The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.

Introduction. The cardiovascular homograft banks in Italy were set up in 1994 in Milan (Lombardia) and Treviso (Veneto) and in 2001 in Bologna, Emilia Romagna. In this study we briefly summarize the data from Emilia Romagna Cardiovascular Tissue Bank.Material and Methods. In Emilia Romagna, vascular homografts were harvested from brain-dead multiorgan donors (aged 15-55 years) by a dedicated vascular surgery team. All donors were virologically screened for human immunodeficiency virus (HIV), hepatitis B and C, Treponema pallidum, cytomegalovirus (CMV), and Toxoplasma. After transferring the vascular homografts to Emilia Romagna Cardiovascular Tissue Bank facilities, the arteries were prepared, classified (class III to 1), and transferred to an antibiotic-containing solution under a laminar flow cabinet. After the decontamination, all homografts were cryopreserved and stored in the vapour phase of liquid nitrogen. Microbiological tests were performed in all phases of preparation. Samples were routinely taken from I vessel and formalin fixed for the histology. Bags with cryopreserved homografts were sent in dry ice to the hospitals when required and thawing protocol of the Bank was included.Results and Conclusions. From January 2002 to October 2004, 543 homografts from 125 heart-beating donors were harvested and transferred to Emilia Romagna Cardiovascular Tissue Bank. After preparation, 459 of 543 (85%) were cryopreserved and stored. Vascular homografts classified class I were discarded. Other criteria of rejection were: (1) positive serology, and (2) persistent positive microbiology after decontamination. From March 2002, 333 cryopreserved homografts were assigned to several vascular surgery departments in Italy. The assessment of 3-year activity of Emilia Romagna Cardiovascular Tissue Bank might be used as an indicator of the efficiency of selecting, cryopreserving, and allocating quality-controlled vascular homografts.