June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Laminin Surface Matrix Promotes Superior Morphologic and Functional Characteristics in Cultured Human Retinal Pigment Epithelium
Author Affiliations & Notes
  • Aya Yanagida
    Department of Ophthalmology, University of Washington, Seattle, WA
  • Jennifer Chao
    Department of Ophthalmology, University of Washington, Seattle, WA
  • Footnotes
    Commercial Relationships Aya Yanagida, None; Jennifer Chao, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1180. doi:
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      Aya Yanagida, Jennifer Chao; Laminin Surface Matrix Promotes Superior Morphologic and Functional Characteristics in Cultured Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1180.

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

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Abstract

Purpose: With the advent of retinal pigment epithelium (RPE) differentiation from human embryonic stem cells and induced pluripotent stem cells, recapitulation of native RPE structure and function in vitro is particularly important. At present, there is no consensus as to the optimal matrix on which to grow RPE cells in culture. We compare two commonly proposed extracellular matrices and determine which better supports human RPE structure and function in vitro.

Methods: RPE cells, dissected from human tissue of gestational age 18-20 weeks, were plated onto laminin or fibronectin coated filters. After culturing for 2-3 months, the expression of appropriate cell surface markers, including bestrophin, ZO-1, occludin, and Na/K ATPase, by immunocytochemistry was examined. Transmission electron microscopy (TEM) and transepithelial resistance measurements characterized RPE cell polarity and the presence of tight junctional complexes. RPE cell function was determined by phagocytosis of 1µm fluorescent polystyrene microspheres and outer segments of NRL-GFP explant retinas co-cultured with the RPE. Apical and basal measurements of VEGF secretion by ELISA were also performed. Finally, deposition of drusen-like deposits in response to human serum exposure was characterized by immunocytochemistry.

Results: RPE cells cultured on laminin coated filters consistently showed better establishment of monolayers and tight junctions as demonstrated by immunocytochemistry, TER, and basolateral VEGF secretion compared to those cultured on fibronectin coated filters. Far fewer apical microvilli and were observed in RPE cells cultured on fibronectin by TEM. In addition, RPE cells cultured on laminin demonstrated phagocytic activity of both microspheres and co-cultured rod outer segments. However, when exposed to human sera, RPE cells grown on both types of matrices deposited drusen-like deposits and immune-mediated complexes.

Conclusions: Human fetal RPE cells cultured on laminin-coated filters established similar morphologic, structural, and functional characteristics to native RPE, in contrast to RPE cultured on fibronectin-coated filters. Our findings provide the basis for culturing RPE cells on laminin and not fibronectin for a higher fidelity in vitro model.

Keywords: 701 retinal pigment epithelium • 694 retinal culture • 504 drusen  
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