Biotechnology and Bioinformatics (1+1)

3.1.5. Requirements of cell culture

3.1.5.1. Serum additives

• Human cord serum is found to be excellent for fish cell culture. But as the cost is higher, calf serum replaces human cord serum.
• Fetal bovine serum can also be used.
• The usual level of serum is 10-15%.

3.1.5.2. Other additives

Other additives used in the culture media are

• products of human ascitic fluid,
• bovine aminoic fluid,
• chick embryo extract,
• lactalbumin hydrolysate,
• serum ultrafiltrate, peptone,
• yeast extract, and
• whole egg ultrafiltrate.

3.1.5.3. pH

Practically the pH range is not so critical.

• Most of the cells can grow well at a pH range of 7.2 – 7.8 (7.4 optimum).
• Although bicarbonate buffered media are usually employed, organic buffers such as HEPES can also be used if desired.
• Also, fish cells require CO₂ either from bicarbonate in sealed vessels or by propagation in a CO₂ incubator.
• Fish cell cultures gene rally do not require periodic changes (feeding) of growth medium between sub-cultures.

3.1.5.4. Antibiotic s

• For routine purposes, media containing 100 IU of penicillin, 100 m g of streptomycin, and 25 IU of mystatin per milliliter and chlortetracycline at 50 m g in lieu have been used.

3.1.5.5. Growth temperature

Fish cell cultures generally retain viability and / or proliferate over a wide range of incubation temperatures. The optimal growth temperature and the temperature range over which a particular culture will grow usually reflect the fish species and its natural environment.

• Temperatures of 15^o–20^oC are usually optimal for cells from “cold water” species such as salmon and trout; however, cells from these species can frequently be maintained and even will proliferate at temperatures ranging from 2^o to 27^oC.
• Intermediate or “cool-water” species have a somewhat higher limit and an optimum between 20^o and 28^oC.
• Most “warm-water” fish cell cultures do not tolerate relatively low incubation temperatures, but may grow even at 37^oC. Generally, the optimum temperature for these cells is between 25^o and 35^oC.

The ability to grow over an extremely broad temperature range makes fish cell cultures uniquely useful for a variety of purposes, particularly studying temperature effects on metabolism, virus replication, and other cell process.

3.1.5.6. Culture vessels

Virtually all fish cell lines are anchorage–dependent and must be maintained as monolayer cultures on some solid substrate like standard culture vessels such as flasks, dishes micro carrier beads, etc.

Microcarrier beads yields two to three times greater per unit volume of medium than standard monolayer cultures. The efficiency of micro carriers in growth vessel and medium requirements provide significant advantages for the large-scale production of fish cell cultures, viruses and cellular products.