Abstract
Current imaging techniques for the characterization of differentiated corneal limbal stem cells are destructive and cannot be used in eye bank for monitoring the regenerated epithelium in culture. We presented a minimally invasive, multimodal, marker-free imaging method for the investigation of epithelia regenerated with cultured human donor corneal limbal epithelial stem cells. Two-photon fluorescence and harmonic generation signals were collected from specimens in culture and used for evaluating the structure and morphology of epithelia cultured on two different bio-scaffolds; in addition, donor human corneal tissues were used as controls. The method provided reliable information on the organization of cellular and extracellular components of biomaterial substrates and was highly sensitive to determine differences between the density packing arrangement of epithelial cells of different biomaterials without relying on inferences from exogenous labels. The present minimally invasive standardized quality control methodology can be reliably translated to eye banks and used for monitoring harvested corneal limbal stem cells growth and differentiation in bioengineered materials.
Highlights
In stem cell research, there is a high demand on techniques for the minimally-invasive, marker-free observation of growth, proliferation, differentiation, and stability of living stem cells under near-physiological conditions[1,2,3,4,5,6]
We present a minimally-invasive approach based on two-photon optical microscopy for investigating the epithelia regenerated from human corneal epithelial limbal stem cells on different bio-scaffold substrates, such as the hemicornea and fibrin gel[7, 9]
To the best of our knowledge, this is the first study reporting the feasibility and reliability of two-photon optical microscopy to investigate the epithelia regenerated by human corneal epithelial limbal stem cells on different biomaterials
Summary
There is a high demand on techniques for the minimally-invasive, marker-free observation of growth, proliferation, differentiation, and stability of living stem cells under near-physiological conditions[1,2,3,4,5,6]. Current imaging techniques for the characterization of differentiated corneal limbal stem cells include immunocytochemistry, immunofluorescence microscopy and electron microscopy. We present a minimally-invasive approach based on two-photon optical microscopy for investigating the epithelia regenerated from human corneal epithelial limbal stem cells on different bio-scaffold substrates, such as the hemicornea and fibrin gel[7, 9]. The hemicornea is a human-derived bioscaffold and consists of a donor anterior corneal stromal lenticule; the fibrin gel is the golden standard for cultivating human limbal epithelial stem cells and treating patients with LSCD7, 9, 10. Data of regenerated epithelia and their substrates were compared with human donor eye bank corneoscleral tissues with intact epithelium and de-epithelialized anterior corneal stromal lenticules, which were used as controls
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
