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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 disperse it with a Screw Tissue Press, Lever Tissue Press,  or MicroMincer.   The semi-solid output of these devices requires no suspension in a buffer or other liquid media and delivers dispersed material having a texture similar to that of toothpaste.  The very small tissue aggregates remain viable. The mechanically dispersed tissue, now having a greatly increased surface area, is ready for enzymatic digestion of the intra-cellular matrices and, as a result,  leaving intact viable single cells. 

Enzymatic Digestion of the Cell Matrix and Trituration

A couple of excellent reviews and guidelines on the classic method for Tissue Dissociation by Enzyme Digestion (see http://www.worthington-biochem.com/tissuedissociation/basic.html  and https://www.chiscientific.com/News1.aspx?type3) emphasize that after the tissue has been incubated in a mixture of select proteolytic enzymes, a final step in primary cell isolation is trituration.  Trituration is defined as a mild shearing technique that involves repeatedly filling and emptying the barrel of a 10 ml pipette at a rate of about 3 ml per sec while being careful to avoid leaving bubbles in the cell suspension. Depending on the tissue type, the optimal trituration rate is determined through trial and error.

Elimination of Trituration

Classic "Trituration" is necessary because a viscous layer consisting of partially hydrolyzed proteins form within the cell aggregates during incubation with the proteolytic enzymes. Release of the single cells is aided by gentle mechanical agitation.  But, too much agitation and the cell are damaged.

The formation of a viscous, partially digested matrix layer during incubation also slows the rate of diffusion of lytic enzymes to their target substrates in the intracellular matrix.

It is suggested that the need for classic trituration with a pipette can be replaced by the addition of low-density Plastic Beads to the enzyme/tissue digestion cocktail.  With densities in the range between one and two g/ml, the beads tend to suspend in the reaction mixture.  The tissue/enzyme reaction mix with beads is incubated with slow rocking or rotating motion, thus causing the beads to make gentle contact with the surface of the tissue fragments. Gently massaged, the gel layer is dispersed into the media as it forms.  In effect, continuous Trituration is added during, rather than after, digestion of the tissue. Faster digestion times and higher yields of viable single-cells are expected.

Other enhancements

Optimization of the above-proposed improvements includes examining variables such as the number of beads used, stirring intensity, incubation time, effect of aeration, and incubation temperature. 

  • What is the effect of Including Poloxamer-188 in the digestion mix?  It is known that this polymer functions as a stabilizer during the classic trituration step. See Narayanappa et. al., Biotechniques, V.67, No.3, p.98-109 (Sep 2019).
  • A study of the incubation temperature may reveal some unexpected results. It is empirically known that low temperatures alter attachment points of cultured cells to artificial surfaces.  Similiar temperature-dependent dynamics might hold true for the native cell to cell contacts.  Would a lower incubation temperature, although prolonging the total incubation time, actually improve the final yield of viable single cells?

 

For a sample of Plastic Beads contact Tim, our Tech Guru... hop@biospec.com.

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