Primary culture of choroidal epithelial cells: characterization of an in vitro model of blood-CSF barrier.
A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier. Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml. The procedure yielded 2-5 x 10(4) cells from pooled plexuses of three to four rats, and a viability of 77-85%. The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2-3 d. The cultures were of distinct choroidal epithelial origins. For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia. Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures. The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 +/- 10 ohm per cm2). The epithelial barriers appeared to actively transport [125I]-thyroxine from the basal to apical chamber. These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood-CSF barrier.
Zheng
W
W
School of Public Health, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
Zhao
Q
Q
Graziano
J H
JH
D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier; D002417:Cattle; D002478:Cells, Cultured; D002831:Choroid Plexus / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology*; D005260:Female; D008297:Male; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley