Enhanced Enzymatic Dispersion of Primary Cells from Tissue

A new PROTOCOL for preparing high yields of viable tissue cells:

If the animal tissue sample is more than 50-100 mg (wet weight), first dispersed it with aTissue Press (TP), MicroMincer (MM),  or Meat Grinder (MG).  The semi-solid output of these devices does not require suspension in buffer or other liquid media and consists of small aggregates of viable cells.  The texture of the dispersed animal tissue varies from "toothpaste-like" for the TP and MM to "hamburger-like" for the MG.

The mechanically dispersed tissue aggregates, now having a greatly enhanced surface area, are ready for the disaggregation to single cells by enzymatic digestion of their intra-cellular matrices.  [For more 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...Tissue Dissociation Tips (1), (2), and (3)].  The reviews emphasize that after the tissue has been incubated with proteolytic enzymes to digest the protein matrix holding the cells together, a crucial part of cell isolation is a process of trituration (e.g., separation of the tissue cell aggregate by repeatedly filling and emptying the softened tissue digest in the barrel of a 10 ml pipette at a rate of about 3 ml per sec while carefully to avoid bubbles in the cell suspension). The object of trituration is to minimize the loss of viability of sensitive cells caused by excessive shearing.  The trituration rate and the number of passes through the pipette will depend on the tissue of choice and must be determined through a tedious process of trial and error.

This present Protocol proposes the elimination of the above manual trituration process.  Instead, trituration is replaced with the mixing of the enzyme/tissue cocktail in the presence of 6 mm diameter low-density Plastic Beads (available from BioSpec Products).  Importantly, these plastic beads have a density similar to the enzyme digestion cocktail.  It is reasoned that during digestion, collisions of the tissue with these low-density beads will be gentle enough to avoid loss of cell viability.

During enzymatic digestion of tissue intracellular matrix, a viscous, gel-like layer forms on the surface of cell aggregates.  This layer decreases diffusion of the added digestion enzyme(s) to their targeted substrates -- the proteins of the intracellular matrix.  With added beads and gentle mixing during incubation, the surface of tissue fragments will be "massaged", the gel layer dispersed, and digestion rates increased, thus speeding up the tissue disassociation and producing higher yields of viable cells.

To maximize viable cell yield, vigorous stirring and excessive exposure to air bubbles in the digestion cocktail must be avoided.  In regard to the latter problem, the inclusion of Poloxamer-188 in the digestion cocktail may be an important stabilizer (for details see Narayanappa et. al., Biotechniques, V.67, No.3, p.98-109 (Sep 2019).

Experimental variables to explore in perfecting this bead-assisted protocol for preparing high yields of viable tissue cells include Amount of beads added to the digestion mix, Stirring intensity, Incubation time, Effect of aeration, and the Effect of incubation temperature.  A study of incubation temperatures may be especially interesting.  Low incubation temperatures may actually improve the yield of viable single cells.  For example, it is known that low temperatures physically alter the attachment points of cultured cells to artificial surfaces.  The same may hold true for "cell-to-cell" contact.   While incubation of a tissue/enzyme digestion cocktail containing stirred beads at 10 deg C will require more time to complete, the proportion of viable cells might be significantly higher.

Contact Tim Hopkins at hop@biospec.com for a sample of Plastic Beads.