Cardiac tissue engineering : bioreactor cultivation parameters

Abstract

Tissue engineering may be useful in fighting heart disease since it offers the possibility of creating functional tissue equivalents for scientific studies and tissue repair. In the present work, we examined how variations in cultivation parameters of a model tissue engineering system influenced cardiac tissue morphogenesis. The central hypothesis was that using a tissue engineering system consisting of isolated cardiac cells, polymer scaffolds, and tissue culture bioreactors, we could engineer cardiac muscle mimicking native tissue in structure and function in the presence of appropriate biochemical and physical signals. The specific objectives were to: ( 1) vary key parameters of the model tissue engineering system, and (2) structurally and functionally characterize engineered cardiac muscle so that effects of parameter variations could be assessed and engineered tissue could be compared to native tissue. Effects of key cultivation parameters, including (I) cell source, (2) cell seeding density, (3) cell seeding vessel, and (4) tissue culture bioreactor on structure and function of engineered cardiac cell-polymer constructs were studied. Advantages of seeding mammalian cells at high densities (6-Sx 106 cells/Smm diameter x 2mm thick scaffold) under mixed conditions and culturing constructs in rotating laminar flow bioreactors were demonstrated, but constructs had interiors (> IOOμm tissue depth) consisting of mostly empty space due to diffusional mass transport limitations. We attempted to overcome diffusional limitations by directly perfusing culture medium through the constructs. Perfusion significantly improved the uniformity of the cell distribution and enhanced expression of a differentiated cell phenotype in comparison to non-perfused (i.e. flask) cultures. Control of the cell microenvironment in the perfusion system was also used to study relationships between oxygen tension and properties of cardiac constructs. Oxygen tension was directly correlated with DNA and protein contents (r=0.88 and 0.89, respectively), aerobic metabolism (r=0.97), muscle protein expression, and ultrastructural differentiation. Characterization of cardiac construct structure, composition, cell phenotype, and in vitro function demonstrated cardiac specific protein expression, metabolic activity similar to that of native tissue, and differentiated ultrastructural features (e.g. sarcomeres). The results support the utility of engineered cardiac muscle as a native tissue model for in vitro studies and eventually for in vivo tissue repair.