Transwell system

Figure 5 The Costar Transwell system with a cell monolayer grown on a porous polycarbonate filter that is mounted onto a removable plastic insert forming the apical chamber. Two other systems, (1) the Costar diffusion chamber system, where a filter-grown (Snap-well) cell monolayer is sandwiched between two chambers of equal volume and the bathing solutions are agitated and/or gassed and (2) filter-grown cell monolayers mounted in a two-chamber rotating cylinder device (Imanidis et al., 1996), are not shown.

Figure 8 Appearance kinetics of radiolabeled solutes that diffuse across Caco-2 cell monolayers via the paracellular pathway. The Transwell system consisted of a donor and receiver solution at pH 7.4. Stirring by planar rotation up to 100 rpm had no effect. The insert with filter, cell monolayer, and donor were transferred to a new receiver chamber at time intervals to maintain sink conditions.

Figure 14 Observed permeability coefficients of urea and mannitol across monolayers of rat alveolar epithelial cells in primary culture in the Transwell system are correlated with transepithelial electrical resistance and days in culture.

Figure 18 Linear fluxes of hydrocortisone across Caco-2 cell monolayers in the Transwell system into a receiver sink as a function of stirring (rotary platform shaker) rate at 25 °C.

Figure 21 Linearized double reciprocal plot of the effective permeability coefficients and corresponding stirring rates to determine the power dependency of the stirring rate and mass transfer resistances for the aqueous boundary layers and the Caco-2 cell monolayer in the Transwell system. 

Table 12 Effective Permeability Coefficients and Thicknesses of the Aqueous Boundary Layer of the Caco-2 Cell Monolayer/ Transwell System as a Function of Stirring by Planar Rotating Shaker3...

Figure 22 Correlation between the log permeability coefficient for a series of peptides across a Caco-2 cell monolayer in the Transwell system and A log PC, which is defined as log PC(n-octanol/water) — log PC (isooctane/water). [Redrawn from Burton et al. (1992) with permission from the publisher.]...

Figure 25 Cumulative fraction of the initial donor concentration of [1-blockers that diffused across Caco-2 cell monolayers as a function of donor pH. Transwell systems were used, and stirring was done using a rotary platform shaker. (A), pH 7.4 (B), pH 6.5.

Figure 29 Intrinsic permeability of the monoester of PNU-82,899 using Caco-2 cell monolayers. A Transwell system was used with receiver sink conditions at 25°C. The initial donor concentration was 199 iM, and donor and receiver solutions were at pH 7.4.

Figure 32 Disappearance and appearance kinetics of transcellular flux of the lipophilic antioxidant PNU-78,517 (pKa 6.5) across MDCK cell monolayers in Transwell systems at 37°C. Donor solutions contained 3% bovine serum albumin (BSA), and receiver solutions contained 0.5-5% BSA at pH 7.4. [Redrawn from Raub et al. (1993) with permission from the publisher.]... 

Figure 38 Correlations between appearance permeability coefficients for a related series of peptides measured in mesenteric blood draining perfused rat ileal segments and Caco-2 cell monolayers in the Transwell system. See Table 14 for identification of the peptides. The Pe for the rat ileum was not corrected for the aqueous boundary layer and blood flow effects. [Redrawn from Kim et al. (1993) with permission from the publisher.]...

Numerous modifications of in vitro culture systems have been developed for the estimation of BBB transfer [52]. Culture systems in use are either primary cultures of brain microvessel endothelial cells (BMEC) or immortalized endothelial cell hues. BMEC may be grown in co-culture with astrocytes or in astrocyte-conditioned medium. Astrocyte-derived factors increase the tightness of the barrier as measured by transendothelial electrical resistance (TEER) and by the permeability of hydrophUic markers such as sucrose. They also up-regulate the expression of BBB-enriched enzymes such as y-glutamyl transpeptidase (y-GTP) and alkaline phosphatase. A setup of the in vitro technique in a transwell system for transport studies is depicted in Figure 2.5. 

Transwell cultures are prepared by adding 500 pL of cell suspension (concentration of 200,000 cells/mL, i.e., 100,000 cells/well) to the apical compartment of a Transwell system, and by adding 1.5 mL of medium to the basolateral compartment. Transwells are then incubated for 9-12 days at 37°C and 5% C02 in the incubator until a confluent cell monolayer is formed on the filter membrane. The cellular resistance across the monolayer steadily increases as the cells differentiate. This time, TEER values are documented under sterile conditions (see Subheading 3.4.), Cultures with TEER values of 600-1200 H-cm2 are used for the experiments. 

Transport studies are performed at 24-well or 96-well formats. Cells grown in 175 cm2 flasks are moved to filter inserts and after 21 days of feeding and cultivation used in transport studies. The following method applies for use of 24-well filters of BD Falcon TM HTS 24-Multiwell Insert System. Alternatively, Costar 24-well filter systems could be used (non-coated, Transwell system). In many pharmaceutical companies cell permeability tests and feeding are performed with automatization equipment. 

In the filterwell or Transwell , systems, cells are grown on a permeable membrane between two separated liquid phases (McCall et al, 1981). In this condition confluent epithelial cells can achieve their full polarized functional state without the stresses induced by doming of epithelial monolayers on glass or plastic surfaces (Rabito et al, 1980). Filterwell culture has proved valuable for modelling the epithelium of the human intestine (Hidalgo et al., 1989). This technique is rapidly becoming the preferred culture system for many studies using epithelial cultures. 

The transwell (Figure 25.2c) or other membrane-based methods also enable isolated cell co-cultures. Membrane filters, with a pore size from 0.1 to 12 pm, are used as cell growth substrates. Generally, one type of cell is seeded onto the membrane, which is immersed into a micro well with a second type of cells cultured on the bottom of the well. Multiwell plate transwell systems are available from manufacturers such as Corning for high-throughput studies. 

The LLC-PKl and MDCK cell lines are pig and dog kidney epithelial cells, respectively, that require shorter culture (4-A days) periods for monolayer formation in the transwell system. These two cell lines present their own set of challenges for data interpretation. LLC-PKl cells have endogenous BCRP activity (Jonker et al., 2000). MDCKI cells have high endogenous P-gp activity (Raggers et al., 2002 Goh et al., 2002). 

SV-40-transformed human bronchial epithelial cells co-cultured (transwell system) with human blood monocytes and exposed to 100 fig chrysotile B per 10 cells showed deoxyribonucleic acid strand lesions induced by the reactive oxygen intermediates released from the mononuclear phagocytes engulfing the fibres (Kienast et al. 2000). The addition of 200 U catalase (EC 1.11.1.6) per ml or 100 /iM desferoxamine to the culture medium blocked almost completely the induction of DNA strand lesions in this system (maximum 85 %). 

This thesis was supported by results reported by De Nijs et al./ who tested in vitro the transformation and possible absorption of D3G in the small intestine. In particular, D3G was submitted to a treatment with human Caco-2 cells in a transwell system. The results showed that Caco-2 cells were not able to convert D3G into DON after 24 h of treatment, while it seemed that when DON was submitted to the same process, a part of it was absorbed by human cells (about 23% of the initial added amount). On the basis of these experiments, it was possible to conclude that D3G is not hydrolyzed or transformed during its passage through the upper tract of the human intestine, while DON can be partially absorbed by human cells in this tract. 

Indirect communication between cells can be studied by physically separating cell types with a semi-permeable membrane. These transwell systems allow the response of cells to be attributed to soluble signals, with signaling occurring in both directions (Domenech et al. 2009). The transwell system allows the cell typ e of interest to be easily isolated and analyzed. Although this transweU technique is limited to the use of just two cell types, a multitude of information has been obtained from this in vitro technique. 

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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