Preparation of single cells from aggregated Taxus suspension cultures for population analysis

Abstract

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.

Culture of Isolated Single Cells from Taxus Suspensions for the Propagation of Superior Cell Populations

“…Taxus cell line maintenance and preparation of single cell suspensions Taxus cuspidata cell line P991 was provided by the USDA, Agricultural Research Service (Ithaca, NY) and maintained in our laboratory as described previously (Naill & Roberts 2004). Single cells were isolated from Taxus cuspidata suspensions by enzymatic digestion with 0.5% (w/v) Pectolyase Y)23 and 0.04% (w/v) cellulase as described elsewhere (Naill & Roberts 2004).…”

“…Single cells were isolated from Taxus cuspidata suspensions by enzymatic digestion with 0.5% (w/v) Pectolyase Y)23 and 0.04% (w/v) cellulase as described elsewhere (Naill & Roberts 2004). …”

“…Plates were wrapped with parafilm and incubated at 23-24°C and 125 rpm. Fresh medium was prepared as previously described (Naill & Roberts 2004) and conditioned medium was isolated from aggregated suspensions on day 14 via filtration through Miracloth (EMD Biosciences, San Diego, CA). No cells were observed with microscopy in conditioned medium following filtration.…”

“…The cell density of single cell cultures was determined via hemacytometry. Samples were stained with fluorescein diacetate (FDA) and viability was assessed by fluorescence microscopy as described previously (Naill & Roberts 2004). For each sample, the viable cell density was reported as the average of at least eight distinct fields (0.1 ll each).…”

“…A method for the isolation of single cells from Taxus cell suspension cultures in high yield has recently been developed (Naill & Roberts 2004). While traditional population-average measurements are insufficient to fully characterize cell culture heterogeneity, isolated single cells allow for the rapid analysis of cell culture population dynamics via flow cytometry (Yanpaisan et al 1999).…”