The use of flow cytometry to study variability of paclitaxel accumulation in Taxus cell suspension cultures

Plants synthesize a wide variety of compounds that are unnecessary for growth, commonly referred to as secondary metabolites. Due to their high specificity of function and wide range of structural variety, many secondary metabolites are valuable commercial products (e.g., dyes, fragrances, or pharmaceuticals). Unfortunately, plant cell culture technology has been underutilized industrially to supply these important compounds. Although improvement has been made in increasing productivity by strategies such as medium optimization, precursor feeding, and enzyme elicitation, many plant cell culture processes are still limited by high variability. One example is the Taxus suspension culture system, which produces the anti-cancer agent paclitaxel (Taxolo ® - Bristol-Myers Squibb). In order to study production variability in plant cell suspension cultures, population information must be collected at the single cell level. The aggregation of plant cell cultures has limited the ability to study plant cell culture population dynamics. Protoplasts, which lack a functional cell wall, are commonly prepared from plant cell suspensions. Unfortunately, protoplasts do not always accurately represent aggregated suspension cultures due to the lack of a functional cell wall. A novel method for the isolation of intact single plant cells has been developed. The population dynamics of Taxus cell cultures was characterized using both isolated single cells and protoplasts. Specifically, protein content, cell cycle distribution, paclitaxel accumulation, and paclitaxel uptake were investigated. Methods were also developed for the sorting and isolation of desired subpopulations that may lead to the establishment of stable cell lines for increased paclitaxel accumulation. Through a characterization of cellular growth, metabolism, and transport, strategies will be presented for the rational improvement of cell culture processes