Establishment of Primary Cell Culture Technique for Liver Tissue of Mandarin Fish Siniperca chuatsi
LUO Hanyu1, LIU Xiao1, ZHENG Feifei1, MAO Tianyu1, FENG Keyao1, SUN Yanhong2, ZHU Dongmei1
1. College of Fisheries, National Demonstration Center for Experimental Aquaculture Education, Huazhong Agricultural University, Wuhan 430070, China; 2. Wuhan Academy of Agricultural Sciences, Wuhan 430345, China
Abstract：The primary cell culture of liver tissue of mandarin fish Siniperca chuatsi with body weight of(33.2±0.2) g was carried out by tissue explants adherent method, enzyme digestion method, and tissue explants adherent combined with enzyme digestion method for screening of the optimal method. The primary cell culture of liver tissue of mandarin fish was in 3 kinds of culture media (M199 culture medium, L15 culture medium, and M199-L15 culture medium), three kinds of culture containers (12 well plate, 6 well plate, and T25 culture bottle) and with or without CO2 were compared to determine the optimal culture conditions of primary cells from liver tissue of mandarin fish. The cells were identified using mitochondrial 16S rRNA gene sequences and chromosome count. The effects of ammonium chloride (0 mmol/L, 1 mmol/L, 5 mmol/L, and 10 mmol/L) on cell morphology and proliferation were also investigated. The results showed that the good cells of the liver tissue of mandarin fish was obtained by 0.25% trypsin-EDTA digested cells under conditions of M199-L15 (volume ratio 1∶1) mixed medium containing 20% fetal bovine serum, which were inoculated into T25 culture flask, and culture at 28 ℃ in an incubator containing 5% CO2. The attached cells accounted for 25%—30% of the bottom of culture flask at 2—3 days, the cells grew 70%—80% of the bottom of culture flask at 4—5 days, and primary monolayer cells were obtained at 6—7 days. The cells were plump and had strong proliferative ability after subculturing, appearing in a mixture of epithelioid and fibroblast-like cells, with fibroblast-like cells being the majority. The liver cells had grown up with 10 passages. The result of mitochondrial 16S rRNA gene sequencing was 99.05% consistent with the gene sequence in GenBank (HQ731435.1), and chromosome count was found to be 48, proving that these cells were from mandarin fish. The significant effects on cell morphology and vitality (P<0.05) were observed at 5 mmol/L and 10 mmol/L ammonium chloride exposure. The primary cell culture technology of liver tissue of mandarin fish was established, and provided an important basis with the development of biology, disease, nutrition and genetics of mandarin fish, and primary culture and establishment of other fish liver cell lines.
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2024, Vol. 43 
