Autofluorescent inclusions in long-term postconfluent cultures of retinal pigment epithelium

To examine the accumulation and morphologic features of fluorescent inclusions in retinal pigment epithelial (RPE) cells in vitro and to determine whether accumulation correlates with parameters of age.

Cultured human RPE cells were maintained undisturbed at confluence for intervals as long as 2 years and then were examined for autofluorescent inclusions by fluorescence and electron microscopy, with comparisons to the lipofuscin of freshly isolated RPE. Autofluorescence was also examined in bovine RPE cultures that were aged in vitro by replicative senescence. In some postconfluent cultures,the activity of the lysosomal enzyme cathepsin D was measured, and the effect of cell growth on autofluorescence was examined by reinducing cell proliferation in late-stage cultures.

Autofluorescent inclusions accumulated in human and bovine RPE cultures after extended postconfluent periods. Accumulation was not accelerated in cultures from older donors or in cultures that were aged in vitro. The number of granules per RPE cell varied for lipofuscin in situ and inclusions in vitro, although the latter were more heterogeneous in size and shape and in ultrastructural appearance of the granule contents. Lipofuscin and autofluorescent inclusions were lost when RPE cells were propagated, but in contrast to lipofuscin, the inclusions became smaller and fainter when growth was reinduced in postconfluent cultures. Cathepsin D activity varied among cultures and showed no significant change as time elapsed after confluence. Activity increased after repropagation of long-term postconfluent cultures.

In the absence of phagocytic challenge with photoreceptor outer segments, postconfluent RPE cultures accumulate heterogeneous materials that show autofluorescence. Accumulation is associated with one marker of age: time at confluence, which simulates a long-term, nonmitotic state similar to that which occurs in situ during aging in postproliferative tissues. The autofluorescent inclusions in vitro predictably differ from autofluorescent lipofuscin in situ, presumably because the inclusions formed in culture are derived from RPE autophagy alone. Postconfluent cultures can be used to examine the molecular and biologic properties of materials originating only from an autophagic source, or coupled with phagocytic challenge to produce a model of developing RPE lipofuscin that has an autophagocytic and a heterophagic component, similar to RPE cells in situ.