ウナギ卵巣由来細胞の単層培養 : 培養細胞の性状と継代技法について

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The main characteristics and efficient subculturing techniques of ovary cells from eel, Anguilla japonica, were clarified in this study. Five female eels were respectively used to start primary culture. The ovaries were digested for 30 minutes at 24°C with pronase-P (0.01% in Eagles MEM) on magnetic stirrer followed by pipetting. The cell suspension was pooied in cold calf serum(CS)immediately after each digestion. After taht the resultant cells were washed three times with Earles BSS by centrifugation, the cells were seeded into glass bottle with Eagles MEM supplemented with 10% fetal bovine serum(FBS). The cells were cultivated at 24°C or 28°C under static and rubber-stoppered condition. Primary monolayers became confluent in 1 to 3 days. The pimary cells were mainly epithelial-like, as the subcultivation passed fibroblast-like cells became dominant. The cells grew optimally at 34°C. At 10°C and 15°C the cells grew scarcely. At 37°C the cells remained viable for at least several days, but the number of cells decreased gradually. Eagles MEM with FBS supported showed better growth than with CS. 10% FBS promoted better growth than 5% an 20% FBS. The supplement with tryptose phosphate broth in medium was somewhat inhibitory. The cells were uniformly dispersed by a short time of rinsing with 0.02%versen at room temperature followed by 5-10 minutes of treatment with 0.2% trypsin and 0.03% versen mixture at 37°C. The cells could be subcultivated 1.5-2 fold in 1-3 weeks at 24°C. A line designated EO-5(15-16 passage)multiplied 4 fold in 7-10 days at 34°C. The nuclei number of cells was successfully counted by the treatment of Sanfords reagents after immersing the cells with a volume of 0.02% versen. The cells could support the replication of IPNvirus, but not support the replication of IHN virus.
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ウナギ卵巣由来細胞の単層培養 : 培養細胞の性状と継代技法について

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