Biotechnology and Bioinformatics (1+1)

3.1.10.1. Long-term storage

• Fish cell cultures can be stored frozen in liquid nitrogen or in ultra cell freezers using standard methodologies for freezing and thawing.
• Salmonid cell lines can be kept at 4^o– 6^oC for a period of 4–6 months.
• Cell lines from warm-water species generally cannot be stored at low temperature as that of cold-water species.
• Cell lines kept at sub-optimal temperatures for extended periods of time can easily be recovered by adding fresh growth medium and incubating at optimal temperature for 24–72 hrs before sub-culturing.

Freezing is done at three stages, first at 0^oC for 30 min, then at -20^oC for 60 min and thirdly at -70^o C for 6 months and finally at -196^oC for one or two years in liquid nitrogen.

In order to protect from damages of cells during storage, DMSO 7.5% and glycerine 10% are used along with medium. Freezing of cells is done mainly for three reasons.

i) During cell line the cells may change their enzyme activity, chromosome number, etc. Therefore it is essential to freeze these cells at a particular stage of cell line and then rejuvenated.

ii) There may be contamination in cell line. To prevent this cells are frozen at periodic intervals.

iii) In an established cell line the cells can be cultured to a maximum of 50 times. In some other cell line, cells are likely to die at any time. Such cell lines can be sub-cultured only for 30 times. Freezing of these cells may extend the period of cell line.