title: In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma.
abstract: OBJECTIVE
  To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.
  
  
  METHODS
  Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.
  
  
  RESULTS
  HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.
  
  
  CONCLUSIONS
  Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital of Jinan University, Guangzhou 510630, Guangdong Province, China.
authors.forename: Xiao-Yong
authors.initials: XY
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Xiao-Ling
authors.initials: XL
authors.lastname: Zhang
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Qin
keywords: amniotic epithelial cells; cornea; keratin; tissue engineering