Human peripheral blood mononuclear cell subfractionation using counterflow centrifugation elutriation.

BACKGROUND

Counterflow Centrifugation Elutriation (CCE) can be used to separate different amounts of cells into distinct subpopulations on the basis of different sedimentation characteristics. It was the aim of this study to evaluate CCE as a technique for fractionation of normal human peripheral blood.

METHODS

Human peripheral blood mononuclear cells (PBMC) were separated into three fractions by means of CCE.

RESULTS

The first two fractions, eluted at a flow rate of 21 ml/min and 25 ml/min, respectively, were composed of lymphocytes with less than 2% and 4% contaminating monocytes. The cells in these fractions were capable of responding to T-cell mitogens, such as phytohemagglutinin (PHA). The third fraction, collected at the termination of the run, was composed predominantly (greater than 85%) of large monocytes that strongly labelled leu-M3 (CD 14); these cells were unable to respond to PHA stimulation. The cell recovery after CCE was considerably high (greater than 93%), and all fractions retained high cell viability (greater than 96%) and were available for further manipulation.

CONCLUSIONS

In conclusion, our data demonstrate that CCE is a technique suitable for large-scale separations that makes highly purified cell fractions available for further manipulation.