Cell culture epithelial cells

Cell cultures are in vitro models used for carrying out cytotoxicity assays. They are generally made up of fibroblasts, which have properties of mesodermal stem cells cultured epithelial cells, which represent those from ecto and endodermal cell layers or blood, spleen, and bone marrow cells, which are used to study leukemia. Cultured cells that have an indefinite life span are called a cell line. In this experiment, we will be using the CHO cell line to study the cytotoxicity of cw-platin. CHO cells are immortal and are easy to maintain in culture. Apparatus and instructions for maintaining a CHO cell culture are given in Appendix 4. 

Transcellular Transport of Protein—Polymer Conjugates in Cultured Epithelial Cells... 

Cell cultures. MDCK cells were seeded in the Transwells at a density of 2.2 x 104 cells/cm. Cells were fed by changing medium in both upper (apical) and lower (basal) compartments periodically. Confluent monolayers were obtained at 5-7 days post-inoculation, when the cell density reached 4.5-5.0 x 105 cells/cm2, and a transepithelial electrical resistance (TEER) of about 2,000 ohms cm2 was measured using an epithelial voltohmmeter (EVOM, World Precision Instruments, West Haven, CT). The amount of FBS in the cell culture medium could be decreased as the cells approached their maximum resistance, and could be maintained at that point for 2 days or longer in medium containing 1% FBS. 

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

Adson A, TJ Raub, PS Burton, CL Barsuhn, AR Hilgers, KL Audus, NFH Ho. (1994). Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers. J Pharm Sci 83 1529-1536. 

Kasumi A, Sako Y, Yamamoto M (1993) Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy. Effects of calcium-induced differentiation in cultured epithelial cells. Biophys J 65 2021-2040... 

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. 

Rabito, C.A. and Ausiello, D.A. (1980). Na+-dependent sugar transport in a cultured epithelial cell line from a pig kidney. J. Membr. Biol. 54 31-38. 

Utoguchi N, Watanabe Y, Suzuki T, Maehara J, Matsumoto Y, Matsumoto M (1997) Carrier-mediated transport of monocarboxylic acids in primary cultured epithelial cells from rabbit oral mucosa. Pharm Res 14 320-324... 

Weinstein K, Kardos P, Strab R, Hidalgo U (2004) Cultured epithelial cell assays used to estimate intestinal absorption potential. In Borchardt RT, Kerns EH, Lipinski CA, Thakker DR, Wang B (Eds) Biotechnology Pharmaceutical Aspects Vol I Pharmaceutical Profiling in Drug Discovery for Lead Selection. AAPS Press, Arlington, pp 197-216. 

Because mucin and/or cilia systems of AIC cultured epithelial cells may work as a barrier for drug transport, lower Papp values are expected in cell layers cultured in AIC than in LCC methods. However, it was interesting to note that no significant differences in Rapp values were observed between the cell layers cultured with the two methods (Table 9.1). This is in contrast to solute permeabilities reported previously for cell layers cultured with LCC versus AIC methods [76, 80], For example, Yang et al. reported that Rapp of lipophilic solutes (e.g., various /3-blockers) across the primary cultured conjunctival epithelial cell layer are about threefold lower when cultured under AIC than LCC conditions, suggesting that the permeability of AIC cultured cell layers generally better reflects that of the excised tissue than LCC counterparts. Mathias et al. [76] also reported that the permeability of hydrophilic solutes across the primary rabbit tracheal epithelial cell layer cultured under AIC conditions was only half of that observed for cell layers cultured under... 

E. coli strains. E. coli 0157 H7 strains lack efal, but do encode toxB, a tnmcated version of the efal gene. A toxB mutant exhibits reduced adherence to cultured epithelial cells (Stevens et ah, 2004). However, toxB had an indirect effect on adherence to epithelial cells by modulating the production and secretion of proteins that play a role for A/E formation in EHEC. The receptors for Efal and ToxB have not been identified and their true role in vivo is unknown. 

Dibb-Fuller, M. P., Allen-Vercoe, E., Thorns, C. J., and Woodward, M. J. (1999). Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis. Microbiology 145,1023-1031. 

The following methods can be used to determine the permeability of a drug substance from the gastrointestinal tract (1) in vivo intestinal perfusion studies in humans (2) in vivo or in situ intestinal perfusion studies using suitable animal models (3) in vitro permeation studies using excised human or animal intestinal tissues or (4) in vitro permeation studies across a monolayer of cultured epithelial cells. 

FIGURE 6.14. Effect of pretreatment with 1% sodium dodecyl sulfate (SDS) on immunostaining of cultured epithelial cells with anti-AE1/2 affinity-purified antibody at a final concentration of 0.45 i,g/ml. (A) In the absence of SDS treatment, AE2 is barely detectable in MDCK cell cultures. (B) After SDS treatment a bright basolateral membrane staining is visible. Reproduced, with permission, from Brown et al. (1996). Copyright 1996 Springer-Verlag. 

Shasby, D.M., M. Winter, and S. Shasby. 1988. Oxidants and conductance of cultured epithelial cell monolayers Inositol phospholipid hydrolysis. Am J Physiol 255 781. 

Immortalised cell cultures, or cell lines, overcome this because they are clones which have been propagated over several hundreds of cell generations. The obvious advantage is a continuous and steady supply of cells in the laboratory. More than 150 continuous cell lines have been established from fish. Most of them are either fibroblast-like or epithelial-like, and originate mainly from the tissues of Salmonid or... 

Hidalgo, I.J. Cultured epithelial cell models. In Models for Assessing Drug Absorption and Metabolism, Plenum Press New York and London, 1996 35 8. 

Kwon, O.J., Collins, P.D., Au, B., Adcock, I.M., Yacoub, M., Chung, K.F. and Barnes, P.J. (1993). Glucocorticoid inhibition of TNFa-induced IL-8 gene expression in human primary cultured epithelial cells. Am. Rev. Respir. Dis. 147, A752. 

Leask A, Stearns T. 1998. Expression of amino- and carboxyl-terminal gamma-and alpha-tubulin mutants in cultured epithelial cells. J. Biol. Chem. 273 2661-68... 

When transfected in cultured epithelial cells, it mediates not only the apical transport of methotrexate and folate but also that of taurocholate and prostaglandin E2. In Hs-inhibition studies, steroids, bile add analogs, and cardiac glycosides were shown to have a high affinity for OAT-K2, suggesting that it participates to the apical transport of hydrophobic anionic compounds in the kidney [52]. 

Aigner J, Kloth S, Kubitza M, Kashgarian M, Dermietzel R, Minuth WW. Maturation of renal collecting duct cells in vivo and under perfusion culture. Epithelial Cell Biol 1994 3 70-78. 

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