title: Cultured epithelial grafting using human amniotic membrane: the potential for using human amniotic epithelial cells as a cultured oral epithelium sheet.
abstract: OBJECTIVE
  Human amniotic cells are a valuable source of functional cells that can be used in various fields, including regenerative medicine and tissue engineering. The aim of this study was to investigate the utility of human amniotic epithelial (hAE) cells as a new cell source for culturing stratified epithelium sheets for intraoral grafting.
  
  
  METHODS
  Enzymatically isolated hAE cells were submerged in a serum-free, low-calcium-supplemented MCDB 153 medium without a feeder layer. The hAE cells were seeded onto a Millicell cell culture plate insert and cultured while submerged in a high-calcium medium for 4 days. Then, they were cultured at an air-liquid interface for 3 weeks. Cultures of hAE cells proliferated at the air-liquid interface.
  
  
  RESULTS
  After 3 weeks, the hAE cells cultivated using the air-liquid interface method lead to almost 10 continuous layers of stratified epithelium without parakeratinization or keratinization. It confirmed immunohistochemically that the presence of CK10/13 and Ki-67 positive cells were spread throughout almost all the epithelial layer, and that CK19 positive cells were expressed throughout the entire epithelial layer in the cultured hAE cell sheets. Cultured hAE cells sheets showed a staining pattern similar to that of uncultured oral mucosa: ZO-1 and occludin were located in the intercellular junctions throughout all the epithelial layers. It was suggested that the hAE sheets consisted of highly-active proliferating cells and undifferentiated cells, and had a barrier function.
  
  
  CONCLUSIONS
  These results suggested that hAE cells may be a promising cell source for the development of stratified epithelium allograft sheets using a human cell strain.
authors.affiliation: Department of Dentistry and Oral Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumato 390-8621, Japan. marianosasi@um.es
authors.forename: Takeshi
authors.initials: T
authors.lastname: Koike
authors.forename: Masanari
authors.initials: M
authors.lastname: Yasuo
authors.forename: Tetsu
authors.initials: T
authors.lastname: Shimane
authors.forename: Hiroichi
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Toshio
authors.initials: T
authors.lastname: Nikaido
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kurita
mesh_terms: D000650:Amnion / Q000166:cytology*; D002118:Calcium / Q000008:administration & dosage; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D016895:Culture Media, Serum-Free; D004847:Epithelial Cells / Q000166:cytology; D004848:Epithelium / Q000033:anatomy & histology; D006801:Humans; D007365:Intercellular Junctions / Q000648:ultrastructure; D053535:Keratin-13 / Q000032:analysis; D053539:Keratin-19 / Q000032:analysis; D007633:Keratins / Q000032:analysis; D019394:Ki-67 Antigen / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis; D009061:Mouth Mucosa / Q000033:anatomy & histology / Q000166:cytology; D062793:Occludin; D010750:Phosphoproteins / Q000032:analysis; D046509:Tissue Culture Techniques; D023822:Tissue Engineering; D062826:Zonula Occludens-1 Protein
chemical_list: D016895:Culture Media, Serum-Free; D053535:Keratin-13; D053539:Keratin-19; D019394:Ki-67 Antigen; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D007633:Keratins; D002118:Calcium