Influence of shear force on ex vivo expansion of hematopoietic model cells in a stirred tank bioreactor.

To evaluate shear stress influence on ex vivo expansion of hematopoietic cell lineages for clinical application, in this study, human pro-monocytic cell (namely U937 cell line) was selected as a hematopoietic stem cell (HSC) model and cultured in suspension mode at two different agitation rates (50, 100 rpm) in the stirred bioreactor. At the agitation rate of 50 rpm, the cells achieved higher expansion folds (27.4 fold) with minimal morphological changes as well as apoptotic cell death, while at 100 rpm the expansion fold decreased after 5-day of culture in suspension culture in comparison with static culture and reached 24.5 fold at the end of the culture. The results of glucose consumption and lactate production were also in agreement with the data of fold expansion and indicated the preference of culture in the stirred bioreactor when agitated at 50 rpm. This study indicated the stirred bioreactor system with an agitation rate of 50 rpm and surface aeration may be used as a potential dynamic culture system for clinical applications of hematopoietic cell lineage. The current experiments shed data related to the effect of shear stress on human U937 cells, as a hematopoietic cell model, to set a protocol for expansion of HSCs for biomedical applications.

Effect of pH, Ionic Strength and Agitation Rate on Dissolution Behaviour of 3D-Printed Tablets, Tablets Prepared from Ground Hot-Melt Extruded Filaments and Physical Mixtures.

With the current focus on 3D-printing technologies, it is essential to understand the processes involved in such printing methods and approaches to minimize the variability in dissolution behaviour to achieve better quality control outcomes. For this purpose, two formulations of theophylline tablets were prepared using hydroxypropyl cellulose (HPC) and ethyl cellulose (EC). Among the two types of tablets, three different methods (physical mixture (PM), hot-melt extrusion (HME) and 3D-printing fused deposition modelling (FDM)) were applied and their dissolution behaviours were studied under various conditions using a biodissolution tester. This was carried out at pH values of 1.2, 2.2, 5.8, 6.8, 7.2 and 7.5, mimicking the medium in the gastrointestinal tract. Dissolution tests under two dipping rates (10 dpm and 20 dpm) and two ionic strengths (0.2 M and 0.4 M) were conducted to mimic fed and fasting conditions. The dissolution efficiency (DE%), release rate, similarity factor (f2) and difference factor (f1) were calculated. When comparing the DE%, the formulation containing EC showed less sensitivity to changes in the dipping rate and ionic strength compared to the HPC formulation. As for the manufacturing method, 3D-printing FDM could improve the robustness of the dissolution behaviour of both formulations to dipping rate changes. However, for ionic strength changes, the effect of the manufacturing method was dependent on the formulation composition. For example, the 3D-printed tablets of the HPC formulation were more sensitive to changes in ionic strength compared to the EC-containing formulation. The release mechanism also changed after the thermal process, where n values in the Korsmeyer-Peppas model were much higher in the printing and HME methods compared to the PM. Based on the formulation composition, the 3D-printing method could be a good candidate method for tablets with a robust dissolution behaviour in the GI tract. Compared to HPC polymers, using hydrophobic EC polymers in printable formulations can result in a more robust dissolution behaviour in fed and fasting states.

Mechanistic Models for USP2 Dissolution Apparatus, Including Fluid Hydrodynamics and Sedimentation.

Drug product dissolution is a key input to Physiologically Based Biopharmaceutics Models (PBBM) to be able to predict in vivo dissolution. The integration of product dissolution in PBBMs for immediate release drug products should be mechanistic, i.e. allow to capture the main determinants of the in vitro dissolution experiment, and extract product batch specific parameter(s). This work focussed on the Product Particle Size Distribution (P-PSD), which was previously shown to integrate the effect of dose, volume, solubility (pH), size and concentration of micelles in the calculation of a batch specific input to PBBMs, and proposed new hydrodynamic (HD) models, which integrate the effect of USP2 apparatus paddle rotation speed and medium viscosity on dissolution. In addition, new models are also proposed to estimate the quantitative impact of formulation and drug sedimentation or "coning" on dissolution. Model "HDC-1" predicts coning in the presence of formulation insoluble excipients and "HDC-2" predicts the sedimentation of the drug substance only. These models were parameterized and validated on 166 dissolution experiments and 18 different drugs. The validation showed that the HD model average fold errors (AFE) for dissolution rate prediction of immediate release formulations, is comprised between 0.85 and 1.15, and the absolute average fold errors (AAFE) are comprised between 1.08 and 1.28, which shows satisfactory predictive power. For experiments where coning was suspected, the HDC-1 model improved the precision of the prediction (defined as ratio of "AAFE-1"values) by 2.46 fold compared to HD model. The calculation of a P-PSD integrating the impact of USP2 paddle rotation, medium viscosity and coning, will improve the PBBM predictions, since these parameters could have an influence on in vitro dissolution, and could open the way to better prediction of the effect of prandial state on human exposure, by developing new in silico tools which could integrate variation of velocity profiles due to the chyme viscosity.

Yield and rheological properties of exopolysaccharide from a local isolate: Xanthomonas axonopodis pv. vesicatoria

Background: The aim of the present study was to evaluate gum productivity of a local strain, Xanthomonas axonopodis pv. vesicatoria, isolated from pepper plant, and its rheological behavior for the first time compared to the standard strain, Xanthomonas campestris DSM 19000 (NRRL B-1459). The influence of operational conditions (agitation rate and inoculum volume) on gum production and rheological properties of gums from the Xanthomonas strains were investigated. Results: The isolated strain of Xanthomonas showed similar xanthan yield compared to the standard strain. Furthermore, this study clearly confirmed that gum yield depended on bacterial strain, agitation rate, and inoculum size. The most suitable conditions for the gum production in an orbital shaker in terms of agitation rate and inoculum size were 180 rpm and 5%, respectively, resulting in an average production of 10.96 and 11.19 g/L for X. axonopodis pv.vesicatoria and X. campestris DSM 19000, respectively. Regarding the rheological properties, Ostwald-de-Waele and power law models were used to describe flow and oscillatory behavior of the gum solutions, respectively. Consistency of the novel gum solution remarkably was much higher than the commercial xanthan gum solution. Flow and oscillatory behavior and their temperature ramps showed that weak gel-like structure could be obtained with less gum concentrations when the novel gum was used. Conclusion: Therefore, yield and technological properties of the aqueous solutions of the exopolysaccharide synthesized by X. axonopodis pv. vesicatoria were observed to be more suitable for industrial production.

Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred-Suspension Culture.

Human induced pluripotent stem cells (hiPSCs) hold great hopes for application in regenerative medicine due to their inherent capacity to self-renew and differentiate into cells from the three embryonic germ layers. For clinical applications, a large quantity of hiPSCs produced in standardized and scalable culture processes is required. Several groups, including ours, have developed methodologies for scaled-up hiPSC production in stirred bioreactors in chemically defined medium. In this study, we optimized the critical steps and factors that affect hiPSC expansion and yield in stirred-suspension cultures, including inoculation conditions, seeding density, aggregate size, agitation rate, and cell passaging method. After multiple passages in stirred-suspension bioreactors, hiPSCs remained pluripotent, karyotypically normal, and capable of differentiating into all three germ layers.

Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc.

Agitation increases expansion of cord blood hematopoietic cells and promotes their differentiation into myeloid lineage.

Mechanical stress caused by agitation is one of the factors that can affect hematopoietic stem cell expansion in suspension bioreactors. Therefore, we have investigated the effects of agitation on umbilical cord blood hematopoietic stem cell (UCB-HSC) growth and differentiation. A comparison was made between various agitation rates (20, 40 and 60 rpm) in spinner-flask and cells cultured in glass petri dish as a static culture. Moreover, the fluid dynamic at various agitation rates of spinner-flask was analyzed to determine shear stress. The spinner-flask contained a rotational moving mixer with glass ball and was kept in tissue culture incubator. To reduce consumption of cytokines, UCB-serum was used which widely decreased the costs. Our results determined that, agitation rate at 40 rpm promoted UCB-HSCs expansion and their colony forming potential. Myeloid progenitors were the main type of cells at 40 rpm agitation rate. The results of glucose consumption and lactic acid production were in complete agreement with colony assay and expansion data and indicated the superiority of culture in spinner-flask when agitated at 40 rpm over to other agitation speeds and also static culture. Cell viability and colony count was affected by changing the agitation speed. We assume that changes in cell growth resulted from the effect of shear stress directly on cell viability, and indirectly on signaling pathways that influence the cells to differentiate.

Control of agitation and aeration rates in the production of surfactin in foam overflowing fed-batch culture with industrial fermentation.

Bacillus amyloliquefaciens fmb50 produces a high yield of surfactin, a lipopeptide-type biosurfactant that has been widely studied and has potential applications in many fields. A foam overflowing culture has been successfully used in the combined production-enrichment fermentation of surfactin. In this study, the agitation and aeration rates were found to have relationships with foam formation and surfactin enrichment. A maximum surfactin concentration of 4.7g/l of foam was obtained after 21h of culture with an agitation rate of 150rpm and an aeration rate of 1vvm in fed-batch culture. By controlling the foam overflow rate (fout) of a fed-batch culture, surfactin concentration in the foam was continuously maintained above 4g/l.

Control de las tasas de aireación y agitación en la producción de surfactina en un cultivo alimentado (fed-batch) en espuma desbordante con fermentación industrial / Control of agitation and aeration rates in the production of surfactin in foam overflowing fed-batch culture with industrial fermentation

Bacillus amyloliquefaciens fmb50 produces a high yield of surfactin, a lipopeptide-type biosurfactant that has been widely studied and has potential applications in many fields. A foam overflowing culture has been successfully used in the combined production-enrichment fermentation of surfactin. In this study, the agitation and aeration rates were found to have relationships with foam formation and surfactin enrichment. A maximum surfactin concentration of 4.7 g/l of foam was obtained after 21 h of culture with an agitation rate of 150 rpm and an aeration rate of 1 vvm in fed-batch culture. By controlling the foam overflow rate (f out) of a fed-batch culture, surfactin concentration in the foam was continuously maintained above 4 g/l.

Bacillus amyloliquefaciens fmb50 produce gran cantidad de surfactina, un biosurfactante de tipo lipopeptídico que ha sido objeto de estudios pormenorizados y tiene aplicaciones en muchos campos. El cultivo en espuma desbordante se ha utilizado con éxito en la fermentación combinada de producción-enriquecimiento de surfactina. En este estudio, se halló que las tasas de aireación y agitación tienen relación con la formación de espuma y el enriquecimiento de la surfactina. Se obtuvo una concentración máxima de surfactina de 4,7 g/l de espuma después de 21 h de cultivo con una tasa de agitación de 150 rpm y una tasa de aireación de 1 vvm en un cultivo alimentado (fed-batch). Al controlar la tasa de espuma desbordante (f out) de un cultivo fed-batch, la concentración de surfactina en la espuma se mantuvo continua por encima de 4 g/l.

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

Drug release from matrix tablets: physiological parameters and the effect of food.

INTRODUCTION: As dissolution plays an important and vital role in the drug-delivery process of oral solid dosage forms, it is, therefore, essential to critically evaluate the parameters that can affect this process. AREAS COVERED: The consumption of food as well as the physiological environment and properties of the gastrointestinal tract, such as its volume and composition of fluid, the fluid hydrodynamics, properties of the intestinal membrane, drug dose and solubility, pKa, diffusion coefficient, permeability and particle size, all affect drug dissolution and absorption rate. There are several dissolution approaches that have been developed to address the conditions as experienced in the in vivo environment, as the traditional dissolution being a quality control method is not biorelevant and as such do not always produce meaningful data. This review also describes the development of a systematic way that differentiates between robust and non-robust formulations by varying the effects of agitation and ionic strength through the use of the automated United States Pharmacopeia type III Bio-Dis apparatus. EXPERT OPINION: With the improved understanding of the physiological parameters that can affect the oral bioperformance of dosage forms, strides have, therefore, been made in making dissolution testing methods more biologically based with the view of obtaining more in vitro-in vivo correlations.

Oxygen limitation favors the production of protein with antimicrobial activity in Pseudoalteromonas sp

This study examined the effect of dissolved oxygen concentration on the production of biomass and metabolites with antimicrobial activity of Pseudoalteromonas sp cultured at 0, 150, 250, or 450 revolutions per minute (rev. min-1). Dissolved oxygen (D.O) was monitored during the fermentation process, biomass was quantified by dry weight, and antimicrobial activity was assessed using the disk diffusion method. The bacterium Pseudoalteromonas reached similar concentration of biomass under all experimental agitation conditions, whereas antimicrobial activity was detected at 0 and 150 rev. min-1 registering 0% and 12% of D.O respectively corresponding to microaerophilic conditions. Antibiotic activity was severely diminished when D.O was above 20% of saturation; this corresponded to 250 or 450 rev. min-1. SDS-PAGE electrophoresis revealed a protein with a molecular weight of approximately 80 kilodaltons (kDa) with antimicrobial activity. Pseudoalteromonas is capable of growing under oxic and microaerophilic conditions but the metabolites with antimicrobial activity are induced under microaerophilic conditions. The current opinion is that Pseudoalteromonas are aerobic organisms; we provide additional information on the amount of dissolved oxygen during the fermentation process and its effect on antimicrobial activity.

Oxygen limitation favors the production of protein with antimicrobial activity in Pseudoalteromonas sp.

This study examined the effect of dissolved oxygen concentration on the production of biomass and metabolites with antimicrobial activity of Pseudoalteromonas sp cultured at 0, 150, 250, or 450 revolutions per minute (rev. min(-1)). Dissolved oxygen (D.O) was monitored during the fermentation process, biomass was quantified by dry weight, and antimicrobial activity was assessed using the disk diffusion method. The bacterium Pseudoalteromonas reached similar concentration of biomass under all experimental agitation conditions, whereas antimicrobial activity was detected at 0 and 150 rev. min(-1) registering 0% and 12% of D.O respectively corresponding to microaerophilic conditions. Antibiotic activity was severely diminished when D.O was above 20% of saturation; this corresponded to 250 or 450 rev. min(-1). SDS-PAGE electrophoresis revealed a protein with a molecular weight of approximately 80 kilodaltons (kDa) with antimicrobial activity. Pseudoalteromonas is capable of growing under oxic and microaerophilic conditions but the metabolites with antimicrobial activity are induced under microaerophilic conditions. The current opinion is that Pseudoalteromonas are aerobic organisms; we provide additional information on the amount of dissolved oxygen during the fermentation process and its effect on antimicrobial activity.