Cell Growth Control by the IRF-1 System in Perfusion Culture

Abstract

Activation of the constitutively expressed interferon-regulatoiy-factor-1/estrogen receptor fusionprotein (IRF-1-hER) in BHK cells was accomplished through the addition of estradiol to the culture medium, which enabled IRF-1 to gain its transcriptional activator function and inhibit cell growth. With the addition of 100 nM of estradiol at the beginning of the exponential phase, IRF-1 activation leads to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, strategies to extend the stationary phase are required. Cycles of estradiol addition/removal were performed in 2 L stirred tank bioreactor operated under perfusion in order to reduce the time span of estradiol exposure by slow dilution after a step addition of 100 nM estradiol (perfusion rate between 0.7 and 1.4 day ⁻¹). Cell growth inhibition of the regulated/non-producer clone was achieved for three consecutive times, showing that the cells were able to respond to estradiol addition independently of the cycle. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM lead to cell growth and viability recovery, independently of the perfusion rate used. These observations led to the definition of strategies to operate IRF-1 regulated cells by pulse estradiol addition followed by the longest possible period withestradiol and by the fast perfusion to low estradiol concentration. The response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with a regulated/Factor VII producer clone, where the time of estradiol exposure and perfusion rate were varied. This clone presents a stronger response to IRF-1 activation without increase on factor VII specific productivity after cell growth inhibition; clearly indicating that the stationary phase obtained is clone dependent. The final conclusion is that it is possible to modulate the IRF-1 effect by the manipulation of cycles of addition/removal of estradiol.