Monolayer cell culture

Quantitative Approaches to Delineate Passive Transport Mechanisms in Cell Culture Monolayers... 

Adson A. (1992). Quantitative approaches to delineate passive transport mechanisms in cell culture monolayers. MSc Thesis, University of Kansas, Lawrence, KS. 

In some cases, cell culture monolayers can not be obtained but whole organs, or pieces of organs, can be cultured. Such organ cultures may still be useful in virus research, since they permit growth of viruses under more or less controlled laboratory conditions. 

The permeability of the drug substance can be determined by different approaches such as pharmacokinetic studies in humans (fraction absorbed or mass balance studies) or intestinal permeability studies (in vivo intestinal perfusion studies in humans or suitable animal models or in vitro permeation studies using excised intestinal tissue or epithelial cell culture monolayers like CaCo-2 cell line). In order to avoid misclassification of a drug subject to efflux transporters such as P-glycoprotein, functional expression of such proteins should be investigated. Low- and high-permeability model... 

Ho, N., Raub, T., Burton, P., Barsuhn, C., Adson, A., Audus, K., and Bor-chardt, R., Quantitative approaches to delineate transport mechanisms in cell culture monolayers, Transport Processes in Pharmaceutical Systems, edited by G. Amidon and P. Lee, Marcel Dekker, New York, 2000, pp. 219 316. 

Fig. 2. Virus growth curve of wild-type WEE virus in A. albopictus (strain C6/36) cells. Cultured monolayers were washed once with PBS and then infected with virus at 5 PFU/cell. After adsorption for 90 min at 28°C the inocula were removed, mono-layers were washed three times with PBS, and culture medium was added. Infected cultures were incubated at 28°C. The samples of culture fluid were taken at the indicated times for plaque assay on CEF monolayers at 34°C. After 10 days, the infected cultures were split weekly at 1 5 or 1 10. The lower panels show uninfected and WEE virus-infected C6/36 cells photographed with phase contrast light microscopy (B. Simizu and S. Maeda, unpublished data).

Animal cell cultures that are initiated from cells removed directly from the animal are called primary cultures (Figure 2). Primary cultures include both explant cultures (i.e., cultures initiated from small pieces of intact tissue), as well as cultures initiated from preparations of individual or dispersed cells (obtained from intact tissue by mechanical or proteolytic dismption). Nerve fiber explant cultures in blood plasma were among the earliest types of tissue cultures (Harrison, 1907). Cells grow out from such tissue explants and form a single layer of cells completely filling the tissue culture vessel surface. Such cell cultures are called confluent monolayers. Confluent monolayers can then be treated with trypsin, so as to remove the individual cells from the culture vessel surface. The resulting cell suspension is then transferred into other culture containers, so that more viable monolayer... 

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

Production of Mucosal Damage 2.3.1.2.1 Cell culture Stimulated neutrophils are known to be cytotoxic to cells in vitro (Dull et al., 1987 Dallegri et al., 1990 Grisham et al., 1990b). Several in vitro systems have been used to demonstrate oxidative damage to intestinal cells. Xanthine/XO increased Cr release and decreased [ H]thymidine uptake by IEC-18 small intestinal epithelial cell monolayers in a dose-dependent manner (Ma et al., 1991). Rat enterocytes show decreased trypan blue exclusion and increased protein release when incubated with neutrophils stimulated... 

Cell cultures. MDCK cells were seeded in the Transwells at a density of 2.2 x 104 cells/cm. Cells were fed by changing medium in both upper (apical) and lower (basal) compartments periodically. Confluent monolayers were obtained at 5-7 days post-inoculation, when the cell density reached 4.5-5.0 x 105 cells/cm2, and a transepithelial electrical resistance (TEER) of about 2,000 ohms cm2 was measured using an epithelial voltohmmeter (EVOM, World Precision Instruments, West Haven, CT). The amount of FBS in the cell culture medium could be decreased as the cells approached their maximum resistance, and could be maintained at that point for 2 days or longer in medium containing 1% FBS. 

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... 

All of these characteristics can be under the regulation of the cell and influenced by the cell culture conditions. The age of the cell monolayer in culture can have a profound impact on the quality of the barrier. In monolayers with actively dividing cells, resistance increases with time in culture as tight junctions form (see Fig. 15, Section III.C.4). Resistance reaches a plateau, then decreases as cell viability declines (Section III.C.4). Time in culture may also be a factor in the expression of polarity, which is related to tight junction formation as well as the state of differentiation of the cells (e.g., differential gene expression). 

Figure 36 Efflux kinetics of PNU-78,517 from the apical membrane of MDCK cells in monolayer culture on a solid plastic surface as a function of bovine serum albumin concentration. [Redrawn from Raub et al. (1993) with permission from the publisher.]...

The identification and characterization of cell culture systems (e.g., Caco-2-cells) that mimic in vivo biological barriers (e.g., intestinal mucosa) have afforded pharmaceutical scientists the opportunity to rapidly and efficiently assess the permeability of drugs through these barriers in vitro. The results generated from these types of in vitro studies are generally expressed as effective permeability coefficients (Pe). If Pe is properly corrected to account for the barrier effects of the filter (PF) and the aqueous boundary layer (PAbl) as previously described in Section II.C, the results provide the permeability coefficient for the cell monolayer... 

Artursson P, C Magnusson. (1990). Epithelial transport of drugs in cell culture. II. Effect of extracellular calcium concentration on the paracellular transport of drugs of different lipophilicities across monolayers of intestinal epithelial (Caco-2) cells. J Pharm Sci 79 595-600. 

Under linear concentration conditions (for a P-C concentration range of 0.12-6pM) at 16h incubation and under cell culture conditions mimicking the in vivo postprandial state, the extent of absorption of all-trans P-C through Caco-2 cell monolayers was 11% a value similar to that reported from different human studies. In humans, the bioavailability of a single dose of P-C... 

Plaques may be obtained for animal viruses by using animal cell-culture systems as hosts. A monolayer of cultured animals cells is prepared on a plate or flat bottle and the virus suspension overlayed. Plaques are revealed by zones of destruction of the animal cells. 

Tavelin, S., J. Taipalensuu, F. Hallbook, K. Vellonen, V. Moore, and P. Artursson. An Improved Cell Culture Model Based on 2/4/A1 Cell Monolayers for Studies of Intestinal Drug Transport. Characterization of Transport Routes., Pharm. Res. 2003, 20, 373-381. 

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. 

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