BBB Cell Culture Models

There are a number of cell-based models available which were developed using endothelial cells from cow, pig, rat and even human brain tissue. There are a number 

Primary cultures developed from pig and cow tissue are the best studied [29-32]. These models closely resemble the BBB, exhibiting many of the key biological properties. However isolation of blood-brain endothelial cells requires relatively complex cell isolation procedures which are labor-intensive and not ideal for screening purposes. Other cell types have been shown or proposed to induce barrier function, for example, astrocytes/pericytes. Significant improvements in barrier function was achieved in these primary culture models by including astrocyte conditioned media or co-culturing with astrocytes [33]. The complexity of primary cultures led to the use of epithelial cell lines not derived from the BBB (e.g., MDCK, MDCK-MDRl or LLC-PKl) [33]. 

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CPT-cAMP, RO-20-1724, and 0.1 aM dexamethasone, TEER increased to 85 0 cm. This effect on TEER was neither observed without astrocytes, nor with other sources of astrocytes (see Note 2). In these conditions, the human BBB model was also sensitive to DT (determined as described earlier) and lipopolysaccharide (EPS, as described in ref. [9]). We were, however, unable to find any indication of P-glycoprotein (P-gp) expression in these cells (determined as described in ref [7]). The relatively low TEER across the BBB model and the possible absence of P-gp expression, however, limits the appUcabitity of this constellation of human endothelial cells and rat astrocytes as a BBB cell culture model for the use for drug transport and effect screening purposes. 

Although both BBMECs and PBMECs are widely accepted as suitable in vitro models of the BBB, there are some limitations to the utility of these models. Over the years, the in vitro cell culture models have proven to be fairly reliable predictors of BBB permeability. However, as with any in vitro model, confirmation of results using an animal model is recommended. One should also be aware that substrate-transporter interactions have been shown to be altered in allelic variants... 

The complexity and plasticity of BBB properties called for experimental dissection of the disrnption process in both in vitro and in vivo conditions. Multiple cell and organ cnltnres, animal models, and measurement techniques have been developed, each of which addresses some of the issues involved. The development of research into BBB characteristics was initially approached in avian embryos, where transplanted endothelial quail cells invaded a developing chick chimera. A simpler cell culture model of the BBB was developed by Rubin and co-workers. More recently, an immortalized cell line created from vascular endothelial cells was used to develop another model of the BBB in co-cultures with glioma cells and was used to demonstrate nitric oxide-induced perturbations of these cells. hi another cell culture model, hypoxia was shown to increase the susceptibility to oxidative stress and intercellular permeability. ... 

Various cell culture models are available for assessing BBB penetration and although they can give some indication of potential brain penetration, they generally do not form sufficiently tight cell junctions. 

Two widely used methods for studying the BBB are a cell culture model using rat, pig, or cow brain endothelial cells [15] and isolated microvessels. 

There are complex interactions among the different cellular components of the neurovascular unit and the extracellular matrix, determining its permeability properties during both physiological and pathological conditions. This highlights the severe limitations of cell culture-based models to mimic neurological diseases associated with BBB disruption. Transwell culture systems of endothelial cells alone rarely achieve adequate transendothelial electrical resistance (TEER). Cocultures of... 

Epithelial barrier models for the skin [48,49], respiratory tract [50], BBB, and intestine [39] are constructed to study and predict the absorption, penetration, and metabolism of drugs or environmental toxins through these barriers. All the models are physically tight structures, and generally involve cells cultured at the air-liquid interface on porous membrane support, such as a porous polycarbonate filter. The use of a permeable support allows cells to be grown in a polarized state under more natural conditions promote Cell differentiation and enhance cell functions. 

Several methods have been reported to measure TEER. Since results are usually expressed as ohm x cm2 obtained by multiplying the measure value by the area of the insert membrane, caution should be made when comparing TEER values obtained in BBB models from different formats. Furthermore, it is important to note that in each experiment, the TEER of the coated insert alone (i.e., without cells) in the same buffer and at the same temperature should also be measured and subtracted from the inserts with cells as parameters such as temperature, medium viscosity, and pore size of the cell culture inserts may influence the TEER values. 

Brain endothelial cell culture requires a number of different factors that are supplied by the added serum. Therefore the selection of the adequate serum is of crucial importance in particular in coculture or tri-culture setup. The replacement of serum could be advantageous, and many factors (e.g., cAMP stimulants or glucocorticoids) have been used to supplement tissue culture media to support the tightness of the in vitro BBB without using serum or coculture with other brain cells. For example, a serum-free porcine BBB model based on primary cells using hydrocortisone-supplemented medium [27] has received much attention. However, each cell culture supplement introduces a new variable in a study which renders the interpretation of toxicity results difficult as the supplement may counteract the effects of the chemical on BBB function. 

After 2 or 3 days, change the medium with DMEM + S or DMEM + S with cAMP remove the medium basolateral and apical, but leave -50 (xl on the cells (otherwise the quality of the cells reduces significantly), then add 200 (xl to the apical side and add 800 pi to the basolateral side. Incubate the cells at 37°C, 10% C02, and use for experiment the second or third day. See Pig. 8.1 for a schematic drawing of the co-culture model of the BBB. 

To characterize the transport properties of in vitro BBB models, the solute permeability P of the in vitro BBB was determined by measuring the flux of the selected tracer. The most commonly used cell culture substrate consists of a porous membrane support submerged in the culture medium (Transwell apparatus). The Transwell system is characterized by a horizontal side-by-side or vertical diffusion system. During the experiment, the flux of tracers into the abluminal compartment of the Transwell system is recorded as a function of the time and the solute permeability P is calculated from the slope of the flux. The tracers used in the transport experiments are labeled by a fluorescent dye or isotope whose intensity can be measured quantitatively. Another index, transendothelial electrical resistance (TEER), or the ionic conductance of the monolayer, is also a measurement of the tightness of the in vitro BBB models. 

So far, two major types of in vitro BBB models have been developed endothelial cell monolayer and coculture of endothelial cells with glial cells (the nonnerve cells in the brain). The cells for these models are basically obtained from primary/subpassaged or immortalized cell cultures. The origins of the cells are also very diverse human, primate, bovine, porcine, rodent, and murine species. 

The in vitro measurements of permeability by the cultured-cell or PAMPA model underestimate true membrane permeability, because of the UWL, which ranges in thickness from 1500 to 2500 pm. The corresponding in vivo value is 30-100 pm in the GIT and nil in the BBB (Table 7.22). The consequence of this is that highly permeable molecules are (aqueous) diffusion limited in the in vitro assays, whereas the membrane-limited permeation is operative in the in vivo case. Correcting the in vitro data for the UWL effect is important for both GIT and BBB absorption modeling. 

Garberg et al. compared a number of in vitro cell models of the BBB with in vivo data (including primary cow and human brain endothelial cells co-cultured with astrocytes, MDCK, MDCK-MDRl, Caco-2, ECV304/C6, MBEC4, SV-ARBEC cocultured with astrocytes). The best correlation, although poor, was seen with cow brain endothelial cells (r 0.43) and MDCK (r 0.46). The correlation was improved with Caco-2 when only passively transported compounds were included in the analysis (r = 0.86), BBEC showing a similar correlation [39]. 

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