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