Three-Dimensional Growth of Endothelial Cells in the Microgravity-Based Rotating Wall Vessel Bioreactor

Abstract

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.

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“…74,75 The horizontally rotating bioreactors, or slowturning lateral vessels, were designed as a rotating cylinder of culture medium, with a gas-permeable membrane for oxygen and carbon dioxide exchange, and fittings for sampling or medium exchange. 75,76 The speed of the rotation could be controlled to match the settling velocity of the tissue constructs, keeping them suspended within the vessel.…”

Human Micro- and Macrovascular Endothelial Cells Exposed to Simulated Microgravity Upregulate hsp70

“…In the case of endothelial cells, conflicting results are reported (Carlsson et al 2003;Cotrupi et al 2005;Sanford et al 2002;Morbidelli et al 2005). While HUVEC and bovine aortic endothelial cells proliferate faster in simulated microgravity than in normal conditions (Carlsson et al 2003;Morbidelli et al 2005), porcine aortic endothelial cells engineered to overexpress vascular endothelial growth factor-receptor 2 are induced to apoptosis in modelled microgravity (Morbidelli et al 2005).…”

Short-Bowel Syndrome in Children — An Update in Management Strategies

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