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Introductionth to Cell and Tissue Culture - Jennie P.

Jennie P. Introductionth to Cell and Tissue Culture - Plenum Press, 2002.
ISBN 0-306-45859-4
Download (direct link): introductiontocellandtissueсulture2002.pdf
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The drawbacks of primary culture are that they are frequently time consuming to prepare and require the use of live animals or fresh tissue. There can be considerable variation from one preparation to another, particularly if prepared by different people. Finally, the cultures are continuously changing from the time the cells are removed from the body until they die or adjust to the culture conditions used. This can include changes in the mix of cell types in the culture, changes in cell shape, changes in cell"Ccell associations, and changes in the factors secreted from the cells and the receptors and other cell surface proteins present on the cells. These events must be taken into consideration in determining how long primary cultures can be studied and in interpreting the results obtained.
Primary culture refers to the cells that are placed in culture directly from the tissue of origin. These are called primary cultures until the first subculture. After the first subculture, they may be called secondary cultures, and thereafter, if continued passage is possible, a cell line. These cultures can contain mixed cell types or consist predominantly of a single cell type. However, given the complex interrelationships of cells in any organ or tissue, primary cultures seldom, if ever, consist exclusively of a single cell type. Attempts may be made to mechanically or enzymatically purify the cell type of interest during the tissue dissociation or to "clean" the cultures by culturing the cells in conditions that specifically inhibit or kill some major contaminant cell type(s). Alternatively, primary cultures can be maintained in conditions chosen to positively select for the survival of only one cell type. This approach is discussed further in Chapter 10.
Page 152
There are undoubtedly more methods for putting cells in primary culture than there are cell types, and that is a great many indeed. In this chapter we will attempt to present general methods for some of the most commonly used types of primary culture and to refer to the volumes available on the isolation and culture of very specialized cell types. In general, the method chosen must take into consideration the desired balance between obtaining a high cell yield and minimizing damage to the cells to be culturedjabetween maximal yield and maximal purity. Considerations of the earliest time the cells are to be studied (after recovery) and the maximum time they are to be kept in primary culture might also determine the isolation method of choice. In preparing cells for primary culture, as in so many other aspects of life, you cannot have it all.
Tumors in vivo are by definition not long subject to the growth controls and regulatory mechanisms of their normal counterparts. When placed in monoculture, in the presence of serum, rodent tumor cells will often rapidly attach, spread, and divide. This may be due largely to autocrine factors produced by these cells that allow for rapid growth or to the activation of oncogenes that release the cells from normal growth control mechanisms. However, there are some rodent tumors, and many human tumors, that fail to grow easily in vitro.
The same biological laws are operative in either normal or neoplastic tissue: The in vitro microenvironment may be insufficient to sustain growth, either due to inappropriate factors contained in the medium, inappropriate substrate or matrix, inappropriate concentration of factors, or all the above. These factors need to be considered if there is difficulty growing a specific tumor in vitro. The special considerations for growing human cells (normal or tumorigenic) will be discussed.
Tumors are usually well vascularized in vivo and are often surrounded by fibrous stroma or connective tissue. Disaggregation of individual cells often requires both mechanical separation and enzymatic digestion. The digestion with certain enzymes (trypsin, crude collagenase) may prove toxic to some epithelial cells, although they are effective in removing and disassociating fibroblasts. In such cases a differential digestion with a crude collagenase containing STI, and then a gentler digestion with collagenase'Cdispase or thermolysin may prove more effective in obtaining viable tumor cells. Many tumor cells are less anchorage dependent than their normal counterpart and may be grown on plastic or substrates with reduced adhesive properties. Although tumor cells are considered mostly to be autonomous from normal growth control in vivo, once they are in monoculture they may depend on paracrine as well as autocrine factors for continued growth and survival and therefore need to be isolated and maintained at high density.
For example, we found this to be the case with a rare human esophageal carcinoma, which we initially isolated from a 1-mm biopsy specimen. We were able to isolate the tumor cells and obtain a cell line by maintaining them at high density in a small surface area initially (a single well of a 96-well microtiter plate) in 100 ^l of medium containing insulin, transferrin, EGF, trace elements, and 0.5% serum. The low serum was sufficient to prevent initial fibroblast overgrowth, and in the presence of insulin, transferrin, and EGF the cells were capable of producing enough autocrine and paracrine growth factors to survive the initial transplant and divide. Conditioned medium from the parent cultures initially aided in passaging these cells.
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