Application Notes
Non-Invasive Oxygen Sensors
Development of a New, Highly Sensitive Sensor for Permeation Measurement: Optical Sensors Used for Permeation Testing of High Barrier Materials
Christian Huber3, Sven Sängerlaub1, and Kajetan Müller2
1TU Munich, Chair of Food Packaging Technology, Freising, Germany; 2Fraunhofer Institute for Process Engineering and Packaging, Freising, Germany; 3PreSens Precision Sensing GmbH, Regensburg, Germany
A new measurement system for testing synthetic materials on oxygen permeability was developed. Unlike the currently used systems with electrochemical or barometrical sensors it was designed for optical measurement. The Fibox 3 LCD trace together with chemical optical sensors was applied to detect trace oxygen concentrations and validate the new measurement system. Apart from an already available sensor type different other types of sensor material were tested to develop an oxygen sensor with an improved limit of detection for high barrier permeation measurements. This sensor type - TAF12 - together with the measurement cells seems to be a promising solution for easy, cost effective permeation measurements in technical applications.
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Non-Invasive Oxygen Sensors
Fibox 3 LCD trace
Perfusion Culture of Cell-seeded 3D Scaffolds for Tissue Engineering: In-line Oxygen Measurement in Perfusion Bioreactors
R. Santoro, B. Pippenger, I. Martin, and D. Wendt, Department of Surgery and Research, University Hospital, Basel, Switzerland
Since oxygen has a low solubility in culture medium it must be supplied to cells within 3D scaffolds via constant perfusion of the medium. The oxygen content of the medium before and after having passed by the cells can then help to determine whether the flow rate applied is sufficient. PreSens flow-through cells with integrated chemical optical sensors can be incorporated in the construct inlet and outlet and allow in-line measurement of oxygen tension. Normoxic oxygen supply creates homogeneous grafts with a uniform distribution of cells.
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Fibox 3
Non-Invasive Oxygen Sensors
Effect of Oxygen Depletion on GDH Activity: Oxygen Monitoring in Shaken Cultures of C. glutamicum and C. diphtheriae
M. Böhme and A. Burkovski; Department of Microbiology, FAU Erlangen-Nuremberg, Germany
The impact of oxygen depletion in cultures of Corynebacterium glutamicum and two different strains of Corynebacterium diphtheriae was investigated under different cultivation conditions. Experiments were conducted to characterize the impact of low oxygen concentrations on a specific enzyme of the nitrogen metabolism - the ammonium transferring enzyme glutamate dehydrogenase (GDH). Oxyen monitoring was performed using the oxygen meter Fibox 3 by PreSens. Growth rates in all tested Corynebacterium cultures were dependent on oxygen supply and increased the more oxygen was available. The investigated activity of GDH seemed not to be affected by low oxygen levels in C. glutamicum. In cultures of C. diphtheriae the GDH activity increased under certain conditions, but only very low activity values could be determined that might not be significant.
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Fibox 3
Non-Invasive Oxygen Sensors
Dissolved Oxygen Determination: Dynamic measurements in wine with a micro-oxygenation system
María del Alamo and Ignacio Nevares, UVaMOX, E. T. S. Ingenierías Agrarias, Universidad de Valladolid, 34071 Palencia, Spain
Micro-oxygenation processes happening while storing wine in barrels give quality red wines the necessary final trait. Dissolved oxygen (DO) levels reached during the usual storage months are so low that measurement systems requirements become very high. There are measurement systems with detection limits at ppb (µg/L) based on electrochemical probes but they consume oxygen during the measuring process which is a problem as carrying out a dynamic measurement, i. e. in a fluid in motion, becomes necessary. Measurement of DO in wine flow has several disadvantages that can be solved but at the expenses of time and money. The optical measurement system Fibox 3 LCD trace allows to deal with the need of measurement in systems with low volume, at different atmospheric pressures, and the advantage of carrying it out in an inerted way.
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Non-Invasive Oxygen Sensors
Fibox 3 LCD trace
SDR / SensorDishes / SensorPlates
Real Time Respiration Measurements: Measuring the aerobic respiration of complex microbial communities
B. R. Roller1, Z. M. Lee1, K. E. Studer-Rabeler2, and T. M. Schmidt1
1Dept. of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA 2Coy Laboratory Products, Grass Lake, MI, USA
Many methods for measuring aerobic respiration, including electrodes and colorimetric assays, limit the capacity for obtaining replicate measurements or the ability to detect oxygen consumption in real time. The OxoDish® - a microtiter plate with integrated oxygen sensors - and SDR SensorDish® Reader offer the potential for making twenty four simultaneous respiration measurements in real time, benefitting ecologists measuring the respiration of complex microbial communities.
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SDR SensorDish Reader
Co-Cultures of Caco-2 cells and E.coli: Optosensoric online monitoring of O2 uptake and extracellular acidification
Martin Brischwein and Daniel Grundl, Heinz-Nixdorf-Lehrstuhl für Medizinische Elektronik, TU München
Oligogalacturonic acids (OGAs), obtained by hydrolysis of pectin, are known to inhibit the adhesion of pathogenic bacteria on intestinal cells and thus to prevent infectious diseases. To test the effects of OGAs, an in-vitro model consisting of Caco-2 cells (human small intestinum) and the E.coli C25 strain was established. The co-cultures were set up in a novel, label-free cell assay platform working with a 24-well plate. The SDR SensorDish® Reader allows to monitor oxygen and pH kinetically in each well with chemical optical sensors to analyse the rates of cellular oxygen uptake and extracellular acidification.
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Process Monitoring in Suspension-Adapted CHO Cell Cultures: Non-invasive online detection of pH and oxygen
Harry Abts and Sarina Arain, Celonic GmbH, Karl-Heinz-Beckurts-Straße 13, Jülich, Germany, PreSens GmbH, Josef-Engert-Str. 11, 93053 Regensburg, Germany
Online detection of oxygen and pH by using SDR at a small-scale can be used as a basis for latter cultivation in large-scale bioreactors to detect optimal growth conditions. Contamination-free online monitoring of culture parameters is not only important for scaling up production in process development, but it also could be valuable for other applications such as tissue engineering, stem cell research, and toxicological tests.
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Online Oxygen Monitoring System: Non-conventional steroid fermentations in microtiter plates
Marques M.P.C.1, Cabral J.M.S. 1, Fernandes P., Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal
A key factor when optimising and developing bioprocesses is the monitoring of the fermentation, in order to quantify in real-time basis parameters like oxygen, pH and carbon dioxide, among others. This monitoring by using SDR allows the time course of the fermentation to be followed and process parameters to be adjusted to desired levels. Online monitoring in stirred vessels, namely fermenters, is a widespread approach but it has seldom been applied in shaken systems due to technical limitations.
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Online Oxygen Monitoring in Cell Culture: Effects of mitochondrial modulators on O2 dynamics of mammalian cells
Lynn S. G. and LaPres J. J., Dept. of Biochemistry, Michigan State University, East Lansing, MI, USA
Real-time measurements of oxygen and pH in mammalian cell cultures are important to understand the metabolic dynamics in cell cultures. The SDR SensorDish® Reader allows the time course of cellular oxygen consumption to be followed in response to environmental conditions or toxicological insults. This type of monitoring could also be valuable for other applications such as tissue engineering and stem cell research.
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Non-Invasive Online Oxygen Measurement: Bacterial toxicity tests for use in pharmacological agent
Anna Vasilevskaya, Institute for Analytical Chemistry, University of Regensburg, Germany
Measurement of oxygen kinetics in respiration inhibition tests is of central importance for pharmacological agent research and toxicology testing of chemicals (REACH). The new SDR SensorDish® Reader makes non-invasive, simultaneous, online recording in 24-well plates possible for the first time ever. The study describes the use of the system in a bacterial-respiratory toxicity test.
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A Novel Online Monitoring System: For Non-Invasive Oxygen Detection in Chondrocyte Cultures
Bettina Giere, Tierärztliche Hochschule Hannover, Germany
For optimisation of cell culture systems it is essential to monitor important metabolic parameters like oxygen and pH quantitatively and to readjust them to physiological values if necessary. Deviations from the physiological limits cause substantial changes for cell growth and differentiation. The new SDR SensorDish® Reader enables non-invasive online monitoring of oxygen and pH in cell culture media. This is exemplified by a novel 3D chondrocyte culture model.
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3D Cutures of Disc-Chondrocytes: Optosensoric Online Monitoring of Oxygen and pH Kinetics
Karin Benz and Jürgen Mollenhauer, Regenerative Medizin II, NMI Reutlingen, Markwiesenstraße 55, 72770 Reutlingen, Germany
Hydrogel-based 3D cell culture systems offer particular advantages for tissue-engineering in cell-based therapy for disc-degeneration. However, light microscopical control of both culture quality and morphological differentiation is not possible within this dense matrix. For a judgement of the quality, alternative parameters have to be considered. In the present study, availability of oxygen within culture medium and kinetics of the pH value are analyzed in this regard by using the SDR. Both parameters are key factors for a successful and reliable in vitro cultivation and differentiation.
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Non-Invasive pH Sensors
pH Regulation for a Liver Cell Bioreactor: Prototype development with a chemical optical pH flow-through cell
Marco Decker, Fachhochschule für Technik und Wirtschaft Berlin (FHTW), Berlin
An important aspect of efficient liver cell bioreactors is the automated regulation of physio-chemical culture parameters. The present study describes the development of the first non-invasive pH regulation device for a perfusion bioreactor. The high performance of the system is based on a chemical optical flow-through cell for pH detection and its combination with precision mass-flow controllers for gas. The new controller allows long time stable and contamination free online pH regulation in complex bioreactor systems - an important technical contribution for future clinical applications.
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Oxygen Microsensors
Hypoxia in Static and Dynamic 3D Culture Systems: Oxygen Gradient Measurements in 3D Culture Systems Designed for Bone Tissue Engineering
Elias Volkmer1, Inga Drosse1, Sven Otto2, Achim Stangelmayer3, Michael Stengele1, Bobby Cherian Kallukalam1, Wolf Mutschler1, and Matthias Schieker1
1)Experimental Surgery and Regenerative Medicine, Department of Surgery, LMU Munich, Germany; 2)Department of Oral and Maxillofacial Surgery, LMU Munich, Germany; 3)PreSens Precision Sensing GmbH, Regensburg, Germany
Gradients in tissue quality from the periphery towards the center are a limiting factor in tissue engineering of sizeable cell-scaffolds. Uneven oxygen supply might be the cause for irregular cellular growth on scaffolds. In this study continuous measurements of oxygen concentration in the center of scaffolds designed for bone tissue engineering and the surrounding medium were performed. In static 3D culture systems the oxygen concentration in the center dropped to 0 % after 5 days and resulted in cell death. Perfusion bioreactors can prevent cell death but still do not entirely eliminate 3D culture-associated oxygen gradients.
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Hypoxic Preconditioning of Human Mesenchymal Stem Cells: Restoring the Differentiation Potential of hMSCs under Hypoxic Conditions
Elias Volkmer1, Booby Cherian Kallukalam1, Josef Maertz1, Sven Otto2, Inga Drosse1, Hans Polzer1, Wolfgang Bocker1, Michael Stengele1, Denitsa Docheva1, Wolf Mutschler1, and Matthias Schieker1
1)Experimental Surgery and Regenerative Medicine, Department of Surgery, LMU Munich, Germany; 2)Department of Oral and Maxillofacial Surgery, LMU Munich, Germany
In this study the osteogenic differentiation potential of hMSCs under hypoxic conditions of 2 % O2 in comparison to standard tissue culture oxygen atmosphere of 21 % was investigated. Further tests were conducted to determine whether hypoxic preconditioning has an effect on the following osteogenic differentiation of hMSCs and if this pretreatment might be beneficial for the differentiation process. It was found that hMSCs proliferate better when cultured at 2 % O2 but constant hypoxia inhibits osteogenic differentiation. Hypoxic preconditioning of hMSCs prior to osteogenic induction restores osteogenic differentiation of the cells under hypoxic conditions.
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New mL-Scale Bioreactor for the Cultivation of Mycelium Forming Microorganisms: Determination of the Volumetric Oxygen Transfer Coefficient (kLa)
Ralf Hortsch1, Ansgar Stratmann2, Dirk Weuster-Botz1
1Institute of Biochemical Engineering, TU Munich, Germany; 2W42 Industrial Biotechnology GmbH, Marl, Germany
A new milliliter-scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms. Applying a newly designed one-sided paddle impeller an enhanced surface-to-volume ratio of the liquid phase was generated where oxygen was introduced in the medium via surface aeration. The fast moving liquid lamella efficiently prevented wall growth and foaming in the mycelium forming culture. In the new bioreactors volumetric oxygen transfer coefficients (kLa) > 0.15 s-1 were measured applying chemical optical oxygen microsensors with fast response time.
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Hydrogels as Cell Carriers for Tissue Engineering: Calcium Peroxide Used as an Oxygen Releasing Additive
Uta Leicht1, Elias Volkmer1,Hinrich Wiese2, Matthias Schieker1
1Experimental Surgery and Regenerative Medicine, Department of Surgery, LMU Munich, Germany; 2Polymaterials AG, Kaufbeuren, Germany
Hydrogels might be a new method for cell-based regenerative therapy. They can be applied in a minimal invasive way but they have to be biodegradable and nontoxic. The biocompatibility of different hydrogels seeded with hMSCs was tested in this study. CaO2 served as an oxygen releasing additive in the gels to overcome oxygen limitations or gradients within the gels. The hydrogels used might be suitable for the future injection of cells in damaged tissue. CaO2 seems to be an outstanding oxygen releasing additive but cytotoxic side effects are a problem.
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SFR / Shake Flasks
Insect cell cultivation in orbitally shaken flasks with sensors: Oxygen mass transfer in insect cell-based high cell density cultivations
N. Riesen, C. Ries, R. Eibl, D. Eibl; Zurich University of Applied sciences, School of Life Sciences and Facility Managment, Institute of Biotechnology, Biochemical Engineering and Cell Cultivation Techniques, Wädenswil, Switzerland
Insect cell-based processes have become increasingly influential for tool protein and Virus-like Particle (VLP) vaccine productions over the last years. At small scale insect cells are normally grown in non-instrumented orbitally shaken disposable flasks, in most cases Erlenmeyers, in order to perform seed train productions and early stage process developments. The Shake Flask Reader with non-invasive optical sensors for pH and DO is an important contribution to easy quantification of oxygen mass transfer in Erlenmeyers recently made. In addition, this allows controlled processing at mL-scale as exemplified for described Sf-9 and Sf-21 cell mass propagation procedures.
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Development of a Shaken Scale-Down Model: Characterization of Critical Parameters in Bioprocess Development
Holger Kley, Jan Kreuzmann, Caroline Schuster, and Robert Puskeiler; Roche Diagnostics GmbH, Pharma Biotech Production and Development, Penzberg, Germany
A shaken scale-down model at approx. 0.1 L scale for a previously established stirred-tank model at 2 L scale was developed. In the shaken scale-down model online monitoring of pH and dissolved oxygen concentration was performed using the SFR Shake Flask Reader by PreSens. Though only a simple, manual control of these two parameters could be realized the shaken scale-down model achieved similar data to the stirred-tank model for viable cell density, specific glycosylation content, and ion exchange chromatography pattern. These results suggest that for this process the importance of pH and DO control might be overestimated and further investigations have to be made for evaluating and comparing the influence of shear stress and power input in both models.
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Development of a Partly Controllable System at Shake Flask Scale: Enhanced data quality obtained from fed-batch shake flask cultivation by minimizing disturbance variables monitored by the SFR
Christian Klinger, Sylvia Baumann, Robert Puskeiler, and Alexander Jockwer, Roche Diagnostics GmbH, Pharma Biotech Production and Development, Penzberg, Germany
The main objective of this study was to develop an easy to handle and partly controllable process cultivation system at shake flask scale. First, it was investigated how disturbances, like taking the flasks out of the incubator for feeding and sampling, affect culture conditions and results. An adapter was constructed that allowed automated feed in the shake flasks. Online monitoring of oxygen partial pressure (pO2) and pH were performed with the SFR Shake Flask Reader by PreSens, so limitations could be detected and adjustments made in time. This "semi-controlled" system clearly reduced the workload and minimized the disturbance variables, thus more reliable and representative data could be obtained for further scale-up processes.
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Characterization of Microbial Growth Profiles: Online detection of oxygen and pH during culture in shake flasks
Konstantin Schneider1, Verena Schütz1, Gernot Thomas John2 and Elmar Heinzle1
1) Biochemical Engineering Institute, Saarland University, 66123 Saarbrücken, Germany; 2) PreSens Precision Sensing GmbH, 93053 Regensburg, Germany
Shake flasks are the most commonly used cultivation systems for process and media optimization, screening for new products and strains as well as for the supply of inoculum for bioreactor cultivation. Oxygen limitation is one of the major though often neglected problems in aerobic shake flask cultivation. The new technology of the SFR Shake Flask Reader is an ideal tool for non-invasive monitoring of the two culture parameters oxygen and pH, and in addition reveals a detailed picture of growth performance and profiles.
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Accurate Insight into Oxygen Content of Shake Cultures
Michael Findeis and Gernot T. John, PreSens GmbH, 93053 Regensburg, Germany
Numerous factors affect growth in cell culture in shake flasks. The free oxygen content plays a decisive role. Insufficient oxygen input from the environment limits growth. With the new SFR measurement system the dissolved oxygen content of the medium can be determined non-invasively and extremely accurately. Continous oxygen monitoring offers advantages for growing new sensitive cell lines, for media optimisation and for QA-related process control.
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CO2 Sensors
Online in Situ Measurements of Soil CO2 Concentrations in Dependence of Ash Roots (Fraxinus excelsior L.)
Ann-Catrin Fender, Christoph Flucke, Dirk Gansert; Department of Plant Ecology and Ecosystem Research, University of Göttingen, Germany
Root-induced temporal and spatial changes of CO2 concentration are of high importance for biogeochemistry of soils, and thus, for their potential of long-term carbon storage. We investigated the effects of ash (Fraxinus excelsior L.) fine roots on CO2 and O2 concentrations in forest soil under constant moisture and temperature conditions in the laboratory. Using chemical optical sensors and monitoring systems by PreSens we measured CO2 and O2 concentrations in the rhizosphere, and 25 mm off the roots in the bulk soil. Further, fine root respiration was estimated by simultaneous measurements of O2 consumption and CO2 production, which reached 19 µmol CO2 g-1 dw s-1 on average at 20 °C soil temperature. Surprisingly, our results indicated a higher CO2 concentration in the bulk soil than in the rhizosphere.
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Non-invasive Online CO2 Measurement with Chemical Optical CO2 Sensors: Chemical optical CO2 sensors provide a versatile tool for online monitoring of dissolved CO2 in various scale and designs
Athanas Apostolidis, PreSens Precision Sensing GmbH, 93053 Regensburg, Germany
Accurate CO2 measurement in the liquid phase has been a challenge for decades. Although off-gas measurement by means of IR-spectroscopy is a proven technology, the correlation of CO2-levels in gas and liquid phase in biological systems is difficult.
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Impact of dCO2 on Process Parameters: Effect of High dCO2 on Viable Cell Density and Product Titer in CHO Cell Culture
Jennifer Couture, Jennifer Lindsay, and Rick Baggio; EMD Millipore Corporation, Bedford, MA, USA
Real-time monitoring of bioprocess parameters is important to ensure reproducible cell culture and optimize product quality. Fast detection of unfavorable conditions in cell culture requires reliable sensor systems. CHO cells - producing and secreting an antibody - were cultivated in bioreactors and viable cell density and product titer were monitored. Then content of dissolved carbon dioxide in the culture medium was monitored with an optosensoric CO2 measurement system.
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