The blood-CSF barrier in culture. Development of a primary culture and transepithelial transport model from choroidal epithelial cells.

The chemical stability of the central nervous system (CNS) is safeguarded by two major barrier systems that separate the systemic circulation from the cerebral compartment. Within the cerebral compartment, the interstitial fluid (ISF) flows between neurons and the cerebrospinal fluid (CSF) circulates among major brain structures and ventricles. The direct continuity of ISF and CSF allows for the free exchange of substances within the extracellular space of the cerebral compartment. Thus, the barrier that separates the systemic compartment from ISF is defined as the blood-brain barrier, while the one that discontinues the circulation between systemic and CSF compartments is named the blood-CSF barrier. The choroid plexus, located within brain ventricles, is the tissue where the blood-CSF barrier is formed (1). Under the microscope, the choroid plexus consists of three cellular layers: (i) the apical epithelial cells; (ii) the underlying supporting connective tissue; and (iii) the inner layer of endothelial cells. These choroidal epithelial cells have the tight junctions near their apical surface, which seal one to another. The tight junctions constitute a structural basis for the blood-CSF barrier. The barrier impedes the diffusion of water soluble small molecules, proteins, other macromolecules, and ions from the blood to the CSF. The barrier also secretes CSF, which comprises approximately 80–90% of the total CSF. Furthermore, the barrier actively participates in the regulation of the homeostasis of the cerebral compartment. For example, the choroid plexus transports, bidirectionally between blood and CSF, a variety of amino acids (e.g., glycine, L-alanine), hormones (e.g., thyroid hormones, melatonin, growth hormone), peptides (e.g., atriopeptin, vasopressin), proteins (e.g., transthyretin), and drug molecules (e.g., β-lactam antibiotics, cimetidine, benzylpenicillin) (1,2). Keeping pace with the rapid growth in blood-CSF barrier research, we have developed a primary choroidal epithelial cell culture derived from rat choroid plexus. The plexus tissues are collected from Sprague-Dawley rats, digested, and then mechanically dissociated. The cells are then cultured. Usually the yield is around 2–5 × 105 cells from pooled plexuses of 3 to 4 rats, and they have a viability of 77–85%. Two days after initial seeding, the culture medium is replaced with medium containing cis-hydroxyproline (cis-HP) for 3–5 d to control the growth of fibroblastic cells. The cells are then cultured in the normal medium without cis-HP. The cultures display a dominant polygonal type of epithelial cells, with a population doubling time of 2 to 3 d. Immunocytochemical studies using rabbit anti-rat TTR polyclonal antibody reveal a strong positive stain of transthyretin (TTR), a protein exclusively produced by the choroidal epithelia in the brain. In addition, reverse transcription polymerase chain reaction (RT-PCR) confirms the presence of specific TTR mRNA in the cultures. We have further adapted this primary culture of choroidal epithelial cells on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurs within 5 d after seeding and can be verified by the presence of a steady electrical resistance across the membrane (100–180 ohm × cm2). This primary cell culture system and the pertinent in vitro model of the blood-CSF barrier have proven useful in studies of thyroxine transport at the blood-CSF barrier and the mechanisms of lead toxicity on CSF transthyretin (3,4,5) (for comments on species other than rat, see Note 1).