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

本研究以TGP为培养基础，对胰管组织块进行三维培养，探讨胰管组织块能否诱导胰腺内分泌细胞新生。方法：取比格犬胰管，切成0.5 mm的方形组织块。组织块在含有8%TGP的RPMI-1640培养液中加入10 ng/mLKGF、10 ng/mLHGF和10 mM烟酰胺进行三维培养。在培养6周时，冷却多孔板中的培养物，使TGP液化，使组织碎片和细胞团沉淀并附着在孔底。然后取出含有TGP的培养液，再用添加40 ng/mLHGF和10 mM烟酰胺的RPMI-1640培养液孵育。结果：细胞从组织块/细胞簇中零星增殖，形成上皮细胞簇。孵育3周及以后，上皮细胞团内可见圆形细胞集落形成。从第8周开始，大小为50~100μm的椭圆形至圆形细胞团从集落中分离出来，并悬浮在培养液中。这些悬浮细胞团生长抑素呈阳性，胰岛素和胰升糖素呈阴性。结论：Somat^+/Insul^-/Gluc^-细胞是分化为胰岛素阳性细胞的前体细胞。进一步改进培养方法将使我们能够诱导分化为胰岛素阳性细胞。

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.