Caco-2 cell – A new vision in cell world

 

Yatri R. Shah¹, Parimal M. Prajapati², Ravi N. Patel³,  Urvi Y. Patel³ and Dhrubo Jyoti Sen³

¹Shree H. N. Shukla Institute of Pharmaceutical Education & Research, Gujarat Technological University, Behind Marketing Yard, Nr. Lalpari Lake, Amargadh (Bhichari), Rajkot, Gujarat

²I. K. Patel College of Pharmaceutical Education and Research, Samarth Campus, Opp. Sabar Dairy, Himmatnagar-383001, Sabarkantha, Gujarat

³Department of Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Hemchandracharya North Gujarat University, Arvind Baug, Mehsana-384001, Gujarat, India

 

ABSTRACT:

 

 

INTRODUCTION:

The Caco-2 cell line is a continuous line of heterogeneous human epithelial  colorectal adenocarcinoma cells, developed by the Sloan-Kettering Institute for Cancer Research through research conducted by Dr. Jorgen Fogh¹. Although derived from a colon (large intestine) carcinoma, when cultured under specific conditions the cells become differentiated and polarized such that their phenotype, morphologically and functionally, resembles the enterocytes lining the small intestine.^2,3 Caco-2 cells express tight junctions, microvilli

, and a number of enzymes and transporters that are characteristic of such enterocytes: peptidases, esterases

The considerable impact of the Caco-2 cell monolayer model can be measured in at least two ways. First, considering that poor pharmacokinetic properties accounted for ~40% of drug failures in development in the early 1990s and only ~10% by 2009, an interval in which Caco-2 monolayers were widely used throughout the pharmaceutical industry to predict absorption, it is not unreasonable to attribute some of that shift to this simple yet powerful model. Second, the 1989 Gastroenterology paper that demonstrated the utility of the model for this application has been cited more than 1000 times since its publication.⁴ The versatility of Caco-2 cells is demonstrated by the fact that, even to this day, they are serving as the basis for the creation of innovative new models that are contributing to our understanding of drug efflux transporters such as P-glycoprotein (ABCB1) and BCRP (ABCG2). RNA interference has been used to silence the expression of individual efflux transporters, either transiently or long-term.⁵ Caco-2 permeability assay Understand the suitability of your compound for oral dosing by using our Caco-2 permeability assay to predict human intestinal permeability and to investigate drug efflux. Caco-2 permeability is one of our Cloe Screen portfolios of in-vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.⁶ Caco-2 permeability assay to investigate intestinal permeability Cloe Screen Caco-2 Permeability assay uses an established method for predicting the in-vivo absorption of drugs across the gut wall by measuring the rate of transport of a compound across the Caco-2 cell line. The Caco-2 cell line is derived from a human colon carcinoma. The cells have characteristics that resemble intestinal epithelial cells such as the formation of a polarised monolayer, well-defined brush border on the apical surface and intercellular junctions.

 

 

Figure-1: Schematic representation of the basic principles of the Monolayer assay system.

Assessing transport in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer enables an efflux ratio to be determined which provides an indicator as to whether a compound undergoes active efflux. The P-glycoprotein (P-gp) inhibitor, verapamil, can be included to identify whether active transport is mediated by P-gp⁷

 

Monolayer assay systems:

Monolayer systems consist of a tight cell layer grown on a porous support to separate two fluid compartments. They are widely regarded as the most sophisticated in-vitro tools for medium to high throughput modelling of important pharmacokinetic barriers, such as intestinal epithelium, blood-brain barrier etc. Two systems that are applied widely in monolayer studies are the human colon carcinoma cell line Caco-2 and transfectant MDCKII cells.

 

Caco2 Monolayers:

Differentiated Caco-2 cells (a human colon carcinoma cell line) express a wide range of transporter proteins on its cell membranes similar to those of intestinal endothelium cells. This makes Caco-2 cells ideal for intestinal absorption simulations. In fact, in the last decade, the utilization of Caco2 cells has become an industry standard for the investigation of intestinal absorption, permeability and drug-drug interactions (DDIs)^9.

 

The Caco-2 monolayer efflux assay is designed to model the net transport events of an important fluid compartment barrier in the organism. This method utilizes a polarized Caco-2 cell layer grown on a supportive membrane surface that separates the two compartments. The unidirectional flux of the TA is determined by applying the TA to either the apical, or to the basolateral side of the cell layer and monitoring the time resolved redistribution of TA between the two compartments. The vectorial transport ratio is determined by applying bidirectional measurements [apical-to-basolateral (A-B) and basolateral-to-apical (B-A)]. In general, a ratio higher than 2 or lower than 0.5 indicates the contribution of an active transport process to the net flux of a compound. In the absence of such transport processes this ratio is around 1. A vectorial transport ratio of 1 does not indicate the absence of active transport. For example, in the case of highly permeable compounds the overall contribution of the active transport process to the net flux might be undetectable. Yet, these compounds might interfere with the transport of other compounds that might result in a clinical drug-drug interaction. This can be assayed by measuring the modification of the vectorial transport ratio of a reporter compound that is known to be affected by some active transport process. A typical example of an indirect assay to estimate P-gp mediated drug-drug interactions is to measure the effect of the TA on the flux of a reporter substrate (i.e., 3H-digoxin).

 

Figure-2: Penetration of molecule through Caco-2 monolayer

 

MDCKII Monolayers:

By now it is common knowledge that transporters can have major effects on the pharmacokinetics of drugs. There is an increasing need to look at interactions on individual transporters. The introduction of transporter transfected cell lines capable of forming tight cell layers brought the possibility of investigating single transporter interactions on monolayers. MDCKII (Madin-Darby canine kidney strain II cells) cell lines have been widely used as hosts for single and/or double transfections. The difference between efflux ratios on the transfected and parental cell lines is regarded as a sign of transporter mediated active uptake or efflux process. Double transfectant cell lines: Introduction of double transfected cell lines, where an apical efflux transporter is located opposite a basolateral uptake transporter with overlapping substrate specificities, allowed the efficient vectorial transport of substrates and thereby experiments on low passive permeability molecules^6,8.

 

Figure 3: Adapted from Kim 2002 Toxicology 181-2:291

 

This set up is able to mimic physiological active vectorial transport processes such as transport of digestive products across the intestinal epithelium from the luminal to the blood side, transport of bile salts and other steroid derivatives from the blood to the biliary side in hepatocytes. Double transfected monolayers are suitable for performing both direct, vectorial transport studies as well as indirect, inhibitory studies (effect of the test articles on the transport of the reporter substrate is measured)⁹.

 

Figure 4: Example of a direct transport measurment on double and single transfectant monolayers

 

Figure 5: Example of an indirect, inhibitory measurement on double transfectant monolayers.

 

Recent advances in caco-2 cells:

Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. When cultured as a monolayer, Caco-2 cells differentiate to form tight junctions between cells to serve as a model of paracellular movement of compounds across the monolayer. In addition, Caco-2 cells express transporter proteins, efflux proteins, and Phase II conjugation enzymes to model a variety of transcellular pathways as well as metabolic transformation of test substances. In many respects, the Caco-2 cell monolayer mimics the human intestinal epithelium. One of the functional differences between normal cells and Caco-2 cells is the lack of expression of the cytochrome P450 isozymes and in particular, CYP3A4, which is normally expressed at high levels in the intestine. However, Caco-2 cells may be induced to express higher levels of CYP3A4 by treatment with vitamin D₃. Caco-2 cell monolayers are usually cultured on semipermeable plastic supports that may be fitted into the wells of multi-well culture plates. Test compounds are then added to either the apical or basolateral sides of the monolayer. After incubation for various lengths of time, aliquots of the buffer in opposite chambers are removed for the determination of the concentration of test compounds and the computation of the rates of permeability for each compound (called the apparent permeability coefficients). Although radiolabelled compounds were used in the original Caco-2 cells monolayer assays, radiolabelled compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS). Mass spectrometry not only eliminates the need for radiolabelled compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells^10,11.

 

REFERENCES:

1.        Fogh J and Trempe G (1975) Plenum, 75: 115-141.

2.        Pinto M et al. (1983) Biol Cell 47: 323–30.

3.        Hidalgo IJ et al. (1989) Gastroenterology 96 (3): 736–49.

4.        Sambuy Y et al. (2005) Cell Biol Toxicol 21(1): 1–26.

5.        Artursson P (1990) J Pharm Sci 79 (6): 476–82.

6.        Artursson P and Karlsson J (1991) Biochem Biophys Res Comm 175 (3): 880–5.

7.        Watanabe T et al. (2005) Pharm Res 22 (8): 1287–93.

8.        Zhang W et al. (2009) Drug Metab Disp 37 (4): 737–44.

9.        Darnell M et al. (2010) Drug Metab Disp 38 (3): 491–7.

10.     Artursson P, Palm K, Luthman K. (2001) Adv Drug Deliv Rev. 46 (1-3): 27–43.

11.     Shah P, Jogani V, Bagchi T, Misra A. (2006) Biotechnol Prog. 22 (1): 186–98.