Three-dimensional growth of endothelial cells in the microgravity-based rotating wall vessel bioreactor

We characterized bovine aortic endothelial cells (BAEC) continuously cultured in the rotating wall vessel (RWV) bioreactor for up to 30 d. Cultures grew as large tissue-like aggregates (containing 20 or more beads) after 30 d. These cultures appeared to be growing in multilayers around the aggregates, where single beads were covered with confluent BAEC, which displayed the typical endothelial cell (EC) morphology. The 30-d multibead aggregate cultures have a different and smoother surface when viewed under a higher-magnification scanning electron microscope. Transmission electron microscopy of these large BAEC aggregates showed that the cells were viable and formed multilayered sheets that were separated by an extracellular space containing matrix-like material. These three-dimensional cultures also were found to have a basal production of nitric oxide (NO) that was 10-fold higher for the RWV than for the Spinner flask bioreactor (SFB). The BAEC in the RWV showed increased basal NO production, which was dependent on the RWV rotation rate: 73% increase at 8 rpm, 262% increase at 15 rpm, and 500% increase at 20 rpm as compared with control SFB cultures. The addition of l-arginine to the RWV cultures resulted in a fourfold increase in NO production over untreated RWV cultures, which was completely blocked by L-NAME [N(G)-nitro-L-arginine-methylester]. Cells in the SFB responded similarly. The RWV cultures showed an increase in barrier properties with an up-regulation of tight junction protein expression. We believe that this study is the first report of a unique growth pattern for ECs, resulting in enhanced NO production and barrier properties, and it suggests that RWV provides a unique model for investigating EC biology and differentiated function.