PMC

An AFM surface scan of the MEA electrode wiring. The scan shows the height of the electrode wirings to be approximately 600 nm. The height was determined by averaging the height profile from an 8-µm-long wiring track.

Expression of troponin T (red) in control (A) and in stimulated (B) samples; blue indicates cell nuclei (scale bars 200 µm).

Experimental design. (A) Experimental groups for long-term stimulation where NRCs were cultured either on gelatin coating (1-3) or on collagen gel (4). Parameters used for electrical stimulation were biphasic 100-ms square pulses at 5.3 V/cm and 1 Hz (1), biphasic 2-ms square pulses at 5.3 V/cm and 1 Hz (2), and monophasic 2-ms square pulses at 5 V/cm and 1 Hz (3 and 4). (B) Experimental timelines for each stimulation group. Control samples were cultured for the same amount of time, but without electrical stimulation. CS, cell seeding; EFS, electrical field stimulation initiation; END, experimental ends.

Cell viability. In addition to cell viability, the orientation of NRCs on gelatin coating parallel to the electrode lines of the MEA chambers is evident in the live/dead stainings (live cells green, dead cells red) in both control (A) and stimulated (B) samples. The tube formation of cells on collagen gel in experiment IV is clearly evident from the live/dead stainings in both control (C) and stimulated (D) samples. Arrows denote tube-like structures, which were observed in both stimulated and control samples. The direction of the electrical field is horizontal in the pictures. Scale bars are 200 µm.

Functional properties of long-term stimulated NRCs. Electrical activity of control NRCs (A) and stimulated samples (B) in experiment II before stimulation (1) and after 5 hours (2), 24 hours (3), and 48 hours (4) of stimulation. (C) Cx-43, MYH-6, and MYH-7 expressions from each experiment determined by qPCR at the end of culturing. Statistical significance is marked with (*p<0.05). Number of control vs stimulated samples analyzed as triplicates: Exp. I n=2 vs n=3, Exp. II n=3 vs n=4, Exp. III n=2 vs n=2, Exp. IV n=3 vs n=3. Error bars are SD. (D) Gene expressions from each experiment determined by RT-PCR at the end of culturing. S, electrically stimulated samples; C, control samples.

(A) The stimulation system. The stimulation sequence is designed with PC software that controls the NI USB-6008 DAQ device. The waveform that the device produces is amplified and delivered to the MEA container, where the cells reside on MEA dishes; (B, C) The simulated result of the electrical field on an MEA dish; (B) If the cells to be stimulated are placed on the MEA electrodes in the middle of the MEA dish, they will experience a fairly uniform 500 V/m electrical field. The image is plotted at the height of 0.1 mm; (C) A lateral view from the center line shows that the electrical field diminishes at the bottom of the MEA close to the electrodes. The MEA dish has been included in the simulation as an insulator at the bottom; (D) The designed stimulation system, consisting of the stimulation electronics, PC, and the MEA dish container; the MEA dish container appears in detail on right, with an individual electrode shown in detail at the bottom.