Skip to main content
PMC home page
Cytotechnology logoLink to Cytotechnology
. 2000 Mar;32(3):253–263. doi: 10.1023/A:1008117410827

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

James W Muhitch 1, Kim C O'Connor 2,✉, Diane A Blake 4,5, Daniel J Lacks 6, Nitsa Rosenzweig 7, Glenn F Spaulding 9
PMCID: PMC3449895  PMID: 19002986

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.

Keywords: aggregation, bovine corneal endothelial (BCE) cells, kinetic model, microcarrier beads, protein expression, rotating-wall vessel (RWV)

Full Text

The Full Text of this article is available as a PDF (135.0 KB).

References

  1. Atkins PW. Physical Chemistry. New York: W. H. Freeman & Company; 1990. [Google Scholar]
  2. Ben-Ze'ev A, Farmer SR, Penman S. Protein synthesis requires cell-surface contact while nuclear events respond to cell shape in anchorage-dependent fibroblasts. Cell. 1980;21:365–372. doi: 10.1016/0092-8674(80)90473-0. [DOI] [PubMed] [Google Scholar]
  3. Bird RB, Stewart WE, Lightfoot EN. Transport Phenomena. New York: John Wiley and Sons; 1960. [Google Scholar]
  4. Blake DA, McLean NV. High-pressure, anion-exchange chromatography of proteoglycans. Anal Biochem. 1990;190:158–164. doi: 10.1016/0003-2697(90)90174-8. [DOI] [PubMed] [Google Scholar]
  5. Blasey HD, Aubry J-P, Mazzei GJ, Bernard AR. Large scale transient expression with COS cells. Cytotechnology. 1995;18:183–192. doi: 10.1007/BF00767766. [DOI] [PubMed] [Google Scholar]
  6. Chan Man Fong CF, de Kee D, Kaloni PN. Advanced Mathematics for Applied and Pure Sciences. Amsterdam: Gordon and Breach Science; 1997. [Google Scholar]
  7. Cheng L, Sun J, Pretlow TG, Culp J, Yang N-S. CWR22 xenograft as an ex vivo humor tumor model for prostate cancer gene therapy. J Natl Cancer Inst. 1996;88:607–611. doi: 10.1093/jnci/88.9.607. [DOI] [PubMed] [Google Scholar]
  8. Cherry RS, Papoutsakis ET. Hydrodynamic effects on cells in agitated tissue culture reactors. Bioprocess Eng. 1986;1:29–41. [Google Scholar]
  9. Cherry RS, Papoutsakis ET. Modeling of contact-inhibited animal cell growth on flat surfaces and spheres. Biotechnol Bioeng. 1989;33:300–305. doi: 10.1002/bit.260330308. [DOI] [PubMed] [Google Scholar]
  10. Clejan S, O'Connor KC, Cowger NL, Cheles MK, Haque S, Primavera AC. Effects of simulated microgravity on DU 145 human prostate carcinoma cells. Biotechnol Bioeng. 1996;50:587–597. doi: 10.1002/(SICI)1097-0290(19960605)50:5<587::AID-BIT14>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  11. Croughan MS, Hamel J-FP, Wang DIC. Effects of microcarrier concentration in animal cell culture. Biotechnol Bioeng. 1988;32:975–982. doi: 10.1002/bit.260320805. [DOI] [PubMed] [Google Scholar]
  12. Davenport EA, Nettesheim P. Type I collagen gel modulates extracellular matrix synthesis and deposition by tracheal epithelial cells. Exp Cell Res. 1996;25:155–162. doi: 10.1006/excr.1996.0069. [DOI] [PubMed] [Google Scholar]
  13. Diaz-Valle D, Benitez del Castillo Sanchez JM, Castillo A, Sayagues O, Moriche M. Endothelial damage with cataract surgery techniques. J Cataract Refract Surg. 1998;24:951–955. doi: 10.1016/s0886-3350(98)80049-7. [DOI] [PubMed] [Google Scholar]
  14. Freed LE, Vunjak-Novakovic G. Cultivation of cellpolymer tissue constructs in simulated microgravity. Biotechnol Bioeng. 1995;46:306–313. doi: 10.1002/bit.260460403. [DOI] [PubMed] [Google Scholar]
  15. Gherardini G, Haegerstrand A, Matarasso A, Gurlek A, Evans GR, Lundeberg T. Cell adhesion and short-term patency in human endothelium preseeded 1.5-mm polytetrafluoroethylene vascular grafts: an experimental study. Plast Reconstr Surg. 1997;99:472–478. doi: 10.1097/00006534-199702000-00024. [DOI] [PubMed] [Google Scholar]
  16. Goodwin TJ, Jessup JM, Wolf DA. Morphologic differentiation of colon carcinoma cells lines HT-29 and HT-29KM in rotating-wall vessels. In Vitro Cell Dev Biol. 1992;28A:47–60. doi: 10.1007/BF02631079. [DOI] [PubMed] [Google Scholar]
  17. Goodwin TJ, Prewett TL, Wolf DA, Spaulding GF. Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. J Cell Biochem. 1993;51:301–311. doi: 10.1002/jcb.240510309. [DOI] [PubMed] [Google Scholar]
  18. Gospodarowicz D, Vlodavsky I, Savion N. The role of fi-broblast growth factor and the extracellular matrix in the control of proliferation and differentiation of corneal endothelial cells. Vision Res. 1981;21:87–103. doi: 10.1016/0042-6989(81)90141-3. [DOI] [PubMed] [Google Scholar]
  19. Hansbrough JF, Dore C, Hansbrough WB. Clinical trials of a living dermal tissue replacement placed beneath meshed, split-thickness skin grafts on excised burn wounds. J Burn Care Rehabil. 1992;13:519–529. doi: 10.1097/00004630-199209000-00004. [DOI] [PubMed] [Google Scholar]
  20. Hawboldt KA, Kalogerakis N, Behie LA. Cellular automaton model for microcarrier cultures. Biotechnol Bioeng. 1994;43:90–100. doi: 10.1002/bit.260430112. [DOI] [PubMed] [Google Scholar]
  21. Hugler W, O'Connor KC, Landry SJ, Bivins JE. Induction of stress proteins in anoxic and hyperthermic Spodoptera frugiperda cells. Cytotechnology. 1995;17:91–101. doi: 10.1007/BF00749396. [DOI] [PubMed] [Google Scholar]
  22. Insler MS, Lopez JG. Microcarrier cell culture of neonatal human corneal endothelium. Current Eye Res. 1990;9:23–30. doi: 10.3109/02713689009000051. [DOI] [PubMed] [Google Scholar]
  23. Jessup JM, Brown D, Fitzgerald W, Ford RD, Nachman A, Goodwin TJ, Spaulding G. Induction of carcinoembryonic antigen expression in a three-dimensional culture system. In Vitro Cell Dev Biol. 1997;33:352–357. doi: 10.1007/s11626-997-0005-6. [DOI] [PubMed] [Google Scholar]
  24. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lelkes PI, Ramos E, Nikolaychik VV, Wankowski DM, Unsworth BR, Goodwin TJ. GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells. In Vitro Cell Dev Biol. 1997;33:344–351. doi: 10.1007/s11626-997-0004-7. [DOI] [PubMed] [Google Scholar]
  26. Lelkes PI, Galvan DL, Hayman GT, Goodwin TJ, Chatman DY, Cherian S, Garcia RM, Unsworth BR. Simulated microgravity conditions enhance differentiation of cultured PC12 cells towards the neuroendocrine phenotype. In Vitro Cell Dev Biol. 1998;34:316–325. doi: 10.1007/s11626-998-0008-y. [DOI] [PubMed] [Google Scholar]
  27. MacCallum DK, Lillie JH, Scaletta LJ, Occhino JC, Frederick WG, Ledbetter SR. Bovine corneal endothelium in vitro. Exp Cell Res. 1982;139:1–13. doi: 10.1016/0014-4827(82)90313-5. [DOI] [PubMed] [Google Scholar]
  28. Molnar G, Schroedl NA, Gonda SR, Hartzell CR. Skeletal muscle satellite cells cultured in simulated microgravity. In Vitro Cell Dev Biol. 1997;33:386–391. doi: 10.1007/s11626-997-0010-9. [DOI] [PubMed] [Google Scholar]
  29. Munjal ID, Blake DA, Sabet MD, Gordon SR. Thrombospondin: biosynthesis, distribution, and changes associated with would repair in corneal endothelium. Eur J Cell Biol. 1990;52:252–263. [PubMed] [Google Scholar]
  30. O'Connor KC. Three-dimensional cultures of prostatic cells: tissue models for the development of novel anti-cancer therapies. Pharm Res. 1999;16:486–493. doi: 10.1023/a:1011906709680. [DOI] [PubMed] [Google Scholar]
  31. O'Neill CW. Flow-mediated NO release from endothelial cells is independent of K+ channel activation or intracellular Ca2+ Am J Physiol. 1995;269:C863–C869. doi: 10.1152/ajpcell.1995.269.4.C863. [DOI] [PubMed] [Google Scholar]
  32. Ryan US, Mortara M, Whitaker C. Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes. Tissue Cell. 1980;12:619–635. doi: 10.1016/0040-8166(80)90017-8. [DOI] [PubMed] [Google Scholar]
  33. Schwarz RP, Goodwin TJ, Wolf DA. Cell culture for threedimensional modeling in rotating-wall vessels: an application of simulated microgravity. J Tiss Cult Meth. 1992;14:51–58. doi: 10.1007/BF01404744. [DOI] [PubMed] [Google Scholar]
  34. Spaulding GF, Jessup JM, Goodwin TJ. Advances in cellular construction. J Cell Biochem. 1993;51:249–251. doi: 10.1002/jcb.240510302. [DOI] [PubMed] [Google Scholar]
  35. Tsao Y-MD, Boyd E, Wolf DA, Spaulding G. Fluid dynamics within a rotating bioreactor in space and Earth environments. J Spacecr Rockets. 1994;31:937–943. [Google Scholar]
  36. Tseng SCG, Savion N, Gospodarowicz D, Stern R. Characterization of collagens synthesized by cultured bovine corneal endothelial cells. J Biol Chem. 1981;256:3361–3365. [PubMed] [Google Scholar]
  37. Tuft SJ, Coster DJ. The corneal endothelium. Eye. 1990;4:389–424. doi: 10.1038/eye.1990.53. [DOI] [PubMed] [Google Scholar]
  38. Unsworth BR, Lelkes PI. Growing tissues in microgravity. Nat Med. 1998;4:901–907. doi: 10.1038/nm0898-901. [DOI] [PubMed] [Google Scholar]
  39. West DC, Sattar A, Kumar S. A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells. Anal Biochem. 1985;147:289–295. doi: 10.1016/0003-2697(85)90274-x. [DOI] [PubMed] [Google Scholar]
  40. Zinke-Allmang M, Feldman LC, Grabow MH. Clustering on surfaces. Surf Sci Rep. 1992;16:377–463. [Google Scholar]

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES