Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line.
OBJECTIVE
ARPE-19 is a spontaneously transformed cell line of human RPE that is widely studied. This report examines its suitability for studying the tight junctions of the RPE.


METHODS
ARPE-19 was maintained in standard medium or one of three reduced-serum medium formulations. The expression and distribution of cytoskeletal and junctional proteins were examined by immunocytochemistry, immunoblot analysis, and the reverse transcription-polymerase chain reaction. Barrier function was measured as the transepithelial electrical resistance (TER) and the transmonolayer diffusion of horseradish peroxidase (HRP).


RESULTS
Unlike the original reports using passage-15 to -20 cells, commonly available strains of ARPE-19 exhibited a heterogeneous mixture of elongate and polygonal cells. Actin was distributed in stress fibers rather than circumferential bands. The TER was low, and the permeability of HRP was high. The expression of claudins and cytokeratins was heterogeneous. Partial differentiation could be induced in subsets of cells by manipulating the growth medium. A common effect was an increase in the expression of JAM-A, AF-6, and PAR-3 that correlated with a redistribution of actin filaments. This effect was accompanied by a 10x decrease in the permeability of HRP, but a minimal effect on TER.


CONCLUSIONS
The properties of ARPE-19 appear to be changing in ways that may depend on how the cells are maintained and passaged. Caution should be exercised in comparing data between laboratories and in interpreting studies in which only a subset of cells may respond to experimental stimuli. Specialized media promoted the maturation of the adherens junction, but only a partial maturation of the tight junctions.
Luo
Yan
Y
Department of Surgery, Yale University School of Medicine, New Haven, CT 06520, USA.
Zhuo
Yehong
Y
Fukuhara
Masayuki
M
Rizzolo
Lawrence J
LJ
D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004553:Electric Conductivity; D017097:Electric Impedance; D004594:Electrophysiology; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D062793:Occludin; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000378:metabolism*
D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D006735:Horseradish Peroxidase