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Abstract

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted 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 vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum 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 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps 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 could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.

Bovine myoblast cell production in a microcarriers-based system

“…Aggregate formation depends on cell type i.e. the ability of cells to grow in multiple layers, and on the dynamic conditions of the culture (Muhitch et al 2000 ). Early observations suggest that cell-microcarrier aggregate formation negatively affects growth of human mesenchymal stem cells (Chen et al 2013 ; Caruso et al 2014 ; Goh et al 2013 ) and we assume that myoblasts behave similarly.…”

Ectopic Osteogenesis of Macroscopic Tissue Constructs Assembled from Human Mesenchymal Stem Cell-Laden Microcarriers through In Vitro Perfusion Culture

“…It should be noted that spontaneous aggregation of hAMSCs-laden CultiSpher S microcarriers in spinner flasks is common, which suggests an intrinsic mechanism of assembling for cell-laden microcarriers [13] . It has been suggested that the aggregation process is cell- and microcarrier-type dependent, possibly relying on cell proliferation, migration, ECM production and microcarrier property [25] – [27] . In addition, by controlling culture condition, aggregates within an appropriate size can be made to ensure high cell viability [15] .…”

Three-dimensional culture systems in cancer research: Focus on tumor spheroid model

“…However, cells experience sheer force generated by continuous motion of the stirring bar, which adversely affects cellular physiology (Lin et al , 2008). Rotating flasks function similarly to the spinner flasks, but instead of using a stirring bar to keep the cells in suspension, the culture flask itself is rotated causing the cells to experience less force (Goodwin et al , 1993; Muhitch et al , 2000). Moreover, rotational speed of the flasks can be adjusted to obtain spheroids of desired sizes.…”