Supercritical CO2-assisted decellularization: advanced pancreatic tissue platform for diabetes treatment

Introduction: Decellularization is a process that often employs harsh chemicals which compromise the extracellular matrix (ECM). This is particularly damaging in case of soft tissues, such as pancreas. Supercritical carbon dioxide (scCO2) is an alternative solvent which presents high transfer rates and diffusivity while being chemically inert and non-toxic [1-3]. The present work explores the use of scCO₂ for decellularizing pancreatic tissue, to preserving its native architecture and biochemical cues, while improving its immune tolerance. This approach aims at establishing an advanced platform for pancreatic cell therapy applications, particularly in conditions like diabetes. Materials and Methods: Porcine pancreatic tissue was decellularized using a conventional detergent-based protocol (Sodium Deoxycholate (SDC), Triton X100, DNase – per cycle) and an alternative scCO₂-assisted method (110-120 bar, 36-38ºC, 1 hour cycle). The samples were collected after the second, fourth, and fifth cycles for further analysis and comparison. The ECM components (glycosaminoglycans, elastin, and collagen) were quantified using Blyscan, Sircol and Fastin assay kits (BioColor life science assays, UK). Results and Discussion: After five cycles of decellularization the obtained tissues were white and clear as observed in Figure 1. The DNA content of the samples was quantified after the second, fourth and fifth cycles of decellularization (Figure 2). The results show that after five cycles of decellularization, the DNA content was considerably reduced. The samples that underwent the scCO2 decellularization protocol achieved complete decellularization, with DNA content below 50 μg/mg. In fact, the scCO₂ protocol was also able to decellularize the samples at the fourth cycle (DNA content of 33.38 ± 6.10 μg/mg), while for the traditional decellularization protocol it was necessary five cycles, to reach a DNA values of 53.44 ± 4.89 μg/mg, in the range of 50 μg/mg threshold suggested by the literature [4]. These results demonstrate the efficacy and the potential to reduce processing time when using scCO2 compared to traditional method. Conclusion: Overall, the scCO₂ contributes to a more efficient cell removal protocol, while it allowed to considerably reduce the use of chemicals and proved to be less time-consuming. This innovative approach highlights scCO₂ as a viable alternative for pancreatic tissue decellularization, opening new opportunities for advancing current cell therapies that lack proper mimetic environments to maintain cell viability and post-implantation functionality.