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Marco Lombardo, Vanessa Barbaro, Giuseppe Lombardo, Marina Bertolin, sebastiano serrao, Enzo Di Iorio; Label-free two-photon imaging of donor corneal lenticules with cultivated human limbal epithelial stem cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1715.
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© ARVO (1962-2015); The Authors (2016-present)
To present a novel, non invasive, optical method for evaluating and comparing two different scaffolds for cultivating human limbal epithelial stem cells, such as the hemicornea and fibrin gel.
Five human hemicorneas and six controls, three of which were fibrin gels with cultivated limbal epithelial stem cells and the other three were human corneal tissues, were imaged by two-photon optical microscopy. A Ti:sapphire laser, tuned at 712 nm and 810 nm, was used to perform two-photon emission fluorescence and second harmonic generation axial scanning measurements in each specimen respectively.
Two-photon signals provided label-free depth sectioning of fresh tissue specimens with high spatial resolution. In the hemicornea, the epithelium resembled the normal architecture of the human cornea; the resulting epithelium was stratified into four to five cell layers, with basal cuboidal cells differentiating upward to winged cells. In all hemicorneas, the basal epithelial plane was firmly attached to the underlying Bowman’s layer and occasionally resembled the digital invasion of the limbal basal epithelium in the palisades of Vogt. The keratocytes in the stroma of hemicornea clearly showed to form a syncytium. The collagen fibrils of the anterior stromal lenticule were regularly arranged and intertwined with each other as in the normal human cornea. Increased stratification and hyperproliferation of epithelial cells were observed when fibrin scaffold was used and the cells were rounded and not firmly attached to each other.
The present label-free two-photon based optical method showed to be reliable for the assessment of different scaffolds for cultivating limbal epithelial stem cells. The stroma of hemicornea represents an active matrix for supporting, through cell-to-cell communication, all the aspects of epithelial cells’ growth and differentiation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
In A) 3D reconstruction of the basal epithelial layer grown on the hemicornea. The stratification and differentiation of epithelial cells are comparable with normal human cornea. In B) 3D reconstruction of the epithelial cells grown on fibrin gel. Fibrin represents the golden standard for cultivating corneal limbal stem cells. However, the stratification and differentiation of epithelial cells on fibrin do not resemble the normal architecture of the human cornea. Scale bars: 50 µm.
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