Table 1 Concepts in cell culture
Isolation of cells | Cells can be isolated from tissues for ex vivo culture in several ways (purified from blood or by enzymatic digestion) |
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Maintaining cells in culture | Cells are grown and maintained at an appropriate temperature, gas mixture and growth media (vary in pH, glucose concentration, growth factors, and the presence of other nutrient components) in a cell incubator. Some times extracellular matrix components (i.e. collagen or fibronectin) are needed to increase its adhesion |
Manipulation of cultured cells | Cells generally continue to divide in culture, this usually lead to nutrient depletion in the growth medium, Accumulation of apoptotic/necrotic cells, cell cycle arrest or promiscuous and unwanted cellular differentiation due to cell-to-cell contact. To avoid these problems cultured cells is manipulated. Most common manipulation: media changes, passaging cells, and transfecting cells |
Media changes | To replenish nutrients and avoid the build up of potentially harmful metabolic byproducts and dead cells by centrifugation or aspiration |
Passaging (splitting) cells | Involves transferring a small number of cells into a new vessel. This can either be done by introducing a small amount of culture containing a few cells diluted in a larger volume of fresh media or by a mixture of trypsin-EDTA, however other enzyme mixes are now available for this purpose |
Transfection and transduction | Involves the introduction of foreign DNA and the cells will express a protein of interest. More recently, the transfection of RNAi constructs have been realised as a convenient mechanism for suppressing the expression of a particular gene/protein |