A Primary Study On Cytological Structures And Primary Cell Cultures Of Liver And Spleen From Hexagrammos Otakii

Liver is considered to be an important organ due to its functions in maintaining the internal homeostasis in fish. The spleen of fish is believed to be involved in haematopoiesis, the clearance of macromolecules, antigen degradation and processing and the production of antibody. The liver cultures and spleen cultures from fish have been proved to be good models for the study of metabolism and physiology as well as for the research of toxicology and carcinogenesis in vitro.Hexagrammos otakii is an important commercial marine teleost fish and its aquaculture has being developed in costar area of northern China since recent years. However, the research on its basic biology is little by now.In this paper, the micro-structure and ultra-strcture of liver and spleen in Hexagrammos otakii were studied by histological and cytological technique. The primary culture technique for the splenic cells and hepatic cells from Hexagrammos otakii is described for the first time. The conditions for primary culture have been determined.The results from liver histology research of H. otakii showed that the connective tissue was less and the hepatic lobule was not distinct. Central vein was irregular, the hepatic cord was radial around central vein, and hepatic sinusoid was irregular. Portal area was not typical. Boundaries of hepatocytes were clear, the hepatocyte was large and polygonal, cytoplasm was plentiful. The results of spleen histology research indicated that the splenic capsule was very thin and the trabeculae were poorly developed. The chief characteristics of the spleen were the pulp and melano-macrophage centers. The spleen parenchyma was composed of white pulp and red pulp, but there was no sharp demarcation between them. The major cellular components were erythrocytes, lymphocytes, reticular cells, granulocytes and macrophages. The melano-macrophage centers were usually located near vessels, but the masses of melano-macrophage were not surrounded by circum adjacent cell layers.The purpose of cell culture study in this paper is to explore proper methods and culture conditions for the initiation of cell cultures from liver and spleen in H. otakii.In order to find out and optimite the culture conditions for the hepatic cells, the orthogonal design experiments which took culture medium (L-15,DMEM,MEM), serum concentration (10%,15%,20%), osmotic pressure (normal NaCl concentration, 10g/L, 15g/L) as the factors and furthermore two-factor three-level experiments which took serum concentration (10%,15%,20%), osmotic pressure (normal NaCl concentration, 10g/L, 15g/L) as the factors were conducted. The results showed that the proper culture condition of hepatic cells was MEM with 15% fetal bovine serum (FBS) without additional NaCl in the medium. Under this culture condition, the cultured cells were highly homogeneous, they grew well and could survive for 14 d or so.During the primary culture of splenic cells from H. otakii, the culture medium (L-15, MEM), serum concentration (10%, 15%, 20%) were also been chosen to screen the culture condition. The results indicated that MEM was the better medium to initiate the culture of splenic cells from H. otakii. Different FBS concentrations supported different types of cells growing. Type C cells (fibroblast-like cells) could grow into monolayer or multilayer in MEM with all FBS concentrations, but they grew best in MEM with 15% FBS. Under this culture condition, Type C cells could outgrow from explants and cover approcimately 80% of the growth surface which was the most among three FBS concentrations. The optimum culture condition for the growth of Type D cells (macrophage-like cells) was MEM with 20% FBS in which the cells could survive longer (50d) than the other two conditions.The hematopoietic foci were observed after the first change of medium in the spleen cultures. The foci were formed by round phase bright cells loosely attached on top of type C cells (fibroblast-like cells), some of which were subsequently released into the medium. It was inferred that type C cells maybe formed a stromal cell layer which played a role in haematopoiesis and the spleen of H. otakii was an important haemopoietic organ as in other teleosts.