Primary culture of choroidal epithelial cells: characterization of an in vitro model of blood-CSF barrier

Abstract

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.

Fig. 1

Growth curves of choroidal epithelial cells in primary culture. Cells were initially seeded at 3 × 10⁵ cells per dish.

Fig. 2

Primary culture of choroidal epithelial cells after 4 d in culture. Note the confluent layer of the cells with predominant polygonal cell type (× 100). The choroid plexuses were obtained from 5-wk-old Sprague-Dawley rats.

Fig. 3

Cultured choroidal epithelial cells possess cytosolic TTR by immunocytochemical staining. a, Cells were treated with anti-TTR primary antibody followed by secondary antibody conjugated with ABC reagent. Note the positive staining in cytosol (× 250). b, Cells were treated with TTR-presaturated primary antibody followed by ABC staining procedure (× 250).

Fig. 4

Choroid plexus tissues and the cultured choroidal epithelial cells express TTR mRNA by reverse transcriptase-PCR analysis. All samples underwent DNase digestion and RT-PCR unless otherwise stated. Arrow indicates bands corresponding to TTR mRNA. Lane I.D.: (1) base pair markers; (2) plexus tissue, total RNA, without RT-PCR; (3) plexus tissue, mRNA with selected primer; (4) liver, mRNA with selected primer; (5) and (6) cultured plexus cells, mRNA with selected primer; (7) same as lane (1).

Fig. 5

Choroidal epithelial cells cultured on Transwell-COL membrane actively transport [¹²⁵I]-thyroxine across a confluent epithelial monolayer. Data represent means ± SD (n = 3).