Studies with Dispersed, Isolated Cells

Various tissue constructs have been reassembled from isolated constituents, including resident cell types whose numbers have been amplified or modified in culture. A three-dimensional co-culture system for human skin keratinocytes layered upon a synthetic mesh infiltrated with dermal fibroblasts, when floated to allow contact of the uppermost keratinocytes with air, exhibits stratification and cornification remarkably similar to in vivo squamous epithelia. This reconstructed epithelial model has been recommended as an in vitro replacement for dermal corrosivity testing. It has been anticipated that this and a similar noncomified model will have application in dermal and ocular irritation testing, but thus far validation studies have yielded mixed results. Reconstructed tissues can also provide context for basic toxicological research on aberrant cellular interactions with cellular and acellular constituents, as illustrated by invasion of cancerous epithelial cells into underlying dermis of a skin equivalent model. 

Suspensions of freshly isolated, dispersed cells commonly are used in short-term incubations for biochemical studies on intermediary metabolism. Similar systems 

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Furthermore, in more recent studies high [Ca2+]mt signals are seen in only a few mitochondria within a given cell, and reports claiming very high [Ca2+]mt under physiological conditions are based on cells isolated by enzymatic dispersion. This, coupled with the uncertainty of calibration of luminescent and fluorescent Ca2+ indicators within the mitochondrial matrix, (for nuclei see Perez-Terzic et al 1997) should raise serious questions about the correct values of [Ca2+]mt. It is unfortunate that, with rare exceptions, very few available studies compare free with total mitochondrial Ca in the same cell type observed under the same condition. 

PANCREAS Membrane potential measurements in pancreatic /3 cells with intracellular microelectrodes, 192, 235 stimulation of secretion by secretagogues, 192, 247 pancreatic secretion in vivo, perfused gland, and isolated duct studies, 192, 256 dispersed pancreatic acinar cells and pancreatic acini, 192, 271 permeabilizing cells some methods and applications for the study... 

As previously mentioned, this behaviour change corresponds to the evolution of cellular structure of the foams from a large amount of cells with lots of connectivities for low densities (and so low conductivities), to isolated and dispersed cells slightly connected for high densities (and so high conductivities). At the transition, data show a foam sensitivity to skip from one structure to the other. The evolution of foam porosity in the transition zone must be confirmed by additional structural study. 

For the study of the thermotropic mesomorphism of membranes the sample preparation is very important. Lipid dispersions are generally prepared by mixing the desired amounts of solid lipid and solvent (H2O, D2O or buffer solution). When using D2O as solvent a closed vessel should be used in order to minimize exchange with atmospheric water. In order to ensure proper hydration the lipid water mixture should be first heated above the temperature of the gel to liquid crystal phase transition of the corresponding lipid, then cooled before commencing the measurements. The study of natural membranes involves re-hydration only if the membranes are isolated from the other cell components and lyophilized (6-8). The experiments on bacterial membranes currently under way in our laboratory utilize live bacteria or isolated membranes which have not been lyophilized, therefore no hydration procedure is involved (9). 

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