Cell Status Monitoring using a Real Time Cell Analyser (label free)

10th August 2020

Category: Bioanalytical / Biosimilars

By: Debbie Allan, PhD - Senior Scientist, Product Development,

The RTCA MP instrument (ACEA Biosciences, Agilent) is designed for label-free cell based assays. A microelectronic biosensor array is incorporated into each well of the 96 well microplate, allowing the RTCA MP (multiple plate) (Figure 1) instrument to measure the activity of living cells in real-time without the use of labels. This instrument reliably detects cellular parameters such as attachment, spreading, growth, death and morphological changes.

Figure 1 RTCA MP Instrument Components

The analyser and control unit are located outside of the incubator, with the RTCA MP Station containing 6x 96 well E plates remaining within the incubator to monitor the cell status. The basis of the system involves cell sensor arrays that are integrated into the bottom of each E Plate which measures electronic impedance of the sensor electrodes. (Image taken from Acea Bioscience User manual) (Figure 2).

Figure 2 Impedance Change

 The Cell Index (CI) is used to measure the relative change in the electrical impedance which represents cell status. In the absence of cells (Figure 2A) the impedance will be small, this is the baseline and is represented by the below equation:

Z= Z0

The more cells that adhere to the electrodes (Figure 2B and C) or the stronger the adhesion, the larger the impedance. Furthermore, cell division over time results in more cells which in turns increases the impedance and CI.

CI = (Zi – Z0 )/ 15 Ω

Zi = impedance of an individual time point

Z0 = impedance at the start of the experiment

The measurement of impedance is a label free technique therefore providing a unique method for bioassay development and performance which is not limited by a traditional end point assay.

Possible applications for this technology include, cell adhesion, cell spreading, proliferation, differentiation, compound mediated cytotoxicity, apoptosis, cell mediated cytotoxicity, quality control of cells and virus mediated cytopathogenicity. Below are examples of how Sartorius Stedim BioOutsource have used the instrument in house for understanding the best passage routine for a cell line (Figure 3) and the best E:T ratio to use for an ADCC assay (Figure 4).

Figure 3 Development of Optimal Passage Routine

An adherent cell line was cultured for 94 hours in PBS alone (red), culture media with no refeed (green) and culture media with a refeed (blue). From the data presented cells seeded in PBS do not adhere or proliferate, as expected. Cells cultured in media adhere and proliferate with an optimal passage after around 60 hours in culture. The data has also shown that replenishing the old media with new media has no effect on proliferation or optimal split time. Using this information we can learn more about the cell lines we use on a routine basis to develop and qualify accurate and precise bioassays with reduced variability.

Figure 4. Determining an Optimal E:T ratio

Here, an ADCC (Antibody Dependent Cellular Cytotoxicity) assay was performed with a dilution of effector cells (dilution A- G) with target cells plus effector cells with no antibody (control). Figure 4A shows the full effector to target (E:T) dilutions investigated. The E:T ratio which reduced the impedance the most is the optimal E:T ratio as this indicated the cells are lifting off the electrodes due to effector cell mediated death. Figure 4B shows the full experiment outcome for the optimal E:T ratio (A) with and without antibody. The data indicated a rapid specific cell death response with E:T (A) which the target cells did not recover from.

The RTCA MP instrument allows for up to 6 independent experiments to be ran in parallel for days or weeks at a time. Data can be obtained throughout the experiment due to the lack of ‘end point’ reagents. This innovative technology provides another exciting tool to the biologists at Sartorius Stedim Biooutsource to aid in the development and performance of robust bioassays to meet client expectations.

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