title: Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells.
abstract: BACKGROUND
  We have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates.
  
  
  METHODS
  Human limbal epithelial cells were dissociated from donor eyes using dispase and gentle pipetting and then seeded onto intact and denuded AM as cell suspension. Limbal epithelial cells on AM were co-cultured with a MMC-treated 3T3 fibroblast feeder layer and epithelial differentiation was promoted by air lifting. Cultures were examined by light, scanning and transmission electron microscopy and differences in cellular attachments and intercellular spacing were quantified. Basement membrane complexes were examined by indirect immunofluorescence.
  
  
  RESULTS
  Limbal cells grown on denuded AM were well stratified and differentiated. Cells were well attached to each other and to the basement membrane. In contrast, limbal cells cultured on intact AM failed to stratify and in places formed a monolayer. The culture on denuded AM had significantly (P<0.001) more desmosomal junctions as well as significantly (P<0.001) more junctional attachments to the carrier than the intact culture. In addition, the intercellular spaces between cells cultivated on denuded AM were significantly (P<0.001) smaller than those between cells grown on the intact substrate. In cultures on both denuded and intact AM, the basement membrane zone displayed a positive staining for collagen VII, integrins alpha-6 and beta-4 and laminin 5.
  
  
  CONCLUSIONS
  We successfully cultivated well-stratified and -differentiated limbal cells on denuded AM, while on the intact AM limbal cells failed to stratify and in places formed only a monolayer of cells. The limbal cells cultivated on denuded AM were well attached to the AM stroma and were morphologically superior to the limbal epithelium cultivated on intact AM. We conclude that for purposes of transplantation of differentiated epithelial sheets, denuded AM is probably the more practical carrier for human limbal epithelial cell cultures when using our cell-suspension culture system.
authors.affiliation: Research Center for Regenerative Medicine, Office for Research Initiatives and Development, Doshisha University, Karasuma-Imadegawa, Kamigyoku, 602-8580 Kyoto, Japan. nkoizumi@ophth.kpu-m.ac.jp
authors.forename: Noriko
authors.initials: N
authors.lastname: Koizumi
authors.forename: Helen
authors.initials: H
authors.lastname: Rigby
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Fullwood
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kawasaki
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tanioka
authors.forename: Kan
authors.initials: K
authors.lastname: Koizumi
authors.forename: Norbert
authors.initials: N
authors.lastname: Kociok
authors.forename: Antonia M
authors.initials: AM
authors.lastname: Joussen
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
mesh_terms: D016475:3T3 Cells / Q000166:cytology; D000650:Amnion / Q000502:physiology*; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism / Q000648:ultrastructure; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000648:ultrastructure; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D013234:Stem Cells / Q000648:ultrastructure
chemical_list: D016326:Extracellular Matrix Proteins