Medium optimization and characterization of cell culture system from Penaeus vannamei for adaptation of white spot syndrome virus (WSSV).

The lack of shrimp cell lines and difficulty in establishing shrimp cell culture systems, with an appropriate medium is a major concern in the aquaculture sector. The present study attempts to address this issue by developing an in vitro cell culture system from various tissues (hemocytes, heart, lymphoid tissue, hepatopancreas, gill, eye stalk, and muscle) of Penaeus vannamei (P.vannamei) using commercially available L-15 medium. The cell culture medium was formulated using five different media such as HBSCM-1, HBSCM-2, HBSCM-3, HBSCM-4, and HBSCM-5 containing L-proline and glucose with fetal bovine serum (FBS) supplements. Among the different media used, the HBSCM-5 medium with supplements showed good attachment and proliferation of cells with fibroblast-like, epithelioid, round, and adherent cell morphology in hemocyte culture. The same medium was further screened using different tissues to enhance the cell growth. The hemocytes, heart, and lymphoid tissue cells were passaged five times and maintained up to 20 days. Hepatopancreas and gill cells initially showed good morphological features and survived for more than ten days following subculture cells. Eye stalks and muscle cells perished within five days and did not show any unique morphology. The primary hemocyte cells were subjected to species identification, using cytochrome oxidase subunit I (COI) gene. To assess the primary hemocyte cell culture, cells were used for in vitro propagation of white spot syndrome virus (WSSV) and confirmed by the conventional polymerase chain reaction (PCR). Similarly, the primary cells were treated with bacterial extracellular products (ECPs) from Vibrio parahaemolyticus and Vibrio harveyi, to evaluate the cytotoxicity.

Ultrasound-mediated transscleral delivery of macromolecules to the posterior segment of rabbit eye in vivo.

PURPOSE: This study aims to determine the in vivo effectiveness of low-frequency ultrasound in mediating the transport of macromolecules to the posterior segment of the eye via transscleral route. It investigates if damage is caused by ultrasound at the tested operation parameters on the posterior ocular tissues and visual function. METHODS: Ultrasound (I(SATA) = 0.12 W/cm(2), center frequency = 40 kHz, 90-second continuous wave) was applied on the sclera of New Zealand white rabbits for one to three cycles. Solution of fluorescent dextran (70 kDa) was placed above sclera during and after ultrasound application to assess transscleral transport of macromolecules. Amount of dextran delivered to vitreous was determined by detection of fluorescence. Visual function of ultrasound-treated rabbits was examined by full-field electroretinography (ffERG). The effect of ultrasound on ocular tissue structures was examined by binocular indirect ophthalmoscope (BIO) and histology. RESULTS: Repeated ultrasound resulted in increasing concentration of dextran, which was otherwise undetectable in the vitreous. Transscleral barrier against dextran transport was restored to original value at 2 weeks postultrasound treatment. Studies from ffERG suggested that electric responses from neural transmission of retinal cells are normal at 1 day, 7 days, and 14 days after ultrasound applications. BIO and histology revealed no structural abnormality in posterior ocular tissues after ultrasound treatment. CONCLUSIONS: Low-frequency ultrasound significantly enhanced the penetration of macromolecules via transscleral route. No undesirable side effects have been found for up to 2 weeks after ultrasound application. The study supports that sonication is a potentially safe and effective method to modulate transscleral barriers for delivering macromolecular therapeutics to posterior segment of the eye.

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

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% CO(2). Although the temperature preference of hemocyte cells was the same as ovarian cells, CO(2) 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.