April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Characterisation of Extended-Life Murine RPE Cell Lines (RPET and DH-1)
Author Affiliations & Notes
  • K. P. Kennelly
    University College Dublin, Catherine McAuley ClinicalResearchCentre, Dublin, Ireland
    Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland
  • T. M. Holmes
    University College Dublin, Catherine McAuley ClinicalResearchCentre, Dublin, Ireland
  • D. J. Hankey
    Institute of Ophthalmology, University College London, London, United Kingdom
  • D. J. Keegan
    Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland
  • Footnotes
    Commercial Relationships  K.P. Kennelly, None; T.M. Holmes, None; D.J. Hankey, None; D.J. Keegan, None.
  • Footnotes
    Support  HRB Project Grant RP/2007/202
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5914. doi:
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      K. P. Kennelly, T. M. Holmes, D. J. Hankey, D. J. Keegan; Characterisation of Extended-Life Murine RPE Cell Lines (RPET and DH-1). Invest. Ophthalmol. Vis. Sci. 2009;50(13):5914.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : All subretinal transplant strategies, including RPE, stem cell and neural retinal transplants, are limited by graft cell loss and failure. There are no commercially available murine RPE cell lines. We sought to complete the characterization of murine RPE cells to current standards for use in our murine allogeneic subretinal transplant model. We have two murine RPE cell lines, RPET and DH-1. RPET was derived from a C57BL/6 mouse harbouring temperature sensitive simian virus 40 T-antigen (SV40T) and DH-1 was derived from a C57Bl/10.R111 mouse and then transformed using SV40T.

Methods: : Both cell lines were grown in DMEM with 1% fetal calf serum at 330C and their basic morphology and growth characteristics recorded. Immunocytochemistry was used to assess ZO-1, β-catenin, cytokeratins and RPE65. Real time PCR was used to identify expression of the RPE specific genes rpe65 and rlbp1. A Meso-Scale Discovery TH1/TH2 multi-spot assay kit was used to quantify cytokine profiles looking at both cell lines under baseline and transplant protocol conditions, as well as in the presence of LPS.

Results: : Cell morphology of both RPET and DH-1 conform to known RPE cell lines such as human ARPE19. DH-1cells gave better epithelioid morphology than RPET, which were more fusiform. DH-1 cells grew as contact inhibited monolayers whereas RPET cells were only partially contact inhibited. Both cell lines were positive for ZO-1, β-catenin and RPE65 immunostaining. PCR confirmed the presence of rpe65 and rlbp1 in both cell lines. Cell stress as occurs with cell preparation for transplantation caused increases in pro-inflammatory cytokines (IL-1β, IFN-γ, TNF-, and IL-6) similar to exposure with LPS though less pronounced.

Conclusions: : These two cell lines show characteristics of RPE cells in culture but they do differ slightly. Preparation of cells for transplantation may prime cells for graft failure. Both cell lines are suitable for our primary purpose of conducting retinal transplant experiments and examining mechanisms of graft cell loss.

Keywords: retinal pigment epithelium • stress response • inflammation 
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