To obtain a maximum yield of functionally viable, single cells, the choice of the most appropriate enzymes in the correct ratios are crucial including the appropriate incubation times with the tissues.  Figure 3 illustrates the relationship between cell viability and yield vs. selection of dissociation enzymes and incubation time.  Aggressive dissociation or poor dissociation of tissues will result in low cell viability.  Outlined below are some possible explanations for sub-optimal cell isolation and the solutions to obtain the most optimal results.

1)  Low yield and low viability: Most likely tissues are dissociated too aggressively which results in cellular damage. Change the type of enzyme and/or decrease the working concentration. (e.g. from trypsin to collagenase/ from Type 2 collagenase to Type 1).

2)  Low yield and high viability: The tissues are digested gently using enzymes that are too mild or the incubation time is too short.  Increase the enzyme concentration and/or the incubation time and monitor both the yield and the viability response.  If the yield remains poor, evaluate a stronger digestive type of enzyme and/or the addition of secondary enzyme(s).

3)  High yield and low viability: The tissues are probably digested with the appropriate enzymes but the enzyme composition is too strong and/or at a high working concentration that causes cellular damage.  Reduce the concentration and/or the incubation time and monitor the yield and the viability response. Alternatively, dilute the proteolytic action by adding bovine serum albumin (BSA) (0.1 - 0.5% w/v) or soybean trypsin inhibitor (0.01 - 0.1% w/v) to the enzyme mixture.

4)  High yield and high viability: These conditions strike the optimal balance for the selected tissues.

In addition to the dissociation procedures mentioned above, trituration can be a crucial part of primary cell isolation. This action of repeatedly pipetting the mixture allows the tissues to break up into fragments, after the tissues have been incubated in the dissociation enzymes.  If done too vigorously, cells will be destroyed lowering viability; if done too weakly, tissue fragments will be left intact lowering yield. The correct method is to use gentle trituration, using a 10 ml pipette by filling and emptying the barrel at a rate of about 3.0 ml per sec.  The best method to determine a suitable trituration rate for the tissue of choice is through trial and error cautious to avoid any bubbles in the cell suspension.