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

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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.

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Author notes

  1. Nitsa Rosenzweig

    Present address: Department of Chemistry, Xavier University, 7325 Palmetto St., New Orleans, LA, 70125, USA

Authors and Affiliations

  1. Department of Chemical Engineering, and Molecular and Cellular Biology Graduate Program, Tulane University, New Orleans, LA, 70118, USA

    James W. Muhitch

  2. Department of Chemical Engineering, and Molecular and Cellular Biology Graduate Program, Tulane University, New Orleans, LA, 70118, USA

    Kim C. O'Connor

  3. Molecular and Cellular Biology Graduate Program, Tulane University, New Orleans, LA, 70118, USA

    Diane A. Blake

  4. Department of Ophthalmology, Tulane University School of Medicine, New Orleans, LA, 70112, USA

    Diane A. Blake

  5. Department of Chemical Engineering, and, Tulane University, New Orleans, LA, 70118, USA

    Daniel J. Lacks

  6. Department of Chemical Engineering, and, Tulane University, New Orleans, LA, 70118, USA

    Nitsa Rosenzweig

  7. Clear Lake Medical Foundation Inc., Houston, TX, 77058, USA

    Glenn F. Spaulding

Authors
  1. James W. Muhitch
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  2. Kim C. O'Connor
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  3. Diane A. Blake
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  6. Glenn F. Spaulding
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Corresponding author

Correspondence to Kim C. O'Connor.

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Muhitch, J.W., O'Connor, K.C., Blake, D.A. et al. Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel. Cytotechnology 32, 253–263 (2000). https://doi.org/10.1023/A:1008117410827

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