The concept of cell lines emerged from the uncontrolled growth of cells called cancer; however cancer cell lines are not easy to develop. In current time a good number of human cancer and non cancer cell lines are available for research to find out a cure for this disease with the development of new therapies and medicines. According to different reports, cases of this disease are increasing day by day. Environmental pollution and different habits in modern life style viz. smoking, tobacco, etc. increasing the risk of cancer. Reports from several countries, including India on this issue are horrendous. The risk, severity and symptoms can be better understood by molecular level analysis of the disease. To date advancement of technology from microarray transcriptome analysis to transcriptome sequencing has been provided a deep insight into cancer. Sample size and ethical issues related to human necessitates the use of in vitro techniques alongwith such molecular techniques to get a thorough understanding of the disease and also provides a good prospectus.
A cell line named BBdF-1, established from the caudal fin of hill stream fish Barilius bendelisis, has been subcultured for more than 52 passages and is being maintained in L-15 media containing 20% FBS. Species origin of the cell line was confirmed using amplification of partial region of 16S and COI mitochondrial gene sequences. The optimum temperature for growth of BBdF-1 cell line was found to be 28°C. Karyotyping revealed diploid chromosome number as 50. Cells exhibited strong binding for cytokeratin marker and thus were found to be epithelial-like. Strong green fluorescence was observed in BBdF-1 cells transfected with phrGFP-II-N vector, indicating its suitability for utilization in gene expression and manipulation studies. Successful assessment of cytotoxicity of two heavy metals, viz. mercury and chromium, was performed. The cell line can serve as a useful resource material for early toxicity screening of pesticides/pollutant and gene expression.
Schizothorax richardsonii, commonly called snow trout is a vulnerable coldwater fish distributed in India and adjoining countries, is an important resource as food, ornamental and game fishery. A cell line, designated as SREM-1, has been established from the eye muscle of S. richardsonii using explant method and cultured in Leibovitz’s L-15 medium. The growth studies at 20-32°C temperatures and 5-20% of fetal bovine serum (FBS) concentrations revealed that SREM-1 cells exhibited optimal growth at 28°C in L-15 medium containing 20% FBS. Cell type was confirmed primarily as epithelial in nature by immuno-phenotyping assay and was sub-cultured for >55 times since its development. The origin of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. SREM-1 cell line is free from mycoplasma contamination and has been cryopreserved at different passage levels with revival efficiency of 70-80% after 6 months. The SREM-1 cell line was successfully transfected and also employed for evaluating in vitro cytotoxicity against a heavy metal, mercuric chloride. The newly established cell line of this vulnerable species would be useful as a model for aquatic toxicological and transfection studies.
Pangasianodon hypophthalmus is an economically important catfish species cultured in India. In the present study, a continuous cell line from the thymus of P. hypophthalmus (PHT) was established using the explant method and subcultured 52 times since development. PHT cells showed optimal growth in L-15 medium containing 20% fetal bovine serum at 28°C. The species of the cell line from striped catfish was confirmed through PCR amplification and sequencing of 16S, and COI genes. The cell line was found to be Mycoplasma free. The modal chromosome number of PHT cells was 60 (2n). Immunophenotyping using different antibodies showed the epithelial nature of the cells. Cytotoxicity of arsenic and mercury was assessed using Neutral red and MTT assay, which revealed reduced cell survival with an increase in toxicant concentration. Cells transfected successfully with the GFP reporter gene using lipofectamine reagent indicated the suitability of the cell line for expression studies. The cell line has been submitted to NRFC, Lucknow with accession no. NRFC-078 at ICAR-NBFGR, Lucknow. The developed cell line will have applications in suspected viral disease investigation, transgenic, and immunological studies.
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