Culturing Cells

Multiple methods of isolating cells for culture exist. These methods generally include either purification from blood or mechanical removal from a tissue and subsequent enzymatic digestion. Cells isolated via these techniques are then incubated in carefully maintained conditions, usually at atmosphere plus 5% CO₂, 37^oC, and 85-95% humidity. The cells are generally grown in plastic or glass vessels. A culture medium provides the cells with most of the nutrients such as amino acids, carbohydrates, vitamins, and minerals they need for metabolism. Additional growth factors and hormones are added to promote growth or attachment to the culture vessel, often via undefined adjuncts such as serum. Most freshly isolated, primary cell cultures undergo senescence, the process by which cells stop dividing after undergoing a certain number of cell divisions. To prevent this from occurring, the new cell line may be immortalized using techniques such as the vinyl chloride immortalization method or transfection with genes such as SV40 T antigen or human telomerase.

Once founded, the cell line can be propagated into several flasks and frozen. At this point, the creator of the cell line must perform some form of authentication and characterization that identifies it as a unique cell line. Depending on whether the cell line is of human, mouse, or another animal short tandem repeat (STR) profiling or cytochrome oxidase (CO)1 barcoding can be used. In addition, a full spectrum of sterility testing should be performed to identify possible mycoplasmal, bacterial, fungal, or viral contamination. The founding laboratory should also institute the seed stock principle to bank the cells. This method ensures the protection of original stock and the consistent availability of low-passage vials of cells via the creation of a master cell bank and working cell banks.