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Br J Ophthalmol 89:1510-1517 doi:10.1136/bjo.2005.072108
  • Laboratory science - Extended reports

The expression of native and cultured human retinal pigment epithelial cells grown in different culture conditions

  1. J Tian1,
  2. K Ishibashi1,
  3. S Honda2,
  4. S A Boylan2,
  5. L M Hjelmeland2,
  6. J T Handa1
  1. 1Michael Panitch Macular Degeneration Research Laboratory, Wilmer Ophthalmological Institute, Johns Hopkins Medical Institutes, Baltimore, MD, USA
  2. 2Departments of Ophthalmology and Molecular and Cellular Biology, University of California, Davis, CA, USA
  1. Correspondence to: James T Handa MD; 3-109 Jefferson Street Building; Johns Hopkins Hospital; 600 N Wolfe Street; Baltimore, MD 21287, USA; jthandajhmi.edu
  • Accepted 6 June 2005

Abstract

Aim: To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE.

Methods: ARPE-19 cells were grown under five conditions in 10% CO2: “subconfluent” in DMEM/F12 + 10% FBS, “confluent” in serum and serum withdrawn, and “differentiated” for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays.

Results: 78% of genes were expressed by native RPE while 45.3–47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells.

Conclusions: While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.

Footnotes

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