tissue-derived decellularized biomaterials are ideal for tissue engineering applications as they mimic the biochemical composition of the native tissue. these materials can be used as hydrogels for cell encapsulation and delivery. the decellularization process can alter the composition of the extracellular matrix (ECM) and thus influence the hydrogels characteristics. The aim of this study was to examine the impact of decellularization protocols in ecM-derived hydrogels obtained from porcine corneas. Porcine corneas were isolated and decellularized with SDS, Triton X-100 or by freeze-thaw cycles. All decellularization methods decreased DNA significantly when measured by PicoGreen and visually assessed by the absence of cell nuclei. Collagen and other ECM components were highly retained, as quantified by hydroxyproline content and sGAG, by histological analysis and by SDS-PAGE. Hydrogels obtained by freeze-thaw decellularization were the most transparent. The method of decellularization impacted gelation kinetics assessed by turbidimetric analysis. All hydrogels showed a fibrillary and porous structure determined by cryoSEM. Human corneal stromal cells were embedded in the hydrogels to assess cytotoxicity. SDS decellularization rendered cytotoxic hydrogels, while the other decellularization methods produced highly cytocompatible hydrogels. Freeze-thaw decellularization produced hydrogels with the overall best properties.The extracellular matrix (ECM) is primarily composed of structural and regulatory proteins and polysaccharides and is generated and maintained by cells. Many cellular functions, such as proliferation, migration or differentiation are regulated by the ECM 1 . Each organ and tissue is composed of a distinctive ECM, in its biochemical composition and structural organization. The properties of ECM are important in the fields of tissue engineering and regenerative medicine, which often aim to replicate the composition and structure of the ECM. By using synthetic or natural materials, three-dimensional scaffolds can be fabricated to repair or restore damaged organs and tissues.One popular approach to generating scaffolds that try to imitate the tissues or organs ECM characteristics is to use decellularization. This technique involves the removal of cellular components from a tissue so that only the ECM remains. Many methods have been examined for performing decellularization and these can be divided into three main categories: physical, chemical and biological 2 . Physical methods include freeze-thawing cycles 3-6 , high hydrostatic pressure 7-9 or supercritical CO 2 10-12 . Chemical agents can involve ionic detergents, such as sodium dodecyl sulphate (SDS) 13,14 or sodium deoxycholate 15 ; non-ionic detergents, such as Triton X-100 16 ; hypertonic or hypotonic salt solutions, such as sodium chloride 17,18 ; and acids and bases, such as peracetic acid 19 or ammonium hydroxide 20 . Enzymes such as trypsin, dispase and phospholipase A2 have been used as biological methods for decellularization 21,22 . Furt...