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A more recent version of this article appeared on August 1, 2008
 (Investigative Ophthalmology and Visual Science. )
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1522
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Article

Characterization of a Spontaneous Mouse Retinal Pigment Epithelial (RPE) Cell Line B6-RPE07

Mei Chen 1, Elizabeth Muckersie 2, Marie Robertson 1, Monika Fraczek 1, John V. Forrester 1, and Heping Xu 3*

1 Ophthalmology, Aberdeen University School of Medicine, Aberdeen, AB25 2ZD, United Kingdom
2 Department of Ophthalmology, Aberdeen University School of Medicine, Aberdeen, United Kingdom
3 Ophthalmology, Aberdeen University School of Medicine, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom

* To whom correspondence should be addressed. E-mail: h.xu{at}abdn.ac.uk.


  Abstract

PURPOSE: A spontaneously arising RPE cell line (B6-RPE07) has been cloned from a primary culture of mouse RPE cells and has been maintained in culture for over 18 months. The morphological and functional properties of this cell line have been characterized. METHODS: The morphology of the B6-RPE07 cells was examined by phase-contrast light-microscopy, electron microscopy and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind and phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy and flow cytometry. Cytokine/chemokine secretion was measured by Cytometric Bead Array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting. RESULTS: In standard culture conditions B6-RPE07 cells display cobblestone morphology. When cultured on 3D collagen gel-coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also express the RPE-specific marker, CRALBP, but not RPE65. Cell junction proteins ZO-1, {beta}-catenin but not claudin-1/3 or occludin-1 were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2. CONCLUSIONS: This is the first report of a mouse RPE cell line with the morphology, phenotype and function similar to in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.

Key Words: retinal pigment epithelium, retinal degeneration, retinal cell culture, mouse






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