3. Multi-parametric monitoring of 3D cultures
Impedance changes are a compilation of multiple processes occurring in cell culture systems. In the case of 2D cultures, significant impedance changes have been successfully correlated with cell proliferation and death. However, in 3D cultures, cellular processes are more complex, thus impedance changes may be influenced by different factors such as matrix degradation, cell spreading, migration or proliferation. This raises the need for a secondary or multi‑parametric approach to further understand, monitor and validate 3D culture systems. In‑line monitoring systems coupled with impedance can lead to better real-time and multi‑parametric biological understanding of complex 3D systems. Zhang et al. developed a multisensor-integrated organ-on-chip platform that contains two organ modules, one physical/chemical sensing module and one bioelectrochemical sensing module, all connected via a perfused system.(Zhang et al., 2017) The physical/chemical sensing system consisted of optical pH and oxygen sensors. Additionally, gold electrodes functionalized with desired antibodies for specific detection of molecules in the media could be incorporated into the bioelectrochemical sensing module. The EIS changed upon binding of the target molecule and detection of different concentrations of the analyte/biomarker was possible. They found that the amount of biomarker that could be captured by the functionalized electrode surface was proportional to the concentration in the solution, with detection levels of 0.1 to 100 ng/ml, similar to many commercial ELISA systems. Thus, biologically relevant concentrations were detected. Even though this is a complex system, it contains fundamental parameters for real-time monitoring of organoids. However, this system is highly modular and specific to the microfluidics system they utilize. This makes it harder to standardize and integrate these systems with current tissue culture consumables and microscopes. Nevertheless, to date, this is the most complete multi-parametric monitoring system for organoids.
Others have also used a multi-parametric monitoring approach coupled with electrical measurements to monitor 3D cell cultures. Misun and colleagues correlated amperometric (current) changes to glucose and lactate concentrations in spheroids, giving real-time information on the metabolic state of these microtissues. For the detection of glucose and lactose in these spheroids, a microfluidic polydimethylsiloxane (PDMS) chip with an enzyme-based hydrogel coated onto sensing electrodes was proposed.(Misun, Hierlemann, & Frey, 2018) However, the sensor chip and well was limited in its use as it is specifically designed for spheroid growth via the hanging-drop culture technique. The sensor also depends on the effective release of glucose from the spheroid to the media. Another approach to monitor glucose concentrations and cell proliferation was shown by Curto et al. They developed an in vitro cell monitoring platform, which monitored the impedance changes of tissue by means of an organic electrochemical transistor (OECT) at the bottom of the well.(Vincenzo F. Curto et al., 2017) Glucose was also monitored with an OECT-based glucose biosensor, but the glucose biosensor was not in situ. Media was collected at the outlet of the perfused system and later measured with the sensor. This multi parameter system allowed the monitoring of kidney cell metabolic state via the glucose sensor as well as changes in resistance and capacitance in real-time while the kidney cell epithelium recovered from an electrical wound.
All of the above systems allow for real-time monitoring of the metabolic activity of cells through measuring glucose, pH and oxygen levels in the media. Even though they are sensitive, they depend on the efficient release of the molecule of interest into the media, which can be limited in a 3D cell culture system. The lack of spatial resolution displayed by most of the monitoring techniques within these devices remains a drawback. Correlating metabolite levels in media with a technique that gives information about spatial resolution would allow for more specialized biological information to be gained from the monitoring system.