Extending In-Plane Impedance Measurements from 2D to 3D Cultures: Design Considerations

Abstract

Three-dimensional (3D) cell cultures have recently emerged as tools for biologically modelling the human body. As 3D models make their way into laboratories there is a need to develop characterisation techniques that are sensitive enough to monitor the cells in real time and without the need for chemical labels. Impedance spectroscopy has been shown to address both of these challenges, but there has been little research into the full impedance spectrum and how the different components of the system affect the impedance signal. Here we investigate the impedance of human fibroblast cells in 2D and 3D collagen gel cultures across a broad range of frequencies (10 Hz to 5 MHz) using a commercial well with in-plane electrodes. At low frequencies in both 2D and 3D models it was observed that protein adsorption influences the magnitude of the impedance for the cell-free samples. This effect was eliminated once cells were introduced to the systems. Cell proliferation could be monitored in 2D at intermediate frequencies (30 kHz). However, the in-plane electrodes were unable to detect any changes in the impedance at any frequency when the cells were cultured in the 3D collagen gel. The results suggest that in designing impedance measurement devices, both the nature and distribution of the cells within the 3D culture as well as the architecture of the electrodes are key variables.

A microfluidic platform with integrated porous membrane cell-substrate impedance spectroscopy (PM-ECIS) for biological barrier assessment

“…The authors suggested this may be due to an increase in conductivity that occurs as the culture medium hydrates the gel; notably though, this effect was less pronounced in hydrogels embedded with stromal cells, which were presumed to act as insulators against the current. Conversely, De Leon et al , using a planar electrode configuration, found that impedance of collagen gel increased with time over a range of 112 Hz to 31.2 kHz, which they attributed to protein and macromolecule deposition onto the electrodes 16 . The use of PM-ECIS, which measures cells directly grown on the electrodes, and is robust to the presence of hydrogel in other compartments ( Figure C ), circumvents these issues.…”

“…The geometries of microfluidic devices exacerbate the non-homogenous current distribution inherent to this technique, with TEER measurements in microfluidic devices tending to be of greater magnitude than in cell culture inserts 7 . Even with the use of geometric correction factors 4,5 , however, simple subtraction of the hydrogel devices fails to take into account the cell distribution and behaviours that may induce changes in the hydrogel, as well as the inherent stability of the matrix 16 . Indeed, there is evidence to suggest that hydrogel contribution to impedance itself changes over time.…”

“…One of the major challenges for incorporating TEER into organ on chip is that electrode size and placement is determined by device geometry, which impacts electric field uniformity; for example channel height has significant impact on the measured TEER values, as demonstrated by Yeste et al . 5 Conversely, planar electrodes penetrate a small volume and distance 16 , which can be leveraged to measure just the cell layer of interest, and suggests that there would be less of an influence of device geometry on ECIS values. Indeed, this idea is supported by data from this study.…”

“…For example collagen, collagen-hyaluronic acid, and Matrigel hydrogels have reported conductivities in the range of 0.02 – 0.6 mS/cm 11–13 , which is 2 to 3 orders of magnitude lower than that of cell culture medium 14,15 . Additionally, the presence of cells 10 , cellular phenomena - like gel contraction and deposition or degradation of extracellular matrix 16 - or even irregularities such as air bubbles 17,18 can all influence the electrical properties of a hydrogel. In such culture systems, with high inherent resistance and where multiple components are interacting over time, interpreting data acquired through electrodes in the standard parallel configuration can be challenging 16 .…”

Bridging barriers: advances and challenges in modeling biological barriers and measuring barrier integrity in organ-on-chip systems

“…272 Many OOC setups employ the use of biomaterials like hydrogels and co-culture with support cells, and simply subtracting hydrogel-only TEER values from cell culture devices does not take into account the effect of cell distribution and behaviours, nor the inherent stability of the matrix on the hydrogel's electrical properties. 273,274 Finally, common practices of reporting TEER, impedance spectroscopy and ECIS outputs as normalized values can mask flaws in the raw data, as well as making it difficult to compare results across publications. 178…”