Brain capillary endothelial cell culture

Freshly isolated or subcultured brain microvascular endothelial cells offer a notable in vitro tool to study drug transport across the blood-brain barrier. Cells can be grown to monolayers on culture plates or permeable membrane supports. The cells retain the major characteristics of brain endothelial cells in vivo, such as the morphology, specific biochemical markers of the blood-brain barrier, and the intercellular tight junctional network. Examples of these markers are y-glutamyl transpeptidase, alkaline phosphatase, von-Willebrandt factor-related antigen, and ZO-1 tight junctional protein. The methods of 

Preparation of Porcine Brain Microvessel Endothelial Cells 

A sufficient plating density has also to be chosen in order to obtain confluent monolayers within the limited time of viability of primary cell cultures. 

An also frequently occurring problem may be the low solubility of test compounds in aqueous solvents. Organic cosolvents, such as DMSO or ethanol can be used however, due to limited cell viability, final concentrations above 1 % have to be avoided. 

Monocultures form the base of most cerebral endothelial cell culture models [105-107], In the case of filter-grown cells, some authors recommend coculture with astrocytes or the use of astrocyte-conditioned media [108-110], The necessity of this approach is presently under discussion, as cells which had been cultured without supplementation with conditioned media show similar features [94, 111], 

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The presence at the BBB of members of the multidrug resistance-associated protein (MRPs) family, whose members preferentially transport anionic compounds, is still controversial. The seven members of the MRP family belong, like P-gp, to the ATP-binding cassette (ABC) protein superfamily. Mrpl has been found at the BBB in isolated rat brain capillaries, primary cultures of brain capillary endothelial cells and in immortalized capillary endothelial cells, but not in human brain capillaries [59]. Another member, MRP2 has been found at the luminal membrane of the brain endothelial cells [60]. However, further studies are required to show that there are MRP transporters at the BBB (Figure 15.5). As for P-gp, a functional Mrpl was found in primary cultured rat astrocytes [56] and it has been shown to take part in the release of glutathione disulfide from brain astrocytes under oxidative stress [61]. 

Primary cultured porcine or bovine brain capillary endothelial cells have been used as an in vitro model for the BBB. Recently, an immortalized cell line has been established from mouse, rat, and human brain capillary endothelial cells by infection with Simian virus 40 or transfection of SV40 large T antigen (45 -7). Tatsuta et al. established an immortalized mouse brain capillary endothelial cell line (MBEC4). The activity of y-glutamyl transpeptidase and alkaline phosphatase, specific marker enzymes for brain capillary endothelial cells, was half that in the brain capillary (45). Also, P-gp was expressed on the apical membrane of MBEC4 cells, which corresponds to the abluminal membrane of the brain... 

The pharmacological effect of L-dopa is affected by diet (362). The off period in Parkinsonian patients treated with L-dopa is a clinical problem, since the efficacy of the drug suddenly fails. Because of the inverse relationship between the plasma levels of large neutral amino acid (LNAA) and the clinical performance of Parkinsonian patients (362) and the fact that the transcellular transport of L-leucine is inhibited by L-dopa (363) across primary cultured bovine brain capillary endothelial cells, the off period may be attributed to the membrane transport of L-dopa via LNAAT at the BBB. In addition to L-dopa, baclofen and melphalan are suggested to be taken up into the brain via amino acid transporter (363,364), and thereby, their brain transport might be also affected by the plasma concentration of large neutral amino acids. 

Tsuji A, Terasaki T, Takabatake Y, et al. P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells. Life Sci 1992 51 (18) 1427-1437. 

II.1.1.1 Primary Cultures of Brain Capillary Endothelial Cells 522... 

Franke et al. (1999) have described an in vitro model for screening of drug entry into the brain using primary cultures of porcine brain capillary endothelial cells (PBCEC). By using serum-free culture conditions... 

Nitz T, Eisenblaetter T, Psathaki K,Galla HJ (2003) Serum-derived factors weaken the barrier properties of cultured porcine brain capillary endothelial cells in vitro. Brain Res 981 30-40... 

CRITICAL ASSESSMENT OF THE METHOD Immortalization of brain capillary endothelial cells is a difficult process and phenotype characterization must be performed very carefully and always in comparison to primary cultured cells. However, an immortal cell line promises higher reproducibility of results, easier handling and finally to be less time-, material- and labour-consuming. 

Terasaki T, Takakuwa S, Saheki A, Moritaui S, Shimura T, Tabata S, Tsuji A (1992) Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier Studies with monolayers of primary cultured bovine brain capillary endothelial cells. PharmRes 9 529—534. 

Ivanov A, Atsumi R, et al. 2001. Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes. Eur. J. Pharm. Sci. 12 215-22... 

Endothelial cells cultured on 35-mm-diameter gelatine-coated dish are harvested at confluence and seeded onto 60-mm-diameter gelatine-coated dishes. After 6-8 days, confluent cells are subcultured at the split ratio 1 15. Cells at the third passage can be stored in liquid nitrogen. Frozen at passage 3, brain capillary endothelial cells are re-cultured on 60-mm-diameter gelatine-coated dishes and trypsinized at confluence before seeding on filters. 

Defrost 24-well filter plates by transferring it into a single-well feeding cell culture plate with 1 ml/well of brain capillary endothelial cell medium. 

Boveri M, Berezowski V, Price A, Slupek S, Lenfant AM, Benaud C, Hartung T, Cecchelli R, Prieto P, Dehouck MP (2005) Induction of blood-brain barrier properties in cultured brain capillary endothelial cells comparison between primary glial cells and C6 cell line. Glia 51(3) 187-198... 

Differential Seeding of Brain Capillaries and Culture of Bovine Brain Capillary Endothelial Cells... 

Mokrzan, E.M., L.E. Kerper, N. Ballatori, and T.W. Clarkson. 1995. Methylmercury-thiol uptake into cultured brain capillary endothelial cells on amino acid system L. J. Pharmacol. Exp. Ther. 272(3) 1277-1284. 

The development of a new coculture-based model of human BBB that enables the prediction of passive and active transport of molecules into the CNS has recently been reported (Josserand et al., 2006). This new model consists of primary cultures of human brain capillary endothelial cells cocultured with primary human glial cells (Megard et al., 2002 Josserand et al., 2006). The advantage of this systan includes the use of human primary cells, avoiding species, age, and interindi-vidual differences since the two cell types are removed from the same human donor and because of the danonstrated expression of functional efflux transporters such as P-gp, MRP-1, MRP-4, MRP-5, and BCRP. Such models have potential for the assessment of permeability of drug and specific transport mechanisms, which is not possible in artificial membrane assays (e.g., PAMPA) or other cell models due to incomplete expression of active transporters. 

Another barrier of interest for drug delivery studies is the blood-brain barrier (BBB). The BBB is formed by the endothelial cells of brain capillaries. The primary characteristics of the BBB are its high resistance to chemical diffusion and transport due to the presence of complex tight junctions that inhibit paracellular transport and its low endocytic activity. Several in vitro models of the BBB have been developed, and several authors have reviewed the models and their possible uses as permeability and toxicity screens (Reinhardt and Gloor, 1997 Gumbkton and Audus, 2001 Lundquist and Renftel, 2002). The most common in vitro BBB model consists of a monolayer of primary isolated brain capillary endothelial cells, primary isolated endothelial cells from elsewhere in the body, or an endothelial cell line cultured on a membrane insert. The endothelial cells are often cocultured with astrocytes or astroglial cells. In cocultures, the barrier properties of the BBB model increase. 

Thole, M., et al. 2002. Uptake of cationized albumin coupled liposomes by cultured porcine brain microvessel endothelial cells and intact brain capillaries. J Drug Target 10 337. 

Cocultures of bovine capillary endothelial cells and astrocytes can be used to study the permeability of drugs (Dehouck et al. 1990). Culture plate inserts are set into six-well plates with 2 ml of buffer added to the upper chamber and 2 ml addded to the plate coating the inserts. Radiolabeled compounds are added to the upper chamber and 100 pi is removed from the lower chamber at various times. The radioactivity can be determined and the permeability can be calculated. Using such techniques ranking of CNS drugs concerning their permeability into the brain can be achieved. 

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