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Isolation, Propagation, Characterization, Cryopreservation, and Application of Novel Cardiovascular Endothelial Cell Line From Channa striatus (Bloch, 1793)

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Abstract

There are only few primary endothelial cell cultures developed from fishes to date, but in this work the development of an endothelial cell line from Channa striatus is described. The vascular explants were plated into fibronectin (5 µg ml−1) and anti-CD31 antibody (100 ng ml−1)-coated flask; after 60 h incubation explants were removed from the flask. The flask contained only endothelial and blood cells. Blood cells were cleared out after subsequent passages. The culture medium used was Leibovitz’s L-15 supplemented with 20 % serum and antibiotics. The cultures were incubated at 28 °C in a normal atmosphere incubator. The plating efficiency was high (53.72 %). The endothelial cells were cryopreserved at different passage levels and revived successfully with 75–85 % survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of C. striatus cardiovascular endothelial (CSCVE) cell line from C. striatus. This cell line was further characterized for chromosome number, transfection, mycoplasma, cell cycle distribution, mitochondrial staining, and phagocytic activity. Cells were analyzed according to morphological appearance and expression of specific endothelial markers by fluorescent staining (von Willebrand Factor, anti-platelet endothelial cell adhesion molecule-1, and anti-Endoglin). The formation of tubules in the Matrigel and endothelial co-cultured with fibroblast like cells was observed. The cytotoxicity of ciprofloxacin on the CSCVE cell line was determined by MTT, AB, and R-123 cytotoxicity end points. Susceptibility of CSCVE cell line to nodavirus was confirmed by cytopathic effect and reverse transcriptase-polymerase chain reaction.

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Acknowledgments

S. Abdul Majeed is a recipient of INSPIRE Fellowship from the Department of Science and Technology, Government of India, New Delhi, India. The authors thank the management of C. Abdul Hakeem College, Melvisharam, India, for providing the facilities to carry out this work. This work was funded by the Department of Biotechnology, Government of India, New Delhi, India.

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  1. OIE Reference Laboratory for WTD, PG & Research Department of Zoology, C. Abdul Hakeem College, Vellore District, Melvisharam, 632 509, Tamil Nadu, India

    S. Abdul Majeed, K. S. N. Nambi, G. Taju & A. S. Sahul Hameed

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Abdul Majeed, S., Nambi, K.S.N., Taju, G. et al. Isolation, Propagation, Characterization, Cryopreservation, and Application of Novel Cardiovascular Endothelial Cell Line From Channa striatus (Bloch, 1793). Cell Biochem Biophys 71, 601–616 (2015). https://doi.org/10.1007/s12013-014-0240-x

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