A. Fontova

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.

Four Versus Two-Electrode Measurement Strategies for Cell Growing and Differentiation Monitoring Using Electrical Impedance Spectroscopy

The aim of this work is to provide optimization tools for cell and tissue engineering processes through continuous monitoring of cell cultures. Structural cell properties can be obtained from non-destructive electrical measurements by using electrical impedance spectroscopy (EIS). EIS measurements on monolayer animal cell cultures are usually performed using a two-electrode strategy. Because of this, the measurement is very sensitive to the electrode covering ratio and to the degree of adherence of cells. Of course, these parameters give useful information but can mask the behaviour of the cell layer above the electrodes. In a previous work, preliminary measurements with commercial microelectrode structures were performed with simulated grow processes using the settlement of cell suspensions with two and four microelectrode strategies to validate the technique. In this work, real cell growths of Vero cells are described and the resulting EIS biomass density estimators are compared to cell counts. The four-electrode impedance spectra are fitted to the Cole-Cole impedance model and the time course of their parameters are extracted and studied

Multiple automated minibioreactor system for multifunctional screening in biotechnology

The current techniques applied in biotechnology allow to obtain many types of molecules that must be tested on cell cultures (high throughput screening HTS). Although such tests are usually carried out automatically on mini or microwell plates, the procedures in the preindustrial stage are performed almost manually on higher volume recipients known as bioreactors. The growth conditions in both stages are completely different. The screening system presented in this work is based on the multiwell test plates philosophy, a disposable multiple minibioreactor that allows reproduction of industrial bioreactor culture conditions: aeration, stirring, temperature, O2, pH and visible range optical absorbance measurements. It is possible to reproduce the growth conditions for both suspended and adherent animal cell types using 1 to 10 ml vol. bioreactors. In the case of bacteria or yeast, it is not possible to achieve a high biomass concentration, due to the reduced head volume air supply

A Multiple Minibioreactor Platform for Parallel and Automated Mammalian Cell Culture

The multiple minibioreactor platform Hexascreen® capabilities to perform screening experiments with adherent and suspension cells were evaluated. In the case of suspension cells using various concentrations of serum, differences in growth and metabolism were observed. In the case of culturing adherent cells with various Neomycin concentrations, due to the impossibility of performing optical measurements of cell concentration, the differences among the cultures were observed using metabolic indicators

Continuous oxygen consumption estimation method for animal cell bioreactors based on a low-cost control of the medium dissolved oxygen concentration

The applications of animal cell cultures are becoming wider every day: protein and vaccine production, toxicity tests, development of tissue and cell therapies, as well as stem cell research. All of these issues, require the use of reliable bioreactors to ensure reproducible culture conditions and data collection. Some common functions of these systems are aeration, stirring, thermoregulation, pH control, so as measurement of variables like biomass density, pCO2, pO2, etc. However, for certain cell species, the traditional probes are not able to provide enough data to evaluate the cells metabolic response, in such cases the study of oxygen consumption becomes a useful tool, where OUR (Oxygen Uptake Rate) is one of the key parameters commonly used.

A Simplified implementation of the O.U.R. stationary liquid mass balance estimation method for On-line monitoring in Animal cell production processes

Many efforts have been invested in the development of culture strategies for animal cell culture process. Several strategies have been studied: batch, fed-batch and perfusion cultures [1]. For implementation of those strategies, a monitoring system for automated, controlled and optimised processes based on simple measurement could be of great interest. Oxygen is a key substrate in animal cell metabolism and its consumption is thus a parameter of great interest for bioprocess monitoring and control. The application of the OUR (Oxygen uptake rate) is investigate here. The main advantages of OUR is that correlates well with the physiological state of cells and also for the prediction of viable cell concentration [2-4]. Different methods for the oxygen uptake rate (OUR) determination in animal cell cultivation have been developed: Dynamic estimation in the liquid phase, the global mass balance in the gas phase, and the Stationary liquid mass balance. Dynamic estimation has a considerable disadvantage because of disturbances suffered by the growing cells because of the necessary variations of the DO concentration. Gas phase balancing has several advantages; knowledge of the kL·a value is not necessary, and yields a higher density of accurate data. However, it has not historically been widely used due to the need for complex and expensive instrumentation like mass spectrometers and extremely accurate DO control systems. The Stationary liquid mass balance method offers minimum cell stress and greatest imation accuracy, but still needs for a significant investment in mass flow controllers as well as some additional instrumentation to determine the oxygen’s molar fraction in the gas phase. Background This works offers a simplified embodiment of the Stationary liquid mass balance method for the continuous estimate of the OUR estimation by means of the use of inexpensive proportional valves and the monitoring of their control signals is introduced and compared with the Global mass balance and Dynamic methods. As far said electrovalves can be considered to be linear, it can be demonstrated through the Mean value theorem for integrals that the mass balance equation for the liquid phase can be expressed as a first order differential equation:

Four Electrode EIS Measurement on Interdigitated Microelectrodes for Adherent Cell Growing and Differentiation Monitoring

Protocols employed in cell and tissue engineering usually involve destructive tests to characterize the cell cultures, mainly in the case of adherent cells. Impedance spectroscopy obtained with planar electrodes is an alternative to perform non-destructive, on-line measurements. The antecedents of this technique use the two-electrode strategy, then providing high sensitivity but also a high dependence on the electrode coverage. In order to improve the accuracy and to separate cell density from coverage and adhesion we use a four electrode-strategy with interdigitated microelectrodes. The preliminary results have been previously reported. In this communication a set of results obtained with animal cell cultures are presented. The measurement set-up consists of miniature bioreactors (10 ml) where a double set of interdigitated microelectrodes (AbTech) has been inserted. They are connected to an impedance analyser through a custom front-end. The set is placed in an incubator together with parallel cultures in order to provide control cell counting along the 7 days cell growing. The four-electrode follow-up method provides higher sensitivity (63%) than the two-electrodes method, showing this last one a reduced response mainly near the culture end.