Establishment of monolayer culture of pig pancreatic endocrine cells by use of nicotinamide.
Establishment of monolayer culture of pig pancreatic endocrine cells by use of nicotinamide.
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DOI：
10.1016/s0168-8227(98)00096-5
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发表时间：
1998-10
影响因子：
5.1
通讯作者：
H. Ohgawara;T. Shikano;K. Fukunaga;M. Yamagishi;S. Miyazaki
中科院分区：
文献类型：
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作者：
H. Ohgawara;T. Shikano;K. Fukunaga;M. Yamagishi;S. Miyazaki
建立了成年猪胰腺内分泌（PE）细胞的分离和原代单层培养方法。在不添加蛋白水解酶的情况下，通过自消化将细胞与胰腺分离，并使用 Histopaque-1077（聚蔗糖和泛影酸钠的混合物）在一次离心步骤中分离成不同的条带。从界面部分收集的细胞悬浮于含有 11 mmol/l d-葡萄糖（含或不含烟酰胺）（0、10、20、40 mmol/l）的 RPMI 1640 中，然后置于培养皿中。当在烟酰胺存在下培养时，胰腺细胞形成单层，而成纤维细胞从培养皿底部脱离。使用酶法对超过 80% 的单层形成细胞进行胰岛素染色，并鉴定为 B 细胞。从形态学上看，PE细胞延伸了多个终止于生长锥状结构的过程，如光学显微镜和扫描电子显微镜所显示的那样。在含有或不含烟酰胺的 RPMI 1640 培养基中孵育 35 天后，胰岛素会分泌以响应葡萄糖刺激。与低葡萄糖 (5.5 mmol/l) 相比，细胞暴露于烟酰胺 35 天，导致高葡萄糖刺激 (16.7 mmol/l) 的胰岛素分泌增加 2-3 倍。使用 fura-2 的 Ca2+ 成像，在 10 mmol/l 烟酰胺存在下培养 35 天的单个细胞中检查葡萄糖诱导的 Ca2+ 反应。这些结果表明，可以在单层培养中制备具有低成纤维细胞污染的猪PE细胞，并在体外维持功能性B细胞相对较长的时间。本方法为胰岛细胞的分化、生长和再生能力的进一步形态学和生理学研究提供了有用的制剂。
A method for the isolation and primary monolayer culture of adult pig pancreatic endocrine (PE) cells was established. Cells were dissociated from the pancreas by autodigestion without addition of proteolytic enzymes and separated into distinct bands in a single centrifugation step using Histopaque-1077 (a mixture of polysucrose and sodium diatrizoate). The cells collected from an interfacial fraction were suspended in RPMI 1640 containing 11 mmol/l d-glucose with or without nicotinamide (0, 10, 20, 40 mmol/l), and then placed in culture dishes. Pancreatic cells formed a monolayer while fibroblasts became detached from the bottom of the dish when cultured in the presence of nicotinamide. More than 80% of monolayer-forming cells were stained for insulin, using an enzymatic method, and were identified as B-cells. Morphologically, the PE cells extended multiple processes terminating in growth-cone-like structures, as visualized by both light microscopy and scanning electron microscopy. Insulin secretion in response to glucose stimulation occurred for 35 days of incubation in the RPMI 1640 medium, with or without nicotinamide. Exposure of the cells to nicotinamide for 35 days resulted in a 2–3-fold increase in insulin secretion in response to high glucose stimulus (16.7 mmol/l) compared with low glucose (5.5 mmol/l). Glucose-induced Ca2+responses were examined in individual cells cultured for 35 days in the presence of 10 mmol/l nicotinamide, using Ca2+imaging with fura-2. These results indicate that it is possible to prepare pig PE cells in monolayer culture with low fibroblast contamination and to maintain functioning B-cells in vitro for relatively long periods. The present method provides useful preparations for further morphological and physiological studies on the differentiation, growth and regenerative capacity of islet cells.