Cryopreservation of whole blood and isolated lymphocytes for B-cell immortalization

Summary

A simple method is described for the cryopreservation of 1 ml of whole blood followed by the establishment of immortal B-cell lines (lymphoblasts). In this procedure, lymphocytes are isolated by gradient centrifugation and red cell debris is removed by panning. Transformations are initiated by Epstein-Barr infection of lymphocytes after cryopreservation of whole blood in 10% dimethylsulfoxide (−180° C) for several weeks to 2 yr. The success rate for the establishment of lymphoblasts from 115 samples of frozen whole blood is 83%. This method is compared to a second group of 126 transformations prepared from 5×10⁶ frozen lymphocytes (90%) and a third group of 154 samples using 1×10⁶ fresh lymphocytes (96%). Full transformation is achieved in an average of 31 days and does not vary significantly between the experimental groups. Fluorescence activated cell sorting of thawed cells from whole blood stored at −180° C for as long as 2 yr reveals normal ratios of B and T cells. The results of this study demonstrate that small volumes of blood are adequate for transformation and that whole blood may be stored at −180° C without significant loss of viability. This protocol is particularly useful for the transformation of B-cells from small blood draws and aliquots of stored blood.