(681b) Isolation and Propagation of High Secondary Metabolite-Accumulating Plant Cell Cultures

Plant cell culture technology is used for the production of many important natural products including pharmaceuticals, flavors, colors and other specialty chemicals. While more than 25% of all the pharmaceutical prescribed in the US are plant derived, less than 2% of all the plant species have been investigated for potential medical applications. A prominent example of plant cell culture technology, in both research and industrial applications, is Taxus spp. for production of anti-cancer agent paclitaxel (Taxol®). There are, however, limitations associated with this technology that prevent widespread industrial use. Cell-cell heterogeneity is a typical feature of these cultures, which results in unpredictable shifts in metabolite accumulation over time and within cultures, thus leading to low and variable yields of these products.

We have developed a novel flow cytometric method for immunostaining and isolating high paclitaxel-accumulating sub-populations from Taxus cell cultures. Florescence-activated cell sorting (FACS) was used to aseptically sort these cultures into high- and low- paclitaxel producing sub-populations. Methods to propagate single cell suspension cultures from these isolated populations were subsequently developed. Sorted suspension cultures were scaled-up, and their growth, viability, size distribution and paclitaxel accumulation were analyzed over time. Aggregate and cell size distribution was determined using Coulter Counter technology and quantification of paclitaxel accumulation in single cell cultures was performed using flow cytometry. Culture conditions (e.g., seeding density, elicitation, media composition, etc.) were optimized for cell growth and production. This technology, through isolation of high-accumulators and establishment of elite cell lines, will provide a more efficient and economically viable method for production of paclitaxel and other plant-derived natural products from suspension culture.