We have developed a method to circumvent the use of exogenous proteolytic enzymes in the isolation of islets of Langerhans from the perinatal rodent pancreas. Advantage is taken of the propensity of fibroblastlike cells to attach and migrate on polystyrene at low-serum concentrations (5%). In contrast, at this serum level, rat islet epithelial cells tend not to adhere to the substrate. At 3 d of culture, islets are visible at the edges of the explants. With further fibroblast outgrowth the majority of islets are freefloating by 7 d. Simple agitation of the medium and centrifugation yields approximately 50 micrograms of islet tissue per perinatal pancreas. Further purification of the islets can be obtained by subculture. Rat islets can be maintained in this manner for several months in Medium F12 supplemented with 25% horse serum in an atmosphere of 5% CO2 and air at 37 degrees C. Hormone content of the islet tissue remains constant during prolonged subculture and such islets continue to exhibit appropriate insulin and glucagon responses to glucose and theophylline. The morphological integrity of the endocrine cells within the cultured islets was confirmed by immunocytochemistry and ultrastructural study. Nonendocrine cells are not identifiable within the long-term cultured islets.
Plasma amine oxidase activities (benzylamine oxidase and spermine oxidase) were determined in the sera of a number of species of various ages. Benzylamine oxidase (BZO) activity, measured spectrophotometrically, was present in bovine, equine, and ovine species examined. Generally its activity in serum increased with the age of the animal. Spermine oxidase activity (SPO) was estimated by a bioassay of in vitro toxicity and did not necessarily correlate with BZO. Cytotoxicity in the presence of spermidine was found only in the sera of the ruminant species examined. Serum activity tended to rise with animal age; however, great variability was found in perinatal bovine sera. The 50% lethal dose (LD50) of spermidine in the presence of 5% serum and 4 X 10(4) NS1 cells/ml was in the micromolar range. Aminoguanidine, a known inhibitor of SPO, could prevent the cytotoxic effects of exogenously added spermidine in vitro. In contrast, raising the ambient oxygen tension in the incubation environment to 95% lowered the LD50 dose of spermidine required for cytotoxicity. The results suggest that a cell line of hematogenous origin is susceptible to the cytotoxic effects of the products of oxidative deamination of spermidine by SPO, an enzyme present in perinatal bovine sera, and that these cytotoxic effects are potentiated in the presence of an oxygen-enriched environment in vitro.
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