Curve -shift analysis of cryopreserved killer T cell function

The conditions for cryopreservation and reconstitution after thawing of cytotoxic effector cells for cell mediated lympholysis (CML) tests are studied. Percentage recovery of functional activity is analysed not by the commonly used method of comparisons at a single concentration of effector cells but by the movement of the curve of functional activity on the axis of cell concentration in culture. By this method the position of the ascending slope of the dose-response curve, at several different effector to target cell ratios, is compared between fresh and frozen-thawed cells. Cooling rates giving optimal recovery were found to vary between experiments. Using simple techniques, optimal cooling rates were found to range from 0.3 to 1.0 degrees C/min, when using dimethyl sulphoxide (10% v/v). Dead cell debris, but not intact dead cells (which take up eosin), were shown to inhibit the lytic ability of functionally active frozen-thawed effector cells. This could be removed by centrifuging the thawed cells over Ficoll-Hypaque. In the recovered population the proportion of cells which excluded the vital dye, eosin, was greater than the proportion of functioning effector cells. This suggested that the cells which excluded eosin contained both functional and nonfunctional, partially damaged effector cells. Thus dye exclusion methods generally over-estimated the functional activity of thawed effector cells. When cells to be used as targets were preserved prior to treatment with phytohaemagglutinin (PHA) no abnormalities were detected in their behaviour to fresh cells.