Effects of cryopreservation on tracheal allograft antigenicity in dogs

Abstract

Background: Prolonged cryopreservation (~8-10 months) has been shown to reduce the antigenicity of tracheal allografts due to subsequent denuding of epithelium. In the present study, tracheal epithelium was assessed after variable periods of cryopreservation. Immunosuppressant-free allotransplantation was then undertaken to evaluate the impact of cryopreservation on tracheal antigenicity in dogs. Methods: Tracheal rings [7][8] were removed from mongrel adult dogs for cryopreservation (1-10 months, −85 ℃) and grafting. Before transplantation, one ring was sectioned from each end for histologic examination. The residual five-ring segments of mediastinal trachea were then transplanted into recipient dogs after 1-7 months (group 1, n=9) or 8-10 months (group 2, n=6) of cryopreservation.Anastomotic sites and allografts were covered by omental pedicles. No immunosuppressants whatsoever were administered.Results: In microscopic views, the ciliated tracheal epithelium of most grafts showed variable loss but was generally intact after cryopreservation, still demonstrating major histocompatibility complex (MHC)-II positivity. By postoperative bronchoscopy, allografts in both groups had largely developed lethal strictures. In group 1, eight dogs were sacrificed or died within 50 days post-transplantation, whereas survival times in group 2 were somewhat longer, with three dogs surviving for >60 days. Upon sacrifice, histologic preparations of grafted tissue in both groups were typically denuded of epithelium, with marked lymphocytic/monocytic submucosal infiltrates. Tracheal cartilage had been absorbed or destroyed.Conclusions: After cryopreservation, some degree of tracheal epithelium loss maybe expected, but complete denudation is not obligatory. Retained epithelial antigenicity is thus capable of triggering rejection, resulting in transplant failures. Although prolonging transplant survival to an extent, fatal rejection of tracheal allografts was not preventable by prior cryopreservation.

Development and In Vitro/In Vivo Comparative Characterization of Cryopreserved and Decellularized Tracheal Grafts

“…CryoT and DecellT grafts were preliminarily compared by H&E for an overall overview on tissue organization and/or evidence of eventual modification/disruptions. Cryopreservation only caused a partial exfoliation of the respiratory epithelium, in accordance with many other studies reported in the literature [ 24 , 60 , 83 , 97 ]. In accordance with Nakanishi et al [ 83 ] it is possible that partial maintenance of epithelial cells in tracheal allografts, eventually capable of survival, may be ascribed to cryoprotection itself.…”

“…The first preclinical study (pig) reporting about cryopreserved tracheal graft implantation in orthotopic position was described by Lenot et al [ 68 ]. Despite surgery failure due to inadequate blood supply, the histologic structures and the mechanical properties were well preserved, stimulating intense research on this substitute type [ 15 , 24 , 26 , 28 , 38 , 60 , 61 , 67 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 ]. Hence, focusing on freezing temperature and liquid nitrogen storage, cryopreserved tracheal graft protocols can be distinguished into two groups: (a) freezing at −60° to −140 °C and storage in liquid nitrogen until use; (b) freezing at −80°/−85 °C, without recurring to liquid nitrogen storage [ 24 ].…”

“…These are intriguing data, considering the limited tissue manipulation required by the method. Cells were still recognizable in each of the tissue three-layers; however, cryopreservation showed its potential in the tissues’ antigenicity modification, suggesting that freezing periods modulation might further decrease the immunogenic potential of the tissue [ 8 , 25 , 74 , 88 , 97 ]. Intermittent immunosuppression was reported in preclinical studies [ 86 ], even though tracheas implant without recurring to immunosuppression, was also successfully assessed [ 72 , 74 , 80 , 89 , 96 ].…”

“…It is very important to confirm the immunomodulatory effect of cryopreservation on tracheal allografts to allow potential clinical application of tracheal transplantation in the future [ 8 ]. Intense research efforts focusing on freezing time, cooling speed, cryoprotectant type/mixture may guarantee for the development of effective, cost-saving and safe allografts [ 23 ], not eliciting adverse immune reaction but also growing without calcifications and remaining patent [ 89 , 90 , 97 ]. Within this scenario, xenografts may represent a valuable resource to bridge the gap between the supply and demand of organs/tissues; however, immunological barriers must be considered as xenotransplants are prone to rejection [ 128 ].…”

Conduits for Right Ventricular Outflow Tract Reconstruction in Infants and Young Children

“…There is no doubt that the cryopreserved homograft is immunogenic as the cryopreserved technology retained lots of allogeneic cells ( 29 ). The graft-related immune response plays an important role in the failure of cryopreserved homograft in young children ( 38 ).…”