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Dirk Weuster-botz - One of the best experts on this subject based on the ideXlab platform.
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Comparative evaluation of Aspergillus niger strains for endogenous pectin-depolymerization capacity and suitability for d-galacturonic acid production
Bioprocess and Biosystems Engineering, 2020Co-Authors: Dominik Schäfer, Dirk Weuster-botz, Kevin Schmitz, J. Philipp BenzAbstract:Pectinaceous agricultural residues rich in d -galacturonic acid ( d -GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in Stirred-Tank Bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, d -GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of d -GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.
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Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses.
Microbial cell factories, 2020Co-Authors: Anna-lena Heins, Jan Reyelt, Marlen Schmidt, Harald Kranz, Dirk Weuster-botzAbstract:Background Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. Results Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated Stirred-Tank Bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in Stirred-Tank Bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. Conclusions Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.
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Two Stirred-Tank Bioreactors in series enable continuous production of alcohols from carbon monoxide with Clostridium carboxidivorans
Bioprocess and Biosystems Engineering, 2018Co-Authors: Kathrin Doll, Anton Rückel, Peter Kämpf, Maximilian Wende, Dirk Weuster-botzAbstract:Microbial batch production of alcohols by fermentation of CO-rich gases with Clostridia is limited by low volumetric productivities due to the need for formation of organic acids first (acidogenic phase) followed by re-consumption of the acids to form alcohols (solventogenic phase). Continuous autotrophic production of alcohols was made possible with C. carboxidivorans by use of two continuously operated Stirred-Tank Bioreactors in series without cell retention. The pH in the first reactor was controlled to pH 6.0 for continuous growth of the cells. Steady-state concentrations of 3.0 g L^−1 acetate and 0.1 g L^−1 butyrate were measured at a mean hydraulic residence time of 8.3 h. The pH in the second reactor was controlled to pH 5.0 for enhancing continuous formation of alcohols resulting in steady-state concentrations of 6.1 g L^−1 ethanol, 0.7 g L^−1 butanol, and 0.1 g L^−1 hexanol at a mean hydraulic residence time of 12.5 h. Continuous formation of alcohols from CO was already observed in the first Stirred-Tank reactor parallel to the formation of acids, whereas re-consumption of acids as well as de-novo syntheses of alcohols from CO was shown in the second Stirred-Tank reactor. Thus, high final alcohol-to-acid ratios of 3.9 g_ethanol g_acetate^−1 and 4.4 g_butanol g_butyrate^−1 were achieved in the continuous syngas-fermentation process with C. carboxidivorans .
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High-cell-density cultivation and recombinant protein production with Komagataella pastoris in Stirred-Tank Bioreactors from milliliter to cubic meter scale
Process Biochemistry, 2016Co-Authors: Andreas Schmideder, Samantha Hensler, Marina Lang, Ansgar Stratmann, Ulrich Giesecke, Dirk Weuster-botzAbstract:Abstract High-cell-density cultivations (HCDC) with methylotrophic yeasts need continuous methanol feeding at controlled process conditions. However, none of the miniaturized bioreactor systems in use for screening studies facilitates continuous feeding. Hence, continuous feeding of methanol in pH-controlled parallel Stirred-Tank Bioreactors on a milliliter scale was ensured by supplying air with varying methanol concentrations in the gas phase. The gas–liquid methanol transfer rates were characterized as a function of the operating conditions. Methanol feeding rates of up to 5.3 g L−1 h−1 were possible without oxygen limitation at the chosen process conditions. HCDC processes with 50–60 g L−1 cell dry weight within 49–69 h were established on the milliliter scale with extracellular production of Candida antarctica lipase B2 variant (CaL-B2) as an example (16,604 U L−1). The scalability was demonstrated by transferring the Komagataella pastoris HCDC processes to the liter and cubic meter scale with the maximum oxygen transfer rate as the scale-up criterion. Biomass concentrations and lipase activities were the same at all scales within the estimation error. Thus, parallel-operated Stirred-Tank Bioreactors on a milliliter scale with continuous methanol feeding via the gas phase will reduce the time and cost of screening methylotrophic yeast strains and cultivation conditions.
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A novel milliliter-scale chemostat system for parallel cultivation of microorganisms in Stirred-Tank Bioreactors.
Journal of biotechnology, 2015Co-Authors: Andreas Schmideder, Timm Steffen Severin, Johannes Heinrich Cremer, Dirk Weuster-botzAbstract:A pH-controlled parallel Stirred-Tank bioreactor system was modified for parallel continuous cultivation on a 10 mL-scale by connecting multichannel peristaltic pumps for feeding and medium removal with micro-pipes (250 μm inner diameter). Parallel chemostat processes with Escherichia coli as an example showed high reproducibility with regard to culture volume and flow rates as well as dry cell weight, dissolved oxygen concentration and pH control at steady states (n=8, coefficient of variation
Dirk Weusterbotz - One of the best experts on this subject based on the ideXlab platform.
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contact free infrared od measurement for online monitoring of parallel stirred tank Bioreactors up to high cell densities
Biochemical Engineering Journal, 2020Co-Authors: Philipp Enne, Samantha Effenberge, Lukas Franzgrote, Tanja Kurzrockwolf, Kai Kress, Dirk WeusterbotzAbstract:Abstract Miniaturized Stirred-Tank bioreactor systems provide a scalable platform for high-throughput bioprocess development. Online measurement of process variables is a major demand to enable efficient process monitoring and control in parallel operated Bioreactors. One miniaturized laser light source and two photodiodes were placed around a cylindrical disposable bioreactor made of polystyrene for individual and contact-free measurement of optical density (OD). One photodiode was positioned at an angle of 28° to the laser for measuring the scattered light, a second photodiode was positioned face to face with the laser for measuring the transmitted light. Miniaturized lasers with wavelengths of 650 nm (orange), 780 nm (NIR) and 850 nm (IR) were evaluated. The best results were achieved with a laser emitting at 850 nm. Both signals (scattered and transmitted light) were influenced by the stirrer speed (usually constant) and the microorganisms under study. After individual calibration, online OD monitoring of pH-controlled fed-batch processes was successfully shown with Escherichia coli, Corynebacterium glutamicum, and Trichosporon oleaginosus. Online OD measurements based on the transmitted light signals showed low standard deviation at low cell densities, whereas scattered light signals were more accurate at higher cell densities. Correlations were reliable up to cell dry weight concentrations of 46 g L−1 in Stirred-Tank Bioreactors on a milliliter scale.
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comparative reaction engineering studies for succinic acid production from sucrose by metabolically engineered escherichia coli in fed batch operated stirred tank Bioreactors
Biotechnology Journal, 2012Co-Authors: Torben Hoefel, Georg Faust, Liv Reinecke, Nicolas Rudinger, Dirk WeusterbotzAbstract:: This study presents a comparative reaction engineering analysis of metabolically engineered sucrose-utilizing Escherichia coli derived from E. coli K12 MG1655 for the anaerobic production of succinic acid. Production capacities of 16 different recombinant strains were evaluated in 48 parallel fed-batch-operated milliliter-scale stirred tank Bioreactors (10 mL) with continuous CO₂ sparging. The effects of recombinant sucrose-utilization systems (csc-operon or scr-operon), enhancements of anaplerotic reactions (pck, ppc, maeA, maeB or heterologous pyc) and gene deletions (ldhA, adhE, ack-pta and ptsG) were studied with respect to the overall process performances of the respective recombinant strains. Both sucrose-utilization systems enabled the production of succinic acid from sucrose in E. coli K12 MG1655. Maximum succinate production was observed by overexpressing the pyruvate carboxylase from Corynebacterium glutamicum resulting in a succinate concentration of 26.8 g L⁻¹ after 48 h and a cell-specific productivity of 0.14 g g⁻¹ h⁻¹. Further experiments in a fed-batch-operated laboratory-scale stirred tank bioreactor (2 L) showed that micro-aerobic conditions preceding the anaerobic phase enhance succinic acid production of E. coli K12 MG1655-derived strains. The work demonstrates the importance of parallel approaches within the scope of applied metabolic engineering studies.
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new milliliter scale stirred tank Bioreactors for the cultivation of mycelium forming microorganisms
Biotechnology and Bioengineering, 2010Co-Authors: Ralf Hortsch, Ansgar Stratmann, Dirk WeusterbotzAbstract:A novel milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter-scale. A newly designed one-sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface-to-volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k(L)a) > 0.15 s(-1) were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter-scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory-scale stirred tank bioreactor with six-bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter-scale stirred tank bioreactor was reduced compared to the laboratory-scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale-up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter-scale (standard deviation < 8%) with up to 48 stirred tank Bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear-thinning non-Newtonian behavior. The newly developed one-sided paddle impellers operated in unbaffled reactors on a 10 milliliter-scale with a magnetic inductive drive for up to 48 parallel Bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically.
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automation of disposable stirred tank Bioreactors
Chemie Ingenieur Technik, 2006Co-Authors: Andreas Kusterer, Klaus Kaufmann, Christian Krause, Matthias Arnold, Dirk WeusterbotzAbstract:dulform der Membranfestbettreaktoren ist in der weiteren Prozessentwicklung ein einfaches Scale-up m glich. Aufgrund der gew hlten Immobilisierungsbedingungen und der verwendeten Membranen werden die Enzyme reversibel gebunden. Bei einem Verlust der Enzymaktivit t oder bei einem gew nschten Wechsel der Enzymaktivit t, kann das bereits immobilisierte Enzym schnell und vollst ndig von der Membran eluiert werden. Nach der Reaktivierung der Membran kann die erneute Enzymbindung erfolgen. Die eingesetzten Membranen zeigen sich f r nachfolgende Immobilisierungen gleiche Bindungskapazit ten. Das vorgestellte Verfahren erm glicht eine zeitsparende Prozessentwicklung.
Ralf Hortsch - One of the best experts on this subject based on the ideXlab platform.
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A new microfluidic concept for parallel operated milliliter-scale stirred tank Bioreactors.
Biotechnology progress, 2011Co-Authors: Gabi Gebhardt, Klaus Kaufmann, Ralf Hortsch, Matthias Arnold, Dirk Weuster-botzAbstract:Parallel miniaturized stirred tank Bioreactors are an efficient tool for “high-throughput bioprocess design.” As most industrial bioprocesses are pH-controlled and/or are operated in a fed-batch mode, an exact scale-down of these reactions with continuous dosing of fluids into the miniaturized Bioreactors is highly desirable. Here, we present the development, characterization, and application of a novel concept for a highly integrated microfluidic device for a bioreaction block with 48 parallel milliliter-scale stirred tank reactors (V = 12 mL). The device consists of an autoclavable fluidic section to dispense up to three liquids individually per reactor. The fluidic section contains 144 membrane pumps, which are magnetically driven by a clamped-on actuator section. The micropumps are designed to dose 1.6 μL per pump lift. Each micropump enables a continuous addition of liquid with a flow rate of up to 3 mL h−1. Viscous liquids up to a viscosity of 8.2 mPa s (corresponds to a 60% v/v glycerine solution) can be pumped without changes in the flow rates. Thus, nearly all feeding solutions can be delivered, which are commonly used in bioprocesses. The functionality of the first prototype of this microfluidic device was demonstrated by double-sided pH-controlled cultivations of Saccharomyces cerevisiae based on signals of fluorimetric sensors embedded at the bottom of the Bioreactors. Furthermore, fed-batch cultivations with constant and exponential feeding profiles were successfully performed. Thus, the presented novel microfluidic device will be a useful tool for parallel and, thus, efficient optimization of controlled fed-batch bioprocesses in small-scale stirred tank Bioreactors. This can help to reduce bioprocess development times drastically. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.
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Process performance of parallel Bioreactors for batch cultivation of Streptomyces tendae
Bioprocess and Biosystems Engineering, 2011Co-Authors: Ralf Hortsch, Harald Krispin, Dirk Weuster-botzAbstract:Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale Stirred-Tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale Stirred-Tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in Stirred-Tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken Bioreactors. The measured product concentrations and product formation kinetics were almost the same in the Stirred-Tank Bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale Bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the Stirred-Tank Bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel Bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.
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new milliliter scale stirred tank Bioreactors for the cultivation of mycelium forming microorganisms
Biotechnology and Bioengineering, 2010Co-Authors: Ralf Hortsch, Ansgar Stratmann, Dirk WeusterbotzAbstract:A novel milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter-scale. A newly designed one-sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface-to-volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k(L)a) > 0.15 s(-1) were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter-scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory-scale stirred tank bioreactor with six-bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter-scale stirred tank bioreactor was reduced compared to the laboratory-scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale-up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter-scale (standard deviation < 8%) with up to 48 stirred tank Bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear-thinning non-Newtonian behavior. The newly developed one-sided paddle impellers operated in unbaffled reactors on a 10 milliliter-scale with a magnetic inductive drive for up to 48 parallel Bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically.
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New milliliter‐scale stirred tank Bioreactors for the cultivation of mycelium forming microorganisms
Biotechnology and bioengineering, 2010Co-Authors: Ralf Hortsch, Ansgar Stratmann, Dirk Weuster-botzAbstract:A novel milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter-scale. A newly designed one-sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface-to-volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k(L)a) > 0.15 s(-1) were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter-scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory-scale stirred tank bioreactor with six-bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter-scale stirred tank bioreactor was reduced compared to the laboratory-scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale-up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter-scale (standard deviation < 8%) with up to 48 stirred tank Bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear-thinning non-Newtonian behavior. The newly developed one-sided paddle impellers operated in unbaffled reactors on a 10 milliliter-scale with a magnetic inductive drive for up to 48 parallel Bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically.
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Milliliter-scale stirred tank reactors for the cultivation of microorganisms.
Advances in Applied Microbiology, 2010Co-Authors: Ralf Hortsch, Dirk Weuster-botzAbstract:This review focuses on recent developments in the field of miniaturized stirred tank Bioreactors for application in high-throughput bioprocess development. Different reactor concepts and their potential for parallel bioprocess development are discussed. A detailed description of important engineering state variables, their measurement at small-scale and their implication for scale-up and scale-down of bioprocesses are given. Examples of two different parallel cultivations at small-scale are presented: one with Escherichia coli and the other one with the filamentous microorganism Streptomyces tendae. It is shown that results obtained in parallelized milliliter-scale stirred tank reactors can be scaled up to the laboratory- and/or pilot-scale in a highly reliable manner. This helps to reduce development times for bioprocesses significantly. Finally, directions for future research are presented.
Robert Zweigerdt - One of the best experts on this subject based on the ideXlab platform.
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Human Pluripotent Stem Cell Expansion in Stirred Tank Bioreactors.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Felix Manstein, Caroline Halloin, Robert ZweigerdtAbstract:This chapter describes a detailed protocol on human pluripotent stem cells (hPSCs) cultivation as matrix-free cell-only aggregates in defined and xeno-free culture medium in stirred tank Bioreactors (STBRs). Starting with a frozen stock pre-expanded on conventional culture dishes (2D), the ultimate process is performed in 150 mL culture scale in stirred tank Bioreactors (3D) and is designed to produce up to 500 million pluripotent hPSC within 7 days. The culture strategy includes perfusion-based cell feeding facilitating process control, automation, and higher cell yields. Ultimately, this detailed protocol describes an important step for generating a defined starting cell population for directed lineage differentiation and subsequently fueling human cell-based assays and regenerative medicine approaches.
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suspension culture of human pluripotent stem cells in controlled stirred Bioreactors
Tissue Engineering Part C-methods, 2012Co-Authors: Ruth Olmer, Axel Haverich, Andreas Lange, Sebastian Selzer, Cornelia Kasper, Ulrich Martin, Robert ZweigerdtAbstract:Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end, we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank Bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption, but in parallel functional integration of all probes mandatory for process monitoring, that is, for pO2 and pH, experiments were performed in 100 mL culture volume in a “mini reactor platform” consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×108 hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluri...
Axel Haverich - One of the best experts on this subject based on the ideXlab platform.
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impact of feeding strategies on the scalable expansion of human pluripotent stem cells in single use stirred tank Bioreactors
Stem Cells Translational Medicine, 2016Co-Authors: Christina Kropp, Caroline Halloin, Henning Kempf, Diana Roblesdiaz, Annika Franke, Thomas Scheper, Katharina Kinast, Thomas Knorpp, Thomas O Joos, Axel HaverichAbstract:: : The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented Stirred-Tank Bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 106 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. SIGNIFICANCE: Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.
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suspension culture of human pluripotent stem cells in controlled stirred Bioreactors
Tissue Engineering Part C-methods, 2012Co-Authors: Ruth Olmer, Axel Haverich, Andreas Lange, Sebastian Selzer, Cornelia Kasper, Ulrich Martin, Robert ZweigerdtAbstract:Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end, we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank Bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption, but in parallel functional integration of all probes mandatory for process monitoring, that is, for pO2 and pH, experiments were performed in 100 mL culture volume in a “mini reactor platform” consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×108 hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluri...