Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07

Mei Chen, Elizabeth Muckersie, Marie Robertson, Monika Fraczek, John Vincent Forrester, Heping Xu

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

PURPOSE. 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.

Original languageEnglish
Pages (from-to)3699-3706
Number of pages8
JournalInvestigative Ophthalmology & Visual Science
Volume49
Issue number8
Early online date17 Apr 2008
DOIs
Publication statusPublished - Aug 2008

Keywords

  • Animals
  • Biological Markers
  • Blotting, Western
  • Carrier Proteins
  • Cell Culture Techniques
  • Cell Line
  • Cell Separation
  • Cytokines
  • Eye Proteins
  • Female
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Confocal
  • Microscopy, Electron
  • Microscopy, Phase-Contrast
  • Phagocytosis
  • Phenotype
  • Pigment Epithelium of Eye
  • Reverse Transcriptase Polymerase Chain Reaction
  • Rod Cell Outer Segment

Cite this

Chen, M., Muckersie, E., Robertson, M., Fraczek, M., Forrester, J. V., & Xu, H. (2008). Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07. Investigative Ophthalmology & Visual Science, 49(8), 3699-3706. https://doi.org/10.1167/iovs.07-1522

Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07. / Chen, Mei; Muckersie, Elizabeth; Robertson, Marie; Fraczek, Monika; Forrester, John Vincent; Xu, Heping.

In: Investigative Ophthalmology & Visual Science, Vol. 49, No. 8, 08.2008, p. 3699-3706.

Research output: Contribution to journalArticle

Chen, M, Muckersie, E, Robertson, M, Fraczek, M, Forrester, JV & Xu, H 2008, 'Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07', Investigative Ophthalmology & Visual Science, vol. 49, no. 8, pp. 3699-3706. https://doi.org/10.1167/iovs.07-1522
Chen, Mei ; Muckersie, Elizabeth ; Robertson, Marie ; Fraczek, Monika ; Forrester, John Vincent ; Xu, Heping. / Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07. In: Investigative Ophthalmology & Visual Science. 2008 ; Vol. 49, No. 8. pp. 3699-3706.
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abstract = "PURPOSE. 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.",
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AU - Fraczek, Monika

AU - Forrester, John Vincent

AU - Xu, Heping

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N2 - PURPOSE. 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.

AB - PURPOSE. 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.

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KW - Eye Proteins

KW - Female

KW - Mice

KW - Mice, Inbred C57BL

KW - Microscopy, Confocal

KW - Microscopy, Electron

KW - Microscopy, Phase-Contrast

KW - Phagocytosis

KW - Phenotype

KW - Pigment Epithelium of Eye

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Rod Cell Outer Segment

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