(648a) Perfusion-Decellularization of Pancreatic Matrix – A Scaffold for Bio-Engineered Pancreas

Introduction: Type I Diabetes affects over 1 million people in the United States. While islet transplantation has proved to be a promising therapeutic strategy, there are still limitations both in maintaining a viable culture of primary islets and a suitable scaffold for transplantation to ensure islet viability and functionality. We hypothesize that a 3-D scaffold recapitulating the native geometry and microenvironment would retain cellular functionality in-vitro and in-vivo. To this effect, we propose that perfusion-decellularization of whole pancreas could yield an acellular scaffold suitable for pancreatic tissue engineering.

Materials and Method: Cadaveric pancreata were isolated from adult, heparinized mice (n=8) and mounted on a perfusion apparatus. Ionic detergent, 0.1% SDS was perfused via the pancreatic duct until tissues were translucent and white in color (24-28 hours). To evaluate the scalability of this technique to a human-sized organ, decellularization of an adult cow pancreas was also performed. The resulting acellular matrices were analyzed for integrity of vasculature tree via trypan blue injections. Cell nuclei post-decellularization was quantified via dapi staining. The cytoskeletal element, actin was examined using phalloidin. Immunofluorescence and histology were done to characterize the retention of the extracellular matrix (ECM) protein after decellularization of the pancreas. For in-vitro culture, acellular matrices were seeded with Beta-TC-6 cells or mouse ES cells (2x10⁵) to examine the proliferation, survival and differentiation (n=3) of the seeded cells. For whole organ recellularization, Beta-TC-6 or mouse ES cells (30x10⁶) were introduced into the acellular matrices via the pancreatic duct in 3 steps, with 20 min interval between each step. Recell constructs (n=3) were then mounted on a perfusion apparatus to allow dynamic culture for 3 days. Both in-vitro and whole organ recell constructs were analyzed using histology, TUNEL, immunofluorescence staining and qRT-PCR.

Results and Discussion: Perfusion via the pancreatic duct with 0.1% SDS resulted in complete decellularization of both murine and cow pancreata. The vascular architecture was retained in acellular pancreas. No dapi staining of intact nuclei or cytoskeletal element were detected in acellular pancreas. Microstructure and major ECM component such as collagen I, collagen IV, fibronectin and laminin were preserved in acellular pancreas. Insulin response assay showed preserved insulin response to glucose challenge in the Beta-TC-6 seeded construct in both in-vitro culture and whole organ culture. In ES cells seeded construct, qRT-PCR results showed elevated gene expression of pancreatic differentiation markers. Immunohistochemistry from whole organ recell demonstrated better viability (<5% TUNEL positive).

Conclusion: Perfusion-decellularization of cadaveric pancreata efficiently removes cells while retaining ECM protein and microstructure. This perfusable 3-D whole organ scaffold yields functional tissue construct with preserved endocrine function and favored endocrine differentiation. Further studies are underway to establish the suitability of acellular pancreatic ECM as a scaffold to transplant donor islets and achieve euglycaemia in diabetic recipients.