Transfection, cytotoxicity, and cell cycle studies on the two newly developed and characterized humpback grouper (Cromileptes altivelis) fin cell lines.

Abstract

The development and characterization of two novel humpback grouper (Cromileptes altivelis) fin cell lines are described in this study. The CA1F3Ex and CA1F4Tr cell lines were developed by explant and trypsinization methods, respectively, in Leibovitz's L15 (L-15) medium supplemented with 20% FBS (fetal bovine serum) and subcultured over 150 times. Cell lines exhibited high stability, as evidenced by the high revival rate (85-95%) and good attachment while seeding after one year of cryostorage. They displayed good seeding (91%) and plating efficiencies (15-25%). The optimum temperature for growth was recorded at 28˚C. Serum requirement decreased with increased passage and lowered to 2% FBS beyond 30-35 passages. However, higher serum concentration (2-20%) caused a concurrent increase in cell growth. Both the cell lines were fibroblast-type, and immunotyping results showed strong reactivity towards the fibroblast marker. Chromosome analysis of these cell lines revealed aneuploidy, and the authenticity was confirmed by mitochondrial Cytochrome C Oxidase Subunit I (COI) genotyping analysis. Cell cycle studies were performed utilizing the flow cytometric technique. CA1F3Ex and CA1F4Tr cell lines showed high transfection efficiency with pEGFP-N1 plasmid using Lipofectamine and cytotoxicity towards heavy metals (Hg and Cd) was also studied. Hence, these continuous cell lines could be employed as in vitro models for aquatic toxicological and genetic manipulation studies.