Retroviral gene transfer represents a potential tool for the treatment of many human genetic diseases. Optimizing vector production improves gene transfer efficiency and may impact clinical outcome. We recently noted significant differences in the kinetics of vector production among currently available packaging cell lines. While each cell line is derived from NIH3T3 cells, the optimal timing for harvest ranges from 8 (for AM12 cells) to 24 hours (for PA317 and PG13) requiring tests of each vector packaging cell line prior to production. To better understand these difference, growth characteristics were studied for the following packaging cell lines: NIH3T3, PA317, PG13, GPE86, and AM12. Growth was initially assessed by determining cell expansion every 2 days over an 8 day period. The growth rate x of these cells, where Ntotal cells=e(x)(#days), was 0.52 for NIH3T3, 0.62 for PA317 0.67 for PG13; and a relatively slower rate of 0.37 for GPE86, and 0.40 for AM12. We next assessed glucose consumption and lactate production in confluent plates of packaging cells at 4, 8, 12, and 24 hours after media change. Glucose consumption at 4 hours (mg/dL per 1x106 cells per hour) was 1.0±0.2 SD for NIH3T3, 1.6±0.4 for PA317, 0.6±0.2 for PG13, 3.4±0.4 for GPE86, and 0.2±0.4 for AM12. Glucose consumption (per cell/hour) did not change significantly at the 8, 12, and 24 hour time points. Lactate production at 4 hours (mg/dL per 1x106 cells per hour) was 1.4±0.4 SD for NIH3T3, 2.7±0.4 for PA317, 1.6±0.4 for PG13, 2.0±0.2 for GPE86, and 1.1±0.2 for AM12. Lactate production was similar at the 8, 12, and 24 hour time points. Interestingly, the cell line with the shortest time to reach maximal vector titer, AM12, had the lowest glucose consumption and lactate production. Finally, each line was tested for its ability to grow in semi-solid media. The parent cell line NIH3T3 exhibited contact inhibition and would not form colonies in soft agar cultures. We found that GPE86 is the only packaging cell line tested which retained this property; colony formation per 1000 cells plated was 0±0 SD for both NIH3T3 and GPE86, 9.0±0.8 for AM12, 25±8 for PA317, and 50±6 for PG13. Our studies demonstrate that packaging cell lines used to generate vector for clinical trials have altered phenotypes compared to the parent NIH3T3 cells. Further understanding of these differences will improve our efforts to optimize production of clinical grade material.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Feb 1999|
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)