A novel serum-free method for culturing human prenatal retinal pigment epithelial cells.
OBJECTIVE
Established techniques for culturing primary human retinal pigment epithelial (RPE) cells have facilitated the laboratory investigation of this multipurpose retinal cell layer. However, most culture methods involve the use of animal serum to establish and maintain RPE monolayers, which can complicate efforts to define and study factors involved in the maturation and function of these cells. Therefore, this study was conducted to develop a simple, serum-free system to propagate and sustain human RPE in vitro.


METHODS
RPE was dissected from human prenatal donor eyes and cultured in serum-free defined medium containing the commercially formulated supplement B27 or N2. Cultures were grown initially as adherent tissue sections or suspended spherical aggregates and later expanded and maintained as monolayers. PCR, Western blot analysis, and immunocytochemistry were used to monitor gene and protein expression in established cultures, followed by examination of secretory products in RPE conditioned medium by ELISA and mass spectrometric analysis.


RESULTS
In medium supplemented with B27, but not N2, RPE could be expanded up to 40,000-fold over six passages and maintained in culture for more than 1 year. In long-term cultures, typical cellular morphology and pigmentation were observed, along with expression of characteristic RPE markers. RPE monolayers also retained proper apical-basal orientation and secreted multiple factors implicated in the maintenance of photoreceptor health and the pathogenesis of age-related macular degeneration.


CONCLUSIONS
Monolayer cultures of human prenatal RPE can be grown and maintained long term in the total absence of serum and still retain the phenotype, gene and protein expression profile, and secretory capacity exhibited by mature RPE cells.
Gamm
David M
DM
Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, T607 Waisman Center, Madison, WI 53705, USA. dgamm@wisc.edu
Melvan
J Nicholas
JN
Shearer
Rebecca L
RL
Pinilla
Isabel
I
Sabat
Grzegorz
G
Svendsen
Clive N
CN
Wright
Lynda S
LS
D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D018929:Cell Culture Techniques; D016764:Cell Polarity; D049109:Cell Proliferation; D016895:Culture Media, Serum-Free; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics; D005333:Fetus / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005865:Gestational Age; D006801:Humans; D013058:Mass Spectrometry; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions; D014019:Tissue Donors
D015415:Biomarkers; D016895:Culture Media, Serum-Free; D005136:Eye Proteins