Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.
OBJECTIVE
Provide a reproducible method for culturing confluent monolayers of hfRPE cells that exhibit morphology, physiology, polarity, and protein expression patterns similar to native tissue.


METHODS
Human fetal eyes were dissected on arrival, and RPE cell sheets were mechanically separated from the choroid and cultured in a specifically designed medium comprised entirely of commercially available components. Physiology experiments were performed with previously described techniques. Standard techniques were used for immunohistochemistry, electron microscopy, and cytokine measurement by ELISA.


RESULTS
Confluent monolayers of RPE cell cultures exhibited epithelial morphology and heavy pigmentation, and electron microscopy showed extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. The mean transepithelial potential (TEP) was 2.6 +/- 0.8 mV, apical positive, and the mean transepithelial resistance (R(T)) was 501 +/- 138 Omega . cm(2) (mean +/- SD; n = 35). Addition of 100 microM adenosine triphosphate (ATP) to the apical bath increased net fluid absorption from 13.6 +/- 2.6 to 18.8 +/- 4.6 microL . cm(-2) per hour (mean +/- SD; n = 4). In other experiments, VEGF was mainly secreted into the basal bath (n = 10), whereas PEDF was mainly secreted into the apical bath (n = 10).


CONCLUSIONS
A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.
Maminishkis
Arvydas
A
National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Chen
Shan
S
Jalickee
Stephen
S
Banzon
Tina
T
Shi
Guangpu
G
Wang
Fei E
FE
Ehalt
Todd
T
Hammer
Jeffrey A
JA
Miller
Sheldon S
SS
D015153:Blotting, Western; D018929:Cell Culture Techniques; D016764:Cell Polarity / Q000502:physiology*; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D004594:Electrophysiology; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism*; D005333:Fetus / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008564:Membrane Potentials; D008854:Microscopy, Electron; D009414:Nerve Growth Factors / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000502:physiology*; D015843:Serpins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D016207:Cytokines; D005136:Eye Proteins; D009414:Nerve Growth Factors; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C467484:VEGFA protein, human