Technique of Increasing the Stability of Porcine Pancreatic Islets Using Biopolymers

Abstract

Pancreatic islet transplantation is considered as the most promising technique for the treatment of type 1 diabetes mellitus. However, the pancreatic islet isolation and transplantation technique have not been optimized yet because of several obstacles in vitro and in vivo works. The clinical problem in human pancreatic islet allotransplantation is the limitation of donor supply and immune rejection after transplantation. Moreover, the researchers related to the porcine islet studies suffer from the instability of porcine islet clusters

thus, it was difficult for them to move on to in vitro experiments. The porcine islets are focused for alternative source to solve the donor limitation problem and the fragility of porcine islet clusters are fixed by re-providing a proper extracellular matrix to the cells. Here, we synthesized adequate biopolymers for islet surface modification and investigated the increase in stability of porcine islet clusters. Rat pancreatic islets, INS-1 cells, and porcine pancreatic islets were used to determine the viability, functionality, and biopolymer coverage on cells. The cytotoxicity and material adherence on the cell surface of biopolymers were confirmed by rat islet ICCs or INS-1 cells before applied to porcine islet clusters. The relative viability of unmodified and biopolymer-grafted cells was determined by CCK-8/DNA assay or LDH cytotoxicity assay. The coverage test of FITC-labeled biopolymer-grafted cells was performed by confocal microscopy. After confirmation of biopolymer candidates, 6-arm PEG-lipid-SH and gelatin-DOPA were selected from candidate and two biopolymers were applied to the porcine islets in multi-layered system. The unmodified and biopolymer-grafted porcine islets were cultured for 7 days for morphology, functionality, viability, material coverage, and stability observations. In result, the relative viability of unmodified and biopolymers-grafted porcine islets for 7-day culture was not shown a significant difference and also there was no significant difference in insulin functionality by dithizone staining in both unmodified and biopolymer-grafted porcine islets. The morphology of unmodified porcine islet clusters were destructed and dissociated into single cells but that of biopolymer-grafted porcine islet clusters were maintained their cluster round-shape. FITC-labeled 6-arm PEG-lipid-SH and gelatin-DOPA, were treated on porcine islets to determine the cell surface coverage of materials. Both materials were not penetrated into the cell cytoplasm but only adhere on the membrane of cells and cover the periphery of clusters. The average size of cell clusters was measured by coulter counter until 7-days of culturing and the distribution of cell size was calculated. The size of unmodified porcine cells on fifth day were decreased to half compared to the first day but the size of biomaterial-grafted groups were decreased less than one-third of the original size. Regeneration of extracellular matrix and cell connection of porcine islets using biopolymers would be advantageous for further porcine works because the islets become easier in handling and also subsequent surface camouflage may be held based on this study. By increasing the stability and cellular interaction in islet clusters, the cellular communication and functionality of porcine islets could be increased and also the viability of the cells could be increased. Moreover, the further surface modification on porcine islets based on our suggestion would lead to inhibition of immune rejection of islets after transplantation.