Segregating Cells - Proteases in Tissue Culture

Abstract

Rous and Johns first introduced the use of trypsin to detach growing cells from explanted tissue pieces (Rous and Johns 1916). For more than 80 years trypsin has remained a favorite enzyme for the primary dissociation of tissues and for detaching cells in monolayers for subsequent replating. The trypsin that was used by Rous and Johns and in many instances still is used is a mixture of various pancreatic enzymes. Low concentrations of crude trypsin usually did not harm cells, but higher concentrations would kill the cells. Tryptic isolation of individual cells from tissue for primary cultures were used to dissociate chick embryonic tissues for cell cultures (Weymouth 1974). Moskona (1952) was using a 3% crude trypsin solution in a calcium and magnesium free solution. The use of calcium and magnesium free solution was based on the evidence that these cations play a role in stabilising the intercellular matrix and thus in controlling mutual cellular adhesiveness. A first exhaustive review on the isolation of cells has been published by Rinaldini (Rinaldini 1958). He also reported that purified trypsin was less effective than crude trypsin preparations pointing out the existence of other enzymes in the mixture of crude trypsin. The intercellular material which has to be digested in order to segregate the cells are mucopolysaccharides and proteins of which collagen is one of the major intercellular proteins. Elastins are another kind of intercellular mucoproteins which are digested by the specific enzyme elastase.