Carvalhal A V, Moreira J L, Carrondo M J
Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica IBET/ITQB, Apartado 12, P-2781-901 Oeiras, Portugal.
J Biotechnol. 2001 Dec 14;92(1):47-59. doi: 10.1016/s0168-1656(01)00365-0.
Activation of the constitutively expressed interferon-regulatory-factor-1/estrogen receptor fusion protein (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 estradiol at the beginning of the exponential phase of a cell suspension culture, IRF-1 activation led to a rapid cell growth inhibition but also to a significant decrease in cell viability. To apply this concept in industry, a reduction of the time span of estradiol exposure is required. Cycles of estradiol addition and removal were performed in 2-l stirred tank bioreactors operated under perfusion, where an initial step addition of 100 nM estradiol was performed, followed, after 48-72 h, by a slow dilution with estradiol-free fresh medium (perfusion rate varying between 0.7 and 1.4 per day). Cell growth inhibition was successfully achieved for three consecutive cycles. Diluting the estradiol by perfusing medium without estradiol to concentrations lower than 10 nM led to cell growth and viability recovery independently of the perfusion rate used. These observations permitted the definition of operational strategies for regulated IRF-1 BHK cell growth by pulse estradiol addition, followed by a period of 48 h in the presence of estradiol and by fast perfusion to estradiol concentrations lower than 10 nM. Cell growth response to IRF-1 activation and following estradiol removal by perfusion was also evaluated with an IRF-1-hER regulated clone expressing constitutively Factor VII, where the time of estradiol exposure and perfusion rate were varied. This clone presented a stronger response to IRF-1 activation without an increase in Factor VII specific productivity after cell growth inhibition; this clearly indicates that the stationary phase obtained is clone dependent. This work proves that it is possible to modulate the IRF-1 effect for cell growth control by the manipulation of cycles of addition and removal of estradiol, potentially representing a new generation of culture procedures for controlled growth production purposes.
通过向培养基中添加雌二醇来实现组成型表达的干扰素调节因子1/雌激素受体融合蛋白（IRF-1-hER）在BHK细胞中的激活，这使得IRF-1获得其转录激活功能并抑制细胞生长。在细胞悬浮培养的指数期开始时添加100 nM雌二醇，IRF-1激活导致细胞生长迅速受到抑制，但细胞活力也显著下降。为了将这一概念应用于工业生产，需要缩短雌二醇暴露的时间跨度。在灌注操作的2升搅拌罐生物反应器中进行雌二醇添加和去除的循环，其中首先添加100 nM雌二醇，48 - 72小时后，用不含雌二醇的新鲜培养基进行缓慢稀释（灌注速率在每天0.7至1.4之间变化）。连续三个循环成功实现了细胞生长抑制。通过灌注不含雌二醇的培养基将雌二醇稀释至低于10 nM的浓度，导致细胞生长和活力恢复，且与所用的灌注速率无关。这些观察结果确定了通过脉冲添加雌二醇、随后在雌二醇存在下维持48小时并通过快速灌注使雌二醇浓度低于10 nM来调控IRF-1 BHK细胞生长的操作策略。还用一个组成型表达因子VII的IRF-1-hER调控克隆评估了细胞对IRF-1激活以及随后通过灌注去除雌二醇的生长反应，其中雌二醇暴露时间和灌注速率有所不同。该克隆对IRF-1激活表现出更强的反应，细胞生长抑制后因子VII的比生产率没有增加；这清楚地表明所获得的稳定期取决于克隆。这项工作证明，通过操纵雌二醇添加和去除的循环来调节IRF-1对细胞生长的控制作用是可行的，这可能代表了用于控制生长生产目的的新一代培养方法。
