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 cultu re medium was tested for osmolality, serum concentration, serum source s 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 Anal osmolal ity at 470-500 mmol kg(-1) allowed for enhanced cell attachment and gr owth; however, cells could not be maintained for more than 5 days. The supplementary nutrients were also tested for carbohydrates, amino aci ds, L-ascorbic acid, Buffalo rat liver(BRL)-condition medium and selen ium. The basic culture medium + 1 g l(-1) glucose were found to enhanc e cell attachment and growth, Our collected lymphoid cells required 3 days of incubation to obtain 80% confluency; however, cells did not gr ow in subcultures. Several growth factors were tested for developing a subculture system of shrimp cells. Epidermal growth factor (EGF) or t ransforming growth factor beta (TGF beta) did not foster cell growth C ells treated with insulin or insulin-like growth factor I (IGF I) were capable of being subcultured, but resultant cells differed in terms o f feeder layer, and were eventually discarded due to yeast contaminati on. Cells treated with basic fibroblast growth factor (bFGF) 20 ng ml( -1) (F-20) could be subcultured for more than 90 passages without a fe eder layer. Some F-20-treated cells were capable of extending their ex tracellular matrices for cell attachment and piled up; some of them be came suspended and lost their anchorage-dependent and contact inhibiti on properties.