新生仔ラット心筋細胞の分離・培養について

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  • A Simple Isolation Method and Primary Culture of Neonatal Rat Cardiac Myocytes

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For long-term maintenance of functional cardiac myocytes in primary culture, we developed a simple method for isolation of cells from neonatal Wistar rat ventricles. The highest viable cell yield was obtained when using 13 to 20 ventricles for one isolation and employing col lagenase as the tissue-digesting enzyme at 200 U/ml. Examination of the seeding density revealed that, at a density of 1.5XlO? cells/35-mm dish, the cells were arranged into long, thick, fibrous masses exhibiting synchronous contraction with a constant rate for up to 20 days in culture, indicat ing that this density was appropriate for maintaining functional cardiac myocytes in culture. The cells at Day 7 of culture were stimulated by addition of 1X10??M isoproterenol, leading to an increase in the beating rate, but the beating rate of the cells at Day 14 decreased somewhat by the addition of isoproterenol. Electron microscopic examinations of the myocytes at Day 3 clarified the reconstitution of the myofibrils with the formation of Z-lines, accompanied by the formation of intercalated discs. The formation of these contractile structures was maintained throughout the 20 days in primary culture. The intermediate filaments, desmin and vimentin, were detected in the cultured myocytes by the immunofluorescence method, showing a cross-striated pattern. It is likely that both intermediate filaments were involved in the reconstitution and maintenance of the striated structures of the myofibrils. These results suggest that the neonatal rat cardiac myocytes isolated and cultured by the present method are useful for an in vitro experimental system of the heart.

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