Proliferation of Pancreatic Endocrine Cells Using Disaggregation–Expansion–Reaggregation Technology in Isolated Rat Islets

• Published in: Transplantation proceedings 2010-04, Vol.42 (3), p.907-910
• Main Authors: Jeong, J.H, Lee, J.-I, Ju, M.K, Joo, D.J, Huh, K.H, Kim, M.S, Kim, J.Y, Cho, Y, Kim, Y.S
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cell Aggregation - physiology; Centrifugation, Density Gradient - methods; DNA Primers; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Glucagon - genetics; Glucose - pharmacology; Homeodomain Proteins - genetics; Insulin - genetics; Islets of Langerhans - cytology; Islets of Langerhans - drug effects; Islets of Langerhans - secretion; Male; Medical sciences; Microscopy, Phase-Contrast - methods; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; RNA - genetics; RNA - isolation & purification; Surgery; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; Tissue, organ and graft immunology; Trans-Activators - genetics
• ISSN: 0041-1345; 1873-2623
• DOI: 10.1016/j.transproceed.2010.02.044

Abstract Donor scarcity is a major obstacle for clinical islet transplantation. Hence, the effective use of the limited number of available islets is necessary for successful islet transplantation. We have developed a new technology that could produce pseudo-islets. Morphologic and functional evaluation was performed to test the feasibility of using these cells for transplantation. A 3-step procedure known as disaggregation–expansion–reaggregation (DER) was employed for pseudo-islet preparation. Islets isolated from 200 to 250-g male Lewis rats by collagenase digestion were separated into single cells by trypsinization. These pancreatic endocrine cells (PECs) were expanded by serial passages in culture before being aggregated at a high cell-density in a suspended state. After DER, cells were morphologically analyzed over time, and gene expression evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). Through expansion by passage for 2 weeks in continuous cultures, approximately 1 million PECs were recovered after aggregation. By phase-contrast microscopy, they presented with spherical shapes and similar sizes compared with naïve islets (50–800 μm). RT-PCR results indicated expression of insulin, glucagon, and pancreatic and duodenal homeobox gene 1, which were observed in primary isolated islets as well. The insulin secretion capacity of pseudo-islets was confirmed by enzyme-linked immunosorbent assay. In conclusion, PECs treated with DER showed potential to serve as a cell source for pseudo-islet generation after in vitro cellular expansion. 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.