Tissue disaggregation

This protocol can be nsed as a fu-st-mn attempt at tissue disaggregation. Fnrther experience with the tissue will probably yield insights as to the best enzyme types, enzyme concentration, digestion time, and digestion temperature. 

For many years a solution of crude acetone powder of bovine or porcine pancreas has been used to disaggregate tissues and to release cultured cells from their substratum. These preparations — referred to as trypsin 1 250 (based on an international standard) — contain not only trypsin but a range of enzymes including chymotrypsin and elastase which are equally important. Purified trypsin seldom is as efficient as the cruder preparations, especially on tissue disaggregation, when mucinous clumps result (Ronaldoni, 1959). 

A conventional flow cytometry procedure for creating single cell suspensions is provided. Methods of tissue disaggregation to create single-cell suspensions from formalin fixed tissue are also given. Chemical, mechanical, and enzymatic multistep methods for separating single cells from tissue are discussed. These procedures are very tissue dependent. 

Key words Flow cytometry, Tissue disaggregation, Single-cell suspension, Mechanical dissociation, Enzymatic dissociation, Chemical dissociation... 

The Scienceware 12-WeU Tissue Disaggregator fits industry standard 12-weU culture plates and is ideal for producing cell isolates for culture or flow cytometry. Scienceware Double-ended Pestle with flat heads maximizes your efforts. Bel-Art Products, www.belart.com, (800) 4BELART. Circle 262... 

Loscalzo J, Vaughan DE. Tissue plasminogen activator promotes platelet disaggregation in plasma. J Clin Invest. 1987 79 1749-1755. 

Weiner, S. and Price, P. A. (1986). Disaggregation of bone into crystals. Calcified Tissue International 39 365-375. 

Costa, A., Silvestrini, R., Del Bino, G., and Motta, R. (1987) Implications of disaggregation procedures on biological representation of human solid tumors. Cell Tissue Kinet. 20,171-180. 

In general, a crude or partially purified extract is electrophoresed on a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) then the protein band is lightly stained and cut out. In the simplest method, the acrylamide gel band is reduced to a pulp, mixed with Freund s adjuvant, and injected. Unfortunately, this technique is not always successful. Its failure can probably be attributed to factors such as the difficulty of disaggregating the acrylamide, the difficulty with which the protein diffuses from the gel, the presence of SDS in large quantities resulting in extensive tissue and cell damage, and finally, the toxicity of the acrylamide... 

Cells can be cultivated in vitro after a tissue is dissected and totally or partially disaggregated by enzymic treatment. Cell, explants, or organ cultures can be established depending on the degree of disaggregation of the tissue, as shown in Figure 2.2. 

Dispase is a neutral protease from Bacillus polymyxa available from Boehringer Corp. Ltd. It is particularly suitable for disaggregation of animal tissues, which seldom suffer from prolonged treatment. It is available alone, or mixed with collagenase. It requires Ca2+ for activity and its action is readily neutralised by EDTA. 

Further trypsinisation cycles (2 or 3) are carried out until all the tissue is disaggregated. 

Efforts to predict the risk of potential QT prolongation over the last decade have resulted in the number of preclinical in vitro and in vivo methodologies. In vitro methods to predict QT liabilities range from simple recombinant cellular assays to disaggregated cells, isolated tissues and perfused heart preparations." ... 

Stamler, J., Vaughan, DE, and Loscalzo, J. 1989. Synergistic disaggregation of platelets by tissue-type plasminogen activator, prostaglandin El, and nitroglycerin. Circ Res 65 796-804. 

Interpretation of the data is complicated by the presence of nonapoptotic cells with damaged membranes. Such cells may have phosphatidylserine exposed on plasma membrane and, therefore, similar to apoptotic cells, bind annexin V. Mechanical disaggregation of tissues to isolate individual cells extensive use of proteolytic enzymes to disrupt cell aggregates, remove adherent cells from cultures, or to isolate cells from tissue mechanical removal of the cells from tissue culture flasks (e.g., by a rubber policeman) and cell electroporation, may affect the binding of annexin V. Such treatments, therefore, may introduce experimental bias in subsequent analysis of apoptosis by this method. 

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