In vitro propagation of primary and extended life span murine corneal endothelial cells.

PURPOSE

To establish primary and extended life span cell cultures of murine corneal endothelial cells for a model system investigating corneal endothelial cell replacement and the immunologic features of corneal graft rejection.

METHODS

The authors have been able to grow corneal endothelium in culture by isolating adult murine Descemet's membrane and allowing the endothelial cells to proliferate from the explants. To isolate extended life span murine corneal endothelial cells, cells were infected with an adenovirus-SV40 hybrid virus (Ad12-SV40).

RESULTS

The primary cells from adult corneas proliferated to passage 3 before growth arrest-senescence was observed. However, the extended life span cells remained proliferative through passage 36 and maintained a structure similar to the primary cells. Immunohistochemical analysis demonstrate that the extended life span cells are SV40 large T antigen positive, and both the primary and extended life span murine corneal endothelial cells exhibit the common expression of several growth factor receptors and TGF-beta.

CONCLUSIONS

This is the first report of the isolation and culture of mouse corneal endothelial cells. Additionally, these cells have been infected with a virus carrying the SV40 large T antigen, which yields extended life span mouse corneal endothelial cells. These cells will be of interest in establishing a murine model for corneal cell transplantation, and the authors are currently establishing protocols for the specific introduction and manipulation of these cells in both in vitro and in vivo systems. These types of analyses may provide an important animal model specific to in vivo corneal endothelial cell replacement for the treatment of endothelial-related keratopathies and can be a useful model in delineating the immunologic parameters of corneal graft rejection.