Simultaneous culture medium perfusion and electrical stimulation improve differentiation of engineered cardiac tissue

The heart is an organ that fails beyond repair because of the intrinsic inability of damaged heart tissue to regenerate after injury. Heart disease is currently the leading cause of death in the United States, and novel treatment methods are needed because drugs therapy cannot reverse the injury process and donor tissue is in scarce supply. Cardiac tissue engineering offers the promise of growing a tissue patch in vitro that could replace or repair damaged myocardial tissue upon implantation in vivo, or serve as a model for testing new treatment modalities.For this thesis we adopt a biomimetic approach to engineered cardiac tissue wherein we attempt to recapitulate important aspects of the native cardiac environment in our in vitro culture systems. Recent studies investigating the role of nutrient supply (by culture medium perfusion) or electrical signaling (by electric field stimulation) in engineered cardiac tissue led us to hypothesize that cardiac constructs cultured with a combination of these conditions would result in engineered cardiac tissue that is homogeneously distributed in a construct of clinically relevant size and is contractile in response to electric stimulation. To validate this hypothesis, we performed a series of studies to optimize a poly(glycerol sebacate) (PGS) scaffold for cardiac tissue engineering and developed a novel bioreactor to provide simultaneous culture medium perfusion and electric field stimulation to cardiac constructs during in vitro culture.First, we optimized scaffold material properties by coating the biomaterial with an extracellular matrix protein and varying the mechanical stiffness. Coating PGS with the extracellular matrix protein laminin resulted in a higher cell seeding efficiency and greater construct contractility, and soft PGS scaffolds (&sim2 kPa compressive modulus) improved cell content, organization, and contractility after culture.We then developed and optimized a technique to seed cardiac cells in PGS scaffolds with an array of small-diameter channels (250 mu.m diameter), which had previously been designed to mimic the role of a capillary network (i.e. shielding cells from hydrodynamic shear during perfusion culture while decreasing transport distance from the culture medium to the cells). By using our perfusion seeding technique, we generated constructs that were homogeneously seeded with a dense population of cardiac cells without causing the small- diameter channels to become blocked during the procedure.Finally, we combined our optimized PGS scaffold and seeding technique with a novel bioreactor designed to provide simultaneous culture medium perfusion and electrical field stimulation. Application of these biophysical stimuli resulted in engineered constructs with greater electrical functionality, contractility, cell survival and spatial homogeneity, and protein organization and expression than was achieved in controls. Therefore, this novel bioreactor could serve as the basis for generating engineered cardiac tissues with clinical utility in the future.