Biphasic Electrical Field Stimulation Aids in Tissue Engineering of Multicell-Type Cardiac Organoids

Abstract

The main objectives of current work were (1) to compare the effects of monophasic or biphasic electrical field stimulation on structure and function of engineered cardiac organoids based on enriched cardiomyocytes (CM) and (2) to determine if electrical field stimulation will enhance electrical excitability of cardiac organoids based on multiple cell types. Organoids resembling cardiac myofibers were cultivated in Matrigel-coated microchannels fabricated of poly(ethylene glycol)-diacrylate. We found that field stimulation using symmetric biphasic square pulses at 2.5 V/cm, 1 Hz, 1 ms (per pulse phase) was an improved stimulation protocol, as compared to no stimulation and stimulation using monophasic square pulses of identical total amplitude and duration (5 V/cm, 1 Hz, 2 ms). This was supported by the highest success rate for synchronous contractions, low excitation threshold, the highest cell density, and the highest expression of Connexin-43 in the biphasic group. Subsequently, enriched CM were seeded on the networks of (1) cardiac fibroblasts (FB), (2) D4T endothelial cells (EC), or (3) a mixture of FB and EC that were precultured for 2 days prior to the addition of enriched CM. Biphasic field stimulation was also effective at improving electrical excitability of these cardiac organoids by improving the three-dimensional organization of the cells, increasing cellular elongation and enhancing Connexin-43 presence.

Perfusable branching microvessel bed for vascularization of engineered tissues

“…The maximum capture rates for the cardiac tissues were similar with or without vascular structures (Fig. 6C) and were comparable to values from our previous studies (46). Troponin T staining demonstrated that cardiomyocytes grown on vascular structures contained betterorganized sarcomeres than those grown on hydrogel coating only (Fig.…”

“…The phenotype of cardiomyocytes in this system could be enhanced further in future studies by applying electrical (46,47) or mechanical (48) stimulation. EC cords previously led to synchronized contraction of cardiomyocytes and increased Connexin-43 expression because of the improved survival and spreading of cardiomyocytes (49).…”

“…The cardiomyocytes were cultivated for 7 d in 1 mL culture medium consisting of DMEM with 1% penicillin/streptomycin, 1% Hepes, and 15% FBS. At the end of the 7-d cultivation, the engineered cardiac tissues were removed from the PDMS substrates, and their functional properties were measured (SI Materials and Methods) as previously described (46). Atomic Force Microscopy.…”

Skeletal myotube formation enhanced by electrospun polyurethane carbon nanotube scaffolds

“…40 Chui et al showed that when symmetrically square biphasic (positive and negative) electrical signals (2.5 V, 1 Hz, 1 msec duration) were applied to cardiomyocyte-seeded polyethylene glycol constructs in vitro, cell elongation was increased. 41 Moreover, in the same study, a higher cell density was observed, along with the presence of cross-striation, and expression of Connexin 43 (a gap junction protein that propagates electrical signaling in cardiac muscle) was increased. Our results suggest that a nonsymmetrical biphasic electrical signal can also be used to augment multinucleated myotube formation from skeletal myoblasts rather than the more common use of symmetrical biphasic (positive and negative) or uniphase pulse signals.…”

Modulation of cell function by electric field: a high-resolution analysis

“…The regulatory effect of EFs has been demonstrated on different cell types such as neurons, osteoblasts, myoblasts, fibroblasts, endothelial cells, muscle cell and epithelial cells [8][9][10][11][12][13][14]. These effects result from manipulation of the native EF in the ionic extracellular environment and across the cell membrane [15 -17].…”