Perfusion Bioreactors Improve Oxygen Transport and Cell Distribution in Esophageal Smooth Muscle Construct

Abstract

Mass transfer is a major issue for producing, in particular, thicker tissue constructs in vitro. Without the support of internal capillary network, oxygen supply is restricted to the superficial layer and the survival of cells deep within the construct could be threatened. In this study, oxygen was postulated as the limiting factor for cellular activity in developing tissue construct and their correlation was investigated. Single- and double-flow perfusion bioreactors were custom designed to provide a controlled tangential laminar flow at a flowrate of 0.545 ml/min over the surface of the tissue construct (3.2 x 2 x 0.2 cm3 porous gelatin scaffold seeded with 2 x 106 cells/cm3 of porcine esophageal smooth muscle cells). Gelatin scaffold of similar size and cell seeding was cultured in Petri dish and served as a static culture control for comparison. At day 5, oxygen profile in-situ was measured across the thickness of tissue construct while cell distribution and total DNA were analyzed. Bioreactors produced higher cell proliferation activity compared to static culture. Under static culture, cells were observed to populate near the surface of tissue construct. While single-flow bioreactor improved cell infiltration/ migration in the scaffold, double-flow bioreactor improved further these cellular events and produced construct with more uniform cell distribution. These cell distributions appear to parallel that of oxygen distribution. In static culture, oxygen concentration dropped abruptly from the construct surface to a depth of about 1000 ìm where it was maintained at a constant level in the core at low cell density but rose near the bottom surface. In the single-flow bioreactor, oxygen level dropped linearly from the top to the bottom surface of construct. In the double-flow bioreactor, oxygen level began to drop at about 2.5 mm above the construct surface but it was maintained at almost a constant level throughout the whole thickness.