Development of an in vitro subculture system for the oka organ (Lymphoid tissue) of Penaeus monodon

We tried to establish a subculture system for cells from the Oka organ (lymphoid tissue) of the grass prawn Penaeus monodon. The basic culture medium was tested for osmolality, serum concentration, serum sources and pH. It was found that Leibovitz's L-15 medium supplemented with 10% fetal bovine serum, 5 g l −1 NaCl, pH 7.63–8.1, with final osmolality at 470–500 mmol kg −1 allowed for enhanced cell attachment and growth; however, cells could not be maintained for more than 5 days. The supplementary nutrients were also tested for carbohydrates, amino acids, L-ascorbic acid, Buffalo rat liver(BRL) -condition medium and selenium. The basic culture medium + l g l −1 glucose were found to enhance cell attachment and growth. Our collected lymphoid cells required 3 days of incubation to obtain 80% confluency; however, cells did not grow in subcultures. Several growth factors were tested for developing a subculture system of shrimp cells. Epidermal growth factor (EGF) or transforming growth factor β (TGF β) did not foster cell growth. Cells treated with insulin or insulin-like growth factor I (IGF I) were capable of being subcultured, but resultant cells differed in terms of feeder layer, and were eventually discarded due to yeast contamination. Cells treated with basic fibroblast growth factor (bFGF) 20 ng ml −1 (F-20) could be subcultured for more than 90 passages without a feeder layer. Some F-20-treated cells were capable of extending their extracellular matrices for cell attachment and piled up; some of them became suspended and lost their anchorage-dependent and contact inhibition properties.