Isolation, culture and characteristics of human foetal and adult retinal pigment epithelium.
BACKGROUND
Current methods for the isolation and culture of adult retinal pigment epithelium (RPE) provide successful primary culture. However, most methods result in contamination with other cell types and low cell yields. The isolation and culture of human foetal RPE presents further problems associated with the limited size of the eye cup and adherence among cells. Reliable methods are necessary for the culture of human RPE and subsequent functional studies.


METHODS
The present procedure is based on mechanical peeling of the whole RPE layer under the dissecting microscope. Dissected pieces are subsequently explanted to a 35 mm culture dish and are cultured with Dulbecco's modified Eagle's medium containing 10% foetal bovine serum. Peroxidase immunohistochemical methods were used to investigate cell phenotypes.


RESULTS
Primary cultures were obtained within 10-14 days with high yields, good viability and purity in subsequent culture. Cultured cells were vimentin and cytokeratin positive and CD31 negative.


CONCLUSIONS
This mechanical dissection technique is recommended for the isolation of foetal and young adult RPE cells, while the enzyme digestion method is preferred for aged adult tissue.
Department of Clinical Ophthalmology, University of Sydney, New South Wales, Australia. meidong@eye.usyd.edu.au
M
M
Zhu
J M
JM
Provis
P L
PL
Penfold
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D003470:Culture Media; D005333:Fetus; D005865:Gestational Age; D006801:Humans; D007124:Immunoenzyme Techniques; D007633:Keratins / Q000378:metabolism; D008875:Middle Aged; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D003470:Culture Media; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D014746:Vimentin; D007633:Keratins