Using Primary Cells

There are several limitations in using primary cell lines from patients with mitochondrial disease (e.g., fibroblasts and myoblasts) (Jl). They are not immortal, and usually grow very slowly in regular culture media. Some of the cell lines do not retain a fully physiological, differentiated phenotype in culture. In addition, they tend to lose mutant mtDNA as a result of mitotic segregation (Jl). Most importantly, one cannot rule out the effect of the nuclear background on the phenotypic expression of mitochondrial dysfunction under examination. 

A primary cell culture often consists of mixed populations of cell types some may continue to proliferate, while others survive without proliferating or exhibit reduced growth rates, leading to unpredictable shifts in the proportions of cells in a population. This possibility should be considered when using primary cells, and adequate controls of the cell population composition should always be used within a single cell preparation and in batch-to-batch preparations. 

Advantages and Disadvantages in Using Primary Cells versus Other Cell Types... 

Even if the concepts are good, the use of primary cells in screening laboratories is not yet widespread. Primary cells are mainly employed in secondary assays to validate effects of compounds identified in initial screens conducted with recombinant cells. Secondary assays using primary cells to validate positive hits found via primary HTS should aid in the selection of meaningful leads with enhanced probability of identifying successful new compounds (Gebrin-Cezar, 2007). 

At present, the use of primary cells in the pharmaceutical industry is not as common as the use of recombinant cell-based assays. A rough estimate shows about 20% of all initial screening is carried out using primary cells and the rate is about the same for secondary screening. The use of primary cells is expected to increase in the next few years as emerging technologies start appearing on the market. 

Multi-Endpoint Analysis. Until recently, in vitro evaluations were typically done using primary cell cultures and a limited number of doses to monitor changes in one or two endpoints. The new paradigm is to evaluate toxicity much earlier in the discovery process. To be useful to discovery teams, these evaluations must be done with a small amount of compound, require no more than 1 or 2 weeks to turn data around, provide information on a new compound s toxicity relative to other chemicals in class, enable the prioritization of new leads based on some parameter of toxicity (e.g., the concentration that produces a half-... 

One primary cell, the gravity cell, has been described in Section 11-5. This cell is called a wet cell, because it contains a liquid electrolyte. A very useful primary cell is the common dry cell, shown in Figure 11-8. The common dry cell consists of a zinc cylinder that contains as electrolyte a paste of ammonium chloride (NH4CI), a little zinc chloride (ZnClg), water, and diatomaceous earth or other filler. The dry cell is not dry water must be present in the paste that serves as electrolyte. The... 

In our laboratory, factors other than the selection of susceptible populations of cells could be controlled to optimize transformation of hamster embryo cells and increase the reliability of the bioassay. All batches of culture medium, plastic vessels, and fetal calf serum were pretested before routine use. Primary cells were planted at a high density (1.3 X 10 /cm ) and cultured for no longer than 2-3 days to minimize the number of cell divisions, since susceptibility to transformation appeared to decrease as the number of cell divisions increased. With certain carcinogens, apparently requiring further metabolic activation... 

FcR-mediated cytotoxicity assays are variable and difficult to perform, especially the ADCC assay, which uses primary cells from peripheral blood. However, recently these assays have been simplified and used as a bioactivity assays for characterization of therapeutic proteins and as a routine potency assays. Some laboratories in industry are able to use cloned NK cells as effector cells and the established cell lines or transfected cells as target cells in simplified ADCC assays. The CDC assay is used as a potency release assay for several therapeutic proteins. The use of cell lines instead of the primary cells in ADCC assays and nonradioactive detection methods, adenylate kinase (AK) or ATP with luminescent readouts, for example, have a potential to improve the performance of these assays and make them more suitable for the routine use. 

The aim of this study was to explore in vitro the control of avUCP expression by isoproterenol and fatty acids by using primary cell cultures of chick myoblasts, and then to investigate the signalling pathways potentially involved in these regulations. 

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