Packaging cell line characteristics and optimizing retroviral vector titer: the National Gene Vector Laboratory experience.
Packaging cell line characteristics and optimizing retroviral vector titer: the National Gene Vector Laboratory experience.
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包装细胞系特征和优化逆转录病毒载体滴度：国家基因载体实验室的经验。
DOI：
10.1089/104303400750001408
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Cornetta,K
中科院分区：
文献类型：
--
作者：
Reeves,L;Smucker,P;Cornetta,K
在临床级逆转录病毒载体上清液的生产过程中，我们注意到提交给国家基因载体实验室（NGVL）的各种生产细胞系的乳酸产量和葡萄糖消耗存在显着差异。由于生长特性的差异对于确定最大化滴度的最佳培养条件可能很重要，因此我们研究了三种常用包装细胞系的生长特性：PA317、PG13 和 GP+envAM12。通过在半固体培养基中形成集落的能力来评估，转化的表型在所有三种测试的包装细胞系中都很明显。在汇合培养物中，测试的三个细胞系的葡萄糖消耗率和乳酸产生率（每个细胞每小时）相似，但生长率和培养物密度不同。 PA317 和 PG13 在达到汇合后继续扩增，导致细胞密度更高，随后在 24 小时观察期内葡萄糖迅速耗尽。当评估细胞系的滴度优化时，生长较慢的包装细胞系 GP+envAM12 通常在汇合滚瓶中培养 8 小时后提供最高滴度，而引入 PA317 和 PG13 细胞的大多数载体在培养 24 小时后产生最佳滴度。我们还发现，之前报道的 PA317 细胞通过在 32°C 培养细胞获得的滴度提高并不适用于其他包装细胞系。特别是，PG13 在较低温度下生长时滴度迅速下降。我们的研究结果表明，效价的优化需要仔细考虑培养条件，应针对载体生产细胞系进行个体化。
During the production of clinical-grade retroviral vector supernatant, we noted significant differences in the lactate production and glucose consumption of various producer cell lines submitted to the National Gene Vector Laboratory (NGVL). Since differences in growth characteristics could be important in determining the optimal culture conditions for maximizing titer, we studied the growth characteristics of three commonly used packaging cell lines: PA317, PG13 and GP+envAM12. A transformed phenotype, assessed by the ability to form colonies in semisolid media, was evident in all three packaging cell lines tested. In confluent cultures, the rates of glucose consumption and lactate production (per cell per hour) were similar for the three lines tested, but the growth rate and culture density varied. PA317 and PG13 continued to expand after reaching confluence, resulting in higher cell densities and subsequent rapid depletion of glucose within the 24-hr observation period. When the cell lines were evaluated for titer optimization, the slower growing packaging cell line GP+envAM12 generally provided the highest titer after 8 hr of culture in confluent roller bottles, while most vectors introduced into PA317 and PG13 cells yielded optimal titers after 24 hr of culture. We also found that the improved titers obtained by culturing cells at 32°C previously reported for PA317 cells do not apply to other packaging cell lines. In particular, PG13 rapidly lost titer when grown at the lower temperature. Our findings suggest that optimization of titer requires careful consideration of the culture conditions, which should be individualized for the vector producer cell line.