An improved method for preparing refrozen rethawed human lymphocytes on plates for microcytotoxicity studies.

This report describes simplified methods for the initial freezing and thawing of human lymphocytes and the subsequent use of these cells after refreezing on cytotoxicity plates, storage, and a second thaw. The proposed initial freeze method eliminates some technical inconveniences required previously such as chilling of cells prior to addition of DMSO, preparing cryoprotective mixtures just prior to freezing, controlled rate of freezing and thawing and the washing of cells after thawing. However, pH of the media, blood freshness, type of storage tube used, and constant storage temperature were found to be very important to maintain good cell viability. Most lymphocytes maintain an average viability of 85 to 95% for at least a year when prepared according to the present freezing and thawing technique.When panels of lymphocytes are prepared for refrozen rethawed cytotoxicity test plates, the thaw time between freezes must be brief. Production of test plates on ice, however, was not found to be necessary. As the period of storage of refrozen cells on plates increases, viability of the cells after a second thaw decreases and treatment with DNase to enzymatically remove the dead cells is useful. With this procedure, refrozen rethawed lymphocytes up to a year old can be prepared on microcytotoxicity test plates with average viabilities of 90 ± 1%.