A new wound healing model of retinal pigment epithelial cells in sheet culture.
OBJECTIVE
To evaluate some RPE cell functions, such as wound healing, in a preparation more similar to in situ conditions, we developed a method to obtain and culture retinal pigment epithelial (RPE) cells as a sheet. And we assessed the effects of fetal bovine serum (FBS) on the rate of RPE wound healing.


METHODS
We prepared RPE sheet cultures by incubating rat eyes in 0.1% proteinase K for 13 min, peeling away the neural retina-RPE complex, and then incubating the tissue for 1 h to promote spontaneous separation of the RPE sheet from the retina. After several days of incubation, the cultured sheets of RPE cells were examined by phase-contrast microscopy, scanning and transmission electron microscopy and immunocytochemistry. We made round defects 1 mm in diameter in cultured RPE sheets and estimated the rate of wound closure in media with different concentrations of FBS (0 to 10%).


RESULTS
The RPE cells cultured in sheets retained their in situ features, including microvilli, tight junctions and gap junctions, and the distribution of actin and cytokeratin filaments. A wound was noted to close with restoration of a polygonal configuration. The rate of wound closure depended on serum concentration in the culture medium; when supplemented with 10% fetal bovine serum, wound closure was complete in approximately 40 h.


CONCLUSIONS
The RPE sheet-culture technique we developed thus provides a suitable model for studying such RPE cell functions as wound healing or phagocytosis.
Kamei
M
M
Department of Ophthalmology, Osaka University Medical School, Japan.
Lewis
J M
JM
Hayashi
A
A
Sakagami
K
K
Ohji
M
M
Tano
Y
Y
D000818:Animals; D001485:Basement Membrane / Q000648:ultrastructure; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D003599:Cytoskeleton / Q000648:ultrastructure; D007365:Intercellular Junctions / Q000648:ultrastructure; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D008954:Models, Biological; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000293:injuries* / Q000502:physiology; D051381:Rats; D014945:Wound Healing
D003470:Culture Media