Pancreatic endorine-like cell cluster generation in vitro from pancreatic ductal tissue fragments using thermoreversible gelation polymer as culture material.

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This study was undertaken to examine whether neogenesis of pancreatic endocrine cells might be induced from pancreatic duct tissue fragments in a three-dimensional culture of pancreatic duct tissue fragments using TGP as a culture base.Methods : The pancreatic duct was isolated by extirpation from a beagle dog, and minced into 0.5-mm square tissue pieces. The tissue fragments were incubated in a three-dimensional culture with 8% TGP-containing RPMI-1640 medium added along with 10 ng/mL of KGF,10 ng/mL of HGF, and 10 mM nicotinamide. At week 6 of incubation, the culture in multi-well plates was cooled to liquefy TGP and to allow the tissue fragments and cell clusters to settle and adhere to the well bottom. The TGP-containing medium was then removed, and the culture was further incubated with RPMI-1640 medium added with 40 ng/mL of HGF and 10 mM nicotinamide.Results : Sporadic proliferation of cells from tissue fragments/cell clusters adhering to the well bottom was observed forming clusters of epithelial cells. At week 3 of incubation and thereafter, there was a formation of colonies of round cells in the epithelial cell clusters. From week 8 onwards, clusters of elliptical to round cells as large as 50-100 μm were detached from the colonies, and remained suspended in the culture medium. These suspended cell clusters were positive for somatostatin, and negative for insulin and glucagon. Differentiation into insulin-positive cells did not occur on further incubation of the culture or despite addition of differentiation inducing factors.Conclusion : Somat^+/insul^-/gluc^- cells represent precursors just antecedent to differentiation into insulin-positive cells. Further modification of the method of culture will enable us to induct differentiation into insulin-positive cells.