Biofabrication of a functional Vascularized Endocrine Pancreas (VEP) for Type 1 Diabetes
Antonio Citro1, Alessia Neroni1, Cataldo Pignatelli 1, Martina Policardi 1, Francesco Campo1, Silvia Pellegrini 1, Erica Dugnani 1, Fabio Manenti 1, Libera Valla1,2, Elisabeth Kemter2, Ilaria Marzinotto1, Cristina Olgasi3, Antonia Follenzi3, Vito Lampasona1, Eckhard Wolf 2, Lorenzo Piemonti1,4.
1San Raffaele Diabetes Research Institute, IRCCS Ospedale San Raffaele, Milan, Italy; 2Department of Veterinary Sciences, LMU , Center for Innovative Medical Models (CiMM), Munich, Germany; 3Department of Health Sciences, School of Medicine, University of Piemonte Orientale, Novara, Italy; 4School of Medicine and Surgery, Università Vita-Salute San Raffaele, Milan, Italy
Background: Intrahepatic islet transplantation in patients with type 1 diabetes is limited by donor availability and lack of engraftment. To overcome these limitations, based on our experience with decellularized rat lung as scaffold for the generation of Vascularized Islet Organ (VIO, lung scaffold repopulated by murine islets and HUVEC cells), we prototyped our platform to engineer an upgrade based on human blood-derived endothelial cells (BOECs - Blood Outgrowing Endothelial Cells) and immature neonatal porcine islet clusters (NPIs).
Material and Methods: NPIs and BOECs phenotype profile was assessed by flow cytometry, insulin secretion test and tubule formation assay. Rat lung was decellularized by vascular perfusion with 1% SDS and 0.1% Triton and seeded with NPIs and BOECs, generating a Vascularized Endocrine Pancreas (VEP). For ex vivo maturation VEP was cultured for 7 days in a customized bioreactor specifically designed to allow cell integration. The NPIs β cell death in mature VEPs was estimated during ex vivo organ maturation evaluating miR-375 expression by droplet digital PCR compared to batch matched NPIs in standard condition. Matured VEPs and control NPIs function were measured by dynamic glucose perifusion and insulin quantification (by ELISA/IF). Once validated in vitro, VEPs were subcutaneously transplanted in diabetic immunodeficient NSG recipients and compared for in vivo function with matched NPIs transplanted in different implantation sites: kidney capsule (KC-NPIs), deviceless (DL-NPIs) and liver (LV-NPIs).
Results: Matured VEPs showed regenerated vascular network (CD31+) with NPIs (insulin+) integrated. miR-375, a marker of β cell death, was expressed in NPIs but not in BOECs, as expected. VEP was able to significantly reduce β cell death: the amount of lost NPIs were ≤ 5% during VEPs maturation, while >71% during in vitro culture (p<0.05). Matured VEPs were able to sustain NPIs engraftment, survival and significantly improve insulin secretion during the maturation process compared to batch matched NPIs cultured in standard conditions (AUC VEPs first phase: 3.765±0.90; NPIs 1.60±0.25 p<0.01). In long-term transplants in diabetic mice, VEPs demonstrated a significant NPIs engraftment with a prompt VEPs function after implantation and the reversal of the glycemia within 2 days until 60 days after implantation. Additionally, VEPs showed a significant superior function compared to all the internal controls (KC-NPIs, DL-and LV-NPIs).
Conclusion: We conclude that ex vivo bio-fabrication of VEPs enables NPIs engraftment, maturation and vascularization before transplantation. VEP is the first organ to our knowledge assembled with relevant source of endocrine and endothelial cells suitable for future clinical translation. Given recent progress in gene editing, this technology may enable assembly of functional personalized endocrine organs to escape immunoreaction against the graft.
This study was supported by a grant from Juvenile Diabetes Research Foundation (JDRF 1-SRA-2019-771-S-B).