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. 2014 Jan 9:3:4.
doi: 10.4103/2277-9175.124630. eCollection 2014.

A proper protocol for isolation of retinal pigment epithelium from rabbit eyes

Noushin Amirpour  1 , Fereshteh Karamali  2 , Shahnaz Razavi  1 , Ebrahim Esfandiari  1 , Mohammad Hossein Nasr-Esfahani  2
Affiliations

A proper protocol for isolation of retinal pigment epithelium from rabbit eyes

Noushin Amirpour et al. Adv Biomed Res. .

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.

Materials and 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.

Keywords: Cytokeratin 18; enzymatic digestion; retinal pigment epithelium; sheet; zonula occludens-1 protein.

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Conflict of interest statement

Conflict of Interest: None declared.

Figures

Figure 1
Figure 1
Phase contrast photomicrographs of retinal pigment epithelium-isolated cells showing cellular morphology according to enzymatic methods 1 (a), 2 (b), and the mechanical method (c). Scale bar: 20, 50, 100 μm, respectively
Figure 2
Figure 2
Immunocytochemistry staining of retinal pigment epithelium cells demonstrates expression of cytokeratin 18 in the mechanical protocol (c), which was not expressed in the enzymatic protocols (a and b)
Figure 3
Figure 3
Retinal pigment epithelium isolated by the mechanical protocol illustrates expression of tight junction protein ZO-1
Figure 4
Figure 4
Reverse transcriptase-polymerase chain reaction analysis showing the expression of rabbit retinal pigment epithelium marker NPR-A in the mechanical protocol (c)
Figure 5
Figure 5
Reverse transcriptase-polymerase chain reaction analysis indicatingthe expression of the specific protein, natriuretic peptid receptor-A on days 0 and 14 in the mechanical protocol
Figure 6
Figure 6
Comparison of the mean absorbance of retinal pigment epithelium in the third protocol on days 0 and 14 after culture (P ≤ 0.05)
See this image and copyright information in PMC

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