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The Impact of De-epithelialization of the Amniotic Membrane Matrix on Morphology of Cultured Human Limbal Epithelial Cells Subject to Eye Bank Storage

Raeder, Sten MD; Utheim, Tor Paaske MD; Messelt, Edward DDS, PhD; Lyberg, Torstein MD, PhD

doi: 10.1097/ICO.0b013e3181ba0c94
Basic Investigation

Purpose: To investigate whether human limbal epithelial cells (HLEC) that have been cultured on intact or de-epithelialized amniotic membranes (AMs) demonstrate differences in morphology after 1 week of eye bank storage.

Methods: HLEC were cultured from limbal explants for 3 weeks on intact AM and AM deprived of the amniotic epithelial cells by incubation with 0.02% ethylene diamine tetra acetic acid followed by mechanical scraping. The HLEC cultures were stored for 1 week in a closed container in a serum-based medium at 23°C. The surface morphology was assessed using scanning electron microscopy, and a quantitative comparison of desmosome and hemidesmosome numbers was performed using transmission electron microscopy.

Results: Although most superficial epithelial cells were closely attached to each other, with tightly opposed cell junctions and distinct cell borders, there was evidence of some cell separation in HLEC that had been cultured on intact and denuded AM after 1 week of storage. In both experimental groups, the epithelia were well stratified, consisting of basal column-shaped cells, suprabasal cuboid wing cells, and flat squamous superficial cells, but dilated intercellular spaces were observed. The total number of desmosomes per micron was 1.39 ± 0.77 in HLEC cultured on intact AM versus 0.98 ± 0.45 in HLEC expanded on denuded AM (P = 0.76). The total number of hemidesmosomes per micron in HLEC cultured on intact AM and denuded AM was 0.87 ± 0.34 and 0.78 ± 0.31, respectively (P = 0.70).

Conclusions: Denuding of AM does not improve the structural integrity of cultured HLEC after eye bank storage.

From the *Center for Clinical Research, Ulleval University Hospital, Oslo, Norway; †Department of Ophthalmology, University of Oslo, Ulleval University Hospital, Oslo, Norway; and ‡Institute for Oral Biology, Dental Faculty, University of Oslo, Oslo, Norway.

Received for publication March 9, 2009; revision received July 6, 2009; accepted July 30, 2009.

This work was supported by the South-Eastern Norway Regional Health Authority.

Tor Paaske Utheim and Sten Raeder have filed a patent application on storage of cultured limbal epithelial cells.

Reprints: Sten Raeder, MD, Center for Clinical Research, University of Oslo, Ulleval University Hospital, Kirkeveien 166, 0407 Oslo, Norway (e-mail:

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