Monolayer culture

The normal rat kidney (NRK) cell line is an untransformed cell line established from rat kidney cells. However, when NRK cells are treated with both TGF alpha and TGF beta, the cells assume a transformed phenotype. In addition to exhibiting an altered morphology in monolayer culture, NRK cells possess the capacity for anchorage-independent growth while being treated with both TGF alpha and TGF... 

In vitro metabolism of pH]PbTx-3 was studi in isolated rat hepatocytes (25). Hepatocyte monolayers cultured in 6-well plates containing 1 ml modified Williams E medium were incubated with 0.1 fig radiolabeled toxin at 37 C for 24 hr. The... 

Tumor cells. EMT6 cells were grown as a monolayer culture in DMEM medium containing 20% fetal calf serum (27). Cells were detached from the plate by trypsin-EDTA treatment and washed in PBS. A total of 5 x 103 cells were injected per mouse via the tail vein of Balb/c mice (6-8 weeks old) to induce experimental lung metastatic tumors. Immunoliposomes were injected iv 2 and 4 days after the tumor cell injection. The survival of mice was followed over the next 60 days. 

Figure 24 Schematic model of passive diffusion of molecular species of a weak base through the transcellular and paracellular routes of a cell monolayer cultured on a filter support.

It is noteworthy that the kinetics viewed from the donor and receiver sides are seemingly independent of each other. As discussed in the next section, this prompts the employment of experimental strategies to understand the events of drug uptake and efflux mechanistically and independently through drug uptake studies with cell monolayers cultured on flat plastic dishes, drug efflux from the... 

Given the low permeability of the antioxidant across MDCK cell monolayers and its large membrane partition coefficient, efflux kinetic studies using drug-loaded cell monolayers cultured on plastic dishes could yield useful information when coupled with the following biophysical model. The steady-state flux of drug from the cell monolayer is equal to the appearance rate in the receiver solution ... 

Figure 36 Efflux kinetics of PNU-78,517 from the apical membrane of MDCK cells in monolayer culture on a solid plastic surface as a function of bovine serum albumin concentration. [Redrawn from Raub et al. (1993) with permission from the publisher.]...

The initial conditions are CD = CD(0) at t = 0 and CR = 0 at t = 0. Efforts to obtain analytical solutions are tedious and unnecessary. By applying the change in concentrations (or mass) in the donor and receiver solutions with time to the Laplace transforms of Eqs. (140) and (141), the inverse of the simultaneous transformed equations can be numerically calculated with appropriate software for best estimates of a, (3, and y. It is implicit here that P Pap, Pbh and Ke are functions of protein binding. Upon application of the transmonolayer flux model to the PNU-78,517 data in Figure 32, the effective permeability coefficients from the disappearance and appearance kinetics points of view are in good quantitative agreement with the permeability coefficients determined from independent studies involving uptake kinetics by MDCK cell monolayers cultured on a flat dish... 

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. 

Fig. 14.1. Schematic representation of drug transport assay in cell monolayers cultured on a culture insert containing permeable membrane.

Seek T75 plastic tissue culture flasks with a minimum of 2.5 x 106 cells in 120ml of Eagle s medium containing 20mM L-glutamine 0.88g l-1 sodium bicarbonate 20 mM HEPES 50 pg ml-1 streptomycin sulphate 50IUml 1 benzyl-penicillin and 7.5% fetal bovine serum. The flasks are incubated for 18-24 h at 37°C in a C02 incubator to establish monolayer cultures. 

For monolayer cultures, the cultures are set up the day before BrdU treatment so that the cells will be in exponential growth before the addition of BrdU or the test compound. After BrdU addition the cells are allowed to undergo the equivalent of two cell cycles before cell harvest. A spindle inhibitor such as colchicine or colcemid is introduced for the final 1-2 h of culture to arrest cells in metaphase, after which the cells are harvested and chromosome preparations are made by routine cytogenetic techniques. 

Cytotoxicity. The liver is the primary target organ for a variety of drugs and chemicals (Hasemen et ah, 1984 Farland et ah, 1985). The prevalence of drug-and chemical-induced liver injury is of concern because some xenobiotics can produce liver damage at dose levels that are magnitudes below that which causes cell death (Plaa, 1976). Environmental and commercial chemicals can increase this effect by as much as 100-fold (Plaa and Hewitt, 1982 Plaa, 1976). Studies of early cell injury caused by exposure to a toxicant can be undertaken easily in monolayer cultures of hepatocytes, whereas early cell injury is very difficult to assess in vivo. 

Cell Lines. Cell lines, derived from tissue of various species, are commercially available from tissue culture banks. These cell populations are immortalized in that they possess the capacity to permanently proliferate in culture. Such cellular models can be studied in short-term suspension (hours) or longer-term monolayer culture (days, weeks, months). Since cell lines have been extensively cultured or passaged for multiple generations, the degree or retention (or loss) of kidney-specific morphology and function is an important limitation that is not thoroughly addressed for a number of renal cell lines. One renal cell line that has been relatively well characterized is the pig kidney cell line, LLC-PK,. 

Bellemann, P. (1980). Primary monolayer culture of liver parenchymal cells and kidney cortical tubules as a useful new model for biochemical pharmacology and experimental toxicology. Studies in vitro on hepatic membrane transport, induction of liver enzymes, and adaptive changes in renal cortical enzymes. Arch. Toxicol. 44 63-84. 

Borenfruend, E. and Puemer, J.A. (1984). A simple quantitative procedure using monolayer cultures for cytotoxicity assays. J. Tissue Culture Meth. 9 7-9. 

Guzelian, PS. and Bissell, D.M. (1974). Metabolic factors in the regulation for cytochrome P-450. Studies in rat hepatocyte monolayer culture. FedProc. 33 1246. 

Holme, J. (1985). Xenobiotic metabolism and toxicity in primary monolayer cultures of... 

A. C. Chao, J. A. Dix, M. C. Sellers, and A. S. Verkman, Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts, Biophys. J. 56, 1071-1081 (1989). 

Cellular micro-arrays are used in the pharmaceutical industry in 96 or 384 well microtiter plates and with 2D cell monolayer cultures [46, 47] that can be easily automated and miniaturized [37],... 

Yamashita F, Mathias NR, Kim K-J, Lee VHL (1996) Dipeptide transport properties of rabbit tracheal epithelial cell monolayers cultured at an air-liquid interface. PharmRes 15 979-983. 

Widdicombe, J. H., D. L. Coleman, W. E. Finkbeiner, and I. Tuet. 1985. Electrical properties of monolayers cultured from cells of human tracheal mucosa. J Appl Physiol 58 1729-35. 

Sulzbacher A, Jarosch A, Schuler R, Acerbi D, Ventura P, Puccini P, Woodcock BG (1998) Validation of a Caco-2 cells monolayer culture for drug transport studies. Int J Clin Pharmacol Ther 36 86-89. 

Costa AK, Trudell JR. 1988. Toxicity of 1,2-dibromoethane in primary hepatocyte monolayer cultures Lack of dependence on oxygen concentration. Toxicol AppI Pharmacol 95 241-247. 

To a log-phase monolayer culture of HeLa cells, add sufficient stock colchicine solution to give a final concenttation of 0.06 pg/mL. 

The isolation and characterization of alveolar Type II cells which transform into alveolar Type I cells has been described, as weh as a monolayer culture of alveolar Type I cells [35,36]. 

Protocol 1.10 Expression of the recombinant protein in the Baculovirus system in monolayer cultures... 

Kate H, Nakazawa Y. 1987. The effect of carbon tetrachloride on the enzymatic hydrolysis of cellular triacylglycerol in adult rat hepatocytes in primary monolayer culture. Biochem Pharmacol 36 1807-1814. 

The cultivation of hepatocytes in a stationary suspension culture is actually ineffective. The hepatocytes lose their differentiation within hours. An improvement was the attachment culture. Thereby, the cells are cultivated either in self- or microcarrier-induced multicellular aggregates or on membranes. When standard monolayer culture was adequate to maintain the cell viability for 1 to 2 weeks the differentiation was lost after a few days. Different modifications as described beneath allowed the maintenance of differentiation for 2 to 3 weeks. 

Cytotoxic effect. Gas phase of mainstream cigarette smoke, in monolayer culture of mouse lung epithelial cells, produced an increase in cytotoxicity in a dose-dependent manner. Cell viability of cultures exposed to gas phase with only the nonorganic components was equivalent to controls. Removal of volatile organic constituents resulted in almost elimination of cytotoxicity of the smoke . Smoke condensate and tobacco extract, at high concentrations in Lewis lung adenocarcinoma cells and mice spleen lymphocytes, were cytotoxic. Smaller doses increased thymidine incorporation in both cell types. Lymphocytes were more susceptible to the toxic effect of tobacco prod-... 

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