Subculture and Cloning, Cell Lines, Monolayer, Normal cells, High cell densities, De-contamination, Antibiotics, Bacteria

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	Subculture and Cloning Mechanically or enzymatically disaggregated tissue pieces or cells, when cultured, attach to the substrate and a monolayer of cells develop from them These are the primary cultures. Many of the cell types present in the tissue will not be available in the primary culture, because they are not able to attach to the substrate and survive. When the medium is poured out during subculture, the dead cells are eliminated. Further selection will occur during the growth of the primary culture and subsequent cell lines derived from them since faster growing cell types will gradually increase and the slower growing types will diminish with time.
After a period of time, the available substrate will be occupied by the monolayer. From the primary culture, cells are harvested and inoculated or seeded into fresh vessel. The new culture thus obtained is called cell line and the process of inoculating cultured cells into fresh culture vessels is termed subculturing. Normal cells are retained in a culture at low densities and under frequent subcultures. But at high cell densities and under prolonged subcultures, the proportion of normal cells gradually declines. They tend to be overrun by transformed cells whose growth is not sensitive to high cell densities. Culture conditions thus exert selection pressure and eliminate certain cell types. In any case, faster growing cells will increase, while slower growing cells will be gradually eliminated.
This is very evident after the first subculture, where many cell types are lost due to their inability to tolerate trypsinization and re-seeding in fresh culture vessels. Subculture may be done every 7 days, but some fast growing lines require a change of medium after 3--4 days. The culture becomes relatively more stable by the third subculture. But the cell lines may still be heterogeneous. Cell lines having specific cell types may be obtained by cloning (culture of cells so as to obtain single cell colonies), selective culture (use of selective media and substrates for the maintenance of specialized cell lines) or by physical cell separation (by centrifugation or flow cytometry).
De-contamination Contamination can take place by microorganisms, (like bacteria, fungi and mycoplasma), viruses and other cell lines. Microbial contamination can be greatly reduced by laminar air flow cabinets and by the use of antibiotics. Contamination can be checked by testing the rapid change in pH, cloudiness of the medium, granular appearance outside the cells and unidentified materials floating in the medium. Contaminated vessels should be autoclaved prior to opening their mouth for cleaning.