Caco-2 cells monolayers

In contrast to previous in vivo models, this in vitro model provides the possibility of dissociating experimentally two important processes of intestinal absorption cellular uptake and secretion. Under conditions mimicking the postprandial state (taurocholate/oleic acid supplementation), differentiated Caco-2 cells were able to (1) take up carotenoids at the apical sides and incorporate them into CMs and (2) secrete them at the basolateral sides associated with CM fractions. Using this approach, the extent of absorption of P-carotene through Caco-2 cell monolayers after 16 hr of incubation was 11.2%, a value falling within the in vivo range (9 to 22%). ° - Of the total amount of P-carotene secreted, 78% was associated with the two CM fractions and 10% with the VLDL fraction. ... 

Since experimental determination of intestinal absorption is quite demanding, Caco-2 cell monolayers have been successfully used to model passive drug absorption. Several models for the prediction of Caco-2 permeability using PSA were developed, including those of van de Waterbeemd et al. [5] and Palm et al. [22] who found that relationships between Caco-2 permeability and PSA is stronger than with Clog D, Krarup et al. [23] who used dynamic PSA calculated for water accessible molecular surface and Bergstrom et al. [24]. 

Hilgers, A. R. Conradi, R. A. Burton, P. S., Caco-2 cell monolayers as a model for drug transport across the intestinal mucosa, Pharm. Res. 7, 902-910 (1990). 

Adson, A. Burton, P. S. Raub, T. J. Barsuhn, C. L. Audus, K. L. Ho, N. F. H., Passive diffusion of weak organic electrolytes across Caco-2 cell monolayers Uncoupling the contributions of hydrodynamic, transcellular, and paracellular barriers, J. Pharm. Sci. 84, 1197-1204 (1995). 

I Hidalgo, K Hillgreen, G Grass, R Borchardt. Characterization of the unstirred water layer in Caco-2 cell monolayers using a novel diffusion apparatus. Pharm Res 8 222, 1991. 

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.

Table 5 Permeability Coefficients of the Paracellular Pathway and Estimation of the Effective Pore Radius and Molecular Restriction Factor for the Caco-2 Cell Monolayer... 

Figure 9 Molecular restriction factor as a function of the ratio of molecular radius to pore radius for paracellular permeants in Caco-2 cell monolayers. Mean pore radius is 1.2 nm.

Interestingly, the permeability coefficients of mannitol in the two cell types are identical, most probably for different reasons, since the physical dimensions of the Caco-2 and MDCK monolayers (Table 8) are markedly different. Compared to the MDCK cell monolayer, the Caco-2 cell monolayer has a taller cell height, a shorter length in tight junctions, longer tortuous path lengths, and smaller slit width in lateral space. One recognizes that... 

Figure 13 Estimated molecular restriction of mannitol in the lateral space of MDCK and Caco-2 cell monolayers. The lateral space was assumed to be a rectangular slit. Molecular restriction functions for slits and cylindrical pores are put into perspective.

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.

Table 11 Delineation of Permeability Coefficients of Steroids in Caco-2 Cell Monolayer System... 

Figure 20 Correlation of appearance kinetics of steroid permeants across Caco-2 cell monolayers in the Trans well system with log partition coefficients (n-octanol/water) and stirring dependency. 

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...

Table 13 Influence of Stirring Flow Rate on the Permeability of [14C] Testosterone Across Caco-2 Cell Monolayer/Filter Support in 02/C02 Gas Lift Side-by-Side Diffusion at 37°C...

Table 14 Permeability Coefficients of D-Phenylalanyl Oligomers Across Caco-2 Cell Monolayers and In Vitro Partition Coefficients... 

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.]...

Because Pe is directly proportional to F(r/R), it should be sensitive to changes in pore radius. It is understood that a change in pore radius compared to control does not result in a proportionate change in Pe among permeants (refer to Table 7). Small molecules will be less influenced than larger molecules. Similar results were obtained in Caco-2 cell monolayers using palmitoyl-DL-carnitine as the per-turbant (Knipp et al 1997). 

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.

In this section we demonstrate the utility of the Caco-2 cell monolayer to provide a systematic and quantitative assessment of passive diffusion coupled with intra-... 

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.

Fig. 37), suggesting that desolvation of the polar bonds in the molecule is a major determinant of permeability. Consistent with this, good correlations were found between the permeabilities of these peptides and their partition coefficients between heptane-ethylene glycol (r2 = 0.87) or the differences in partition coefficients between n-octanol-buffer and isooctane-buffer (r2 = 0.82) both these buffer systems provide experimental estimates of hydrogen-bonding potential. These results are qualitatively identical with those described earlier for the permeability of these peptides across Caco-2 cell monolayers. 

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.]...

Buur A, N Mprk. (1992). Metabolism of testosterone during in vitro transport across Caco-2 cell monolayers Evidence for beta-hydroxysteroid dehydrogenase activity in differentiated Caco-2 cells. Pharm Res 9 1290-1294. 

Knipp GT, NFH Ho, CL Barsuhn, RT Borchardt. (1997). Paracellular diffusion in Caco-2 cell monolayers Effect of perturbation on the transport of hydrophilic compounds that vary in charge and size. J Pharm Sci 86 1105-1110. 

Figure 2 The permeability in Caco-2 cell monolayers as a function of distribution coefficient (octonol/water, pH 7.4) for 5-fluorouraciI and prodrugs. A sigmoidal relationship can be observed. (Replot from Ref. 26, with kind permission from Elsevier Science-NL, Amsterdam, The Netherlands.)...

A Buur, L Trier, C Magnusson, P Artursson. Permeability of 5-fluorouracil and prodrugs in Caco-2 cell monolayers. Int J Pharm 129 223-231, 1996. 

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... 

FIGURE 17.3 Kinetics of P-C transport through Caco-2 cell monolayers as a function of (a) the incubation time at a fixed P-C concentration (1 pM) and (b) the initial P-C concentration for 16h incubation. (Modified from During, A. et al., J. Lipid Res., 43, 1086, 2002.)... 

Differential Absorption of Individual Carotenoids through Caco-2 Cell Monolayers... 

In conclusion, the Caco-2 cell monolayer model has given original data on the competition effect of several nutrients on carotenoid uptake. Most of these data have been confirmed in several in vivo studies, including clinical studies, confirming that this model is a valuable tool to study competition effects on carotenoid absorption. 

---