Bright Singh, I S; Rosamma, Philip; Jayesh, P; Seena, Jose(Springer, September 30, 2012)
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Abstract:
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
8C. 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 (29), 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’’.
Description:
Cytotechnology (2013) 65:307–322
DOI 10.1007/s10616-012-9491-9
Bright Singh, I S; Rosamma, Philip; Mohandas, A; Seena, Jose(Elsevier, August 31, 2010)
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Abstract:
Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of
investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context,
a method of developing primary hemocyte culture from this crustacean has been standardized by
employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose,
MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM),
0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph
drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium
the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its
incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence
assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient
method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture.
Expression of viral genes and cellular immune genes were also investigated. The cell culture could
be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining
its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and
cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs
and chemicals
Description:
Journal of Invertebrate Pathology 105 (2010) 312–321