Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures

Plant cell aggregates have long been implicated in affecting cellular metabolism in suspension culture, yet the rigorous characterization of aggregate size as a process variable and its effect on bioprocess performance has not been demonstrated. Aggregate fractionation and analysis of biomass-associated product is commonly used to assess the effect of aggregation, but we establish that this method is flawed under certain conditions and does not necessarily agree with comprehensive studies of total culture performance. Leveraging recent advances to routinely measure aggregate size distributions, we developed a simple method to manipulate aggregate size and evaluate its effect on the culture as a whole, and found that Taxus suspension cultures with smaller aggregates produced significantly more paclitaxel than cultures with larger aggregates in two cell lines over a range of aggregate sizes, and where biomass accumulation was equivalent before elicitation with methyl jasmonate. Taxus cuspidata (T. cuspidata) P93AF cultures with mean aggregate sizes of 690 and 1,100 μm produced 22 and 11 mg/L paclitaxel, respectively, a twofold increase for smaller aggregates, and T. cuspidata P991 cultures with mean aggregate sizes of 400 and 840 μm produced 6 and 0.3 mg/L paclitaxel, respectively, an increase of 20-fold for smaller aggregates. These results demonstrate the importance of validating experiments aimed at a specific phenomenon with total process studies, and provide a basis for treating aggregate size as a targeted process variable for rational control strategies.     

A population balance model to modulate shear for the control of aggregation in Taxus suspension cultures

Cellular aggregation in plant suspension cultures directly affects the accumulation of high value products, such as paclitaxel from Taxus. Through application of mechanical shear by repeated, manual pipetting through a 10 ml pipet with a 1.6 mm aperture, the mean aggregate size of a Taxus culture can be reduced without affecting culture growth. When a constant level of mechanical shear was applied over eight generations, the sheared population was maintained at a mean aggregate diameter 194 μm lower than the unsheared control, but the mean aggregate size fluctuated by over 600 μm, indicating unpredictable culture variability. A population balance model was developed to interpret and predict disaggregation dynamics under mechanical shear. Adjustable parameters involved in the breakage frequency function of the population balance model were estimated by nonlinear optimization from experimentally measured size distributions. The optimized model predictions were in strong agreement with measured size distributions. The model was then used to determine the shear requirements to successfully reach a target aggregate size distribution. This model will be utilized in the future to maintain a culture with a constant size distribution with the goal of decreasing culture variability and increasing paclitaxel yields.     

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.     

Characterization of plant suspension cultures using the focused beam reflectance technique

Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R ²>0.99) while the mean of the cube-weighted FBRM chord length distribution was nonlinearly related to aggregate size.     

Characterization of aggregate size in Taxus suspension cell culture

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 to 2,000 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 (R ² > 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.     

Sphingomyelinase-to-LDL molar ratio determines low density lipoprotein aggregation size: biological significance

The subendothelial retention of low density lipoproteins (LDL) is believed to be the central pathogenic event in atherosclerosis, as stated by the response-to-retention hypothesis. Sphingomyelinase, an enzyme present in the arteries, has been proven to promote LDL aggregation. This study investigates the hypothesis that the extent of LDL aggregation is determined by the molar ratio of sphingomyelinase (SMase)-to-LDL, rather than the absolute concentrations. A mass action model is used to describe the aggregation process, and binding and dissociation rate constants are determined by fitting of dynamic light scattering data. The model predicts aggregate sizes that agree well with experimental observations. This study also tests the hypothesis that monocyte uptake of LDL correlates with aggregate size. LDL aggregates of three specific sizes (75, 100, and 150 nm) were incubated with J774A.1 cells and the net accumulation of LDL was monitored by measuring changes in the cellular cholesterol and protein content. Relative to a control sample, cholesterol accumulation was enhanced for aggregate sizes of 75 and 150 nm. The intermediate size aggregates, 100 nm, led to a very striking result demonstrating that cholesterol accumulation was markedly greater than the other samples, and was sufficient to cause cell death. These results underscore an important role of colloidal aggregation, and the influence of LDL aggregate size, in atherosclerosis.     

Improved assessment of aggregate size in Taxus plant cell suspension cultures using laser diffraction

In suspended culture, most relevant for biotechnological application, plant cells form aggregates. This phenomenon is of importance as it is related to productivity, leads to local heterogeneities, and might be a reason for the considerable shear sensitivity of these cultures. The valid measurement of plant cell aggregates, however, is not trivial, due to a rather large size distribution and measurement artifacts implied by the measuring method. In this study, laser diffraction was used as a novel method for characterization of Taxus chinensis cells, a major source for the antitumor agent paclitaxel. Aggregate size measured in shaking flask cultivations over 10 days revealed an increase during the growth phase of a batch cycle and a decrease during the stationary phase. During growth, the increase in bio dry weight was proportional to aggregate size. Laser diffraction was found superior to microscopy and image analysis, which had a tendency to underestimate aggregate size up to 20%. This novel approach provides a practicable, rapid, robust, and reproducible way to analyze a 100‐fold more samples in considerably less time than image analysis and is therefore of especial value for quality control in industrial plant cell cultivation.     

Rheological characteristics of cell suspension and cell culture of Perilla frutescens

Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stres. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size. (c) 1992 John Wiley & Sons, Inc.     

中国红豆杉悬浮培养细胞聚集体异质性的初步研究

通过梯度浓度的蔗糖溶液（0－1mol/L）的筛选,分拣出7种不同物理学密度的红豆杉细胞聚集体,并对它们进行了木质素含量及紫杉醇含量测定。结果显示：不同的细胞聚集体在物理学密度、木质素含量、紫杉醇含量、生长速度等方面存在着差异,其中密度最小的细胞聚集体木质素及紫杉醇含量分别是密度最大的细胞聚集体的5倍和8倍,并且在一定密度范围内（大于0.2mol／L蔗糖溶液密度）,细胞聚集体的木质素含量与紫杉醇的含量呈平行关系,表明不同聚集体紫杉醇含量一细胞分化有一定的关系,首次提出了红豆杉悬浮细胞聚集体培养中存在着异质现象,并对其可能机理及意义给予阐述。     

Gradient of anthocyanin in cell aggregates of Daucus carota in suspension cultures

Summary The size distribution of cell aggregates, and the effect of cell aggregate size on anthocyanin content of Daucus carota cells in suspension cultures, was studied. The profile of biomass distribution in various size groups of cell aggregates indicated that over 92% of biomass was present in the aggregates of 500–1500 m in diameter. The anthocyanin content increased initially with the increase in cell aggregate diameter up to 500–850 m, and decreased rapidly with the increase in the cell aggregate size above this critical diameter. On the other hand, the surface colour intensity showed a steady increase with the increase in cell aggregate size, indicating a steep radial gradient of anthocyanin content along the radius of the larger cell aggregates.     

Hydrodynamic effects on BHK cells grown as suspended natural aggregates

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John Wiley & Sons, Inc.     

Chemically controlled aggregation of pluripotent stem cells

Heterogeneity in pluripotent stem cell (PSC) aggregation leads to variability in mass transfer and signaling gradients between aggregates, which results in heterogeneous differentiation and therefore variability in product quality and yield. We have characterized a chemical‐based method to control aggregate size within a specific, tunable range with low heterogeneity, thereby reducing process variability in PSC expansion. This method enables controlled, scalable, stirred suspension‐based manufacturing of PSC cultures that are critical for the translation of regenerative medicine strategies to clinical products.     

Application of imaging tools to plant cell culture: Relationship between plant cell aggregation and flavonoid production

Summary Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin-producing cell suspension cultures of ohelo (Vaccinium pahalae). The ex situ imaging system could image cell aggregates from 30 μm to 2 mm in diameter. The image analysis algorithm was based on extracted geometric features and morphological methods for biomass volume estimates, and hue, saturation, and intensity color characteristics for pigmentation estimates. Detailed information available from sampled cell culture images was validated by comparison to standard destructive manual measurements. Image analysis measurements revealed that pigment accumulation was negatively correlated with aggregate size. Although a substantial proportion of small aggregates remained colorless, the highly-pigmented small aggregates, 18 to 238 μm in breadth, contributed over 70% of the culture anthocyanin production (mg L⁻¹), despite their minor contribution to the overall biomass. The relative frequency of pigmented aggregates was higher in large-size aggregate classes; however, the pigmented sectors were mostly confined to only the periphery of the aggregates. As a result, large aggregate classes had only a minor contribution to overall culture anthocyanin yield.     

Aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. for triptolide, wilforgine, and wilforine production

The relationships between aggregate cell types, cell growth, and the triptolide, wilforgine, and wilforine content in aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. were examined. Aggregate cells larger than 2?mm grew quickly and constituted the majority of the white aggregates. The accumulation of triptolide was strongly correlated with the size of the aggregates and the length of the culture period. The aggregates 0.5?C2?mm in diameter accumulated higher triptolide content than those with other sizes throughout the culture. However, the size of the aggregate cells did not significantly affect on the wilforgine and wilforine content. Two other kinds of aggregate cells, the brown and green aggregate cells, also formed in the suspension cultures. The smallest aggregates (0.1?C0.5?mm) had a lower biomass and growth rate and had more chloroplasts and higher alkaloid content. The results of this study can be used to improve the selection process for the mass production of triptolide, wilforgine, and wilforine from cell suspension cultures.     

Periodic cell aggregation in suspensions of Dictyostelium discoideum

Abstract. Periodic activities of Dictyostelium discoideum can be observed in cell suspension as two types of oscillations in the light-scattering properties, spike-shaped and sinusoidal. Responses of suspended cells to applied chemoattractants are also reflected by transient changes in light scattering. Alterations in the light-scattering properties are due to structural changes such as changes in cell shape and/or changes in the size of cell aggregates. Therefore, changes in the aggregation state during autonomous oscillations and during attractant-induced responses were investigated. In order to be able to withdraw multiple samples and larger sample volumes from optically monitored cell suspensions, a photometer comprising glass fiber optics immersable in a cell suspension was constructed. Samples were fixed with formaldehyde and photographed. The aggregation state of the samples was quantified by counting the number of particles (cells and cell aggregates) per volume. Folic acid elicited in suspensions of undifferentiated cells a transient decrease in the number of particles per volume as did cAMP in suspensions of preaggregation cells. Periodic changes in the number of particles per volume occurred synchronously with spike-shaped and sinusoidal oscillations. The relative amplitude of the oscillations in particle number was larger during sinusoids than during spikes. Photographs showed periodic changes in the aggregate size during sinusoidal oscillations. In each cycle, the cell-aggregation phase was followed by a phase of partial disaggregation. The recurring loosening of cell-cell contacts may be relevant for sorting out the different cell types. The potential role of contact site as synchronizer and as constituent of an oscillator is discussed.     

Focussed beam reflectance measurement (FBRM) monitoring of particle size and morphology in suspension cultures of Morinda citrifolia and Centaurea calcitrapa

Laser light scattering technology, as applied in the Lasentec focussed beam reflectance measurement (FBRM) system, was used to characterise two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa. Shake-flask suspensions were analysed in terms of biomass concentration and aggregate size/shape over the course of typical batch growth cycles. For the heavily aggregated C. calcitrapa, biomass levels [from 10-160 g fresh weight (fw) l(-1))] were linearly correlated with FBRM counts. For M. citrifolia, which grows in unbranched chains of 2-10 elongated cells, linear correlation of biomass concentration with FBRM counts was applicable in the range 0-100 g fw l(-1); at higher levels (100-300 g fw l(-1)), biomass was non-linearly correlated with FBRM counts and length-weighted average FBRM chord length. For both cell systems, particle morphology (size/shape) was quantified using semi-automated digital image analysis. The average aggregate equivalent diameter (C. calcitrapa) and average chain length (M. citrifolia), determined using image analysis, closely tracked the FBRM average chord length. The data clearly demonstrate the potential for applying the FBRM technique for rapid characterisation of plant cell suspension cultures.     

The influence of plant species,fertilization and elevated CO2 on soil aggregate stability

We tested the effects of plant species, fertilization and elevated CO₂ on water-stable soil aggregation. Five annual grassland species and a plant community were grown in outdoor mesocosms for 4 years, with and without NPK fertilization, at ambient or elevated atmospheric CO₂ concentrations. Aggregate stability (resistance of aggregates to slaking) in the top 0.15 m of soil differed among plant species. However, the more diverse plant community did not enhance aggregate stability relative to most monocultures. Species differences in aggregate stability were positively correlated with soil active bacterial biomass, but did not correlate with root biomass or fungal length. Plant species did not affect aggregate stability lower in the soil profile (0.15–0.45 m), where soil biological activity is generally decreased. Elevated CO₂ and NPK fertilization altered many of the factors known to influence aggregation, but did not affect water-stable aggregation at either depth, in any of the plant treatments. These results suggest that global changes will alter soil structure primarily due to shifts in vegetation composition.     

Viability characterization of Taxus chinensis plant cell suspension cultures by rapid colorimetric- and image analysis-based techniques

For the commercially established process of paclitaxel production with Taxus chinensis plant cell culture, the size of plant cell aggregates and phenotypic changes in coloration during cultivation have long been acknowledged as intangible parameters. So far, the variability of aggregates and coloration of cells are challenging parameters for any viability assay. The aim of this study was to investigate simple and non-toxic methods for viability determination of Taxus cultures in order to provide a practicable, rapid, robust and reproducible way to sample large amounts of material. A further goal was to examine whether Taxus aggregate cell coloration is related to general cell viability and might be exploited by microscopy and image analysis to gain easy access to general cell viability. The Alamar Blue assay was found to be exceptionally eligible for viability estimation. Moreover, aggregate coloration, as a morphologic attribute, was quantified by image analysis and found to be a good and traceable indicator of T. chinensis viability.     

Modeling aggregation and growth processes in an algal population model: analysis and computations

 Aggregation, the formation of large particles through multiple collision of smaller ones is a highly visible phenomena in oceanic waters which can control material flux to the deep sea. Oceanic aggregates more than 1 cm in diameter have been observed and are frequently described to consist of phytoplankton cells as well as other organic matter such as fecel pellets and mucus nets from pteropods. Division of live phytoplankton cells within an aggregate can also increase the size of aggregate (assuming some daughter cells stay in the aggregate) and hence could be a significant factor in speeding up the formation process of larger aggregate. Due to the difficulty of modeling cell division within aggregates, few efforts have been made in this direction. In this paper, we propose a size structured approach that includes growth of aggregate size due to both cell division and aggregation. We first examine some basic mathematical issues associated with the development of a numerical simulation of the resulting algal aggregation model. The numerical algorithm is then used to examine the basic model behavior and present a comparison between aggregate distribution with and without division in aggregates. Results indicate that the inclusion of a growth term in aggregates, due to cell division, results in higher densities of larger aggregates; hence it has the impact to speed clearance of organic matter from the surface layer of the ocean. Received 1 July 1994; received in revised form 23 February 1996     

Cell aggregation on agar as an indicator for cell-matrix adhesion: effects of opioids

The slow aggregation assay is generally used to study the functionality of cell–cell adhesion complexes. Single cells are seeded on a semisolid agar substrate in a 96-well plate and the cells spontaneously aggregate. We used HEK FLAG-MOP cells that stably overexpress the mu opioid receptor and the mu-opioid-receptor-selective agonists DAMGO and morphine to study whether other factors than functionality of cell–cell adhesions complexes can contribute to changes in the pattern of slow aggregation on agar. HEK FLAG-MOP cells formed small compact aggregates. In the presence of DAMGO and morphine, larger and fewer aggregates were formed in comparison to the vehicle control. These aggregates were localized in the center of the agar surface, whereas in the vehicle control they were dispersed over the substrate. However, in suspension culture on a Gyrotory shaker, no stimulation of aggregation was observed by DAMGO and morphine, showing that opioids do not affect affinity. A dissociation experiment revealed that HEK FLAG-MOP aggregates formed in the absence or presence of opioids are resistant to de-adhesion. We demonstrated that the larger aggregates are neither the result of cell growth stimulation by DAMGO and morphine. Since manipulations of the substrate such as increasing the agar concentration or mixing agar with agarose induced the same changes in the pattern of slow aggregation as treatment with opioids, we suggest that cell–substrate adhesion may be involved in opioid-stimulated aggregation.