Pancreatic islet culture for islet transplantation

胰岛移植是替代胰岛素注射治疗糖尿病的一种新方法。胰岛细胞在体外培养过程中有时会丧失其功能，因此胰岛细胞的体外培养是胰岛细胞的关键过程。在本研究中，我们将重点放在基质涂层，以改善培养和延长其胰岛素分泌。为了研究基质的作用，将胰岛细胞培养在涂有几种基质的培养皿中，并测量细胞从它们中脱离和胰岛素释放对葡萄糖水平的响应。将分散的细胞接种在涂有I型胶原、IV型胶原、纤连蛋白、层粘连蛋白或不涂有层粘连蛋白的培养皿上。通过在20次移液后计数脱离的细胞数来测定细胞与基质的脱离强度。在富糖培养基和贫糖培养基中进行葡萄糖刺激试验，用ELISA法测定培养上清液中胰岛素的含量。移液后，在I型胶原和层粘连蛋白上培养的胰岛细胞最容易分离，在IV型胶原上培养的细胞也容易分离，但在纤维连接蛋白和无涂层塑料上培养的细胞不能分离。在层粘连蛋白上培养的细胞释放最多的胰岛素，并显示出最高的刺激指数，但在纤连蛋白或无涂层塑料上培养的细胞释放最少的胰岛素，并显示出最低的刺激指数。提示贴壁培养可抑制胰岛细胞的胰岛素释放，胰岛细胞应在易分离的培养皿上培养。

Islet transplantation is a novel diabetic therapy instead of insulin injection. In vitro culture of islet is pivotal process, because islet cells sometimes lose their function during in vitro culture. In this study, we focused on matrices coating so as to improve the culture and prolong their insulin secretion. In order to investigate the effect of matrices, the islet cells were cultured on dishes coated with several matrices, and measured the detachment of the cells from them and insulin release in response to glucose level.The islets were isolated from male Lewis rats and then dispersed with trypsin. Dispersed cells were seeded on the dishes coated with type I collagen, type IV collagen, fibronectin, laminin or without. Intensity of detachment of the cells to the matrices was assayed by counting the detached cell number after 20 times pipetting. Glucose stimulation test was carried out in glucose-rich medium and glucose-poor medium, and the insulin amount in the culture supernatant was determined by ELISA. The islet cells cultured on type I collagen and laminin were most easily detached after pipetting, and cells on type IV collagen were also easily detached, but cells on fibronectin and noncoated plastic were failed to detach. Cells cultured on laminin released most amount of insulin and showed highest stimulation index, but the cells on fibronectin or noncoated plastic released the least insulin and showed the lowest stimulation index. These results imply that attachment on the culture dish could inhibit insulin release from islets cells and that islet cells should be cultured on easily detachable dish.