Tracheal abnormalities, congenital or acquired, represent a currently unmet clinical need. Tissue engineering has recently advanced and has been used to engineer hollow organs, including tracheae, for clinical use on a compassionate basis. However the current clinically used method for tracheal decellularisation has received mixed success and requires development to achieve GMP translation, quality manufacturing standards and ultimately routine clinical use. This thesis first examined the current clinically used detergent-enzymatic method (DEM) of decellularisation, a highly manual process that takes twenty-eight days to complete. Although the method achieved full decellularisation of non-cartilaginous regions of the tracheae, it failed to reduce the donor nuclear material sufficiently and resulted in the loss of key biochemical components, glycosaminoglycans (GAG) and collagen Type II, and the loss of biomechanical strength. A novel method for rapid, effective and non-detrimental tracheal decellularisation was required. Pressurised transmural flow was hypothesised to meet those requirements. A dual chamber bioreactor system was designed, fabricated and optimised to enable pressurised transmural decellularisation (PTD) to be investigated. Optimal PTD process parameters were ascertained and shown to produce tracheal scaffolds that achieved full decellularisation of the non-cartilaginous regions of the tracheae, a reduction of donor nuclear material (95%) which met the currently recommended levels of residual donor DNA for tissue engineered products, as well as maintaining GAG, collagen and biomechanical strength at comparable levels to the native tracheae. Additional to this, the new PTD process achieved this effective and non-detrimental decellularisation of tracheae in five working days with a ten-fold cost of goods reduction. With further development, the PTD method could become a fully automatable and closed process which could progress tissue engineered tracheae towards becoming a validated and regulated advanced therapy medicinal product (ATMP) and enable the advancement of tissue-engineered tracheae into regular clinical use.