Growth of human fetal retinal pigment epithelium as microspheres

The aim was to develop a three-dimensional cell culture system for human fetal retinal pigment epithelial (HFRPE) cells for in vitro cellular studies and for possible application in subretinal transplantation.

Pieces of freshly isolated HFRPE monolayer tissue were grown on crosslinked fibrinogen (CLF) films. The growth pattern and morphologic characteristics of the implanted tissue were studied using phase-contrast microscopy, photography, and light and electron microscopy. The cells were screened immunohistochemically for HLA-ABC, HLA-DR, ICAM-1, B7, and Cytokeratin. Cell proliferation was studied using 5-bromo-2-deoxyuridine incorporation.

After attachment to CLF, HFRPE monolayer tissue formed small tumor-like formations, i.e. microspheres. HFRPE microspheres survived and proliferated in a floating state for at least 4 months. After attachment of the microspheres to the culture dish floor, formation of a confluent HFRPE cell monolayer with high proliferative activity was noted around the microspheres. HFRPE cells stained positive for HLA-ABC, ICAM-1, and cytokeratin and negative for B7 and HLA-DR. The microspheres could be easily detached from the dish and they were able to initiate similar growth after reattachment.

HFRPE grown on CLF resemble a three-dimensional culture system with high yield of pure cells that can be useful for a wide variety of in vitro studies. Because of their adjustable size, spherical shape, and ability to initiate growth of cells with a high proliferative potential, HFRPE microspheres may be successfully utilized as a source of donor cells for subretinal transplantation.