Effect of Continuous Shear Stress and Enzymatic Disaggregation on Cell Health and Secondary Metabolite Production in Taxus Plant Cell Culture

Rauber, Caroline

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Effect of Continuous Shear Stress and Enzymatic Disaggregation on Cell Health and Secondary Metabolite Production in Taxus Plant Cell Culture

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Paclitaxel (or TaxolTM) is a highly effective FDA-approved chemoterapeutic drug due to its effective cytotoxicity against different types of human and animal cancers. However, the limitation in the widespread use of paclitaxel is its very low production yield in nature, given the slow growth of the yew tree and the very low content of paclitaxel stored in the bark of the tree. Although there are currently different alternative methods to produce paclitaxel for clinical use, plant cell culture (PCC) is the one that is the most viable, stable and attractive commercially as it offers uniform and continuous paclitaxel quality and a renewable resource. PCC, however, faces some challenges such as aggregation of cells in culture, which induces cellular heterogeneity resulting in unpredictable changes in metabolism and growth. Previous research has demonstrated that smaller aggregated cultures accumulate higher levels of paclitaxel. Several techniques have been developed to reduce aggregate sizeusing either physical or chemical factors. This thesis focused on the use of an automated shearing device to study both short-term and long-term shearing with varied parameters. Results were compared with previously established enzymatic digestion methods. The impact of long-term shearing combined with methyl jasmonate elicitation on cell health, production of paclitaxel and its precursors, global secondary metabolite synthesis and mean aggregate size was studied. Both techniques of disaggregation were very effective in reducing aggregate size when applied for four hours, but continuous shearing via the automatic device was shown to have the most significant results in reducing aggregate size while still maintaining healthy and metabolically active cells. Preliminary data on the automated device showed that continuous shearing at the highest flowrate (8.6 mL/s) did not impact negatively cell metabolic activity and resulted in a significant decrease of the mean aggregate size. Long-term shearing showed increased levels of paclitaxel over time, the highest concentrations being achieved for sheared cultures elicited with methyl jasmonate. These promising results support the further study and adaptation of the automated shearing device for promotion of paclitaxel synthesis in PCC.

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