Enrichment of vital adult cardiac muscle cells by continuous silica sol gradient centrifugation
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Summary
A major improvement in the isolation of vital adult cardiocytes was achieved by isopycnic preformed continuous silica sol gradient centrifugation after perfusion of the heart with collagenase. Vital rat cardiocytes were enriched to 90–95% vital cells reproducibly and constantly by one- or two-step gradient centrifugations. The isolated cardiocytes were tolerant to calcium concentration up to 0.03 mmol/l, to diluted human serum, and to human complement. Gentamycin (50 μg/ml) exerted a cytotoxic effect on myocytes, whereas Penicillium and Streptomycin in concentrations of 50 IU/ml did not induce cytolysis of cital cells. Digoxin 15 ng/ml) decreased the natural decay of myocytes of 20% in 24 hours to 8%.
Enriched of vital cardiocytes by silica sol gradient centrifugation following their isolation by perfusion with collagenase may be helpful for investigations depending on a high yield of vital myocardial cells.
Key Words
Percoll™ silica sol continuous gradient collagenase treatment isolated rat cardiocytes calcium tolerance gentamycin cytotoxicity digoxin penicillium streptomycinZusammenfassung
Mit der isopyknischen kontinuierlichen Silica-Sol-Gradientenzentrifugation (Percoll™, Pharmacia) konnte eine wesentliche Verbesserung bei der Isolierung vitaler adulter Herzmuskelzellen erzielt werden. Nach einer Perfusion des nach Thorakotomie entnommenen Rattenherzens mit einer kollagenasehaltigen Krebs-Ringer-Lösung wurde eine ein-oder zweifache Percoll-Gradientenzentrifugation durchgeführt, bis über 90% vitale Herzmuskelzellen in Suspension waren.
Die isolierten Kardiozyten tolerierten Ca2+ bis 0,03 mmol/l ebenso wie Humanserum und Komplement. Gentamycin (50 μg/ml) erwies sich als zytotoxisch, während Penicillin und Streptomycin in Konzentrationen von 50 IU/ml keine Zytolyse vitaler Kardiozyten induzierten. Digoxin (15 ng/ml) verbesserte die Überlebensrate der Kardiozyten um 20% in 24 Stunden.
Mit dieser Methode konnte eine höhere Ausbeute vitaler Herzmuskelzellen als mit den bisher beschriebenen Verfahren erzielt werden.
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