Characterization of aggregate size in Taxus suspension cell culture

Abstract

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 μm to 2000 μm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R2 > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.

Lignan accumulation in two-phase cultures of Taxus x media hairy roots

“…intracellular + extracellular: both in aqueous and PFD phases) yield of paclitaxel was obtained in PFD-degassed carrying cultures after two weeks of elicitation. Under the conditions of the current study we also observed in most of the cases higher lignan contents after two week elicitation, however, the PFD-aerated supported elicited cultures seemed to be more favourable for lignan accumulation (Tables 3, 4) likely due to generated to a higher extent than in PFD-degassed supported cultures oxidative stress which is believed to affect plant secondary metabolism (Shilpa et al 2010;Kolewe et al 2010). PFD application in the function of in situ extrahent could also contribute to the lower metabolite feedback inhibition and diminished cytotoxic metabolites accumulation in aqueous phases of the medium which seemed to be often the reason of limited secondary metabolites production (Malik et al 2013;Kolewe et al 2010).…”

Methyl jasmonate represses growth and affects cell cycle progression in cultured Taxus cells

“…A Multisizer 3™ Coulter counter equipped with a 2000 µm aperture (Beckman Coulter, Brea, CA, USA) was used to determine total biomass dry weight based on previously published correlations (Kolewe et al 2010). For analysis, two × 2 mL samples of well-mixed culture broth (cells plus media) were taken from each flask.…”

Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures

“…For elicitation, 200 μM methyl jasmonate (MeJA) was added on day 7 post-transfer during the mid-exponential phase of growth, as described previously [34]. A Multisizer 3 ™ Coulter counter equipped with a 2,000 μm aperture (Beckman Coulter, Brea, CA) was used to measure culture aggregate size distributions, as described previously [30]. As the total aggregate volume was previously shown to correlate directly with biomass [30], this measurement was also used to determine dry weight (DW), and all DW data presented here were based on this correlation.…”

“…In Taxus suspension cultures, aggregates ranging from less than 100 μm to well over 2 mm have been observed [30]. Cells within these aggregates are subject to different microenvironments, leading to cell-cell differences in morphology [31] and metabolism [32].…”