Caco tight junctions

Tanaka, Y. Taki, Y. Sakane, T. Nadai, T. Sezaki, H. Yamashita, S., Characterization of drug transport through tight-junctional pathway in Caco-2 monolayer Comparison with isolated rat jejunum and colon, Pharm. Res. 12, 523-528 (1995). 

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.

To estimate the relative importance of the tight junction and the lateral space composing the paracellular route, let us consider the permeability of mannitol across Caco-2 and MDCK cell monolayers. The results taken from earlier examples are presented below ... 

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... 

Table 9 Theoretical Assessment of Lateral Space and Tight Junction Contributions to the Paracellular Permeability of Mannitol in Caco-2 and MDCK Cell Monolayers3...

JM Anderson, CM Van Itallie, MD Peterson, BR Stevenson, EA Carew, MS Mooseker. (1989). ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells. J Cell Biol 109 1047-1056. 

Lindmark, T., Y. Kimura, and P. Artursson. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty adds in Caco-2 cells, J. Pharmacol. Exp. Ther. 1998, 284, 362-369... 

The paracellular pathway, between the epithelial cells, is both size- (MW, volume) and charge-dependent [60, 109, 110]. In general, compounds that are limited to paracellular transport are not efficiently absorbed due to the small available absorptive area and the restriction by tight junctions. The molecular weight cut-off seems to be around 400 g mol-1 and 300 g mol-1 for the small and large intestine respectively, and 300 g mol-1 for the Caco-2 cell monolayers [60], which shows the more colonic nature of the Caco-2 monolayer model. Compounds with a... 

The oral administration of large proteins and peptides is limited due to their low membrane permeability. These compounds are mainly restricted to the para-cellular pathway, but because of their polar characteristics and their size the pore of the tight junctional system is also highly restrictive. An additional transcellular pathway has therefore been suggested for these peptides, i.e., the transcytotic pathway, which involves a receptor-mediated endocytosis in Caco-2 cells [126],... 

Another drawback is the lack of correlation for paracellularly transported compounds. For example, some low molecular weight hydrophilic compounds (e.g., ranitidine, atenolol, furosemide, and metformin) showed poor permeability in the Caco-2 model despite an absorption larger than 50% in humans [168], This is due to the smaller size of the paracellular channels (controlled by tight junctions) in the Caco-2 model compared to human small intestine [146],... 

Reduced Culture Time Caco-2 cells are usually grown for at least 20 days to form differentiated monolayers on a porous filter support, forming an apical and a basolateral compartment. The 20-day culture time is required to obtain tight junctions, cell polarity, and an adequate expression of drug efflux mechanisms. For economical reasons, various attempts have... 

MDCK Madin-Darby canine kidney (MDCK) cells have received attention as an alternative to Caco-2 cells for permeability measurements. When grown under standard culture conditions, MDCK cells develop tight junctions and form monolayers of polarized cells. The main advantage over Caco-2 cells is the shorter culture time to confluence (3-5 days). The transep-ithelial electrical resistance of MDCK cells is lower than that of Caco-2 cells and thus, closer to the TEER of the small intestine in vivo. The permeability coefficients of hydrophilic compounds are usually lower in Caco-2 cells than in MDCK cells, which is consistent with the lower TEER values for MDCK cell monolayers. The nonhuman (canine) and nonintestinal (renal) origin of MDCK cells is considered as a disadvantage. They have low expression levels of transporter proteins and low metabolic activity [34], MDCK cells that are stably transfected with P-gp/MDRl are often proposed as an alternative for Caco-2 cells to study bidirectional transport of compounds and, more... 

There are several cell monolayer models that are frequently used for the evaluation of drug permeability and absorption potential (Table 18.1). For a more detailed discussion, please refer to Chap. 8. Caco-2 cells (adenocarcinoma cells derived from colon) are the most extensively characterized and frequently used of the available cell lines [5-9], A unique feature of Caco-2 cells is that they undergo spontaneous enterocyte differentiation in cell culture. Unlike intestinal enterocytes, Caco-2 cells are immortalized and replicate rapidly into confluent monolayers. When the cells reach confluency during culture on a semi-porous membrane, they start to polarize and form tight junctions, creating an ideal system for permeability and transport studies. During the past decade, use of... 

Ranaldi G, Marigliano I, Vespignani I, Perozzi G, Sambuy Y (2002) The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line. J Nutr Biochem 13 157-167... 

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

Other cell lines used in permeability studies include the T84 human colonic adenocarcinoma colonic crypt cell model. This line has a reduced carrier expression, secrets mucus, and has very high resistance [31, 32], The IEC cell line is a rat fetal intestinal epithelium cell with higher permeabilities than Caco-2 cells [33], LLC PKi is a pig kidney epithelial cell line with low expression of efflux systems, but expression systems for transport proteins [32], 2/4/A1 cells are a conditionally immortalized rat fetal intestinal epithelium line with crypt cell-like morphology and temperature-sensitive differentiation [34], They form differentiated monolayers with tight junctions, increased brush border enzymes when grown on extracellular matrices with laminin. Transport of drugs with LP in 2/4/A1 monolayers was comparable to that in the human jejunum and up to 300 times faster than that in Caco-2 monolayers. In contrast, the permeability of HP drugs was comparable in both cell lines [34],... 

Boulenc, X., Roques, C., Joyeux, H., Berger, Y., and Fabre, G., Biophosphonates increase tight junction permeability in the human intestinal epithelial (Caco-2) model, Int. ]. Pharm., 123,13,... 

---