Development and characterization of primary cell cultures from the hematopoietic tissues of the Dublin Bay prawn, Nephrops norvegicus.

Improved maintenance in vitro of the hematopoietic tissue of the Dublin Bay prawn Nephrops norvegicus (L.) resulted by using 10% (v/v) 2x Leibovitz's medium prepared in seawater (salinity = 25 per thousand), and supplemented with 10% (v/v) heat inactivated fetal bovine serum plus 5% (v/v) Nephrops serum or 5% (v/v) Nephrops muscle extract, and 0.06 g/l of L-proline and 1 g/l of glucose. Pronase at 100 microg/ml improved tissue dissociation and subsequent spreading of hematopoietic cell cultures. The addition of epithelial growth factor (EGF), based fibroblast growth factor (bFGF) or insulin growth factor 1 (IGF-I) did not enhance cell growth. Cell culture contained several types of maturing hemocytes, in the size range of 6-24 microm diameter. Acid phosphatase, alpha-naphthyl butyrate esterase, alpha-naphthyl acetate esterase, naphthyl AS-D chloroacetate esterase activity and phenoloxidase activity was demonstrated, but not so alkaline phosphatase or peroxidase. Small PAS (= Periodic acid Schiff) positive granules, unsaturated lipids and phospholipids were observed. Cultures remained functional for over two weeks. Mitosis was noticed occasionally; however, cell proliferation was not recorded by use of nuclear proliferation markers.