(B-63) Electrochemical Impedance Spectroscopy for Monitoring Cell Growth and Maturation in Hydrogel-Based Microfluidic Platforms

Introduction:: To monitor cell growth and stimulation without interfering with the biological response of cells, a non-invasive method is needed. Electrochemical impedance spectroscopy (EIS) can be used to measure cell resistance label free system [1]. Integrating electrodes into microfluidic platforms makes it able to detect the electrochemical signal with high sensitivity and faster analysis. A continuous process that eliminates the need for post-processing and integrates electrodes during microfluidic platform fabrication is critical for high-throughput screening applications [2, 3]. We propose using PEDOT:PSS, a tunable biocompatibile polymer, for electric stimulations in our hydrogel-based microfluidic devices. PEDOT:PSS is a conductive material for our light-assisted 3D-printed microfluidic design and can be translated into large scale biofabrication [3].

Materials and Methods:: We recently optimized the process of making 3D-printed microfluidic chips. We fabricated electrodes 3D constructs on a metal-coated glass substrate using used a multi-material bioprinter [4]. We used our recent publication for the ink formulation (e.g., E = 0.5 W/cm²) [4]. For introducing conductivity, we incorporated poly (3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOT:PSS) particles doped into polyethylene glycol diacrylate (PEGDA), with a concentration of 3.0-4.0% in water, and of high-conductivity grade purchased from Sigma-Aldrich, MO) doped into polyethylene glycol diacrylate (PEGDA). We tested the ink’s printability and mechanical properties for a selected range of PEDOT:PSS mass concentrations: 0, 0.5, 1, 3, and 5% w/v in PEGDA (30% v/v). The conductivity of the ink was measured using the four-probe method in different concentrations of conductive particles from 0.5 to 5% (w/v) in the hydrogel ink. The study utilized impedance analysis to investigate the electrochemical biosensing capabilities of a hydrogel matrix with varying densities of mouse myoblast cells (C2C12, ATCC) in the frequency range of 1kHz to 1MHz. Impedance measurements have been performed during AC electrical stimulation to measure the change in cell resistance in the system.

Results, Conclusions, and Discussions:: The physical testing of the 3D printed conductive bioink in different concentration shows homogenous distribution of PEDOT:PSS is different concentration. Our conductivity data indicated the lowest value at 0.5% w/v PEDOT:PSS and the highest at 5% PEDOT:PSS, with no predictable change in conductivity when varying the number of layers in the light-assisted 3D printing process (Fig.1 ). Electrochemical impedance spectroscopy was finally performed to measure charges transfer resistance (R2) in the GelMA system bioreactor. The results showed an increase in cell density resulting in an impedance increase most significantly in the frequency below 25 KHz (Fig.1 D). Electrical Stimulation as expected showed an increase in myoblast cell maturation and impedance increase for stimulated cells in comparison to the control condition.

We optimized the printability of the hydrogel with various concentrations of conductive particles, finding that concentrations above 5% w/v reduced the elastic modulus of the printed structure. The observed increase in impedance with cell density has implications for the potential use of the system as a biosensor. Biosensors typically rely on changes in electrical properties to detect and quantify biological analytes. The observed relationship between cell density and impedance suggests that this system could potentially be used as a biosensor to detect changes in cell density or other biological markers. This system can enable the measurement of cell proliferation dynamics, the investigation of cellular responses to various stimuli, and the application of impedance spectroscopy and electrical stimuli for disease treatment [5]. Cell alignment that is due to the electrical stimulation plays role in contraction of matured myoblast cells.


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References (Optional): : [1] A. Olmo, Alberto, et al. Sensors, 2020; [2] X. Wang., et al, Analytical Methods, 2022; [3] D. Heo., et al, Mater Sci Eng C, 2019; [4] A. Bhusal., et al. ACS appl bio mater, 2022; [5] C.Yang., et al, Nat commun, 2021.