Cell monolayer assay

Figure 7 Schematic drawing of a cell monolayer assay with the cell monolayer on a filter support separates the apical and basolateral chamber from each other (reprinted from Reference 15).

Methods to Detect and Quantitate Viral Agents in Fluids. In order to assess the effectiveness of membrane filtration the abihty to quantitate the amount of vims present pre- and post-filtration is critical. There are a number of techniques used. The method of choice for filter challenge studies is the plaque assay which utilizes the formation of plaques, localized areas in the cell monolayer where cell death caused by viral infection in the cell has occurred on the cell monolayer. Each plaque represents the presence of a single infectious vims. Vims quantity in a sample can be determined by serial dilution until the number of plaques can be accurately counted. The effectiveness of viral removal may be determined, as in the case of bacterial removal, by comparing the vims concentration in the input suspension to the concentration of vims in the effluent. 

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

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... 

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

Very few published data exist on the evaluation of automated systems, though one report has been made of an automated absorption assay using Caco-2 cells cultured on both sides of polycarbonate membranes [93], The concept of culturing cells on the lower sides of the membranes was investigated as a means of improving the opportunity to study transport in the secretory basolateral to apical direction. However, this approach resulted in increased variability and impaired active transport properties of the cell monolayers, and was therefore not recommended. 

Two main factors have guided the need for optimization of the early screening techniques on one hand the use of simple, quick and high-capacity cell monolayer methods, e.g., Caco-2 cell and MDCK and on the other hand the increased synthesis of more lipophilic, insoluble compounds from combinatorial libraries. This has created a vast number of different variants of cell-based assays and has resulted in variability among the data obtained. A need for optimization of as many as possible of the different parameters in order to increase the predictivity and throughput of the model has been suggested in the literature [98-100]. 

Fig. 14.1. Schematic representation of drug transport assay in cell monolayers cultured on a culture insert containing permeable membrane.

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

Berkowitz FE, Levin MJ. Use of an enzyme-linked immunosorbent assay performed directly on fixed infected cell monolayers for evaluating drugs against varicella-zoster virus. Antimicrob. Agents Chemother. 1985 28 207-210. 

HTS plates permit to determine drug permeability across a cell monolayer with a throughput similar to that of the parallel artificial membrane permeation assay (PAMPA), which measures rate of diffusion across a lipid layer.46 As is the case with PAMPA, the tiny surface area of the filters of the 96-well HTS presents an analytical challenge for compounds with low-to-moderate permeability. 

Substrates are usually identified using transfected MDCK, Caco-2, or endothelial cell lines that express the transporter of interest. These cell types are grown in a monolayer on a membrane separating two chambers of culture medium (i.e., the TransweU Cell Culture Assay, Coming Costar Corp., Cambridge, MA). Drag is administered into one chamber, and drag transport across the monolayer is... 

The wound assay [49] is another method of measuring endothelial cell migration. This assay is based on damaging or wounding a confluent monolayer of endothelial cells and the subsequent repair or closing of the wound by migration of endothelial cells. This assay can be carried out using different matrix components. 

Variations on the filter-based assay have been designed to approximate more physiological contexts. Such assays include tumor cell invasion across a confluent cell monolayer (e.g., endothelial cells (EC) as a surrogate for intravasation or extravasation during hematogenous metastasis (24)) and ovarian carcinoma invasion of mesothelial cell monolayers (25). Additionally, 1 mm thick slices of human brain tissue have been used as a tissue barrier on Transwell filters with invasion of GFP-labeled glioma cells measured by confocal microscopy (26). 

A confluent fibroblast cell monolayer in 78-cm2 plates, cultured in fresh DMEM (see section 6.1.4.1, subheading Specimen ), is incubated with recombinant human INF-y (2.5 pi per 5 ml DMEM 1250 U) and TNF-a (2.5 pi per 5 ml DMEM 500 U). After stimulation for 24 h, cells are harvested by trypsinization, washed with PBS, and immediately lysed for neopterin and biopterin measurements and for GTPCH activity assay. 

Interferon solutions in MEM (30 lU/mL) were preincubated with the indicated gan-glioside concentrations at 37°C for 30 min before addition to the L-cell monolayer. Antiviral assays are described in Materials and Methods. Y EMC titer in the absence of interferon. This titer was unchanged in the presence of both ganglioside preparations up to a concentration corresponding to 100 p.M sialic acid. 

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