Poster BB10 - Multi-Well Bioreactor for Electrical Stimulation during Cardiomyocyte Differentiation

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Lauren K. Mabe (she/her/hers)

Student
University of North Carolina at Chapel Hill and North Carolina State University, United States

Co-Author(s)

JT

Jack Twiddy, MR

Research Assistant
University of North Carolina at Chapel Hill and North Carolina State University, United States
Suh Hee Cook, MS, PhD (she/her/hers)

Postdoctoral Fellow
University of Maryland
College Park, Maryland, United States
JG

Jessica Gluck, MS, PhD

Assistant Professor
North Carolina State University
Raleigh, North Carolina, United States

Primary Investigator(s)

Michael Daniele, PhD

Professor & University Faculty Scholar
Joint Department of Biomedical Engineering UNC-Chapel Hill / NC State University, United States

Introduction: According to the American Heart Association, 1 in 5 heart attack victims will have a second heart attack within 5 years [1]. This danger comes from the inability of the heart to remodel itself after injury, revealing a vital need to develop regenerative cell therapies. As such, researchers have been attempting to develop cardiomyocytes from stem cells. The contractile function of cardiomyocytes relies upon electrical signals, and previous studies have demonstrated that when electrical stimulation was provided during differentiation, coordinated depolarization and contraction was enhanced [2]. We have engineered a bioreactor that supplies electrical stimulation to cell cultures in a 12-well plate using carbon paper electrodes, affixed using a custom 3D-printed adapter [3]. To create a more robust system, the signal output was tested in varied loading conditions in an attempt to improve reliability for future versions. The goal of the improved bioreactor is to encourage research among other groups concerning electrical stimulation during cardiomyocyte differentiation through dissemination of our technology.

Materials and

Methods: The bioreactor includes on-board regulation, overcurrent protection, and reverse polarity protection to provide a precise stimulation voltage and to protect the circuit. To generate the signal, a relaxation oscillator built using a 555 timer creates a square wave with an adjustable frequency and duty cycle, set to 200 Hz and 20% on-time, respectively, for this experiment. The signal is then separated into four channels, each attenuated to a distinct stimulation amplitude, chosen for this experiment to be 10 V, 1 V, 0.1 V, and 0 V. Laser-cut carbon paper electrodes deliver the stimulation waveform to the cells, with a “clip-in” system allowing for easy electrode replacement in the case of damaged electrodes or contamination. Each channel applies its voltage simultaneously across three parallel wells of the culture plate. A custom-fabricated 3D-printed lid acts as an interface between the bioreactor and the 12-well plate (Figure 1). Notches in the lid as well as slits for the carbon paper electrodes allow for secure, reliable, and precise placement of the stimulation hardware relative to the culture wells.

Results, Conclusions, and Discussions: Phosphate-buffered saline (PBS) pipetted in 1 mL increments into culture wells within the (nominal) 10 V channel resulted in a measured voltage ranging from 7.68-9.84 V (Figure 2). These tests revealed the range of output loading over which the bioreactor can reliably deliver stimulation. These results informed areas in which the bioreactor’s performance can be further improved, including minimizing output impedance and improving rail-to-rail operation. Under unloaded conditions, we observed an error of 160 mV which was isolated to the 555 stage. This error was found to scale with output amplitude. Loading-related error was identified predominantly in the 10 V output channel. Future versions of the bioreactor will account for both sources of error, improving stimulation reliability. With these improvements, our bioreactor promises to aid in research concerning the electrical stimulation of tissues, including both cardiac and non-cardiac cell types. Adjustable frequency, voltage, and duty cycle along with temperature compensation, reverse polarity protection, and visual stimulation feedback are expected to enhance ease of use, enabling dissemination of this impactful technology to other research groups.

Acknowledgements (Optional): The authors would like to acknowledge the American Heart Association, the UNC-NCSU Joint Department of Biomedical Engineering, and the NCSU Comparative Medicine Institute for their financial support.

References:
[1] American Heart Association. "Proactive Steps Can Reduce Chances of Second Heart Attack." 16 Aug. 2021, www.heart.org/en/news/2019/04/04/proactive-steps-can-reduce-chances-of-second-heart-attack.
[2] 10.1016/j.yjmcc.2014.05.009
[3] 10.1021/acsomega.2c01807