Laboratoire des Sciences du Génie Chimique, CNRS-INPL, BP 451, F-54001, Nancy Cedex, France.
Cytotechnology. 1999 Jan;29(1):71-84. doi: 10.1023/A:1008014909474.
In this paper, we propose an alternative strategy to the ones proposed before (Oh et al., 1993; Øyaas et al., 1994a) to get real increases of global final antibody titer and production at hyperosmotic stress, by reducing the detrimental effect of such a stress on cell growth, and conserving the stimulating effect on antibody production. It consists of cultivating the cells in continuous culture and increasing the osmolality stepwise. In this way, the cells could progressively adapt to the higher osmolality at each step and antibody titers could be nearly doubled at 370 and 400 mOsm kg-1, compared to the standard osmolality of 335 mOsm kg-1. Surprisingly, the stimulation of antibody production was not confirmed for higher osmolalities, 425 and 450 mOsm kg- 1, despite the minor negative effect on cell growth. Intracellular IgG analysis by flow cytometry revealed at these osmolalities a significant population of non-producing cells. However, even when taking into account this non-producing population, a stimulating effect on antibody production could not be shown at these highest osmolalities. It seems to us that osmolality has a significant effect on the appearance of these non-producing cells, since they were not observed in continuous cultures at standard osmolality, of comparable duration and at an even higher dilution rate. The appearance of the non-producing cells coincides furthermore with modifications of the synthesised antibody, as shown by electrophoretic techniques. It is however not really clear if these two observations reflect actually the same phenomenon. Hyperosmolality affects the cell behaviour in continuous culture in multiple ways, independently of the growth rate, counting all at least partially for the observed stimulation of antibody production: acceleration of the amino acid, and in particular the glutamine metabolism, increase of the cell volume, increase of the intracellular pH and accumulation of cells in the G1 cell cycle phase.
在本文中，我们提出了一种替代之前提出的策略（Oh 等人，1993 年；Øyaas 等人，1994a），以在高渗胁迫下获得真正增加全球最终抗体滴度和产量的方法，即减少这种胁迫对细胞生长的不利影响，并保持对抗体产生的刺激作用。它包括在连续培养中培养细胞并逐步增加渗透压。通过这种方式，细胞可以逐步适应每一步的更高渗透压，与 335 mOsm kg-1 的标准渗透压相比，在 370 和 400 mOsm kg-1 时，抗体滴度可几乎增加一倍。令人惊讶的是，尽管对细胞生长的负面影响较小，但对于更高的渗透压（425 和 450 mOsm kg-1），抗体产生的刺激作用并未得到证实。通过流式细胞术对细胞内 IgG 的分析表明，在这些渗透压下，存在大量非生产性细胞。然而，即使考虑到这种非生产性群体，在这些最高渗透压下也无法显示出对抗体产生的刺激作用。在我们看来，渗透压对这些非生产性细胞的出现有显著影响，因为在标准渗透压、持续时间相当且稀释率更高的连续培养中没有观察到这些细胞。非生产性细胞的出现与合成抗体的修饰同时发生，电泳技术显示了这一点。然而，尚不清楚这两个观察结果是否实际上反映了同一现象。高渗透压通过多种方式影响连续培养中的细胞行为，与生长速率无关，至少部分解释了观察到的抗体产生的刺激作用：加速氨基酸，特别是谷氨酰胺代谢，增加细胞体积，增加细胞内 pH 值以及将细胞积累在 G1 细胞周期阶段。
