Purpose: Patients with tracheal lesions that exceed half of the trachea’s total length require a tracheal substitute. Tissue engineering, using either synthetic materials or decellularized tracheal tissue, opens up new possibilities for generating tracheal substitutes. Decellularization, a procedure in which the tissue's immunogenic cellular material is removed while the extracellular matrix (ECM) is preserved, seems the most promising approach. The majority of tracheal decellularization methods, however, are reliant on harsh chemicals and require lengthy wash procedures, resulting in damage to the ECM. To address these issues, a supercritical carbon dioxide (scCO2) decellularization approach has been suggested as an alternative solution due to its ability to both decellularize and sterilize tissues while leaving no toxic residues and requiring less treatment time. Therefore, the aim of this thesis was to compare scCO2 treatment with a chemical decellularization method, which is the current gold standard reported in literature, for creating a decellularized porcine tracheal scaffold with good cytocompatibility.  Methods: A total of five different protocols were tested that varied in decellularization and sterilization methods used. Decellularization efficiency was evaluated in terms of DNA content, histological appearance, and retention of ECM components. Additionally, mechanical tensile testing and scanning electron microscopy were used to assess the effects of the different decellularization protocols. Further, decellularized scaffolds were recellularized with fibrin-encapsulated porcine adipose derived stem cells to assess the cytocompatibility of the scaffolds. Results: The highest reduction in DNA content was observed when samples were subjected to the detergent-enzymatic protocol, followed by sterilization with gamma irradiation, and when samples were subjected to scCO2 treatment, followed by washing with sodium hydroxide. The latter protocol, however, also negatively impacted the ECM, whereas good preservation of ECM components was seen with the DEM protocol. DNA and histological analysis showed that treatment with scCO2 in combination with a hydrogen peroxide (H2O2) washing step was unable to completely decellularize porcine tracheas. Static surface seeding of the decellularized scaffolds led to poor cell adherence. Cells encapsulated in fibrin and seeded on the tracheal scaffolds were able to adhere and survive, showing the cytocompatibility of the decellularized scaffolds.  Conclusions: Decellularization with scCO2 in combination with a H2O2 washing step was not successful in completely decellularizing the porcine tracheas, and possibly requires the use of a co-solvent or secondary agent for successful decellularization. For recellularization of decellularized tracheal scaffolds, the use of fibrin as a cell carrier is an effective and simple seeding method. The findings in this thesis open up new avenues for potential optimizations in future research.