Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity.
Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.
Naill
Michael C
MC
University of Massachusetts-Amherst, Department of Chemical Engineering, 159 Goessmann Laboratory, 686 North Pleasant Street, Amherst, MA 01003, USA.
Roberts
Susan C
SC
D001973:Bromodeoxyuridine / Q000032:analysis; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects; D017239:Paclitaxel / Q000494:pharmacology; D013395:Sucrose / Q000378:metabolism; D020946:Taxus / Q000166:cytology*
D013395:Sucrose; D001973:Bromodeoxyuridine; D017239:Paclitaxel