Cerra et al. 1990

Cerra R, Zarbo RJ, Crissman JD. Dissociation of cells from solid tumours. Methods in Cell Biology 33: 1-12.

These authors describe methods to produce cell suspensions from a range of cancer tumour types collectively described as “solid”. Many tumours are relatively easy to extract individual cells in suspension; this paper describes methods suitable for tumours in which the cells are bound to each other and to extracellular materials. Existing methods for flow cytometry analysis of these tumours have focused on enucleation techniques, which avoid the problems associated with cell membrane bonds to other cells by rupturing cell membranes. This causes the loss of potentially informative cell-membrane-bound and cytoplasmic materials, and these authors state they are not convinced enucleation is the best option.

Dissociating cells can be accomplished by a vast range of mechanical and enzymatic techniques, many of which can be used in combination, such that the optimum technique to use on a given tissue must be determined empirically for each case. Mechanical dissociation techniques typically start with cells in the early stages of cell culture. The authors recommend the use of two scalpel blades rather than scissors to slice tissues because scissors tend to mash the tissue rather than cut. The resulting semi-dissociated tissue should be sieved through something called a “100-mesh cell sieve”, whatever that is. I think this step removes clumps of cells and large pieces of extracellular debris. Other mechanical dissociation methods include the use of tissue attached to a wine cork and vibrated with a mechanical engraver to release cells (Eade et al. 1981).

Enzymatic dissociation methods tend to focus either on the use of proteases to disrupt intercellular desmosomes, or collagenases to disrupt the extracellular matrix. Each category of enzymes may damage or remove cell-membrane features that may be useful in diagnosis during flow cytometry. In all cell dissociation techniques, DNAases are routinely used to remove the very sticky free DNA released by dead cells. Liotta et al. (1982) provide a review of the use of collagenases in cell dissociation.

Enucleation techniques can be applied to a wider range of tissues than can cell dissociation techniques, including fixed and frozen tissues. I get the impression that fixed and / or frozen tissues are routinely used in cancer flow cytometry with a range of human tissues. The authors caution that enucleation typically causes high levels of background debris compared to cell dissociation. Thornthwaite et al. (1980) and Vindeløv et al. (1983) provide protocols for one-step isolation and staining of nuclei; these authors provide a more complicated method for the use of paraffin-embedded tissues.