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

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      Publication Details (including relevant citation   information):

      Authors: Vishal Gaurav, Rohan A Patil, Susan C Roberts

      Publication date: 2008

      Journal name: American Institute of Chemical Engineers (AIChE)   Proceedings

      Publisher: 2008 Annual Meeting, Philadelphia, PA

      Abstract:

      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   (Taxol®) 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.

      Address (URL): https://sites.google.com/site/vishalpubs/A_Facs-Based_Approach_to_Characterizing _Culture_Heterogeneity_Based_on_Accumulation_of_the_Anti-Cancer_Agent_Paclitaxel _In_Plant_Cell_Suspensions.pdf