Monitoring cell behaviour through real-time impedance analysis

Supporting biologically relevant potency assay design and implementation with label-free techniques in advanced in vitro models

The development of highly innovative medicinal products, including those containing or derived from living cells, has raised the need to establish robust and reliable methods to assess their biological activity, ensuring consistent potency across batches and throughout their lifecycle.

The biological activity/functionality describes the specific ability or capacity of a product to achieve a defined biological effect. Potency is a quantitative measure of biological activity based on the product attribute and is linked to the relevant biological properties (ICH 6QB). Due to their complexity, biological medicinal products (MP) cannot be fully characterised through well-defined chemical properties, as their activity is intrinsically linked to interactions with living systems. As a result, assays designed to demonstrate biological activity should be based on the intended biological effect and ideally be predictive of a clinical response.

A combination of multiple methods may be used to adequately define the potency of a biological MP. Among these, in vitro cell-based assays enable the measurement of biochemical or physiological responses at the cellular level, providing functional insights into the product’s activity.

If the intended biological effect targets tissue repair and regeneration, these responses often depend on complex and progressive cellular behaviours, including cell adhesion, proliferation, migration, and barrier formation. Traditional endpoint assays may not fully capture these dynamic processes, potentially limiting their ability to reflect the true functional activity of the product. In this context, there is a growing need for analytic capabilities better capable of assessing the full extent of the MP potency.

Electric Cell-Substrate Sensing (ECIS)

The ability to capture dynamic changes in cell behaviour over time is becoming essential, driving the need for analytical capabilities that enable continuous, real-time monitoring of cellular responses in non-invasive and label-free approaches for more robust Quality Control (QC).

Electric Cell-Substrate Impedance Sensing (ECIS) is a real-time, label-free, impedance-based technique that enables the monitoring of several cellular behaviours, including proliferation, adhesion and barrier formation, as well as detecting cell alterations (including changes in morphology). By applying a low, non-intrusive alternating current through gold thin-film electrodes located at the bottom of tissue culture wells, ECIS measures changes in impedance associated with cell attachment and morphological alterations, using the electrodes as substrates.

When an electric potential is applied, current flows between the electrodes through the conductive culture medium. As cells proliferate, they attach and spread across the well, partially obstructing the electrode surface and impeding current flow, resulting in measurable changes.

The extent of this impedance change is influenced by several factors, including cell number, size, morphology, and the quality of substrate attachment. As a result, impedance measurements can be directly correlated with dynamic cellular behaviours and allow to determine real-time functional activity.

The gold microelectrode surface and the applied electric potential (22 mV) do not affect cell health and behaviour.

Impedance-based measurements provide functional insight into cellular activity by enabling continuous monitoring of biologically relevant processes in real-time. As mentioned, impedance changes reflect the interaction between cells and the electrode surface and can therefore be directly associated with dynamic cellular responses and functional states.

This approach allows the assessment of fundamental cellular processes such as adhesion and proliferation, as well as more complex functional behaviours including barrier formation, cytotoxicity, morphological remodelling and cell spreading, and cellular responses to external stimuli. By monitoring these events continuously, ECIS generates kinetic data that can reveal both the onset and progression of a biological response, offering a more comprehensive functional profile than conventional endpoint assays.

Integration of ECIS in QC potency assay development

When the intended biological effect of an MP involves tissue repair/barrier restoration, the selection of a biologically relevant in vitro model becomes critical for establishing a functional potency strategy. Epithelial cell models can provide a suitable system for evaluating regenerative responses, given their role in tissue repair, barrier maintenance, and cellular reorganisation following injury or in the case of an underlying condition.

In this context, the integration of impedance-based monitoring enables the continuous assessment of key cellular processes involved in regeneration, including adhesion, proliferation, and migration, generating functional readouts that can be directly linked to the intended biological claim of the MP.

To further assess regenerative capacity, an in vitro injury model such as a scratch assay can be integrated with ECIS monitoring to evaluate barrier recovery over time. By tracking impedance changes throughout the repair process, it becomes possible to characterise the kinetics of cellular recovery and quantify the effect of the active substance on tissue regeneration-related cellular behaviour.

ECIS readout

In ECIS-based systems, cellular impedance of electron flow caused by adherent cells is reported using a unitless parameter designated Cell Index (CI), representing a relative change in impedance caused by cell interaction with the electrode surface. The CI at each time point is defined as the normalisation of the difference between the cell-electrode impedance of the well when it contains cells and the background impedance of the well with the media alone.

Rather than representing an absolute physical quantity, the CI reflects relative variations in cell adhesion, proliferation, morphology, and barrier formation over time, thereby providing a comparable representation of impedance changes across wells and experimental conditions.

Developing potency assays for ATMPs requires more than analytical tools: it requires the understanding of the underlying biology and the expertise to translate that understanding into robust, measurable functional readouts.

At Stemmatters,real-time cellular analysis (RTCA) capabilities support the development, optimisation and implementation of advanced in vitro functional assays,  with the incorporation of the xCELLigence RTCA system (Agilent).

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