Evaluation of different decellularization protocols on the generation of pancreas-derived hydrogels.

Different approaches have investigated the effects of the different extracellular matrices (ECMs) and 3D culture on islets function, showing encouraging results. Ideally, the proper scaffold should mimic the biochemical composition of the native tissue as it drives numerous signaling pathways involved in tissue homeostasis and functionality. Tissue-derived decellularized biomaterials can preserve the ECM composition of the native tissue making it an ideal scaffold for 3D tissue engineering applications. However, the decellularization process may affect the retention of specific components, and the choice of the proper detergent is fundamental in preserving the native ECM composition. In this study, we evaluated the effect of different decellularization protocols on the mechanical properties and biochemical composition of pancreas-specific ECM (pECM) hydrogels. Fresh porcine pancreas tissue was harvested, cut in small pieces, rinsed in water and treated with two different detergents (SDS or Triton X-100) for one day followed by 3 days in water. Use of a protease inhibitor was also evaluated. Effective decellularization was confirmed by PicoGreen assay and H&E staining, showing no differences among groups. Triton-treated samples were able to form a firm hydrogel under the appropriate conditions, while the use of SDS had detrimental effects on the gelation properties of the hydrogels. ECM biochemical composition was characterized both in the fresh porcine pancreas and all decellularized pECM hydrogels by quantitative mass spectrometry analysis. Fibrillar collagen was the major ECM component in all groups, with all generated hydrogels having a higher amount compared to the fresh pancreas. This effect was more pronounced in the SDS treated hydrogels when compared with the Triton groups, showing very little retention of other ECM molecules. Conversely, basement membrane and matricellular proteins were better retained when the tissue was pretreated with a protease inhibitor and decellularized in Triton X100 making the hydrogel more similar to the native tissue. In conclusion, we showed that all the protocols evaluated in the study showed effective tissue decellularization, but only when the tissue was pre-treated with a protease inhibitor and decellularized in Triton detergent, the biochemical composition of the hydrogel was closer to the native tissue ECM.