Stirred Systems Research Articles

Expanding T-cells using stirred-tank and a novel biaxial rotary bioreactor

Background & Aim Expansion of T cells is a crucial step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant cells. However, it remains a major translational and commercial bottleneck due to the manual, small scale culturing systems used for their production. One of the main concerns is that primary T cells are shear sensitive and therefore do not grow as effectively in more scalable, agitated systems, such as stirred tank bioreactors, as compared with static conditions. However, the use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems. Methods, Results & Conclusion In our study, we explore a robust and practical platform for adoptive cell culture using stirred-tank bioreactor and novel biaxial rotary bioreactor for large-scale and high-quality cellular production. We have investigated various factors, such as bioreactor parameters, media, supplements and stimulation, with a suitable structure of scaling-up the bioreactors with robustness and reproducibility of the process. T cell quality was monitored using surface markers and intracellular cytokines as the critical quality assessment criteria in early, middle and late stages of cell production. Our approach addresses the fundamental scaling challenges in the manufacturing process.

Fibers and Particles in Stirred Systems: Influence on the Flow Velocities

AbstractDie Auslegung und Optimierung von Mischprozessen faseriger Suspensionen erfordert eine umfassende Quantifizierung vorhandener Zusammenhänge zwischen den Zustandsgrößen der Suspension, den zu wählenden Prozessgrößen und dem Mischergebnis. In experimentellen Untersuchungen zeigt sich, dass die faserigen Bestandteile die Strömungsgeschwindigkeiten wesentlich beeinflussen. Partikelgröße und ‐form nehmen Einfluss auf die Höhe der Geschwindigkeiten, so dass die Verwendung von vereinfachten, partikelfreien Modellstoffsystemen für die Nachbildung von Mischprozessen faseriger Fluide nicht oder nur bedingt geeignet ist.

Scale-up of mixing processes of highly concentrated suspensions using electrical resistance tomography

Qualification and quantification of mixing processes are crucial requirements for process engineering and energetic optimization in chemical and pharmaceutical industry as well as in wastewater treatment and biogas production. The analysis of mixing processes in stirred systems becomes a challenging task, especially when using opaque substrates. With Electrical Resistance Tomography (ERT), a powerful measuring technique is provided to allow a comprehensive and non-intrusive quantification of mixing processes of complex suspensions. Combined with advanced cross-correlation techniques, ERT offers the possibility to derive the axial flow velocity profile inside a stirred system. Investigations in different scales are an essential prerequisite regarding the evaluation and optimization of large-scale mixing processes under consideration of similarity laws. The experimental tests presented in this paper are carried out in reactor systems with volumes of 0.1m3 and 1m3. The validity of scale-up methodologies was ensured by comparable flow conditions and velocity distributions between the lab and pilot plant scale. For biogas plants, as an example of the importance of efficient mixing, the scale-up principles ‘geometric similarity’, ‘constant impeller tip speed’, ‘similar viscosity and flow characteristics’ as well as ‘scale-up of particles and fibers of the dispersed phase’ are proved to be valid by the Fraunhofer Institute for Ceramic Technologies and Systems (IKTS). Within the scope of further investigations, reliable information related with Computational Fluid Dynamics (CFD) are ought to be derived for the continuing evaluation of mixing processes at any scale to establish a foundation for the dimensioning and operation of stirring systems, especially for highly concentrated, non-Newtonian fluids.

Clinical-Grade Manufacturing of Therapeutic Human Mesenchymal Stem/Stromal Cells in Microcarrier-Based Culture Systems.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) has triggered the need for high cell doses in a vast number of clinical applications. This demand requires the development of good manufacturing practices (GMP)-compliant ex vivo expansion protocols that should be effective to deliver a robust and reproducible supply of clinical-grade cells in a safe and cost-effective manner. Controlled stirred-tank bioreactor systems under xenogeneic (xeno)-free culture conditions offer ideal settings to develop and optimize cell manufacturing to meet the standards and needs of human MSC for cellular therapies. Herein we describe two microcarrier-based stirred culture systems using spinner flasks and controlled stirred-tank bioreactors under xeno-free conditions for the efficient ex vivo expansion of human bone marrow and adipose tissue-derived MSC.

Robust Expansion of Human Pluripotent Stem Cells: Integration of Bioprocess Design With Transcriptomic and Metabolomic Characterization.

: Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well-characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct-4, Nanog, SSEA-4, and TRA1-60 and the ability to spontaneously differentiate into the three germ layers. Whole-genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred-tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low-oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect-like phenotype, with no evidence of hypoxic stress response, in contrast to two-dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the "omics" tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred-tank bioreactors, which can contribute to improved scale-up production systems. The clinical application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust protocols able to sustain production of high cell numbers, as required for regenerative medicine. In this study, a strategy was developed for the expansion of human embryonic stem cells in well-defined culture conditions using microcarrier technology and stirred-tank bioreactors. The use of transcriptomic and metabolic tools allowed detailed characterization of the cell-based product and showed a phenotypic convergence between both hESC lines along the expansion process. This study provided valuable insights into the metabolic hallmarks of hPSC expansion and new information to guide bioprocess design and media optimization for the production of cells with higher quantity and improved quality, which are requisite for translation to the clinic.

Filtration methodologies for the clarification and concentration of human mesenchymal stem cells

Currently human mesenchymal stem cells (hMSC) are expanded using microcarrier-based stirred culture systems from one to hundreds of liters of culture volume to guarantee the required cell numbers to be delivered to the clinic. Such culture volumes need to be clarified, ensuring efficient removal of microcarriers, and concentrated without compromising the cells׳ characteristics. The aim of this work was to evaluate the applicability of filtration methodologies, as dead end filtration and tangential flow filtration, for the clarification and concentration of hMSC, respectively.Different process variables and their impact on hMSC quality were evaluated, showing that polypropylene filters with pore sizes higher than 75μm can ensure the removal of microcarriers from the cell suspension bulk, without compromising cells׳ recovery or viability. Furthermore, hMSC could be successfully concentrated up to a factor of ten while maintaining their identity, potency and high cell viability, allowing for the recovery of over 80% of viable cells; an initial cell concentration higher than 2×105 cell/mL, and polysulfone membranes with pore sizes higher than 0.45μm were identified to be key conditions to obtain such concentration factors; shear rate and permeate flux were also shown to impact the cells׳ recovery yields, viability and quality.

Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints

There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers’ expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity.

Modeling human neural functionality in vitro: three-dimensional culture for dopaminergic differentiation.

Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca(2+) transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons (Vm=-70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.

Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.

The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations.

Designing clinical-grade integrated strategies for the downstream processing of human mesenchymal stem cells

Background During the past decade, human stem cells have been the focus of an increased interest due to their potential in clinical applications, as a therapeutic alternative for several diseases. Within this context, human mesenchymal stem cells (hMSCs) have gained special attention due to their immune-modulatory characteristics, as well as in secreting bioactive molecules with anti-inflammatory and regenerative features [1]. In order to face the high demands of hMSCs (from 10 to 10 cells per patient) [2] to be used in therapies, the establishment of robust manufacturing platforms that can ensure the efficient production, purification and formulation of stem cell-based products is still a challenge. Although substantial efforts have been performed on the development of clinical-grade bioprocesses for the expansion of hMSCs in microcarrier-based stirred culture systems, the incorporation of downstream strategies that assure efficient cell-bead separation and consequent hMSC concentration (i.e. volume reduction) and washing is required to deliver safe hMSCs to the clinic [3,4]. Therefore, the main aim of this work was the design of integrated methodologies (filtration and membrane technology approaches) [5] for the robust and clinicalgrade downstream processing of hMSC.

Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O2 concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O2 solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O2 concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34+ cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O2 for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34+ cells at 5% O2, using 21%-O2 cultures as a control. In hypoxia we found that CD34+ cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level.

Flotation in a novel oscillatory baffled column

This paper presents an evaluation of an oscillatory baffled column (OBC) as a novel flotation device. The cell is based on a standard column design but employs a novel agitation mechanism where a series of baffle plates are oscillated sinusoidally through the fluid. This type of agitation has been shown to produce a more evenly distributed shear rate in the cell and allows the effect of agitation on particle–bubble contacting to be decoupled from gas dispersion effects. The column was first characterised in terms of mixing and gas dispersion, before being flotation tested using a quartz-amine system. Results indicated that the OBC was able to improve the flotation rate constant by up to 60% for fine particles (<30 μm) and by between 30% and 40% for coarser particles, relative to a standard flotation column. Interestingly, optimal flotation performance was obtained at power intensities orders of magnitude lower than those found in similar studies in stirred systems. This is believed to arise from the even distribution of shear in the OBC together with the oscillatory motion of the fluid in the cell. This oscillatory motion does not contribute to the average power intensity in the fluid and therefore results in more fluid motion per unit energy than would be obtained in a conventional stirred system. The OBC was therefore able to significantly improve flotation rates at power intensities orders of magnitude lower than those found in conventional cells.

Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors

Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies and for drug screening. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications. However, conventional techniques such as hanging drop or static suspension culture are either inherently incapable of large scale production or exhibit limited control over cell aggregation during EB formation and subsequent EB aggregation. For standardized mass EB production, a well defined scale-up platform is necessary. Recently, novel scenario methods of EB formation in hydrodynamic conditions created by bioreactor culture systems using stirred suspension systems (spinner flasks), rotating cell culture system and rotary orbital culture have allowed large-scale EB formation. Their use allows for continuous monitoring and control of the physical and chemical environment which is difficult to achieve by traditional methods. This review summarizes the current state of production of EBs derived from pluripotent cells in various culture systems. Furthermore, an overview of high quality EB formation strategies coupled with systems for in vitro differentiation into various cell types to be applied in cell replacement therapy is provided in this review. Recently, new insights in induced pluripotent stem (iPS) cell technology showed that differentiation and lineage commitment are not irreversible processes and this has opened new avenues in stem cell research. These cells are equivalent to ES cells in terms of both self-renewal and differentiation capacity. Hence, culture systems for expansion and differentiation of iPS cells can also apply methodologies developed with ES cells, although direct evidence of their use for iPS cells is still limited.

Hematopoietic reconstitution of CD34+ cells grown in static and stirred culture systems in NOD/SCID mice

The hematopoietic reconstitution of cord blood (CB) CD34(+)cells grown in static and stirred system was studied. Static cultures were better than stirred cultures for cell expansion. Engraftment of stirred-culture hematopoietic stem cells (HSCs) was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs.

Sonochemical synthesis of polyaniline nanofibers

Conventionally, micro-sized irregular polyaniline (PANI) particles were synthesized by dropwise addition of the ammonium persulfate (APS) solution into the aniline (ANI) solution with mechanical stirring. By replacing the mechanical stirring with an ultrasonic irradiation, PANI nanofibers in diameters of ∼50 nm and lengths of 200 nm to several micrometers were prepared. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that at the early stage of polymerization, the polymers formed in both the mechanical stirred and ultrasonicated systems are in the form of nanofiber. However, with continuing of the reaction, these primary nanofibers grow and agglomerate into irregular shaped PANI particles in the mechanical stirred system, while in the case of the ultrasonic irradiation, the growth and agglomeration are effectively prevented, preserving thus the PANI nanofibers in the final product. By increasing the APS/ANI molar ratio from 0.5 to 2.5, the aspect ratios of the PANI nanofibers decreased. The PANI nanofibers exhibit higher solubility than the irregular shaped PANI particles. Although the yield, as well as the conductivity of the ultrasonic synthesized PANI nanofibers, was slightly lower than the irregular shaped PANI particles, the ultrasonic synthesis approach is one of the facile and scalable approaches in synthesizing PANI nanofibers in comparison with other ones without use of templates (e.g., the interfacial polymerization and rapid mixing polymerization). UV–Vis and Fourier transformed infrared (FTIR) spectra indicated ultrasound had no significant effect on the chemical structure of the PANI.

Erratum: A comparative gene-expression analysis of CD34+ hematopoietic stem and progenitor cells grown in static and stirred culture systems.

[This corrects the article DOI: 10.2478/s11658-006-0039-x.].

A comparative gene-expression analysis of CD34+ hematopoietic stem and progenitor cells grown in static and stirred culture systems

Static and stirred culture systems are widely used to expand hematopoietic cells, but differential culture performances are observed between these systems. We hypothesize that these differential culture outcomes are caused by the physiological responses of CD34+ hematopoietic stem and progenitor cells (HSPCs) to the different physical microenvironments created in these culture devices. To understand the genetic changes provoked by culture microenvironments, the gene expression profiling of CD34+ HSPCs grown in static and stirred culture systems was compared using SMART-PCR and cDNA arrays. The results revealed that 103 and 99 genes were significantly expressed in CD34+ cells from static and stirred systems, respectively. Of those, 91 have similar levels of expression, while 12 show differential transcription levels. These differentially expressed genes are mainly involved in anti-oxidation, DNA repair, apoptosis, and chemotactic activity. A quantitative molecular understanding of the influences of growth microenvironments on transcriptional events in CD34+ HSPCs should give new insights into optimizing culture strategies to produce hematopoietic cells.

Modelling corn starch swelling in batch systems: effect of sucrose and hydrocolloids

Hydrocolloids are used in starch-based products to improve stability and to obtain specific textural characteristics. In stirred batch systems with constant jacket temperature, xanthan gum, guar gum and sodium alginate (1% w/w) were added to corn starch (10% w/w) and sucrose (15% w/w) in aqueous systems to test their effects on granule swelling, starch gelatinization temperatures and viscoelastic behavior of the hot paste. As heating is not instantaneous, time–temperature relationships cannot be avoided. In this regard, heat penetration was faster in viscoelastic systems with lower complex dynamic modulus ( G *) values, while pastes containing gums showed lower heat transfer rates during gelatinization. Microscopy and image analysis were applied to describe the granule swelling process in starch gelatinization. Starch granule swelling was considerably reduced by the presence of gums due to the lower heating rates and the decreased mobility of water molecules. The presence of sucrose increased starch gelatinization temperatures, measured by differential scanning calorimetry; gum addition did not show a significant influence on these temperatures. The effect of time and temperature on the starch swelling process was modelled considering, as driving force, the difference between the instantaneous mean curvature of the granule (reciprocal of the diameter) and the asymptotic curvature. Gelatinization rate constants were calculated for the different pastes and the lowest values were observed for starch pastes added with gums. Activation energies ranged from 80 (±14) to 119 (±3) kJ mol −1, being comparable to values reported in the literature for starch gelatinization measured by different methods.

Culture of human T cells in stirred bioreactors for cellular immunotherapy applications: shear, proliferation, and the IL-2 receptor.

Ex vivo expansion of T cells is a key step of many cellular immunotherapy protocols, which require large numbers of immune cells to eradicate malignant or virally infected cells. The use of stirred culture systems for T cell expansion offers many potential advantages over the static culture systems commonly used today, including homogeneity of culture conditions, ease of sampling, and implementation of control systems. Primary human T cells as well as the transformed TALL103/2 T cell line were cultured in 100-mL spinner flasks as well as 2-L bioreactors to investigate the effects of shear forces produced by agitation and sparging-based aeration on the expansion of T cells. Primary T cells could be successfully grown at agitation rates of up to 120 rpm in the spinner flasks and to 180 rpm in the bioreactors with no immediate detrimental effects on proliferation. Exposure to agitation and sparging did, however, cause a significantly increased rate of downregulation of the interleukin-2 receptor (IL-2R), resulting in lower overall expansion potential from a single stimulation as compared to static controls, with faster IL-2R downregulation occurring at higher agitation rates. For the primary T cells, no significant effects of agitation were found on expression levels of other key surface receptors (CD3, CD28, or CD62L) examined. No significant effects of agitation were observed on primary T cell metabolism or levels of cellular apoptosis in the cultures. The TALL103/2 T cell line was found to be extremely sensitive to agitation, showing severely reduced growth at speeds above 30 rpm in 100-mL spinner flasks. This unexpected increased fragility in the transformed T cell line as compared to primary T cells points out the importance of carefully selecting a model cell line which will accurately represent the characteristics of the cell system of interest.