Surface-patterned electrode bioreactor for electrical stimulation

Abstract

We present a microscale cell culture system with an interdigitated microarray of excimer-laser-ablated indium tin oxide electrodes for electrical stimulation of cultured cells. The system has been characterized in a range of geometeries and stimulation regimes via electrochemical impedance spectroscopy and used to culture primary cardiomyocytes and human adipose derived stem cells. Over 6 days of culture with electrical stimulation (2 ms duration, 1 Hz, 180 microm wide electrodes with 200 microm spacing), both cell types exhibited enhanced proliferation, elongation and alignment, and adipose derived stem cells exhibited higher numbers of Connexin-43-composed gap junctions.

Figure 1. Indium-tin-oxide patterned substrate

(A) Schematic of the laser-ablation process of the indium-tin-oxide substrates. (B) Closeup image of patterned electrode array with 200 μm electrodes and 200 μm spacing (scale bar corresponds to 200 μm). (C) Atomic force micrograph of a 50 μm-wide electrode patterned via laser ablation (scale bar: 50 μm) (D) Photograph of slide patterned with two interdigitated electrode arrays. (E) Photograph of bioreactor with two culture wells (employed for all studies reported here).

Figure 2. Electrical characterization of patterned ITO substrate

(A–B) Bode plot of (A) electrode impedance and (B) phase versus frequency for a new (solid) and aged (dotted) ITO electrode array (used for 6 weeks of continuous stimulation) with 180 μm electrodes and 200 μm spacing. (C) Graph of current for ITO electrode array with 180 μm electrodes and 200 μm spacing and input stimulus of 250 mV. (D) Equivalent circuit for modeling the electrochemical behavior of the ITO electrode array.

Figure 3. Finite-element modeling

(A,B,D) A colormap of electric potential and (C) potential vs. position plot are shown for an ITO electrode array with 200 μm electrodes and 200 μm spacing with an applied voltage of 1 V. (A) top view of five pairs of positive and negative electrodes. (B) close-up of the space between a single positive and negative electrode (dashed lines indicate electrode edges). (C) plot of potential vs. position at different heights above the glass surface between the electrodes. (D) cross-sectional view of the electrical field lines between electrodes. Scale bars: (A) 300 μm, (B) 100 μm, (D) 200 μm.

Figure 4. Electrical stimulation of neonatal rat cardiac cells on an ITO electrode array with 180 μm electrodes and 200 μm spacing

Bright field images of cells at day 4 of culture either with (A) or without (B) electrical stimulation. Arrow in section A shows the direction of the applied electric field. Graph (C) represents the percent area beating per well for cardiac cells cultured with (blue) or without (pink) electrical stimulation for a range of testing stimuli. Scale bar corresponds to 200 μm.

Figure 5. Effects of electrical stimulation on hASCs

(A,B) F-Actin (red) and DAPI (blue) of ASC culture with (A) or without (B) electrical stimulation. (C,D) Connexin-43 (green) and DAPI (blue) of ASC culture with (C) or without (D) electrical stimulation. Arrows in sections A and C show the direction of the applied electric field. Graphs (E, F, G) show (E) the number of measured gap junctions per cell, (F) the number of cells measured per mm² and (G) the direction of actin fibers for ASCs cultured with (+ES) or without (−ES) electrical stimulation. *represents statistically significant difference (p<0.01). Scale bar corresponds to 100 μm.