Primary cell cultures

Primary cell cultures, which are prepared directly from tissues. 

Established cell lines, cell strains or primary cell cultures may be used. The most often used are Chinese hamster cell lines and human peripheral blood lymphocytes. The merits of these two cell lines have been reported (Ishidate and Hamois, 1987 Kirkland and Gamer, 1987). The cell system must be validated and consistently sensitive to known clastogens. 

Established cell lines, primary cell cultures of rodents, may be used. Detailed information on in vitro and in vivo assays may be obtained in reviews of SCE methods by Latt et al. (1977, 1981), Perry and Thompson (1984) and Perry et al. (1984). The in vitro methods will be briefly explored here. 

Williams, G.M. (1976a). Carcinogen-induced DNA repair in primary rat liver cell cultures a possible screen for chemical carcinogens. Cancer Lett. (Shannon, Ire.) 1 231-236. Williams, G.M. (1976b). The detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37 1845-1851. 

Sakagami M, Omidi Y, Campbell L, Kandalaft LE, Moris CJ, Barar J, Gumbleton M (2006) Expression and transport functionality of FcRn within rat alveolar epithelium a study in primary cell culture and in the isolated perfused lung. Pharm Res 23 270-279. 

For in vitro toxicity studies and assessment of the barrier function, drug transport, cell physiology, and metabolism as well as the development of delivery systems, cell culture models provide powerful systems for scientific research. As the corneal epithelium is the main barrier for ocular penetration, various corneal epithelial cell cultures were established besides the corneal constructs that mimic the whole cornea and serve as reductionist models for the ocular barrier. In general, two types of cell culture models are available primary cell cultures and immortalized, continuous cell lines. 

A sufficient plating density has also to be chosen in order to obtain confluent monolayers within the limited time of viability of primary cell cultures. 

Based on the results from those cell calibration assays, the routine incubation period was set at 3 days. During that interval, some of the cells will proliferate, some will be quiescent but metabolically active, and some will die, which is the case for most primary cell cultures. Because these cells are cultured for periods far shorter than their typical population doubling time (about 6 to 7 days), these cells should not fully adapt to the tissue culture conditions and, therefore, maintain their primary cell phenotype. 

A variety of cell lines, strains, or primary cell cultures, including human cells, may be used (e.g., Chinese hamster fibroblasts, human or other mammalian peripheral blood lymphocytes). Cell cultures are exposed to the test substance both with and without metabolic activation and at predetermined intervals after exposure, they are treated with a metaphase-arresting substance (e.g., colchicine), harvested, stained, and metaphase cells are analyzed microscopically for the presence of structural chromosome aberrations. At least three concentrations should be used. 

The simplest and most sensitive assays for detecting clastogenic (i.e. chromosomal breaking) effects involve the use of mammalian cells. Cultures of established cell lines (e.g. Chinese hamster ovary) as well as primary cell cultures (e.g. human l)nnphocyte) may be used. After exposure to a range of chemical concentrations in the presence and absence of an appropriate metabolic activation system, the cell cultures are treated with a spindle inhibitor (e.g. vinblastine) to accumulate cells in a metaphaselike stage of mitosis. Cells are harvested at appropriate times and chromosome preparations are made, stained with DNA-specific dye and the metaphase cells are analysed under the microscope for chromosome abnormalities. 

Williams GM. 1977. Detection of chemical carcinogens by unscheduled DMA synthesis in rat liver primary cell cultures. Cancer Res 37 1845-1851. 

Teuscher, N. S., Shen, H., Shu, C., Xiang, J., Keep, R. F., and Smith, D. E. (2004). Carnosine uptake in rat choroids plexus primary cell cultures and choroids plexus whole tissue from PEPT2 null mice. ]. Neurochem. 89, 375-382. 

McDowell, E. M., Ben, T., Newkirk, C., Chang, S., and De Luca, M. (1987b). Differentiation of tracheal mucociliary epithelium in primary cell culture recapsulates normal fetal development and regeneration following injury in hamsters. Am. J. Pathol. 129,511-522. 

Surprisingly, a growth inhibiting and pro-apoptotic function has been demonstrated for oncogenic Ras mutants. In primary cell cultures, activation of the Ras pathway is linked to an increase in the concentration of the tumor suppressor proteins p53 and pl9ARF (Serrano, 1997), which both promote programmed cell death, or apoptosis (see Chapter 15). This example shows that, according to the cellular context, the Ras protein can promote both cell death and cell survival via interactions with distinct effector proteins. 

Reinhardt CA (1993) Neurodevelopmental toxicity in vitro Primary cell culture models for screening and risk assessment. Reprod Toxicol, 7(Suppl 1) 165-170. 

Zhao, Q., Zhang, W., Jin, M., Yu, X., and Deng, M. (2005). Formulation of a basal medium for primary cell culture of the marine sponge Hymeniacidon perleve. Biotechnol. Prog. 21, 1009-1012. 

Over the last four decades, there has been substantial progress in the development of dedicated, highly specialized delivery systems for nucleic acids in vitro as well as in vivo. Numerous different approaches addressing multiple established cell lines and primary cell cultures have been developed and refined. For most of the... 

Cannabinoids may also cause effects via mechanisms distinct from the cannabinoid receptor pathways. The most extensively investigated compound is (+)-HU 211, a synthetic cannabinoid with a stereochemistry opposite to that present in the naturally occurring compounds. It does not produce THC-type effects in animals and shows insignificant binding to the CB, receptor. However, HU 211 blocks A-methyl-n-aspartate (NMDA) receptors and calcium uptake through the NMDA-receptor-ion channel in primary cell cultures. HU 211 is a potent blocker of NMDA-induced tremor, seizures, and lethality in mice. It may therefore prove useful as a nonpsychoactive drug that protects against NMDA-receptor-mediated neurotoxicity. This is supported by the potent attenuation of NMDA-receptor-mediator neurotoxicity in cell cultures by HU 211. 

Different hypotheses have been raised to explain these purported beneficial effects. Ono et al. have shown that the neuroprotective effects of various polyphenols (e.g., myricetin, morin, and, to a lesser extent, quercetin) may be due to their ability to inhibit amyloid fibrils and to destabilize fibrilized forms of Ap,20 suggesting that they could be considered as new therapeutic agents for the treatment of AP-associated diseases.21 Resveratrol, a red-wine polyphenol, has been proposed to promote the intracellular degradation of Ap by a proteasome-dependent and secretase-independent activity.22 Based on these findings, we compared the effects of polyphenols found in teas and red wine, using the model of AP-induced toxicity in rat hippocampal primary cell cultures. 

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