In vitro culture and characterisation of a new brain cell line from the spine cheek anemone fish Premnas biaculeatus (Bloch, 1790)

Priyanka Poulose; K. S. Sobhana; K. Madhu; Rema Madhu; C. Kalidas; K. K. Joshi; Grinson George; A. Gopalakrishnan

doi:10.21077/ijf.2024.71.2.144886-09

Authors

Priyanka Poulose CENTRAL MARINE FISHERIES RESEARCH INSTITUTE and CUSAT
K. S. Sobhana
K. Madhu
Rema Madhu
C. Kalidas
K. K. Joshi
Grinson George
A. Gopalakrishnan

https://doi.org/10.21077/ijf.2024.71.2.144886-09

Keywords:

Clownfish, Brain cell line, Fish cell lines, Leibovitz's L15 medium, Trypsinization

Abstract

In this study, a new brain cell line designated PB1BrTr was derived from the maroon clownfish Premnas biaculeatus (Bloch, 1790) and characterised. The PB1BrTr cell line was developed by trypsinisation method using Leibovitz’s L15 (L-15) medium supplemented with 20% FBS (foetal bovine serum) and subcultured over 100 times. Characterisation encompassed studies on optimal growth kinetics, chromosomal analysis and genotyping of the mitochondrial CO1 gene. A high revival rate (85-95%) and good attachment during seeding after a year of cryostorage demonstrated the high stability of the cell line. This cell line exhibited good seeding efficiency of 84% at 1.25 x 105 cells ml-1 and a range of plating efficiencies from 14-23% at varying cell densities. It was observed that 28˚C was the ideal temperature for its growth. Serum requirement decreased with increased passage and lowered to 2% FBS beyond 60 passages. However, higher serum concentration (2-20%) caused a concurrent increase in cell growth. The cell line displayed a fibroblast-type morphology, with immunotyping results revealing robust reactivity towards the fibroblast marker. Chromosome analysis of this cell line revealed aneuploidy and its authenticity was validated by mitochondrial Cytochrome C Oxidase Subunit I (COI) genotyping analysis. This brain cell line demonstrated notably high transfection efficiency with pcDNA3-EGFP plasmid using Lipofectamine 3000 transfection reagent. This continuous cell line presents a valuable in vitro tool for diverse research applications, including gene transfer and expression studies.

Keywords: Brain cells, Fish cell lines, Leibovitz’s L15 medium, Maroon clownfish, Trypsinisation

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