Roberts:Research

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Research Overview The Roberts lab is interested in exploring the use of aggregated cell cultures from both mammalian and plant sources. Optimizing the growth and functionality of these cell types to gain desired products is achieved by manipulating cell behavior using metabolic engineering, bioprocess engineering, and polymer science. Plant Metabolic Engineering The supply of pharmaceuticals is often met through in vitro fermentation and cell culture systems, which can be easily manipulated to yield high quantities of the product of interest. The goals of our program focus on the development and optimization of processes for the production of pharmaceuticals in plant-based systems, with an emphasis on the understanding and control of cellular metabolism. Traditional chemical engineering is combined with modern biotechnology to facilitate the development of such processes. Our research is directed towards understanding how cellular metabolism can be effectively controlled at both the genetic and cellular level. We are developing novel methods (both experimental and theoretical) for studying poorly characterized (both genetic and metabolic) cell systems (e.g., Taxus for Taxol®, paclitaxel, production). We utilize a variety of techniques to characterize cellular metabolism including enzyme elicitation, utilization of metabolic inhibitors, application of radio-labeled and fluorescently-labeled substrates, HPLC-MS for metabolite identification, transcript profiling, 2D-gel electrophoresis, molecular transformation via Agrobacterium, flow cytometry (FCM) and fluorescence-activated cell sorting (FACS). We are working in several additional areas to optimize the plant cell culture process for paclitaxel including design of liquid-liquid extraction processes for improved selectivity and recovery, and characterization of paclitaxel degradation in cell culture. Metabolic Control of Paclitaxel Accumulation in Taxus Cell Cultures Sarah is measuring paclitaxel accumulation using UPLC The application of molecular approaches to studying plant secondary metabolism is very limited. We are studying the Taxus cell system for production of the anti-cancer agent paclitaxel. The majority of research performed thus far has been directed towards identification and characterization of specific pathway genes with an ultimate goal of enhancing paclitaxel accumulation through specific genetic transformation of Taxus cultures, with no a priori information on whether the particular gene is important in pathway control. We are using significantly different approach, one that combines the use of transcript profiling and proteomics to identify genes involved in global pathway control. We are currently evaluating gene expression in both high- and low-paclitaxel producing states. We utilize the biotic elicitor methyl jasmonate to upregulate expression of secondary metabolic genes, resulting in increased accumulation of paclitaxel. Plant Cell Population Dynamics While the production of secondary metabolites by plant cell culture has been studied extensively, most work has been done using whole-flask averages as opposed to studies on individual cells. The isolation of single cells for analysis allows for the rapid collection of information about the behavior of individual cells within a culture population. This data can then be used in the modeling of population dynamics, with an ultimate goal of understanding the regulation of secondary metabolism in plant cell cultures. We are developing novel single cell preparation methods for aggregated plant cell cultures to allow for population analysis via flow cytometry. Current efforts are aimed at characterizing cell populations and sorting these populations for subsequent culture and analysis. Our model system of study is paclitaxel accumulation in Taxus suspension cultures. Additionally, we are concurrently determining the complement of metabolites via HPLC-MS for comparison between distinct cell populations under different conditions (e.g., enzyme elicitation). Mammalian Cell in vitro Systems Whitney is emulsifying samples The collection of scientific and engineering data for in vivo applications is highly dependent on animal studies. To better design such studies so as to minimize animal testing, we are utilizing in vitro systems for the high-throughput testing of multiple designs/parameters as well as interactions between variables. An effective in vitro system will mimic human physiology. We are developing novel reactor configurations and cell culture techniques as well as applying relevant metabolic assays to evaluate cellular responses. We utilize a variety of equipment/techniques in our studies including UV-vis spectroscopy, fluorescence microscopy, TEM, application of radio-labeled and fluorescently-labeled substrates, HPLC-MS for metabolite identification, and flow bioreactors. Cell Encapsulation Technologies This project is aimed at developing new approaches for the formulation and design of structured materials for cell encapsulation, specifically for signal-responsive cells with a high metabolic rate and oxygen demand. Our efforts are focused specifically on characterizing cell growth and metabolism and cell-material interactions in encapsulated systems through the application of novel in vitro techniques. We are specifically studying hydrogel systems (e.g., alginate) with oxygen reservoirs to promote high cell viability and functionality over extended periods of time.

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