4. 3D impedance tomography
In 3D cell culture, obtaining spatial resolution is of interest as it allows researchers to determine the location of cells over time, monitor the specific cell health and helps to highlight differences in cell health and function in different areas of the tissue. Electrical impedance tomography (EIT) is a relatively new technique that has emerged as a potential solution to provide better spatial resolution within 3D tissue cultures. However, most recent efforts in EIT have been focused on its use in vivo and as a medical diagnostic, where electrodes are placed on the body to obtain an image and health status of an organ of interest.(Fernandez-Corazza et al., 2018; Schwartz, Chauhan, & Sadleir, 2018; Sun, Yue, Hao, Cui, & Wang, 2019)
Similar to the implementation of EIS, implementing EIT involves the application of a small current to a sample, such as the human body, tissue model or inanimate objects with different conductive properties (generally used for prototyping), and the voltage is measured in order the obtain the electrical properties of the sample.(Chitturi & Nagi, 2017) Different electrode designs, current injection strategies and image reconstruction algorithms have been recently reviewed by Chitturi and colleagues.(Chitturi & Nagi, 2017) In general, the set up involves a matrix of electrodes that is placed around the sample, a current is injected through a pair of electrodes while the voltage changes are measured through a different pair of electrodes. The values obtained are then used to calculate the resistivity of the sample and reconstruct the image using the chosen algorithm.
The successful use of EIT as a medical diagnostic and its ability to obtain in vivo information, suggests that it may be an effective monitoring technique for future 3D cell cultures. Researchers have demonstrated the measurement of 3D tissue impedance and subsequently reconstruction of the respective images using EIT.(Jamil et al., 2016; Wu, Yang, Bagnaninchi, & Jia, 2017, 2018; Wu, Zhou, Yang, Jia, & Bagnaninchi, 2018; Yang et al., 2017; Yin, Wu, Jia, & Yang, 2018; Yin, Yang, Jia, & Tan, 2017) For monitoring 3D cell aggregates or spheroids, two electrode distribution designs were implemented. In one study, circular electrodes were radially distributed at the bottom of a well, in which case current was injected through a pair of electrodes and voltage was measured at the adjacent pair, while the reference electrode was connected to ground (Figure 7a). In another study, current was applied to rectangular electrodes placed at the boundary of the well and voltage was measured as shown in Figure 7b. Using both electrodes designs the authors were able monitor the presence and growth of spheroids(Yin et al., 2018) as well as spheroid disintegration upon the addition of Triton-X 100 (Figure 7c). However, the resolution of the output is not yet at the cellular level as shown by the heat maps in Figure 7c. Nevertheless, compared to EIS, additional information can be gathered using EIT including the position and potentially the size of spheroids and tissues in culture systems. Given the issue of contraction in some collagen-based 3D models, as well as the presence of contraction in wound healing models,(Lotz et al., 2017) a potential application for EIT could be to monitor contraction over time or after wounding in these models. Others have designed similar set ups to those demonstrated in Figure 7, with the eventual aim of monitoring cell cultures, but have not yet tested it using cells.(Z. Xu et al., 2018)
Likewise, a more complex setup of electrodes was designed with the intention of monitoring 3D cultures using EIT.(E. J. Lee et al., 2014) The quality of the designed device (Figure 8) was assessed using performance metrics such as crosstalk, amplitude stability error, total harmonic distortion and signal to noise ratio. While this system was not tested using cells, the high quantity of electrodes positioned all over the well (Figure 8) could potentially increase the final image resolution and subsequently biological output, as more data points may be obtained from the well. Conversely, having electrodes in such close proximity may also increase the crosstalk and artefacts such as stray capacitance. A fine balance between the number of electrodes and electrode design is needed to obtain optimal resolution in the presence of 3D cell cultures.
The above studies have demonstrated a prospective use for EIT in 3D cell culture monitoring, however current applications do not offer a clear advantage over EIS. EIS is comparatively simpler and may be more cost effective in obtaining the electrical properties of a biological 3D sample. While EIT may be an attractive solution for better spatial resolution in the monitoring of 3D cultures, the current sensitivity obtained can only give qualitative information at the tissue level as opposed to information at the cellular level. Achieving resolution at the cellular level will give a better indication of what is occurring as cell cultures grow over time, after treatments and within different regions of the 3D culture.
 
5. Future directions
3D culture is expanding across research and industrial laboratories, as it offers the possibility of more physiologically relevant models for drug testing and replication of diseased tissues. Therefore, there is a need to characterize and standardize monitoring techniques across different laboratories. This will increase the scope of 3D culture as well as ease its implementation.(Verjans et al., 2017) Further it will allow researchers to select matured tissues that are physiologically relevant in their contexts and allow them to perform experiments in a more controlled environment where variability within cultures can be monitored. However, a lack of monitoring techniques represents a limitation for further growth. Developing monitoring techniques targeted for 3D cultures will allow for easier implementation and standardization of these cultures. The development of reproducible, scalable and validated 3D cell culture systems that can handle a diverse range of cell types and co-cultures whilst providing real-time feedback on culture health will improve our fundamental understanding of biology. The ability to monitor the cultures as they grow and assess their viability prior to testing them with an external stimulus will accelerate the rate of discovery. 
Multiple approaches to integrating electrodes into 3D cell culture systems have enabled the monitoring of cell health over time. To date, researchers have developed in situ sensors using EIS to monitor cell proliferation and viability. Furthermore, they have created devices with functionalized electrode surfaces to enable the measurement of the metabolic activity of 3D cell cultures. However, each of these devices are highly customized for the specific application and 3D culture technique, making standardization difficult. In future, EIT may provide better spatial resolution, but the currently achieved resolution remains low and the data gathered can also be obtained with EIS, which utilizes simpler hardware and data analysis. An ideal monitoring system for 3D cell culture would be able to monitor multicellular models in situ and in real-time. Having an ideal spatial resolution at the cellular level (~50 µm) will allow monitoring of specific cells in a multicellular construct and their state within the system (live, death differentiated). Impedance alone can achieve real-time monitoring of cell growth and proliferation, however, it should be complemented and correlated with measurements from the monitoring of other cell functions such as metabolic activity which can be detected downstream in the media using functionalized surfaces as transducers or sensors of interest. 
 
Acknowledgements
The authors gratefully acknowledge funding from the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Probing Biosystems Future Science Platform, CSIRO Research+ Science Leader Scheme and Swinburne University of Technology for the PhD stipend and scholarship for SDL.