title: A proper protocol for isolation of retinal pigment epithelium from rabbit eyes.
abstract: BACKGROUND
  Retinal pigment epithelium (RPE) is a hexagonal monolayer of pigmented cells located between the neural retina and the choroid with an essential role for visual function. So, isolation, propagation and maintenance of their functional integrity of RPE are crucial for research in vitro which next used for cell transplantation. The evaluation of features of RPE cells as a sheet after 14 days has not been reported yet. This study aimed to examine and compare three protocols for RPE isolation from rabbit eyes and obtain a proper protocol, which illustrated isolated RPE cells as a sheet cause to preserve their characterize even after 2 weeks.
  
  
  METHODS
  RPE cells were prepared from eyes of 24 rabbit eyes. After enucleating of eyes, anterior segment discarded and posterior segment cut to small pieces. Two of these procedures are based on the enzymatic digestion, but third protocol based on mechanical dissection. The culture cells harvested and morphological feature of cells assessed by phase-contrast microscope and then analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry.
  
  
  RESULTS
  Evaluation of morphological feature showed that isolation of RPE cells as a sheet lead to preserve their hexagonal morphology. Immunocytochemistry and RT-PCR assessment demonstrated RPE cell cultured in sheet maintained their phenotypic feature, tight junction and the distribution of actin and cytokeratin filament. Comparison of three protocols showed that dissociation of RPE cells as a sheet was superior in the preserve of RPE characteristic.
  
  
  CONCLUSIONS
  Isolation of RPE cells as a sheet maintains the integrity of these cells, this procedure promising a therapeutic approach, which is important for some retinal diseases.
authors.affiliation: Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran.
authors.forename: Noushin
authors.initials: N
authors.lastname: Amirpour
authors.affiliation: Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Fereshteh
authors.initials: F
authors.lastname: Karamali
authors.affiliation: Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran.
authors.forename: Shahnaz
authors.initials: S
authors.lastname: Razavi
authors.affiliation: Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran.
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Esfandiari
authors.affiliation: Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Mohammad Hossein
authors.initials: MH
authors.lastname: Nasr-Esfahani
keywords: Cytokeratin 18; enzymatic digestion; retinal pigment epithelium; sheet; zonula occludens-1 protein