Development and characterization of a new muscle cell culture system from Clarias magur (Hamilton, 1822).

The cell line has been used as a novel in vitro tool for executing several studies in life sciences. The current study aimed to develop and characterize a muscle cell culture system derived from Clarias magur. The primary muscle cell cultures derived from the caudal peduncle muscle have been successfully sub cultured up to 13 passages to establish a new muscle cell culture system known as CMM. At a temperature of 28 °C, L-15 medium supplemented with 20% FBS produced the maximum growth of muscle cells. However, muscle cells were optimized to grow at 10% FBS. To enhance the proliferation capacity of the CMM cells, a growth-promoting factor bFGF (10 ng/ml) was added, thereby reducing the time interval of passages for the subsequent cultures. DNA barcoding of the CMM cell culture system authenticated the species of origin. The cell culture system was successfully cryopreserved by a slow freezing procedure at - 80 °C with a revival efficiency of 60%.

Establishment and characterization of a new fibroblast-like cell line from the skin of a vertebrate model, zebrafish (Danio rerio).

BACKGROUND: The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discovery & screening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio. METHODS AND RESULTS: A novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz's -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28 °C, respectively. Cells were cryopreserved and revived with 70-75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24-48 h post-transfection. CONCLUSION: The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.

Cellular Aquaculture: Prospects and Challenges.

Aquaculture plays an important role as one of the fastest-growing food-producing sectors in global food and nutritional security. Demand for animal protein in the form of fish has been increasing tremendously. Aquaculture faces many challenges to produce quality fish for the burgeoning world population. Cellular aquaculture can provide an alternative, climate-resilient food production system to produce quality fish. Potential applications of fish muscle cell lines in cellular aquaculture have raised the importance of developing and characterizing these cell lines. In vitro models, such as the mouse C2C12 cell line, have been extremely useful for expanding knowledge about molecular mechanisms of muscle growth and differentiation in mammals. Such studies are in an infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines, except a few fish muscle cell lines that have not yet been used for cellular aquaculture. The Prospect of cell-based aquaculture relies on the development of appropriate muscle cells, optimization of cell conditions, and mass production of cells in bioreactors. Hence, it is required to develop and characterize fish muscle cell lines along with their cryopreservation in cell line repositories and production of ideal mass cells in suitably designed bioreactors to overcome current cellular aquaculture challenges.

Development and characterization of a new gill cell line from the striped catfish, Pangasianodon hypophthalmus (Sauvage, 1878).

Cell lines as an in vitro model developed from different target organs of fish find their use in virus susceptibility, cytotoxicity, gene expression studies. The striped catfish, Pangasianodon hypophthalmus, is one of the main species in aquaculture, especially in Southeast Asian countries like Thailand, Indonesia, China, India, Bangladesh, and Vietnam. The present study reports the development of a new permanent cell line from the gills of P. hypophthalmus designated as PHG and its application in toxicological research. Leibovitz's L-15 cell culture medium supplemented with 15% fetal bovine serum (FBS) was used to maintain cell line PHG. The morphology of the PHG cell line was observed fibroblastic-like. PHG cells grew well at varying temperatures ranging from 24 to 30 °C with an optimum temperature of 28 °C. The PHG cell line was characterized using a sequence of mitochondrial cytochrome C oxidase subunit I, which authenticated the species of origin of the cell line. The cell line was transfected with a pEGFP-C1 plasmid, and the transfection reporter gene was successfully expressed 48 h post-transfection with 9% transfection efficiency. The toxicity assessment of two organophosphate pesticides, chlorpyrifos, and malathion using the PHG cell line revealed that the two organophosphate pesticides were cytotoxic to the cell line at varying concentrations.