Separation of neonatal rat ventricular myocytes and non-myocytes by centrifugal elutriation.
The preparation of pure cardiac myocyte cultures from neonatal rats is hampered by the presence of non-myocytes, which can proliferate during culturing, thereby causing a progressive decrease in the proportion of myocytes. In order to obtain myocyte cell suspensions of high purity, a method based on centrifugal elutriation was developed. Cardiac cells, isolated from neonatal rat heart ventricles, were subjected to elutriation using flow rates that increased step-wise from 20 to 80 ml/min. The cell fraction obtained at 80 ml/min consisted of 68-90% myocytes. Still, upon culturing, the remaining non-myocytes proliferate, causing the proportion of myocytes to decrease to 60 +/- 2% at day 5. A second elutriation protocol was developed in which myocytes and non-myocytes were separated after a period of co-culturing for 4-5 days. By this approach a fibroblast-rich cell fraction (87 +/- 5%) and a myocyte-rich cell fraction (82 +/- 6%) were obtained. In conclusion, centrifugal elutriation creates the opportunity to separate neonatal rat myocytes from non-myocytes, either freshly isolated or after a period of culturing. Particularly, cell separation after a period of culturing ventricular cells offers an advantage to analyse the experimental effects on myocytes and non-myocytes separately.
LUMC, Dept. of Clinical Oncology, Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
M
M
Boerma
C G C
CG
van der Wees
J
J
Wondergem
A
A
van der Laarse
M
M
Persoon
A A
AA
van Zeeland
L H F
LH
Mullenders
D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002498:Centrifugation; D006352:Heart Ventricles / Q000166:cytology; D032383:Myocytes, Cardiac / Q000166:cytology*; D051381:Rats; D017208:Rats, Wistar