An Ultrastructural Study of Retrocorneal Fibrous Membrane-Like Corneal Endothelial Metaplasia In vitro

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Abstract
The pathogenesis of many corneal diseases involves alteration in endothelium and subjacent Descemet’s membrane. A common manifestation of these disorders is the appearance of a retrocorneal fibrous membrane (RCM) composed of dissociated endothelial cells interposed between layers of Descemet’s membrane-like materials. The stimulus for the endothelium to lose its epithelioid characteristics and assume this more fibroblastic behavior is unknown. In the present study we describe tissue culture conditions which stimulate isolated corneal endothelial cells to undergo a transition similar to that seen in RCM formation. Accordingly, a monolayer of hexagonal endothelial cells become multilayered metaplastic cells and produce abundant Descemet’s membrane-like material. Cells were isolated by enzyme digestion and modification of thiol groups to yield single or small 2- to 3-cell aggregates which could be evenly dispersed on plastic tissue culture dishes. Contaminating stromal fibroblasts were not present in these preparations as verified morphologically. Cultures were confluent within 5–7 days after plating and at 28 days of incubation, cultures displayed features similar to regenerating rabbit endothelium. These included loss of contact inhibition leading to multilayering of cells, intracytoplasmic densities and extracellular matrix (ECM) which accumulated not only between the basal layer of cells and plastic substratum but also in the cellular interstices. The fibrillar and amorphous components of this ECM accumulated in thin layers and were ultrastructurally similar to the anterior banded and posterior unbanded portions of Descemet’s membrane, respectively. This in vitro lamination of cells and their ECM is highly reminiscent of the RCM accompanying various corneal diseases. Therefore, our in vitro system, leading to the formation of multilaminar corneal endothelial cells, provides an advantageous experimental model for studies of cellular activity leading to RCM formation.

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