Establishment of monolayer culture of pig pancreatic endocrine cells by use of nicotinamide.
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.
Ohgawara
H
H
Medical Research Institute, Tokyo Women's Medical University, School of Medicine, Japan. hisakooh@lab.twmc.ac.jp
Shikano
T
T
Fukunaga
K
K
Yamagishi
M
M
Miyazaki
S
S
D000818:Animals; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D009536:Niacinamide / Q000494:pharmacology; D013552:Swine
D007328:Insulin; D009536:Niacinamide; D005947:Glucose; D002118:Calcium