(306e) Oxygen Gradients Correlate with Decrease in Cell Density and Viability in Engineered Cardiac Tissue

For clinical utility, cardiac grafts should be thick and compact, and contain physiologic density of metabolically active and differentiated cells. This involves the need to control the levels of nutrients, and most critically oxygen, throughout the construct volume. Most culture systems involve diffusional transport within the constructs, a situation associated with gradients of oxygen concentration, cell density, cell viability, and function. The goal of our study was to measure diffusional gradients of oxygen in engineered cardiac tissue, and to correlate oxygen gradients to the spatial distributions of cell number and cell viability.

Using microelectrodes, we measured oxygen distribution in a disc-shaped constructs (3.6 mm diameter, 1.8 mm thickness) based on neonatal rat cardiomyocytes cultured on collagen scaffolds for 16 days in dishes. The oxygen profile measurements were performed in unconfined constructs placed on a layer of agarose in a stagnant culture medium (to reflect static culture conditions) or suspended on a needle with superficial medium flow (to reflect the culture in mixed flasks or rotating vessels). To asses cross-sectional cell viability the live constructs were stained using Reduced Biohazard Cell Viability Kit (Molecular Probes), cross-sectioned and imaged using fluorescent microscope. The viability was determined using Image Analysis. Cell distribution was determined from DAPI counterstained histological cross-sections using image analysis. To rationalize experimental data, a mathematical model of oxygen distribution was derived as a function of cell density, viability, and spatial position within the construct.

At the end of cultivation, live cells expressing cardiac markers (sarcomeric α-actin, cardiac troponin I and connexin-43) were confined to the 250μm thick surface layer. Oxygen distribution (175.6 μM to 22.4μM) and viability (60%-5%) decreased linearly and live cell density decreased exponentially with construct thickness with the decay constant of 236μm. . Physiological density of live cells in the range 1.6-1×10^8cells/cm3 was present only within the first 128μm of the construct thickness corresponding to the decrease in oxygen concentration from 176 to 158 μM in this layer. Beyond the first 1000μm of construct thickness, oxygen concentration was below 74 μM and the live cell density was 3 to 4 orders of magnitude lower than in the native heart, yielding a non-functional piece of tissue. This observation is consistent with the report that cardiomyocytes begin to change their metabolism in response to hypoxia by down-regulating energy using processes at oxygen concentration of 70 μM [1]. Application of superficial medium flow during measurement significantly increased oxygen concentration in the construct cross-section, and reduced the thickness of the mass transport boundary layer. This correlated with the improved tissue properties in mixed flasks and rotating vessels.

One way to overcome diffusional limitations is to cultivate cardiac constructs with medium flow in perfusion bioreactors. Recently, we described cultivation of channeled cardiac tissue constructs perfused with oxygen carrier supplemented culture medium [2]. In the channels, the transport was governed by convection and in the tissue it was governed by diffusion. The described correlations between life cell density and oxygen profile can be used in conjuction with mathematical modeling to define a scaffold geometry and flow rate that would yield compact tissues with physiological cell density.

References: 1. Casey, T.M. and P.G. Arthur, Hibernation in noncontracting mammalian cardiomyocytes. Circulation, 2000. 102(25): p. 3124-3129. 2. Radisic, M., et al., Mathematical model of oxygen distribution in engineered cardiac tissue with parallel channel array perfused with culture medium containing oxygen carriers. American Journal of Physiology- Heart and Circulatory Physiology, 2005. 288(3): p. H1278-H1289.