title: Standardized limbal epithelial stem cell graft generation and transplantation.
abstract: OBJECTIVE
  To describe a standardized, xenogenic-free protocol for the manufacture of limbal epithelial stem cell grafts and a "no touch" surgical technique for its standardized transplantation.
  
  
  METHODS
  Antwerp University Hospital, Antwerp, Belgium.
  
  
  METHODS
  The limbo-amnion composite graft is generated by cultivating limbal epithelial stem cells on a standardized (thermolysin treated and spongy layer removed) amniotic membrane, stretched within an interlockable amnion ring. The cells are cultured in CnT-20 medium with the addition of 1% human AB serum for a period of 2 weeks. Fibrin glue is applied to the surgically prepared recipient's cornea and in one fluid motion, the composite graft within the amnion ring construct is transferred from culture and positioned onto the graft bed. The required size is cut out at the level of the limbus by means of a trephine and/or microsurgical scissors.
  
  
  RESULTS
  The lightweight, plastic interlockable ring offered stability to the graft during culture, transport, and transplantation. The use of the standardized amniotic membrane, within the amnion ring construct, improves reproducibility of the results and therefore heralds elective surgery. Rapid transplantation of a wrinkle-free graft, using a sutureless, “no touch" technique was achieved and this allowed precise tailoring of the graft to the recipient bed.
  
  
  CONCLUSIONS
  This is the first time a standardized, clinical grade protocol has been described for manufacturing limbal epithelial grafts with an efficient surgical technique that prevents postsurgical graft shrinkage and improves corneal integration. The quick, sutureless, and manipulation-free technique ensured transplantation of viable, proliferating limbal epithelial stem cells.
authors.affiliation: Department of Ophthalmology, University Hospital Antwerp, Antwerp, Belgium. nadia.zakaria@uza.com
authors.forename: Nadia
authors.initials: N
authors.lastname: Zakaria
authors.forename: Carina
authors.initials: C
authors.lastname: Koppen
authors.forename: Viggo
authors.initials: V
authors.lastname: Van Tendeloo
authors.forename: Zwi
authors.initials: Z
authors.lastname: Berneman
authors.forename: Andrew
authors.initials: A
authors.lastname: Hopkinson
authors.forename: Marie-José
authors.initials: MJ
authors.lastname: Tassignon
mesh_terms: D000650:Amnion / Q000166:cytology; D003470:Culture Media; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D015203:Reproducibility of Results; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*
chemical_list: D003470:Culture Media