Transwell

In this in vitro system, the presence of serum in cell culture medium is not necessary, but the type of transwell is important (the total amount of H-triglycerides secreted was two-fold higher when using 3 pm versus 1 pm pore size transwells), and oleic acid supplementation is required for the formation and secretion of CMs as well as the transport of 3-carotene through Caco-2 cells. Finally, the presence of Tween 40 does not affect CM synthesis and secretion in this in vitro cell culture system. Thus, CMs secreted by Caco-2 cells were characterized as particles rich in newly synthesized H-triglycerides (90% of total secreted) containing apolipoprotein B (30% of total secreted) and H-phospholipids (20% of total secreted) and with an average diameter of 60 nm. These characteristics are close to those of CMs secreted in vivo by enterocytes. ... 

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. 

The in vitro system we have been using to study the transepithelial transport is cultured Madin-Darby canine kidney (MDCK) epithelial cells (11). When cultured on microporous polycarbonate filters (Transwell, Costar, Cambridge, MA), MDCK cells will develop into monolayers mimicking the mucosal epithelium (11). When these cells reach confluence, tight junctions will be established between the cells, and free diffusion of solutes across the cell monolayer will be markedly inhibited. Tight junction formation can be monitored by measuring the transepithelial electrical resistance (TEER) across the cell monolayers. In Figure 1, MDCK cells were seeded at 2 X 104 cells per well in Transwells (0.4 p pore size) as described previously. TEER and 14C-sucrose transport were measured daily. To determine 14C-sucrose... 

Figure 3. Transcellular transport of HRP-S-PLL in filter-grown MDCK cell monolayers. Confluent MDCK monolayers in Transwells were treated at the basal compartment (closedsquares)or the apical compartment (open squares) with 3 pg/mL HRP-S-PLL conjugate.

Measurements of Pe in fixed-pH solutions but at various different stirring speeds need to be made. The double-reciprocal analysis, HPe versus 1/v , for Caco-2 permeability measurements in the Transwell (Corning Costar) system produced a linear plot for x- 0.8 [514]. The intercept yields the membrane permeability for the particular pH value in the study the slope determines the k constant. From the analysis of testosterone transport, for the stirring speed of 25 rpm (planar rotating shaker), the thickness of each UWL (assuming symmetric geometry) was calculated to be 465 pm at 150 rpm, haq= 110 pm [514], Karlsson and Artursson [512] found x = 1.0 to best represent their stirring-based analysis of the UWL permeability. 

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 17 Appearance kinetics of sucrose across untreated Transwell filters (0.4 Um pore size) and those coated with a cross-linked rat tail collagen matrix. In both cases the systems were stirred at 100 rpm.

In Section III, emphasis was placed on flux kinetics across the cultured monolayer-filter support system where the passage of hydrophilic molecular species differing in molecular size and charge by the paracellular route was transmonolayer-controlled. In this situation, the mass transport barriers of the ABLs on the donor and receiver sides of the Transwell inserts were inconsequential, as evidenced by the lack of stirring effects on the flux kinetics. In this present section, the objective is to give quantitative insights into the permeability of the ABL as a function of hydrodynamic conditions imposed by stirring. The objective is accomplished with selected corticosteroid permeants which have been useful in rat intestinal absorption studies to demonstrate the interplay of membrane and ABL diffusional kinetics (Ho et al., 1977 Komiya et al., 1980). 

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.]...

Previously, the rate-determining step in the Caco-2 cell transmonolayer/ Transwell flux of testosterone was shown to be convective diffusion across the ABLs. Using the values for the 50 rpm situation in Table 11, Eqs. (78) and (79) become... 

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.]...

For AIC conditions, the apical surface of the epithelial cell layer is exposed to air after the nasal cells reached confluence on the Transwell insert, while the basolateral side is fed with culture fluid. Figure 9.3 shows TEER changes in epithelial cell layers cultured up to 20 days in LCC versus AIC methods [46], In AIC condition (initiated from day 3 after seeding), TEER peaked on day 5 and maintained above the TEER values observed for LCC counterparts. By contrast, TEER observed for LCC conditions peaked on day 2 and declined toward zero by day 15. These data indicate that human nasal epithelial cells at an air interface culture exhibit better electrophysiological characteristics than those cultured by the conventional liquid-covered conditions. 

Either Transwell inserts or side-by-side diffusion chambers can be used for transport studies. Bode et al. have provided an excellent review on this subject [60], Briefly, cells are incubated for 30-60 min with a buffer solution. To initiate the transport study, a transport buffer containing the drug under investigation is added to either the apical or the basal chamber depending on the transport direction of interest. At predetermined time points, the respective receiver chamber is sampled and the withdrawn volume is replaced with the same volume of fresh buffer. The permeability coefficient (Papp) is calculated and the ratio of /apP in the basolateral-to-apical direction versus that in the apical-to-basolateral direction gives the efflux ratio. These sort of transport experiments are well suited to determine if drugs/xenobiotics are substrates of the placental efflux proteins. 

The Caco-2 permeability assay is usually performed in a Transwell device (Figure 18.1). The Transwell contains two compartments a donor and a receiver compartment. The apical donor compartment contains a porous membrane that supports the growth of the Caco-2 monolayer. Caco-2 cells are seeded on the porous membrane. Upon confluency of the cell culture, the compound is added into the donor compartment at a concentration range from one to several hundred micromolar. Samples are collected from the receiver compartment for up to 2 h, then LC-UV or LC-MS methods are used to quantify compound in each sample. The permeability coefficient of the compound is calculated based on the following equation ... 

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