A second generation flow contraction device was developed and modeled which allows cells to be subjected to well-defined hydrodynamic forces. Studies were conducted with this system on wild-type Chinese Hamster Ovary cells (CHO-K1) and a strain of CHO cells which expresses the human Bcl-2 triangle gene (CHO-bcl-2). In this study, the following questions were asked: (1) Does an acute hydrodynamic force induce apoptosis in wild-type CHO and CHO-bcl-2 cells? (2) Does the type of culture media make a difference with respect to the induction of apoptosis or necrosis? and (3) Does culture history affect induction of apoptosis or necrosis? The results obtained with this new flow contraction device and corresponding computer simulations are consistent with previously published studies with respect to the level of energy dissipation rate (EDR) required to create significant cell lysis. Second, while detectable relative to the control in the T-flask experiments, only a small fraction of the cells become apoptotic when exposed to a sub-lysis level of EDR (<10(8) W x m(-3)). Third, cells cultured in suspension with serum free media do not exhibit any higher or lower sensitivity (with respect to apoptosis) to various levels of EDR when compared to control cultures grown in T-flask and serum containing media; on the other hand, necrosis is significantly increased in experiments performed on suspended cells without serum. Fourth, the addition of the Bcl-2 gene product might slightly reduce the occurrence of apoptosis in T-flask culture; however, the baseline response is so low that the difference is insignificant.

Fluorescence activated cell sorting, FACS, is a widely used method to sort subpopulations of cells to high purities. To achieve relatively high sorting speeds, FACS instruments operate by forcing suspended cells to flow in a single file line through a laser(s) beam(s). Subsequently, this flow stream breaks up into individual drops which can be charged and deflected into multiple collection streams. Previous work by Ma et al. (2002) and Mollet et al. (2007; Biotechnol Bioeng 98:772-788) indicates that subjecting cells to hydrodynamic forces consisting of both high extensional and shear components in micro-channels results in significant cell damage. Using the fluid dynamics software FLUENT, computer simulations of typical fluid flow through the nozzle of a BD FACSVantage indicate that hydrodynamic forces, quantified using the scalar parameter energy dissipation rate, are similar in the FACS nozzle to levels reported to create significant cell damage in micro-channels. Experimental studies in the FACSVantage, operated under the same conditions as the simulations confirmed significant cell damage in two cell lines, Chinese Hamster Ovary cells (CHO) and THP1, a human acute monocytic leukemia cell line.

A majority of the previous investigations on the hydrodynamic sensitivity of mammalian cells have focused on lethal effects as determined by cell death or lysis. In this study, we investigated the effect of hydrodynamic stress on CHO cells in a fed-batch process using a previously reported system which subjects cells to repetitive, high levels of hydrodynamic stress, quantified by energy dissipation rate (EDR). The results indicated that cell growth and monoclonal antibody production of the test cells were very resistant to the hydrodynamic stress. Compared to the control, no significant variation was observed at the highest EDR tested, 6.4 x 10(6) W/m(3). Most product quality attributes were not affected by intense hydrodynamic stress either. The only significant impact was on glycosylation. A shift of glycosylation pattern was observed at EDR levels at or higher than 6.0 x 10(4) W/m(3), which is two orders of magnitude lower than the EDR where physical cell damage, as measured by lactate dehydrogenase release, was observed. While not as extensively investigated, a second monoclonal antibody produced in a different CHO clone exhibited the same glycosylation change at an intensive EDR, 2.9 x 10(5) W/m(3). Conversely, a low EDR of 0.9 x 10(2) W/m(3) had no effect on the glycosylation pattern. As 6.0 x 10(4) W/m(3), the lowest EDR that triggers the glycosylation shift, is about one order of magnitude higher than the estimated, maximum EDR in typically operated, large-scale stirred tank bioreactors, further studies in a lower EDR range of 1 x 10(3)-6.0 x 10(4) W/m(3) are needed to assess the glycosylation shift effect under typical large-scale bioreactor operation conditions. Follow-up studies in stirred tanks are also needed to confirm the glycosylation shift effect and to validate the repetitive hydrodynamic stress model.

Production of biofuels and bioenergy precursors by phototrophic microorganisms, such as microalgae and cyanobacteria, is a promising alternative to conventional fuels obtained from non-renewable resources. Several species of microalgae have been investigated as potential candidates for the production of biofuels, for the most part due to their exceptional metabolic capability to accumulate large quantities of lipids. Constraint-based modeling, a systems biology approach that accurately predicts the metabolic phenotype of phototrophs, has been deployed to identify suitable culture conditions as well as to explore genetic enhancement strategies for bioproduction. Core metabolic models were employed to gain insight into the central carbon metabolism in photosynthetic microorganisms. More recently, comprehensive genome-scale models, including organelle-specific information at high resolution, have been developed to gain new insight into the metabolism of phototrophic cell factories. Here, we review the current state of the art of constraint-based modeling and computational method development and discuss how advanced models led to increased prediction accuracy and thus improved lipid production in microalgae.

The effect of hydrodynamic forces on animal cell cultures, while extensively studied, still lacks significant, fundamental understanding. A previous manuscript reported on the acute exposure of CHO cells to hydrodynamic forces in a second generation convergent-divergent microfluidic device (Mollet et al., 2007). In this study, the use of this device is extended in a proof of concept system in which suspended animal cells, grown in a typical bioreactor, are subjected to chronic exposure of moderately high levels of hydrodynamic forces by way of a continuous recycle loop between the bioreactor and the microfluidic device. A strain of CHO cells (CHO-6E6) was grown in a batch culture under controlled pH, temperature, and dissolved oxygen conditions. At mid exponential stage of growth in the bioreactor the recycle flow was initiated. The cells either stopped growing or started dying at EDR values that were significantly lower (one to two orders of magnitude) than those previously reported to kill cells from a single, acute exposure. These observations allow further refinement in the design of bioprocess equipment since it provides a more accurate threshold, above which one does not want to subject animal cells to continuous exposure to specific levels of hydrodynamic forces.

Mixing and aeration are normally used to provide a homogeneous, suitable environment for cells in a bioreactor. In spite of the large cumulative experience in the use of bioreactors in the biotechnology industry, there are still challenges when scaling‐up animal cell cultures. Remarkable advances have been achieved over the last two decades in designing and applying mixing and aeration strategies, as well as understanding the effects of hydrodynamic forces on cell cultures. This section reviews the basic concepts and current practices for mixing and aeration in bioreactors, with focus on stirred tank bioreactors (STR), as most of the processes for recombinant proteins, antibodies, and vaccines involve the use of STR. Current methods of oxygen supply in STR include surface aeration and gas sparging, although the use of perfluorocarbons, oxygen carriers and membranes has been investigated. Sparger design and the role of CO 2 accumulation and removal are discussed. Also, a summary of the main aspects involved in mixing is presented, including tank characteristics, impeller geometry, and power drawn. Finally, this review explores several different approaches that have been taken in order to unveil the response of animal cells to the hydrodynamic forces generated inside bioprocessing devices.

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Cells can sense changes in their extracellular environment and subsequently adapt their biomass composition. Nutrient abundance defines the capability of the cell to produce biomass components. Under nutrient-limited conditions, resource allocation dramatically shifts to carbon-rich molecules. Here, we used dynamic biomass composition data to predict changes in growth and reaction flux distributions using the available genome-scale metabolic models of five eukaryotic organisms (three heterotrophs and two phototrophs). We identified temporal profiles of metabolic fluxes that indicate long-term trends in pathway and organelle function in response to nitrogen depletion. Surprisingly, our calculations of model sensitivity and biosynthetic cost showed that free energy of biomass metabolites is the main driver of biosynthetic cost and not molecular weight, thus explaining the high costs of arginine and histidine. We demonstrated how metabolic models can accurately predict the complexity of interwoven mechanisms in response to stress over the course of growth.