TY - JOUR
T1 - Improved primary cell culture and subculture of lymphoid organs of the greasyback shrimp Metapenaeus ensis
AU - Han, Qian
AU - Li, Pengtao
AU - Lu, Xiongbin
AU - Guo, Zijuan
AU - Guo, Huarong
N1 - Funding Information:
We thank Professor Xiaohang Huang (First Institute of Oceanography, State Oceanic Administration, China) for his assistance in the osmolarity test. This work is supported by the National Natural Science Foundation of China (grant no. 31172391 ), National High-tech R&D Program of China (863 program; grant no. 2012AA10A402 ), Scholarship Foundation for Excellent Scientists of Shandong Province (grant no. BS2011SW054 ) and Fundamental Research Funds for the Central Universities of China (grant no. 201122005 ).
PY - 2013/10/10
Y1 - 2013/10/10
N2 - 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.
AB - 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.
KW - Lymphoid organs (Oka organ)
KW - Metapenaeus ensis
KW - Primary cell culture
KW - Shrimp
KW - Subculture
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U2 - 10.1016/j.aquaculture.2013.06.024
DO - 10.1016/j.aquaculture.2013.06.024
M3 - Article
AN - SCOPUS:84884177266
SN - 0044-8486
VL - 410-411
SP - 101
EP - 113
JO - Aquaculture
JF - Aquaculture
ER -