Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

This study proposes an easy to use in situ device, based on multi-frequency permittivity measurements, to monitor the growth and death of attached Vero cells cultivated on microporous microcarriers, without any cell sampling. Vero cell densities were on-line quantified up to 10⁶ cell mL⁻¹. Some parameters which could potentially impact Vero cell morphological and physiological states were assessed through different culture operating conditions, such as media formulation or medium feed-harvest during cell growth phase. A new method of in situ cell death detection with dielectric spectroscopy was also successfully implemented. Thus, through permittivity frequency scanning, major rises of the apoptotic cell population in bioreactor cultures were detected by monitoring the characteristic frequency of the cell population, f_c, which is one of the culture dielectric parameters. Both cell density quantification and cell apoptosis detection are strategic information in cell-based production processes as they are involved in major events of the process, such as scale-up or choice of the viral infection conditions. This new application of dielectric spectroscopy to adherent cell culture processes makes it a very promising tool for risk-mitigation strategy in industrial processes. Therefore, our results contribute to the development of Process Analytical Technology in cell-based industrial processes.     

Physical mechanisms of cell damage in microcarrier cell culture bioreactors

The negative effects of excessive agitation on tissue cells in microcarrier culture have often been ascribed to "shear." Analysis of the fluid mechanics occurring suggests that there are actually three potential damage mechanisms: collisions of a cell-covered microcarrier with other beads, collisions with parts of the reactor (primarily the impeller), and interaction with turbulent eddies the size of the microcarrier beads. Review of the available quantitative information on agitation effects in cell cultures does not establish which mechanism is predominant; the range of experimental variables reported emphasizes power input over the other reactor and impeller parameters. The bead-bead collision model is tentatively supported by the available data, but the other mechanisms may still be significant in some systems. The formation of bead aggregates by cellular bridging provides a parallel means of damaging cells. Breaking of these bridges by any of the three means identified earlier can cause cell destruction and/or the net transfer of cells to formerly bare beads. High concentrations of bridges are favored by lower agitation rates, presumably because the bridges are not as quickly destroyed after formation.     

Electrical impedance spectroscopy measurements using a four-electrode configuration improve on-line monitoring of cell concentration in adherent animal cell cultures

This paper describes the improvement in the use of electrical impedance spectroscopy (EIS) for animal cell concentration monitoring of adherent cultures by using a four-electrode configuration instead of the commonly used two-electrode configuration. This four-electrode configuration prevents cell concentration measurements from external masking effects such as the electrode covering ratio, the degree of cellular adherence to the electrodes and the impedance of the measuring electrodes. Cell concentration was monitored using both four-electrode and two-electrode configurations in vero cell and human mesenchymal stem cell cultures in order to analyze the attained improvement in two cell lines with opposite growth characteristics. The experiments performed with vero cell cultures evidenced that the four-electrode configuration enables cell concentration measurements along all culture phases, even once the culture reached cell confluence (over 2×10(5) cells/cm(2)), confirming that this configuration is less effected by all the external influences. The experiments performed with human mesenchymal stem cells demonstrated good sensitivity of the measurement at very low cell concentrations, as well as a very good robustness all over the 12-days experiment. Finally, off-line cell measurements during cell cultures proved good accuracy of impedance measurements carried out with a four-electrode configuration along all cell growth phases, enabling determination of relevant cell growth parameters.     

应用生物反应器培养Vero细胞制备EV71病毒初探

目的应用生物反应器培养Vero细胞制备EV71病毒。方法以3 L生物反应器采用4 g/L、8 g/L Cytodex-1微载体培养比较Vero细胞比生长率,并以4 g/L微载体培养EV71病毒。结果 4 g/L微载体培养Vero细胞3～4 d微载体细胞密度达2.3×106/mL,按0.001的感染复数(MOI)接种EV71病毒,病毒收获液的滴度最高达7.90 lgPFU/mL,较静置培养平均高出0.92 lgPFU/mL。结论初步建立了3 L生物反应器微载体培养Vero细胞制备EV71病毒的工艺,为进一步放大生产规模奠定了基础。     

Hydrodynamic behaviour of animal cell microcarrier suspensions in split-cylinder airlift bioreactors

Hydrodynamic characteristics of suspensions of microcarriers used for culturing anchorage dependent animal cells are reported in split-cylinder internal-loop airlift bioreactors. Cell culture media are simulated using salt solutions that duplicate the ionic strengths of typical media. Effects of solids loading (0–30 kg·m^–3), microcarrier particle size (150–300×10^–6 m diameter) and density (1030–1050 kg·m^–3) on gas induced circulation of the slurry, mixing time, gas holdup and gas velocity requirements to attain complete suspension of solids are discussed for two reactors with aspect ratios of 7.6 and 14.5, but equal riser-to-downcomer cross-sectional area ratios of 1.0, aerated at low air flow rates (0–8×10^–6 m³·s^–1) through a sintered glass sparger with 110×10^–6 m diameter pores. The study covers the ranges of solids concentrations, types, densities, particle sizes and aeration rates that are of relevance in animal cell culture applications.Airlift bioreactors displayed suitable hydrodynamic characteristics for potentially supporting anchorage dependent cell cultures on microcarriers at carrier loadings similar to those that are currently used in stirred tank bioreactors. The minimum gas flow rates and the induced liquid circulation rates necessary to achieve and maintain suspension of the heaviest and the largest microcarriers were well within practicable limits, limits which have been shown to be withstood by animal cells in non-anchorage dependent suspension culture in airlift bioreactors. No floatation problems were encountered with the carriers, nor was sedimentation a problem so long as the identified minimum suspension criteria were met. Chisti's liquid circulation equation, originally intended for two-phase flow, applied to the three-phase gas-liquid-microcarrier systems.     

A flow injection flow cytometry system for on-line monitoring of bioreactors

For direct and on-line study of the physiological states of cell cultures, a robust flow injection system has been designed and interfaced with flow cytometry (FI-FCM). The core of the flow injection system includes a microchamber designed for sample processing. The design of this microchamber allows not only an accurate on-line dilution but also on-line cell fixation, staining, and washing. The flow injection part of the system was tested by monitoring the optical density of a growing E.coli culture on-line using a spectrophotometer. The entire growth curve, from lag phase to stationary phase, was obtained with frequent sampling. The performance of the entire FI-FCM system is demonstrated in three applications. The first is the monitoring of green fluorescent protein fluorophore formation kinetics in E.coli by visualizing the fluorescence evolution after protein synthesis is inhibited. The data revealed a subpopulation of cells that do not become fluorescent. In addition, the data show that single-cell fluorescence is distributed over a wide range and that the fluorescent population contains cells that are capable of reaching significantly higher expression levels than that indicated by the population average. The second application is the detailed flow cytometric evaluation of the batch growth dynamics of E.coli expressing Gfp. The collected single-cell data visualize the batch growth phases and it is shown that a state of balanced growth is never reached by the culture. The third application is the determination of distribution of DNA content of a S. cerevisiae population by automatically staining cells using a DNA-specific stain. Reproducibility of the on-line staining reaction shows that the system is not restricted to measuring the native properties of cells; rather, a wider range of cellular components could be monitored after appropriate sample processing. The system is thus particularly useful because it operates automatically without direct operator supervision for extended time periods.     

Transfer of an adherent Vero cell culture method between two different rocking motion type bioreactors with respect to cell growth and metabolic rates

Rapid and simple cell and virus cultivation can currently be carried out using disposable bioreactors. The CELL-tainer^® (CELLution Biotech BV) disposable bioreactor is a rocking-type bioreactor which not only has vertical movement but horizontal displacement as well. Due to this two-directional movement relatively high mass-transfer capacities can be reached when compared with conventional rocking motion-type bioreactors.Using the design of experiments (DoE) approach we have developed models for the mixing times in both the CELL-tainer^® and the BIOSTAT^® CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type). The conditions for cultivation of Vero cells in the CELL-tainer^® bioreactor were chosen based on comparable mixing times.Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer^® and in the BIOSTAT^® CultiBag RM. Both bioreactors were controlled with regard to temperature, pH and % dissolved oxygen. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, polio virus production in both bioreactors was shown to be similar.     

Thermal profiling for parallel on-line monitoring of biomass growth in miniature stirred bioreactors

Recently we have described the design and operation of a miniature bioreactor system in which 4-16 fermentations can be performed (Gill et al., Biochem Eng J 39:164-176, 2008). Here we report on the use of thermal profiling techniques for parallel on-line monitoring of cell growth in these bioreactors based on the natural heat generated by microbial culture. Results show that the integrated heat profile during E. coli TOP10 pQR239 fermentations followed the same pattern as off-line optical density (OD) measurements. The maximum specific growth rates calculated from off-line OD and on-line thermal profiling data were in good agreement, at 0.66+/-0.04 and 0.69+/-0.05 h(-1) respectively. The combination of a parallel miniature bioreactor system with a non-invasive on-line technique for estimation of culture kinetic parameters provides a valuable approach for the rapid optimisation of microbial fermentation processes.     

Towards on-line monitoring of cell growth in microporous scaffolds: Utilization and interpretation of complex permittivity measurements

Here we demonstrate the ability to characterize microporous scaffolds and evaluate cell concentration variation via the utilization and interpretation of complex permittivity measurements (CP), a direct and nondestructive method. Polymer-based microporous scaffolds are of importance to tissue engineering, particularly in the promotion of cell adhesion, proliferation, and differentiation in predefined shapes. Chitosan gel scaffolds were seeded with increasing concentrations of macrophages to simulate cell growth. Complex permittivity measurements were performed using a dielectric probe and a vector network analyzer over a frequency ranging from 200 MHz to 2 GHz. An effective medium theory was applied to interpret the data obtained; respectively, Looyenga and Maxwell-Wagner-Hanai functions were used to retrieve the porosity and the variation of the cell concentration from the CP measurements. Calculated porosities were in agreement with experimental evaluation-porosity ranged from 81-96%. Changes in cell concentration inside the scaffolds upon injection of differing cell concentrations into the scaffold were detected distinguishably. Variations resulting from the cumulative injection of 400-1800 microL of 10(6) cells/mL solution into the scaffold were monitored. Results suggest that CP measurements in combination with an appropriate effective medium approximation can enable on-line monitoring of cell growth within scaffolds.     

Analysis of Vero cell growth behavior on microcarrier by means of environmental scanning electron microscopy

By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesion and spreading stage.     

Analysis of Vero cell growth behavior on microcarrier by means of environmental scanning electron microscopy

By using environmental scanning electron microscopy, the morphological changes of Vero cells attached to and grown on the microcarrier Cytodex-3 were observed, and their behavior of adhesion, spreading and proliferation was analyzed. The effect of exogenous fibronectin/ laminin on adhesion and spreading of MCC/Vero cell was studied. The images of ESEM showed that expansion of cell growth was directed toward vacancy space. The growth curve and cell concentration change during the whole culture process were obtained from the statistical counting method based on ESEM images and the crystal violet method. The growth rate of Vero cells increases with increasing the concentration of cell inoculation, that is, the specific growth rate increases quickly with increasing the concentration of cell inoculation. When serum concentration in medium #199 ranged from 5% to 10%, experimental results indicated that serum concentration is one of the important factors influencing cell growth, particularly in the cell adhesio     

Online- and offline- monitoring of stem cell expansion on microcarrier

In the biopharmaceutical industry, adherent growing stem cell cultures gain worldwide importance as cell products. The cultivation process of these cells, such as in stirred tank reactors or in fixed bed reactors, is highly sophisticated. Cultivations need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time on-line monitoring tools are recommended. A tool meeting the new requirements may be the dielectric spectroscopy for online viable cell mass determination by measurement of the permittivity. To establish these tools, proper offline methods for data correlation are required. The cell number determination of adherent cells on microcarrier is difficult, as it requires cell detachment from the carrier, which highly increases the statistical error. As an offline method, a fluorescence assay based on SYBR^®GreenI was developed allowing fast and easy total cell concentration determination without the need to detach the cells from the carrier. The assay is suitable for glass carriers used in stirred tank reactor systems or in fixed bed systems, may be suitable for different cell lines and can be applied to high sample numbers easily. The linear dependency of permittivity to cell concentration of suspended stem cells with the dielectric spectroscopy is shown for even very small cell concentrations. With this offline-method, a correlation of the cell concentration grown on carrier to the permittivity data measured by the dielectric spectroscopy was done successfully.     

Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements

The culture of HEK-293S cells in a stirred tank bioreactor for adenoviral vectors production for gene therapy is studied. Process monitoring using oxygen uptake rate (OUR) was performed. The OUR was determined on-line by the dynamic method, providing good information of the process evolution. OUR enabled cell activity monitoring, facilitating as well the determination of the feeding rate in perfusion cultures and when to infect the culture. Batch cultures were used to validate the monitoring methodology. A cell density of 10 × 10⁵ cell/mL was infected, producing 1.3 × 10⁹ infectious viral particles/mL (IVP/mL).To increase cell density values maintaining cell specific productivity, perfusion cultures, based on tangential flow filtration, were studied. In this case, OUR measurements were used to optimize the dynamic culture medium feeding strategy, addressed to avoid any potential nutrient limitation. Furthermore, the infection protocol was defined in order to optimize the use of the viral inoculum, minimizing the uncontrolled release of particles through the filter unit mesh. All these developments enabled an infection at 78 × 10⁵ cell/mL with the consequent production of 44 × 10⁹ IVP/mL, representing a cell specific productivity 4.3 times higher than for the batch culture.     

Hydrocyclones as cell retention device for CHO perfusion processes in single-use bioreactors

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20–25 days duration at cell concentrations up to 50 × 10⁶ cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.     

Growth behavior of number distributed adherent MDCK cells for optimization in microcarrier cultures

An assay for measuring the number of adherent cells on microcarriers that is independent from dilution errors in sample preparation was used to investigate attachment dynamics and cell growth. It could be shown that the recovery of seeded cells is a function of the specific rates of cell attachment and cell death, and finally a function of the initial cell‐to‐bead ratio. An unstructured, segregated population balance model was developed that considers individual classes of microcarriers covered by 1–220 cells/bead. The model describes the distribution of initially attached cells and their growth in a microcarrier system. The model distinguishes between subpopulations of dividing and nondividing cells and describes in a detailed way cell attachment, cell growth, density‐dependent growth inhibition, and basic metabolism of Madin‐Darby canine kidney cells used in influenza vaccine manufacturing. To obtain a model approach that is suitable for process control applications, a reduced growth model without cell subpopulations, but with a formulation of the specific cell growth rate as a function of the initial cell distribution on microcarriers after seeding was developed. With both model approaches, the fraction of growth‐inhibited cells could be predicted. Simulation results of two cultivations with a different number of initially seeded cells showed that the growth kinetics of adherent cells at the given cultivation conditions is mainly determined by the range of disparity in the initial distribution of cells on microcarriers after attachment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

Long-term continuous monitoring of dissolved oxygen in cell culture medium for perfused bioreactors using optical oxygen sensors

For long-term growth of mammalian cells in perfused bioreactors, it is essential to monitor the concentration of dissolved oxygen (DO) present in the culture medium to ascertain the health of the cells. An optical oxygen sensor based on dynamic fluorescent quenching was developed for long-term continuous measurement of DO for NASA-designed rotating perfused bioreactors. Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) chloride is employed as the fluorescent dye indicator. A pulsed, blue LED was chosen as the excitation light source. The sensor can be sterilized using an autoclave. The sensors were tested in a perfused rotating bioreactor supporting a BHK-21 (baby hamster kidney) cell culture over one 28-day, one 43-day, and one 180-day cell runs. The sensors were initially calibrated in sterile phosphate-buffered saline (PBS) against a blood-gas analyzer (BGA), and then used continuously during the entire cell culture without recalibration. In the 180-day cell run, two oxygen sensors were employed; one interfaced at the outlet of the bioreactor and the other at the inlet of the bioreactor. The DO concentrations determined by both sensors were compared with those sampled and measured regularly with the BGA reference. The sensor outputs were found to correlate well with the BGA data throughout the experiment using a single calibration, where the DO of the culture medium varied between 25 and 60 mm Hg at the bioreactor outlet and 80-116 mm Hg at the bioreactor inlet. During all 180 days of culture, the precision and the bias were +/-5.1 mm Hg and -3.8 mm Hg at the bioreactor outlet, and +/- 19 mm Hg and -18 mm Hg at inlet. The sensor dynamic range is between 0 and 200 mm Hg and the response time is less than 1 minute. The resolution of the sensor is 0.1 mm Hg at 50 mm Hg, and 0.25 mm Hg at 130 mm Hg.     

Evaluation of membranes for use in on-line cell separation during mammalian cell perfusion processes

In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristics under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristics of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (<30 KD) are produced.Abbreviations PP Polypropylene - PTFE Polytetrafluoroethylene