Toward a greener approach for next-generation functional pancreatic ECM in diabetes treatment

Current decellularization methods often rely on harsh chemicals compromising the extracellular matrix (ECM) biochemical structure, particularly in soft tissues like pancreas. Supercritical carbon dioxide (scCO₂) offers a sustainable, non-toxic alternative, preserving structural and biochemical integrity, while improving immune tolerance [1]. This study establishes an integrated scCO₂-based workflow for porcine pancreas decellularization and sterilization, aiming at developing an immunocompetent decellularized ECM (dECM) platform for diabetes cell-based therapies. Pancreas decellularization was achieved using a scCO₂-assisted method (scCO2-dECM) and compared with a conventional detergent-based protocol. Its efficiency and ECM preservation were assessed through DNA quantification, histology (H&E, Masson’s Trichrome, Alcian Blue), SEM/TEM, biochemical assays (glycosaminoglycans (GAGs), collagens) and proteomics. ScCO2-assisted sterilization was validated through turbidity assay. Pyrogenic safety was studied through endotoxin quantification. Cytotoxicity was evaluated following ISO 10993-5:2009 through metabolic activity, proliferation assays, and immunofluorescence imaging. Both methods achieved DNA levels below 50 ng/mg of dry tissue [2]. However, scCO2-dECM preserved higher GAGs content, with enhanced collagen-network preservation. Proteomics revealed that scCO₂-dECM better preserves functional, pancreas- and insulin pathway-related proteins, along with elastic fibers, indicating enhanced retention of tissue-specific biomolecules. ScCO₂-assisted decellularization was more effective in reducing endotoxin content (0.027 EU/mg in scCO₂ vs. 0.413 EU/mg in detergent-treated samples). ScCO₂ sterilization ensured Bacillus subtilis inactivation. Non-cytotoxicity was confirmed by metabolic activity and cell proliferation over time. DAPI/Phalloidin staining confirmed normal morphology and cytoskeleton organization. Ongoing study investigates whether Damage-Associated Molecular Patterns (DAMPs) trigger macrophage inflammation. Overall, scCO₂ enables fast and efficient decellularization, preserving dECM integrity and showing cytocompatibility. This greener approach delivers functional pancreatic dECM, as potential platform towards pancreatic tissue engineering for diabetes.