An improved method of cell culture system from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei
An improved method of cell culture system from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei
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DOI：
10.1007/s11626-010-9343-x
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发表时间：
2010-09
期刊：
影响因子：
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通讯作者：
S. George;A. Dhar
中科院分区：
文献类型：
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作者：
S. George;A. Dhar
以南美白对虾（Penaeus vannamei）的眼柄、肝胰腺、肌肉、卵巢和血细胞为材料，采用合成培养基和虾肌肉提取物（SME），建立了改良的细胞培养方法。对于血细胞和卵巢细胞培养物，补充有10%（v/v）胎牛血清和10%SME（v/v）的Grace昆虫培养基显示增强的细胞附着和增殖。血细胞和卵巢细胞培养物可分别维持48和66天，并分别传代培养4次和6次。卵巢和血细胞培养物主要含有上皮样细胞。来源于卵巢组织的细胞优选在26°C和28°C与5%CO2之间生长。虽然血细胞和卵巢细胞的温度偏好性相同，但补充CO2对血细胞的生长没有任何影响。当注射脂多糖（8 μg/g虾）并于注射后24 h抽取血淋巴时，血细胞的体外多样性显著提高。在上述条件下，眼柄、肝胰腺和肌源性细胞的生长与卵巢细胞和血细胞相比要少得多。卵巢细胞和血细胞的最适培养条件也与眼柄、肝胰腺和肌肉细胞的最适培养条件不同。肝胰腺、眼柄和肌肉细胞原代培养物的增殖效率分别约为30、12和<7天。本文所述的改进的培养条件，特别是对血细胞和卵巢的培养条件，将对涉及感染对虾的病毒的体外研究和对虾基因组研究非常有用。
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, CO2supplementation 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, thein vitromultiplicity 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 forin vitrostudies involving viruses infecting shrimp and in shrimp genomic studies.