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Title: Development of a three-dimensional, all-human in vitro model of the blood-brain barrier for cancer metastasis studies
Author: Fry, Kathryn Emily
ISNI:       0000 0004 2678 187X
Awarding Body: University of Portsmouth
Current Institution: University of Portsmouth
Date of Award: 2010
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Background: Around 20-40% of cancers will spread to the brain by passing through the physical blood-brain barrier and thereby worsen prognosis. Unfortunately, there is not a reliable model that closely mimics the in vivo situation which can be used in cancer cell invasion studies. In vitro models of the blood-brain barrier generally utilise murine or porcine brain endothelium and rat astrocytes. In addition, these models are grown in animal-derived serum supplemented conditions which modify cell growth rates and adhesive properties. Aims: To develop three-dimensional invitro models of the blood-brain barrier from human brain-derived cells under human serum supplementation to examine the metastatic behaviour of malignant non-central nervous system neoplasms (cancer cells). Materials & Methods: The blood-brain barrier model comprised of human immortalised cerebral microvascular endothelial cells (hCMECID3), human astrocytes (CC-2565 and SC-1810), and human pericytes (HBVP) grown on Transwell® Burn polycarbonate membrane filters under human serum supplementation. Different cell permutations were tested to find the optimum model in terms of high trans-endothelial electrical resistance values. Non-small cell lung carcinoma, breast adenocarcinoma, melanoma, and a cerebellar metastatic adenocarcinoma were chosen to add to the model for cancer cell invasion studies. All cells were characterised with appropriate immunomarkers using flow cytometry and immunocytochemistry, while population doubling times were established. An electrical cell-substrate impedance sensing system was investigated to monitor endothelial tight junction resistance with the aim of improving the Transwell" model. A novel modelling system that incorporates flow, the dynamic in vitro bloodbrain barrier model, was investigated as a potential model to replace Transwell" filters. Results: Growth curves, antigenic expression, adhesive properties, cultivation on Transwell® models and trans-endothelial electrical resistance measurements were established. The electrical cell-substrate impedance sensing system demonstrated the potential of hCMEC/D3 to form a tight barrier and the positive influence extracellular matrices and conditioned media have on the junction resistance values. The dynamic in vitro blood-brain barrier model showed it had potential to more closely mimic the in vivo situation. The addition of cancer cells to the hCMEC/D3 monolayer was also assessed to monitor invasion. The cancer cells were shown to invade through the monolayer using live-cell imaging. Discussion: An all-human invitro model of the blood-brain barrier has been developed using Transwell® filters and the endothelial tight junction resistance monitored with the use of an electrical cell-substrate impedance sensing system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available