Flow Cytometric Identification of Paclitaxel-Accumulating Subpopulations

Abstract

An immunofluorescence procedure was developed for paclitaxel quantification at the single cell level via flow cytometry in Taxus cuspidata suspension cultures. Intracellular staining was validated via fluorescence microscopy. Paclitaxel content of isolated cells and protoplasts was compared to total paclitaxel levels measured via HPLC. Paclitaxel accumulation was significantly increased by elicitation with methyl jasmonate (100 microM) on day 7 post-transfer as compared to unelicited cultures. Maximum accumulation was observed by day 12 post-transfer in both total paclitaxel (approximately 0.25 mg/L) and the percentage of paclitaxel-accumulating cells (approximately 95%). A similar trend was observed with isolated protoplasts, although protoplasts accumulated only ca. 40-75% of the paclitaxel present in single cells. In unelicited cell cultures, a small subpopulation (ca. 3-5%) of single cells was shown to accumulate paclitaxel. Although nearly all cells were observed to accumulate paclitaxel in methyl jasmonate-elicited cell cultures, a high degree of cell-to-cell variation was observed in paclitaxel content. The identified subpopulations represent targets for cell sorting, which may be applied to develop higher-accumulating cell lines. The quantification of single cell paclitaxel content is useful for characterizing production variability in cell cultures and can be utilized to develop rational strategies to increase paclitaxel production.

Development of a Culture Sub‐population Induction Model: Signaling Pathways Synergy and Taxanes Production by Taxuscanadensis

“…In the case of 200 µM MJ elicitation, the fraction of X ET,JA elicited cells approached 1 after 13 h. The sinusoidal curve shape of X ET,JA further explains why product inhibition terms of higher taxanes were not required by this comprehensive model. As further support to the derived model, similar trends (curve shapes) were observed between model-predicted sub-populations with induced ET and JA-signaling pathways (resulting in higher processed taxanes production), X ET,JA , of this research and the experimentally measured time-dependent accumulation of intracellular pac by flow cytometric identification of paclitaxelaccumulating sub-populations, as shown by Naill and Roberts (52). …”

“…Most notably, the enzyme kinetics approach assumes a homogeneous Taxus culture, which has been shown to be untrue (16,(49)(50)(51)(52)(53). In addition, even though raw data points were effectively predicted, the enzyme kinetics-derived taxane models failed to provide further introspection into governing intracellular mechanisms resulting in the mixture of taxanes observed in Figure 1.…”

“…Recent research has identified heterogeneous aggregates in Taxus cuspidata, and a method was developed to enable single cell analysis using flow cytometry (16,49,50). Results have revealed a high degree of variability in cellular protein content (51), intracellular paclitaxel accumulation (52) and the percentage of cells with an active cell cycle during the mid-exponential culture growth phase (65% noncycling) (53).…”

“…Intracellular taxanes were excluded from the model because of the large increase in paclitaxel production observed from a semi-continuous reactor configuration with total cell recycle (15); in this system approximately 90% of total taxanes were extracellular. However, the model structure is amendable to enable inclusion of Golgi-mediated release of intracellular taxanes (16,52) as a future development. This modeling approach was greatly aided by the use of real-coded genetic algorithms (RCGAs) for model parameter optimization.…”

Development of a Particle Bombardment-Mediated Transient Transformation System for Taxus spp. Cells in Culture

“…In Taxus spp. cell culture, addition of methyl jasmonate often increases taxane accumulation or facilitates a state shift from nonpaclitaxel-accumulating to paclitaxel-accumulating (2,3). However, even with these advances in the Taxus spp.…”