Introduction Oesophageal atresia has an incidence of 1:3000–1:5000 births and is associated with significant morbidity after conventional surgery (30–40%). Tissue engineering could represent a safer alternative. Animal oesophagi can be decellularised to create scaffolds that maintain the 3D structure and the extracellular matrix (ECM) information. Acellular scaffolds are biocompatible and thought not do provoke chronic rejection reactions. They can be used as a matrix for reseeding prior to implantation, but a storage protocol is required to allow off-the-shelf production and increase their availability for transplant.

Aim Optimise oesophageal decellularization in large animals and assess different storage techniques.

Materials and methods Rabbit oesophagi (n = 30) were decellularized with different cycles of detergent-enzymatic treatment (DET). Scaffolds were evaluated by histology, immunofluorescence, scanning electron microscopy (SEM), synchrotron imaging, biomechanical testing and ECM component quantification. Scaffolds were stored in: i) 4°C in PBS (4C), ii) -20°C after freeze-drying (FD), iii) -80°C after snap-freezing (SF), iv) medium with 10%DMSO cooled at -1°C/min then stored in liquid nitrogen (MD). Samples were analysed at 2 and 4 weeks, 3 and 6 months with ECM quantification, SEM and biomechanical testing.

Results 3 DET cycles preserve the microarchitecture while removing cellular material. 6 months storage at 4C ruined scaffolds architecture. Oesophageal architecture was preserved in SF and MD groups, although elastin fibres were hyper-contracted in SF group. Oesophageal layers in FD group were completely collapsed.

Conclusions Optimal decellularization is achieved after 3 DET cycles. MD is the optimal storing method.