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. Research in our laboratory focuses on two major topics discussed below: Engineering of Taxus Plant Cell Cultures and Engineering Mammalian Cell Microenvironments. Engineering of Taxus Plant Cell Cultures Development and optimization of plant cell culture processes is vital for the future commercialization of valuable plant-derived natural products. We address this in our laboratory through the study of cellular aggregation and the development of both metabolic engineering approaches and flow cytometric methodologies. Plant cell culture technology impacts several important areas in bioprocessing, gene discovery, and human health. Specific applications include U.S. and world-wide supply of plant-derived therapeutics, platforms for metabolic engineering of novel natural products, propagation and genetic engineering of superior plant species, and identification of new plant biosynthetic pathways or regulatory genes. The goals of our research program center on development and implementation of novel tools in plant cell culture systems to both gain fundamental insight into plant biosynthesis and to directly influence the design and engineering of culture systems for commercial use. Our primary system of study is Taxus suspension cell cultures for production of the secondary metabolite paclitaxel (Taxol™, Bristol-Myers Squibb), an FDA approved anti-cancer agent. Results in this field of research broadly influence the metabolic engineering of non-model plants and more specifically a significant industrial process to supply a potent therapeutic agent for the treatment of cancer and other diseases. Metabolic Engineering of Taxus Cell Cultures Sarah uses UPLC to determine the paclitaxel concentration in her Taxus suspension cultures Genetic engineering of Taxus cells is essential to assess gene function and to evaluate the effectiveness of overexpression or silencing strategies on cell metabolism. Taxus is highly recalcitrant and resistant to genetic transformation. We developed the first methodology to enable transient transformation using particle bombardment technology. This technology is being applied in evaluation of putative transcription factors, shown to increase expression of taxane biosynthetic pathway genes in collaboration with Elsbeth Walker in the Biology Department at UMass Amherst. We provided the first evidence for taxane biosynthetic pathway regulation at the mRNA level that suggested two candidate biosynthetic genes for metabolic engineering. To extend this work to a more system-wide analysis, we performed differential expression studies to identify genes that were upregulated in cultures that accumulate higher amounts of paclitaxel, including several putative novel pathway genes. In collaboration with Jennifer Normanly in the Biochemistry and Molecular Biology Department at UMass Amherst, we are developing heterologous transformation systems (e.g., tobacco) to study the function of these genes. Working with Joyce Van Eck at the Boyce Thompson Institute for Plant Research, we are investigating Agrobacterium-mediated stable transformation of both Taxus callus and suspension cultures, which opens up a plethora of opportunities with regards to characterizing the cultures in response to overexpression or silencing of putative regulatory genes. This metabolic engineering work represents a significant future focus for our laboratory. Aggregated Culture Heterogeneity Taxus aggregation profiles and dynamics in culture fluctuate over time as a result of factors which are poorly understood. We have demonstrated that cellular metabolic variability in secondary metabolite accumulation results from this aggregation phenomenon. Our lab has recently established experimental techniques to rapidly and reliably characterize the size distribution of plant cell aggregates in culture using a specially equipped Coulter counter system. This methodology is currently being applied to explore the effects of aggregation on culture properties such as growth and paclitaxel accumulation. Recent exciting data have shown increases of up to 20-fold in paclitaxel levels when aggregate size is effectively manipulated in culture and hence represents an important focus of future research efforts. Current studies are directed towards manipulation of aggregate size using controlled fermentation systems, development of tools to investigate aggregate physiology, and study of cell growth within aggregates using flow cytometry. In collaboration with Mike Henson in the Chemical Engineering Department at UMass Amherst, we have developed the first population balance equation (PBE) model that can be used to simulate aggregate dynamics in plant cell suspension culture. Techniques for Analysis of Plant Cell Cultures Our lab has invested significant resources into the development of new methodologies for investigating the metabolic activity and viability of aggregated plant cell cultures. Techniques include the creation of single cells from aggregates for analysis and sorting using flow cytometry and the analysis of aggregated culture heterogeneity using a Coulter counter. These studies provided the first characterization of cell-cell heterogeneity in Taxus suspensions and serve as a model for the study of other plant culture systems. Research is ongoing to further characterize these populations, with a focus on multi-parameter staining to determine the relationship between primary and secondary metabolism. These studies will inform strategies to optimize bioprocesses for both growth and paclitaxel accumulation. Engineering Mammalian Cell Microenvironments Understanding the role of physiological and mechanical properties on aggregated mammalian cell cultures grown within 3-dimensional biomaterials is the second major research thrust in the Roberts Lab. Recognizing the role of oxygen tension in tissue engineered constructs has lead to the investigation of synthetic oxygen carries (perfluorocarbons) in both 2- and 3-dimensional cell culture. Whitney is creating an emulsion to create a composite biomaterial. We have demonstrated enhanced oxygen transport and cell function using immobilized PFC oxygen vectors in hydrogel matrices for encapsulated cells. The use of synthetic PFC oxygen carriers and characterization of the mechanism behind increased oxygen transport is an important step in enabling the design of tissue engineering devices and in sustaining viability and functionality of large-scale devices. Our new technology has recently received a Commercial Ventures and Intellectual Property Award to enable collection of in vivo data for wound healing applications Surita Bhatia in the Chemical Engineering Department at UMass Amherst. We have also recently extended our strategies to focus on aggregated cell culture systems, such as the clinically relevant application of islet cell transplantation. To broaden the impact of this work, we are directing research towards the fundamental study of protein transport in hydrogels containing microemulsions and have recently shown that protein transport is influenced by composite hydrogels. To effectively design a functional encapsulation system the effects of additives must be taken into consideration when optimizing transport of specific proteins (e.g., insulin) within hydrogel matrices. Ongoing and future work is directed towards understanding the effect of inclusion of these additives on both small molecule and protein transport as well as matrix properties. A new collaboration with Kimberly Tremblay in the Veterinary and Animal Science Department at UMass Amherst and Alfred Crosby in the Polymer Science and Engineering Department at UMass Amherst will lead to further investigation of the role of matrix microstructure and extracellular environment on the growth and function of aggregated cell types in composite hydrogels.

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