Mechanical shearing Taxus plant suspension cultures reduces aggregation without affecting cell health

A high degree of cellular aggregation within Taxus suspension cultures results in increased culture heterogeneity, as well as variable and reduced paclitaxel yields. Here, a method was developed to mechanically shear Taxus suspension cultures through manual pipetting of cells through a small aperture. In this manner, the mean aggregate diameter of a culture can be effectively reduced. The method was applied to cultures over multiple generations, showing that cell viability and growth were not affected by mechanical shearing. For the first two generations, mean aggregate size of the sheared populations was not statistically different from the unsheared controls, with each population having a mean aggregate diameter below 375 μm. As the unsheared population increased in mean aggregate diameter to ∼475 μm and additional shear was employed to disaggregate cultures, significant differences in mean aggregate size between the sheared and unsheared cultures were observed. Throughout the experiment, the unsheared cultures had a range in mean aggregate size from 240-500 μm, whereas the sheared cultures had a range in mean aggregate size from 218-384 μm. This experiment demonstrates the promise of mechanical shearing for reducing culture heterogeneity by lowering the mean aggregate size distribution without affecting cell viability or growth.