: They cannot be used to determine cell origin on location. Thus, the presence or absence of donor and recipient cells can be confirmed in allograft explants but morphology, number distribution and localization of these cells will remain unknown. The above listed disadvantages can be overcome with the aid of in situ hybridization. If a sex mismatch between donor and recipient is present, DNA in situ hybridization for the Y chromosome can reliably distinguish between donor and recipient cells on location, that is in the tissue sections. Sensitivity and specificity of this technique are high. As this method can be applied on location, the morphology and distribution of donor and recipient cells can be established. Semi-quantitative estimation of both donor and recipient cell populations can be obtained. Using a DNA probe for the porcine Y chromosome we were able to determine accurately the origin and localization of both donor and recipient porcine cells in explanted porcine cryopreserved aortic allograft valves.16 Analysis of human allograft explants with the aid of in situ hybridization for the Y chromosome has started more recently in our department. The technique of ISH in itself cannot determine whether a cell is viable or not. Hybridization requires an intact DNA, but the fact that hybridization occurs does not necessarily mean that the cells are capable of protein synthesis. In our studies, cellular morphology was used to support the probability of viable cells.