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

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% 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.

Establishment of Shrimp Cell Lines: Perception and Orientation

“…Among the commercial media used, Leibovitz's-15 (L-15) has been the most popular one for shrimp cell culture. Of the 50 selected publications 32 (64 %), were based on L-15 as the basal medium [2-4, 7-11, 13, 15, 18, 25, 37, 40-42, 46, 51, 56-58, 62-64, 69, 73, 74, 78, 80-83, 89], six (12 %) selected Grace's Insect Medium [26,27,49,63,82,89], five (10 %) M199 [28,39,48,74,82], and three (6 %) MPS [19,36,80]. A couple of other media such as Pj-2 [55], NCTC 135 [89], MM Insect medium and TC 100 medium [63], were also tested for the development of cell lines from shrimp ( Fig.…”

“…Of the 50 selected publications, 17 reported (34 %) P. monodon [2-4, 7, 8, 10, 15, 23, 35, 41, 42, 44, 58, 69, 83, 89, 90] as the species of choice, eight researchers (16 %) used Penaeus japonicus [10,39,47,48,[55][56][57]72], seven (17 %) selected Penaeus chinensis [13, 19, 36-38, 40, 80] and Penaeus vannamei [18,26,27,49,51,63,82]. Moreover, six authors (16 %) selected Penaeus stylirostris [49,51,63,73,74,78] as the donor animal of tissues and organs.…”

“…[27,46,62,80]. Besides, testis [62,81], heart [9,10,48,62,80], hepatopancreas [9,28,48,55,58,62,64,81,89], gill [9,62], nerve [9,13,48,62,80,81], muscle [9,27,48], hematopoietic tissue [11,62,90], embryonic tissue [19,80,82], epidermis [81,82], gut [9,62] and Y organ [81] were also widely used for cell culture development (Fig. 3).…”

“…Embryonic cell cultures could be maintained for several months [82] and attained 10 passages [19]. Moreover, researchers could maintain nerve cells from 15 days [13] and to up to 3 months [63], heart tissue for 4 days [10] and hepatopancreas for 30 days [27]. The striking observation was that there existed no consistency in the number of days which a cell culture could be maintained by different workers.…”

The scope of the crustacean immune system for disease control

“…In part this is attributable to the early development of permanent cell lines for insects. Whilst recent significant progress has been made in sustaining primary cultures of crustacean haemocytes and ovarian cells (George & Dhar, 2010) homari and Hematodinium spp.). In almost all cases it is very unlikely that intervention and control strategies, such as treatment by prophylaxis, will be applicable in these cases.…”