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ePaper created 2016-03-17, 15:06:18 | version 1.45.01
8 0 HUMAN IN VITRO BLOOD-BRAIN-BARRIER MODELS FOR DRUG DEVELOPMENT Antje Appelt-Menzel, Alevtina Cubukova, Heike Walles, Marco Metzger Dense barriers between blood and brain The blood-brain barrier (BBB) presents one of the densest and most important barriers between the blood circulation and the central nervous system (CNS). The BBB consists of special- ized microvascular endothelial cells, which coat the cerebral capillaries and are connected through very tight junctions. Together with pericytes, astrocytes, neurons, microglial cells, and the extracellular matrix of the basal membrane of the brain capillaries, they form a dynamic and complex regula- tory system – the so-called neurovascular unit [1]. The main function of the BBB can be divided into three subgroups: the physical, metabolic, and transport barrier. The BBB serves principally to maintain the homeostasis of the CNS and for protection against neurotoxical substances and pathogens such as germs and viruses. Demand for BBB models for drug development For the development of drugs applied for the treatment of neurodegenerative diseases such as Alzheimer’s, Parkinson’s disease, and multiple sclerosis or even brain tumors, the denseness of the BBB models towards substances and the high metabolic activity of the endothelial cells poses a prob- lem. Numerous medications/drugs cannot overcome the BBB in sufficient concentration to reach the target location or they are metabolized before transportation and thus become less effective. Due to the high demand for test systems in basic and preclinical research of medication/drug development and infection studies, a range of different BBB models have been developed. Besides the in silico, acellular in vitro, and in vivo models, numerous cell-based BBB models have been developed. However, standardized models based on immortalized cell lines show only inhomogeneous tight-junction expression and possess low barrier integrity which is detected through tran- sendothelial electric resistance T R below cm2 [2]. In comparison, the TEER values in animal tests reached more than cm2 in the blood-brain barrier [3, 4]. The availabil- ity of human primary BBB cells is extremely limited. Moreover, using human primary BBB cells is an very serious matter not only in respect of ethical aspects. Human brain cells can, for instance, be isolated from biopsy or autopsy material obtained from patients suffering epilepsy or brain cancer. However, the risk remains that the isolated cells are contaminated with cells that are changed due to diseases, which may significantly change the features of the BBB models. Novel models from induced pluripotent stem cells In order to provide human BBB models, an innovative alter- native to avoid such problems is the application of human induced pluripotent stem cells (hiPSC). We are able to dif- ferentiate hiPSC in vitro – under reproducible methods and with established and reproducible methods – into endothelial cells of the blood-brain barrier [5, 6] and to use them for establishing models. The endothelial cells were examined for the e istence of endothelial-specific markers, tight- unctions markers as well as specific transporters ig by means of protein- and gene-based detection methods. The establish- ment of the BBB models includes using transwell inserts as quadrupel culture with pericytes, astrocytes, and neural cells, which can also be differentiated from hiPSC (Fig. 2). The barrier integrity, detected through TEER measurements, is 1 PHARMACY A GLUT1 CL5B 80