A Facs-Based Approach to Characterizing Culture Heterogeneity Based on Accumulation of the Anti-Cancer Agent Paclitaxel In Plant Cell Suspensions

Vishal Gaurav, Rohan A. Patil, and Susan C. Roberts. Chemical Engineering, University of Massachusetts Amherst, 686 North Pleasant Street, 159 Goessmann Lab, Amherst, MA 01003

Plant cells culture provides an alternative production source for plant-derived pharmaceuticals in which environmental conditions can be more easily controlled, manipulated, and optimized to yield high quantities of these valuable natural products. Our laboratory focuses on development and optimization of bio-processes for production of the anti-cancer agent Paclitaxel (TaxolŽ) in Taxus cell suspension cultures with an emphasis on understanding cellular metabolism at both the molecular and cellular level. Most studies concerning metabolite production via cell culture technology rely on culture-average parameters, which are often insufficient to describe culture heterogeneity. We have developed novel methods to isolate single cells from aggregated Taxus cultures, thereby allowing analysis of single cell phenotypes in a culture population. In this talk, we will present our recent work aimed at characterizing Taxus cell suspension sub-populations with respect to Paclitaxel accumulation. By analyzing cell populations with varying levels of Paclitaxel accumulation, we can understand the inherent molecular and metabolic differences amongst cells in culture and begin to understand the phenomena that underlay culture heterogeneity and production variability.

We have established a novel live-cell based indirect immunofluorescence assay for Paclitaxel based on PE fluorescence. Paclitaxel is primarily stored in the plant cell wall, and because our studies necessitate the sorting and selection of live cells both for mRNA expression analysis and re-culturing, it is critical that cells remain intact and not permeabilized or damaged throughout the staining procedure. This immunoassay was sufficient to stain respective amounts of cell wall-associated Paclitaxel in different sub-populations. Concomitantly, cells were also stained using fluorescein diacetate (FDA) to monitor viability. Multiparameter flow cytometry was performed and different Taxus cell populations were examined based on both PE and FITC fluorescence. To further validate the efficacy of this method, time-based viability tests were performed and demonstrated that cell viability remained high both during and after the staining procedure. We detect a broad range of Paclitaxel accumulation amongst Taxus cells in culture, providing an excellent target for fluorescence-activated cell sorting (FACS).

FACS can be effectively used to sort cells based on a variety of parameters including size, growth rate, protein production and metabolite production. Plant cells are challenging to sort due to their aggregated nature in suspension and relatively large size when compared to microbial and mammalian cells. We developed a new technique for high-throughput plant cell sorting using the BD FACSVantage equipped with a MacroSort option to accommodate our larger Taxus cells. A larger 200 μm nozzle with optimized optics and fluidics conditions was used in analysis and sorting of Taxus cells based on Paclitaxel accumulation. The instrument was calibrated with 25 micron beads and Taxus cells were first sorted on the basis of size. Following a successful (~80-90% purity) sort by size, cells were stained for Paclitaxel using the procedure mentioned above and sorted based on fluorescence. Cells were gated into two populations based on low and high Paclitaxel accumulation and sorted accordingly. We were able to attain a high level of purity (~90%) for both sorted populations, demonstrating that live Taxus cells can be effectively sorted according to Paclitaxel content. The new technologies developed here not only allow recovery of low and high accumulating cells for metabolic analysis, but also allow for re-culture and initiation of potentially superior cell lines with both enhanced Paclitaxel accumulation and stability.