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An improved method of cell culture system from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei

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Abstract

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO2. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO2 supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.

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Acknowledgment

This project received support from the Defense Advanced Research Projects Agency (DARPA) and from the Defense Threat Reduction Agency (DTRA) Chemical and Biological Technologies Directorate Transformational Medical Technologies Initiative under DTRA contract HDTRA1-07-C-0078. The authors would like to thank Krista N. Kaizer, Yelena Betz and Elena Artimovich, Advanced BioNutrition Corp., Columbia, MD for their technical help. The authors would like to thank Dr. F. C. Thomas Allnutt for reviewing the manuscript and providing us with valuable comments.

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Correspondence to Sunil K. George.

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Editor: J. Denry Sato

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George, S.K., Dhar, A.K. An improved method of cell culture system from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei . In Vitro Cell.Dev.Biol.-Animal 46, 801–810 (2010). https://doi.org/10.1007/s11626-010-9343-x

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