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Enhanced Enzymatic Dispersion of Primary Cells from Tissue

 - a proposed protocol for improved dissociation of tissue into viable single cells -

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Dispersing whole tissue samples

If the amount of animal tissue sample is more than 50-100 mg (wet weight), first dispersed it with a Tissue Press (TP), MicroMincer (MM),  or Meat Grinder. The semi-solid output of these devices requires no suspension in a buffer or other liquid media and delivers small aggregates of viable cells.  The texture of the dispersed material is similar to that of toothpaste for the Tissue Press and MicroMincer and is more course, hamburger-like from the Meat Grinder.

Enzymatic Digestion of the Cell Matrix

The mechanically dispersed tissue, now have a greatly increased surface area, is ready for enzymatic digestion of its intra-cellular matrices, thus freeing intact, viable single cells.  [ For excellent reviews and guidelines on classic methods for Tissue Dissociation by Enzyme Digestion see http://www.worthington-biochem.com/tissuedissociation/basic.html  and https://www.chiscientific.com/News1.aspx?type=3Links ].  These reviews emphasize that after the tissue has been incubated in a solution of special dissociation enzymes, a crucial final step in primary cell isolation is trituration.  Trituration is a mild shearing technique the involves repeatedly filling and emptying the barrel of a 10 ml pipette at a rate of about 3 ml per sec. Depending on the tissue type, the optimal trituration rate for the tissue of choice should be determined through trial and error while being careful to avoid leaving bubbles in the cell suspension.

Elimination of Trituration

An untested protocol enhancement that eliminates labor- and time-dependant Trituration is proposed..the ... Replace manual Trituration with gentle mixing of the enzyme/tissue cocktail using special 6 mm diameter Plastic Beads. The Plastic Beads, would have a density of about 1 g/cc - a density similar to the enzyme/tissue digestion cocktail and would impart a gentle physical mixing while minimizing loss of cell viability caused by shear.  It is reasoned that during the digestion process a viscous layer forms on the surface of cell aggregates. This gel-like layer would interfere with the diffusion of lytic enzymes to its target substrate - the intracellular matrix. With the added beads and gentle mixing during incubation, the surfaces of tissue fragments will be "massaged" - thus, hastening dispersion of the gel layer. Significantly faster digestion times and higher yields of viable cells are to be expected.

Other enhancements

Include Poloxamer-188 during trituration as a stabilizer. It maximizes viable cell yield by dampening vigorous stirring and its detrimental shearing effects and also helps prevent excessive exposure to air bubbles in the digestion cocktail.  For details see Narayanappa et. al., Biotechniques, V.67, No.3, p.98-109 (Sep 2019).

Optimization of these improvements includes examining the number of beads used, stirring intensity, incubation time, effect of aeration, and incubation temperature.  A study of the later variable, for example, may reveal some useful but unexpected results.  While increased incubation temperature is expected to increase the rate of enzyme digestion of tissue, a lower temperature, while prolonging the incubation time, may improve the net yield of viable single cells.  It is empirically known that low temperatures physically alter attachment points of cultured cells to artificial surfaces.  The same temperature-dependent dynamics may hold true for native "cell to cell" contacts.

          For a free sample of low density Plastic Beads contact Tim Hopkins at hop@biospec.com.

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