Mercille S, Massie B
Groupe d'Ingénierie des Cellules Animales, Institut de Recherche en Biotechnologie, Conseil National de Recherches du Canada, 6100 Avenue Royalmount, Montréal, PQ, Canada, H4P 2R2.
Biotechnol Bioeng. 1999 Jun 5;63(5):529-43. doi: 10.1002/(sici)1097-0290(19990605)63:5<529::aid-bit3>3.0.co;2-x.
We have shown previously that recombinant NS/0 myelomas expressing sufficient amounts of E1B-19K were resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. However, no significant increase in monoclonal antibodies (MAb) was observed during the prolonged stationary phase of these batch cultures. Here, we show that E1B-19K can enhance cell survival and improve MAb productivity in high cell density perfusion culture. Typically, lymphoid cells grown under steady state in perfusion exhibit decreasing viabilities with concomitant accumulation of apoptotic cells. By modulating the ability of these cells to resist to induction of apoptosis in low nutrient environment, a 3-fold decrease in specific death rate from 0.22 day-1 for NS/0 control to 0.07 day-1 for E1B-19K cells was achieved, resulting in a significant improvement in cell viability throughout perfusion. E1B-19K cells at the perfusion plateau phase also exhibited a 3-fold reduction in specific growth rate concomitant with a lower percentage of S and higher percentage of G1 phase cells. This was associated with a 40% decrease in specific oxygen consumption rate, likely related to a reduction in the specific consumption rates of limiting nutrient(s). Expression of E1B-19K consequently had a significant impact on the steady-state viable cell density, allowing maintenance of 11.5 x 10(6) E1B-19K cells/mL versus 5.9 x 10(6) control NS/0 cells/mL for the same amount of fresh medium brought into the system (half a volume per day). Whereas MAb concentrations found in perfusion culture of control NS/0 myelomas were almost 3-fold higher than those found in batch culture; in the case of E1B-19K-expressing myelomas, the MAb concentration in perfusion was more than 7-fold higher than in batch. This was attributable to the 2-fold increase in viable cell plateau and to a 40% increase in the perfusion to batch ratio of specific MAb productivity (2.2-fold for E1B-19K myelomas versus 1.6-fold for NS/0 control).
我们之前已经表明，表达足够量E1B-19K的重组NS/0骨髓瘤细胞对分批培养后期以及在无谷氨酰胺培养基中培养或热休克等应激条件下发生的细胞凋亡具有抗性。然而，在这些分批培养的延长静止期，未观察到单克隆抗体（MAb）有显著增加。在此，我们表明E1B-19K可提高高细胞密度灌注培养中的细胞存活率并改善MAb产量。通常，在灌注中稳态生长的淋巴细胞活力下降，同时凋亡细胞积累。通过调节这些细胞在低营养环境中抵抗凋亡诱导的能力，将比死亡率从NS/0对照的0.22天-1降低至E1B-19K细胞的0.07天-1，降低了3倍，从而在整个灌注过程中显著提高了细胞活力。处于灌注平台期的E1B-19K细胞比生长速率也降低了3倍，同时S期细胞百分比降低，G1期细胞百分比升高。这与比氧消耗率降低40%相关，可能与限制营养物质的比消耗率降低有关。因此，E1B-19K的表达对稳态活细胞密度有显著影响，对于每天加入系统的相同量新鲜培养基（每天半体积），可维持11.5×10⁶个E1B-19K细胞/mL，而对照NS/0细胞为5.9×10⁶个/mL。虽然对照NS/0骨髓瘤的灌注培养中发现的MAb浓度几乎比分批培养高3倍；但对于表达E1B-19K的骨髓瘤，灌注中的MAb浓度比分批培养高7倍以上。这归因于活细胞平台增加2倍以及比MAb产量的灌注与分批培养比率增加40%（E1B-19K骨髓瘤为2.2倍，NS/0对照为1.6倍）。
