Enhanced Enzymatic Dispersion of Primary Cells from Tissue
Research Opportunity -
Developing an enhanced dispersion of viable single cells from animal tissue fragments
First draft PROTOCOL for preparing a highly dispersed tissue sample:
If the animal tissue sample is more than a 50-100 mg (wet weight), first dispersed it with a Tissue Press (TP), MicroMincer (MM), or Meat Grinder (MG). (see www.biospec.com for these devices). The semi-solid output of these devices does not involve suspension in buffer or other liquid media and consists mostly of small aggregates of viable cells. The texture of the dispersed material varies from that of toothpaste for the TP and MM to hamburger-like material from the MG.
The mechanically dispersed tissue, having a greatly enhanced surface area, is now ready for the enzymatic digestion of its intra-cellular matrices. [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), for excellent reviews and guidelines on classic methods for Tissue Dissociation by Enzyme Digestion]. The reviews emphasize that after the tissue has been incubated in the dissociation enzymes, a crucial part of primary cell isolation is trituration using a 10 ml pipette by repeatedly filling and emptying the barrel at a rate of about 3 ml per sec. The optimal trituration rate for the tissue of choice is determined through trial and error while being careful to avoid bubbles in the cell suspension.
The present Protocol eliminates manual trituration and substitutes that method with inclusion of 6 mm diameter Plastic Beads (available from BioSpec Products in the enzyme/tissue cocktail during or after matrix digestion. , The density of the plastic beads have a density similar to the enzyme digestion cocktail, thus being gentle enough to avoid loss of cell viability by excessive shearing effects.
During enzymatic digestion of tissue intracellular matrix, a viscous, gel-like layer forms on the surface of cell aggregates. This layer decreases the rate of diffusion of the added hydrolytic enzyme to its target substrates - the intracellular matrix. With added beads and gentle mixing during incubation, the surface of tissue fragments will be massaged - quickly dispersing the gel layer. The net benefit will be faster digestion times and higher yields of viable cells.
To maximize viable cell yield, avoid vigorous stirring and excessive exposure to air bubbles in the digestion cocktail. In this regard, the inclusion of "good" Poloxamer-188 is an important stabilizer (for details see Narayanappa et. al., Biotechniques, V.67, No.3, p.98-109 (Sep 2019).
Experimental variables to explore are the amount of beads, stirring intensity, incubation time, the effect of aeration and incubation temperature. A study of the later variable may reveal unexpected results. While increasing incubation temperature is expected to increase the rate of enzyme digestion of the tissue, lower temperatures may improve the yield of viable single cells. For example, it is known that low temperatures physically alter attachment points of cultured cells to artificial surfaces. The same may hold true for original "cell to cell" contact. While incubation of the tissue/enzyme digestion cocktail containing stirred beads at 10 deg C will clearly require more time to achieve high tissue digestion yields compared to enzyme digestion at room temperature or native-tissue-context temperatures (typically 37º C), the fraction of viable cells may be significantly higher.
To request a sample of Plastic Beads contact Tim Hopkins at firstname.lastname@example.org.