(42b) Perfusion-Decellularized Pancreas As a Scaffold for Pancreatic Tissue and Whole-Organ Engineering

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 suitable scaffold for transplantation to ensure islet viability and functionality. We hypothesize that a 3D 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 and organ engineering.

Materials and Method: Cadaveric pancreata were isolated from adult mice (n=8) and decellularized via detergent (0.5% SDS) perfusion. Resulting pancreatic matrix was characterized for acellularity, preservation of ECM and feasibility to transplant for in vivo use. For recellularization, β-cell (MIN6, 30x10⁶ cells) was first seeded into decellularized pancreas. Insulin gene expression was evaluated after 5 days of ex vivo culture in perfusion bioreactor (n=3). For hESC differentiation, decellularized pancreas was seeded with hESC-derived definitive endoderm (DE) cells (30x10⁶ cells) and differentiated into pancreatic progenitor (PP) cells by sonic hedgehog inhibition (cyclopamine) and retinoic acid induction. The PP cell-laden scaffolds were then implanted under kidney capsule of immunodeficient mice for in vivo maturation. PP cells implanted without scaffold served as controls (n=3). The recellularized constructs were evaluated via quantitative RT-PCR and immunostaining for pancreatic maturation markers and human C-peptide respectively.

Results and Discussion: Perfusion-decellularization of pancreas with 0.5% SDS resulted in complete removal of cells. Characterization of the decellularized pancreas revealed preservation of ECM and 3D architecture. Implantation of decellularized pancreas induced tissue remodeling but did not elicit adverse immune response. MIN-6 cultured in pancreatic constructs demonstrated higher upregulation of INS1 and INS2 gene expression compared to MIN-6 cultured on fibronectin, collagen I and IV surfaces (P<0.05). Repopulation and in-vitro differentiation of hESC-derived DE cells in decellularized pancreas showed 52.7±10.8 fold increase in PDX1 compared to Matrigel (P<0.05). In vivo maturation under kidney capsule after 8 weeks supported full differentiation of the seeded hESC into C-peptide expressing cells. Immunostaining showed that engrafted cells were also positive for NKX6.1, PDX-1 and co-localized with human nuclear antigen staining – confirming the matured β-cells were derivatives from the implanted hESC seeded pancreatic matrix graft.

Conclusion: Perfusion decellularized pancreas promotes β-cell function, and provides a favorable microenvironment for pancreatic differentiation when seeded with hESC-derived DE cells. The cell-laden matrices can be transplanted in vivo and successfully matured into insulin-producing cells. Transplantation into immunodeficient mice with streptozotocin-induced diabetes is underway to evaluate the therapeutic potential of the hESC-matured endocrine pancreas to achieve normoglycemia.