May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
Ex–vivo Generation of Corneal Epithelial Cell Sheets on Temperature Responsive Surfaces
Author Affiliations & Notes
  • S. Khormaee
    Ophthalmology, University of Washington, Seattle, WA
  • B.D. Ratner
    Bioengineering, University of Washington Engineered Biomaterials, Seattle, WA
  • T.T. Shen
    Ophthalmology, University of Washington, Seattle, WA
  • Footnotes
    Commercial Relationships  S. Khormaee, None; B.D. Ratner, None; T.T. Shen, None.
  • Footnotes
    Support  NSF–ERC EEC–0529161, NESAC/BIO EB NIBB 002027, Howard Hughes Medical Institute Undergraduate Research Grant
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 2716. doi:
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      S. Khormaee, B.D. Ratner, T.T. Shen; Ex–vivo Generation of Corneal Epithelial Cell Sheets on Temperature Responsive Surfaces . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2716.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : To test the efficacy of plasma polymerized N–isopropyl Acrylamide (ppNIPAM) in generating a confluent, carrier–free, corneal epithelial cell monolayer as part of a construct for artificial corneas.

Methods: : ppNIPAM was recently characterized as a temperature–responsive surface for cell culture. Above a lower solution critical temperature (LCST) of 31 degrees Celsius, cells adhere to ppNIPAM, and below the LCST, cells detach without the use of trypsin or other damaging digestive enzymes. PpNIPAM surfaces were prepared as previously described (1), and were analyzed using X–ray Photoelectron Spectroscopy. Immortalized human corneal epithelial cells were seeded at a density of 2 x 105 cells on a ppNIPAM–coated, 35mm TCPS culture dish. Cells were allowed to form a confluent monolayer over a two week period. Once confluent, the ppNIPAM cell culture surfaces were held below ppNIPAM’s LCST at 20 degrees Celsius, and the epithelial cells spontaneously detached, forming a free monolayer. (1) Pan YV, Wesley RA, Luginbuhl R, Denton DD, Ratner BD. Plasma polymerized N–isopropyl acrylamide: synthesis and characterization of a smart thermally responsive coating. Biomacromolecules 2001; 2: 32–36.

Results: : Reducing the temperature of a ppNIPAM surface supporting confluent cells results in reliable detachment of a carrier–free corneal epithelial cell monolayer.

Conclusions: : ppNIPAM’s temperature–responsive properties are well–suited to the generation of carrier–free corneal epithelial cell monolayers, which may be transferred to other substrates for stratification in corneal tissue engineering.

Keywords: cornea: epithelium 

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