Biotechnology and Bioinformatics (1+1)

3.1.3.1. Primary cell culture

The methodologies and growth media for the preparation and maintenance of fish cell cultures gene rally do not differ from those used for the culture of cells from homeotherm vertebrates.

• The selection of fish species and appropriate tissues for the initiation of primary cell cultures is usually dictated by the cell type or function to be studied and/or the ultimate use of the cell culture.
• The main sources are embryonic cells and reproductive cells or gonadal tissue, due to their rapid multiplication.
• Other sources are gill tissue, connective tissues, skeletal, cardiac, epithelial cells, neural cells, heart, kidney, liver and spleen and endocrine cells.
• In many ways, the initiation of cell cultures from fish is actually easier than from hoemotherm vertebrates. Unlike mammalian cells, which must be kept near 37^oC, most fish cells easily tolerate or even prefer wide range of temperatures < 37^oC. Therefore, tissue samples can be collected at field sites, placed in growth media, and transported on ice or even at ambient temperatures to the laboratory for preparation.

Cell suspensions for monolayer cultures are usually prepared by standard methods of

• enzymatic dissociation, usually Trypsin – EDTA.
• Fish tissues are usually dissociated at very low temperatures or at temperatures approximately those of the species natural environment.
• In some cases when enzymatic dissociation has failed to produce actively dividing cells or when relatively small volumes of tissue are available and the number of viable cells can be expected to be relatively small, success in initiating monolayer cultures has been achieved starting with explants cultures.
• Naturally, for certain types of study, tissue explant and organ culture are the methods of choice.
• Migration of dividing cells out of the explants frequently results in foci of small cell monolayers surrounding the explant, which can usually be sub cultured following enzymatic duration.

Primary fish cell cultures usually consists of a variety of cell types including both epithelial-like and fibroblast-like cells as well a variety nondividing cells. Following several subcultures, however, one cell type usually becomes predominant.

The ratio at which subcultures can be made from primary cultures varies considerably. Few passages are made at relatively low ratios such as 1:2 or 1:3.