Animal Cell Engineering Group, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave, Montréal, P.Q., Canada, H4P 2R2.
Cytotechnology. 1998 Nov;28(1-3):189-203. doi: 10.1023/A:1008054403470.
Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAbtrade mark. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAbtrade mark accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAbtrade mark resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAbtrade mark. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAbtrade mark with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells.
表达足够水平的 Bcl-2 或 E1B-19K 的淋巴细胞已知能够抵抗在无谷氨酰胺或营养有限的分批培养中诱导凋亡。然而，尽管在分批培养中增加了存活率并延长了静止期，但产物产量不一定会提高。在这里，我们发现，在存在某些细胞周期调节剂的情况下培养的 NS/0 骨髓瘤细胞中表达 E1B-19K 可导致与未转染的对照细胞相比，MAb 产量显著增加。E1B-19K 的使用可显著提高渗透压剂（山梨醇、NaCl）、DNA 合成抑制剂（羟基脲、过量胸苷）和细胞培养添加剂 OptiMAbtrade mark 存在时的细胞存活率。在 NaCl 或 OptiMAbtrade mark 存在下培养的 E1B-19K 骨髓瘤细胞在 G1 期积累，而用过量胸苷阻滞的细胞则在所有阶段被阻滞。有趣的是，用这些药物处理的对照 NS/0 细胞被发现以细胞周期特异性方式死亡。因此，虽然所有 G1 和大多数 S 期细胞很快发生凋亡，但如果提供足够的营养，G2/M 期细胞仍保持存活并维持 MAb 分泌超过 10 天。对于对照和 E1B-19K 细胞，用山梨醇或羟基脲孵育对 MAb 分泌有害，而添加 NaCl、过量胸苷和 OptiMAbtrade mark 与分批培养相比导致特定 MAb 生产率增加。然而，只有在 OptiMAbtrade mark 的情况下，这种增加才导致最终 MAb 产量的提高。E1B-19K 赋予的存活能力延长允许使用 OptiMAbtrade mark 进一步提高最终 MAb 产量，与对照 NS/0 细胞相比，E1B-19K 细胞增加了 3.3 倍，而对照 NS/0 细胞增加了 1.8 倍。
