Cell Culture Volume Research Articles

Optimization of protein electroextraction from microalgae by a flow process

Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5μm, we used repetitive 2ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size.

Bioimpedance monitoring of 3D cell culturing—Complementary electrode configurations for enhanced spatial sensitivity

A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance spectroscopic (EIS) characterisation were used to determine the configurations’ sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics at the corners of the 3D culture chamber. By combining a number of electrode configurations, complementary spatially distributed information on a large 3D cell culture can be obtained with maximised sensitivity in the entire 3D space. The experimental results show that cell proliferation can be monitored within the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering.

Modulation of mAb quality attributes using microliter scale fed-batch cultures.

A high-throughput DoE approach performed in a 96-deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO-S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed-batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N-glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines - some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small-scale bioreactors. Being fast and resource effective, this integrated high-throughput approach can provide information which is particularly useful during early stage cell culture development.

Electrical conditioning of adipose‐derived stem cells in a multi‐chamber culture platform

In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration.

Purification and biological characterization of soluble, recombinant mouse IFNβ expressed in insect cells

Interferon β (IFNβ) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNβ utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNβ. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNβ purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNβ than any other reported eukaryotic-based expression system. Recombinant murine IFNβ produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNβ activity in vivo. Recombinant IFNβ also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNβ.

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell-culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell-carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications.

Integration of stochastic simulation with multivariate analysis: Short-term facility fit prediction

This article describes a decision-support tool to help pinpoint the potential root causes of sub-optimal short-term facility fit issues in biopharmaceutical facilities. This was achieved by creating a tool that integrated stochastic simulation with advanced multivariate statistical analysis. Process fluctuations in product titers in cell culture, step yields, and chromatography eluate volumes were mimicked using Monte Carlo simulation data derived using a stochastic discrete-event simulation model. The resulting stochastic datasets, with the computed consequences on key metrics such as product mass loss and cost of goods, were examined using advanced multivariate statistical techniques. Principal component analysis combined with clustering algorithms was used to analyze the complex datasets from complete industrial batch processes for biopharmaceuticals. The challenge of visualizing the multidimensional nature of the dataset was addressed using hierarchical and k-means clustering as well as stacked parallel co-ordinate plots to help identify process fingerprints and characteristics of clusters leading to sub-optimal facility fit issues. Industrially-relevant case studies are presented that focus on technology transfer challenges for therapeutic antibodies moving from early phase to late phase clinical trials. The case study details how sub-optimal facility fit can be alleviated by allocating alternative product pool tanks. The impact of this operational change is then assessed by reviewing an updated process fingerprint.

Optimization of tri-expression of human CYP3A4 with POR and cyt b5 in Sf 9 cells

To investigate the optimal conditions of tri-expression of CYP3A4, POR and cyt b5 in Sf 9 cells. The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfactant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. When the temperature and rotation speed of the shaker were 27 degree and 90 r/min, the cell density and culture volume were 5X105 cells/ml and 80-120 ml per 250 ml shaker flasks, respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf 9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1:1:1, the expression condition was optimal, under which the Km, Vmax, and CLint for testosterone metabolism were 119.6 μmol/L,0.52 μmol/(min*g protein) and 4.34 ml/(min*g protein), respectively. The conditions of tri-expressing of CYP3A4, POR and cyt b5 have been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

Reaction–diffusion modelling for microphysiometry on cellular specimens

Using modeling and simulation, we quantify the influence of spatiotemporal dynamics on the accuracy of data obtained from sensors placed in microscaled reaction volumes. The model refers to cellular reaction (i.e. proton extrusion and oxygen consumption) in complex, buffering solutions. Whole cells or viable tissues cultured in such devices are monitored in real time with integrated sensors for pH and dissolved oxygen. A 3D finite element model of diffusion and metabolic reaction was set up. With respect to pH, the effect of buffering species on proton diffusion is analysed in detail. To account for the delayed time response of real sensors, the sensor impulse response time was implemented by linear convolution. A validation of the model has been achieved by an electrochemical approach. The model reveals significant deviations of measured pH and O2, and values of these parameters actually occurring at different sites of the cell culture volume. It is applicable to any setting of (bio-) sensors involving reaction and diffusion of dissolved gases and particularly H(+) ions in buffered solutions.

The Crystal Structure of the Catalytic Domain of the NF-κB Inducing Kinase Reveals a Narrow but Flexible Active Site

The NF-κB inducing kinase (NIK) regulates the non-canonical NF-κB pathway downstream of important clinical targets including BAFF, RANKL, and LTβ. Despite numerous genetic studies associating dysregulation of this pathway with autoimmune diseases and hematological cancers, detailed molecular characterization of this central signaling node has been lacking. We undertook a systematic cloning and expression effort to generate soluble, well-behaved proteins encompassing the kinase domains of human and murine NIK. Structures of the apo NIK kinase domain from both species reveal an active-like conformation in the absence of phosphorylation. ATP consumption and peptide phosphorylation assays confirm that phosphorylation of NIK does not increase enzymatic activity. Structures of murine NIK bound to inhibitors possessing two different chemotypes reveal conformational flexibility in the gatekeeper residue controlling access to a hydrophobic pocket. Finally, a single amino acid difference affects the ability of some inhibitors to bind murine and human NIK with the same affinity.

Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.

Electrochemical detection of catecholamine release using planar iridium oxide electrodes in nanoliter microfluidic cell culture volumes

Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20μm×300μm) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0–400μM, with a sensitivity of 23.1±0.5mA/Mmm2. The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50mM) containing Tyrode's solution at a flow rate of 1nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ∼52μM. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells.

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.

Enzyme-coated microelectrodes to monitor lactate production in a nanoliter microfluidic cell culture device

Monitoring the degree of anaerobic respiration of cells in high density microscale culture systems is an enabling key technology and essential for cell-based biosensors. We have fabricated and incorporated miniature amperometric lactate sensing electrodes with working areas from 3 to 5 × 10 −2 mm 2 into a microfluidic-based microscale cell culture system to measure the lactate production rate of fibroblasts in nanoliter volumes. Planar thin film platinum electrode arrays on glass substrates were spin coated with lactate oxidase and a protective Nafion layer. The lactate electrodes had a high enzymatic activity described by a Michaelis–Menten constant of 2.6 ± 0.1 mM, a linear response in the range 0.01–2.5 mM and a sensitivity of 7.3 × 10 −2 mA/mM cm 2. A replica-molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. We trapped fibroblasts in the cell culture volume and measured the lactate production rate using a stop–flow protocol. The average lactate production rate was 0.011 ± 0.0049 mM/min. The lactate production was suppressed with the addition of 2-deoxy- d-glucose, which binds to hexokinase. The blocking of hexokinase prevents the generation of pyruvate, the intermittent substrate required for lactate production even in the presence of glucose.

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

pHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH(in)) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH(in) for three prototrophic wild-types. The new construct was also used to determine the pH(in) in strains differing in the activity of the plasma membrane Pma1 H(+)-ATPase and the influence of glucose on pH(in). We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

A highly efficient pipeline for protein expression in Leishmania tarentolae using infrared fluorescence protein as marker

BackgroundLeishmania tarentolae, a unicellular eukaryotic protozoan, has been established as a novel host for recombinant protein production in recent years. Current protocols for protein expression in Leishmania are, however, time consuming and require extensive lab work in order to identify well-expressing cell lines. Here we established an alternative protein expression work-flow that employs recently engineered infrared fluorescence protein (IFP) as a suitable and easy-to-handle reporter protein for recombinant protein expression in Leishmania. As model proteins we tested three proteins from the plant Arabidopsis thaliana, including a NAC and a type-B ARR transcription factor.ResultsIFP and IFP fusion proteins were expressed in Leishmania and rapidly detected in cells by deconvolution microscopy and in culture by infrared imaging of 96-well microtiter plates using small cell culture volumes (2 μL - 100 μL). Motility, shape and growth of Leishmania cells were not impaired by intracellular accumulation of IFP. In-cell detection of IFP and IFP fusion proteins was straightforward already at the beginning of the expression pipeline and thus allowed early pre-selection of well-expressing Leishmania clones. Furthermore, IFP fusion proteins retained infrared fluorescence after electrophoresis in denaturing SDS-polyacrylamide gels, allowing direct in-gel detection without the need to disassemble cast protein gels. Thus, parameters for scaling up protein production and streamlining purification routes can be easily optimized when employing IFP as reporter.ConclusionsUsing IFP as biosensor we devised a protocol for rapid and convenient protein expression in Leishmania tarentolae. Our expression pipeline is superior to previously established methods in that it significantly reduces the hands-on-time and work load required for identifying well-expressing clones, refining protein production parameters and establishing purification protocols. The facile in-cell and in-gel detection tools built on IFP make Leishmania amenable for high-throughput expression of proteins from plant and animal sources.

Roles of Protein-disulfide Isomerase-mediated Disulfide Bond Formation of Yeast Mnl1p in Endoplasmic Reticulum-associated Degradation

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.

Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b6f Complex from Nostoc sp. PCC 7120

The crystal structure of the cyanobacterial cytochrome b(6)f complex has previously been solved to 3.0-A resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b(6)f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b(6)f complex. Purified b(6)f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b(6)f complex, determined to a resolution of 3.0A (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b(p) that is rotated 180 degrees about the alpha- and gamma-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme c(n) is similar to that previously found in the b(6)f complex from other sources.

Cellular Recognition of Trimyristoylated Peptide or Enterobacterial Lipopolysaccharide via Both TLR2 and TLR4

Evidence for specific and direct bacterial product recognition through toll-like receptors (TLRs) has been emphasized recently. We analyzed lipopeptide analogues and enterobacterial lipopolysaccharide (eLPS) for their potential to activate cells through TLR2 and TLR4. Whereas bacterial protein palmitoylated at its N-terminal cysteine and N-terminal peptides derived thereof are known to induce TLR2-mediated cell activation, a synthetic acylhexapeptide mimicking a bacterial lipoprotein subpopulation for which N-terminal trimyristoylation is characteristic (Myr(3)CSK(4)) activated cells not only through TLR2 but also through TLR4. Conversely, highly purified eLPS triggered cell activation through overexpressed TLR2 in the absence of TLR4 expression if CD14 was coexpressed. Accordingly, TLR2(-/-) macrophages prepared upon gene targeting responded to Myr(3)CSK(4) challenge, whereas TLR2(-/-)/TLR4(d/d) cells were unresponsive. Through interferon-gamma (IFNgamma) priming, macrophages lacking expression of functional TLR4 and/or MD-2 acquired sensitivity to eLPS, whereas TLR2/TLR4 double deficient cells did not. Not only TLR2(-/-) mice but also TLR4(-/-) mice were resistant to Myr(3)CSK(4) challenge-induced fatal shock. d-Galactosamine-sensitized mice expressing defective TLR4 or lacking TLR4 expression acquired susceptibility to eLPS-driven toxemia upon IFNgamma priming, whereas double deficient mice did not. Immunization toward ovalbumin using Myr(3)CSK(4) as adjuvant was ineffective in TLR2(-/-)/TLR4(-/-) mice yet effective in wild-type, TLR2(-/-), or TLR4(-/-) mice as shown by analysis of ovalbumin-specific serum Ig concentration. A compound such as Myr(3)CSK(4) whose stimulatory activity is mediated by both TLR2 and TLR4 might constitute a preferable adjuvant. On the other hand, simultaneous blockage of both of the two TLRs might effectively inhibit infection-induced pathology.

DEVELOPMENT OF A NEW PROBE FOR IN-SITU OXYGEN UPTAKE RATE (OUR) MEASUREMENT IN MAMMALIAN CELL CULTURE PROCESSES

Abstract We have developed a new probe that measures the oxygen uptake rate (OUR) of mammalian cells, an important indicator for the metabolic state, inside the reactor (in-situ) and in real-time without the need for sampling or complicated analytics. The probe isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time and releases it again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard DO probe for process monitoring when it is not performing OUR measurements. This new probe was successfully tested in BHK perfusion cell cultures.