Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel

Rotating-wall vessels are beneficial to tissue engineering in that the reco nstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall v essel (HARV) attached to collagen-coated Cytodex-3 beads as a representativ e monolayer culture to investigate factors during HARV cultivation which af fect three-dimensional growth and protein expression. A collagen type I sub stratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 bead s each, but it did not promote multilayer growth. A kinetic model of microc arrier aggregation was developed which indicates that the rate of aggregati on between a single bead and an aggregate was nearly 10 times faster than b etween two aggregate and 60 times faster than between two single beads. The se differences reflect changes in collision frequency and cell bridge forma tion. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls p erhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it c ould be useful in modeling aggregation in other cell systems, propagating h uman corneal endothelial cells for eye surgery and examining the response o f endothelial cells to reduced shear.