Ultrastructural Changes in Cryopreserved Tracheal Grafts of Sprague-Dawley Rats

Abstract

It has been reported that immunosuppressant-free tracheal transplantation can be achieved by using cryopreserved grafts. However, the ultrastructural changes of cryopreserved tracheal tissue remain unclear. This study investigated this issue in cryopreserved tracheal grafts of 30 Sprague-Dawley (SD) rats, divided into five groups (n = 6 rats/group). Tracheal rings removed from them were stored in a deep freezer at -85 degrees C for 1 week, 1 month, 3 months, or 6 months. Never frozen tracheal tissues were obtained from the control group. Ultrastructural changes in the cryopreserved tracheas of each group were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As the duration of cryopreservation lengthened, tracheal grafts were observed by SEM to develop an obviously speckled depletion of epithelium, accompanied by a loss of cilia in the remaining epithelium. TEM examination of chondrocytes that had been cryopreserved for 3 months revealed obvious injury. Cell membranes and nuclei were frequently observed to be ruptured, and fibrils appeared disorganized and randomly oriented. As the time of cryopreservation increased, ultrastructural injury to tracheal chondrocytes became more severe. The data indicated that the optimal maximum time for cryopreservation of tracheal grafts of SD rats might be <3 months with our methods.

Effects of cryopreservation on tracheal allograft antigenicity in dogs

“…In a related experiment, we investigated the ultrastructural changes of cryopreserved tracheal rings in rats. As cryopreservation time increased, ultrastructural injury to tracheal chondrocytes became more pronounced (26). In particular, we consider tracheal epithelial separation as one manifestation of thermal damage.…”

Is Tracheal Transplantation Possible With Cryopreserved Tracheal Allograft and Hyperbaric Oxygen Therapy? An Experimental Study

“…[6]. However, ultrastructural damage has been reported with tracheal cryopreservation periods longer than 3 months [5]. On the basis of these observations, the cryopreservation period in this study was limited to 6 weeks, and epithelium denudation was observed in the cryopreserved trachea in our study.…”

“…Allografts have come into prominence due to their natural physiologic and anatomic structure, which preserve the flexibility of the respiratory tract and enable lumen patency [1,2]. In addition, previous studies have shown that the immunomodulatory effects of cryopreservation prevent rejection of tracheal allografts, thus reducing antigenicity [1,[4][5][6].…”

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

“…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 ].…”

“…Freezing and thawing may induce a depletion and loss of class II antigen expression amenable to respiratory epithelium [ 24 , 76 , 79 , 91 , 96 , 127 ]. Together with these promising data, contrasting evidence also arose suggesting that no effect was exerted by prolonged periods of cryopreservation on the chondrocytes’ viability and thus tracheal allogenicity [ 83 , 85 , 92 ]. 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 ].…”