title: Rapidly constructed scaffold-free cornea epithelial sheets for ocular surface reconstruction.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  The scaffold-free corneal epithelial sheets were rapidly constructed using optimal centrifugation procedure, which was demonstrated to reconstruct ocular surface with LSCD.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Jianhui
authors.initials: J
authors.lastname: Xiao
authors.forename: Chaoyang
authors.initials: C
authors.lastname: Li
authors.forename: Pengxia
authors.initials: P
authors.lastname: Wan
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wu
authors.forename: Minghai
authors.initials: M
authors.lastname: Huang
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Wang
authors.forename: Zhichong
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002498:Centrifugation; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects; D019573:Epithelium, Corneal / Q000187:drug effects / Q000601:surgery*; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D016850:Limbus Corneae / Q000187:drug effects / Q000473:pathology; D011817:Rabbits; D019651:Plastic Surgery Procedures / Q000379:methods*; D013234:Stem Cells / Q000187:drug effects / Q000473:pathology; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D005990:Glycerol