Caco-2 cultures

Finally, addition of Tyv-specific antibodies to Caco-2 cultures significantly inhibits larval moulting (McVay et al., 2000). It seems likely that the inhibitory effect is an indirect result of antibody-mediated encumbrance of larvae in the monolayer rather than a direct effect on the process of moulting. Nevertheless, the observation provides further support for the conclusion that antibodies specific for Tyv interfere with the niche of the parasite. 

Among these testing methods, the small animal GI model and the Caco-2 cell culture model have shown the best correlation with oral absorption in vivo. The Caco-2 culture system consists of a monolayer of human intestinal epithelial cells grown on semipermeable supports such as polycarbonate membranes. Because the cells are human in origin, they exhibit many characteristics of the human small intestinal epithelium. The permeability coefficients relative to the extents of human drug absorption are listed here ... 

The most commonly used cell culture model today is the 21-day Caco-2 human colonic cell line derived from a human adenocarcinoma. They can be cultured in special transwell cell culture plates (Figure 9) that enable the investigation of passive diffusion (apical to basolateral side) and active transport (basolateral to apical side). Although the system has its limitations, for many compounds it can give a good indication of likely in vivo absorption. An alternative cell culture, which has been shown to correlate well with absorption in vivo and permeability in Caco-2 cultures, is the 3-day Madin-Darby canine kidney cell line. Also, the expression of transporter proteins in cell cultures has led to new screens being established for identifying transporter substrates. 

In-vitro models can provide preliminary insights into some pharmacodynamic aspects. For example, cultured Caco 2 cell lines (derived from a human colorectal carcinoma) may be used to simulate intestinal absorption behaviour, while cultured hepatic cell lines are available for metabolic studies. However, a comprehensive understanding of the pharmacokinetic effects vfill require the use of in-vivo animal studies, where the drug levels in various tissues can be measured after different dosages and time intervals. Radioactively labelled drugs (carbon-14) may be used to facilitate detection. Animal model studies of human biopharmaceutical products may be compromised by immune responses that would not be expected when actually treating human subjects. 

LIU Y and HU m (2002) Absorption and metabolism of flavonoids in the caco-2 cell culture model and a perfused rat intestinal model. Drug Metab Dispos. 30 (4) 370-77. 

In culture, the human colon carcinoma cell hne Caco-2 spontaneously differentiates at confluency into polarized cells with enterocyte-like characteristics. The principle of this approach consists of following the passage of the compound of interest from the apical or lumen-like sides to the basolateral or lymph-hke sides of Caco-2 cells, thus following the absorption of the compound per se. One obhgate step for fat-soluble nutrients such as carotenoids to cross the intestinal barrier is their incorporation into CMs assembled in the enterocytes. Under normal cell culture conditions, Caco-2 cells are unable to form CMs. When supplemented with taurocholate and oleic acid, Caco-2 cells were reported to assemble and secrete CMs. ... 

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. ... 

Liu, C.S., Glahn, R.P., and Liu R.H., Assessment of carotenoid bioavailability of whole foods using a Caco-2 cell culture model coupled with an in vitro digestion, J. Agric. Food Chem., 52, 4330, 2004. 

Garrett, D.A., Failla, M.L., and Sarama, R.J., Estimation of carotenoid bioavailability from fresh stir-fried vegetables using an in vitro digestion/Caco-2 cell culture model, J. Nutr. Biochem., 11, 574, 2000. 

Fahy, D.M., O Callaghan, Y.C., and O Brien, N.M., Phytosterols lack of cytotoxicity but interference with 3-carotene uptake in Caco-2 cells in culture, FoodAddit. Con-tarn., 21, 42, 2004. 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

Cell culture Damage to small intestinal epithelial cells by XO can be prevented by SOD and desferrioxamine (Ma et al., 1991), whilst that to rat enterocytes, CaCo cells or rabbit colonic epithelial cells by XO can be decreased by catalase (Baker and Baker, 1990 Baker and Campbell, 1991 Kawabe etal., 1992). 

We define this permeability as apparent, to emphasize that there are important but hidden assumptions made in its derivation. This equation is popularly (if not nearly exclusively) used in culture cell in vitro models, such as Caco-2. The sink condition is maintained by periodically moving a detachable donor well to successive acceptor wells over time. At the end of the total permeation time f, the mass of solute is determined in each of the acceptor wells, and the mole sum mA (t) is used in Eq. (7.10). Another variant of this analysis is based on evaluating the slope in the early part of the appearance curve (e.g., solid curves in Fig. 7.14) ... 

Since the widely accepted in vitro permeability model in the pharmaceutical industry is based on the use of cultured cells, such as Caco-2 or MDCK, it was appropriate to analyze the regression correlation coefficients based on the comparisons of Caco-2 log Pe and the log Pe values based on the human jejunal measurements [56]. 

Lentz, K. A. Hayashi, J. Lucisano, L. J. Polli, J. E., Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classification system, Int. J. Pharm. 200, 41-51 (2000). 

Artursson, R, Epithelial transport of drugs in cell culture. I A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells, J. Pharm. Sci. 79, 476-482 (1990). 

Hilgendorf, C. Spahn-Langguth,H. Regardh,C. G. Lipka, E. Amidon,G. L. Langguth, P., Caco-2 vs Caco-2/HT29-MTX co-cultured cell lines Permeabilities via diffusion, inside- and outside-directed carrier-mediated transport, J. Pharm. Sci. 89, 63-75 (2000). 

Figure 2 Comparison of intestinal epithelial cells in culture and in situ. (A) Human colon Caco-2 cells grown in culture for 16 days on a semiporous filter. (B) Epithelial layer of rat jejunum. AP, apical or luminal membrane B, basal or abluminal membrane BM, basement membrane G, goblet cell LS, lateral space mv, microvilli Nu, nucleus TJ, tight junction. Bars equal 10 pm. 

Figure 6 An electron micrograph of intercellular junctions between two human colon Caco-2 cells in culture. D, desmosome LS, lateral space mv, microvilli ZA, zonula adherens ZO, zonula occludens (i.e., tight junction). Bar equals 200 nm.

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... 

Kim DC, PS Burton, RT Borchardt. (1993). A correlation between the permeability characteristics of a series of peptides using an in vitro cell culture model (Caco-2) and those using an in situ perfused rat ileum model of the intestinal mucosa. Pharm Res 10 1710-1714. 

Royleanone, horminone, and acetyl horminone isolated from the roots of Salvia officinalis L. abrogated the survival of colon carcinoma cell Caco-2 and human hepatoma cell HepG2, cultured in vitro with induction of DNA breaks (48). 

Reboul et al., 2007a,b). As mentioned earlier the competitive uptake occurs also in the presence of a mixture of carotenoids where absorption of lutein is inhibited by [1-carotene but not by lycopene (Reboul et al., 2005). This indicates that the presence of a mixture of different lipophilic substrates can strongly influence the uptake of certain carotenoids. It has also been demonstrated that cultured Caco-2 cells secrete (3-carotene, preferentially within micelles rich in long fatty acids (Yonekura et al., 2006), suggesting that carotenoids can be stored in the cell or secreted depending on the absence or presence of appropriate carotenoid acceptors. 

Despite the feasibility of using cultured RPE cells for studies similar to those performed using Caco-2 cells, the role of the RPE in carotenoid uptake and dynamic regulation has only just begun to be investigated. As carotenoids are carried in blood by lipoproteins, lipoprotein-rich serum seems to be the most appropriate vehicle for carotenoid delivery to cultured RPE cells. Indeed, recent studies comparing carotenoid delivery from fetal calf serum and from organic solvents showed that delivery in the presence of serum was superior to tetrahydrofuran (Shafaa et al., 2007). 

Thus, in contrast to previous in vivo models, this in vitro model provides the possibility of dissociating experimentally two important processes of the intestinal carotenoid absorption cellular uptake and secretion. Under conditions mimicking the postprandial state (TC OA supplementation), differentiated Caco-2 cells were able (1) to take up carotenoids at the apical side and to incorporate them into CM and (2) to secrete them at the basolateral side, associated with CM fractions. In this model, no attempt has yet been made to reproduce the in vivo physiochemical conditions occurring in the intestinal lumen, such as carotenoid release from the food matrix and solubilization into mixed lipid micelles. Carotenoids were delivered to Caco-2 cells in aqueous suspension with Tween 40 (During et al., 2002). Using this cell culture system in conjunction with an in vitro... 

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... 

The Caco-2 cell line was isolated from a human colon carcinoma, and has been characterized as one of the best in vitro models of intestinal epithelium. Indeed, in contrast to other intestinal cell lines, Caco-2 cells are able to constitute a homogenous monolayer and to spontaneously differentiate into polarized cells, highly similar to human mature enterocytes, after approximately 2 weeks of culture. Furthermore, the Caco-2 cells present microvillosities at the apical side and have a high transmembrane resistivity, which confirms the fact that the cells are confluent and link to one another via gap junctions. Finally, they can absorb different compounds, express many enzymes involved in intestinal metabolic pathways (Pinto et al. 1983, Musto et al. 1995, Salvini et al. 2002), and give reproducible in vitro results consistent with results obtained in in vivo studies (Artursson and Karlsson 1991). 

Pinto, M. et al. (1983). Enterocyte-like differentiation an polarizationof the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47 323-330. 

Fig. 6.2. Caco-2 epithelial cell monolayers cultured with T. spiralis L1 larvae in (A) the absence or (B) presence of 1 mg ml 1 rat monoclonal, tyvelose-specific antibody 9D4 (McVay etal., 2000). Monolayers were fixed and stained with trypan blue as described in ManWarren etal. (1997). (A) Serpentine trails of nuclei in dead cells are evident, revealing the paths travelled by larvae. (B) Tyvelose-specific antibody has inhibited the migration of the larva such that it is encumbered in cell debris and has pulled up a large area of the monolayer, creating a plaque (P). Bar = 50 urn. Photomicrograph prepared by C. McVay, TTUHSC, Lubbock, Texas. 

Pade, V., Stavchansky, S., Estimation of the relative contribution of the transcellular and paracellular pathway to the transport of passively absorbed drugs in the Caco-2 cell culture model, Pharm. Res. 1997, 34, 1210-1215. 

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