Caco culture time

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... 

Briske-Anderson MJ, Finley JW, Newman SM (1997) The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells. Proc Soc Exp Biol Med 214 248-257. 

Another technical limitation of Caco-2 cells is the long culture time required to obtain full differentiation of the cells. It takes 3 weeks to obtain fully differentiated cell monolayers of Caco-2 cells on filter inserts [1, 116, 121]. It has recently been suggested that 2 weeks of culture on filters is sufficient for obtaining a full expression of transporters and integrity [23], but these claims require solid experimental confirmation. 

These models consist of cells grown on permeable inserts. Transport of compounds across the cell monolayer can be used to quantitate the permeability of a new chemical entity in a rapid manner. One of the most popular cell lines is Caco-2, derived from human colon adenocarcinoma cells. The monolayer exhibits ion conductance and possesses transepithellal electrical resistance indicative of fully formed tight junctions that restrict the paracellular transport of a chemical entity. Although Caco-2 cells are the most commonly used cells, Madin-Darby Canine Kidney (MDCK) cells are becoming more widespread in use, in part because of the shorter culture time (4-7 days versus 21-30 days for Caco-2 cells) needed for their use in permeability experiments. 

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. 

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) ... 

The expression of the active transport systems is time-dependent and may vary with nutritional conditions [53, 54]. The culturing conditions, e.g., the passaging process, can dramatically alter the biological characteristics and transport properties of Caco-2 cell monolayers [55-58]. 

In addition to screening molecules for intestinal absorption, Caco-2 cells have also been used to study mechanisms of drug transport. For many compounds, intestinal permeation involves a transporter to either aid or limit transepithelial transport. The value of Caco-2 cells in this type of studies is due to the fact that these cells express various membrane transporters relevant to drug absorption.1719-23,28,30 However, when interpreting results of studies that involve carrier-mediated transport, discretion, and scaling factors may be required because of the difference in expression level of transporters between in vitro and in vivo systems.12 Another important consideration in carrier-mediated transport studies is that some transport systems in Caco-2 cells may achieve maximal expression level at different days in culture.17,21,38,74 Thus, validation of Caco-2 cells for mechanistic studies should include the identification of the time for optimal expression of transporters as well as the qualitative evaluation of the transporters to establish that they are representative of the native intestinal transporters. 

Unlike some classes of polyphenols such as flavonols and flavones, flavanols are almost always present in the nonglycosylated form. Removal of glycoside from flavonoids, usually necessary before the transport across the intestinal barrier, is not required in the case of flavanols [Scalbert and Williamson, 2000]. The absorption of procyanidins by the small intestine was investigated by studying 14C-( + )-catechin, dimer, trimer, and procyanidin polymers permeation through Caco-2 cell cultures [Deprez et al., 2001]. There was little difference in permeability between monomer, dimmer, and trimer, based on the measurement of radioactivity present on the basal side of the cultures, whereas the permeability of the polymers was 10 times lower. The authors reported the absence of catechin metabolism but did not determine whether the radioactivity measured on the basal side of the cultures was from the parent dimers to polymers or from their products of degradation or metabolites, which could have resulted from instability of the parent compounds in the culture... 

Figure 4 Time profiles of the transcellular transport of vinblastine in Caco-2 cells and the effect of verapamil on this transport. The transcellular transport of vinblastine in the presence (+verapamil) and absence of verapamil (100 pM) was measured across a monolayer of Caco-2 cells cultured on a porous filter for 14 to 15 days. B->A corresponds to the transport from the basal-to-apical and A—>B is in the opposite direction. Source From Ref. 44.

Peters WHM, Reolofs HMJ (1989) Time-dependent activity and expression of glutathion-S-transferases in the human adenocarcinoma cell line CACO-2. Biochem J 264 613-616 Rosenberg DW, Leff T (1993) Regulation of cytochrom P450 in cultured human colonic cells. Arch Biochem Biophys 300 186-192... 

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