Background: The aim was to develop a three-dimensional cell culture system
for human fetal retinal pigment epithelial (HFRPE) cells for in vitro cellu
lar studies and for possible application in subretinal transplantation.
Methods: Pieces of freshly isolated HFRPE monolayer tissue were grown on cr
osslinked fibrinogen (CLF) films. The growth pattern and morphologic charac
teristics of the implanted tissue were studied using phase-contrast microsc
opy, photography, and light and electron microscopy. The cells were screene
d immunohistochemically for HLA-ABC, HLA-DR, ICAM-1, B7, and Cytokeratin. C
ell proliferation was studied using 5-bromo-2-deoxyuridine incorporation.
Results: After attachment to CLF, HFRPE monolayer tissue formed small tumor
-like formations, i.e. microsphere. HFRPE microspheres survived and prolife
rated in a floating state for at least 4 months. After attachment of the mi
crospheres to the culture dish floor, formation of a confluent HFRPE cell m
onolayer with high proliferative activity was noted around the microspheres
. HFRPE cells stained positive for HLA-ABC, ICAM-1, and cytokeratin and neg
ative for B7 and HLA-DR. The microspheres could be easily detached from the
dish and they were able to initiate similar growth after reattachment.
Conclusion: 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 v
itro studies. Because of their adjustable size, spherical shape, and abilit
y to initiate growth of cells with a high proliferative potential, HFRPE mi
crospheres may be successfully utilized as a source of donor cells for subr
etinal transplantation.