Tissue plasminogen activator production from cells cultured in milk extract

Abstract

Mammalian cells are conventionally cultured in nutrient medium containing 10% foetal calf serum (FCS). The aim of the project was to investigate whether bovine milk extract could be used to replace or minimise the use of serum in cell culture without affecting the level of product expression, thereby significantly reducing the overall cost and risk of microbial contamination. Cells producing tissue plasminogen activator (t-PA) were cultured in nutrient medium containing varying concentrations of either milk extract or FCS or different combinations of both. Cell number, cell morphology and product expression were monitored with time in comparison with the control culture grown in 10% FCS. The results obtained indicate that the milk extract, in itself, was a poor substitute for serum in terms of cell growth. However, it was found that with a combination of 2% milk extract and 1% FCS in nutrient medium, the cells remained attached to the substrate over longer periods and the cell number reached about 75–85% of that observed in the control cultures. Further reduction of FCS below 0.5% in milk+serum supplemented culture medium had a detrimental effect on cell growth. t-PA expression, as measured by chromogenic assay, from cells grown in 2% milk+1% FCS supplemented medium was similar to the control culture over a 12 day period. One of the advantages of this system is that it can avoid the need to change the culture medium from serum-containing to serum-free, before harvesting the product. These data indicate that milk extract, combined with low concentrations of serum, has a beneficial effect on cell attachment and growth with little or no effect on expression. Thus, the use of milk extract may prove to be economically advantageous in large scale cell culture for natural products, such as t-PA and recombinant proteins.