Dry Preservation Of Tissue Engineering Of Skin With Trehalose

Tissue engineering of skin (TES) was thought to have such obvious clinical benefits that it led to inspiring clinical and commercial expectations. However, there is a huge gap between demands of patients and short "shelf time" of already slowly made products. Exploring new and more effective preserve methods are urgently needed. Nowadays to preserve living cells in engineering skin substitutes is one of the main problems and cryopreservation cannot avoid the inevitable damage caused by ice crystals formed in thawing process.Cryopreservation has been successfully adopted in the preservation of suspended cells. However, it is difficult to gain similar achievement when it comes to storage of TES because of the inevitable damage of ice crystals. Trehalose, a non-reducing disaccharide, is thought to have special property to confer desiccation tolerance on organisms that withstand desiccation. We presumed that by increasing the desiccation tolerance of fibroblasts though trehalose loading we can introduce freeze-drying preservation as a new way to storage TES with living cells, avoiding cellular damage by ice crystals.In this paper, experiments were performed to indentify the optimal conditions to introduce trehalose into fibroblasts and then evaluate the performance of trehalose-loaded cells when suffering freeze-drying and air-drying. Subsequently, we constructed TES with trehalose-loaded fibroblasts and collagen of rat tails and explore the possibility of drying preservation by structure observation and viability assessment in vitro and in vivo.1. The trehalose loading conditions were measured as a function of extracellular trehalose concentration, time and temperature. The optimal conditions were turned out that fibroblasts were loaded in medium with200mM as extracellular trehalose concentration for8h at37℃. Two methods were tried in this paper to dehydrate cells, including freeze drying and air drying. According to MTT assay, cells suffered freeze drying retained30%viability, while cells suffered air drying revived40%. Membranes were mostly maintained intact by fluorescence staining and SEM.2. We successfully constructed TES with trehalose-loaded fibroblasts and rat-tailed collagen. Suffering from freeze drying and rehydration, TES with trehalose maintained26%viability according to MTT assay and kept structure intact by SEM. In vivo analysis was then performed by transplantation into C57BL/6J rats with dlcm full thickness skin defect at the back. Results showed that the recovery rate of TES with the present of trehalose was30%faster than that of hydrogel without trehalose. There is no significant difference between TES with trehalose and TES without freeze drying. With the present of trehalose, we introduce freeze drying into the preservation of TES. The results suggested that the viability of TES were indeed being preserved to some extent and therefore provided a new way to preserve TES.