Transplantation of human corneal limbal epithelial cell sheet harvested on synthesized carboxymethyl cellulose and dopamine in a limbal stem cell deficiency.

This study aimed to evaluate the efficacy and safety of transplantation with human corneal limbal epithelial (HCLE) cell sheets cultured on carboxymethyl cellulose (CMC)-dopamine (DA)-coated substrates and harvested via enzymatic digestion of CMC with cellulase in a rabbit animal model of limbal stem cell deficiency (LSCD). Synthesized CMC-DA was pretreated onto the surface of culture plates. Then, HCLE cells were cultured on precoated CMC-DA and HCLE cell sheets were harvested using cellulase-containing cell culture medium. HCLE cell sheets were evaluated using a live/dead assay, histological examination, and immunofluorescence staining. For in vivo assessment, HCLE cell sheets were transplanted in a rabbit model of LSCD for 2 weeks to determine the effectiveness of the repair. Primary culture of HCLE cells stained positively for p63, cytokeratin (CK)15, and CK12. HCLE cell sheets were generated with a well-preserved morphology and transparency ranging in size from 15 to 19 mm after cellulase-assisted cell sheet generation. HCLE cell sheets uniformly stained positively for human mitochondria, p63, CK15, CK12, CK3/2p, and zonula occludens (ZO)-1. HCLE cell sheet transplantation in a rabbit model of LSCD improved the corneal opacity and neovascularization scores. Transplanted HCLE cell sheets stained positively for p63 and CK12. Transplantation of HCLE cell sheets harvested on CMC-DA coating combined with cellulase is a safe and efficient procedure for corneal epithelial regeneration in a rabbit model of LSCD. This system could enable a promising strategy to regenerate corneal epithelium by transplantation in ocular surface disorders.