Routine development of long-term primary cell culture and finite cell line from the hemolymph of greasyback shrimp (Metapenaeus ensis) and virus susceptibility

Long-term and continuous shrimp cell cultures can offer an indispensable tool for studies on the isolation, multiplication and pathogenic mechanism of shrimp causative viruses at cellular and molecular levels. However, they are hard to be developed routinely and continuous shrimp cell line is still unavailable. This can be attributed to a great degree to the unsuitability of medium used. In this study, we have successfully developed a long-term culture condition for the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. Using the newly developed gelatin-containing optimal basic medium (SBM-12), MeH cell monolayer could be routinely developed and maintained healthily beyond 275 days. In the novel optimal growth medium (SGM-10), the proliferation potentials of MeH cells have been markedly improved, evidenced by the presence of dividing cells in the primarily cultured 30-day hemocytes, suggesting that the mitosis-arrest of MeH cells has been tentatively broken. It was also found that among the 4 types of hemocytes (granulocyte, semi-granulocyte, hyalinocyte and prohaemocyte), the hyalinocytes could not survive after a long-term in vitro culture, but the prohaemocytes could survive well and thus could serve as a good cell source. The addition of gelatin could significantly alter the attachment and cellular morphology of MeH cells and result in a loose attachment and round shape, which made it easy for the cells to be passaged by physical flushing for up to 32 times and a finite shrimp cell line can be developed routinely. Virus susceptibility test showed that the MeH cells cultured in the optimal growth medium were sensitive to shrimp covert mortality nodavirus (CMNV), and obvious cytopathic effects and viral multiplication were detected in the infected cells. In a word, the success in the routine development of long-term primary cell culture and finite cell line from shrimps in this study will greatly facilitate the establishment of shrimp continuous cell lines and studies on shrimp viruses.