Optimized decellularization and sterilization method of whole porcine renal scaffold
Nafiseh Poornejad, Blake J. Cannon, Spencer J. Baker, Beverly L. Roeder, Alonzo D. Cook
Brigham Young University, Provo, Utah
Chronic kidney diseases are one of the prominent causes of morbidity and mortality that affect people’s lives all over the world. The combination of a patient’s own cells with an appropriate scaffold to achieve a healthy functional kidney without any potential for immunogenic rejection would be the most promising alternative for end stage renal failures. The first step towards this goal is choosing the appropriate scaffold for cell culture. Perfusion of native kidneys with detergents or other cell lysing solutions removes cells and leaves a non-immunogenic collagenous structure with critical morphology and essential chemical components suitable for cell growth and differentiation.
In an effort to achieve intact acellular scaffolds, both decellularization and sterilization steps were optimized. We were able to reduce SDS exposure time to less than 5 h with our novel tonic cycle method of decellularization. More than 95% of DNA was removed and collagen and glycosaminoglycans were preserved 3 times more than with common SDS-only methods (p-value<0.05). Reduced SDS exposure time also led to significantly higher human cell adhesion and growth potential of the acellular ECM. To achieve an optimized and reproducible sterilization method, four different methods were tested, including: 70% ethanol, 0.2% peracetic aicd in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma irradiation. The time and intensity required to have complete sterile tissue was 1 h for the first three methods and 3 kGy for gamma-irradiation. Gamma irradiation was observed to be the most damaging to the collagen structure and GAGs content, while peracetic acid in salt solution had the least damage. Human cell growth was approximately the same for all methods except for gamma irradiation, which induced programmed cell necrosis after 24 h.
In summary, we believe that the tonic cycle method for decellularization combined with peracetic acid sterilization is a promising and reproducible method to obtain acellular whole porcine kidneys ready to be recellularized with human cells.