Culture and Transport Experiments

This equation for calculation of Papp is easily improved by taking into account the change of donor concentration (Cd) during the experiment, which affects the concentration gradient and the driving force for passive diffusion (Equation 7.2)  

A general equation that does not require sink conditions can also be applied [118,119] (Equation 7.3). In this nonsink analysis, Papp is determined by nonlinear curve fitting of 

The recovery should be sufficient to assure that reliable Papp values are obtained and reported (Equation 7.4). Common limits for recovery are 80-120%. Sometimes, 

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In the selection of an appropriate cell culture system, a number of criteria must be considered (Table 3). These include not only the characteristics of the cell type but also the controllable parameters of the complete transport system such as the permeants, the filter properties, and the assay conditions. In general, most transport experiments employ the experimental design shown schematically in Figure 4 with modifications as discussed below. Typically, the desired cell is seeded onto some sort of semipermeable filter support and allowed to reach confluence. The filter containing the cell monolayer separates the donor and receiver... 

There is one major caveat of using the tissue culture transport experiment to study P-gp efflux that cannot be overlooked—P-gp efflux is not directly determined in this experiment. Rather, the effects of P-gp-mediated efflux activity and changes to this activity are inferred from the resulting overall transport data. Particularly with regards to substrate identification, there is the potential for false negatives. For a compound to be affected by P-gp-mediated efflux, it must reach P-gp s binding site that is within the cell. Compounds with poor membrane (transcellular) permeability are not likely to be identified as substrates (395,397). Conversely, compounds with very high passive membrane permeability can saturate P-gp efflux at low micromolar concentrations and are often not identified as substrates... 

To measure the transport of drugs across the BBB in vitro 2.5 iCi of 3H-labelled drug and 14C-sucrose are applied to each Transwell (in case of 14C-labeled substances, permeability studies are performed with 3H-sucrose). This concentration is high enough to ensure sufficient excess to neglect the decrease of tracer in the donor (apical) compartment during the experiments. Volumes of 1.5 ml in the donor (apical) and 2.5 ml in the acceptor (basolateral) compartment avoid hydrostatic pressure. After addition of the radiolabeled compound, samples of 50 til are taken in duplicate from the basolateral acceptor compartment every 20 min and replaced by 100 xl of fresh assay medium. Cells are kept under culture conditions during the whole transport experiment. Radioactivity is measured after addition of liquid scintillation cocktail in a counter. 

Lithium uptake experiments in erythrocytes may have value in the prediction of those patients who are most likely to respond to treatment (114-117). Other studies have used squid axon, hepatocytes, 3T3 fibroblast cell cultures, and liposomes to investigate lithium transport across the plasma membrane (77, 118). 

In contrast, in tighter epithelia such as Caco-2 and in artificial membranes such as HDM, the permeability of the ionized forms of the drugs and the paracellular permeability are lower or insignificant, respectively. These findings will have implications in the experimental design and data interpretation of pH-dependent drug transport experiments in cell culture models as well as in artificial membrane models, such as HDM and PAMPA. 

Figure 4.12 Schematic drawing of a cell culture model, Caco-2 cells. Cells are seeded on filter support and are left to differentiate for 1-3 weeks before the transport experiments. Experiments are started by adding the compound to the donor side and taking out samples from the receiver side at times up to 2 h. The incubation with the compound is done with good stirring and at 37°C.

Primary hepatocytes or liver slices can be used to measure metabolism, but only for a short period of time after the liver sample has been removed from the body however, both models have problems associated with their use. In liver slices, cell-to-cell contact and three-dimensional structure are maintained with a full compliment of cell types (including Kupffer cells) primary hepatocytes have lost the orientation, organization, and nonhepatocyte cells which may contribute to the metabolic activity of the whole liver. Liver slices may suffer from the presence of damaged or dead cells, restricted access of culture media to internal cells, thereby reducing oxygen and nutrient supplies, and from a build-up of toxic products that may result in impaired metabolism. Perfusion of tissue in situ can ameliorate these problems, but of necessity such experiments can normally only be carried out in animals, and these will have different metabolic profiles due to differences in enzyme and transporter expression. 

Further studies using NeuSAc have been related to the metabolism of extracellular CMP-NeuSAc and the detection of ectosialyltransferase activity. On the basis of a lag period observed for NeuSAc uptake and incorporation into glycoconjugates, and the absence of such a lag for CMP-NeuSAc incorporation, two routes have been proposed. The uptake of NeuSAc and metabolism as described above has been studied in hamster and mouse fibroblasts, and cell surface labelling of glycoprotein and glycolipid demonstrated (Datta 1974). The breakdown of CMP-NeuSAc was shown, and incorporation due to NeuSAc uptake rather than direct CMP-NeuSAc transfer proposed (Hirschberg et al. 1976). The uptake of CMP-NeuSAc into the cells (NIL, BHK and 3T3 fibroblasts) could be ruled out, and the K , for NeuSAc uptake was estimated to be 10 mM. Other experiments with CMP-NeuSAc and intact cell cultures (Painter and White 1976, Cerven 1977, see section III.9) pointed to surface sialyltransferase. Further studies by Fan and Datta (1980) provided evidence that both transfer and transport occur, by localization of acceptors within the cell and on the cell surface (plasma membrane), and direct demonstration of the presence of a plasma membrane sialyltransferase. The sialylation due to NeuSAc uptake occurs (at least initially) with different acceptors in comparison with CMP-NeuSAc plasma membrane sialylation. 

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. 

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