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Rapidly constructed scaffold-free cornea epithelial sheets for ocular surface reconstruction.

PURPOSE
To develop a centrifugal cell seeding method for rapid and efficient reconstruction of ocular surface with limbal stem cell deficiency (LSCD) in rabbits.


METHODS
The orthogonal design method was used to optimize centrifugation parameters for cell seeding. Methylthiazol tetrazolium proliferation assay, colony-forming efficiency, and flow cytometry were used to study cell viability. Histology, electron microscopy, and immunocytochemistry were evaluated for centrifugation-constructed cornea epithelial sheets (CCCESs). The rabbit eyes with LSCD were treated with or without CCCES for in vivo evaluation.


RESULTS
The 80.04% attached cells with 98.04% viability were achieved using optimal cell seeding density at 9 × 10(5) cm(-2) with centrifugation at 1800 rpm for 4 min. The 0.4% glycerin was added in the medium to increase the surface tension and osmotic pressure to optimal condition for obtaining higher cell density. The three-layer epithelial sheets were rapid constructed, which displayed the characteristics of normal corneal epithelium. In vivo transplantation, labeled cells of CCCES were detected at 30 days. CCCES reconstructed the LSCD corneal epithelia without conjunctivalization and neovascularation, evidenced by positive K3 and negative K4, Muc5AC.


CONCLUSION
The scaffold-free corneal epithelial sheets were rapidly constructed using optimal centrifugation procedure, which was demonstrated to reconstruct ocular surface with LSCD.