Proliferation and functional assessment of pseudo-islet cells using pancreatic endocrine cells
췌장 내분비 세포를 이용한 유사 췌도의 제작 및 기능평가
Dept. of Medicine/박사
Pancreas islet cell transplantation is a new therapy for type 1 diabetes mellitus. However, there are many obstacles precluding the use of islet transplantation as a conventional treatment. Particularly, insufficient islet mass may limit the use of this approach. We have developed a new technology that could produce pseudo-islets. Recently researches concerning co-culture with pancreatic islet and bone marrow mesenchymal stem cells (BMSCs) have been reported that low differentiated BMSCs demonstrated the positive effect on pancreatic islet functions. The purpose of this study is Morphologic and functional evaluation was performed to test the feasibility of using these cells for transplantation and to prove the mutual effect between pancreatic islet cells and BMSCs. A three-step procedure known as disaggregation-expansion-reaggregation (DER) is the method employed for pseudo-islet preparation. Islets were isolated from 200-250 g male Lewis rats by collagenase digestion and separated into single cells by trypsinization. These pancreatic endocrine cells (PECs) were expanded by serial passages in culture and were then aggregated at a high cell-density in a suspension state. For BMSCs collection, bone marrow was aspirated from the rat femur and adherent mononucleated cells were further expanded by passaged cultures. Subsequently, PECs and BMSCs at a high density were placed on low cell binding culture dish, and kept suspended state using shaking culture and maintained. The mixed cell complex were evaluated its function and characteristics with Glucose Challenge Test, insulin ELISA Analysis, RT-PCR and Immunohistochemistry. Through expansion for 2 weeks in continuous culture passages, approximately one million PECs were recovered after aggregation. They presented with a spherical shape and a similar size when compared to naïve islets (50–800 μm) by phase-contrast
microscopy. RT-PCR results indicate expression of insulin, glucagon, and PDX-1, which were observed in primary isolated islets as well. The insulin secretion capacity of pseudo-islets was confirmed by ELISA. The cellular aggregates of pancreatic islet cells and BMSCs were retrieved. These cellular spheroids showed the fortified function and maintained viability. In conclusion, PECs treated with DER were found to have the potential to serve as a cell source for pseudo-islet generation following in vitro cellular expansion. These cells are both morphologically and genetically similar to naïve islets. And, we suggested the manufacture method of mixed cellular complex from two different origins. Using this construct, cell-cell interactions can be examined in vitro such as improvement of secretion ability and cell differentiation. BMSCs can be used as a replacement or additives for bioengineered pseudo-islets. As these cells were both morphologically and genetically similar to naïve islets, our new technique could be a potential method to overcome the scarcity of donor islets in the near future.