Background: Whole organ decellularization is gaining a lot of popularity since its development in tissue engineering. It is a process which allows to create complex 3D extracellular matrix bioscaffold with preservation of intrinsic vascular network. Bioscaffolds can be potentially used in regenerative medicine because of their ability to cells reseeding in bioreactor. We report and compare different approaches of methods used in pancreas decellularization and also their advantages and limitations.

Methodology: In our research four different approaches have been established. Two types of detergents were combined with two types of working solutions and addition of DNase I or heparin were used. Time of flow was varied between 12 hours up to 72 hours. Every process of decellularization was performed under sterile conditions in temperature of 4[deg]C. First method consisted of [micro]Q water (24h) followed by 0,5% SDS (48h) and [micro]Q water (72h). The second method was as above but instead of 0,5% SDS we used 1% Triton X-100/0,1% ammonium hydroxide. Third method comprised of heparin wash in PBS (15 min), 0,5% SDS (24h) followed by PBS solution (72h). Fourth method: heparin wash in PBS (15 min), 1% Triton X-100/0,1% ammonium hydroxide, PBS solution (72h) and DNase I with Ca²⁺ (12h). To assess the accuracy of the decellularization process histopathological evaluation was performed (H&E staining of frozen and paraffin scaffold specimens) as well as evaluation of residual DNA content.

Results: Preliminary studies have shown that addition of [micro]Q water causes strong edema of pancreatic cells while PBS solution with heparin works great. 0,5% SDS solution is not successful in decellularization of pancreas while 1% Triton X-100 with 0,1% ammonium hydroxide causes no precipitation and is the most effective detergent in pancreas decellularization. H&E staining confirmed complete loss of dense cellularity in the last method. Electrophoresis shows significant loss of DNA

Conclusion: Analysis of results showed that use of 1% Triton X-100/0,1% ammonium hydroxide followed by PBS is the most efficient method for pancreas decellularization. Preliminary studies of our research are the basis for further tests involving DNA amount.

CITATION INFORMATION: Berman A., Karzynski K., Serwanska-Swientek M., Pilka N., Klak M., Kosowska K., Cichon J., Ambrozkiewicz F., Wszola M. Pancreas Decellularization Protocol – Comparison of Methods Am J Transplant. 2017;17 (suppl 3).

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