The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry (Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO4, NO3, NH4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d(-1) based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest Bat the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

A physiological product-release model for baculovirus infected insect cells

Existing models describe the product release from baculovirus infected insect cells as an unspecific protein leakage occurring in parallel with protein production. The model presented here shows that the observed product release of normally non-secreted proteins can be described through cell death alone. This model avoids the implicit non-physiological assumption of previous models that cells permeable to recombinant protein as well as trypan blue continue to produce protein. (c) 2005 Wiley Periodicals, Inc.

A phase-segregated model for plant cell culture: The effect of cell volume fraction

Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases, Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus, Large errors in the simulated results were observed if the phase-segregation was not considered.

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and similar to 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1 --> residues or the disaccharide Galp-beta-(1 --> 2)-Galp-alpha-(1 --> linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 --> 4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating --> 4)-D-Manp-beta-(1 --> and --> Lt)-D-Glcp-beta-(1 --> branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1 --> 4)-[D-Galp-beta-(1 --> 2)-D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> 4)-D-Glcp-beta-(I --> 4)-[D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure. (C) 1997 Elsevier Science Ltd.

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG(4) monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 mu g 10(6) cells(-1) mL(-1) at a PEI nitrogen: DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from similar to 13 to similar to 39 mg L-1. Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended activated hypothermic synthesis.

The pearl oysters, Pinctada maxima and P margaritifera, respond in different ways to culture in dissimilar environments

Growth, Condition Index (CI) and survival of the pearl oysters, Pinctada maxima and R margaritifera, were measured in three size groups of oysters over 14 months at two dissimilar environments in the Great Barrier Reef lagoon. These were the Australian Institute of Marine Science (AIMS) in a mainland bay and Orpheus Island Research Station (OIRS) in coral reef waters. Temperature, suspended particulate matter (SPM) and particulate organic matter (POM) were monitored during the study. Temperature at AIMS fluctuated more widely than at OIRS both daily and seasonally, with annual ranges 20-31 degrees C and 22-30 degrees C, respectively. Mean SPM concentration at AIMS (11.1 mg l(-1)) was much higher than at OIRS (1.4 mg l(-1)) and fluctuated widely (2-60 mg l(-1)). Mean POM level was also substantially higher at AIMS, being 2.1 mg l(-1) compared with 0.56 mg l(-1) at OIRS. Von Bertalatiffy growth curve analyses showed that P. maxima grew more rapidly and to larger sizes than P. margaritifera at both sites. For the shell height (SH) of R maxima, growth index phi'=4.31 and 4.24, asymptotic size SHinfinity = 229 and 205 mm, and time to reach 120 mm SH (T-(120))= 1.9 and 2.1 years at AIMS and OIRS, respectively. While for P margaritifera, phi'=4.00 and 4.15, SHinfinity = 136 and 157 mm, and T-(120) = 2.5 and 3.9 years at AIMS and OIRS, respectively. R maxima had significantly lower growth rates and lower survival of small oysters during winter compared with summer. There were, however, no significant differences between the two sites in growth rates of P. maxima and final Cl values. In contrast, P. margaritifiera showed significant differences between sites and not seasons, with lower growth rates, survival of small oysters, final Cl values and asymptotic sizes at AIMS. The winter low temperatures, but not high SPM at AIMS, adversely affected P. maxima. Conversely, the high SPM levels at AIMS, but not temperature, adversely affected P. margaritifera. This was in accordance with earlier laboratory-based energetics studies of the effects of temperature and SPM on these two species. P maxima has potential to be commercially cultured in ca. > 25 degrees C waters with a wide range of SPM levels, including oligotrophic coral reef waters with appropriate particle sizes. It is possible to culture R margaritifera in turbid conditions, but its poor performance in these conditions makes commercial culture unlikely. (c) 2005 Elsevier B.V. All rights reserved.

The effect of organisational culture perceptions on the relationship between budgetary participation and managerial job-related outcomes

The impact of managers' perceptions of their organizational culture (OC) on the relationship between budgetary participation (BP) and managerial job-related outcomes, operationalized as managerial performance and job-related tension (JRT) is examined. Data supported predictions that increasing BP would lower JRT for managers perceiving a high emphasis on innovation within their OC, regardless of their perceptions of an emphasis on attention to detail. When managers perceived low innovation, however, their perception of level of attention to detail had a significant effect on the relationship between BP and JRT.