Eric Olmos

Numerical description of flow regime transitions in bubble column reactors by a multiple gas phase model

The study of the hydrodynamics of bubble column reactors reveals that three main flow regimes can be distinguished. The present study is an original application of standard numerical multiphase models aiming at a better prediction of the transitions and a better description of these regimes. This approach consists of a two-step calculation with, in a first time, the determination of bubble size distributions using coalescence and break-up laws, and in a second time, the implementation of this distribution in a multiple gas phase Euler–Euler model. Calculations are compared with experimental data: liquid velocities and global gas hold-ups. They were obtained in a cylindrical bubble column in which local or uniform aerations were performed. The comparison shows that our approach allows a good prediction of both flow regime transitions and flow characteristics such as hydrodynamics or turbulence. Furthermore, two bubble-induced turbulence models consisting, respectively, of an added viscosity and of two turbulence source terms added in the k–e transport equations have been compared.

Description of flow regime transitions in bubble columns via laser Doppler anemometry signals processing

This work aims at studying the non-linear dynamics and the flow regime transitions in bubble column reactors. For this purpose, various signal processing techniques e.g. frequency analysis, fractal analysis and deterministic chaos analysis have been applied to laser Doppler velocimetry signals. The system considered is a two-dimensional reactor allowing LDV measurements at higher void fractions than in three-dimensional systems. Each signal processing technique presents a specific capacity to describe a regime transition or a feature of the flow structure. Use of these various techniques have highlighted the occurrence of two states in the transition regime and yielded detailed information on the physical mechanisms responsible for these transitions.

Limiting cell aggregation during mesenchymal stem cell expansion on microcarriers.

Mesenchymal stem cells (MSC) are known to be a valuable cell source for tissue engineering and regenerative medicine. However, one of the main limiting steps in their clinical use is the amplification step. MSC expansion on microcarriers has emerged during the last few years, fulfilling the lack of classical T‐flasks expansion. Even if the therapeutic potential of MSC as aggregates has been recently highlighted, cell aggregation during expansion has to be avoided. Thus, MSC culture on microcarriers has still to be improved, notably concerning cell aggregation prevention. The aim of this study was to limit cell aggregation during MSC expansion on Cytodex‐1®, by evaluating the impact of several culture parameters. First, MSC cultures were performed at different agitation rates (0, 25, and 75 rpm) and different initial cell densities (25 and 50 × 106 cell g−1 Cytodex‐1®). Then, the MSC aggregates were put into contact with additional available surfaces (T‐flask, fresh and used Cytodex‐1®) at different times (before and after cell aggregation). The results showed that cell aggregation was partly induced by agitation and prevented in static cultures. Moreover, cell aggregation was dependent on cell density and correlated with a decrease in the total cell number. It was however shown that the aggregated organization could be dissociated when in contact with additional surfaces such as T‐flasks or fresh Cytodex‐1® carriers. Finally, cell aggregation could be successfully limited in spinner flask by adding fresh Cytodex‐1® carriers before its onset. Those results indicated that MSC expansion on agitated Cytodex‐1® microcarriers could be performed without cell aggregation, avoiding a decrease in total cell number.

Impact of shear stress and impeller design on the production of biogas in anaerobic digesters

Today, intensification of anaerobic digestion is still a scientific and technical challenge. The present study proposed combined experimental and computational fluid dynamics simulations to characterize the impact of shear stress and impeller design on the biogas production after sequential additions of substrate. Liquid phase (cattle manure digestate) rheological law was experimentally determined and input in numerical simulations. The results showed that the original use of a double helical ribbon in digester allowed a significantly faster dispersion of fresh substrate than the use of a classical Rushton turbine, leading to a 50% higher methane production rate. However, with both impellers, too high agitation rates entailed a clear slow-down of production rate and a decrease in CH4 content. To avoid this loss of productivity, it was shown that the maximal value of shear stress, determined by numerical simulations, was a consistent parameter to set the upper agitation conditions in digesters.

Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement

Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates.

Lactate production as representative of the fermentation potential of Corynebacterium glutamicum 2262 in a one-step process

The fermentative properties of thermo-sensitive strain Corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. In particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. In both processes, lactate was produced in significant amount, 27 g/L in batch culture, and up to 55.8 g/L in fed-batch culture, but the specific production rate in the fed-batch culture was four times lower than that in the batch culture. Compared to other investigated fermentation processes, our strategy resulted in the highest yield of lactic acid from biomass. Lactate production by C. glutamicum 2262 thus revealed the capability of the strain to produce various fermentation products from pyruvate.

Coupling Between Cell Kinetics and CFD to Establish Physio-Hydrodynamic Correlations in Various Stirred Culture Systems

According to the recent increase of the culture volumes of industrial processes using animal cells, it becomes necessary to precisely study the reactor hydrodynamics. Indeed, scale-up rules have to integrate hydrodynamic stress distributions to be more reliable. The aim of our work was to establish new correlations between pertinent hydrodynamic parameters and cellular physiology data obtained by suspension cell cultures study. Global kinetic response of recombinant CHO cells to various agitation rates, in spinner flasks and in stirred tank reactors, were studied. Reactors hydrodynamics was described using Computational Fluid Dynamics (CFD) and validated by Laser Doppler Velocimetry (LDV). The results revealed, contrary to the commonly admitted idea, a beneficial effect of increased agitation rates on cell growth. Moreover, by coupling experimental and numerical results, original physio-hydrodynamic correlations were established for both cell culture systems. It is thus possible to propose an integrated and innovative method for reactor scale-up by using the Kolmogorov theory of turbulence and CFD simulations.

Revisiting the determination of hydromechanical stresses encountered by microcarriers in stem cell culture bioreactors

Background Expansion of mesenchymal stem cells (MSC) is one of the key steps for their use in tissue engineering or cell therapies. Today, expansion processes are mainly based on the use of microcarriers to allow large interfacial adherence areas [1]. However, this culture technology is known to be practically limited to low agitation intensity and microcarrier concentrations due to possible cell damage arising from particle hydromechanical stress or collisions between microcarriers [2]. Unfortunately, the description of the relationship between bioreactor hydrodynamics, microcarrier suspension and occurrence of collisions was neither clearly established in the case of stem cell cultures, nor based on a local description of the bioreactor hydrodynamics heterogeneity. Thus, in the present study, it is proposed to use numerical simulations to describe not only the liquid phase but also the microcarrier dispersion and the occurrence of hydromechanical stress encountered by the microcarriers. Two kinds of hydromechanical stress can be distinguished: (i) fluid-solid interactions (fluid shear stress) arising from turbulent eddies and (ii) solid-solid interactions arising from collisions between microcarriers or between microcarriers and bioreactor walls [2].

Serum-free media for mesenchymal stem cells expansion on microcarriers

Background Expansion of mesenchymal stromal cells (MSC) is one of the key steps for their use in tissue engineering or cell therapies. To increase cells expansion yields, two milestones have to be achieved that will allow a wider MSC therapeutic use, namely an optimal serum-free medium and a process intensification via 3D suspension culture on microcarriers [1]. Indeed, one of the major obstacles to obtain a reliable manufacturing process is that most of MSC cultivation methods still rely on media being supplemented by a significant volume of fetal calf serum. While efforts have been made to develop serum-free media (SFM) for MSC expansion, they were systematically designed for planar plastic cultivation systems. The aim of this study was to compare usual serum alternatives on 2D and 3D cultures. However, these alternatives can be ill-defined, either when the medium formulation is proprietary or when it has been humanized with blood derivatives. Thus several supplements were also investigated to design a more defined serum-free formulation that could be used to expand stem cells on microcarriers.

Adhesion and colonization of mesenchymal stem cells on polylactide or PLCL fibers dedicated for tissue engineering

Background Tissue engineering covers a broad range of applications dedicated to the repair or the replacement of part or whole tissue such as blood vessels, bones, cartilages, ligaments, etc [1]. Practically, a bio substitute, made with cells cultivated on scaffold, is needed. Mesenchymal stem cells (MSC) are generally the most suitable cells for such application since they are self-renewable with a great potential for differentiation and immuno suppression [2]. However, materials used for bio functional scaffold synthesis have to meet several criteria, such as biocompatibility and biodegradability. Thus, the aim of the study was to screen several biopolymers differing in their composition for their capability to promote adhesion and growth of MSC.