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KMID : 0378019870300100049
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New Medical Journal
1987 Volume.30 No. 10 p.49 ~ p.58
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Dedifferentiation of differentiated cells by means of cell fusion in culture
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Abstract
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To induce the dedifferentiation of myocardial cells in culture, yentricles of new-born rat hearts we e removed, pretreated with cold trypsin overnight and digested with warm collagenase. Myocardial cell s were mixed with HEP-2 cells and suspension-fused with PEG solution
Fused cells were suspended in complete EHM supplemented with 10% FCS and seeded in 96-wall tissue culture flasks. After 5 days of incubation in 37°C CO, inubator, healthy hybrid cells were harvests and subcultured in Leight on tubes without cover-slips. After 4 weeks of cultivation, cells were harvested again and subcultured in 25m1 falcon flasks. After another 4 weeks of culture, cells were seeded in Leighton tubes with cover-slips.
These cells were used for live observation, cytochemistry and autoradiography using 3H-thymidinto investigate the interspecific hybrid formation and their identification and subsequent isolation. The results are summarized as follows:
1. The viability of myocarolia cells after segregation was approximately 85% while that of HEP-2 was 95%. 2. During 5 consecultive fusion experiments, mononucleate hybrid cells were observed in approximatel 12 wells out of each 96-wells.
3. The 3H-thymidine labelling indices of myocisdial, HEP-2 and hybrid cells were 3%, 38% and 16´ respectively. The generation times of HEP-2 and interspecific hybrid cells were 17 hrs and 42 hrs respec tively.
4. The intensity of succinate dehydrogenase reaction was strongest in myocardial cells and strong n hybrid cells while that of HEP-2 cells was very weak.
5. The methods applied in this investigation was confirmed to be useful in isolation of interspecific hybri cells.
6. The differentiated function of interspecific hybrids were decreased and the proliferative ability was restored.
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