Purpose: To test the hypothesis that a level of chemical and electrical stimulation exists that allows differentiation of progenitor cells into organized contracting myocytes.
Methods: A custom-made bioreactor with the capability of delivering electrical pulses of varying field strengths, widths, and frequencies was constructed. Individual chambers of the bioreactor allowed continuous electrical stimulation of cultured cells under microscopic observation. On day 0, 1% dimethylsulfoxide (DMSO), known to differentiate cells into myocytes, was added to P19 progenitor cells. Additionally, for the next 22 days, electrical pulses of varying field strengths (0-3 V/cm), widths (2-40 ms), and frequencies (10-25 Hz) were continuously applied. On day 5, the medium containing DMSO was exchanged with regular medium, and the electrical stimulation was continued. From days 6-22, the cells were visually assessed for signs of viability, contractility, and organization.
Results: P19 cells remained viable with pulsed electrical fields <3 V/cm, pulse widths <40 ms, and pulse frequencies from 10 to 25 Hz. On day 12, the first spontaneous contractions were observed. For individual colonies, local synchronization and organization occurred; multiple colonies were synchronized with externally applied electrical fields.
Conclusion: P19 progenitor cells progress to organized contracting myocytes after chemical and electrical stimulation. Incorporation of such cells into existing methods of producing endothelial cells, fibroblasts, and scaffolds may allow production of improved tissue-engineered vascular grafts.