Date of Award

5-13-2011

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemical Engineering

First Advisor

Susan C. Roberts

Second Advisor

Michael A. Henson

Third Advisor

Kalidas Shetty

Subject Categories

Chemical Engineering

Abstract

Plant cells culture provides a 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. The Roberts 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. There are several limitations associated with this non-model system; and adapting optimization strategies similar to other cellular systems (e.g., yeast and bacterial) do not necessarily translate into productive and economical processes. The two primary challenges for plant cell culture processes are low yields and variability in productivity. One of the primary sources of variability is due to culture heterogeneity, which is characterized by metabolic differences amongst cells, induced by cell aggregation. To study this heterogeneity we optimized methods to isolate single cells from aggregated Taxus cultures, thereby allowing analysis of single cell phenotypes in a culture population. In this work, flow cytometric characterization of Taxus cell subpopulations with respect to paclitaxel accumulation is presented. By analyzing cell populations with varying levels of paclitaxel accumulation, the inherent molecular and metabolic differences amongst cells in culture can be explored; and the phenomena that underlay culture heterogeneity and production variability can be more completely understood.

Following a statistically optimized enzymatic digestion procedure to yield intact single cells in high yields, a live cell-based indirect immunofluorescence assay for paclitaxel based on PE fluorescence was developed. Paclitaxel is primarily stored in the plant cell wall, and because our studies necessitated the sorting and selection of live cells both for metabolic analysis and reculturing, it was critical that cells remained intact and not permeabilized or damaged throughout the staining procedure. This immunoassay was sufficient to stain cell wall-associated paclitaxel in different subpopulations. A broad range of paclitaxel accumulation amongst Taxus cells was detected, which provided an excellent basis for fluorescence-activated cell sorting (FACS). FACS on plant cells is challenging due to their aggregated nature in suspension and relatively large size when compared to microbial and mammalian cells. A new technique for high-throughput plant cell sorting using BD flow cytometers was developed to accommodate large-sized Taxus cells. A BD FACSVantage® equipped with 200 μm nozzle with optimized optics and fluidics conditions was employed in analysis and sorting of Taxus cells based on paclitaxel accumulation. Additionally, a BD FACSAria®, with 100 μm nozzle was also used to sort Taxus cells. Following a successful (~80-90% purity) sort by size, cells were stained for paclitaxel and sorted based on fluorescence. Cells were gated into two populations based on low and high paclitaxel accumulation and sorted accordingly. A high level of purity (~90%) was obtained for both of the sorted populations using both instruments, demonstrating that Taxus cells could be effectively sorted according to paclitaxel content. These subpopulations were maintained in culture for several months and analyzed for growth and paclitaxel accumulation. The new technologies developed here not only allow recovery of diverse cell subpopulations for metabolic analysis, but also for establishing superior cell lines for use in bioprocesses.

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