Amino acid supplementation enhances urokinase production by HT-1080 cells

Abstract

Medium optimization is an important strategy that can lead to several fold increase in the production of proteins in cell culture. However, the usual methods of medium optimization are complex and time consuming. Urokinase is a widely employed thrombolytic drug for the treatment of stroke. We describe here medium optimization for maximizing urokinase production by HT-1080 cells using supplementation of specific amino acids. The new specifically designed method resulted in 240 % increase in urokinase productivity.

Fiber-Reinforced Silk Composite for Enhanced Urokinase Production Using High-Density Perfusion Culture and Bioactive Molecule Supplementation

“…In context, the growth phase (day 1 to day 5) and the stationary phase (day 5 to day 9) of cells cultured on scaffolds were postulated from the cell proliferation analysis. Many investigators have reported that maintenance of the stationary phase results in a significant enhancement in urokinase productivity. , Moreover, successful media manipulation leads to a several-fold increase in metabolite production without creating nutrient depletion for the cells . Interestingly, uPA production from mammalian cells depends on two major factors: (i) regulation of uPA expression and (ii) addition of supplements like amino acids to enhance the uPA synthesis .…”

“…Many investigators have reported that maintenance of the stationary phase results in a significant enhancement in urokinase productivity. , Moreover, successful media manipulation leads to a several-fold increase in metabolite production without creating nutrient depletion for the cells . Interestingly, uPA production from mammalian cells depends on two major factors: (i) regulation of uPA expression and (ii) addition of supplements like amino acids to enhance the uPA synthesis . Several studies have reported the effect of serum concentration on HT1080 cell growth and the uPA production. , On reducing the serum concentration from 10% FBS to 2% FBS, the stationary phase of the cells cultured on scaffolds was retained, which is also considered as the production phase.…”

“…The absorbance was recorded at 405 nm in a multi-plate reader (Tecan Infinite Pro 200), and the urokinase activity in units (U/L) was calculated from the standard graph as reported in earlier protocols . One unit of urokinase activity is defined as the amount of enzyme required to produce one mole of p -nitroaniline ( p NA) in 1 min from the chromogenic substrate under standard conditions (37 °C, pH 7.4) …”

“…To date, uPA has been harvested from a considerable number of mammalian cells, like HT1080 (human kidney fibrosarcoma cells), TCL-598 (human epithelial kidney tumor cells), CAKI 1 and CAKI 2 (human kidney carcinoma), HUVEC (human umbilical vein endothelial cells), and CALU-3 (human lung carcinoma) . Among all the cell types, HT1080 cells have been reported as a good candidate for the large-scale production of urokinase in static as well as dynamic culture conditions . Several in vitro studies have been focused on media formulation to accomplish optimal cell growth that aids in maximum production of urokinase. − High serum concentration is reported to have an inhibitory effect on urokinase production, while at lower serum concentrations an increase in urokinase synthesis and subsiding cell proliferation has been observed. , Notably, urokinase synthesis was found to be maximum during the cells’ stationary phase as compared to their exponential growth phase .…”

“…Among all the cell types, HT1080 cells have been reported as a good candidate for the large-scale production of urokinase in static as well as dynamic culture conditions . Several in vitro studies have been focused on media formulation to accomplish optimal cell growth that aids in maximum production of urokinase. − High serum concentration is reported to have an inhibitory effect on urokinase production, while at lower serum concentrations an increase in urokinase synthesis and subsiding cell proliferation has been observed. , Notably, urokinase synthesis was found to be maximum during the cells’ stationary phase as compared to their exponential growth phase . Alterations in media composition, for instance, reducing the serum concentration and adding external supplements (growth factors and amino acids), during the stationary phase have presented manifold increases in urokinase yield .…”