Cryopreservation preserves the morpho-structural and mechanical integrity of human decellularized tracheas and supports a reduced immunogenic profile.

Background: A suitable tracheal substitute must support revascularization and regeneration, while resisting necrosis and infection. Decellularized tracheas are promising candidates; however, degradation limits their shelf life, making tissue banking essential. Although various decellularization methods have been developed, few studies assess the impact of storage, particularly cryopreservation, on tissue quality, limiting their clinical translation. Methods: In this study, decellularized tracheas and decellularized + cryopreserved tracheas were developed from three human donors, segmented and processed accordingly. These were compared to native tracheas in terms of macroscopic appearance and structural integrity. Thus, residual nuclei/DNA were assessed using DAPI staining and DNA quantification. Histological stains (Hematoxylin and Eosin, Alcian Blue, Masson's Trichrome, Weigert Van Gieson) assessed tissue and extracellular matrix architecture. Glycosaminoglycans and elastic fibers were quantified through both quantitative and semiquantitative methods. Immunostaining and semi-quantification for Human Leukocyte Antigen-DR (HLA-DR) was performed to preliminarily evaluate residual immunogenicity reduction. Ultrastructure and mechanical properties were analyzed using scanning electron microscopy and compression tests. Results: The decellularization protocol effectively reduced DNA content to <50 ng/mg, confirmed after cryopreservation. Native tracheas showed normal respiratory epithelium, whereas decellularized tracheas and decellularized + cryopreserved tracheas retained only the basal lamina. Submucosa was similar across groups, except for the absence of nuclei in treated samples. Glycosaminoglycans and collagen were well preserved, as showed by Alcian Blue and Masson's trichrome stainings. Elastic fibers integrity and content were reduced in accordance with Weigert Van Gieson staining and morphometric analysis. Only few HLA-DR immuno-positive elements were recognized after treatments, as confirmed by semi-quantitative analysis. Scanning electron microscopy revealed epithelial cell removal with preserved basal lamina and adventitial collagen, although less compact. Treated cartilage showed empty lacunae. Mechanical testing revealed no significant differences in stiffness between groups. Conclusion: Study results indicate that combining decellularization with cryopreservation effectively preserves tracheal structure and further reduces HLA-DR-immunopositive elements compared with decellularized trachea. These findings support the clinical potential of decellularized + cryopreserved grafts for tracheal replacement.