Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.
OBJECTIVE
A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic 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/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 three-dimensional (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 expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and 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 morphology, phenotype, and function similar to those of 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.
Chen
Mei
M
Department of Ophthalmology, School of Medicine, Institute of Medical Sciences, University of Aberdeen, Scotland, United Kingdom.
Muckersie
Elizabeth
E
Robertson
Marie
M
Fraczek
Monika
M
Forrester
John V
JV
Xu
Heping
H
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D010587:Phagocytosis / Q000502:physiology; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012374:Rod Cell Outer Segment / Q000378:metabolism; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D015415:Biomarkers; D002352:Carrier Proteins; D016207:Cytokines; D005136:Eye Proteins; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C020932:11-cis-retinal-binding protein