Oxygen Transport, Shear Stress, And Metabolism In Perfused Hepatocyte-Seeded Scaffolds With Radial Pore Architecture: Experimental And Computational Analyses

Several modalities have been proposed as treatments or temporary stop-gap for patients suffering from liver failure until a suitable organ is available. However there is still an urgent need for an off-the-shelf device that can accommodate clinically relevant cell numbers, be cultured at physiological oxygen tensions and, can be fully integrated into and heal the injured hepatic space. In this study we investigated the effects that convective and direct oxygenation had on hepatocyte functionality, morphology and viability while cultured in bulk 3D chitosan scaffolds and perfusion bioreactor systems. Cylindrical chitosan scaffolds with radial directed pore structures were fabricated by a thermal gradient directed from the center to the periphery. Capillary-like direct oxygenation was facilitated by embedding gas permeable silicone tubing throughout the scaffold body. Three iterations of bioreactor design and optimization produced a perfusion system that could enable direct oxygenation, accommodate high density hepatocyte seeding (8x10-7 to 1x10-8 cells), ensure adequate mass transfer and induce sustainable metabolic outputs for a least 7 days at a flow rate of 10 ml/min. A computational fluid dynamics model of the internal scaffold pore structure infused with spheroids that resembled hepatocyte aggregates was utilized to understand how varying flow rates (5, 10, 15, 20 and 25 ml/min) effected fluid flow profiles, shear stress imposed on the cells and oxygen consumption within the microenvironment. The results showed that the volumetric flow rate 15 ml/min at the scaffold’s central port inlet produced the best oxygen consumption profile with no damaging effects due to shear stress or eddies flow. The simulation was validated and showed good correlation to empirically derived data. Experimentally the flow rate of 15 ml/min induced the most favorable hepatic response out of the five experimental flow conditions and a static culture (only direct oxygenation). We also looked at how increasing cellular compactness, via reduced scaffold dimensions, would affect phenotypic expression and viability. It was discerned that increasing the cell packing density by 14% increased the rate of albumin and urea production by 79% and 40% respectively. In total the results show that the experimental measures conducted in this study enhanced hepatocyte metabolic performance, viability and morphological appearance