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为培养基质，对胰管组织碎片进行三维培养，探讨胰管组织碎片诱导胰腺内分泌细胞新生的可能性。方法：取比格犬胰管，切除胰管，切成0.5 mm方形组织块。组织片段在含8% tgp的RPMI-1640培养基中三维培养，加入10 ng/mL KGF、10 ng/mL HGF和10 mM烟酰胺。在培养第6周时，将多孔板中的培养物冷却以使TGP液化，并使组织碎片和细胞团沉淀并粘附在孔底。然后除去含tgp的培养基，用添加40 ng/mL HGF和10 mM烟酰胺的RPMI-1640培养基进一步培养。结果：观察到组织碎片/细胞簇粘附在井底的细胞零星增殖，形成上皮细胞簇。培养第3周及之后，上皮细胞簇中形成圆形细胞集落。从第8周开始，50-100 μm大小的椭圆到圆形细胞团从菌落中分离出来，悬浮在培养基中。这些悬浮细胞簇对生长抑素呈阳性，对胰岛素和胰高血糖素呈阴性。分化为胰岛素阳性细胞没有发生进一步的培养孵育或尽管添加分化诱导因子。结论：Somat^+/胰岛素^-/葡萄糖^-细胞是胰岛素阳性细胞分化前的前体。进一步修改培养方法将使我们能够诱导分化成胰岛素阳性细胞。

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.