May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Nanotechnology Enables Real Time Observation and Tracking of Limbal Epithelial Stem Cells in Culture and Post-Transplantation
Author Affiliations & Notes
  • A. J. Shortt
    Institute of Ophthalmology, London, United Kingdom
    Cells for Sight Transplantation and Research Programme,
    Ocular Repair and Regeneration Biology Unit,
  • G. A. Secker
    Institute of Ophthalmology, London, United Kingdom
    Cells for Sight Transplantation and Research Programme,
    Ocular Repair and Regeneration Biology Unit,
  • P. T. Khaw
    Institute of Ophthalmology, London, United Kingdom
    Ocular Repair and Regeneration Biology Unit,
    Moorfields Eye Hospital, London, United Kingdom
  • S. J. Tuft
    Institute of Ophthalmology, London, United Kingdom
    Cells for Sight Transplantation and Research Programme,
    Moorfields Eye Hospital, London, United Kingdom
  • J. T. Daniels
    Institute of Ophthalmology, London, United Kingdom
    Cells for Sight Transplantation and Research Programme,
  • Footnotes
    Commercial Relationships A.J. Shortt, None; G.A. Secker, None; P.T. Khaw, None; S.J. Tuft, None; J.T. Daniels, None.
  • Footnotes
    Support Medical Research Council, UK and Moorfields Eye Hospital Special Trustees
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4440. doi:
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      A. J. Shortt, G. A. Secker, P. T. Khaw, S. J. Tuft, J. T. Daniels; Nanotechnology Enables Real Time Observation and Tracking of Limbal Epithelial Stem Cells in Culture and Post-Transplantation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4440.

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

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Abstract

Purpose:: The transplantation of ex-vivo cultured limbal epithelial cells has become an established treatment for total corneal limbal stem cell deficiency. Existing methods used to determine survival of transplanted cells have poor specificity and sensitivity. The aim of this study was to develop novel methods of detecting and tracking cells in-vivo post transplantation.

Methods:: Ex-vivo cultured primary human limbal epithelial cells were labelled with quantum dot nanocrystals. These nanoparticles behave as fluorophores, absorbing photons of light then re-emitting photons at a different wavelength. The toxicity of quantum dots was assessed using live-dead assays. The effect on limbal stem cell function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured human limbal epithelial cells labelled with quantum dot nanocrystals were transplanted onto decellularized human corneo-scleral rims in an ex-vivo organ culture model and observed to investigate the behavior of transplanted cells.

Results:: Quantum dot labelling had no detrimental effect on limbal epithelial stem cell integrity or function in-vitro. There were no differences in the results of live-dead assays between labelled and unlabelled cells at 1 and 4 days post labelling. Proliferation resulted in a gradual reduction in quantum dot signal but sufficient signal was present to allow tracking of cells through multiple generations. There was no statistically significant difference between the colony forming efficiency of labelled and unlabelled cells and expression of CK3, P63alpha and ABCG2 were identical in both groups. Cells labelled with quantum dots could be reliably detected and observed using confocal microscopy for at least 1 week post transplantation in our ex-vivo organ culture model. In addition it was possible to label and observe epithelial cells in intact cadaveric human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA.

Conclusions:: Quantum dots are a useful tool for studying limbal epithelial stem cell biology. This novel combination of nanotechnology with cutting edge imaging devices may in the future allow us to observe and monitor transplanted ex-vivo cultured limbal stem cells in-vivo in human subjects.

Keywords: cornea: epithelium • cornea: basic science • microscopy: confocal/tunneling 
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