Culturing conditions, animal cell lines

For positive controls, it is extremely important to have a tissue culture cell line with an abundant amount of target nucleic acid. Varieties of tissue culture cell lines are available from American Tissue Culture Center, Rockville, MD. Rapidly growing tissue culture cell lines transfected with the target nucleic acid could be successfully used for positive controls. For example, formalin-fixed paraffin-embedded foreskin fibroblast tissue culture cell-line FS4 can be used as a positive control for human lysyl oxidase mRNA detection and c-W-ras transformed RS-485 cell line for normal ras message detection. Thin sections (4-5 im) of paraffin-embedded positive control cell lines could be placed simultaneously near the side of the experimental human or animal tissue. Similarly, one can also use freshly cut animal or human tissue (with the target sequences) preserved, sectioned, and mounted under standard laboratory conditions. For negative controls, choose a cell line (or a tissue) that completely lacks the target nucleic acid see Note 12). 

Cell culture systems must provide the physiological conditions for cell survival and proliferation. In vitro, animal cell growth is dependent on several factors, such as pH, temperature, osmolality, gas concentration (oxygen and CO2), available surface substrata, and state of the cells at inoculation (Freshney, 2005). Other factors that impact the culture are medium composition, which can differ extensively between cell lines and is discussed in detail in Chapter 5, as well as susceptibility to hydrodynamic stress, as discussed in Chapter 7. 

Primary thyroid cells from rat, dog, pig, cow, sheep, and human have also been used for in vitro studies of thyroid follicle cells [70], They act similar to the cell lines described above in culture, and cultured conditions, media, and additives are the same. Fewer reagents are available for porcine or bovine cells however, thyroid tissue is fairly readily obtained and provides more cells than most common preclinical animals (e.g., rat, mouse). We have not detected T3 or T4 in culture medium of either porcine or bovine thyroid cells, but they respond to TSH or forskolin with concentration-dependent increases in intracellular levels of cAMP. Decreased expression of TSH-R and increased NIS and Tg expression were measured in the presence of TSH or forskolin (our unpublished data). 

The complexity and plasticity of BBB properties called for experimental dissection of the disrnption process in both in vitro and in vivo conditions. Multiple cell and organ cnltnres, animal models, and measurement techniques have been developed, each of which addresses some of the issues involved. The development of research into BBB characteristics was initially approached in avian embryos, where transplanted endothelial quail cells invaded a developing chick chimera. A simpler cell culture model of the BBB was developed by Rubin and co-workers. More recently, an immortalized cell line created from vascular endothelial cells was used to develop another model of the BBB in co-cultures with glioma cells and was used to demonstrate nitric oxide-induced perturbations of these cells. hi another cell culture model, hypoxia was shown to increase the susceptibility to oxidative stress and intercellular permeability. ... 

Our laboratories have been carrying out a large series of studies on the effect of various antioxidant micronutrients on both animal and human cancer cell lines in culture (Schwartz and Shklar, 1994). These studies were carried out to augment our extensive animal studies. Various molecular parameters can be evaluated more easily in cell culture studies, and one can study many human cancer cell lines and compare them to cell lines derived from animal studies. In vivo studies have shown that ascorbic acid s primary biochemical activity is as an aqueous antioxidant that interacts with other antioxidants. Fundamental to the activity of ascorbic acid is the formation of the ascorbate radical, which forms under various oxidizing conditions. The development of the ascorbate radical during carcinogenesis could then replace... 

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