Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue

Abstract

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue, but due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation, and unconstrained (i.e., not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate in concert these three key factors. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modeling studies. We then culture cardiac cells obtained from neonatal rats in porous, channeled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After eight days of culture, constructs grown with the simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23±0.10% vs. 0.14±0.05, 0.13±0.08, or 0.09±0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization than either control group. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs.

A Bioreactor to Apply Multimodal Physical Stimuli to Cultured Cells

“…At this point, their clamps are not attached yet. Next, fit the carbon electrodes ( Figure 3A#4) to the brackets with a medical suture, ensuring antiparallel current flow (see Note 13 ). Make sure the wiring is fitted firmly into its opening from the inside of the chamber.…”

“…Contamination is not only prone to waste of resources and time, but may yield misleading results. 13 The carbon electrodes have to be knotted to the PEEK brackets, and electrically connected in an antiparallel fashion, to allow for a rather homogeneous electrical field distribution (14).…”

“…Stimuli applied include cyclic stretch (7), electrical pacing (8), "electro-tensile" stimulation (where electrical and mechanical stimuli are applied in controlled sequence) (9), constant fluid perfusion (10)(11)(12), oxygenation via carriers through channelled scaffold (12), and more recently, concerted medium perfusion in combination with electrical stimulation (13).…”

Engineering Cardiovascular Regeneration

“…Vascularized cardiac grafts can be produced in vivo by casting hydrogels on an AV-loop [113,114] or multistacking of cell sheets on a vascularized area of the body [115]. For in vitro vascularization, cell sheets have been layered onto a surgically explanted and artificially perfused vascular beds [116•], or constructs are perfused through artificial channels [101,117,118]. Approaches using the biological grafts have the advantage that the vascular bed of the cardiac graft can be anastomosed after transplantation [114,117].…”

Biomimetic Cardiac Microsystems for Pathophysiological Studies and Drug Screens

“…Their functional role is verified with the expression of actin, myosins, and troponin, among other sarcomeric contractile proteins. [23][24][25][26]36,[41][42][43][44] Their responses to digitizer drugs, such as digoxin, are of interest when studying CMSs as well. 6,23,24 Pacemaker cells are difficult to isolate, and so far no CMS can replicate their ability to generate spontaneous action potentials.…”