Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line, especially continuous crab cell line, can provide us a useful tool for studies on the virology, immunology, and molecular biology of crabs. However, no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest. In this study, long-term in vitro culture conditions for both two- (2D) and three-dimensions (3D) were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus , designated as PTH cells. In 2D culture, a novel crab basic medium in osmolarity of 990–1 100 mOsm/kg was optimized for the first time, which is different from Leibovitz’s L-15 medium in mainly the components of amino acids, containing double strengths of the contents of free amino acid mixture in the crab serum. Then an optimal crab growth medium was developed by supplementing 5% fetal bovine serum, 50-g/L yeast extract powder, 20-µg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium, and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells. These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen. In 3D culture, using the same crab growth medium, the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60% on Day 103. This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.