Improved primary cell culture and subculture of lymphoid organs of the greasyback shrimp Metapenaeus ensis

• A 1.5×L-15 based shrimp medium III has been newly formulated and verified.
• Primary lymphoid cell monolayer could be rapidly formed in medium III within 24 h.
• Lymphoid cells in medium III could maintain their susceptibility to shrimp virus.
• Routine and successful development of primary lymphoid cell cultures was achieved.
• HyQTase and ECDS along with cold-PBS pretreatment are effective subculture methods.

Despite numerous attempts, no continuous shrimp cell lines have yet been established. Here we report on an improved primary cell culture system as evidence of the routine and successful development of primary cell monolayers from the lymphoid organs of greasyback shrimp Metapenaeus ensis, using a newly formulated 1.5 × L-15 based shrimp medium III. The shrimp medium III showed significantly better results than the other two shrimp media of I (0.5 × L-15 based, hemolymph-imitated) and II (2 × L-15 based) did. In medium III, the migration of lymphoid cells from the explants was initiated at 2–3 h after seeding, and a 70%–80% confluent cell monolayer was formed within 16–24 h and remained viable for over 20 days. Moreover, the lymphoid cells cultured in medium III maintained their susceptibility to shrimp white spot syndrome virus (WSSV) and the WSSV could replicate in the infected lymphoid cells, an important feature for primary cell activity. In the attempts to passage the lymphoid cell culture, non-mammalian-derived enzyme complex of HyQTase, enzyme-free cell dissociation solution (ECDS), and the physical shocks with cold PBS (phosphate-buffered saline, 4 °C) are important to markedly increase efficiency for both detachment (90%–100%) and reattachment (54.1 ± 2.0% for HyQTase and 60.2 ± 2.7% for ECDS) in comparison with other enzymes and methods tested. Using HyQTase and ECDS, we successfully passaged the lymphoid cell monolayers twice and then the cell density became too low to be further sub-cultured due to the absence of active proliferation of the in vitro cultured shrimp cells. The tested digestive enzymes of trypsin, collagenase type II, dispase, hyaluronidase, elastase and pronase were highly toxic to shrimp cells, and produced unfavorable subculture results in our procedures. The establishment of the above-mentioned primary cell culture and subculture systems will lay a solid foundation for further investigation of shrimp cell immortalization and development of stable shrimp cell lines.