The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Commemorative Booklet for CAAT 20th Anniversary Symposium

Development of an In Vitro Cell Model of the Human Blood-Brain Barrier

M. Fabre, E. Gonzalez, G. Ruiz, S. Gonzalez, R. Casaroli, and S. Vilaro
University of Barcelona, Spain

The development of efficient therapeutic drugs not only requires the optimization of specific and potent pharmacological activity at the target site, but also the efficient delivery of the drug to this site. The main obstacle for efficient drug targeting to the brain lies within the blood-brain barrier (BBB). The barrier, created where endothelial cells meet cerebral capillaries, strictly controls the concentration and entry of solutes into the central nervous system. An in vitro model would allow academic groups and pharmaceutical companies to more easily -- and more humanely -- assess the potential toxicity and bioavailability of new drugs in development.

In response to this urgent need, and as an alternative method to animal testing, the aim of our proposal is to produce stable, immortalized lines of human brain endothelial cells, which will retain their functional properties, in order to further develop an in vitro cell model of the human BBB for pharmacological and toxicological testing of compounds. Based on the co-culture of astrocytes and immortalized endothelial cells from human brain microvessels, this in vitro cell model will be fully characterized by testing the endothelial cell response to a battery of drugs with known BBB profiles.

To optimize our method for obtaining primary cultures of human cells, our work initially focused on the collection and culture of endothelial cells from bovine corneas, due to the ready availability of this tissue. Once successful in this effort, we then performed preliminary experiments to isolate microvessel endothelial cells from human brain biopsies according to the same method. However, due to the very small amounts of human brain tissue available, the number of microvessels isolated was insufficient for subsequent endothelial cell clones recovery. We currently are reproducing these experiments using total brain samples from post-mortem, multi-donor patients.

In addition, we obtained a retroviral construct (pWZLblast3:hTERT) expressing the human telomerase catalytic subunit sequence, an immortalizing but non-transforming gene, and are currently preparing a viral stock by transfection into Phoenix-Ampho packaging cells. Once obtained, the next step will be to optimize immortalization of these primary cultures to generate stable human endothelial cell lines.

We are confident that a BBB model will serve as an alternative to reduce, or even replace, the use of large numbers of animals in drug screening and will significantly accelerate the CNS-oriented drug discovery and development processes.