Developing a decellularization process for tuna corneas

Introduction: One of the main challenges of Tissue Engineering is to obtain a biomaterial that can reproduce, at the same time, the physicochemical, mechanical, and biological characteristics of the extracellular matrix of organic tissues. The use of decellularized extracellular matrix (dEM), i.e., a native tissue from human or animal sources that undergoes the process of cell removal, preserving the extracellular polymers, has proven to be a promising alternative. The use of dEM aims to preserve as much of the polymeric diversity as possible, both for the delivery of the biochemical signals that cells need to develop on the scaffold and to avoid the decrease of mechanical and physicochemical properties of these biomaterials due to extraction and purification processes. Objectives: The main purpose of this work was to develop a decellularization process for tuna (Thunnus albacares) corneas. Methods: Thus, we studied four decellularization strategies: (M1) a physical method (5 freeze-thaw cycles at -80°C, F/T); (M2) a chemical method (immersion in Triton X-100 for 72h); (M3) 5 cycles of F/T combined with immersion in Triton X-100 for 24h and (M4) 5 cycles of F/T combined with enzymatic treatment (DNAse in 10mM Mg2+) for 1h. The efficiency of the decellularization strategies was analyzed by DNA quantification and histology (hematoxylin-eosin). Results: M1, M2, M3, and M4 treatments were able to reduce tissue DNA content by 60%, 75%, 61%, and 91%, respectively. Histological analysis did not detect intact cell nuclei in any of the treatments. Regarding the density of collagen fibers, M1 resulted in the greatest loss and separation of collagen fibers, followed by M2 and M3 which had similar results, and by M4, with the greatest preservation of matrix proteins. Conclusions: Thus, M4 was the best method tested for the decellularization of tuna corneas as it fulfilled both prerequisites for efficient decellularization: eliminate DNA and preserve the tissue matrix. This is the first work to develop a method for the decellularization of tuna corneas and represents the first step to developing a decellularized matrix for human cornea regeneration from fish sources.