ARPE-19, A HUMAN RETINAL-PIGMENT EPITHELIAL-CELL LINE WITH DIFFERENTIATED PROPERTIES

The retinal pigment epithelium (RPE) plays a critical role in the deve lopment and maintenance of adjacent photoreceptors in the vertebrate r etina. This study describes the development and characterization of AR PE-19, a spontaneously arising human RPE cell line with normal karyolo gy which forms polarized epithelial monolayers on porous filter suppor ts. The cell line was established by selective trypsinization of a pri mary RPE culture resulting in a uniform population of highly epithelia l cells which exhibit a strong growth potential. To determine the exte nt of biochemical differentiation, the expression of the RPE-specific markers CRALBP and RPE65 was examined by Northern analysis. A single 1 .6 kb CRALBP mRNA transcript and a single 2.8 kb RPE65 transcript were detected in samples of ARPE-19 total mRNA. The expression of CRALBP p rotein in ARPE-19 cell lysate was detected by Western blot analysis an d immunocytochemistry was used to detect CRALBP throughout the cytopla sm of most, but not all, cells in confluent cultures. The essential cr iteria for monolayer formation were determined experimentally and it w as found that ARPE-19 cells exhibit morphological polarization when pl ated on laminin-coated Transwell-COL filters in medium with a low seru m content. The time course of tight-junction formation was determined by recording the transepithelial resistance of monolayers and reached a maximum of 50-100 Omega cm(2) after 4 weeks of culture. Barrier prop erties of ARPE-19 monolayers were evaluated by measuring the flux of H -3-inulin from the apical to the basolateral compartment of cell cultu re chambers. Finally, ARPE-19 clonal sublines were generated by serial dilution in an attempt to produce a subline with a high transepitheli al resistance (TER). The morphology of the sublines was variable and t he cloned cells exhibited a tendency to senesce in culture, confirming that this cell line is not transformed. No subline monolayers develop ed a TER greater than those recorded for the parent cells. Our results demonstrate that ARPE-19 has structural and functional properties cha racteristic of RPE cells in vivo and suggest that this cell line will be valuable for in vitro studies of retinal pigment epithelium physiol ogy. (C) 1996 Academic Press Limited