Madin-Darby canine kidney cell culture

S. J., Goldenberg, S., Wiliams, C., Pasatan, I., Gottesman, M. M., Handler, J., Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epifhelia, J. Biol. Chem. 1989, 264, 14880-14884. 

The in vitro system we have been using to study the transepithelial transport is cultured Madin-Darby canine kidney (MDCK) epithelial cells (11). When cultured on microporous polycarbonate filters (Transwell, Costar, Cambridge, MA), MDCK cells will develop into monolayers mimicking the mucosal epithelium (11). When these cells reach confluence, tight junctions will be established between the cells, and free diffusion of solutes across the cell monolayer will be markedly inhibited. Tight junction formation can be monitored by measuring the transepithelial electrical resistance (TEER) across the cell monolayers. In Figure 1, MDCK cells were seeded at 2 X 104 cells per well in Transwells (0.4 p pore size) as described previously. TEER and 14C-sucrose transport were measured daily. To determine 14C-sucrose... 

The purpose of this chapter is to present overviews of a selection of the major endothelial and epithelial barriers to drug delivery for which there are either primary culture or cell line systems that recapitulate the characteristics of the in vivo barrier. Our objective is to define some general characteristics of cell culture models and highlight the more commonly applied primary cell cultures and cell lines in use today. Specifically, we focus on cell culture models for the intestinal epithelium, blood-brain barrier, pulmonary and nasal epithelium, ocular epithelium, placental barrier, and renal epithelium. Renal epithelium was included here primarily because some cell lines derived from this tissue [e.g., Madin-Darby canine kidney cells (MDCK)] are often used as surrogates for other barriers by pharmaceutical scientists. We have arbitrarily chosen to exclude the skin and liver from the scope of this overview. However, it should be noted that hepatocyte cell culture models, for example, are becoming more widely available and have been the subject of recent reviews.1,2... 

Such direct basolateral-apical sorting has been investigated in cultured Madin-Darby canine kidney (MDCK) cells, a line of cultured polarized epithelial cells (see Figure 6-6). In MDCK cells infected with the influenza virus, progeny viruses bud only from the apical membrane, whereas in cells infected with vesicular stomatitis virus (VSV), progeny viruses bud only from the basolateral membrane. This difference occurs because the HA glycoprotein of influenza... 

Nonintestinal Cell Lines. Madin Darby canine kidney cells (MDCK [50]) differentiate into columnar epithelial cells forming tight Junctions when cultured on semi-permeable membranes they are commonly applied to study cell growth regulation, metabolism, toxicity, and transport at the level of the distal renal tubule epithelia [51]. MDCK cells, like Caco-2 cells, are suitable for molecular permeability screening studies MDCK cells have an advantage over Caco-2 cells in that they do not need 3 weeks in culture before differentiation, but a disadvantage is that they do not express P-gp. 

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. 

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.]. 

MDCK = Madin-Darby canine kidney cultured epithelial cells [563]. 

To reach such a site, a molecule must permeate through many road blocks formed by cell membranes. These are composed of phospholipid bilayers - oily barriers that greatly attenuate the passage of charged or highly polar molecules. Often, cultured cells, such as Caco-2 or Madin-Darby canine kidney (MDCK) cells [1-4], are used for this purpose, but the tests are costly. Other types of permeability measurements based on artificial membranes have been considered, the aim being to improve efficiency and lowering costs. One such approach, PAMPA, has been described by Kansy et al. [5],... 

Some laboratories have found an alternative to the short-term cultures by using cell lines other than Caco-2 cells. The most popular of these is Madin-Darby canine kidney (MDCK) cells, an epithelial cell line from the dog kidney. MDCK cells have been suggested to perform as well as Caco-2 cells in studies of passive drug permeability [56]. These cells have also been used to optimise the conditions for studies of low-solubility drugs [53]. However, as noted previously, the active transport processes of this cell line can be quite different to those of Caco-2 cells [28-30], Another cell line that only requires short-term culture is 2/4/A1, which is a conditionally immortalised rat intestinal epithelial cell line [86]. The 2/4/A1 cell line is discussed in Section 4.3.2.2 below. 

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... 

In this contribution a population balance model of influenza A vims replication during vaccine production in Madin-Darby canine kidney (MDCK) cell cultures is developed. Differentiation on the population level is described by a degree of infection, which is proportional to the amount of intracellular viral proteins. This can be measured directly using flow cytometry. It is shown that the model shows reasonable agreement with experimental data, although not all details of the inner dynamics can be fully reproduced. 

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. 

Media and reagents for cell culture are purchased from Gibco Biocult and Bio-chrom. Growth medium for Madin-Darby canine kidney strain II (MDCKII) cells consists of EME with Earle s salts (E-MEM) supplemented with 10 mM Hepes, pH 7.3, 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. MDCKII cells are grown and passaged as described previously (Matlin et al., 1983). 

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