A novel medium for the development of in vitro cell culture system from Penaeus monodon
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Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg−1 and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”.
KeywordsShrimp cell line Penaeus monodon Shrimp cell culture medium (SCCM) MTT assay Lymphoid cell culture
This research was supported by Department of Biotechnology, Government of India (BT/PR8050/AAQ/03/289/2006). The first author thanks DBT for Fellowship.
- Chen SN, Jong KJ, Kou GH (1989) Cell cultures derived from tissues of penaeid shrimp, Penaeus penicillatus and hard clam, Meretrix lusoria. In: Mitsuhashi J (ed) Invertebrate cell system applications. CRC Press Inc., Florida, pp 253–262Google Scholar
- Freshney RI (2000) Culture of animal cells: a manual of basic technique. Wiley-Liss, New YorkGoogle Scholar
- Jose S (2009) Cell culture from Penaeus monodon: development and application. Ph.D. Thesis. Cochin University of Science and Technology, IndiaGoogle Scholar
- Lu Y, Tapay LM, Loh PC, Brock JA, Gose RB (1995) Distribution of yellow-head virus in selected tissues and organs of penaeid shrimp Penaeus vannamei. Dis Aquat Organ 23:67–70Google Scholar
- Lynn DE (2001) Novel techniques to establish new insect cell lines. In Vitro Cell Dev-An 37:319–321Google Scholar
- Machii A, Wada KT, Awaji M, Nakamura HK, Townsley SJ (1988) Some characters of cells of the midgut gland and chytrids from primary cultures of the prawn Penaeus japonicus. In: Kuroda Y, Kurstak E, Mararnorosch K (eds) Invertebrate and fish tissue culture. Japanese Scientific Societies Press, Tokyo, pp 11–14CrossRefGoogle Scholar
- Mitsuhashi J (2001) Development of highly nutritive culture media. In Vitro Cell Dev-An 37:330–337Google Scholar
- Shimizu C, Shike H, Klimpel KR, Burns JC (2001) Hemolymph analysis and evaluation of newly formulated media for culture of shrimp cells (Penaeus stylirostris). In Vitro Cell Dev Biol Anim 37:322–329Google Scholar
- Tapay L, Lu Y, Brock JA, Nadala ECB, Loh PC (1995) Transformation of primary cultures of shrimp (Penaeus stylirostris) lymphoid (Oka) organ with simian virus-40 (T) antigen. Proc Soc Exp Biol Med 209:73–78Google Scholar