When agitating mesenchymal stem cells adhered on microcarriers in bioreactors, a compromise has to be found between sufficient particle suspension and limitation of hydromechanical stresses. The ...present study proposes a strategy to improve the design of an ‘elephant ear' impeller at the just‐suspended state by varying its relative size, blade slope angle, and position in the reactor. To do that, computational fluid dynamics simulations were coupled with multi‐objective optimization to minimize the hydromechanical stress encountered by the microcarriers. Two minimization criteria were considered: (P/V)@p and the energy dissipation function EDC. On the basis of 31 conditions, an optimal impeller geometry is proposed.
A computational fluid dynamics‐based optimization strategy was developed to improve the design of an impeller for stem cell culture on microcarriers. Based on local integration of the turbulent energy dissipation rate within the bioreactor, two criteria were defined to minimize the hydromechanical stress encountered by the particles.
This study investigates the development of a scale-down approach for anaerobic digesters. Geometric similarity, flow regime, dimensionless cavern size and power consumption per unit volume (P/V) were ...identified as scaling criteria to design the pilot digester. At this pilot scale, a Computational Fluid Dynamics (CFD) model was validated using Particle Image Velocimetry (PIV) measurements and extended to the industrial anaerobic digester to describe its hydrodynamics and evaluate the robustness of the scaling criteria. The pilot scale CFD simulations were able to capture the main features of the flow, and model P/V and cavern diameter (Dc) with an average error of 4% and 3%, respectively. The comparison between the pilot and the industrial scale showed a deviation of 9% for P/V and about 5% for Dc and similar distributions of the dimensionless velocities inside the cavern, validating the scaling approach.
•A scaled down approach to viscoplastic flows in anaerobic digesters was developed.•The numerical model was validated at pilot scale by Particle Image Velocimetry data.•The model was successfully applied to industrial scale to describe its hydrodynamics.•Constant Re, P/V and dimensionless cavern diameter were found to be robust criteria.
•Zwietering’s model is revisited in the case of microcarrier suspension.•Njs was better predicted using dimensional analysis proposed model.•CFD simulations of microcarrier Njs were ...validated.•Dimensionless number of particles should be preferred to dp/ D ratio.•Impact of particle concentration on Njs was dependent on flow structures.
Large-scale mesenchymal stem/stromal cells culture uses 3D culture systems involving spherical solid particles, called microcarriers. Cells adhere on these spheres, which are then set in suspension in stirred tank bioreactors. This work was more particularly focused on the determination of the critical impeller agitation rate Njs, allowing complete beads suspension. It is indeed generally assumed that this value is a good compromise between sufficient nutrients homogenization, mass transfer and minimization of hydromechanical stress encountered by the cells. However, no robust correlation predicting Njs in the case of microcarriers can be found in literature. To fill this lack, a set of various operating conditions was carried out, dealing with geometrical variables and two different microcarriers, and Njs were experimentally determined for 140 conditions. An empirical correlation was established and a dimensional analysis was performed, showing that the impact of the particle concentration on Njs was function of the impeller design. Moreover, two dimensionless numbers characterizing the number of particle and an Archimede number applied on the particle cloud were found to better describe the impact of particle diameter and density on Njs. Simultaneously, a strategy based on Computational Fluid Dynamics simulations was conducted in order to predict Njs and was validated with the Njs experimental values.
Les cellules souches mésenchymateuses (CSM) interviennent de plus en plus dans le domaine de la médecine régénérative, notamment pour traiter des maladies aujourd’hui difficilement curables avec les ...moyens actuels. Deux verrous scientifiques limitent pourtant leur utilisation et leur commercialisation. D’une part, de grandes quantités de cellules sont nécessaires pour répondre à la forte demande médicale. D’autre part, les cellules étant elles-mêmes le médicament final, délivré chez le patient, leur qualité doit être préservée (phénotype souche, capacité de différenciation). La mise en culture de ces cellules, sur des microporteurs, en bioréacteur agité, semble répondre à ces enjeux. Cependant, une connaissance plus précise de l’impact, sur la réponse physiologique des cellules, des technologies utilisées et de l’hydrodynamique générée est nécessaire pour améliorer les lois d’extrapolation des bioréacteurs de culture de CSM. Dans ce contexte, des travaux ont été mis en œuvre pour étudier l’influence du mode d’agitation (orbital ou mécanique) sur l’attachement, l’expansion et le détachement de CSM issues de la gelée de Wharton (GW-CSM) de cordons ombilicaux, sur des microporteurs de différentes compositions. Pour contribuer à la quantification de l’expansion cellulaire, une méthode de comptage automatique in situ a été développée pour estimer le nombre de cellules par microporteur, ainsi que leur répartition, sans avoir à procéder à leur détachement. Des microporteurs commerciaux ont ensuite pu être comparés à des microporteurs synthétisés dans un laboratoire partenaire, en termes d’attachement et expansion cellulaire, ainsi que de facilité de détachement. En parallèle de ces travaux, l’impact de la conception du mobile d’agitation, en bioréacteur mécaniquement agité, sur la mise en suspension de microporteurs a été analysé. A l’issue de cette étude, une analyse dimensionnelle et des simulations CFD ont été mises en place et deux modèles reliant la fréquence minimale de juste mise en suspension (Njs) avec la géométrie du mobile d’agitation (forme, taille, position dans la cuve) et les propriétés matérielles des particules et de la phase liquide ont été proposés. Une stratégie d’optimisation des paramètres géométriques d’un mobile en minibioréacteur, dédié à la culture de CSM sur microporteurs, a été mise en place, à partir de paramètres caractérisant les contraintes hydromécaniques perçues par la phase solide, judicieusement choisis et intégrés lors des simulations CFD. Selon un plan d’expérience, et les résultats extraits des simulations, des surfaces de réponse ont été construites et une optimisation multi-objective a été réalisée afin de déterminer la géométrie minimisant les contraintes perçues par les particules, et donc par les cellules adhérées. Des cultures de GW-CSM en minibioréacteurs équipés de différents mobiles ont finalement été validées, avec une comparaison préliminaire de l’impact de ces géométries sur l’expansion cellulaire
Mesenchymal stem cells (MSC) are becoming increasingly involved in the regenerative medicine field, particularly to treat diseases that are not effectively curable with the current therapies. Two scientific barriers are nevertheless responsible for MSC use and commercialization limitations. On one side, large amounts of cells are needed to reach the high cell dose requirements. On the other side, cells being the final product themselves, directly injected into the patient, their quality have to be controlled (stem cell phenotype, differentiation capability). MSC cultivation on microcarriers in a stirred bioreactor seems to meet these challenges. However, a precise knowledge about the impact of the technologies and the hydrodynamics generated, on the physiological cell response, is necessary to improve the scale-up of MSC cultures in bioreactors. In this context, present work is dedicated to the study of the impact of the agitation mode (orbital or mechanical) on the cell attachment, expansion and detachment on various microcarrier types, in the case of MSC derived from the Wharton’s jelly (WJ-MSC) of umbilical cords. To quantify more precisely cell distribution and expansion on microcarriers, an automatic and in situ counting method was developed, which need no detachment step. This allowed the identification of commercial microcarriers suitable for WJ-MSC cultures, which were then compared to home-made microcarriers, synthesized by a partner laboratory, in terms of cell attachment and expansion, and detachment efficiency. In parallel to these works, the impact of the impeller design on the microcarrier suspension in stirred tank bioreactors was investigated. Based on a dimensional analysis and CFD simulations, it resulted in the establishment of two models relating the minimal agitation rate to ensure all particle suspension (Njs) with the impeller geometrical characteristics (design, size, off-bottom clearance) and the material properties of both the solid and the liquid phases. CFD models validation allowed then to develop a strategy to optimize the geometrical configuration of an impeller, dedicated to MSC cultures on microcarriers in a minibioreactor. Parameters characterizing the hydromechanical stress encountered by the solid phase were wisely chosen and integrated into CFD simulations. Based on a design of experiments, and the hydrodynamics data recovered from simulations, response surfaces were built and a multiobjective optimization was achieved in order to determine the geometry minimizing the particle stress, and also by adhered cells. WJ-MSC cultures in minibioreactors equipped with impellers displaying various geometries were finally validated, with a preliminary comparison of the impact of these geometries on the cell expansion
The objectives of this study were to quantify lymphocytes and eosinophils in the mucosa of the duodenum and rectum in asthmatic horses.
8 healthy and 10 asthmatic horses.
Asthmatic horses were ...evaluated in a symptomatic (after 6 weeks of exposure to moldy hay) and asymptomatic status (3 and 7 months after being fed alfalfa pellets n = 4 or treated with inhaled fluticasone 6). Duodenal and rectal biopsies were endoscopically (n = 4 to 6) taken in each horse. Eosinophils were counted on slides stained with hematoxylin, eosin, phloxine, and saffron, and immunohistochemistry was used to evaluate T and B lymphocytes using CD3 and CD20, respectively.
The duodenal and rectal epithelium of asthmatic and control horses contained exclusively T lymphocytes (CD3). Symptomatic asthmatic horses, compared to controls, had a significantly higher number of T lymphocytes (CD3) in the duodenal epithelium (P = .016) and the adjacent lamina propria of the villi (P = .04). Compared to symptomatic asthmatic horses, the fluticasone-treated group had significantly fewer T lymphocytes in the total lamina propria of the rectal mucosa (P < .01).
Taken together, these results suggest that asthmatic horses have greater infiltration of T lymphocytes in the duodenal and rectal mucosa, indicating a certain degree of inflammation, which could be due to a systemic inflammatory effect and/or a local effect of ingested hay allergens in asthmatic horses. Systemic markers of inflammation have not been investigated to better qualify if the infiltration noted is due to a local and/or systemic effect.
•Experimental and numerical analysis of microcarrier suspension in STR.•Cytodex-1 microcarriers with dP = 162.2 μm and ρS = 1020 kg m−3 at 10%v.•Solid spatial distributions are obtained from light ...attenuation measurements.•Analysis of spatial distribution enables the determination of Njs.•CFD simulation predicts Njs but not the solid spatial distributions.
Solid-liquid suspensions in stirred tank reactors are common operations in many processes, including bioprocesses such as animal or stem cell cultures. These cells are often anchorage-dependent, i.e. they need to adhere to a surface to grow. Typically, they are cultivated on the surface of small spherical microbeads, the so-called microcarriers, suspended in stirred-tank bioreactors.
As far as we know, no extensive experimental characterization, and thus no validated simulation approach, of microcarrier suspensions in stirred-tank reactor exists in the literature. Therefore, the first aim of this work is to develop an experimental technique based on light attenuation to characterize the spatial distribution of particle concentration for various particle suspension states. The second aim is to determine the validity of Euler-Euler CFD simulations to predict the spatial distribution of low density particles, such as microcarriers, in a stirred tank bioreactor.
Experiments and simulations were performed in a small hemispherical bottom bioreactor stirred with a down-pumping axial impeller. The particles used were Cytodex-1 microcarriers (dP=162μm and ρS=1020 kg m−3) at a solid concentration of 10% in volume.
The light attenuation technique enabled the characterization of the spatial distribution of the solid phase in the whole bioreactor, even for very dense suspensions by advantageously using the optical properties of the Cytodex-1 microcarriers. The analysis of the solid spatial distribution shows that the bioreactor volume can be divided in three distinct zones: a clear layer below the free surface where αS,layer tends to 0, a bulk zone where the solid phase is homogeneously distributed (αS,bulk⩽αS) and a packed bed of motionless particles at the bottom (αS,bed=αS,max). One very interesting finding is that the evolution of the bulk solid concentration is directly proportional to the agitation rate and its value equals the averaged volume fraction αS,bulk=αS at N=Njs.
Concerning the simulation results, the modeling approach presented in this work enables a reasonable estimation of the just-suspended agitation rate Njs. Nevertheless, the solid spatial distributions are not well predicted for agitation rates below or above Njs. The solid concentration at the vessel bottom is underestimated for N<Njs but overestimated for N⩾Njs.
The present study proposed to compare the impact of agitation mode (static, orbital, and mechanical) on the culture of mesenchymal stem cells extracted from the Wharton's jelly of umbilical cords ...(WJ‐MSC), in a clinical grade culture medium, using human platelet lysate and different xeno‐free microcarriers. Attachment, expansion, and detachment performances were characterized by a new dedicated tool of microscopic image posttreatment, allowing an in situ cell counting without detachment step. Results showed that performances in static mode were not necessarily representative of those obtained in dynamic mode. Moreover, impacts on nutrient consumptions and metabolite productions were identified, such as a higher glutamine consumption when Cytodex‐1 microcarriers were used. The detachment strategy used was relatively efficient for Star‐Plus, Plastic‐Plus, and Hillex II, but not sufficient for Cytodex‐1. Despite Cytodex‐1 presented promising attachment and expansion performances, Star‐Plus and Plastic‐Plus showed a better compromise, respectively, for the orbital and the mechanical agitation modes.
The performance of bioleaching stirred tank reactors (STR) is related to the homogeneity of biomass, substrates and dissolved gases. This work was focused on the characterization of the impeller ...design on bioreactor hydrodynamics and, more specifically on power, mixing efficiency and particle stress. Few studies addressed the issue of the impact of the impeller design on these, especially for multi-stage bioreactors which are the most commonly used at the industrial scale. To fill this lack, a two-stage solid-liquid computational fluid dynamics (CFD) model was simulated on more than 50 conditions to assess power consumption, dissipated power, suspension quality and particle stress. A dual impeller configuration was chosen using Rushton turbines, R600, Hydrofoil, Elephant Ear and HTPG impellers. Grinded pyrite-rich materials (average particles size: 80 μm) were considered as the solid phase at 3 different solid concentrations (10, 18 and 26% w/w). Considering the impeller power number (Np), the configuration with an axial impeller consumed less energy than a radial impeller in concordance with literature data. The results show that the impeller design had few to no effect on mixing efficiency considering a given power dissipation per unit volume. Independently on the impeller used, unique relationships were found between particle stress and mixing efficiency. This study gives new insights for reactor design and scaling of bioleaching stirred tank reactor and more specifically on the reduction of shear stress for the attached bacterial communities.
•50 CFD simulations were applied in various designs of bioleaching reactors.•Suspension quality was independent of the reactor design at a given power dissipation.•Particle stress was independent of the reactor design at a given suspension quality.•Coupling between solid loading, particle mixing and particle stress was determined.
Mesenchymal stem cell (MSC) products are promising therapeutic candidates to treat a wide range of pathologies. The successful commercialization of these cell therapies will, however, depend on the ...development of reproducible cell production processes. For this, using microcarriers as growth supports within controlled conditions may be a viable process option. Although increasing microcarrier concentration may be associated with greater productivity due to the increased available culture surface, additional friction or shocks between microcarriers are likely to lead to undesired cell death. However, data detailing the impact of microcarrier collisions on MSC growth remains scarce. The following work demonstrates that MSC growth on microcarriers is greatly influenced by particle concentration even when little impact is observed on the apparent growth rate. It is suggested that the apparent growth rate may result in an equilibrium between growth and death kinetics which are independently affected by particle concentration and that certain MSC quality attributes may be progressively degraded in parallel. In addition, the theoretical reduction of the MSC growth rate was modeled according to the ratio between the average interparticle distance and the Kolmogorov scale. This study is an original contribution toward understanding the hydrodynamic effects in microcarrier‐based stem cell cultures.
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