April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
An organotypic culture model of porcine neuroretina supplemented with porcine retinal pigment epithelium (pRPE) cells to simulate an ex vivo subretinal space
Author Affiliations & Notes
  • Salvatore Di Lauro
    Ophthalmology, Instituto Universitario de Oftalmobiologia Aplicada (IOBA), University of Valladolid, Valladolid, Spain
    Ophthalmology, Hospital Clinico Universitario de Valladolid, University of Valladolid, Valladolid, Spain
  • Ivan Fernandez-Bueno
    Ophthalmology, Instituto Universitario de Oftalmobiologia Aplicada (IOBA), University of Valladolid, Valladolid, Spain
    CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), Valladolid, Spain
  • David Rodríguez-Crespo
    Ophthalmology, Instituto Universitario de Oftalmobiologia Aplicada (IOBA), University of Valladolid, Valladolid, Spain
  • Girish K Srivastava
    Ophthalmology, Instituto Universitario de Oftalmobiologia Aplicada (IOBA), University of Valladolid, Valladolid, Spain
    CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), Valladolid, Spain
  • Amar K Singh
    Ophthalmology, Instituto Universitario de Oftalmobiologia Aplicada (IOBA), University of Valladolid, Valladolid, Spain
  • Maite Garcia-Gutierrez
    Ophthalmology, Instituto Universitario de Oftalmobiologia Aplicada (IOBA), University of Valladolid, Valladolid, Spain
    CIBER de Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN), Valladolid, Spain
  • Manuel Gayoso
    Cellular Biology, Histology and Pharmacology, University of Valladolid, Valladolid, Spain
  • Jose-Carlos Pastor
    Ophthalmology, Instituto Universitario de Oftalmobiologia Aplicada (IOBA), University of Valladolid, Valladolid, Spain
    Ophthalmology, Hospital Clinico Universitario de Valladolid, University of Valladolid, Valladolid, Spain
  • Footnotes
    Commercial Relationships Salvatore Di Lauro, None; Ivan Fernandez-Bueno, None; David Rodríguez-Crespo, None; Girish Srivastava, None; Amar Singh, None; Maite Garcia-Gutierrez, None; Manuel Gayoso, None; Jose-Carlos Pastor, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 6314. doi:
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      Salvatore Di Lauro, Ivan Fernandez-Bueno, David Rodríguez-Crespo, Girish K Srivastava, Amar K Singh, Maite Garcia-Gutierrez, Manuel Gayoso, Jose-Carlos Pastor; An organotypic culture model of porcine neuroretina supplemented with porcine retinal pigment epithelium (pRPE) cells to simulate an ex vivo subretinal space. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6314.

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

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Abstract

Purpose: To develop a new physically separated co-culture model of neuroretina with retinal pigment epithelium (RPE) cells that simulates an ex vivo subretinal space to recreate some of the conditions that follow retinal detachment.

Methods: RPE cells from porcine eyes (pRPE) were isolated and maintained in culture as previously described by our group (Srivastava et al., 2011). pRPE cells were seeded (30,000 cells/cm2) on the bottom of Transwell culture plates for 24 hrs for adherence and growth. Neuroretina explants (5x5 mm), from the porcine area centralis, were obtained as previously described by our group (Fernandez-Bueno et al. 2008). Explants were cultured over Transwell membranes with the photoreceptor layer facing the supporting membrane. Neuroretina explants supplemented with pRPE cells were co-cultured in Neurobasal A/DMEM (1:1) medium during 9 days. In parallel, neuroretina explants were cultured alone as controls. Specimens were processed for epoxy-resin embedding and cryosectioning. Light and immunofluorescence microscopy were performed, using toluidine blue staining and antibodies against GFAP, CRALBP, calbindin (CB) and synaptophysin (SYP) to evaluate retinal modifications. Specimen thickness was evaluated over neuroretinal images using the software Image J (version 1.47v, NIH Image, National Institute of Health, EE.UU).

Results: Neuroretina explants co-cultured with pRPE better preserved tissular architecture and cellular organization than controls at 9 days of culture. Co-cultured explants thickness was statistically significant maintained than in controls after culture (109,35±4,24 μm vs 92,28±14,94 μm; p<0.05). Protein immunoexpressions in co-cultures revealed less reactive gliosis (GFAP and CRALBP) and preserved cones (CB) and synapses (SYP) compared to controls.

Conclusions: A double-layer co-culture model of neuroretina and RPE cells was successfully developed and standardized. In this model, RPE cells presence reduced retinal degeneration changes, probably related to the secretion of neuroprotective factors by RPE cells. Physically separation between neuroretina and RPE could recreate an ex vivo subretinal space. Thus, this model may be useful to evaluate potential therapies for retinal detachment and other retinal degeneration processes.

Keywords: 694 retinal culture • 701 retinal pigment epithelium • 697 retinal detachment  
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