filipin br Experimental Procedures br Author Contributions b

Experimental Procedures
Author Contributions
Acknowledgments We thank Qian Yu for assistance with the induction of pluripotent stem cells and Dr. Bing Song for assistance with the flow cytometry assays. We thank Profs. Xiaofang Sun and Yong Fan of the Key Laboratory for Major Obstetric Disease of Guangdong Province, The Third Affiliated Hospital of Guangzhou Medical University for their valuable advice and comments on the manuscript. This work was supported by funding from the National Natural Science Foundation of China (nos. 81271401, 81471280).
Introduction The blood-brain barrier (BBB) is the most important biological barrier between the blood circulation and the central nervous system (CNS), consisting of specialized blood endothelial cells (ECs) that line the cerebral capillaries and are connected by very dense tight junctions (TJs). Anatomically, the BBB is part of the neurovascular unit, which maintains the physiological function of the filipin capillary ECs and includes cellular components such as pericytes, astrocytes, neurons, and microglia (Hawkins and Davis, 2005). The main functions of the BBB are the maintenance of CNS homeostasis and the prevention of penetration of neurotoxic substances as well as pathogens, such as bacteria and viruses. Besides functioning as a physical barrier, the BBB plays a major role as a transport and metabolic barrier (Neuhaus and Noe, 2010). Models of the BBB serve as very strong tools in drug development and are important to elucidate further physiological and pathophysiological molecular mechanisms. Besides in silico and in vivo models, a variety of cellular in vitro BBB models are available, such as transwell models, dynamic flow-based hollow-fiber models, or microfluidic devices (Avdeef et al., 2015). So far, primary porcine, bovine, and rodent ECs are characterized by the best functionality, tightest barrier integrity, and lowest permeability (Vastag and Keseru, 2009). Disadvantages associated with the use of primary cells are the time- and cost-intensive isolation processes, the variabilities between cells of different isolations, and the high consumption of animals for each new isolation. Access to human primary brain material is very limited and restricted to biopsy or autopsy material from patients with diseases such as epilepsy or brain tumors. The use of EC lines for BBB modeling helps to circumvent the disadvantages of primary cells. Immortalized cells of different species, such as murine EC lines (MBEC4, b.END3, b.END5, cEND, cerebEND) as well as cell lines from rat (RBE4), cow (t-BBEC-117), pig (PBMEC/C1-2), and human (hCMEC/D3, hBMEC, TY10, and BB19) exist (Eigenmann et al., 2013; Avdeef et al., 2015). These cell lines have the advantage of being usable over many passages with a higher reproducibility of the results compared with primary cells. Notably, almost all immortalized cell lines form barriers with a transendothelial electrical resistance (TEER) below 150 Ω cm2 (Deli et al., 2005). For drug transport and barrier functionality studies, a minimal tightness of the BBB models with TEER values between 150 and 200 Ω cm2 has been defined (Gaillard and de Boer, 2000). However, compared with physiological TEER values of more than 1,500 Ω cm2, which have been measured in capillaries of rat or frog brains (Crone and Olesen, 1982; Butt et al., 1990), the discrepancies with current in vitro models are significant. Another important aspect is the species differences that exist between humans and other mammalian subsets. In particular, the expression and functionality of important BBB transporters such as P-glycoprotein are described (Takeuchi et al., 2006; Warren et al., 2009). Therefore, there is a significant need for adequate human BBB models for academic research and the pharmaceutical industry. Minimal requirements would be the reproducibility of results, characteristic permeability of reference components, expression of main BBB transporters, and physiological cell morphology (Cecchelli et al., 2007).