Fibroblast cultures, mammalian cell

Since the brain expresses many mRNAs that are also found in non-nervous tissue and are therefore of little interest to the psychopharmacologist, it is necessary to isolate only those cDNAs that, for example, encode for a specific enzyme or receptor protein. Several techniques have been developed to achieve this. For example, a specific cDNA plasmid may be inserted into cultured mammalian cells such as fibroblasts that can express the specific receptor or enzyme. Once this has been expressed in the culture medium, the receptor or enzyme can be identified by adding a specific ligand or substrate. This enables those cells that expressed the specific macromolecule of interest to be identified and... 

Despite the central role of oxygen in the synthesis of ATP via oxidative phosphorylation it is now clear that oxygen can have inhibitory effects on the proliferation of cultured mammalian cells. For example, as oxygen concentrations are raised, cultured human fibroblasts begin to manifest reduced proliferation which results from an increase in non-cycling cells [22]. 

Cultured mammalian cells and isolated hepatocyte studies. There have been several recent reports of the damaging effects of visible light exposure (>400 nm) on various microorganisms (9,10) and cultured mammalian cells (11-13). We have reported that human diploid cells on exposure to visible light (14) and oxygen (>10%) (15) lost the ability to proliferate, while ultrastructural studies showed the presence of numerous damaged mitochondria in the illuminated cells (16). WI-38 human fibroblasts show a grad-... 

Specific cDNAs (for example a receptor) inserted in plasmid that transfects a mammalian cell (e.g. fibroblasts) in culture... 

Cytotoxicity assays employ mammalian celk in culture to measure cellular metabolic impairment and death resulting from exposure in vitro to soluble and particulate toxicants. Mammalian cells derived from various tissues and organs can be maintained as short term primary cultures or, in some cases, as continuous cell strains or lines. The cytotoxicity assays, incorporated as part of Level 1 analysis, employ primary cultures of rabbit alveolar (lung) macrophages (RAM) and maintenance cultures of strain WI-38 human lung fibroblasts. 

Mammalian cells in culture are exposed to the test substance. Established cell lines are treated both with and without metabolic activation. Cells are incubated for an appropriate length of time, then rinsed, fixed, and dried. Slides are developed, stained, and exposed silver grains are counted. The endpoint of UDS is measured by determining the uptake of labeled nucleosides in cells that are not undergoing scheduled (S-phase) DNA synthesis. The most widely used technique is the determination of the uptake of H-TdR by autoradiography. Primary cultures (e.g., rat hepatocytes), human lymphocytes, or established cell lines (e.g., human diploid fibroblasts) may be used in the assay. Multiple concentrations of the test substance over a range adequate to define the response, should be used. 

Dichloromethane induced DNA single-strand breaks in mammalian cell cultures, but inconclusive or negative effects were reported for induction of gene mutations. It did not induce unscheduled DNA synthesis either in vivo in rodents or in human fibroblast cultures. It was genotoxic in fungi but not in Drosophila in the sex-linked recessive lethal assay. 

DOPE containing CL/DNA complexes in mammalian cell cultures exhibit the ii structure rather than the found in DOPC containing complexes. As revealed by optical microscopy, complexes remained stable inside cells and attached to giant anionic vesicles, whereas Hjj complexes showed fusion of their lipid with the mouse fibroblast cell membranes (Figure 10.12) or giant anionic vesicles that results in DNA release. 

The concept behind the design of this testing protocol is that the plastic sample is delivered to a mammalian fibroblast culture. Once this exposure is made, the culture is evaluated for signs that the cells are interacting with the plastic or its leachants. Although some reaction is permissible, a subjectively defined upper limit is set beyond which the plastic or elastomeric container or closure will fail the test. The test protocol includes a positive control and a negative control in addition to the sample to assure that the system is valid. 

An altmialive method to measming mutation induction within mammalian genes involves the examination of mammalian chromosomes microscopically for the presence of visible damage. Cultures of established cells (e.g., Chinese hamster fibroblasts) or primary cells (e.g., human periphraal l3rmphoc3rtes) can be used. 

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