Immunogenicity, Antigenicity, and Endothelial Viability of Aortic Valves Preserved at 4°C in a Nutrient Medium

In patients undergoing aortic valve replacement, allograft valves stored at 4OC in a nutrient medium have been associated with excellent immediate and long-term results. The effects of this method of prolonged storage on the antigenic, immunological, and cellular characteristics of these grafts are incompletely understood. This study was designed to study these phenomena in rat aortic valves subjected to antibiotic sterilization and stored for up to 3 weeks in RPMl containing 10% fetal calf serum. Selected valves from Brown Norway rats were implanted heterotopically into the abdominal aorta of Lewis rats. Other valves were studied prior to transplantation. Antigenicity was determined by immunocytochemical staining using monoclonal mouse antibodies directed at Class I and Class II rat antigens. lmmunogenicity was determined by duration of second-set skin graft survival following heterotopic aortic valve implant. Endothelial cell viability was determined by flow cytometric analysis of endothelial cells harvested from aortic valve allografts by collagenase digestion. Only fresh valves and valves stored for 1 day were positive for Class I antigens; no valves were positive for Class II antigens. Duration of skin graft survival was prolonged with greater duration of storage, but grafts remained immunogenic after 21 days of storage. Endothelial cell viability declined from 95% in the fresh valves to 64% after 21 days of storage. With prolonged duration of allograft valve storage at 4 "C, there is an attenuation of antigenicity, immunogenicity, and endothelial cell viability. Loss of endothelial cells may contribute to the changes in immunological responses to the valve allografts. The expression of antigens on the endothelial surface is not a reliable predictor of immunological response. Human valve allografts have been successfully used for a variety of cardiac surgical proceSupported by grant R01 HL 42426 from the National Heart, Lung, and Blood Institute. Presented at the V International Symposium on Cardiac Bioprostheses, Avignon, France, May 24-27, 1991. Address for correspondence: Flavian M. Lupinetti, M.D., University of Michigan Hospital. Section of Thoracic Surgery, 2120 Taubman Center, Box 0344, Ann Arbor, MI 48109 Fax: (313) 764-2255. dures. One limit on the application of these useful materials is their potential for degeneration. Methods of allograft treatment and storage may exert a powerful influence on ultimate allograft longevity and function. Storage of allografts at 4OC is a time-tested method of preservation that has proven acceptable in achieving reliable clinical results.',* However, other methods of graft treatment and storage have been devised that could improve the quality of the grafts and ex454 Journal of Cardiac Surgery Vol. 6, No. 4 , 1991 VIABILITY OF AORTIC VALVES PRESERVED IN NUTRIENT MEDIUM tend the duration of storage that the grafts will tolerate. The effects of the various preservation techniques on specific characteristics of the allografts are not well defined. Among these characteristics that may influence the pathological fate of allografts are the intrinsic antigenicity of the graft, the immunological response of the host to the allograft, the viability of the allograft, the site of origin of the allograft (pulmonary or aortic), and probably other factors not yet defined. The purpose of this investigation was to examine three potential determinants of allograft function: antigenicity, immunological response, and endothelial cell viability, in experimental aortic allografts subjected to various durations of storage at 4OC. One goal of this study was to discern correlations among these factors, with the intent of learning how to predict the in vivo response to allograft insertion from features that could be observed prior to implant.